IE863335L - Gm-csf protein, derivatives and preparation - Google Patents

Abstract

CSF proteins which are biologically active are obtained by expression of a gene coding for human granulocyte macrophage colony stimulating factor (CSF) in bacteria. Biologically active derivatives with a modified amino acid sequence are obtained by altering the natural or synthetic gene structure.

Description

59V79 I Human granulocyte macrophage colony-stimulating factor (GM-CSF) is a glycoprotein with a molecular weight of about 23,,000 dalton* The cDMA sequence and the expression of the glycoprotein in mammalian cells have already been 5 disclosed (<3..G. Wong et &i. Science 228 (1985), 810-815,. D- Metcalf, Science 229 (1985), 16-22).

The invention relates to human granulocyte macrophage colony-stimulating factor proteins (GM-CSF) of the formula Pro- (As) s-CSF (12-126) -2 10 in which (As)s denotes all or some of the first 11 amino acids of the natural GM-CSF sequence, and E denotes Glu or Asp, hereinafter referred to aa CSF. Further aspects of the invention and preferred embodiments thereof are illustrated in further detail below ar are defined in the 15 patent claims.

The invention furthermore relates to the preparation of CSF -by expression in bacteria, in particular in E- coli. In particular, it is possible to use for this purpose the published cDNA sequences which can be obtained in a 20 manner known per se, preferably by synthesis.

The invention additionally relates to expression vectors for use in bacteria,, in particular in S. coll, which contain, in a suitable arrangement ("operatively linked to"), a DN& coding for CSF or a CSF fusion protein,. 25 The invention additionally relate© to biologically active derivatives of CSF which can be obtained by modifications, which are known per s&, ox the DMA sequences. Thus, for example, it is possible in the construction of vectors for fusion proteins to incorporate cleavage sites which, 30 after elimination of the CSF protein, have C-terminal and N-tenninal modifications in the amino acid sequence. Furthermore, the invention relates to the use of proteins of this type in medical treatment and to their use for 3 the preparation of medicaments, and to medicaments which contain CSF protein and its biologically active derivatives, in particular medicaments for the stimulation of proliferation of hemopoietic cells and for promotion of the 5 formation of granulocytes and macrophages.

The invention is furthermore illustrated by Figures 1 to 15, each of which explains, mostly in the form of a flow diagram, the processes of the examples of the same numbers „ These figures ar© not to scale, in particular the scale 10 has been "expanded" in the region of the polylinkera.

Thus, Figure 1 and its continuations la and lb show the preparation of the vector pW 225 which is used for the direct expression of (Met-)CSF. This product can be converted by acid cleavage into CSF according to the 15 invention. The figures which follow relate to vectors which result in the expression of fusion proteins in which a "ballast" protein, which is derived from a part-sequence of hiu&an interleukin~2, hereinafter "IL-2" or "aIL~2 ", is located at the terminal end in front of the 20 CSF amino'acid sequence: Figure 2 and its continuations 2a and 2b show the preparation of the vector pW 216 which codes for a fusion protein from which is obtained.,, by acid cleavage, a CSF derivative which is extended at the M-terminal end by the 25 amino acid proline- Figure 3 shows the synthesis of the vector pW 240 which codes for a fusion protein which results, after acid cleavage, in a CSF derivative which has proline in place of the first amino acid falanine). 30 Figur® 4 relates to the preparation of the vector 241 which codes for a fusion protein which results, after acid cleavage, in a CSF derivative in which the first aiaino acid {alanine) is Missing.

Figure 5 demonstrates the preparation of the tree-tor pW 242 which codes for a fusion protein which, results, after acid cleavage, In a CSF derivative in which the first five amino'-, acids have been eliminated.

Figure 6 relates to the preparation of the vector pW 243 which codes for a fusion protein which results, after acid cleavage, in a CSF derivative in which the first seven amino acids are missing.

Figure 7 shows the synthesis of the vector pW 244 which codes for a. fusion protein with which is obtained, after acid cleavage, a CSF derivative in which the first 11 amino acids have been eliminated.

Figure 8 and its continuation 8a show the synthesis of the vector pW 246. This codes for a fusion protein in which two modified sequences, denoted "CSF'", follow the IL-2 part-sequence. Acid cleavage results in a CSF derivative in which proline is located at the N-temninal end JLn front of the first amino acid proline and in which the last amino acid has been replaced by aspartic acid.

Figure 9 shows the synthesis of the vector p¥ 247 which codes for a fusion protein in which three CSF" sequences follow the IL-2 part-sequence. Acid cleavage results in the CSF derivative characterized in Figure 8 being obtained.

Figure 10 and its continuation Figure 10a show the preparation of the hybrid plassnids pS 200 to 204 which contain synthetic CSF DNA part-sequences, the plasmid pS 200 containing "synthesis block 1% shown in Appendix I, plasmid pS 201 containing "synthesis block II" shown in Appendix II, plasmid pS 202 containing "synthesis block III" shown in Appendix III, pi&ssaic! pS 203 containing the entire synthetic gene, and pS 204 representing an expression plasmid which likewise contains the entire synthetic CSF DMA sequence-Expression and acid cleavage result in the same CSF 5 derivative as described in Figure 2 being obtained- Figure 11 and its continuation Figure 11a show the synthesis ''of the expression plaemid pS 207 which codas for a fusion protein which provides, after cleavage with 5 N-bromosuccinimide, a CSF derivative in which Trp in each of positions 13 and 122 has been replaced by His.

