AU617095B2 - Molecular cloning and expression of human il-3 - Google Patents

Abstract

A cDNA encoding human interleukin-3, also designated herein hIL-3 or hmulti-CSF, has been disposed in expression systems and caused to produce hIL-3 substantially free of impurities normally accompanying this protein as it is produced $i(inter alia) by peripheral blood lymphocytes in nature. The resulting hIL-3 can be produced in practical amounts and is useful in a variety of therapeutic and diagnostic protocols.

Description

r, AU-Al -10577/88 WORLD INTELLECTUAL PROPERTY ORGANIZATION PCT Internationlal Bureau INTER NATIONAL APPLICATION PUBI' 13HED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 C 12N 15/00, 1/20, 1/16 C12N 5/00, C12P 21/02, 21/00 (C12P 211:00, C12R 1:91) (C12N 1/20, C12R 1:19,1:07) (Cl2N 1/16. C12R 1:85) (11) International Publicatioh Number: (43) IternonaQublgon 31pe: WO 88/ 04691 30 June 1988 (30.06.88)

I

(21) International Application Number: PCT/NL87/00037 (22) International Filing Date: 16 December 1987 (16.12.87) (31) Priority Application Numbers: 86202285.2 EP 87201322.2 EP (32) Priority Dates: (33) Priority Countries: 16 December 1986 (16.12.86) 3 July 1987 (03.07.87) Nl, et aL NL-2907 MB Capelle a/d Ijssel VAN LEEN, Robert, Willem [NL/NL], Heeskesacker 23-23, NL- 6546 TW Nijmegen (NL).

(74) Agents: HUYGENS, Arthur, Victor et al.; Gist-Brocades Patent and Trademarks Department, Wateringseweg 1, P.O. Box 1, NL-2600 MA Delft (NL).

(81) Designated States: AU, DK, Fl, JP, NO, US.

Published Withi international search report, A.0. J. P. 1 8 AU G '1988

AUSTRALIAN

15 JUL 1988 PATENT OFFICE (71) Applicant (for all designated States exccept US): GIST- BROCADES NY. [NL/NL]; Wateringseweg 1, PO.

Box 1, NL-2600 MA Delft (NL).

(72) Inventors; and Inventors/Applicants (for US only) DORSSERS, Lambertus, Christiaan, Johannes Kerkstraat 17, NL-6668 AN Randwijk WAGEMAKER, Gerard Ananasstraat 146, NL-2564 GT Den Haag VOS, Yvonne, Johanna ENL/NL]; Zadkinerade 1, (54)Ttle- MOLECULAR CLONING AND EXPRESSION OF HUMAN IL-.i (57) Abstract A cDNA encoding human interleukin- 3, also designated herein hIL,3 or hmulti- CSF, has been disposed in expression systems and caused to produce hIL-3 substantially free of impurities normally accompanying this protein as it is, produced inter a/ia by peripheral blood lymphocytes in nature. The resulting hIL-3 can be produced In practical amounts and is us~ful in a variety of therapeutic atid diagnostic protocols.

SmKS E E

M

FSm 1 hui&mrt Multl-CSF eDNA

L

SO 8/04691 PCT/NL87/00037 1 MOLECULAR CLONING AND EXPRESSION OF HUMAN IL-3 Field of the Invention The present invention relates to cDNA encoding human interleukin-3 (hIL-3) and its use, inter alia, in the cloning and expression in various organisms, including microorganisms, in particular yeasts, bacteria and fungi, tissue culture cells and transgenic animals and plants.

Background of the Invention Hemopoiesis involves the active process of proliferation and differentation of pluripotent progenitor cells into all types of mature blood cells and some specialized tissue cells. Production of functional blood cellis regulated by specific proteins, the hemopoietic growth factors (HGFs). Some of the HGFs control maturation of a specific maturation lineage, whereas others stimulate proliferation and differentiation of progenitors along multiple pathways. Much of our knowledge of the hemopoietic differentiation process has been obtained from mouse studies in vitro and in vivo, using purified growth factors. The murine growth factor interleukin-3 (mIL-3), also termed Multi- CSF, mast cell growth factor, stem cell activating factor or several other designations, stimulates the proliferation of developmentally early, multipotent cells (CFU-S) as detected by the spleen colony assay, resulting in the production of progenitor cells along the erythroid, megacaryocyte, granulocyte/macrophage, osteoblast and several other lineages.

Furthermore, mIL-3 has been implicated in replication of pluripotent stem cells, probably in synergism with other HGFs.

In recent years, several groups have succeeded in cloning mIL-3 cDNA. No results have been reported sofar of identifying homologous sequences in human DNA using mIL-3 DNA i 4 WO 88/04691 PCT/NL87/006437 2 as a prot,,. Presumably, the human gene has diverged extensively from the mIL-3 gene or has lost its function during prima.te evolution. However, human leukocytes were found to produce a HGF(s) which can replace mIL-3 in supporting the proliferation of murine CFU-S. Thus, the existence of a human HGF was postulated, which shares biological properties with mIL-3 and therefore could be the human homolog. Yang, Y-C, et al, Cell (1986) 47:3-10, dated 10 October discloses cDNA encoding a protein having IL-J like activity from gibbon Tcells, and retrieval of a genomic DNA which encodes the human counterpart. The sequence of a cDNA encoding human IL-3 can be deduced from the human gene sequence published by Yang et al.

However, said article does neither disclose ror teach a method for isolation of a cDNA encoding human IL-3, nor was the production of hIL-3 achieved. This invention describes for the first the isolation of a cDNA comprising the entire coding sequence for human IL-3.

Human IL-3 protein has never been prepare& in purified form, nor have its characteristics, other than its activity in certain in vitro proliferation assays and deduced primary structure, been disclosed. The present invention permits the recovery of purified human IL-3, and identification of its characteristics through recombinant production from a cDNA clone.

Summary of the Invention As stated above, the present invention for the time describes the isolation of a cDNA comprising the entire coding sequence for human IL-3. The low degree of homology between the DNA sequences coding for murine and human IL-3 does not permit the retrieval of a cDNA for hIL-3 by hybridization with the mIL-3 coding sequence. Unexpectedly, the hIL-3 cDNA clone could be isolated by exploiting the rather high degree of homology in the 3' noncoding part of the cDNA's. The availability of the cDNA clone permits the production of hIL-3 by wide range of host organisms. Subsequent to large scale production the protein may be purified and used therapeutically.

-2a- The present invention permits production of recombinant human IL-3 protein in a wide range of host cells by transcription and translation from a cDNA sequence encoding the human IL-3 protein. The production of the protein is illustrated in several hosts, including E. coli, COS cells, C127 cells, B. subtilis and B. licheniformis, S. cerevisiae and K. Iactis, hereinbelow. Production in other hosts using appropriate expression systems is also made possible by provision of the intronless cDNA. More generally, the availability of antihuman IL-3 antibodies which permit identification of colonies exhibiting successful production of the recombinant protein aids in production of human IL-3 from any recombinant system.

In one aspect, therefore, the invention is directed S to a recombinant, intronless, DNA encoding human IL-3 protein.

lvn In another aspect, it is directed to a transformed living host cell containing genetic material derived from recombinant DNA material without introns and coding for human S IL-3 having a Pro at position 8 of the mature protein molecule, wherein the host cell is selected from the group consisting of yeasts, bacteria, fungi and tissue culture cells, "DMW/3283U e *e I I Wq 88/04691 Pr/NL87/00037 The present invention permits pr<e recombinant human 1L-3 protein in a wide range of host cells by transcription and translation from a cDNA sequence en ding the human IL-3 protein. The production of the protei is illustrated in several hosts, including E. coli OS cells, C127 cells, B. subtilis and B. licheniform S. cerevisiae and K. lactis, hereinbelow. Production other hosts using appropriate expression systems is a o made possible by provision of the intronless cDN More generally, the availability of antihuman IL-3 nibodies which permit identification of colonies exhi ting successful production of the recombinant protei aids in production of human IL-3 from any recombinant s em.

n one aspect, therefore, the invention is directed to a combinant, intronless, DNA encoding human IL-3 protein.

In another aspect, it is directed to expression systems capable of effecting the expression of said DNA n equ e nc d-cn n-q-L i In other aspects, the invention is directed to recombinant human IL-3 protein in glycosylated or unglycosylated form, to human IL-3 free of substances normally accompanying said protein, and to antibodies specifically reactive with these recombinant or purified proteins.

Brief Description of the Drawings Figure 1 shows a comparison of DNA and protein sequences of human multi-CSF and mouse IL-3. The hmulti-CSF protein and DNA sequence (clone Dll, top lines) were aligned with the mIL-3 DNA (11, 35) and protein sequence Identical nucleotides are indicated by a vertical line, identical amino acids are shown in boxes. Black dots indicate a polyadenylation signal sequence and horizontal bars mark ATTTA repeat units.

Figure 2 shows the construction of plasmid pLB4 containing human IL-3 cDNA. E EcoRI, Sm SmaI, B BamH!, S SstI, K KpnI.

Figure 3 shows the biological activity of COS/pLB4 WO 88/0469 1 PCT/NL87/00607 Cm on human bone marrow progenitors. The -nean numbers of erythroid (BFU-E), granulocyte -macrophag e (CFU-GM), granulocyte (CFU-C), eosinophil (CFU-Eo), macrophage (CFTJ-M) and mixed (CFtJ-MIX) colonies are shown for duplicate cultures stimulated with 9 raded volumes of COS/pLB4 CM.

Figure 4 shows induction of AML proliferation by COS/pLB4 CM as assessed in a colony culture assay (panel A) and in a DNA synthesis (3H-TdR incorporation) assay (panel B), Figure 5 shows a construction diagram of the H, coli expression vectors pGB/IL-301, GB/IL-302, pGB/lL-303, PGB/IL-304, pGB/IL-305 and pGB/IL.-306. In this Figure X stands for XhoI, E for EcoRI, B for BamEII and A for Aval site.

Figure 6 shows the sequence of the rulticloning site in pTZl8R (Pharmacia) and its derivative pTl.

Figure 7 shows a schematic presentation of hmulti- CSF expression clones. For the eucaryote expression plasmids pLB4 and pLl'ionly the hmulti-CSF cDNA insert is shown.

I

4 eader peptide and mature hrnulti-CSF' protein coding regions are indicat ed in boxes. Bacterial expression clones of hrnulti-CSF (derived from pLHI) contain the 1acZ and multi-linker protein coding region 5' terminal nonccding region of hrnulti-CSF and the hmulti-CSF coding region. The arrow mark's the ATG startcodon used in the particular vector.

Figure 8 shows the sequences of fusion regions of lacZ/hmulti-CSF DNA for various bacterial expression vectors.

The seq~uence of clones is given from the start of the lacZ protein in either pUC8 or pTZl8R (lower case letters) and of hmulti-CSF DNA sequence up to the cdal site at position 158. Mutations in the hmulti-CSF DNA sequence are underlined, resulting in: trpl 3 __;argl 3 (pGfl/IL-302); leu 9 ,pro 9 and trp 1 3 arg 1 3 (pGB/IL-3Q3); ret 3 thr 3 and a silent change (pGB/11,-304). The superscripts denote the amino acid residue number of the mature protein.

Figure 9 show~g polyacrylamide ge1-electrophoresis of bacterial hrnulti-CSF produced from bacteria containing pGB/II..

303. and pGB/IL-302.

Figure 10 shows the titratioti of hwiulti-cSF fusion I i S0O $8/0469! PCT/NL87/00037 protein on AML blast cells.

Figure 11 shows a Western blot demonstrating the IL- 3 specific reaction of rabbit antisera raised against the 21 kd protein isolated from a lysate of E. coli transformed with pGB/IL-301.

Figure 12 shows the effect of the antisera of Figure 11 on IL-3 activity.

Figure 13 shows a schematic representation of plasmid pGB/IL-307. The box (Ta) indicates the human IL-3 coding sequence. The N-terminal amino acids of the fusion protein are depicted below the drawing.

Figure 14 shows a schematic representation of plasmid pGB/IL-308. The nucleotide sequence of the promoter region is depicted below the drawing.

Figure 15 shows the construction of plasmid pGB/IL- 309. The first box indicates a part of the human IL-3 sequence, viz. the signal sequence plus 20 amino acids of the mature protein. The other box 1) indicates part of the 3' noncoding region of the IL-3 cDNA sequence.

Figure 16 is a schematic representation of plasmid pGB/IL-310.

Figure 17 shows the nucleotide sequence of plasmid pBHAl.

Figure 18 shows the construction of the plasmids pGB/IL-311 and pGB/IL-312. The box indicates the precursor human IL-3 coding region.

Figure 19 shows the construction of the plasmid pGB/IL-313. The sequence at the 5' side of the IL-3 sequence is depicted below the drawings.

Figure 20 shows a schematic representation of plasmid pGB/IL-317.

Figure 21 shows a schematic representation of plasmid pGB/IL-316.

Figure 22 shows the nucleotide sequence of plasmid pGB/IL-316 between the unique SacII site in the lactase promoter and the HindIIl site behind the terminator (residues 4457 to 7204).

Figure 23 shows the nucleotide sequence of plasmid 1 WO 88/04691 PCT/NL87/00037 pGB/IL-318 between the unique SacII site in the lacatse promoter and the HindIII site behind the terminator (residues 4457 to 7190).

Figyure 24 shows the nucleotide sequence of the EF-lo0 promoter, SalI-BglII-XhoI linker and actin terminator as present on plasmid pGB/TEFact.

Detailed Description of the Invention A. Definitions As used herein, "human IL-3", "hIL-3, "human multi- CSF", and "hmulti-CSF" are used interchangeably, and designate a protein preparation which exhibits the following activities: 1. The protein stimulates colony formation by human hemopoietic progenitor cells wherein the colonies formed include erythroids, granulocytes, granulocyte macrophages, and mixed.

2. The protein stimulates DNA synthesis by human acute myelogenous leukemia (AML) blasts, as evidenced, for example, by labeled thymidine uptake.

To fit the definition of hmulti-CSF, the activity in the foregoing assay must not be substantially inhibited by antibodies raised in response to, and immunospecific for, GM- CSF, unless these antibodies also inhibi, these activities by the illustrative hmulti-CSF below.

One illustrative form of hmulti-CSF is shown in Figure 1 as a 133 amino acid mature protein, having a 19 amino acid signal sequence. The amino acid sequence of Figure 1 is identical with that disclosed by Yang, et al., Cell (1986) 47:3-10 (supra) except at position 8 of the mature protein wherein the Ser of the Yang protein is replaced by Pro herein. As shown herein, this amino acid sequence is effercive in its nonglycosylated form. However, it contains two glycosylation sites, and the glycosylated form is also included within the scope of the invention. It is also recognized that the protein may exist in acid addition salt

I:

088/04691 PC/NL87/0037 7 form, basic salt form, or may be neutral, depending upon the pH of its surroundings. Derivatization by phosphorylation, acetylation, and so forth, to the extent that activity is not destroyed, also results in a protein included within the scope of the invention.

It is also recognized that the entire sequence may not be necessary for activity. Parts of the amino acid sequence may be deleted or replaced, while retaining biological activity. As illustrated herein, the alanine at position 1 may be deleted, as may as many as the first fourteen amino acid residues if replaced by a sequence of residues of a fused peptide sequence. In addition, it is believed that the murine form of the protein requires only the first 79 residues for activity; this corresponds approximately to the first 83 residues of the human counterpart. Accordingly, fragments which comprise only the first 83 amino acid residues of the protein, and the N-terminal replaced forms thereof are also included within the scope of the invention.

Furthermore, it should be considered that the N-terminus of mature hIL-3 is formed by the residues ala-pro-met etc. (see Figure It is known that the protein, when secreted by a yeast host, may in some instances be shortened by two amino acids (ala-pro), due to the interaction with a dipeptidylaminopeptidase The hIL-3 without the N-terminal alanine and proline still retains its biological activity. Yeast strains carrying a null mutation of the X-prolyl dipeptidylaminopeptidase gene will produce complete hIL-3 (amino acids 1-133). Accordingly, included in the multi-CSFs of the invention are hose which contain and those which do not contain the N-terminal alanine and proline, produced by Xprilyl dipeptidylaminopeptidase mutants and wild type hosts, respectively.

When produced as a mature protein in a procaryotic host, the coding sequence for the mature protein will be prefaced by an ATG start codon. The resulting N-terminal methionine may then be removed, or partially removed, by processing within the bacterial host, depending on the nature of the subsequeit amino acid sequence. Again, both forms of WO 88/04691 PCT/NL87/0037 hIL-3 are biologically active. Therafore, included in the hmulti-CSFs of th( ,nvention are those which contain and those which do not contain the N-terminal met'hionine.

From the above it is clear that amino acid changes may be introduced into the human IL-3 protein, without affecting its biological function. It is recognized that minor changes in amino acid sequences by chemical modification of the encoded residue, substitution of a different residue, or deletion or addition of one or more, but preferably only one, residue results in proteins which retain activity. Accordingly, these nondestructive mutations are also included within the invention, in particular, the naturally occurring allelic Variations and other mutations which are nonlethal to the activity.

On the other hand it should be considered that amino acid changes in the human IL-3 protein may be beneficial to the therapeutic use of the protein. As recognized herein, the mature protein has four conserved domains at residues 15-36, 54-61, 74-91, and 107-118. Proteins containing single and multiple amino acid changes in the nonconserved regions, 1-14 (which are, in any event, replacable by the sequences of host derived fusion proteins), 37-53, 62-73, 92-106, and 119-133 are possible. However, it appears that the cysteine residues at positions 16 and 84 may be necessary for disulfide bridge formation as they are conserved between species. Changes in the conserved domains mention above may influence biological properties of the protein, such as receptor binding and signal transduction, It is envisaged that hIL-3 having altered properties are of therapeutic use. Such derivatives of hIL-3, whicht may be made by known protein engineering techniques, are to be understood within the scope ot the present invention.

