DK176057B1 - Secreted ligand-binding receptor analogues e.g. PDGF receptor - used in assays, in purifications and as or with therapeutic agents - Google Patents

Abstract

The following are claimed: (A) methods for producing (i) a secreted human PDGF receptor (PDGF-R) analogue, (ii) a secreted, biologically active peptide dimer, by (a) introducing into a host cell a DNA construct contg. a transcriptional promoter operatively linked to a secretory signal sequence followed downstream in proper reading frame by (i) a DNA sequence encoding at least a portion of the extracellular domain of a human PDGF-R, the portion including a ligand-binding domain, or (ii) a DNA sequence encoding a peptide requiring dimerisation for biological activity fused to a dimerising protein, (h) growing the host cell in appropriate growth medium and (c) isolating the prod.; the dimerising protein may comprise a portion of an immunoglobulin light chain, an immunoglobulin heavy chain or yeast invertase; the peptide joined to a dimerising protein may be a ligand-binding receptor analogue; (B) a human PDGF-R analogue consisting of the extracellular domain of a human PDGF-R, the portion being capable of binding human PDGF or isoforms; (C) a pharmaceutical compsn. comprising a human PDGF-R analogue fused to a dimerising protein in combination with a carrier or diluent; (D) a diagnostic compsn. comprising a human PDGF-R analogue fused to a dimerising protein, tagged with a label capable of providing a detectable signal.

Description

DK 176057 B1

Process for preparing a secreted, biologically active peptide dimer.

The present invention relates to the preparation of a secreted, biologically active peptide dimer.

In higher eukaryotic cells, the interaction between receptors and ligands (eg, hormones) is of central importance in the transmission of and response to a variety of extracellular signals. It is widely accepted that hormones and growth factors trigger their biological functions by binding to specific recognition sites (receptors) in the plasma membranes of their target cells. Following ligand binding, the receptor undergoes a conformational change, eliciting secondary cellular responses that result in the activation or inhibition of intracellular processes. The stimulation or blocking of such interaction by pharmacological agents has an important therapeutic significance for a wide variety of diseases.

Ligands fall into two classes: the ligands that have stimulating activity, called agonists, and the ligands that block the effects triggered by the original ligands, called antagonists. The discovery of agonists, which have a different structure and composition than the original ligand, may be medically useful. In particular, agonists smaller than the original ligand may be particularly useful. The bioavailability of these minor agonists may be greater than the bioavailability of the original ligand. This may be particularly important in topical applications and in cases where diffusion of the agonist to its target sites is inhibited by poor circulation. Agonists may also have slightly different spectra for biological activity and / or different strengths, allowing them to be used in very specific situations. Agonists which are smaller and chemically simpler than the native ligand can be produced in greater quantities and at a lower cost. The identification of antagonists, which specifically e.g. blocks growth- DK 176057 B1 2 factor receptors, has important pharmaceutical applications. Antagonists that block receptors against the action of the endogenous native ligand can be used as therapeutic agents in conditions which include atherosclerosis, autocrine tumors, fibroplasia and keloid formation.

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The discovery of new ligands that can be used in pharmaceutical applications has been centered on the design of compounds by chemical modification, complete synthesis and screening of potential ligands by complex and expensive screening procedures. The process of forming a new ligand usually begins with the change of structure of the original effector molecule. If it is known that the original effector molecule is chemically simple, e.g. a catecholamine or prostaglandin, the task is relatively straightforward. However, if the ligand is structurally complex, e.g. a peptide hormone or growth factor, the work of finding a molecule that is functionally equivalent to the original ligand becomes extremely difficult.

Currently, potential ligands are screened by radioligand binding methods (Lefkowitz et al., Biochem. Biophys. Res. Comm. 60: 703-709, 1974; Aur-bach et al., Science 186: 1223-1225, 1974; Atlas et al. , Proc Natl Acad Sci.

USA 71: 4246-4248, 1974). Potential ligands can be directly assayed by binding the radiolabeled compounds to responding cells, to disrupted cell membrane fractions, or to solubilized receptors. Alternatively, potential ligands may be investigated by their ability to compete with a known, labeled ligand for cell surface receptors.

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The beneficial result of these procedures depends on the availability of reproducible, high-quality preparations of membrane fractions or receptor molecules as well as the isolation of responding cell lines. The generation of membrane fractions and soluble receptor molecules involves extensive manipulations and complex purification steps. The isolation of membrane fractions requires careful treatment of the preparation, a procedure which is not suitable for commercial production. It is very difficult to maintain a high biological activity and biochemical purity of receptors when purified by classical protein chemical methods. Since receptors are integral membrane proteins, they require cumbersome purification procedures which include the use of detergents and other solvents that interfere with their biological activity. When these membrane preparations are used in ligand binding assays, they typically result in low reproducibility due to the variations of the membrane preparations.