Figure 12 shows a synthetic DMA part-sequence which permits the preparation of a CSF derivative in which lie in position 100 has been replaced by Thr- 10 Figure 13 and its continuation Figure 13a show the synthesis of the expression plasmid pS 210 which codes for a fusion protein which provides, after cleavage with cyanogen bromide f a CSF derivative in which ail methionine residues have been replaced toy neutral amino acids, 15 namely by lie in position 36 and by Leu in positions 46, 79 and 80.

Figure 14,shows a synthetic DMA sequence which permits, in accordance with the synthesis scheme in Figure 13, the preparation of a CSF derivative in which Met In position 20 36 has been replaced by lie, and Met In position 46 has been replaced by Leut» and a single Leu residue is present in place of amino acids 79 and 30.

Finally, Figure 15 shows a synthetic DBA whose use in the synthesis scheme shown in Figure 13 permits the prepara- 25 tion of a CSF derivative In which Met in position 36 has been replaced by lie and in position 46 has been replaced by Leut* and in which the two casino acids In positions 79 and 80 have been deleted.

The possible variations explained la these figures and 30 examples are, of course, merely examples of the large numbers of modifications which are possible'according to the invention. Thus, it is also possible in a manner known per se t© use other protein sequences, especially 6 bacterial, as the "ballast" portion of the fusion proteins, and it is possible to use all customary method© for the linkage and cleavage of the fusion proteins, it being possible for other CSF derivatives with a modified amino acid sequence in the molecule or at both ends ox the molecule to result- The choice of the IL-2 sequence and the synthetic DMA sequences and the cleavage of the fusion proteins should thus be viewed merely as preferred embodiments of the invention which can be varied in a manner known par se.

It has emerged that the "open reading frajne"'.comprising a DNA which codes for interleukin-2 is particularly advantageous as an expression aid for the expression of peptides and proteins, and that an N-terminal portion of IL-2 which essentially corresponds to the first 100 amino acids is particularly well suited for the preparation of fusion proteins. The primary product obtained in this way is a fusion protein which is composed entirely or very predominantly of eukaryotic protein sequences - Surprisingly , this protein is apparently not recognized as being a foreign protein by the protaases which are intrinsic to the host, nor is it immediately degraded again- Another advantage is that the fusion proteins according to the invention are sparingly soluble or insoluble and thus can easily be removed, appropriately by centrifugation, from -the soluble proteins - Since, according to the invention,, the functioning of the "ballast portion" of the fusion protein does not depend on the IL-2 portion being a biologically active molecule, it likewise does not depend on the exact structure of the IL-2 portion. It suffices for this purpose that essentially the first 100 H-terminal amino acids are present. Thus, it is possible, for example# to carry out at the N-terminal end modifications which permit cleavage of the fusion protein in the ease where the desired protein is located N-terminal thereto. Conversely# modifications at the C~terminal end can be carried out in order to permit 7 or facilitate the elimination of the desired protein.

The natural DMA sequence coding for human XL-2 is disclosed in the European Patent Application with the publication muaher 0,091,539. The literature quoted there 5 also relates to mouse and rat IL-2. These mammalian DMAs can be used for the synthesis of the proteins according to the invention« However, it is more appropriate to start from a synthetic DNA, particularly advantageously from the DMA for human IL-2 which has been described in 10 German Offenlegungsschri£t 3,419,995 and in Patent Specification No- 1319/85. This synthetic DNA. not only has the advantage that in its choice of codons it is suited to the circumstances in the host 'tjhich is used most frequently, £. coli, but it also contains a number of 15 cleavage sites for restriction endonucleases at the start and in the region of the 100th triplet, it being possible to make use of these according to the invention. However, this does not rule out modifications to the DMA being carried out in the region lying between them,, it being 20 possible to make use of the other cleavage sites.

If use is made of the nucleases Ban II, Sac I or Sst I, then the IL-2 part-sequence which is obtained codes for about 95 amino acids- This length is, in general,, sufficient to obtain an insoluble fusion protein. If the lack 25 of solubility is still inadequate, for example in the case of a desired hydrophilic CSF derivative,, but it is not wanted to make use of cleavage sites located nearer to the C-terminal end - in order to produce as little "ballast" as possible - , then the DNA sequence can be 30 extended at the N-terxain&l and/or C-terminal end by appropriate adapters or linkers and thus the "ballast" portion can be "tailored" to requirements. Of course,, it is also possible to use the DNA sequence - more or less -up to the end and thus generate biologically active 35 IL-2 - modified where appropriate - as "by-product".