The protein preparation may contain the hmulti-CSF peptides in monomeric or aggregated form, provided the aggregates retain activity as above-defined.

As used herein, "expression system" refers to a ONPA sequence which contains both a coding sequence. whose expression is desired and appropriate control sequences in WO,88/04691 PCT/NL87/00037 9 operable linkage with it which permits its expression when the control sequences are compatible with the host into which the expression system is placed. As is generally understood, "control sequences" refers to DNA segments which are required for or regulate the expression of the coding sequence with which they are operably linked.

Control sequences for all hosts include promoters, which may or may not be controllable by regulation of their environment. Typical promoters suitable for procaryotes include, for example, the trp promoter (inducible by tryptophan deprivation), the lac promoter (inducible with the galactose analog IPTG), the beta-lactamase promoter, and the phage-derived PL promoter (inducible by temperature variation). Additionally, especially for expression in Bacillus, useful promoters include those for alpha-amylase, protease, Spo2 and synthetic promoter sequences. Suitable promoters for expression in yeast include the 3-phosphoglycerate kinase promoter and those for other glycolytic enzymes, as well as promoter regions for alcohol dehydrogenaos and yeast phosphatase. Also useful are the transcription elongation factor (TEF) and lactase promoters. Mammalian expression generally employs promoters derived from viruses such as the adenovirus promoters and the SV40 promoter systems, but they also include regulatable promoters such as the metallothionein promoter, which is controlled by heavy metals or glucocorticoid concentration. There are also now available viral-based insect cell expression systems, as well as expression systems based on plant cell promoters such as the nopaline synthetase promoters.

In addition to the promoter DNA sequence, which is necessary for the transcription of the gene by RNA polymerase, a variety of control sequences, including those regulating termination (for example, resulting in polyadenylation sequences in eucaryotic systems) are also useful in controlling expression. Some systems also contain enhancer elements which are desirable but not necessarily necessary in effecting expression.

Translation controls include a ribosome binding site WO 88/04691 PCT/NL87/000G37 10 (RBS) in procaryotic systems, whereas in eucaryotic systems translation may be controlled by the nucleotide sequence around the AUG codon.

As implied above, recombinant protein production can be effected in a wide variety of hosts, including bacteria (predominantly E. coli, Bacillus, and Streptomyces), in yeast and fungi (such as Saccharomyces, Kluyveromyces, and Aspergillus), and in mammalian and other cell cultures such as COS cells, C127 cells, Chinese hamster ovary cells, Spodoptera frugiperda (Sf9) cells, and so forth. The protein may be produced as an intracellular mature or fusion protein, or may be secreted when the DNA encoding an appropriate compatible signal is included in the gene.

The present invertion for the first time enables large scale production of recombinant human IL-3, so that this protein in purified form can now be used as a therapeutic agent. The methods described herein provide means for producing glycosylated as well as unglycosylated forms of the protein, which can be purified to substantially pure human II.- 3. "Purified" human IL-3 refers to human IL-3 as defined above which is free of other proteins which normally accompany it, B. Retrieval of cDNA Encoding Human IL-3 Human IL-3 was isolated according to the following strategy: 1. A procedure was developed which allowed for reproducible production of hemopoietic growth factors (HGFs) by human leucocytes.

2. mRNA was prepared from such producing cells and transcribed into double-stranded DONA.

3. The cDNA was screened with a complete mIL-3 cDNA which contained both the coding and untranslated 3' downstream portions to obtain DII.

4. The hybridyzing cDA clone DII was inserted into an expression vector pLO to obtain pLB4 which was expressed in COS cells to confirm the presence of the sequence encoding human IL-3. Conditioned media from these cells showed the i i i I oW088/04691 PCT/NL87/00037 11 biological activity expected of hIL-3.

The human cDNA was retrievable using this procedure because despite considerable lack of homology with the murine coding sequence, a surprising degree of homology was present in the 3' untranslated region. Applicants are unaware of any prior disclosure of the use of a 3' untranslated sequence homology to retrieve an alternate species gene.

In more detail, conditioned medium of lymphocytes cultured in the presence of 12-0-tetradecanoylphorbol-13 acetate (TPA) and concanavalin A (Con A) is a suitable source for human HGFs as determined by assay of the medium using stimulation of mouse CFU-S in suspeusion cultures, proliferation of mIL- 3 dependent DA-l cells, human hemopoietic progenitor assays by colony fornation in vitro, and in vitro stimulation of acute leukemia blasts, A cDNA library from human lymphocytes was constructed in lambda gt-10 phage and screened using the HindIII-Xbal fragment of mlb-3 cDNA, for the occurrence of mXIL-3 related sequences. No hybridizing clones were identified, However, when complete murine IL-3 cDUA was used as probe, four clones were identified. Restriction enzyme analysis of the largest clone (Dj1) indicated a 9 %ert containing an internal EcoRI site (at position 4l1, m i1).

(It was investigated whether this EcoRI sit iad arisen by ligation of two i enependent ODNA fragments or was a naturally occurring site. Southern analysis of restriction enzyme digested human DNA using labeled 5' and 3' EcoRI fragments of clone D11 as probe, revealed identical DNA fragments following digestion with HindIII (15 kb) and BamHI (4.6 kb). Furthermore, the DNA sequence around the Eccc'. site doa not correspond to linker sequence (pCCGAATTCoQ) used for inserting cDNA into phage OtA, indicating that these aoRI fragments are derived from a inglo inRNA.) From hybridization and sequencing experiments it was concluded that the small clones (II, IV and VX) nre identical to the 3' nucleotide sequence of clone Dll and eiaved from the same mRNA species.

Computer assisted alignment (Figure i) ,he DII WO 88/04691 PCTNL87/0037 12 cDNA and the mIL-3 cDNA revealed sequence homology in the terminal 100 bp, between nucleotides 236-269 and between nucleotides 598-803 in the 3' terminal region 71% and 73% homology, respectively). In particular, the region between nucleotides 706 and 763 is highly conserved (93% homology) and contains repetitive AT-rich sequences. The low homology in the terminal 600bp of the human cDNA precludes detection by hybridization with the HindIII-XbaI fragment of mIL-3.

Analysis of the human cDNA clone for an encoded protein shows an open reading frame up to the termination zodon TGA at position 495-497 (Figure The first ATG triplet is probably the actual initiation codon of the encoded polypeptide. The putative encoded protein consists of a hydrophobic leader peptide of 19 amino acids, which is probably cleaved between the glycine and alanine residues (22, 23).

The alignment of the predicted amino acid residues of the human and mouse IL-S (Figure 1) reveals a homology of for the leader peptide (residues -26 to and 28% for the mature protein (residues 1 to 133). Within the leader peptide, there are two conserved regions of four amino acids (residues -13 to -10 and -3 to of which the second one encloses the processing site. The mature protein is 133 amino acids long ang has a molecular weight of 15 kd, The mature protein has four conserved domains (residues 15-36, 54-61, 74- 91 and 107-118) and contains two potential glycosylation sites (residues 15-17 and 70-72). Both cysteine residues present in the human protein (positions 16 and 84) are conserved and may play an essential role in protein folding by disulfide bridge formation.

In order to verify that this human cTNA encodes a functional protein that resembles mIL-3, the Dll cDNA was inserted in an eucaryotic expression vector (pLO, containing a SV40 transcription unit) to htain the expression vector pLB4 and transfected to COS i cells. The COS/pLB4 conditioned medium (CM) was tested for its capacity to stimulate colony formation by human bone marrow cells, and to stimulate human acute myelogenous leukemia (AML) blasts.

In vitro colony growth of human hemopoiotic j -ii i i 1 1. r WOo 88/04691 1 PCI'/NL87/00037 13 progenitors ,.%leted of myelomonocytic (Vim-2 positive) and Tlymphocytic (T-3 positive) accessory cells, was efficiently stimulated by COS/pLB4 CM. The data demonstrate stimulation of progenitors of several 1'mopoietic differentiation lineages and of a subpopulation of BFU-E by COS/pLB4 CM.

In a separate experiment, bone marrow was enriched for progenitor cells by density centrifugation, E-rosette sedimentation to remove T-lymphocytes and adherence to remove mononuclear phagocytes and cultured iji enriched medium containing fetal calf serum. Under these conditions, the majority of the colonies obtained upon stimulation with COS/pLB4 CM contained two or more hemopoietic differentiation lineages; all contained macrophages, approximately half immature blasts and/or immature erythroid cells and/or neutrophilic granulocytes and a minority, in addition, basophilic or eosinophilic granulocytes. These results demonstrate the multilineage stimulatory properties of the protein encoded by the human cDNA clone Dll and its action on developmentally early, multipotent hemopoietic cells.

With respect to AML stimulation, AML blasts of five patients were stimulated with the COS/pLB4 CM and assayed f£r a response by measuring 3H-TdR incorporation and colony formation. Three of the five leukemia cell samples responded to the (OS/pLB4 CM in both assays; characteristic doseresponse relationships for colony formation and DNA synthesis of AML blasts of different patients were obtained. The responses to GM-CSF demonstrated further phenotypic differences among the leukemias responding to the COS/pLB4

CM.

These data demonstrate that the Dll cDNA clone contains the complete genetic information for a biologically active protein which is exported into the culture medium in the transformed COS cells. Despite the apparent lack of homology with respect to the protein sequence between the human protein and mIL-3 (only the proteins are comparable with respect to their biological function.

Both proteins exert their effect on developmentally early hemopoietic progenitors of various lineages. The low homology WO 88/04691 PCT/NL87/00037 14 at the amino acid level is also reflected by a low homology in the coding nucleotide sequence. However, very unexpectedly, a rather high degree of homology sufficient for retrieval of the human cDNA clone occurred in the 3' untranslated region.

Southern analysis of human DNA revealed a single hybridizing gene indicating that this cDUA does not belong to a family of closely related genes.

From the foregoing results we conclude that the human cDNA insert in DlI encodes the human homolog of mIL-3.

We decided to use the operational term hmulti-CSF for the protein encoded by the cDNA clone Dll in view of its major biological effect and assay.

The identification of hmulti-CSF cDNA clones by virtue of hybridization with the 3' termiial region of the mIL-3 cDNA was unexpected. Whereas homologous DNA sequences are in general predominantly found in the coding region, the hmulti-CSF sequence has extensively diverged homology) in this part of the gene. Analysis of the highly conserved domain in the 3' terminal non-coding region reveals the occurance of 5 ATTTA repeat units which are all preserved in the mIL-3 cDNA (Figure 1).

hMulti-CSF and mIL-3 display considerably less protein homology than other murine and human growth factors or lymphokines such as GM-CSF interleukin-2 interleukin-i (26) and interferons (27-29). The biological activity of the mature mIL-3 appears to be contained in the first 79 amino acids, including an absolute requirement for the cysteine residue at position 17 This cysteine residue is conserved in hmulti-CSF (Fig. 1, pos. 16) and may play an essential role in protein folding. The occurrence of a potential glycosylation site around this cysteine residue may interfere with disulfide bridge formation.

C. Production and Formulation of hmulti-CSF Applicants have provided a representative variety of expression systems capable of producing human IL-3 protein in a variety of forms as fusion proteins, as mature intrai i W,88/04691 PCF/NL7/00037 15 cellular proteins, and as secreted proteins. Applicants are unware of availability anywhere in the art of recombinant forms of human IL-3, or, indeed, of any human IL-3 in a preparation which is free of proteins normally accompanying this desired protein. Accordingly, the invention herein provides, for the first time, the human IL-3 protein in a manner which is capable of adaptation to therapeutic and diagnostic uses.

The human IL-3 can be produced as a fusion protein with sequences heterologous to the human IL-3 amino acid sequence. By "heterologous" is meant a sequence which is not found in human IL-3 itself, but is an unrelated sequence. This heterologous sequence may be derived from a bacterial protein, a yeast protein, a mammalian protein, or any of variety of miscellaneous fortuitously encoded sequences such as, for example, those encoded by polylinkers. It is clear from the results hereinbelow that at least the first 14 amino acids of the N-terminus of the human IL-3 sequence can be replaced by heterologous sequence, at least if the fusion protein is further extended past the N-terminus.

The protein can also be obtained as a mature intracellular protein by constructs in which the ATG start codon is placed immediately upstream of the desired N-terminus. These intracellular proteins, whether mature or fusion proteins, can be recovered by lysing the cells and purifying the human IL-3 using standard protein purification techniques.

Protein purification is simplified if the human IL-3 is secreted into the medium. When produced in mammalian cells with which the native signal sequence is compatible, this native signal sequence can be used to effect secretion into the medium. In bacterial or yeast systems, signal sequences compatible with these hosts, such as the penicillinase or alpha-amylase sequence in bacteria or the alpha-factor signal sequence in yeast can be used.

hen produced recombinantly, the human IL-3 is free of proteins normally accompanying it, and can be purified from the proteins and other materials indigenous to the recombinant WO 88/04691 PCT/NL87/00037 16 host using, for example, chromatographic methods, gel filtration, ammonium sulfate precipitation, and so forth.

As described hereinbelow, the protein in useful for therapeutic and diagnostic purposes. For therapeutic uses, the protein may be formulated in ways standard for pharmaceutical compositions which are used for the administration of proteins. Suitable excipients include, for example, physiological saline, Ringer's solution, and so forth. Alternate formulations, including solid formulations lyophilized), can also be employed.

D. Preparation of Antibodies The availability of recombinant IL-3 protein or parts thereof will permit production of antibodies directed against the protein or parts thereof, as demonstrated hereinbelow. Such antibodies are useful, inter alia, for in vitro detection of colonies producing hIL-3, for therapeutical use, and for the purification of both natural and recombinant hIL-3.

Statement of Utility The nucleotide sequence of the whole or parts of the cDNA of human IL-3, or closely-related DNA sequences will advantageously enable the detection of genetic abnormalities, including genomic rearrangements, restriction fragment-length polymorphisms, mutations and altered gene expression with the use of such techniques as the analysis of chromosomal DNA using restriction enzymes, DNA and RNA blotting as well as hybridization techniques (Maniatis et al. 1982) and twodimensional gel electrophoresis (Fisher and Lerman, 1983).

The recombinant hmulti-CSF as provided by the present invention will facilitate a detailed analysis of its role in human hemopoiesis, in particular the possible synergism of hmulti-CSF and various other HGFs. Furthermore, hmulti-CSF is of considerable interest because of its applicability for in vitro diagnosis of human diseases in 1; WO,18804691 PCT/NL87/00037 17 which hemopoietic progenitor cells are involved, which include the leukemia, as well as potential therapeutic applications aimed at expansion of hemopoiesis in vivo. The effect of hmulti-CSF on various hemopoietic malignancies with respect to terminal differentiation oi the leukemic cells also needs to be explored. In addition hMulti-CSF may be required for establishing a proliferative state of human stem cells in gene therapy protocols, since stimulation with mIL-3 was shown to be required for succesful infection of mouse stem cells with recombinant, replication defective retroviruses.

IL-3 protein can also advantageously be used for the detection of early hemopoietic precursor cells in standardised in vitro cultures (Wagemaker and Visser, 1980; Metcalf et al.

1982; Merchav and Wagemaker, 1984, Metcalf, 1986).

IL-3 protein and variants can further be used for the multiplication of hemopoietic stem cells in vitro, possibly in conjunction with other growth factors, for bone marrow transplantation and the genetic manipulation of stem cells (Lowenberg and Dicke, 1977; Wagemaker and Petem, 1978; Lemischka et al, 1986).

The IL-3 protein can be used for the determination of the response pattern of malignant hemopoietic cells in in vitro tests (Touw and Lowenberg, 1985; Griffin et al, 1986; Griffin and Lowenberg, 1986).

The IL-3 protein can further be used for the detection of remaining leukemic cells by in vitro methods (Touw and Lowenberg, 1986; Griffin et al, 1986; Griffin and Lowenberg, 1986).

Furthermore, the IL-3 protein can be used in vivo for the treatment and prevention of malignant and nonmalignant disorders, either by itself or in combination, in which an obtained specific response by the hemopoietic system can result in a clinical benefit.

These applications include: cytopenias and/or immunosuppression due to infections such as AIDS cytopenias due to chemotherapy and/or irradiation bone disorders such as bone fractures and osteoporosis WO 88/04691 PCT/NL87/00037 18 immunodeficienties due to general anaesthetic procedures recovery following bone marrow transplantation adjunct to vaccinations and adjunctive therapy of infections.

The cloned human IL-3 DNA sequence or closelyrelated DNA can be used for gene therapy in genetic deviations from the normal IL-3 gene.

To facilitate the above-described analysis, a large quantity of human IL-3 is required. The easiest way to obtain sufficient amounts of the protein is the production with microorganisms, in particular yeasts, bacteria and fungi, e.g.

Saccharomyces, Kluyveromyces, Aspergillus, Streptomyces, Bacillus and E. coli species. Production in mammalian and other eucaryotic systems, such as C127 cells, Spodoptera cells and transgenic animals and plants, is also possible for skilled persons following the teaching of the present invention. These possibilities are all included within the scope of this invention.

As an illustration how to obtain living cells that produce the human IL-3 protein by expression of the hIL-3 cDNA, a number of plasmids were constructed and transferred to E. coli, B. subtilis, B. licheniformis, S. cerevisiae, K.

lactis and C127 cells. Using these host strains the production of recombinant human IL-3 was achieved. The products were tested for their capacity to stimulate human AML blasts as described above for the COS/pLB4 conditioned medium. From these experiments it appeared that the proteins made were biologically active.

The following examples are intended to illustrate I 30 but not to limit the invention.