As noted above, ligand binding assays require the isolation of responding cell lines. Often, only a limited subset of cells respond to a particular agent, and such cells may respond only under certain conditions. Furthermore, it may be difficult to grow these cells in culture or they may have a low number of receptors. The currently available cell types that respond to a platelet-derived growth factor (PDGF) contain, e.g. only a low number of receptors per cell (up to 4 x 105; see Bowen-Pope and Ross, J. Biol.

Chem. 257: 5161-5171, 1982), which requires a large number of cells to analyze PDGF analogs or antagonists.

At present, only a few naturally occurring secreted receptors have been identified, e.g. interleukin-2 receptor (IL-2-R). Rubin et al. (J. Immun. 135: 3172-3177, 1985) have disclosed the release of large amounts of IL-2-R into the culture medium from activated T cell lines. Bailon et al. (Bio / Technology 5: 1195-1198, 1987) has disclosed the use of a matrix-bound interleukin-2 receptor (IL-2-R) for the purification of recombinant interleukin-2.

Three other receptors have been secreted from mammalian cells. The insulin receptor (Ellis et al., J. Cell Biol. 150: 14a, 1987), the cellular HIV-1-30 envelope glycoprotein receptor CD4 (Smith et al., Science 238: 1704-1707, 1987) and the epidermal growth factor receptor (EGF). receptor) (Livneh et al., J. Biol.

4 DK 176057 B1

Chem. 261: 12490-12497, 1986) have been secreted from mammalian cells using truncated cDNAs encoding portions of the extracellular domains.

Therefore, in the art, a process for producing secreted receptors is needed. Furthermore, there is a need for an assay system that allows for comprehensive screening of compounds acting on higher eukaryotic cells via specific surface receptors. This analysis system must be fast, inexpensive and adaptable to a comprehensive screening.

The present invention provides such a method and assay system and further related benefits are provided.

Briefly, the present invention relates to processes for the preparation of secreted, biologically active peptide dimers.

According to one aspect, the invention relates to a method for producing a secreted, biologically active peptide dimer, characterized by introducing into a host cell a DNA construct capable of directing the expression and secretion of a biological agent. active peptide dimer, which DNA construct contains a transcriptional promoter operably linked to at least one secretion signal sequence downstream and in proper reading frame followed by a DNA sequence encoding a peptide requiring dimerization for biological activity and which is fused to a dimerization protein, that the host cell is grown in a suitable growth medium under conditions which allow dimerization and secretion of the peptide dimer, and that the peptide dimer is isolated from the host cell.

In one embodiment of the present invention, the host cell is a yeast cell carrying a defect in a gene whose product is necessary for the addition of outer chain oligosaccharide units to glycoproteins.

In a preferred embodiment, the host cell is a yeast cell containing a genetic defect in the MNN9 gene or an interruption in the MNN9 gene.

In another embodiment, the secretion signal sequence is selected from the group consisting of the MFa1 pre-pro sequence, PH05 signal sequence, BAR1 signal sequence, SUC2 signal sequence, PDGF receptor signal sequence and mouse immunoglobulin V1 signal sequence.

In a third embodiment, the dimerization protein comprises at least a portion of a protein selected from the group consisting of a light immunoglobulin chain, a heavy immunoglobulin chain, and yeast invertase, wherein this portion associates as a dimer in a covalent or non-covalent manner.

In a fourth embodiment, the dimerization protein is selected from the group consisting of a hinge region of a heavy immunoglobulin chain and yeast invertase.

In a fifth embodiment, the peptide requiring dimerization for biological activity is selected from the group consisting of a polypeptide comprising. the amino acid sequence shown in Figure 1 from isoleucine # 29 to lysine # 531, and a polypeptide comprising the amino acid sequence shown in Figure 1 from isoleucine # 29 to methionine # 441.

In a sixth embodiment, the host cell is a yeast cell or a cultured mammalian cell.

These aspects and other aspects of the present invention will become apparent upon reference to the following detailed description and the accompanying drawings.

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Figure 1 shows the nucleotide sequence of the PDGF receptor cDNA and the primary translation product derived amino acid sequence. Numbers above the lines refer to the nucleotide sequence; numbers below the line refer to the amino acid sequence.

5 Figure 2 shows the construction of pBTL10, pBTL11 and pBTL12.

Figure 3 shows the construction of pCBS22.