The cleavage of the fusion protein can be carried out 8 chemically or ©nzymatieally in a manner known per se. The choice of the suitable method depends, in particular, oa the amino acid sequence of the desired protein. If there is tryptophan or methionine at the carboxyl terminal end 5 of the bridge member Y, or if Y represents Trp or Met, then chemcal clea-vage with N~bromosuccinimide or cyanogen halide can be carried out in the cases where the particular CSF derivatives which are synthesized do not contain these amino acids. 10 CSF and those of its derivatives which contain in their amino acid sequence Asp - Pro and are sufficiently stable to acid can, as already shown above, be cleaved proteolytically in. a manner known, per 15 se. This results in proteins %?hieh contain proline at th© ^-terminal end or aspartic acid at the C-terminal end "being obtained. Thus, it is possible in this ^ay also to synthesize modified proteins.

The Asp-Pro bond can be made even more labile to acid if 20 this bridge member is (Asp)„-]Pro or Glu-(Asp)n-Pro, n denoting 1 to 3.

•Examples for. enzymatic cleavages are likewise known, it also being possible to use modified enzymes having improved specificity j?e£» C.5. Craik et al., Science 228 25 (1985) 291-297)- The fusion protein is obtained by expression in a bacterial expression system in a roasaer known per se. Suitable for this purpose are all known host-vector systems, such as bacteria ox the varieties Streptoo&yces, 30 B. subtilis, Salmonella typhimuriusa or Serratia saarees-cens, in particular 3- coli.

The DNA sequence which codes for the desired protein is I incorporated in a known maimer in a vector which ensures good expression in the selected expression, system.

It is appropriate for this to select the promoter and operator from the group comprising trp, lac, tac, PLor Pa of phage x, hsp, oinp or a synthetic; promoter as proposed inp for example, German Offenlegungsscrift 3,430,683 or Patent Specification IMo. 2048/85,, The tac promoter-operator sequence is advantageous, and this is now commercially available (for example expression vector pKK223-3, Pharmacia, "Molecular Biologicals t Chemicals and Equipment for Molecular Biology™, 1984, page 63).

It may prove to be appropriate in the expression of the fusion protein -according to the invention to modify individual triplets for the first few amino acids after the ATG start codon in order t'o prevent any base-pairing at the level of the mRNA. Modifications of this type, such as deletions or additions of individual amino acids, are familiar to the expert, and the invention likewise relates tq them.

Particularly advantageous CSF derivatives are those containing N-termin&l proline, since proteins of this type are more stable to attack by proteases. The CSF derivative which has the entire CSF amino acid sequence following the proline added to the Si-terminal end is particularly preferred- However, it has emerged, surprisingly , that the variants of the CSF molecule obtained by elimination of the first 11 amino acids also have biological activity. variants of the invention which are also advantageous are those which initially result in fusion proteins which contain the CSF sequence more than once, advantageously twice or three times. By their nature, the ballast portion in these fusion proteins is reduced, and thus the yield of the desired protein is increased- 10 The plasmid pHG 23 which was obtained by incorporation of the CSF cDNA sequence into the Pst I cleavage site of BR 322 has bean deposited, in 2. coli^ at the American Type Cultu.ce Collection under number A.TCC 39900* The DMA 5 sequence of this corresponds to the variant described in Figure 3 (B) of Wong et al. The incorporation made use of the Pet I cleavage site near the 5'' end, on the one hand, and of a Pst I site introduced at the 3' end by GC tailing (EP-A 0,183,350)- 10 Example 1 Direct Expression of CSF The commercially available vector pUC 12 is opened with the restriction enzymes Sma I and Pst I, and the large fragment (1) is isolated. 15 By cutting the cDNA sequence for CSF with the enzymes Sfa MI and Pst I is obtained the fragment (2) which is ligated with the synthetic linker (3) and then with the pUC 12 fragment (1)- The hybrid plasmid pW 201 (4) which is thus obtained contains the CSF DNA sequence following 20 the start codon, ATG.

The hybrid plasmid (4) is opened with Nco I, and the protruding ends are filled in to give the blunt-ended fragment (5). The vector pUC 12 is opened with the enzyme Eco RI, whereupon the protruding ends are filled in. This 25 is followed by treatment with bovine alkaline phosphatase , the pUC 12 derivative (6) being obtained.

Ligation of the fragments (5) and ffi) results in vectors which contain the CSF DNA sequence in both orientations being obtained. They are called pW 203 (7). 30 Using Eco RI and Rsa I on the vector |7) results in isolation of the fragment (9) which contains the codons for amino acids 63 to 127 of CSF. On the other hand, 11 cutting the vector (4) with Nco I and Hsa I results in isolation of the fragment (9) which contains the codons for amino acids 1 to 61 of CSF.

The plasmid pH 131/5 (German Offenlegungsschrift 5 3,514PT13 or Patent Specification No. 1031/86, Example 1f Figure 1) (10) is cut 'with Pvu II, the small fragment is removed, and the larger one ia ligated to give the plasmid pPH 160 (11) which is present in £. coli cells in a higher copy number than pH 131/5. The plasmid (11) is opened with Nco I and 10 Eco RI, and the large fragment (12) ia isolated.