""Y,0,88/04691 PCT/NL87/00037 19 Example 1 Retrieval of cDNA Encoding Human multi-CSF (hmulti-CSF) Human leukocytes stimulated with TPA (5 ng/ml) and ConA (10 ug/ml) produced considerable amounts of HGFs as measured by the murine stem cell proliferation assay and various other colony assays. Cells were harvested 24 hrs after stimulation, because mRNA production is often transient following stimulation with phorbol esters and lectins. Already after 24 h-s, HGFs were easily detectable in the CM.

mRNA Preparation Cells were harvested, washed with PBS and homogenized in guanidinium isothiocyanate solution RNA was pelleted through a cesium chloride cushion. Oligo(dT)cellulose chromatography was used for selection of mRNAs (36).

cDNA Synthesis cDNA was synthesized essentially according to Gubler and Hoffman using oligo(dT) as primer and AMV reverse transcriptase. Second strand was synthesized with RNaseH and E. coli DNA polymerase I. Gaps were closed with T4-DNA ligase and ends were flushed by T4-DNA polymerase. To protect internal EcoRI restriction sites, the cDNA was methylated with EcoRI methylase. Subsequently, the cDNA was ligated to phosphorylated EcoRI linkers with T4-DNA ligase. After digestion with EcoRI, excess linkers were removed by Sepharose CL-4B chromatography. The material recovered in the void volume of the column was larger than 250bp and was used for construction of the libraries.

Construction of the Phage cDNA Library.

The cDUA was ligated to lambda gtl0 phage arms and packaged with commercial packaging extracts (Gigapack, Vector Cloning Systems). The recombinant phages were propagated in E. coli C600 hfl.

~e A WO 88/04691 20 PCT/NL87/00037 Screening of the Phage Library.

Of each plate containing 1-5000 plaques, two nitrocellulose filter replicas were made according to standard procedures. Filters were then hybridized with radiolabeled mIL-3 probe from the HindIII-XbaI fragment of mIL-3 cDNA or with the complete mIL-3 cDNA clone radiolabeled with random primers. The mIL-3 cDNA clone (pL01) was isolated from a IEHI-3B cDNA library. WEHI-3B mRNA was isolated using the guanidinium isothiocyanate CsC1 method, size fractionated on sucrose gradient and injected into Xenopus laevis oocytes.

RNA fractions inducing the oocytes to produce a factor capable of supporting murine stem cell proliferation, were used for synthesis of cDNA as described above, cDNA was tailed with dC residues and inserted in the PstI site of pUC9. mIL-3 clones were identified using synthetic oligonucleotides (from published mIL-3 sequence, 11). Insert of pLl01 was purified on polyacrylamide gel and used for screening of the human cDNA library. Probe DNA was labeled using the random primer method Potential positive plaques were rescreened and plaque purified. In this way four clones were identified, including phage Dll.

Sequencing of cDNA Clo. as.

Recombinant phages were grown at large scale and purified, cDNA inserts were removed from the phage arms by digestion with EcoRI and purified on polyacrylamide gel. The purified fragments were ligated into M13mpl8 and pTZ18R DNA digested with EcoRI and used for transformation of E. coli JM109. Single strand DNA was prepared and sequenced according to established procedures Sequence data were analyzed using various computer programs (40-43).

The sequence obtained for the insert in phage 011 is shown in Figure 1. This 910 bp sequence contains the entire coding region for hmulti-CS and its signal sequence, and exhibits high homology to the murine clone pL01 in the 3' untranslated region. The homology upstream in the coding sequence is relatively more limited. As described above, the protein has a putative 19 arano acid signal sequence followed LYle L i i _r IL Ir_ WO,88/04691 PCT/NL87/00037 21 by a 133 amino acid mature protein containing two glycosylation sites (15-17 and 70-72) and two cysteine residues at 16 and 84.

The deduced amino acid sequence is the same as that encoded by the genomic DNA dizclosed by Yang, Y-C, et al.

(supra), except for one amino acid that at position 8 of the putative mature protein, th.e Yang DNA encodes Ser, the cDNA herein encodes Pro.

The intronless sequence obtained in the phage DI1 can be used for procaryotic expression, as well as for expression in eucaryotic systems, as illustrated below.

Example 2 Expression in Mammalian Cells A. Construction of the eucaryote expression vector pLB4 Phage D11 (containing the longest cDNA insert) was diiested with Hind III and BglII and subcloned in plasmid pTl (a derivative of pTZ18R, containing some additional restriction sites in the multilinker, see Example 3A). Clones containing the phage fragment containing the cDNA insert were identified by restriction analysis. The cDNA insert was removed from this plasmid by partial digestion with EcoRI and purified by polyacrylamide gel electrophoresis. The appropriate fragment was inserted in a eucaryote expression vector (pLO) in an SV40 transcription unit.

pLO comprises: EcoRI (filled in) PstI of pBR322 (1-755), PstI-Aval of pBR329 (756-1849), AvaI-PvuII adapter (1850-1868), PvuII-HindII (filled in) of SV40 (promoter) (1869-2211), PvuIt-BamHI adapter containing the unique EcoRI site (2211-2251), MboI "splice fragment" of SV40 (2252-2862), BclI-BamHI (filled in) "poly A fragment" of SV40 (2862-3098), PvuII-HindIIi promoter fragment of SV40 (3099-3440), HindIII- BamHI Eco gpt gene (3441-4501), Mbol "splice fragment" of (4502-5111) and the Bcll-BamHI (filled in) "poly A fragment" of SV40 (5112-5348).

The Eco gpt transcription unit is of no importance

A.,

WO 88/04691 PCT/NL87/00637 22 in transient expression of proteins in COS 1 cells. The resultant expression plasmid for hmulti-CSF was termed pLB4 and was purified on CsCl. This plasmid in E. coli was deposited with the Centraal Bureau of Schimmelcultures

(CBS),

Baarn, the Netherlands, under the provisions of the Budapest Treaty on December 12, 1986 under CBS 568.86. The construct is shown in Figure 2.

B. Expression of hmulti-CSF in COS 1 Cells and Bioassays.

pLB4 DNA was transfected to COS 1 cells using the calcium phosphate coprecipitation method Cells were cultured for 48-72 hours in alpha medium containing 10% fetal calf serum. The culture medium was recovered, filtered and used in assays for establishing its biologic activity. Human bone marrow progenitor colony assays and acute myeloid blasts colony and proliferation assays were performed as follows.

Bone marrow was obtained from hematologically normal adult volunteers by posterior iliac crest puncture following informed consent. The mononucleated cells were separated by density gradient centrifugation on a Ficoll gradient (Nijegaard and Co., Oslo, Norway), washed and resuspended in Hanks balanced salt solution (HBSS). Myeloid cells and Tlymphocytes were then removed, For this purpose, marrow cells were lysed following incubation with monoclonal antibodies OKT-3 (CD3; Ortho, Ravitan, and Vim 2 (myelno .onocytic cells, at saturating concentrations in the presence of rabbit complement 30 minutes, 25 0 C) according to established procedures The cells were washed two times in HBSS, resuspended in Islcove's modified Dulbecco's medium (IMDM) and cultured in the presence of autologous plasma according to Fauser and Messner as described before at a concentration of 1.5-3 x 10 4 /ml. Erythropoietin 1 U/ml (sheep, step III, Connaught, Willowdale, Canada) and COS/pLB4 CM were added as growth stimulating activities, Results of standard cultures with phytohaemagglutinin stimulated leukocytes CM (PH-LCM) in direct comparison with COS/pLB4 CM are also given. Sixty percent of the colonies were plucked and identified by microscopical analysis. The CM from WO 88/04691 PCr/NL8700037 23 COS cells transfected with the vector without insert (pLO) failed to stimulate colony formation by itself.

The results are shown in Figure 3. As shown in the figure, the mean numbers of erythroid (BFU-E), granulocytemacrophage (CFU-GM), granulocyte (CFU-G), eosinophil (CFU-Eo), macrophage (CFU-) and mixed (CFU-MIX) colonies are shown of duplicated cultures stimulated with graded volumes of COS/pLB4 CM.

Induction of AML Proliferation (see Figure 4) AML blasts were purified using a bovine albumin (BSA) density gradient. Residual T-lymphocytes were removed from the AML samples by E rosette sedimentation (17, 49, AML (patient 1) colony formation was determined not only in the established PHA leukocyte feeder (PHA 1.f) system, but also in a modified version of the technique in which the leukocytes were replaced by COS/pLB4 CM, permitting assessment of its colony-stimulating activity (17, 18, 49, 50) as shown in Figure 4A. All experiments were performed in triplicate.

DNA synthesis of AML, blasts (patient 2) was assayed by thymidine uptake as described (51) with results shown in Figure 4B. Both assays showed a dose dependent relationship to CQS/pLB4 CM added. Addition of control COS medium did not affect AML proliferation in either assay.

C. Construction of eucaryotic expression vector pLB4/BPV In order to establish stable cell lines expressing human tL-3, C127 cells (ATCC CRL 1616) were transfected with a derivative of pLB4. This derivative was constructed by insertion of the entire BPV-1 genome (69) into pLB4 by the following strategy. The BPV-l BamHI fragment was excised from the vector pdBPV-MMTneo(342-13) The BamHI sticky ends were filled in using Klenow polymerase. Then the vector pLB4 was cleaved at the unique tECOV site within the Eco gpt gene.

Subsequently, the blunt-ended 8PV-1 fragment was cloned into the EcORV cleaved pLB4, resulting in the vector pLB4/BPV which is able to replicate in C127 cells. pLB4/BPV was transfected WO 88/04691 PCT/NL87/0037 24 to C127 cells using the calcium phosphate precipitation method The transfected cells were cultured for 16 days, after which foci were picked from the culture dishes. Several independent cell lines were established. The pLB4/BPV vector appears to be stably maintained within the cells, as judged by Southern blotting of Hirt extracts (71) of several cell lines.

Conditioned culture medium was tested for IL-3 activity using the AML proliferation assay. The stable cell lines produce active human IL-3.

Example 3 Construction of E. coli Expression Vectors A. Construction of pGB/IL-301 (see Figures 6, 7 and 8) For construction of E. coli expression vectors, the following modifications were performed according to standard procedures (36).

1. The 3'-terminal noncoding sequences between the Aval site (position 541) and the Xhol site (position 856) in pL44 were deleted by fusion of the DNA fragments following filling of the sticky ends with Klunow enzyme (Figure 2. For introduction of the hmulti-CSF insert into a bacterial expression vector, the following steps were performed. The pLHI vector was digested with AvalI and the recessed ends filled with Klenow polymerase, Following ligation of a BglIX linker (CAGATCTG), the DNA was digested with BglII and BamHI. The BglIt-BamHI hmulti-CSF fragment was purified on polyacrylamide gel and subcloned in the BglI site of pTl, a derivate of pTZS8R (Pharmacia) modified in the multiple cloning site (see Figure Two clones were obtained, which had the insert in the opposite orientation with respect to the lacZ promoter (see Figure Inserts of these two clones were isolated on polyacrylamide gel following digestion with BglII and EcoRV and subcloned in pTI digested with BglII and HindIt. The junction of the BglI linker and the hmulti-CSF DNA was verified by sequence analysis and showed a fusion of the linker to the AvalI site located at

I

WO,88/04691 PCT/NL87/00037 nt 1 of the cDNA clone (this AvaIX si't.e hbd arisen by ligation of the EcoRI linker to the cDNA o u).Since this con~struct (pGB/IL-300) was not in l with the lacZ protein, the BglII-EcoRV insert was subcloned into mliI and HindlI digested pUCS The resulting onatruct (pGB/IL-301, see Figures 5, 7 and 8) was tested for product,',n of a lacZ/hmulti-CSF fusion protein.

B. Construction of pGB/IL-302, pGB/IL-303, pGB/IL-304 and pGB/IL-305 (Figures 5, 7 and 8) Several base changes were intr~oduced into the coding sequence for the N-terminal part of the fusion proteins by introduction of synthetic oligo nucleotides into The new expression vectors, calledJ pGB/IL-3021 pGB/IL-303 and pGB/IL-3024 were constructed as follows-. the H-ind-HindIuI fragment of pGB/IL-300 was isolated on agarose gel and ligate to a synthetic oligonucleotides comprising the nucleotides 99- 137 of hmulti-CSF and a 5' terminal SalII recognition, sequence and inserted into pTZI8R digested with Sall and HiindIl. The sequence of several clones was established. Indeed, several base changes were observed, resulting in modifications of the hmulti-CSV protein. inserts of several clones were transferred to pUC8 for expression of the laoZ fusion protein (p013/t-302, pGB/IL-303), Clone pOB/IL-304 Was made in fase with lacZ by 2 5 ligation of the Salt site following filling of recessed ends with Klenow. Construction was verified by PvuI digestion.

Several clones lacked a synthetic olligonucleotide and were found to be tilsed in frame to the 2tacZ protein. One example of these clones wgas Called pGH/IL-305, C. Construction of pGB/IL-30G (sae Figures 5, 7 and 0) An expression vector coding for a protein lacking the lacZ 0'-terminal amino acids was made from VGB/Ib-300 by deletion le:oping as described in The synthetic otiqonucleotide com~prised 22 nUcleoti4dos Upstream. of the PTZ lacz gene including the ATG start codon and the first 24 nucleotidep coding for mature IL-3, This plasmid was called pGB/It.-306 (Figures S# 7 and 8), 11W

I

i 4

'I

IJ

O

88/04691 PCT/NL87/00037 26 E. coli strains containing the plasmids pGB/IL-300, pGB/IL-301 and pGB/IL-302 were deposited with CBS on July 13, 1987 under CBS 377,57, CBS 379.87 and CBS 378.87, respectively.

Figure 8 shows the sequence of fusion regions for the various plasmids constructed. The sequence of the clones is given from the start of the lacZ protein coding region in either pUC8 or pTZS18R (lower case letters) and of the hrulti- CSF coding region (upper case letters) up to the ClaI site at position 158. Mutations in the hmulti-CSF DNA sequence are underlined, resulting in tpl 3 ->argl 3 pGB/IL-302); leu 9 pro 9 and trpl 3 -4argl 3 (pG0/It-303); met 3 thr 3 and a silent change (pGB/IL-304), In the priority application EP 67201322.2, filed on July 13, 1987, other designations were used for these plasmids as follows: 2 pGB/IL- 300 pGB/Il-301 pGB/IL-302 pGoB/L- 33 pGB/Ib-304 pGB/IL-305 pGB/IL-306 =pT-hIL3t pUC/hmulti pUC/hmulti L 1A =pUC/hmulti LBt puC/hmultiZ CC pUC/hmultiLA2; pTZ/hmulti; D. Expression of lacZ/hmulti-CSF Fusion Proteins and Mature hmulti-CSF in L. coli E. coli strains (JM 109) carrying various expression vectors were grown in LB medium containing 50 pg/mi of ampicillin at 37 0 C until an optical density of 0.5 at 550 nm 30 was reached. Subsequently IPTO (isoproyl beta-D-thiogalactoside, Pharmacia) was added to the culture to a final concentration of 1 mM and incubation was continued for 3-4 hours.

Plasmids pGO/IL-306 and pGB/XL-302 were also transformed to E. coli Dbl (wild type lacZ operon), Those strains were grown in LB medium or 2 x TY medium containing g/ml of ampicillin at 37 0 for 16 hours.

Bacteria were collected by centrifugation and WO 88/04691 PCT/NL87/00037 27 sonicated in buffer containing 0.1 M Tris/HC1, pH 8.0; EDTA 0.2% Nonidet P40 (NP-40) and 1 mM phenylmethylsulfonyl fluoride (PMSF) and centrifuged for 30 min at 20,000 x g.

Polyacrylamide gel electrophoresis of the pellet and supernatant fractions showed that the bulk of the bmulti-CSF proteins is stored in the bacteria in an insoluble form.

The pellet was re-extracted with 0.5% NP-40 buffer and finally solubi.ized with 8 M urea 0.1 M Tris/HC1, pH and 5mM dithiothreitol. Thus, an extensive purification of the fusion prote 4 ns was achieved (Figure 9).

As shown in the figure, inclusion bodies from bacteria coli) containing pGB/IL-301 and pGB/IL-302 were isolated as described. Lanes 1 show the 0.2% NP40 supernatant (sample corresponds to 0.1 ml of the original bacterial culture). Lanes 2 show the 0.5% NP40 supernatant (0.2 ml) and lanes 3 the pellet solubilized in 8M urea buffer 0.05 ml; B: 0.2 ml). The proteins were separated on a 13.5% SDSpolyacrylamide gel and stained with Coomassie Brilliant Blue, Molecular weights (in kd) of marker proteins (lane M) are denoted on the right. The human multi-CSF fusion proteins are indicated by arrows. The fusion protein encoded by pGB/IL-301 has a MW as expected of about 20 kd; that produced from pGB/IL-302, of about 16 kd.

E. Determination of Biological Activity of Bacterial hmulti- CSF Preparations.

Bacterial protein preparations were diluted in alpha medium containing 1% bovine serum albumin, filter sterilized and assayed in the AML blast proliferation assay. Diluted samples were added to purified AML blasts and cultured for four days. DNA synthesis was measured using 3 H thymidine as described One unit per ml is defined as the amount of hmulti-CSF required for half maximal proliferation of AML blasts. Figure 10 shows this titration. Various dilutions of the urea extractei protein preparation of bacteria containing the plasmid pGB/IL-302, were assayed for the stimulation of AML blast proliferation using 3 H-thymidine. The fusion protein concentration of this protein preparation was 33 pg/mi. Based WO 88/04691 PCT/NL87/00037 28 on the presented titration curve, the activity of this preparation is 16,000 units/ml.

The amount of bacterial fusion protein in the preparations was estimated from polyacrylamide gelelectrophoresis and used for calculating specific activities.

The results are shown in the following table: Table 1 Bioloqical Activity of Bacterial hmulti-CSF Preparations Mr (x 10 3) ug protein units Specific lacZ/hmulti per ml per ml activity units per mg IL-3 pGB/IL-301 20 20 45 4,500 pGB/IL-302/303 16 5 2400 400,000 pGB/IL-304 i8 ND(4) 18 pGB/IL-305 16 1 300 300,000 pGB/IL-306 15 ND 70 ND 1. Approximate molecular weights are estimated from the DNA sequence of the fusion protein (Figure 8).