Figure 4 shows the construction of pBTL13 and pBTL14.

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Figure 5 shows the construction of pBTL15.

Figure 6 shows the construction of pBTL22 and pBTL26.

Figure 7 shows the construction of pSDL114. The symbols used are: S.S., signal sequence, C ^, sequence for constant region of light immunoglobulin chain, μ-prom, μ-promoter, μ -enh, μ-amplifier.

Figure 8 shows the construction of pSDLB113. Symbols used are: SS, 20 signal sequence, C | _1, C [_ | 2, C | _ | 3, heavy immunoglobulin chain constant domain sequences, H, heavy immunoglobulin chain hinge region, M, immunoglobulin membrane anchoring sequences, CY1M, constant sequences area of heavy immunoglobulin chain and membrane anchoring site.

Figure 9 shows the constructs of pBTL15, pBTL14, Pgl-Neo, pICOSV ^ HuC ^ - neo. The symbols used are listed for Figures 7 and 8 and also include Ι_μ |, heavy mouse immunoglobulin chain signal sequence, V | _ | heavy mouse immunoglobulin chain variable region, E, heavy mouse immunoglobulin chain amplifier, L ^, light mouse immunoglobulin chain signal sequence, DK 176057 B1 de, Οδνν, light mouse immunoglobulin chain variable region,

Neo ^, neomycin resistance gene.

Before describing the invention, to understand it may be helpful to provide definitions of certain terms which are used herein.

DNA Construction: A DNA molecule or clone of such a molecule that is either single or double stranded and has been modified by human intervention to contain segments of DNA which are combined and juxtaposed each other in such a way that otherwise it would not exist in nature.

Sequencing signal sequence: A DNA sequence encoding a secretion peptide. A secreting peptide is an amino acid sequence. which directs the secretion of a mature polypeptide or protein from a cell. Segmentation peptides are characterized by a core of hydrophobic amino acids and are typically found at the amino-terminal end of newly synthesized proteins. The secreting protein is very often cleaved from the mature protein during secretion. Certain secretion peptides can be used together to control the secretion of polypeptides and proteins. One such secreting peptide that can be used in combination with other secreting peptides is the third domain of the yeast barrier protein.

Analog to blood oil-derived growth factor receptor (PDGF-R analogs) Part of a PDGF receptor capable of binding antibodies to the PDGF receptor, PDGF, PDGF isoforms, PDGF analogs or PDGF antagonists.

Dimeriserin Protein: A polypeptide chain which has affinity for another polypeptide chain such that the two chains associate to form a dimer which has an additional activity independent of both polypeptide chains in the form of monomers. The other polypeptide chain may be the same chain or another chain.

Biological activity: A function or set of activities exhibited by a molecule in a biological context (ie, in an organism or in vitro facsimile). Biological activities may include the induction of extracellular matrix secretion from responding cell lines, the induction of hormone secretion, the induction of chemotaxis, the induction of mitogenesis, the induction of differentiation, or the inhibition of responding cell division.

Ligand: A molecule other than an antibody or immunoglobulin, which molecule is capable of being bound by a receptor's ligand-binding domain. The molecule can be chemically synthesized or can occur in nature.

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Coupled with: Two or more coding DNA sequences are said to be joined when the coding DNA sequences are translated as a result of fusion frame readings between the coding DNA sequences or as a result of removal of intervening sequences by normal cellular processing. And a polypeptide fusion.

With the present invention, as mentioned above, methods are provided for the preparation of secreted, biologically active peptide dimers. Such peptide dimers can be used to investigate novel compounds that act as agonists or antagonists when interacting with cells containing membrane-bound receptors. The method of the present invention further provides peptides which have therapeutic value and which are only biologically active as dimers.

An example of a receptor that can be secreted from a host cell is a platelet-derived growth factor receptor (PDGF-R). A complementary DNA encoding a PDGF-R with a primary translation product of 190 kDa has been cloned (Gronwald et al., Proc. Natl. Acad. Sci. USA 85: 3435-3439, 1988 ). The receptor comprises an extracellular domain implicated in the ligand binding process, a transmembrane domain, and a cytoplasmic domain containing a tyrosine kinase activity. PDGF-R is capable of binding any native PDGF or any combination of native PDGF or its isoforms. (PDGF is a disulfide bonded two chain containing molecule consisting of an A chain and a B chain. These chains may be combined as AB heterodimers, AA homodimers or BB homodimers. These dimer molecules are referred to herein as "isoforms". ").