The fragments (8)tr (9) and (12) are now? ligated to give the hybrid plasmid pW 206 (13). This restores the codon for amino acid 62.

The commercially available plasmid pKK 65-10 (PL Bio-15 chemical Inc») is cleaved with Eco RI, and the fragment (14) which contains the two terminators Tl and T2 is isolated.. This fragment (14) is inserted into the plasmid (13 )^ which has been opened with Eco RI, the plasmid pW 225 (15) being obtained. 20 E. coli 24 bacteria which contain the pis,sudd (15) are cultured in LB medium (J-B. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, 1972) containing 30 to 50 ^g/sal ampicillin at 37 ®C overnight., The culture is diluted in the ratio 1:100 with M9 medium 25 (J.M. Miller, op. cit-) which contains 200 pm/1 casamino acids and 1 pg/1 thiamine, and the mixture is incubated at' 37*C with continuous agitation- At an ODeoo - 0-5 or 1 indolvl-3-acrylic acid is added to a final concentration of 15 jug/1, and the mixture is incubated for 2 to 3 hours 30 or 16 hours respectively- The bacteria are then removed by eeimtrif ligation.. The bacteria are boiled for five minutes in a buffer mixture (7M urea, 0.1% SDS, 0-1 M sodium phosphate, pH 7.0), and samples are applied to an SDS gel electrophoresis plate. It emerges that the 35 protein pattern of cells whose trp operon has been 12 induced contains a new protein, in the range: of about 14,000-18,000 dalton, which is not found with non-induced cells- The induction conditions which have been indicated apply 5 to shake cultures? for larger fermentations appropriately modified OD imlnes and, where appropriate, slight variations in the inducer concentrations are advantageous.

Example 2 Pro°-CSF 10 The vector pUC 12 is opened with Eco RI and Pst I, and the large fragment (16) is isolated. This fragaisnt (16) is ligated with the synthetic DNA fragment (17) and the fragment (2) (Example 1; Figure 1)„ Competent cells of E„ coli JM 103 are transformed with the ligation mixture, 15 and the desired clones which contain the plasmid pW 212 (18) are selected- The fragment (19) which contains the CSF sequence is cut out of the plasmid DNA using Pvu I and Pst I.

Insertion of the lac repressor (P-J. Farabaugh, Nature 20 274 (1978) 765-769) into the plasaiici pKK 177-3 contain •the puC 8 polylinker (Amann et al., Gene 25 (1983) 167; Patent Specification No. 1930/84) results in the plasmid pJF 118 (20) being obtained (Fig. 2a; cf. German Patent Application P 35 26 995.2, Example 6, Fig. 6). The latter is opened at the 25 unique restriction site for Ava I, and is reduced in size by about 1,000 bp by exonuclease treatment in a manner known per se. Ligation results in the plasmid pEH 1000 (21) being obtained, in which the lac repressor gene is completely retained but which, because of the reduction 30 in size, is present in a markedly higher copy number than the initial plasmid.

In place of the plasmid pKK 177-3, it- is also possible to 13 start from the sboveraentioned a otzme r c i a 11 y available plasmid pKK 223-3, to incorporate the lac repressor, and to shorten the resulting product analogously.

The plasmid pEW 1000 (21) is opened with the restriction 5 enzymes EcoR I and Sal I, and the fragment (22) is isolated.

The plasmid pl59/6 (23), prepared as described in German Offenlegungsschrift 3 ,,419^995 (Patent Specification No. 1319/85), Example 4 (figure 5)# is opened with the restriction enzymes 10 Eco RI and SA1 I, and the small fragment (24), which contains the IL-2 sequence, is isolated.

The hybrid plasmid pEW 1001 (25) is obtained by ligation of the fragments (22) and (24).

On. the one hand? the plasmid (25) is opened with Eco RI 15 and Pvu I, the fragment (26) which contains the largest part of the IL-2 sequence being obtained.. This part-sequence is denoted ®aIL2m in the figures.

On the other hand,, the plasmid (25) is opened with Eco RI and Pst I, and the large fragment (27) is isolated. 20 Ligation of the fragments (19)# (26) and (27), transformation of competent S. coli 294 cells, and selection results in clones which contain the plasmid pW 216 (28) being obtained. The plasmid DMA is characterized by restriction analysis and DNA sequence analysis - 25 An overnight culture of S. coli cells which contain the plasmid (28) is diluted with LB medium (J. H- Miller, op., cit-), which contains 50 pg/otl ampicillin, in the ratio of about 1:100, and the growth is followed via measurement of the OD. At OD = 0.5, the culture is adjusted to 30 1 mM in is©propyl 0-galactopyranoside (IPTG) and, after 150 to 180 minutes, the bacteria are removed by centrifu-gation. The bacteria are boiled for five minutes in a 14 buffer mixture (7M urea, 0*1% SDS£, 0.1 M sodium phosphate,, pH 7.0), and samples are applied to an 80S gel electrophoresis plate- Following electrophoresis, a protein Shaiad which ' corresponds to the size of the ex-5 pected fusion protein is obtained from bacteria which contain the plasmid (28). After disruption of the bacteria (French press? *Dyno Mill) and centrifwgation^ the fusion protein is located in the sediment so that it is possible already to remove considerable amounts of the 10 other proteins with the supernatant. Isolation of the fusion protein is followed by acid cleavage to liberate the expected CSF derivative which contains an additional N-terminal proline™ This shows activity in the biological test. 15 The induction conditions which have been indicated apply to shake cultures; for larger fermentations appropriately modified OD values and, where appropriate, slight variations in the IPTG concentrations are advantageous.