2. IL-3 concentrations were estimated on SDS-polyacrylamide gel and calculated per ml of starting culture, 3. Activity of urea solubilized protein was determined in the AML proliferation assay and is expressed par ml of starting culture.

4. Not determined.

From these results it was concluded that human multi-CSF expressed as a fusion protein in E. coli was obtained in biologically active The results show that changes introduced into the N-terminus of the fusion proteins may influence the specific activity of these proteins.

I I O, 88/04691 PCT/NL87/00037 29 Example 4 Preparation of Antibody Preparations Capable of Immunospecific Reaction with human IL-3 Protein A. Polyclonal Rabbit Anti-Human IL-3 Antiserum.

A preparative gel was made from a lysate of E. coli containing the plasmid pGB/IL-301. The 20 kd band with the IL- 3 fusion protein was sliced out, minced in saline with a mortar and emulsified in a 1:1 ratio in Complete Freund's Adjuvant containing 1 mg of Mycobacterium tuberculosis H37RA per ml. New Zealand White rabbits (spf) were immunized with 1 ml of the emulsion (with 100 pg IL-3 fusion protein) divided over 5 injection sites (2 x i.m. in the thighs, 3 x s.c. on the back). Booster injections of the same antigen in Incomplete Freund's Adjuvant were given at week 2, 4 and 6.

Serum was collected at week 8 by venapuncture from the ear.

One volume of serum was absorbed with 9 volumes of sonicated pUC8 containing E. coli (overnight at 4 0 C) to remove nonspecific antibodies. Immunoblotting of all IL-3 constructs made in E. coli, B. licheniformis, B. subtilis, S. cerevisiae and K. lactis showed immunospecific reaction with the absorbed sera at a dilution of 1 in 6500.

Some of these results are shown in Figure 11. The proteins were isolated from the recombinant hosto as described above and were separated on a 13.5% polyacrylamide gel and blotted onto a nitrocellulose membirane. Lane 1. 2. coli containing pTZlSR (control); Lane 2: pGB/IL-301; Lane 3: pGB/IL-301; Lane 4: pGB/IL-302; Lane 5: pUC19 (control); Lane 6: pGB/IL-301; Lane 7: pGB-IL-302. Lanes 6 and 7 show proteins present in the pellet after the sonification of the bacteria. Lanes 3, 4 and 5 show proteins present in the pellet after the first washing step. Lanes 1 and 2 show the final urea-solubilized protein fractions.

The arrows show the fusion proteins (of the expected size) expressed from pGB/IL-301 and pGB/IL-302.

Figure 12A shows the inhibition of IL-3 dependent proliferation of AML blast cells by anti-IL-3 antiserum.

I

WO 88/04691 PCT/NL87/00037 30 Figure 12B shows that the preimmune serum does not affect the action of IL-3 on AML blast cell proliferation. In both panels, A IL-3 at 10 U/ml; IL-3 at lU/ml; control, no addition.

Figure 12A shows IL-3 dependent growth in the AML blast proliferant assay (51) was inhibited by the sera in a dose dependent manner; Figure 12B shows preimmune sera do not have this effect. As control, GM-CSF dependent growth was unaffected by these sera in the same assay (Figure 12A where GM-CSF at 100 U/ml.) B. Monoclonal Mouse Anti-Human IL-3 Antibodies Balb/C mice were immunized with 3 x 0.1 ml of the same emulsion as used for the rabbits. A booster (0.1 ml of antigen in Incomplete Freund's Adjuvant was given at week 2 and three days later spleen lymphocytes were fused with SP2/0 myeloma cells according to standard procedures Hybridoma supernates were screened in the Enzyme Linked Immunosorbent Assay, using a lysate of E. coli pGB/IL-302 (containing the 17 kd IL-3 fusion product) as a positive control and a lysate of E. coli pUC8 as negative control. In j total, 29 IL-3 hybridoma cultures secreting antibodies specific for IL-3 were selected and stabilized.

Example Construction of Bacillus expression vectors General cloning techniques were used (36).

A. Constuaction of pGB/IL-307 (Figure 13) For construction of pGB/IL-307 the Smal fragment of pLB4 carrying tho hmulti-CSF gene, was ligated into PvuII digested pUB110 After transformation to competent cells (56) of DB105 (a spo- derivative of the protease deficient strain DB104 two clones were obtained, as expected: the fragment was cloned in both orientations. The plasmid that harbored the fragment in the correct orientation with respect HNNMM Ir WOp88/04691 PCT/NL87/0037 31 to the so-called "Hpa II promoter" (57) was called pGB/IL-307.

In this case a fusion protein will be made (see Figure 13).

B. Construction of pGB/IL-310 A hmulti-CSF expression plasmid was prepared as described below.

1. Promoter cloning (Figure 14).

For expression in Bacillus a synthetic r 4 3 promoter as described (58) is used (the promoter used to be called q Plasmids pPROM55s the promoter containing plasmid, and pGPA14 (59) were digested with EcoRI and XbaI.

The promoter fragment was ligated into the vector fragment, which had been purified on an agarose gel. After transformation to E. coli (JM 101), the correct plasmid was obtained and called pGB/IL-308 (Fig. 14).

2. Introduction of a synthetic oligonucleotide into pGB/IL-308 (Figure A synthetic oligonucleotide comprising the nucleotides 39-158 and 484-546 of hmulti-CSF, a 5' terminal SalI recognition sequence and a 3' terminal XmaIII site was ligated into Sall-XmaIII digested pGB/IL-308. The ligation mixture was introduced into JM101. After analysis of a number of transformants, the correct plasmid was found, pGB/IL-309.

3. Introduction of hIL3 (Figure 16).

Af'ter transformation to and isolation from B. subtilis DB105, the plasmid pGB/IL-309 was digested with XmaIII. The recessed ends were filled in with Klenow polymerase, and the plasmid was cleaved with Clal. The plasmid pGB/IL-307 was digested with Aval, the ends filled in with Klenow and then digested with Clal. Subsequently, the hmulti-CSF containing fragment was ligated into the pGB/IL-309 fragment and transformed to JM101. The resulting plasmid was called pGB/IL-310 (Figure 16). This plasmid

L

WO 88/04691 PCT/NL7/00OJ7 32 harbored the hIL-3 gene with its own signal sequence. After isolation of the correct plasmid, it was also introduced into B. subtilis DB105.

C. Construction of pGD/IL-311 and pGB/IL-312 (Figures 17, 18) pGB/IL-310 was partially digested with HindIII and totally with PvuII. The two hmulti-CSF containing PvuIIdigested with HindIII and SmaI.

Figure 17 sh6ws the nucleotide sequence of plasmid pBHAl. The plasmid consists of positions 11-105 and 121-215; bacteriophage FD terminator (double): positions 221-307; a part of plasmid pBR322 (viz. positions 2069-2153): positions 313-768; bacteriophage Fl, origin of replication (viz.

positions 5482-5943): positions 772-2571; part of plasmid pBR322, viz. the origin of replication and the beta-lactamase gene: positions 2572-2685; transposon Tn903, complete genome: positions 2719-2772; tryptophan terminator (double): positions 2773-3729; transposon Tn9, the chloramphenicolacetyltransferase gene. The nucleotides at position 3005 3038 330? and 3409 differ from the wild type cat coding sequence. These mutations were introduced so as to eliminate the Ncol, Ball, EcoRI and PvuII sites: positions 3730-3804; multiple cloning site: positions 3807-7264; part of plasmid pUBllO, viz. the replication function and kanamycin resistance gene (EcoRI-PvuII fragment) (66, G7):'positions 7267-7331; multiple cloning site. The fragments wer put together by known cloning techniques, e.g. filling in of sticky ends with Klenow, adapter cloning, etc. All data were derived from GenbankR, National Nucleic Acid Sequence Data Bank, NIH,

USA.

After transformation to JM101 and analysis of a number of ampicillin resistant colonies, two different plasmids were found: pGB/IL-312, which harbored the complete gene with complete control sequences, and pGB/IL-311, which contained the complete gene and the promoter lacking the region in the other orientation (see Figure 18).

pGB/IL-311 has been transformed to B. subtilis DB105 and B. licheniformis strain T399 (Lamy, spo-, exo- I I i 1 vO?88/4691 PCT/NL87/00037 33 protease negative, rifr, see ref. 68).

D. Construction of pGB/IL-313 (Figure 19).

In order to obtain a smaller plasmid, with the hmulti-CSF gene behind the "HpaII promoter", pGB/IL-312 was digested with BamHI and religated. The ligaton mixture was transformed into DB105 competent cells. A number of neomycin resistant colonies were analysed and the correct plasmid was obtained. The plasmid was called pGB/IL-313.

E. Construction of pGB/IL-317 (Figure In order to clone the hmulti-CSF gene behind the B.

licheniformis alpha-amylase transcriptional and translational initiation region and signal sequence, one of the earlier described pOL5-delta vectors (68) was used, viz. pOL5-2 delta.

Besides the alpha-amylase signal sequence (29 amino acids long) this plasmid harbors one amino acid of the alpha-amylase mature sequence (an Ala) followed by a multiple cloning site: EcoRI-XmaIII-XmaI-SalI-HindIII (68).

The SalI-PvuII fragment of plasmid pGB/IL-310 containing the hmulti-CSF gene was ligated into the SalI-PvuII digested pOL5-2 delta vector and transformed to DB105. The resulting plasmid was called pGB/IL-317 (Figure 20). The hIL-3 gene still harbors its own signal sequence on this plasmid.

The plasmid was also introduced into B. licheniformis T399.

F. Expression of Five Expression Plasmids in Bacillus Strains B. subtilis and B. licheniformis strains carrying the expression plasmids mentioned below were grown in TSB medium containing 20 pg/ml neomycin or 10 pg/ml erythromycin at 37*C (for 16-24 hours); 300 pg/ml of the culture was centrifuged. The pellet was resuspended in sample buffer and analyzed using polyacrylamide gel-electrophoresis followed by Western blotting. The supernatant was TCA precipitated, and the pellet was resuspended in sample buffer. Both supernatant and pellet were analyzed for IL-3 protein (see Table 2).

To determine the biological activity of the produced proteins, the following steps were carried out: The WO 88/04691 PCT/NL87/00037 34 cellpellets were resuspended in a buffer containing 0.1 M Tris/HCl pH 8.0 and 10 mM MgC1 2 Lysozyme was added to a final concentration of 1 mg/ml and PMSF to a final concentration of 1 mM. The solution was incubated for 30 min. at 37*C.

Subsequently DNase (final concentration 20 ig/ml) was added and the solution was incubated for 15 min. at 20*C. Finally, the biological activity of this preparation as well as of the supernatant of the cultured cells was determined as described.

The results are shown in Table 2.

Table 2 Expression of the Bacillus Vectors Plasmid Strain MW IL-3 Biological Pellet supernatant activity (kd) pellet supernatant pGB/IL-307 DBI05 21 pGB/IL-310 DB105 1517 1517 pGB/IL-311 DB105 12.5;15 T399 pGB/IL-313 DB105 15;17 12.515 T399 pGB/IL-317 D8105 12.515 12.5?15 17;20 17 T399 12.5;15 12.515 17;20 17 It can be concluded, that in B. subtilis, using pGB/IL-307, a fusion protein is made that has IL-3 activity.

!Then the human IL-3 gene only contains its own signal sequence no significant secretion of human IL-3 is obtained. All IL-3 activity is found intracellularly. In those cases it seems that besides precursor IL-3 mature IL-3 (15 kd) has been formed in the cell. Thus, some transport across the membrane might have taken place, but the protein is not transported I 88/04691 W0,88/04691 PCT/NL87/00037 35 across the cell wall. However, using the alpha-amylase regulation and secretion signals (pGB/IL-317) most of the IL-3 activity appeared to be secreted into the culture medium.

Besides a degradation product, two proteins are detected in the supernatant, one of about 15 kd and one of about 17 kd, most probably mature IL-3 and precursor IL-3, respectively.

These data indicate that both processing sites, viz. the alpha-amylase and the hmulti-CSF processing site, are used. In the cell the most abundant product is precursor IL-3 containing the alpha-amylase signal sequence (the 20 kd protein) as shown by Western blotting. Sometimes a degradation product is detected.

Example 6 Construction of Kluyveromyces lactis expression vectors A. Construction of pGB/IL-316 A DNA fragment comprising the Tn5 gene (61) conferring resistance to gentamycin G418, under the direction of the alcohol dehydrogenase I (ADHI) promoter from S.

cerevisiae, similar to that described by Bennetzen and Hall was inserted into the SmaI site of ptUI9 An E.

coli strain containing the obtained plasmid, pUC-G418, was deposited with CBS on December 4, 1987 under CBS 872.87.

Into the XbaI-HindIII cleaved pUC-G418 vector a Xbal-HindIIJ fragment from plasmid pGB903 (64) containing the K. lactis lactase promoter and calf prochymosin DNA was inserted, resulting in plasmid pGB/IL-314.

The SalI-HindIII fragment from this plasmid was replaced by a synthetic DNA fragment containing a small multiple cloning site and the lactase terminator (see Figures 21, 22). The resulting plasmid is designated poG/IL-315.

In the SacII.XhoI cleaved pGB/IL-315 vector the following fragments were ligated: 1. The SacII-XbaI fragment from pKSlC5 Pat.

Appln. Ser. 078,539, 64), carrying the 3' part of the lactase promoter and the 5' part of the alpha-factor signal sequence

I

is ii

H

I!

U

H

WO 88/ 9 1 PCT/NL87/0007 36 of S. cerevisiae.

2. A synthetic oligonucleotide comprising the 3' part of the alpha-factor signal sequence starting at the XbaI site and the 5' part of the mature hIL-3 cDNA sequence upto the 5' half of the Hpal site (aa-residue 14).

3. The HpaI-XhoI fragment carrying most part of the hIL-3 cDNA sequence (residue 15-133 plus the 3' non-coding region). The resulting plasmid, designated pGB/IL-316, is depicted schematically in Figure 21. The complete vector sequence from the SacXI site in the lactase promoter sequence up to the HindIII site at the end of the synthetic terminator is given in Figure 22.

Figure 22 shows the nucleotide sequence of plasmid pGB/IL-316 between the unique Sac 11 site in the lactase promoter and the Hind III site behind the terminator (residues 4457 to 7204). Residues 4457 to 6100 comprise the lactase promotor sequence. Residues 6101 to 6355 comprise the alpha factor signal sequence. Residues 6356 to 7115 comprise the sequence for mature human IL-3 plus the 3' noncoding CDNA sequence. Residues 7116 to 7204 comprise the synthetic terminator sequence.

B. Construction of pGB/IL-318 An expression vector similar to pGB/IL-316 was constructed in which the coding information for the alpha factor signal sequence of S. cerevisiae was replaced by the alpha-factor signal sequence of K. lactis The remaining part of the plasmid is identical to pGD/IL-31,. The sequence of pGB/IL-318 between the SacII site in the lactase promoter and the HindIIl site behind the terminator (residues 4457 to 7190) is given in Figure 23.

Residues 4457 to 6087 comprise the sequence of the lactase promoter and a small linker sequence. Residues 6068 to 6342 comprise the K. lactis alpha factor signal sequence.

Residues 6343 to 7102 comprise the sequence for m4ture human IL-3 plus the 3 0 noncoding cDNA sequence. Residues 7103 to 7190 comprise the synthetic terminator sequence.

WO 88/04691 5/26 PCT/NL87/00037 i I: W088/04691 PCT/NL87/00037 37 C. Transformation of Kluyveromyces Lactis and Analysis of Secreted hIL-3 Plasmids pGB/IL-316 and pGB/IL-318 were digested at the unique SacII site in the lactase promoter region, and used to transform K. lactis strain CBS 2360 (see 64). Integration of the plasmids is thus targeted to the chromosomal lactase gene promoter region. The resulting G418 resistant transformants were grown to saturation in liquid YEPD medium, and the culture supernatants and cell lysates were assayed for IL-3 activity using the AML cell DNA synthesis assay.

Virtually all IL-3 appeared to be secreted into the culture medium, and to be active. The proteins from the culture supernatant were precipitated using ethanol and analyzed using denaturing polyacrylamide gel-electrophoresis followed by Western blotting, The predominant product has an apparent MW of about 21 kd, whereas also a distinct band at about 15 kd is observed. The latter product most probably corresponds to the mature unglycosylated IL-3, whereas the 21 kd product is the product carrying core glycosylation at the two potential glycosylation sites. Incubation with Endoglycosidase H results in a protein migrating in the 15 kd range, suggesting that all IL-3 is processed correctly during the secretion process and that the bulk of the protein is being glycosylated.

Example 7 Construction of a Saccharomyces Cerevisiae Expression Vector A. Construction of pQB/IL-319 First an expression vector called pGB/TEFact was constructed, On this pTZl8R (Pharmacia) derived plasmid the St cerevisiae translation elongation factor (EP-lalpha) promoter sequence, which was cloned and sequenced as described (73,74), is coupled by ireans of a small SalI-BglII-XhoI linker to the S. cerevisiae actin transcription terminator sequence which was synthesized using an Applied Biosystems DNA synthesizer. The sequence of the expression cassette is given wv~ F WO 88/04691 PCT/NL87/000 7 38 in Figure 24. Residues 1 to 949 comprise the EF-lalpha promoter. Residues 950 to 967 comprise the sequence of the SalI-BglII-Xhot linker. Residues 968 to 1113 comprise the actin terminator sequence.

The unique SmaI site in pGB/TEFact was used to introduce the G418 resistance cassette described in Example 6.

The resulting plasmid was called pGB/TEFactG418.

Finally, the hIL-3 expression vector pGB/IL-318 was constructed by introduction of the following DUA sequences into the SalI-XhoI cleaved pGB/TEFactG418 plasmid: The SalI-Mru fragment from pGB/IL-316 carrying the S.

cerevisiae alpha factor signal sequence and the hIL-3 coding sequence upto the Nrul site.