Example 3 20 Pro'-CSF (2-127) Ligation of the fragments (2) (Figure 1) and (16) (Figure 2) with the synthetic DNA sequence (29) results in the hybrid plasmid (30) which corresponds to the plasmid (18) apart from the synthetic DNA sequence. 25 Pvu I and Pst I are used to cut out of the plasmid (30) the fragment (31) which contains the CSF DNA. sequence in which, however, the codon for the first amino acid has been replaced by a codon for proline. Ligation of the fragment (31) with the fragments (26) and (27) results in 30 the hybrid plasmid pW 240 {32) being obtained. Expression in 2. coli, which is carried out as in Sxample 2, provides a CSF derivative in which the first amino acid has been replaced by proline. This derivative also shows biological activity.

* Trade Mark 15 Example i CSF (2-127) A plasmid which contains the CSP DNA sequence with a Pst I restriction site at its 3' end, for example the plasmid 5 pHG 23 (ATCC 39900), is cleaved with Sfa NI, and the linearized plasmid (34) is partially filled in using Klenow polymerase and GTP. The protruding nucleotide A is eliminated using SI nuclease, and then the fragment (35) is cat out with Pst I. 10 Ligation of the fragment (35) with the synthetic HHA sequence (36) and the fragment (IS) (Figure 2) results in the plasmid (37)f which is analogous to plasmid (18), being obtained.

Pvu I and Pst I are used to cut the fragment (38) out of 15 the plasmid (37). This fragment is ligated with the fragments (26) and (27)» by which means the plasmid pW 241 (39) is obtained.

Expression as in Example 2 results in a fusion protein which, after acid cleavage, provides a CSF derivative 20 missing the first amino acid. This derivative is biologi cally active.

Example 5 CSF (6-127) The plasmid (33) (or a cor responding plasmid which 25 contains the CSF DNA sequence) is first totally cleaved with Pst I and then partially cleaved with Bst NX, . and the fragment (40) is isolated.

The synthetic DNA sequences (41) and (36) (Figure 4) are first ligated to give the sequence (42), and the latter 30 is then ligated with the fragment (15) (Figure 2) and the 16 fragment (40)t the plasmid pW 212 (43) being obtained- Pvu I and Pst I are used to isolate from the plasmid (43) the fragment (44) which contains the DMA sequence for the CSF derivative. This fragment (4#) is ligated with the 5 fragments (26) and (27), which resalts in the hybrid plasmid pW 242 (45).

Expression as in Examples 2 results in a fusion protein from which is obtained, after acid cleavage, a CSF derivative missing the first five amino acids. This 10 product is also biologically active., Example 6 CSF (8-127) When first the synthetic DMA sequence (36) (Figure 4) is ligated with the synthetic DMA sequence (46), and there-1S after the resulting DMA fragment (47) is ligated with the fragments (40) and (IS)f then the hybrid plasmid (48) is obtained. Pvw I and Pst I are used to cut out of the latter the fragment (49) which contains the DNA sequence for the CSF derivative. Ligation of the fragments (49), 20 (26) and (27) provides the hybrid plasmid pW 243 (50) which corresponds to the plasmid (45) apart from the shortened DMA sequence for the CSF derivative.

Expression as in Example 2 results in a fusion protein which, after acid cleavage, provides a CSF derivative 25 missing the first seven amino acids. This derivative ia also biologically active.

Sxamole 7 CSF (12-127) 30 When the synthetic DMA sequence (51) is ligated with the fragments (33) and (16) then the hybrid plasmid (52) is 17 obtained- When Pvu I and Pst I are used to cut out of the latter the sequence (53), which contains the DNA sequence for the CSF derivative, and this fragment (53) is ligated with the fsr&gments (26) and (27) then the hybrid plasmid 5 pW 244 (54) which corresponds to the plasmid (45) apart from the shortened CSF sequence is obtained- Expression as in Example 2 results in a fusion protein which„ after acid cleavage, provides a CSF derivative from which amino acids 1 to 11 have been eliminated® This 10 shortened molecule is also biologically active.

Example 8 Pro°~CSF( 1-126)-Asp The DNA sequence (19) (Figure 2) is partially cleaved with Bst HI, and the fragment (55), which contains the 15 largest part of the CSF sequence, is isolated- Cleavage of the plasmid (33) (figure 4) (or of a corresponding plasmid -hieh contains the CSF DNA sequence) first with Pst I and then partially with Bst HI results in the DNA sequence (56) which comprises the largest part 20 of the CSF sequence being obtained.

-The DNA sequence (57) is synthesized which together with the sequence (56) provides a DNA sequence which codes for a CSF derivative in which the C-texminal glutamic acid has been replaced by aspartic acid. 25 The vector pUC 13 is opened with Pst I and Sana X, and the large fragment (58) is isolated. Wbea this linearized plasmid (58) is ligated with the fragments (56) and (57), then the hybrid plasmid pW 245 (59) with the modification of the C-terminal sequence ia obtained,, 30 Sfa NI and Pst I are used to cut out of the plassd.cl (59) the fragment (60) which contains the modified CSF DNA 18 sequence. This fragsaeat (60) is ligated with the synthetic DMA sequence (61) and the fragment (55), the DMA sequence (62) being obtained. The latter is ligated with the DMA fragments .(26) -and (27) (Figure 2), the hybrid 5 plasmid pW 246 (63) being obtained. This plasmid is shown twice in Figure 8a, the lower representation indicating the amino acid sequence of the coded fusion protein.

Expression as in Example 2 results in a fusion protein frosn which, after acid cleavage, is derived a CSF deriva-10 tive which is extended by an N-terrain&l proline and in which, additionally, the final amino acid has been replaced by aspartic acid- This derivative is biologically active.

Example 9 15 Pro°-CSF( 1-126) -Asp The hybrid plasmid (63) (Figure 3) ia cleaved with Eco RI and Pst I, and the fragment which contains the two modified CSF sequences following the IL-2 part-sequence is isolated. This sequence (64) is partially cleaved with 20 Rsa I, and the two fragments (65) and (66) are isolated.

The fragment (66) is cleared with Bet MI,, and the fragment (67) is isolated- Ligation of the DNA sequences (27), (65), (67), (&1) and (60) results in the hybrid plasmid pW 247 (68) in which the ligated sequences are 25 arranged in the specified sequence.

Expression as in Example 2 provides a fusion protein from which results, after acid cleavage, the same CSF derivative as in Bxample 8.

Example 10 30 Synthetic gene (for Pro°-CSF} Processes known per se, for example the phosphite method 13 (German Of fenlegungs schriften 3,327,007, 3,328,793, 3,409,966, 3,414,831 and 3,419,995) are used to synthesize the three "synthesis blocks69 I (CSF-J) , designated (59) in the figures, II (CSF-II), (70) in the 5 figures,,, and III (CSF-III)r (71) in the figures., The synthesized oligonucleotides la to Iaa, lie. to Ilf and Ilia to III1 are indicated in the nucleotide sequence of these synthesis blocks (Appendix).

The choice of the nucleotides for the synthetic gene 10 entailed provision not only of unique cleavage sites at the points of union of the three synthesis blocks but also of a number of unique restriction sites inside the gene fragments., These are listed in the tables below. These unique restriction sites can be used, in a manner 15 known per se, to exchange, add, or delate codons for amino acids.

Synthesis Block I (CSF I) Cut after nucleotide no. (coding strand) 1 4 4 5 13 24 25 26 48 line II 65 R® 89 95 101 128 Enzyme Recognition sequence 20 25 H&r I Hpa II Bae II Nae I "Pvu I Sal I Acc I Hinc II Hsa 1/ GG4CGCC C 4CGG GGCGC 4C GCC *QC~C CGAT4CG 64fCGAC C-T COAC GTC 0TT4AAC 30 Hha I GCGC Hinf I &M6JC Ira I tC0*SSA Ima III C4GGCCG Sae II ocecce See If GATJAIC 20 Synthesis Block II ICSF-III Enzyme Recognition sequence Gat after nucleotide no. (coding strand) A£1 III ACAlSf 157 Mlu I A4SQGQT 169 Xm 1 cmmc 175 Taq I fmm 176 Hga T «fa» 6ACGC (5/10) 17 7 Mrs, 1 C vTCGAG <» The re-isolated synthesis blocks (69), (70) and (71) are now ligated in the vector pUC 12 (77) which has been linearised with Eco HI and Sma I, the result being the S plasmid pS 203 (78'). This hybrid plasmid is - as the plasmids with the individual synthesis blocks - amplified in E. coli 79/02, and the synthetic gene is characterized by restriction analysis and sequence analysis.

The plasmid (78) is cleaved with Pvu I partially and with 10 Bam HIt, and the 3UB.11 fragment (79) with the complete CSF sequence is isolated.

The expression plasmid (21) is opened with Beo RI and Bam HI, and the large fragment (80) is isolated. This fragment (80) is now ligated with the fragment (26) which IS contains the IL-2 part-sequence and the synthetic gene (79). This results in the plasmid pS 204 (81) which codas for a fusion protein in which the IL-2 part-sequence is followed first by the bridge member which, permits acid cleavage and then by the amino acid sequence of CSF. 20 Thus, acid cleavage results in a CSF derivative which is extended by proline at the N-tenninal end.