A synthetic NruI-XhoI pA fragment comprising the remaining nucleotides coding for hIL-3 and the XhoI recognition sequence immediately following the TGA stopcodon.

B. Transformation of Saccharomyces Cerevisiae and Analysis of Secreted hIL-3 Plasmid pGB/IL-319 was cleaved at the unique EcoRI site in the E-10 promoter. Integration of the plasmid is thus targeted to the chromosomal EF-lo( region, S, cerevisiae wild type strain D273-103 (alpha; ATCC 25657) was transformed as described for K. lactis The 0418-resistant colonies were picked and transformants were given to saturation in liquid YEPD medium. The culture supernatant was assayed for hIL-3 activity using the AML assay. The protein produced by So cerevisiae was found biologically active.

The proteins from the supernatant were precipitated using ethanol and subsequently analyzed by polyacrulamide gelelectrophoresis followed by Western blotting. Two prominent products could be distinguished on the Western blot, a 21 kd glycosylated product and an unglycolysed product of about kd.

Wo,88/04691 PCT/NL87/00037 39

REFERENCES

1 Metcalf Blood 67, 257-267 (1986).

2 17hetton A.D. and Dexter T.M. TIBS 11, 207-211 (1986).

3 Wagemaker In "Bone Marrow Transplantation" (eds. Van Bekkum D.W. and Lowenberg B. Marcel De1ker Inc. New Yor} 1-72 (1985).

4 Dorssers L. et, al., Exp. Hematol, 12, 357, 1964, Till J.E. and McCulloch Radiat. Res, 14, 213-222 (1961).

6 Hape] AiJ. at, al, Blood 1453-1459 (1985).

7 Scheven Nature 321, 79-81 (1986), 8 Garland I,M. and Crompton S. Exp, Hematol. 11, 757-761 (1983).

9 Stanley E.R. et. al., Cel] 45, 667-674 (1986).

.0 Kreigler A.B. et. al,, Blood 60, 503-508 (1982).

11 Fung NC, et, NatUre 307o 233-237 (1984).

12 Yokata T. et. al., Proc. Nb4*J, Acad. Sci, USA# 81, 1070- 1074 (1984).

13 Lowenberg B, and Dicke Exp. Hemato1, 5, 319-331 (1977).

1.4 11agemale.r G. and Peters MF.j Coll, Tiss, Kinet. 11, 45-56 (1978).

jh]e Jd4,# et4 al., In "advances in viral oncdology', vol. 4 (ed Klein 95-137o RaVen Press, NeW Yor% 1984.

16 FaUser A.A, and Messner H.A. Blood 52, 1243-1248 (1978), 17 Lowenber B, et. al., Res, 4, 143-140 (1980).

18 Lowenberg B. et, all. tBlood 59, 64-G45 (1982).

19 Buick R.O. et, Blood 54, 95-104 (1979).

20 Huynh T.V. ot. al., in "DNP doning", Vol. 1 (Ed. Glover D.Ms) IRL press, oxford 45-70 (1985).

21 Koza M. Call 44, 283-292 (1986)o 22 Von lieijne a. Eur 1. 5iochem. 133, 17-21 (1983), 23 Perlman and Halvorson 14B. t. Mo1. Biol. 167, 391-409 (1983).

24 Shaw 0. and Karern R. Cll 46, 659-67 (1906).

Schrader a.3.11. et, 4L,, Proc, Natl. Acad. Sci. USA 83, 2458-246 (1986).

ar I I I ~L WO 88/04691 26 March C.J.

27 Higashi Y.

28 Dijkema R.

29 Zwarthoff (1985).

i 30 Clark-Lewi 31 Kindler V.

100i (1986 PCT/NL87/0007 40 et. al., Nature 315, 641-647 (1985).

et. al., J. Biol. Qhem. 258, 9522-9529 (1983).

et. al., EMBO J. 4, 761-767 (1985).

E.C. et, al., Nucleic Acid Res. 13, 791-804 s I. et. al., Scie.,ce 231, 134-139 (1986).

et. al., Proc. Nati, Acad. Sci. USA 83, 1001i i :i 32 DeLamarter J.F. et. al., EMBO J. 10, 2575-2581 (1985).

33 Lemischka I.R. et. al., Cell 45, 917-927 (1986).

34 Yu-Chung Yang et. al., Cell 47, 3-10 (1986).

35 Miyatake S. et. al., Proc. Aatl, Acad. Sci. USA 82, 316-320 (1985).

36 Maniatis T. et. al., In "Molecular Cloning, A. laboratory manual", Cold Spring iHarbor Laboratories, New York (1982).

37 Gubler U. and Hofmann Gene 25, 263-269 (1983).

38 Feinberg A.P. and Vogelstein B. Anal. Biochem. 132, 6-13 (193).

39 Sanger F, et, al., Proc. Natl. Acad. Sci. USA 74, 5463 (197).

Queen C. and Korn L.J. Nucleic Acid Res. 12, 581-599 (1984).

41 Staden R, Nucleic Acid Res, 10, 2951-29G1 (1982).

42 Devereux et, al., Nucleic Acid Res. 12, 387-395 (1984).

43 Lipman D.J. and Pearson W.R. Science 227, 1435-1441 (1985..

44 Subramani S. and Southern P.J Anal., Bioch. 135, 1-15 (1983).

45 Wigler et. Cell 14, 725-731 (1978), 46 Majdic et. al., Int. J. Cancer 33, 617-623 (1984).

47 Lowenberg 8. and Bauman J.GiJ. Blood 66, 1225-1232 (1984).

48 Daiwe] eti a1., Blood 68, 41-45 (1986) 49 Swart K. et. al., Bloo 59, 816-821 (1982).

50 s .r-t K. and Lowenberg B. Cancer Res. 44, 657-660 (1q984).

51 Touw 1. et, al., Blood 68, 1088-1094 (1986), 52 Vieira, J. and Messing Gene 19, 259-268 (1982) 53 Osinga, K,A. et al., Nuclic Acids Res. 11, 8595-8608 (1983) *WC 88/04691 PCI'/NL87/00037 41 54. Gryczan, T.C. et al., J. Bacteriology 134, 318-329 (1978) Kawamnura, F. and Doi, J. Bacteriology 160, 442-444 (1984) 56. Bron, S. and Venema, Mutat. Res. 15, 1-10 (1972).

57. Zyprian, E. and Matzura, DNA 5, 219-225 (1986).

58. EPA 0224294, published June 3, 1987.

59. EPA 0244042, published November 4, 1987.

Stanssens P. et al., In "Protein Engineering and Site- Directed Mutagenesis". Twenty-Fourth Harden Conference.

Program and Abstracts (1985) (Fersht, A.R. and Winter, G., eats 61. Reiss, B. et al., EMBO J. 3, 3317-3322 (1984).

62. Bennetzen, J.L. and Hall, J. Biol. Chem. 257, 3018- 3025 (1982).

63. Yanisch-Perron, C. et Gene 33, 103-119 (1985).

64. U.S. Appi. Ser. No. 078,539, filed July 28, 1987.

Saifre, S. -and Milstein, Meth Enz 73, 3-75 (1981), 66. McKenzie, T. et al.# P3.asrid 15, 93-103 (1986), 67. Mc~(enzie, T. et al., Plasmid 17, 83-85 (1987).

68. European Patent Application 87201379.2, filed July 69. Chen, E.Y. et al., Nature 299, 529-534 (1982).

Law, M-F. et Cell B~iol. 3, 22110-2115 (1983) 71. Hirto J. Mol. Biol. 26, 365-367 (1967).

72. Suarez Rendueles, M4.P. et FEBS Lett. 131, 296-300 (1981).

72st Najata, S. et EMIBO J. 3, 1825-1830 (1984).

74. Nagashimna, K. et Getia 45, 265-273 (1986).

GalJlwitz, D. and Sures, I1., Proc. Nat).. Acad. "i USA 77, 2546-2550 (1980).

Claims (15)

1. A transformed living host cell containing genetic material derived from recombinant DNA material without introns and coding for human IL-3 having a Pro at position 8 of the mature protein molecule, wherein the host cell is selected from the group consisting of yeasts, bacteria, fungi and tissue culture ceils.

2. A transformed yeast host cell according to Claim 1, which is selected from the group of Saccharomyces and Kluvveromvces.

3. A transformed bacteria host cell according to Claim 1, which is selected from the group of Bacillus.

4. A transformed tissue culture host cell according to Claim 1, which is selected from the group C127 and insect cells.

5. An expression system operable in a recombinant host S which expression system consists essentially of a DNA sequence containing no introns encoding human IL-3 having a Pro at position 8 of the mature protein molecule operably linked to control sequences effective in said host. An expression system according to Claim 5 wherein S. control sequences comprise a promoter selected from the group consisting of the lac promoter, the HpaII promoter, the J43 promoter, the alpha-amylase promoter, the EF-1 alpha promoter and the SV40 promoter.

7. A recombinant host cell according to any one of Claims 1 to 4, transformed with the expression system according to Claim 5 or 6.

8. A vector selected from the group consisting of pGB/IL-300 to pGB/IL-319 as hereinbefore defined.

9. A vector selected from the group of pLB4 and pLB4/BPV as hereinbefore defined. A recombinant DNA sequence containing no introns which encodes human IL-3 having a Pro at position 8 of the mature protein molecule,

11. The DNA of Claim 10 which comprises the nucleotide sequence shown as encoding amino acids 1-133 in sequence "H" Figure 1. -LL- r i i 11.;- V "t -43-

12. A method for isolating a DNA sequence encoding a CSF, comprising screening a DNA library with the use of the 3' untranslated region of the IL-3 gene, having a Pro at position 8 of the mature protein molecule obtained from another species, or of the cDNA derived from such a gene, as a probe.

13. A method according to Claim 12, wherein the DNA library is derived from mammalian DNA.

14. A method according to Claim 12 or 13, wherein the DNA library is a cDNA library. A recombinant human interleukin-3 DNA sequence having a Pro at position 8 of the mature protein molecule obtained by isolating a DNA sequence identified according to Claim 12.

16. A method for producing human IL-3 by a host cell, said method comprising: introducing into said host cell a DNA construct comprising an expression cassette which comprises in the direction of transcription a transcriptional initiation S regulatory region functional in said host cell; a DNA sequence containing no introns encoding human IL-3 having a Pro at position 8 of the mature protein molecule S" which has been isolated according to Claim 17, A human IL-3 having a Pro at position 8 of the mature protein molecule purified by the method according to Claim 16, DMW/3283U L WO ,88/O4691 PC/NL87/OO37 1/26 M ETSER' AR G LEV PRO VAL~ LE U LE L U t'uCLN LEUrLEUl- H CACCACAACAACACAGA GTC CCT OCT CCC GAT CCA AAC ATO AGO COC CTC CCC CTC OT TO CTC T CTC CAA CTC CTO II1111111 IIii I IIII II II II IIIIIIIII ii Iii II I M :1 GAACCCCTCAOCACCAGAACCACACA.. ATC OTn OTT CCC ACC TOT ACC ACC ACC ATC CAC ACC AlaCT CTC CT CTC CTC AIG dCT MET VAL. LEU ALA SER SER THR T8RISERILE H1IS 1118 METtLEU LEV LEU LILOU METILEtI 1 VAL. ARC PRo'CLY LEU GLH ALAIPRO MEOT THR GLN THR THRI PRO LEU LYS THRISER TRP VAL-ASN CIS SER ASH HOT ILE ASP H:t78 OTC CCC CCC GOA 010 CAA OCT CCC ATC ACC CAG ACA ACC CCC TTC AAG ACA ACC TOG OTT AAC TOO TOT A.AC ATG ATC GAT TTC CAC 010 OCA CTC CAA OCT TCA ATC ACT CCC cc COAT ACC CAC OT TTA ACC AGA ACO TO AAT TCC AGO TCT ATT 0TC AAO M:89 P110 HIS LEViCLY LEU GLN ALA SER ILE S08 CLY ARO ASPITHRIHIS ARC LEUtTIRtARO 111 LEV ASH 015 SER SER ILE VAL. LYS 1 10 'GLU ILE ILE T1R HIS 7OU LYS GLN =PRO PRQO MOPRO LEU LEU ASP P11E ASH ASH LEU ASH GLY CLV ASP GLH ASP L TO E 14:159 GAA An7 ATA ACA CAC TTA AAG CAG CCA COT TIC COT TIG CTo CAC TIC AAC AAC OTC AAT COG GAA GAO CAA GAC Arr 010 ATO M;173 GAG ATT ATA COG AAC CTC CCA GAA CCT CAA CTC AAA' ACT CAT OAT CAA GOCA CCC TOT 010 AGO ICLU ILE ILE OLY LYSgLOUjPRO GLUtPR0ICLUILEUILYS THR ASP I ASP, G LU CLY PRO ,ER LEU I ARG 60 70 CLU ASH ASH L EU A RG-A-R GPR'0S MNLEU L U A LA PH-E' ASH ARC ALA VLt. LYS 508 LEV CLN ASH ALA SER ALA r 1 -'ClLU H :243 OAA AAT AAO onT OA AGO OCA AAO 010 GAG CCA no AAO AGO OCT OTC AAQ ACT nTA CAG AACOGCA TOA OCA AT? OGO M.236 AAT AAG AOC ITT COG AGA CIA AAO C10 TCC AAA noC GTC OAA AGO CAA CCA OAA 010 CAT OCT GAG GAO AGA TACOTn ATO AAC ASH LYS 5CR PHEARG ARC VALIASN LEV SER LYSIP1OpVAL GLU SER 01.11 CLT CLV VAL ASP PRO CLU ASP ARC TYR VALIILEILYS so 60 80 90 100 SER ILOELOUILYS AS'LOULEU PRO'CIS LEU PRO LEV ALA ITH11IALArALAPRO THR ARC HIS5 PRO I1., HIS5 ILE LYS ASP OLT AS 1 H 321 AGO ATI onT AAA AAT CTC CTC COA TOT 010 CCC OTGOCCC AC CO G0k 000 ACC OA CAT OCA ATO OAT ATC AAG GAO COT GAC I I I t11 111 1 it II I M.320 TOO AAT onT 0kG AAA onT Ako TOT TOOC CO CT ACA TCT GC AAT CAC TOT GCO O 10 00 COG OTC TO ATT OA OAT ISERIASNtLOUIOLN LYSILEUIASN 015CY LEVU PROi ITHRISERIALAI ASH ASP SER ALA LEU PRO GLT VAL. P110 ILE ARGIASPI "0 120 130 TRP ASH GLIJ'p11E ARCIARC'LYS LEU'T11RIPHE TYR LEV LIS TIHRr 1 -E-GLU ASH ALA CLN ALA CLX CLN 5 R LEV SER LEVl ALA H :405 TOG AAT GAA TIC COG AGO 04A CO AC TO TAT OTG AA.A AOC CTn GAG AAT CG OAG OCT CAA 0kG ACG ACT TTC AGO 010 000 1 111 1 11 111 1l~l~ll 111 11 11 1 111 1 11 11 ii 1 M:39 COAT GAO TTT CO AAC AAA 010 AGA noC TAO ATO OT0 CAC OTT AAC CAT CTO GAG ACA OTC; OTA ACC TOT AGA OCA COT,. LEU ASP ASPONIE ARC LYStLYS LEV ARWiP1E TYRHET VAL HISILEIiASN ASP LEV1 CLJ 111 VAL LEVITHRISER ARO PRO PRO 100 110 120 ILE PH110 H;489 ATO TTT TGAOTCCAACGTCCAGCTCGTTCTCTGCGCOOTTCTCACCACAGACCCTOGAOATCAAAAACACC~dAACTTCTOAAACOTCGGOTCATTTACACAT I I lii111 11111 11 111 1ii III III 1 l~l 1 1 1111111ifI I if M.1479.... CACCCOCATCTGOCTCCCTCTCT OCTACCOTGOAACCCTOOAATGTTAA. AACAG ,ArOCArACAOOAAAQTCTO, AATGTTCCTCATOC OLNHROALASEOLVSERVALSER PR0ASNARGLTR VLLUCSI 00 130 14o H -597 TCCACGACCAGAACAnTTCACCTTTTCCTCCOGCATCACATCAATT0TTAA. 1-rATCTAATrICTGAAATGTGCAOOTCCCATIrrCCCTGTCGOTTGTGr-:CTCAT III III III 11111if11 1 1 11111 111 111 iiIIIII1IIIIIII 11111111 1111I M 1571 CCCATCCTCAAAAOAnTTTACATTCCTTTATGCCATCAAATGTCTTATCAATTI ATCTACTTICTGAAATTACAAC'6-CTTTGGCTTACAATATtJTIC.CTA H t706 nTrTATCCOArtTOACTArITATTTATGTATOIATCTATTTATTTAmrATOCTGOAO.,.TGAACTGTATTTATTTTAGCAOAGGACCrCATGTCCTGCTGCTTCT H :81 4 OCAAAAAACTCAGAGTCC0GTGO0GAGCAITTrCATrrGTACCTCGAOrTT.AAA CTOGTTCCTAGOGATCTGTIA0CAAACTAGACTCrfCAACA 91 C 1 1111 it 11 1 111 l~llll~ 111 1111 1I 1 1 1 M 1782 AAAOAAAATCCAAGAIGACTGGGCCATTTCATTGTCCCrrIIGAAATAA ATAACTTTOAACAAA 852 FIGURE 1 SUBSTITUTE SHEET W0488/04691 2/26 PCT/NL87/00037 Sm K S E E Sm B human Alutti-C5F cDNA FXGURE 2 SUBSTITUTE SHEET WOi 88/04691 PC/NL87/00037 3 /2 6 100 Lt 0.1 1 COS/PLB4CM 0BFU-E U CPU-MIX oCFU-CM CFU-CM 9CFU-Eo XiI PHALC (sadadsytm !Li 0 FIGURE 3 200 100 0 AL. r*1

20.1 0.3 0.6 1.25 245 5 rOS/rLB4 CM V/V) 10 PHA L,r. system 6.10. 0~ FIGURE 4 I I I I I I I JI III 0 1 2.5S 5 10 20 C0S/PLB4 CM V/V) SUBSTITUTE SHEET 9: E E I I I -I 1L plB14 pLH1 X Av al K len ow 3gill linker ligation X BgllI X BamHI Fragment isolation iLigation pTl x Bgl11 B gl SFragment isolation B g ll-Eco RV Ligation fragment in RV E r pT1/3Llh pTlx B g]I!H in dl pUG 8 xBarnHix Hin d]I Hindil BglI[ pG B/I L-3 00 Ideletion looping pG BIL-3 06 HindMl L.Lj PstI I-pGB/IL-301 Hindil-HindilI fragment oligonucleotide pG B/I L-3 02 pG B/IL-a o pGB I IL-3 04 pG B/I L-3 Pst I I I WO 88/04691 5/26 PCT/NL87/00037 EcoRI Sacl KpnI SinaI BaxnHI XbaI Sall PstI SphI Hindlli pTZ18R gggaattcgagctcggtacccggggatcctctagagtcgacctgcaggcatgcaagcttg EcoflI SadI EcoRV HindIII BglII BamHI XbaI Sall Pstl pT1 gggaattcgagctcgatatcaagcttagp tctcgagggggatcctctaggtcgao tgcag SphI NdeI gcatgoaagotgcatatgcagcttg FIGURE 6 SUBSTITUTE SHEET pLB4 _E7. pLH1 1 "..aI pGB/lL-302 'I pGBIIL-305 pGB/IL-306 FIGURE7 A4" 1 4 WO 88104691 WO. 8804691PCT/NL87/00037 7/26 pGB/IL-301 pOB/IL-302 pMf/IL-303 pOB/IL-304 Met Tht' atg acc Giu Gin GMA CAA Ser Arg AGC CGO Val Arg GTC COC Thr Pro ACG CCO Met-.lie ATG ATC Met Thr atK ace Val Asp gtc gac Ser Arg AGOCOO Met Thr atg acc Val Asp gtd gac Ser Arg AGO COO Met Thr atg ace Gly Asn ggg aat S.x Ile tog aec Met Ile Thr atg att acg Asp Arg Vat GAO AGA GTO Leu Pro Val CTG COO OTC Pro Gly Leu CCC OA OTO Leu Lys Thr TTG MAG ACA Asp GAT Met Ile Thr atg att acg Pro 2 Met Thr COO ATG ACO Val Asn' Cys OTT MOC TGC Met Ile Thx, atg att acg Pro 2 Met Thr CCC ATa ACC V41 Asn Cys OTT MAC TGC met Ile Thx, atg att ecqg Set Ser Ser tog agc tog Asp Pro 2 Th, gaxc CCC ASG Asn Ser Arg eat tcc cgg Pro Pro Aia CCT COT 0CC Leu Lou Leu OTG OTO CTG Gln Aia'Pro CMA OT CCC Ser Trp Val AGO TOO OTT Asn Ser Arg sat tcc cgg Gin Thr Thr CAG ACA ACO Sex, Asa Met TOT MOC ATG, A n Sex, Arg eat tcc qgg Giri Thr Thr CAG ACA ACO Ser Asn Met TCT MAC ATG Asn Leu le eat tta ata Val Pro Oty gta cqc gg9 Thr Giu Thx, ACC CAO ACA Giy gga Asp GAT Lou OTC Met ATG Asn AAC Gly Ega Pro CCC le ATO Oly 9ga Pro CCC Arg Asp gat AO Gly Pro gGA OCA Asn Met MOC ATG Leu Lou CTO OTO Gin Thr CAG ACA, Ser Asn TOT MAC Ser Arg tct aga Lys Thr MGO ACA Ser Arg tot age Lys Th, MAG ACA Thr Ie act ata Leu Giu eta gag Lou Lys CTO MAO Tx, Sex, Trp Val Asn ACA AGO, TOO GO." AAC Cys Sex, Asn Mot ile Asp TOO TCT AC ATO NrC OAT SUBSTITUTE SHEET F'IGURE 8 WO 38/04691 WO 3894691PCT/NL87/00037 8/26 pGB/IL-305 Met Thr Met Ile Thr Asn Leu le atg acc atg att acg aat tta ata Arg Leu Thir Ile cga ctc act ata pGB/IL-306 Gly Asn Ser Ser Ser Val Pro Gly Asp Pro Leu Giu ggg aat tcg agc tcg gta ccc ggg gat cct cta gag Asn 15 CyS Ser Asn Met Ile Asp AAC TGC TOT MAC ATG ATO GAT Met Ala'Pro Met Thr Gin Thr Thr Pro Leu Lys Thr atg OCT CCC ATO ACC CAO ACA 400 CCC TT( MOG ACA Ser Trp Val. Asn Cys Ser Asn Met le Asp AO TOO G'IT MAC TGC TOT MAC ATO ATC CAT FXGURR 8 (cont'd) SUBSTITUTE SHEET WO, 88/04691 WO' 8804691PCT/NLS7/00037 9/26 pGB/IL- 301 1 2 A 3 BM pGB/IL-3O2 A 3B 2 1 "Nw i~n am-fu -mo 3 w 68 ~1 a 14 FIGURE '0 SUBSTITUTE SHEET dpm 3 16 '12 8, 4 0 pGB/IL-302 0\ '0 H 1'J 1 FIGURE -a 0.001 0.01 0.1 Percentage,., hmulti-CSF in culture WO 88/04691 "t I- 11/26 PCT/NL87100037 0 @1 9 1' Ii SUBSTITUTE SHEET B. 4 0 T- 0 0 -4 -i Ln 0 1 fI 0 800 400 200 I~~ 100 FIGURE 12 EcoRT (I) C (I) A A C A hi CD :r. rr' A !Q M0 EcoRi *Sequence of the N-terminus of the fusion protein: Met Ser Tyr Ala Val Cys Arg Met Glu Lys Vat Lys Ser Gly Val Pro Ser Ser Asn Ser Gly Pro Glu Gin Asp Arg Val Pro Pro Ala Asp Pro Asn Met Ser Arg Leu Ala Pro hIL-3 signal sequence hlL-3 mature sequence FIGURE 13 CI) Hiiidlll w p'romne 9 EcoPi Hindu xig I 'GAATTC'TTGACAAAGCTVfTCGAG' m ACTGAT.ATAAT'GAGCT-C Smal-BamHI Shine Dalgarno Xbal..SaII 0~ 00 00 0~ ~0 H -J FIGURE 14 Sall Clal Sail~ mla aTI[ Aval N r ul oligonucleotide Ca CO) -4 C -4 in 2i EcoRI Saill CF 4 3 S X~ Xm al p~Tre IL-3 a.a.-1 9-+20 X Sall X X m aTl T4 igase 3' noncoding stop sequence mm~1 Ecorl Sal I I Cial NruI Aval XmaIII IL-309 FIGURE F pTA 1060 ori 32le pGBIIL-310 ori 5.1 kb p IL-3 P vull ndMl HindMl Xhol-10 SstI G4 3 pIL-3:precursor gene human ISinai BainHi SD Xbal Saill IL-3. FIGURE 16 WO 88/04691 17 /2 6 PCT/NL87/00037 20 30 40 50 60 70 s0 90 100 110 120 130 140 IS0 160 170 100 190 2VA 210 220 230 240 250 260 270 200 290 310 320 330 340 IS0 360 370 360 390 400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550 560 570 5o0 590 600 Gl~=AA= =M W~=TTMM M=GT== GIIU 'eo 610 620 630 640 650 660 670 680 690 700 710 720 730 740 750 760 770 709 790 800 610 020 030 840 650 060 67Q 8O0 890 900 TAO T175ACAAT TApu.iiiA.i1 910 920 930 940 950 960 970 900 990 1000 0010 0020 1030 1040 0050 100 1070 1000 1090 1100 1110 1120 1130 1140 1150 1160 t170 1180 1190 1200 1210 1220 1230 124 1250 1M6 1270 1200 1290 1300 1310 1320 1330 1340 1350 CCG~ TIT ACACTTGGOk AAA M TC-COCGA r=hA TACGOG A~AX COTCTAtT= 136 1370 i1"5 1390 1400 1410 1420 1430 1440 1450 1460 0470 1/00 1490 1500 1510 1520 1530 154Q 1550 1560 1570 1500 0509 1600 1610 1620 1630 1640 1650 OAAAMCMTCQAOAM UUnMW M ATMATM TA~LA 064 1670 1600 1690 1700 1710 1720 17341 1740 1750 0760 1770 1790 1790 1800 0810 1620 0030 1840 1850 100 1870 0800 1890 1900 1910 1920 1930 1940 1950 ATC~ O=TTA=X U 1960 1970 1900 1900 2000 201Q 2020 2036 1.040 2050 2060 2070 2060 2099 2100 2110 2120 2130 2140 2150 2060 2170 2000 2190 2200 2210 222 2230 2240 2250 2160 2270 2200 2290 230 2310 2320 233 2340 23$0 2360 2370 2%01 2390 2400 24i0 2420 2430 2440 240 240 2479 2400 2490 2509 2510 2520 2530 254 2550 2560 2570 z500 2590 260 2610 2620 2639 2640 2650 2660 2670 2w0 2690 2700 2710 2720 2730 2740 2750 2760 2770 2709 2790 2000 2010 2020 2830 2040 2050 2060 2070 2000 289 20D0 2910 2920 2930 2940 2950 2960 2970 2900 290 3000 3600 3020 2030 5040 3050 3060 2070 300 34190 3100 3100 3120 3100 3140 3150 FIGURE 0, SUBSTITUTE SHEET WO 88/04691 18/26 PCT/NL87/00037 3160 3170 3180 3190 3200 3210 320 3220 3240 320 3260 3270 3280 32m 3300 CAM MAACGA~T M T= ArCAAAWA AWMM AAG CXa=AACM A= UT=A 3310 3320 3330 3340 3350 3360 3370 3300 3390 3400 3410 3429 3430 344 3450 346 3470 3400 3490 N500 3510 40o 3530 3540 3550 3560 3570 3580 3590 3600 3610 3620 3630 3640 3650 360< 3679 3600 369a 3700 3710 3720 3730 3740 3750 TGA MW ACAO~CT OU M D AUC=CAA MM~ifATTOIX A ATGWAG= 3760 3770 3700 3790 3800 3810 3820 303 3840 3850 306 3870 38W0 3890 300 OGGATCOK CAAWT=O AAGA~IACA AAUAUACWTAA~e4 MGA AAOA3AT W CTACAA A 3910 3920 3930 3940 395 3960 3970 3900 3990 4000 4010 4020 4030 4040 AMWhU AACGOAOMTAAM OW=A G= tTUW AC T= ACTAATTZ TAMTAM AM 4060 4070 4080 4090 4109 4110 4120 4130 4140 4150 4160 4170 4109 4190 4200 4210 4220 4230 4240 4250 4260 4270 4200 4299 4300 4310 4320 4330 4340 4350 AMATA 0?0G ACAAAMM 0AAGTA AUM MT0 GA AMTG0MWATAATG AT0M7CTCCATAC;I tCAA77MA0GT0MA4GTAACt1G, 4360 4370 4380 4390 440Q 4410 4420 4430 4440 4450 4460 4470 4400 4490 4500 :40 4520 4530 4540 4550 4560 4570 4500 4590 4600 4610 4620 43 40 4660 4670 4600 4653 4700 4710 4720 4730 4740 4750 4760 4770 4700 4799 4800, 4080 4020 6830 404 4050 486 4870 4w00 4090 4900 400 497'.' 4930 4940 4950 80MAT0MTtA0TAT0ATOCAATGTAACMWTGAr AAMTATATAA7C=A1A1ATT AAT A ,TCAkM TAt M ,..AATM7AT =%TIAM AMA 4900 4970 4990 4999 590 5010 502 50P9 504 5050 w06 5070 5080 W39 5100 5110 5139 5130 5140 5150 5160, 5170 SIMo $190 5200 5210 5220 5239 5210 525 5249 S270 5200 5290 5300 5300 $320 W3 534 535 534 5370 030 S3%k S400 TMATMTM =WC Q TATT ATTTMAA MATCTAAMTTA? A~23AAT ATAQTI3AT AAAAT MG MMTCJ.MTM3GAA M 5410 542Q 5430 5440 5450 5460 5470 540 490 559 550 552 5530 I*A 550 5570 554Q 559Q0 5600 510 5620 5630 544 5650 5660 $670, 50 69 $700 5720 5770 5740 5750 5769 577 5700 579 5000 s010 WO3 r. $840 $00 5w8 507 5000 00 5909 5919 5920 5939 5940 5954 5060 5970 00 5990 6000 6010 6020 6030 6040 6050 6060 6070 6000 6090 6100 6110 WO62 6130 6140 6150 6160 6070 000 6190 6200 6210 6220 6230 6240 625 6269 6270 6209 6290 00 FIGURE 17 (cont'd) SUBSTITUTE SHEET wn, JaRmA401 PC-rl YA-7/00AI7 19 26 631 0 6320 6330 6340 6350 6360 6370 6300 6390 6400 6410 6420 64 30 6440 6450 6460 6470 640 6490 650 6510 6520 653 6540 6550 6560 6570 6500 6590 I000 4NTT= M AA GAT~t= K= W M AM n A WTAGATTT4CTTka 6610 6620 6630 6640 6650 6660 6670 6600 6690 6700 6710 6720 6730 6740 6750 A CTV AATACATCATIAACAG ITOA A W r A 1104! =UACC AtIIAOCTI132A WAMG AAT ?ATTATTAAGTQTA0W4ATCOKX* 6760 6770 6780 6790 6000 6010 6820 683 684 6850 604 6870 68w 690 6900 6910 6920 6930 6949 6950 6*Ci 6970 6900 6990"~ 7010 7020 7030 7640 I T 0~0C 0I0A' I IL I I I I j A I C IA 7060 7070 7080 7090 7100 7110 7120 7130 7140 7150 7160 7170 7100 7190 7200 MAW W 7210 7220 7230 7240 7250 7260 7270 7200 729 7300 7310 7320 7330 FIGURE~ 17 (cont'd) SUBSTITUTE SHEET 4,- I I I I pGB/ IL-310 'Sal_ I -7 7 7 7771 EcoRi XhoI i I HindMl I Pvull 3 1I 1 Hful-1 plL--3 PvuUl partial HindHLi x P v ulfH "HpaUL promoter 0 /~ne o m~2HindlI ibaI Kp~ m.d two Pvull-Hindff[ Tragments X 'r-indI[ X Sinai nI SphT EcoRV coRI FIGURE 163 pIL-3 pGB/ IL-312 partial 043 promoter pGB/I IL-31 1 hft m.c.1 S-ail aI SD Bam-l Sinai X BmHJl 1 10 Xhol H-indIlI-35 EcoRI T4 ligase Neo Hpf1 promoter' 'HindHlh pGB/IL-313 Sequence; S.D.