Example 11 CSF(1-12)His(14-121)His(123-127) When the nucleotides in synthesis block 1 up to Bo. 48 25 (cleavage sit© for Hpa I) are replaced by the synthetic sequences (82) and (83), then the result is a modified synthesis block I which codes for a CSF I analog ia which there is Trp is. front of the first amino acid (Maj * and Trp in position 13 has been replaced by His.. 30 The plasmid (12) (Figure 10) is opened with Eco SI and Hpa I, and the large fragment (84) is isolated. The latter is now ligated with the synthetic fragments |82) 23 and (83) , the plasmid pS 205 (85) which codas for this modified CSF I (C5FI') being obtained- The plasmijd (76) (Figure 10) is opened with aind III and Sal I j. and the small (86) find large (87) fragments are 5 isolated. The small fragment (86) is then cut with Taq I, and the fragment (88) is isolated - The large fragment (87) is now ligated with (88) and with ths synthetic fragment (89) in which the codon for Trp in position 122 has been, replaced by His, the plasmid pS 206 10 (90) which codes for the modified CSF III (CSF III') being obtained., This plasmid is transformed into 2- coli# amplified, re-isolated, cut with Hind III and Sal I, and the small fragment (91) which codes for CSF III' is isolated. 15 The plasmid (85) is cut with Pvu I partially and with Pst I,, and the small frsgsnent (92) which codes for CSF I' is isolated.

When the fragments (22) , (26), (92) ^ (70) and (91) are now ligated then the plasmid Ps 207 (93) is obtained -20 This codes for a fusion protein in which the IL-2 part- sequence is followed by a. bridge member which contains Trp immediately in front ox the first amino acid of CSF .(Ala) . Since Trp ia positions 13 and 122 of the CSF molecule have been replaced by His, it is now possible to 25 cleave the fusion protein, with N-bromosuccinimide. This results in ths CSF derivative in which tryptophan in both positions has been replaced by histidine- 24 Example 12 CSF(1-99)Thr(101-127) When, in the synthesis of the synthesis block III, oligonucleotides IlJe and lllf are replaced by the 5 synthetic sequence (94) and the process ia otherwise carried out as in Example 10, than a CSF derivative in which lie in position 100 has been replaced by Thr is obtained., Example 13 10 CSF(1-35)He(37-45)Leu(47-78)Leu-Leu(81-127) First the oligonucleotide (95) which contains in position 36 the codon for lie in place of Met,# and the oligonucleotide (96) in which the codon for Met in position 46 has been replaced by a codon for Leu, are synthesized. 15 The - plasmid (72) (Figure 10) is then opened with Pvu I and Xnta III,, and the fragment (97) is isolated.

In addition, the sequence (98) in which the codon for Met is located in front of the codon for the first amino acid is synthesized. 20 When the fragments (16), (98), (97), (95) and (96) are now ligated then the plasmid pS 208 (99) is obtained. This corresponds to the plasmid (72) but contains in position 0 of the CSF I sequence the codon for Met, in position 36 a codon for lie, and in position. 46 a codon 25 for Leu.

In addition, the sequence (100) which in positions 79 and 80 codes for Leu in place of Met is synthesized.

When the plasmid (76) (Figure 10) is opened with Hind III and Nhe I, and the large fragment (101) is isolated and 25 ligated with the synthetic sequence (100)# then the plasmid pS 209 (102) which corresponds to the plasmid (76) apart from replacement of the two codons ia posi-. tions 79 £^d 00 in the CSF III sequence is obtained. 5 The plasmid (93) (Figure 11a) is now partially cut with Pvu I and with Sal I, and ths large fragment (103) is isolated. The plasmid (99) is likewise partially opened with Pvu I and with Pst I, and the small fragment (104), which contains the modified CSF I sequence is isolated-10 In addition, the plasmid (102) is opened with Hind HI and Sal I, and the small fragment (105) £, which' comprises the modified CSF III sequence is isolated- The fragments (103), (104),, (70) and (105) ar a now ligated,, there being obtained the plasmid pS 210 (106) 15 which corresponds to the plasmid (93) (Figure 11a) but codes for a CSF derivative which has Mat in position 0 and in which, on the other hand, the four Met residues have been replaced by other amino acids.

When E. coli is transformed with the plasmid (106) then, 20 after induction, a fusion protein is obtained which can be cleaved with cyanogen halide resulting in a CSF derivative which contains lie in position 36 and Leu in positions 46, 79 and 80.

Example 14 25 CSF(1-35)lie(37-45>Leu(47-78)Leu(81-127) When the process is carried out as in Example 13, but the synthetic sequence (107) is used in place ox the synthetic sequence (100), then a deletion product which has lie in position 36 and Leu. in position 46, .and in which 30 the amino acid Leu is present in place of amino acids 79 and 80, is obtained.