- Hpall promoter-AAAGGAGCGATTTACAT ATG AGT met ser TAT GCA GTT TOT AGA ATG CAA AAA GTG tyr ala vat cys arg met gin lys val -S.D. AAA TCA GGG GGA TCC AAG GAG GTG ATO lys ser gly gly ser lys glu val lie TAG AGT CGA CATG...pl.L-3 stop met FIGURE 19 pGB/IL-317 4.8 kb Nool. P vullj BglU1 PstI Sall HindMl -i PVU CO C C B. ori Neor 0 a-amylase promoter, signal sequence ©a-amylase terminator PstI I1 *:CT GCA GCG EcoRI XmaTU .XmaI Sall -1 +1 GCG GCA I' I I I GAA T-TC GCG GOC GCG CGG GTC GAC a-amylase signal sequence a-amylase maturation site FIGURE Hindlff XbaI Hindil Xhol Hpal SacI[ Xbaii F 7 pUG 19 G418' lactase promoter aft IL-3 te FIGURE 21 WO088/04691 2/6PCT/NL87/00037 4.11111 1 44111 449t, 4',06 4F,16 012(6 013G 4!.40G 4V.01 1:CCOU(WU11AT :(lA(,1I1A AA 11ATAOI''AArOC TT'TA ATLI1 M1 TATU.A1TTGA CTACC.AAUCA TTA11TCTrkiT TGCAGAAI'Gd; GAAT13:T-cIu L.CTcTT1(AA 011116 0 4 A 4 1-(1 46116 4616 4626 4636 -1,4 41156 rIIVAIITTOWL' 1-i'TTIIi'i' TAT'rr13;1AT TTITI(A(1TTT fTTrlTTGT r.AATTTAI]C CA077(ICTTC T(1CAAGATrA AAI'AAACIC 1(M (71At"ITT 4 1 C,0r .11171: 401"1t 4 S N 4706 4710 4720 47,1 7F 47!.6 T(;T(1.'TVGIAA (IArr1IlrAATC ;(ITTTTmIlA Ill; 1(hl.A(AA AAr1'AAATGT TTrCTTr11,TTA (!ATTrCiGT(; CrTAGCTA(uI' TCI IAtT(.. Ti.AAAtI',GACT 4 7 116 4'i (1 4 7 Is6 4 719 1 486 0(16 4826 4 83r. 48(40 4 0 56 TA1(GACTI ACTAVA1I(!At; A(TTrTA f11111TT1;1 TI1(1'G1TTT GAAAGAAAAC, A1ICGIIOAACT CGCGGGTT(XI CGC1(:11AATAA rCA5111,A'rA 4866 4:1711 43813 409G 4906 4016 4926 4136 49146 49f.6 1(IT'rrriOc. (1TT(:CAAtITI (1(ATCITA(UAC TAGA(:AACAr, ACAGU;GAUIIG IIAIXAAGGAT CTTTCACTGA GATCCTGTAT CTrr'r;;oT AAIIT(NiiAT0i 49G6 11117 1; 4118 4tM1 b.011 60116 6026 5030 W 5 C. AAAGI.I(GAAI' WTGA TOIAWiAT G nr 0 A ArOAiM TAACOGCCTTT TGTTAA(1TTII TTTAATTATT ATGGGCAGCIG MCAGiA(1011 1 AGIIAAT11'TAT W.166 f-07f1 t01161 11911 516 11116 .12p6 f, 36 141. 11"1!. r GTuTGTGAOII COGlII(01,17IAGI1CAT(C A(IGCCAIIO TA GAAATAGA()A AAGCCGAA1'G TTAGACAATA TGGCAGICGTA CITAGNITAIMO TAtInrlA(W(A 61663 f141(i 111i 10196 52,06 $26 52 230 1, MITA()T(CTA rLAAAi;.A (1AA(IMMTAG CT(1AITCTTr, (1CATTCCACA CCI3TTAGTC.T GTCAGTTTAI AIIAAAAAAAC AACTAITATA 'CAA1'TAITA 5266 52811 f.2110 5306 5310 63?0 s 3;A s '.3415 !,3111 (1AC1rriTAAC. 71AO11r'l'IV.l AA(!(;U(TTTT 11(ACTOIUOA TTATTCGTCA (81AATCAAI TA(IGAATTTI1 GTCATATTTA CLII1fAIA1AIAG T111111TO .11T0 WIWI1 tl. I I n 11, .1911 r,4111G 5,4 10 5426 f.471 11- ,1(61.a U(1ATAT(W(AG, TAOU1AAAA' I Al;ATCATGOIT ATC( TrA1AT AT11TTU(!OGA ATTCTI.TTI1A cCa1CAAAIITT (CGGJTUIIT(1 17Tl811'TT (,G'rcrl F,,I11!0 F,.0 1-00( VMS0 55 11 5526 .5 1 r 1'r, TTCGTTTIICT TOC17ITT-il IITTIMIAT(1TT AATAAT*Atl((1 'AGrlrT(ITrA tIC1IAAACTT AIGr.T ArtCT TAnrIITTrIIA A(CGr5ArAAT (ITA'rd:K;T1I' hF'.1I S.11 50111 1.11' 'A10 '.06N ,111 t .4 r, .1 1 r IlArIl'Tv1(iTTT AACCA64C#A 1311I1t1it (IW Al11l! A1M1 AV('AIIM'IITTT TTOTATTt;T TO!AI;rATAIIT 'rlvrI(AAAA1T 1JTAI;1l1I1IAAA TAT(;T(IIIIThC r. r.11 (1111 1. .f;i b7 G 16 r572 0 1773 .1 0I I.71, ;5,;1AAA1 GTCrrTkrCr AITr11bOO; TIMVIIT'lAI'T (IQTTiIAATT 111V1'ATTTrCIA (.TT'F(ITTG(:T ArArIC1ATTA MIA!T'NAAI Tr(;A1t;TQ 57111 .51 1! r740 f.1111 t C 5829 f1836 A0;(TAT(iAT TT(1AGTG~r OWAIIIItT A(1TIA11AA T(VV'TTToATT (,rTGTTTTA(i TT(!AIIA'TTC' 1ATTCIA0tiA ll 1 'Al ITA ATA((lVT(11iAA 1A.80 1111 Animl 013110 19011 1.916 !11:26 '.4,16 ,,.6!10 TCAATGTIITT AVI11*1T1T4 AIIA'YI'TrTT VrOOTAAf1T1!l1 AAAOI3TATAT (AO(ICTTGT(( TTT((TTAGCIA GAATTATTAT TCTTrTv;TTA T(ITTIIc!(TT b96 1n 1.11 96 8006 6016 6026 Mar11 0oI. VM11 (iTAGTTIIGAA AMI7(II'AGA IAI(AAA1IT TAA(!AGTT11A AATTTAIUGAA A(1MACIAAT TTGIIAAAAM AAATAAAAAA AAAAThAAACA (-,TQ1ACI!TI 0066 61171i o1011 (1006 006 (;I1I6 6126 0130 61156 ((I56 TCAaCGGA'TA ACAAT((!A('A f!ATA(VI.AT(I r.A(,AA(T3AA MIATA3'GA(A TTTCCATI6IA TTTTTACTG;C AqTTTTATTO CAIV(ATIII.1' (1331ATTAMI? 6106 .1ill CM1( 111110 62.011 0216 0226 6323c0 024R 'rGCTCCAGTO AAATACAII UAAANA AA((ltAIIAA ATTrrC .rTG ((AGCTGTCAT COOTTACTT& IIATTTA1IAAG fOGGATTTrUA 713771111707 0266 l1!!(1 C, ,086 61,96 0300 6316 0326 U3311; 61346 G.156 TTOCCATTTT COAAAIl(AP AAATAMt.41IC, TTATTIITTTA TAAATACTAC TATTGCCAIC AT'fGCTGCTA AA(*,AAGAAIIII r.GTA'TCTA C.ATAAAAIIAQ 0300 1:111: 0300 (1390 6406 010 0420 04.30 01110 04116 CTCCCATOAC COAI1AWo- CICTTGAA(1A CAA(ICTOMI.T TAAC.TUCTGT AAUATGA1CG ATaAAATTAT AACACA(1IA AACOAIII'IAC CTT-rGC1TTT C,466 (1470- 641:111111 6~r, S1.06 6516 652f; 111136 fl11.1 1111.9 Gr.T01Q1W'rr AACAACTC* P.'jt. I(l1AA~l(( ('IAA11A('A'T lT1IAT1IIAA(, AW. Aee'TTW11 A61111rh(AAr PI11A111r'M' TI'Aio., vajo1 TO'TVAA'1;0T 6506 W 6 0580 6516 696 6610 8620 003 1111 16 811. TTACAQOAACI3 CATCAMMIAT T~oAGAGCATT CTTAAAAATC TCCT(IOCATG TCTGCCCQTG GCOACG(JCCG CACCOC7IQO ACAT(NOATC CATrATQAIIGII $661 1166 S610 67116 0716 6720 0 7,10C 047.6 1 ACGGTGACTU GAA'IIIAATTti CGIJAGGAAAe TCA(UTTCQA T(1TIAAAACC CTTQAG;AAT0i COCAGGC7rCA ACAGACOACT TTIOO'TC CIJATCTTTT(- AGTCCAAIGT CIIAG1TCGTT CTCTGGOT TCTcAC.CACA OAGCCTCCGG ACATCAA/IM OAGCAGAACT TCTGAAACOT OTCUMM1TATC! TCTCACAVXT 6866 68711 6066 0696 6906 6916 6920 1:136 l194(1 111150 TOCAGGACCA GAAV,(1N1TTC AI)CT'TTCCT GCrQGOAT((AG ATIIAATTOTT AATTATCTAA TTTUTGAMAT (4TOCAOUTCC CATTTM1ICICT I'ITGC(3'TTU 696 (111 0986 Cq9q6 7009 7016 7026 7030 71)40 TGTTCTCATT 7775,71(((12' TGAGACTATT TATTTATGTA TGTA7GTATT TATTTATTTA TTCTGGAG TGTaAACTOT ATTTATTTTA GCAG;A(ICAG]C 7066 7107G 7000 70196 7106 7110 7126 71,16 1140 7 ll(1 CATUTCCTGC T007TlirUC1A AAAAACTCAG ACTGtIGGT7GG (JAGCATGTT CATTTGTACC TUGAaAATTT ATACTTAGAT AAQTATI'1AC TTACAGUITAT 7166 71711; 11,6 7196 5,777075,705 GATACTO15,T( TATACATOCA 3GATAATATT TAAA(ICTT FIGURE 22 SUBSTITUTE SHEET WO 88/04691 25/26 4466 4476 4486 4496 4506 CCGGGGGAT CGACTCATAA AATAGTAACC T'rCTAATGCG TATC7AT77OA 4566 4576 4586 4596 46,06 TTC,4GCTTGC TT'rTCATTTT TATTTTCCAT TTTTCAGTTT 77677TT676 4666 4076 4686 4696 4706 'FGTGTCGCAA GATCCTAATC GACTTTMU~ CCC100CACAA AAGTAAATGT 4766 4776 4786 4796 4806 TAGGGACTGC ACTACATCAG AGTGTGTTC A CCTOGTTTGC TGCQTGGOTT 4866 4876 4866 4896 4906 GTCCTTTaOC CTTCCAAGTC OCATOTAGAG TA13ACAACAG ACA(IAGAG6G 4966 4976 4986 4996 5006 AAAG(;GGAAT CGTATUAGAT TGGAGAGGAT CCGGAAGAOG TAACGCCTTT 5066 5076 5086 5096 5106 GTGTGTGAGO CGGGCGAGAC GGAGCCATCC AGCCCAGGTA GAAATAGAGA 5166 5176 5186 5196 5206 AGTACTGCTA GCAAAGAGGA GAAGGGTAAC C-TCACTCTTC GCATTCCAOA 5266 5270 5286 5296 5306 CACTOGAAC TGACTTTTOO AACGGCTTTT CGGACTGCGA TTATTCGTGA 5366 5376 5386 5306 640G CCATATGGAG TAGGAAAACG AGATCATrGT ATCCTCAGAT ATOTTGCGOA 45 16 rTA:r:AArc.!A 4F.6 CGAAT'Ak;C 47 1 r TTCTTTC6TITA 4816 OAAAGAAAAG 4016 CAGGAAQGAT TGTTrAA('TTn 5110 AAGCCGOAATG 5216 CCTI'AGTOT 5316 6GAATCAAMI 'A416 ATTC0TCTTCA PCT/NLS7/00037 4526 4 f,36 454G 4 '.r6 7rTAG7TTGG TaCAGAA001O ilAATTc'-r(x fTT01"rc:VAA 4626 4630 4640 4G650 VAUT6CTTC TCCAAGATGA AAAAACCCCT G(CC'ArTTU 4726 4730 4746 4756 CATTC66CCI' OGTOCTAOC TrCCC6OAATC TCAAAGGACT 4026 4830 4646 4856 AGCGGGAACT CGOGTTCC CGGCC.9TAA TOATOGrATA 492G 4936 4946 4956 CTT7CACTGA GATUCTGTAT r6TTqTTOCCGT AAUTCGGATr 5026 5036 5046 5050 TTTAATTATT ATGGGGCAGO6 CGAGAGO0G6 AGGAATGTAT 5126 5136 5146 W156 TTAGACAATA TOCCAGCOTA GTAG;AGTAGUI TAGC6TAGGCA 5226 5236 524G GTCAGTTTAa ACAAAAAAAC AACTAOTATA CCAATTA63TA 5326 5336 5346 5356 TAGGAATTTG GTCATATTTA CJ;GACAACAOi TGC1GTGATTC 5426 54136 544C 5450 CCOCAAAGTT CAGGGTGCTC TGGTGGGTTT CGGTTQGCTT 5526 5536 5546 6550 AGQGTAGGOT TAC7O7TTOUA ACGTAViATAT QTATC:ACQVT 5626 5636 5046 5r,6 56r TQAGTATAQT TGTGAAAAfiT QTAG WQGAAA TATCTGQTCC~ 6720 V730 G746 51It56r CTTTGTTQCI' ACA6CCATTCA CTAVTTGAAC TC1IAUTriTf-A 5826 5836 5846 ".356 TTGAGATTTC GATTUAGAAA AA0GTATTTA ATAO6.TC6AA soad 5936 44v 5.956 TTTP'rTAGOA GAATTATTAT TCTTTTC1TTA TGTTQC!(,CTT 6026 6036 0046 006 TTOCAAAAA AAATAAAAAM AAAV)'AAACA CGiT(GACT111 0126 6136 6146 6150a ACTGCTTTAA MTCCOTTT 'rATI100TrGT C:CArTTTCTA v 116 0236 0246 6 0G TTGAOTTAAC COdOGATUOA OTT716TTOT TOTOJ(TTAA 5466 5476 5406 5496 5506 5 5l16 TTGCTTTGCT TCTCCCTTOT CTTGCATGTT AATAATAOCC TAqCCTGTGCA GcCCiAMNrT 5566 5876 5566 5596 506 56 GACTTGOTTT MACCAGGCGA QCTQGTACCC AGCCATACCC ACA(1A6ITTTr TTTOTATT1T 5666 5676 5686 5696 706 C-7 16 GAGCAACAGC GTCTTTTTCT AGTAGT.-CGG TCGGTTACTT O6TTOACATT OGiTATTT6'IA 5766 5776 5786 5796 500r, fG. AGGGTATGAT TTCTAGTGGT GAACAqCTTT AOTTACGTAA TGTTTTC.ATT' QrTTT-rTAV. Saco 5876 5886 5896 S900 50)16 TCAATGTGTT ATCATTOTGA AGATGTTCTT CCCTAACTCO MAAGGTATAT ()A666TTOTO 5966 5976 6986 5996 6006 r.010 GTAGTTGGAA AAGGTGAAGA GAcAMAAGCT TAACACTTGA AATTTAIIGAA AI(RA(ItAA' 6066 0076 0086 6096 610)1 TGAGCGGATA ACACTCGAGO GATCTTCATT ATOAAATTCT CTACTATATT 6166 6176 6186 6196 6266 CCGAAACTGA CATCGACCAT 0TTCCAATTT CGUTTCCAGA AGAAOC.TTQ 1;1 10 AGCCCAI*UT 6210 ATlTGOTTOA 626r' 6270 626 TAACGGAAC.. CACACTGOTA TTCTATTCTT 6386 6376 636 ACAACGCCCT TGAAGACAAG CTGGGTTAAC 6466 6476 6466 ACCTCAATOG GGAAGACCAA WAATTOTOA 6568 6576 6586 AGCAATTGAG AOATTCTTA AAAATCTCCT 6666 6676 666 GIAATTCCGOA GGAAACTGAO GTTOTATCrO 6766 6776 6786 CTCGTTOTCT GaGCCTTCTC ACCACAGAac 6866 6076 686 CATTrCACCT TTTdCTGCOO CATCAGATGA 6966 6976 G986 TCCCATTGA.. ACTATTTATT TATOTATGTA 7066 706 7086 TCTGCAAAAA AVTCAGAG'rU GGGTGGGGAG 7166 7176 7186 CTGATGTATA CATUCATGAT AATATTTAAA 6296 AMOCACCACC 6396 TOCTCTAACA 6496 TnOAAAATAA 6596 CCATGTCTG 6696 AAAACCCTTG 6796 CTCOGACAT 6896 ATTGTTA.ATT 6996 TGTATTTATT 7096 CATGTTCATT GC'rr 04300 ATOlTO W, 6406 T(1ATCGATOA 6060 rTMcIAAMl; 8006( 6CCOTUCCA 0706 AqAAT6606QA 6006 CAAAAAGAM'C 0906 AT CTAATT 7 C (006 TATTTATTGC 7106 TGTAUCTCOA (;J1G 6326 6330 6340 G IIr C r.TC*C:T'rTCIICr TGACAAGAT GATTTCIAAOA AGO(WOOUTCC ATGACUAr, G418 6426 04361 0446 64561 AATTATAA1A QACTTAAAOOC AGCCACOTT OOCCTTT(IrTG GACTTCAACA ofI r 652.8 6536 0540 rr 1 rNWAMrrl(M A6I16ATTt:AA VA(1) lflTii71f AA6AeiTTTAV AIIAAO'* 1'. 001r, 66H6 6038 6 66,6 55 .11 C(;rlCMCO~ CAQGCGACAT CCAATCCAI'A TCAAUGACOU TOACTGGAAT 0716 f. 6726 6736 61740 97566 dc1(1TCAACMl ACCACTTTC1A COUTCUCOAT C'rTTTrT(ArTC CACOTCUAII eta16 6826 60l360 0.14C 6656 r AGAA6TITO AAACllTCTGG LUTd:ATCTOTO ACACATTUC(A G6IACrAGAM-1

0151. 69216 0036 6940 0956U TUAAANIT(16 AGOTCCCATTr TU0(CCTTOTO C.(;,TTGTUTT OTCATTTTTA 70110 7026 70311 7046 70f0 CTOOMUTITU AACTaTATTT ATTTTACCAQ, A(WAUCCATO TCCT6CTuQT It II V 7126 7136 7146 71501 'IAATT1TATrAO TTAGATAAGT ATOTACTlTAC AWITATATTT CTATOAGATA FIGURE 23 SUBSTITUTE SHEET TCGAATTTGC 110 CACACTGiGA, 4210 AGTCAGAGGC 310 GAGGC"TCAAA 410 CTTTGThCGT 510 GAGGACCcICG 610 TTCTCGGACT 710 CTAAAGGTTT 810 TTTTTTTGAT 910 TACAACTTTT If0n GGGGAGAAGA 120 CCTCATAGAG 2 20 AGGAACAGCC 320 ACGAAATTAT 420 TCAAAATACA 520 AATCCTTACA 620 CCGCGCATCG 720 GGAAAAGAAA 820 TTTTTTCTC'1 920 TTTACTTCTT 30 TGGATrCTATG 110 (1A(XGGTGTC 230 (3TCAAGGG( 330 TGACAGCCTA 430 ATGCAGTAGA 530 TroACACCCAA 630 CCGTACCACT 7-30 AAAGA(IACCG 830 TTCGATGAGG 930 GCTCATTAGA 40 CT-AAATC-TAA 140 U(iCCATAA 240 CATrAAGACTA 340 G~ACATCAATA '140 TfANTTATG 540 TCCCCCACAA 640 TCAAAACACC 740 CCTCGTTTrCT 840 TCCCATTGA1 940 AAGAAAGCAT 5 0 60 ATACMA(3CAT1'T (2AAAAACCGAC 1!)0 160 ATTTTTCAAC Q(CYATAATTrA I!250 26;0 C(#(;TCATCCC CATC'r(CGcrC 350 360 (3TCATACAAC A(;AAA3CXAC 4 0 460 CATATTACAT ATAATACAT A 5,50 560 (TGATCCCCC ACACACCATA 650 660 CAA(3CACAGC ATACTAAATT 7!5O 760 TTTTCTTC(1T CGAAAAAGGC 850 860 A'TTTAAG4TTA ATAAACGGTC 9 5 0 960 AGCAATCTAG TOGACAGATC *70 GACUAGTTAC 170 A(.3ACGACAA(1 270 TI'CGTCCACX4(. 370 CACCCAACTT 470 TCACATAGGA 570 GCTTCAAAAT 670 TCCCCTCTTT 770 AATrAAAAATT 8170 TTCAATTTCT 970 TCTCGAGTG3C 10,10 TAAGGAAAAG go ACG;A(CArA'VC A A(,1G'TTCA(( III a 0~ CT'T(3'WCAA('A 380 ,ri('UrtGATAA 480 A(ACAACAGiUC 580 GTTTCTAC IC 600 CTTCCTCTAG ?80 *TTTATCACGT 880 CAAGTTTC AG 980 TTTI'TQGC' (JCCATCTTTA 19 0 AAATc'rTTAA GG(IAGITCT'r 'VAGCVGTAT(AA 490 GCCITTGGACT 590 CTTTTTTACT 690 GGTGTCGTTrA 790 TTCTTTTTCT 890 TTTCATT'TT 990 GTATGTT TAT 10 0 AATrGAcCAA(! AC.AATG T'TfiATT'TTC 601) CTTCCA(GA'I' 700 ATTACCC(3TP TGAAAATTT1 C'rTGTTCT'ATr 1000 GTAITGTrAGc,, 1010 1020 1030 J0410 1W00 AAACCACCCT GTCAATAAAA TAAAAATAAT 1060 AAAGATr'TT 1080 1090 1.100 ACGTGTTTAA GCACTGACTT TATCTACTTT CTCTCTCTAT 1110 TTGTACGTCT TTCTATTTTT AGA FIGURE 24 WO 88/04691 PCTINL87/00037 PCT Aalicantls (7,nide '/olurre I Annex M3 Internatilonal Aopiicatlon No,. PCTI I MtCROCIRGANIS MS ontonai sheet in connection wit,th Min.mcoorgonlamn rotor'/od to an 26 4 a the dSSCflotlIf t A. 11KHTIFICATION OF 04POI1T'1 Furtlist deomile are identiAmd an anI ddIlloflal sn..4-X Narma at 4.0061tary nletilt Centraal Bureau voor Schimmelcultux'es (CBS) Addra., of 0.oalary irIMtU1I011 (InCtUding 0011111 Code and CoUnlryl Oosterstraat 1, 3742 SK BAARN, the Netherlands Oa16.3 0110001s t Accession murnoor July 13, 1987 CBS 378.87 Il. ADDITIONAL I,4'.2ICATIONS t (leave blame if mot 4olcatils( This inlotematiofl is comnto an 4 sagefels enlacned onet C. DESIGNATZO STATt3 FOR WHICH INOICATIONS ARtK MADE S (Ij in* InlCaltoflC not 101 411 4*6 Inai*n States) 0, 391PARATIC FURlll3141MG OF 1?4 11CATIOHS 6 (leave OlInx it not 400110401et The cinaili. 11;1@4 *od ill tUC suormnl 14 Ioris Inletralionel llutaU le( (specifyIn vistlnatur flat t Ac seot n til M It at oitoosil no lnoicaitoa 0,0.j 'I IThis aIl wee .4 ecilwoolf -In:1 intallontil soolicailln '-nan Mlogo ea dro;%#a con the ftooint;Officol T-i ho data of teceat [taorn few. mnoill or tIC Iitelirn Gureict I Poenm PcTRopi3t wtat y isoij WOI/i6 AD oI c ant's GU I e IlIunie I PCT/NL.87/00037 MAl EX-M3- lnternatIa,,al Aopilcatiao No: PCT/ MICROORGANIS MS Octonl See I com-llc -11 I o,9018mlm rstsf?, 10 On 06ls1 :Ins 2 at the aecrioant A. u011I SeT IFICA I COflfl 11lI SITi Funrior doctits are goenlled an itm eddilanal sneelt 17 Nae at docoltary Inanitunor,- Centraal Bureau voor Schimmelcultures (CBS) Addleca of decosgiay Initution (lIcln costal coop anid count~ry) Oosterstz-aat 1, 3742 SK BAARN, the N'etherlIands Daa tasnetAcceion CBSo 87.8 December 4, 1987 1CS828 11. A001T1ONAL IMIDICATIONS I Ieave Olana ,i mat socillmiO. Tis inlnrmallon is canunu*q an a ascsil onsicmod oset C, OI1II0NATtO STAr15 FOR~ WHICH- imwOcATIONS AIOI MADIE 1 (1114 idli. ailofl is not lot 1 all 0onam~te Stats) 0. 31PAIOArZ FURNISHING40 OF IN D1CATIONS Ii (lean ~t m0 11 not agaCIlcotal Thle n~imiansistned t14..0 d *1 O.UOMltted to Ins IntatnaloflI 8ufsaw lt 6 (SoocitY Ins, geerl nalli. 61 the. lIIci~iOn$ 0. 1Accodsiofl HIImnet of OeoOSII1t '*This Ose .44 tegaieo Ina lits tSIioflul Aiti~ork emom rildso is vmsle~d cy Ins iscsivlll Ofts)u The sk at50 reizoll lOwn kits A00Iliano ot In. Inloomnsionl. Buteou Io Fair"~ PC7,AROIUI i.Jnstey mil4( WO 88/04691 PCT/NL87/0037 FICT Applicant's Guide lY a r k rinex 113 International Application No: PCT/ MICROORGANISMS actuarial Shoat iIn coninection tn trio mscroor'anism rotarred to arn line 1 ,at he doecgtiof A. IOINTIFICATION OF OKIPOSIT I Further uisoosits are Idmttinad On' an additional oneealX Name of diaolat InetitiltiOn Centraal Bureau voor Schimmelcultures (CBS) Adore.. a 4, 'Zotten Itltitjtof (IUitf n~hlg Postal code aM4 Cotrltt4 Oosterstraat I, 3742 SK BAARN, the Netherlands Oas at deost 6 Accessionl Numor a July 13, 1987 CBS 379.87 4.U ACOMlONAL INDICATIONS T (leave 0anit 11 not aeptteatef. Thie mitrmation as contnued ant m, eeoae onocnod inset 1 C. 010tIG4ATIOD STATtS FOR~ WHICH INDICATION4S ARK MADAI fit tMe nuicalians 410 nat tar 411 44tignaled $4146)s 0, 3111ARATt FURNISHING 00 INOICATIONS 4 ~iaae ans it n ot taAtet The inuicalttane tistd 0tis oi delt a uarntieu to kio tfltelestlonal Outetil Wt 1 (30041"1 the jeneat natuts r i indicttations 6.4,, Attasaln IMunnel Of 0epoelt tC]This inse wit togataed W1tiIn l nike 16#14110014 d00jiatan wteit 11144 (to 06 0ie4114 byi 111e 104au1voto MIds tee tAutltted Offitel Ptenm PCTtI-003A wtitt, tsill) 'JC7 A001lCant'S Guide volume, WO 88/04691 PCT/NL87/00037 International Aopilcafllon No; PCT/ MICROORGANISMS Octional Sii.ot in connicllom wsi, ins mikiroargenini roets o n *1tedncotr A, i0fIXT1FICATIC10 OP OKPOSl?'I Pwnif CoCaitt ioSn1111.a011 m AM4014 Ad.In.q nIt 7 Monte at ocoollary Iriltuiloi Centraal Bureau voor Schimmelcultures (CBS) Addr614 01 0.0onsikry Institution (Inctuainq gamt Coo, Aa 4:oUntfl Oosterstraat 1, 3742 SK BAARN, the Netherlands 0*.040011* December 4, 1987 AC1 60 CBS 872.87 S. ADDITIONAL. INDICATICIPS 7 AA itI oploglcllh Thia iinoan is con#unuton4 on coio acfe onest C, DIESIGa4ATIE ZTATVI FOR~ WHICH INUICA11,421 '.at MACK III th fmlcldiqfs 4I 10 tot all dationstoo 31isl 0, 395IATIE FUR~NISHI$NG OFl IMOICATIOa4S 11641.6 01ano 11 not 400114thiow Tie 1l414:40taO 1s4ed 041C'. -111 its h10Mtsn111 Iri onlsnal auteou IspeGiI'l trio gonqsi nawuto 41 the fntitattano 4., Ace a n "nn aumor Q 1 0Osonsit t, PTi onest '490" q,.0 t~o 'fli1lrl1ttano sa00hI@O1t om A144lis (to is 0,10,4.1 01 irk* 1441a111Q OM401 The 44te of foetbi 1ol tlt e V.Aaa',4anl al in. InIstfolo~nei awd.lw to SI *44 IAIdIo,,104 OI 14..I Form OVIAW14. WeAtieff voilt, INTERNATIONAL SEARCH REPORT International Application No PCT/NL 87/00037 1. CLA~rSIFICATION OF SUBJECT MATTER (it several classiflcation symbols apply, indicate all) According cInternational Patent Classif'ication (IPC) or to both National Classification and IPC IPC 4 C 12 N 15/00; 1/20; 1/16; 5/00; C 12 P 21/02; 21/00;/! (C 12 P 211/00; C 12 R 1:91); (C 12 N 1/20; C 12 R 1:19;1 It. FIELDS SEARCHED Minirrum Documentation Searched I Classification System ICiassitication Symbols IPC 4C 12 N Documertation Searched other than Minimum Documentation to the Eylsrit that such Documents are Included In tihe Fields SearchedI Ill. DOCUMENTS CONSIDERED TO NE RELEVANTI Category I Citation of Document, I' with Indication, where appropriate, of the relevant passages 12 Relevant to Claim No, 13 X EP, A, 0138133 (SCHEPING BIOTECH CORP.) 1-9,12-19 24 April 1985 see claims; page 14, lines 8-12), page 21, line 8 page 23, line 12 X Cell, volume 47, 10 October 1986, Cell 12,13 Press, Yu-Qhung Yang et a "Human IL-3 (Multi-CSF): Identification by expression cloning- of novel hema- topoietic growth factor related to murir TL-31", pages 3-10 see the whole document cited in the application A chemical Abstracts, volume 103, no. 23, 1-3,12 9 December 1985, (Columbus, Ohio, US), T. Yokota et "Cloning and expression of lymphokine genes from mouse and human T-cell lines", see page 187, abstract 190859q, Short rep. 1985, 2(Adv. Gene Technol.), 49-52 Specal Categories of cited documental to 4T.. later document published afer the international tfling data IA4 document defining the general state ol the art which Is not or Priority date and not in conlict with the application but consdere tobe o paticuar elevnceCited to understand the principle or theory underlying the consdere tobe o paticuar elevnceinvention oirler document but, published on or alter the International, "X document of particular relevance:, the claimed invention filing data cannot be considered novel or cannot be consideredl to "Ll' document which may throw doubts on priority claim(s) of Involve an Inventive step Which Is Cited to esltblis11h the publication date Of &nothes( oueto irlua elvne h lie neto cittio orothr ecAveaon las specified) cannot be considered to Involve an Inventive stop when the 0" document referring to an oral disclosure, use, exhibition or document is Combined with one or more other such docu-. other means menta, such combination being otivious to a person skilled "P11 document Published prior to the International fing date but InI the art, later than the Priority date Claimad document member of the same patent family IV. VttRiFICATION D004 of the Actual Completion of the Iitrntlonal $earth. Dotet of Mailing Of this liyntilonat Search Report 9th March 1988 13 APR International SearchIng Authority Signature of Authorliad 0M~ EUROPEAN PATENT OFFIZ M. 'VAN HYL Form, PCT/15A1210 (*stcand sheet) (January 1045) 7 p INTERNATIONAL SEARCH REPORT International Aooiicaton No PCT/NL 8 7 0 0 037 1. CLASSIFICATION OF SUBJECT MATTER (it %severaI clkuaiiction symools aoply, indicate alil) According to international Patent Classification (iPC) ori0t Natior-i Classncation and IPC IPC 4 1:07); (C 12 N 1/16; C 12 R 1:85) 11. FIELDS SEARCHED Minimum Documentation SearchedI Classif'ication Syatem I Classiflication Symbol* Documentation Searched other than Minimum Docu.mentation to the Extent that such Documents are tnctuded In the Fields SearchedI Ill. DOCUMIENTS CONSIDEr4E0 TO 1111 RELEVANT$ Category q Citation ot Document, 11 with Indication, where ePorooriate, ot the relevant passagesit 12ewn toClam No. 13 P,X Gene, volume 55, no. 1, 1987, Elsevier 1251 Science P'ublishers B. V. (ANsterdarr. NL), L. Dorssers et "Characterization of a human rnUltilineage-colony- stimulating factor cDNA clone identified by a conser~ved noncoding seqxuence in mouse intetleukin-311, pages 115-124 see the whole-*documnent special categories of cited dtocumrents; to Way doyijmeit published atesr the intetnational riting 4ls document donining the general slate of the art which tono or Priority 04ale and not in conflict with the 3oitaio hiut Contidered IQ be of particular teloeance cited to un~dertandI too Prinmii at ihepry under yI ma the ivention earlier document but published on of after the international mxi document Of OArtidular telenence; the Climed invention filingccannot be COnsered novet or qannol be Oonsidered to dricumont vqhich may throw- Ibis on priority Claimfa() or Ivly ari Inventive step which is Citeud to atablisth, the publication date Of Another document of partir-.:1t relevant*; the claimed ivvehtort Citation or Othier special reason (es ipeciied) cannot be roiyidered to Involve aml Invenivey step win Iiiq I0'" document reterring to an oral disclosure, use, oshibition or dodumvl' to oomoinA so with one of mole other. och_ ctoeu. other means ments, 4uCh Combintationi beinlg Qbvios to a persoli skiled IIP" document oublisheqk Orior to the International Miing data but~ in tho sit, later then the PripliY date Claimed "ill dodumtie membar of th# 6am a ptent, fail~y IV. CERTIFICATION Date of Ihe Acruel Completion ot the litteflatitionat Search O0a* o Meling of this International Saafg O W e~l 9th Match 1988 Iiferational Searching Authority Sintr of Authoried Oftier -2- Form PC1Ii210 (6ecOnd 61104t) l44anuery 1056), a IA ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. NL 8700037 SA 19865 This annex lists the patent famnily members relating to the patent documents cited in the above-nientioned international search report. The menmbers are as contained in the lFuropean Patent Office EDM' ile on 25/03/88 The Europcan Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent ocutnent Publication Patent family Publication cited in s airch report datemebr!)d-t EP-A- 0138133 24-nl4-85 G2-A- 2147585 15-05-85 AU-A- 3375684 18-04-85 JP-A- 60176597 10-09-85 US-A- 4695542 22-09-87 For miore details about tis anw, §X see Official Journal of the European l'atlit office, No. 12/82 t 1 WO 88/04691 PCT/NL87/00037 PCT A~olicant's Guide Volume r Anne% r- International Application No:, PCT/II Centraal b~ureau voor Schimmelcultures (CBS) Address W1 deposition, Institution vinCIUw,,4004441a Code 4a country) Oogtu'straat 1, 3742 SK BAARN, the Netherlands Doe0 1041"July 13, 1987 Aceso uor4 CBS 377.87 a. A1O01IOPAL INDICATIONS 1 ticca. ollank it not oiai~ This information it conlifluc o a iscafes eacried onaii C, 0113I014ATIED STATIS Iron 'WHICH I?4112CATIONS ARIE MAOF I I i In Iasorts a not [or all dosla rao Stalfe*4 0, 3IPARATt FILIFNISHIM0 Of INDICATION4S I leave 416n% it not acolicsolal Tire tol callont !Istod 0iir.0 1 nol 0sUimited to in* interational duo&u litief (Socrl liie 9011119,aure 01 lie 16131C4114111 I' A94464114 NU'ink t 000111 9. This saiet was t'eqai 'aln Ina mttne~lnaI aoolicduoar n (rer rIt 40 4tected tiv in* lorsivii; 0 'iil lAulflottlec Ol~etl thlie dotet of l..era iftorm Ina eooih4aMil of in. Iintatonal tduferu to *1 ha lr- .rI Omri WO 88/04691 PC-TJNL37/00037 PCT Ao lint;2 Guide -Voiume r Annex ti3 International Appilcation No: PCTI 00ijonal teaot in connection -tin (Aieroi. ran raefdt nPg- 2 ie3 -6 h ecito A. 1ITIFICATION OF DEPOSIT I Further docoeita aItsilntild anan addittonal onset L Hams at 000641osIis~iltiof Centraal Bureau vooc Schimmelcultures (CBS) Address of doosi~qte Iistillution (Including Post coos a countin) Oosterstraat 1, 3742 SK BAARN, the N~etherlands oats at osoasit IAccoasion NUrntor December 12, 1986 1CBS 568.86 2, ADDITIONAL INOICATOMSIP3 (1*ii. Piano it not sopiicsolet. This intarrnerion is Cont;as an a soas;.,. ertacned Inset C. OL5IGP4ATEO STATES FOR~ WHIttCH ttOICATIONS ARX MADF. i1 ith ind nications 40 not lt 41 sitsgnated State%) 0. !f9PA!tATt FUtttItSttMO OF INDICATIONS I leav. otanit it not &oot,,coi~ The tilaicattans IteI tai00 -Itii 9isomiite to tile internittianel fluseau tater (Sositity ;njs generaltmature at the Indications Accessiotn H Urno 1t 00000it 1 1.,17 This onset s aceinca uom the iiitsotiainat £oAaiian te ticatommAs (to *a 4.oci* ai iy(oscaining Q~nlt (A titcc Offcosi