Example 15 CSF(1-35) lie (37-45) Leu. (47-78) - (81-127) When the process is carried out as in, Example 13 but the synthetic sequence (108) is used in place of the syn-5 thetic sequence (100)t then a deletion product which has He in position, 36 and Leu in position 46, and ia which the amino acids in positions 79 and 80 have been deleted,, in obtained. fil APPENDIX Synthesis block (CS3 MI ICS AfC SAC GAG CCS SOU CCS SOCjCGA TCG CCO TCT COS ©C TAG CTG CTG GGC SSO SSO CGG GCT AGcjoGG AGA GGC se HI) II© Asp Asp Pro Ala Pro Ala Arg Ssr Pro Ser Pro -Lh- i?®o % J f- -*< ± d it -le • • <* 8 TCG ACC CAG CGC ||TGG GAA CAC GST AAC 6CG ATC CAG GAjjft GCG GTG SM TTG CGC AGC TGG GTC GGG ACC CTTj Trp SIq His M Asa Alu r la, ■ % " *>■' . 1 TAG SIC CTT CSC His Glu Ala C20) T TP — 4 100 « # « CGG GST CTG CTG AAC CTG AGT 56C jsAC ACS Set 60A BAG GAC TT® SAC TCA ©CG St© TGC 1 Arg Arg leu lew Ass Leu Ser Arg Asp Thr SCO GCG §AA ATG Cm CGC CTT TAC Ala Ala Glu Met Ik- r k it- :t- 149 • • TTC SAC CTG CA AAC §AA ACC GTT §AA {GTG ATA TCT SAG ATG tTG CTT TGG CAA CTT CAC TAT|AGA CTC TAC AAS CTG G (Pst Asa 61a Thr Val Glu 7al lie Ser Sla Set fin- ft » 9 0 Synthesis bloek IX (CSF Hi (70) ^ ■la. >Xt m • m * CPst 1)6 9AA CCG ACA TGJ CTC OAS ACQ|COT CTC GAG CTC TAC AC GTC Off GGC TGT ACA GAG GTS TGC SCI GAlfcTG GAG ATG i' 31n)Glu Pro Thr €rs Xea Sin Shr Arg Zoa Gin lew !50) C55) ill #*■ -* JL d 200 214 —t — ** * —-lie r AAA CIA GGC|CTT CGT GGT TCT CTG AOS A (Hind III) 1' j, TTT GTT SCO GAA GCA|CCA AGA SAO TGG "TO SA Lys 01b Gly Lew Arg Gly Sor hen Thrdyn) (65) (70) I* —~^r{- 2 ^> <£> 1 c Ieia) __ _ i'JS Gly Pro %&"& Wir «1£ 0» Cso) (85) TI7" klL^ • • # P • lb fq fl«3 /ft Li*J TOC CCG CCG ACf CCG 'SAG ACOjTCT TGC ,>HSj aWB ffi 'AC6 SAG GTC GIG ACS GGC GGC TGA GGC v«L v TGC AGA ACS?! TGC GTC Gin His Cys w?l <*>&« « Sin if* % ii *o; i (95) TTTf 300 1 • m • —A 4 ,$ ATC unr I acc AAV re A A -401 tr&idTb do tf> AAA qaa aac CTG mg #■*1 ® « epipip IJI gQ, TAO tgg aagJ CTT aga A ft f MM& CTT TTG oao «£» «£> ,T; tf\ « 11® Tl A <*t« uio ''»> HB A« ■ it t

Claims (1)

1. 30 nt Claims Human granulocyte macrophage colony-stimulating factor proteins (GM-CSF) of the formula Pro-(As).-CSF( 12-126 )~Z in ^rhicb. (As)x denotes all or some of the first 11 amino acids of the natural GM-CSF sequence, and Z denotes Glu or Asp, hereinafter referred to as CSF. A process for the preparation of CSF as claimed in claim If which comprises incorporation of a gene coding for CSF in a bacterial expression vector, transformation of bacteria, especially E. coli? therewith, and bringing about expression therein™ The process as claimed in claim 2, wherein the CSF is expressed in the form of a fusion protein which is then cleaved enzymatically or chemically. The process as claimed is claim 3, wherein the fusion protein contains N-terminal adjacent to the CSF the amino acid sequence {Glu)n-(Asp)n-Pro in which m is sero or 1, and n is 1, 2 or 3, and is proteolytically cleaved. A bacterial expression vector containing at least one gene coding for CSF as claimed in claim L A bacterial cell, especially S. coli., coatainiag a vector as claimed in claim 5« A medicament containing or composed of CSF as claimed in claim 1- 31 CSF as claimed in claim 1 for use in medical treatment. The use of CSF as claimed in claim 1 for the preparation of medicaments. 1 o. Human granulocyte Macrophage colony-stimulating factor proteins according to claim l, substantially as hereinbefore described and exemplified. 11. A process according to claim 2 for the preparation of CSF,, substantially as hereinbefore described and exemplified. 12. CSF whenever prepared by a process claimed in any one of claims 2 - 4 or claim 11 . 13. A bacterial expression vector according to claim 5 e substantially as hereinbefore described and exemplified. 14. A bacterial cell according to claim 5, substantially as hereinbefore described and exemplified. 15» A medicament according to claim 7 „ substantially as hereinbefore described. F. R. KELLY & CO. , AGENTS FOR THE APPLICANTS.