A secreted PDGF-R (sPDGF-R) can easily be used in studies to characterize PDGF-R. PDGF-R can e.g. are characterized by identifying ligands other than PDGF and its isoforms, by competitive analysis 15 using various ligands, and by modifying sPDGF-R to define receptor domains critical for ligand binding. These studies are necessary and lead to the systematic design of novel, PDGF-like agonists and antagonists. sPDGF-R is also a source of large amounts of the receptor protein for use in ligand screening procedures. sPDGF-20 R can also be used in radioligand binding assays to compete with samples containing PDGF. The use of sPDGF-R as a therapeutic agent has the advantage of high receptor affinity and specificity for PDGF. As an antagonist, sPDGF-R is a potential drug for atherosclerosis. PDGF, which is implicated in the pathogenesis of atherosclerotic plaques, can be blocked by therapeutic use of sPDGF-R, thereby preventing plaque formation. sPDGF-R can also be used to prepare a battery of new antibodies that can be used both in vivo and in vitro. Examples of in vivo use of PDGF-R antibodies include the use of PDGF-R blocking antibodies in atherosclerosis therapy or the use of antibodies having an agonist character in wound healing. In vitro, sPDGF-R antibodies can be used in a variety of studies and assay procedures, e.g. Western blots, ELISA assays and immunostaining. The sPDGF-R molecules can also be used in the purification of PDGF by taking advantage of the ligand-receptor affinity interaction.

As stated above, the present invention provides methods for producing peptide dimers which require dimerization for biological activity or for enhancing biological activity. Peptides requiring dimerization for biological activity include nerve growth factor, colony stimulating factor-1 (CSF-1), transforming growth factor β (TGF-β), PDGF and γ factor XIII. Nerve growth factor is a non-covalently coupled dimer (Harper et al., J. Biol. Chem. 257: 8541-8548, 1982). CSF-1, which specifically stimulates the proliferation and differentiation of cells of a single-core phagocytic line, is a disulfide-linked homodimer (Retternmier et al., Mol. Cell. Biol. 7: 2378-2387, 1987). TGF-β is biologically active as a disulfide bonded dimer (Assoian et al., 15 J. Biol. Chem. 258: 7155-7160, 1983). Factor XIII is a plasma protein that exists in its activated form as a two-chain homodimer (Ichinose et al.,

Biochem. 25: 6900-6906, 1986). PDGF is, as stated above, a disulfide-linked, two-chain molecule (Murray et al., U.S. Patent No. 4,766,073).

The present invention provides methods by which receptor analogs comprising ligand binding receptor analogs and PDGF-R analogs requiring dimerization for activity can be secreted from host cells. The methods described herein are particularly advantageous in that they allow the production of large amounts of purified receptors in the form of a peptide dimer. The peptide dimers can be used in assays to screen potential ligands, in assays in binding studies, as imaginative agents, and as agonists and antagonists in therapeutic drugs.

A DNA sequence encoding a human PDGF receptor can be isolated as a cDNA using known methods (see, e.g., Okayama and Berg,

Moth. Cell. Biol. 2: 161-170, 1982; Moth. Cell. Biol. 3: 280-289, 1983). EtcDNA, 11 encoding 176057 B1 encoding a PDGF-R, has been cloned from a diploid, dermal fibroblast human cDNA library using oligonucleotide probes complementary to sequences of the mouse PDGF receptor (Gronwald et al., ibid.). A DNA sequence encoding a PDGF-5 receptor analog may be used, consisting essentially of the extracellular domain of a PDGF receptor, although smaller DNA sequences encoding regions of at least 400 amino acids may also be used. of the extracellular domain.

In the present invention, DNA sequences encoding EGF-R (Ullrich et al., Nature 304: 418-425, 1984), the insulin receptor (Ullrich et al., Nature 313: 756-761,1985) can also be used. ), nerve growth factor (Ullrich et al., Nature 303: 821-825, 1983), colony stimulating factor-1, (Rettenmier et al., ibid.), transforming growth factor β (Derynck et al., Nature 316: 701-15705, 1985), PDGF (Murray et al., Ibid.) And factor XIII (Ichinose et al., Ibid.).

In order to direct peptides requiring dimerization for biological activity into the secretory pathway of the host cell, at least one secretion signaling sequence is used together with the DNA sequence of interest. Preferred secretion signals include the alpha factor signal sequence (pre-pro sequence) (Kurjan and Herkowitz, Cell 30: 933-943, 1982; Kurjan et al., U.S. Patent No. 4,546,082; Brake EP No. 116,201,1983 ), The PH05 signal sequence (Beck et al., WO 86/00637), the BAR1 secretion signal sequence (MacKay et al., U.S. Patent No. 4,613,572, MacKay, WO 87/002670), and mouse immunoglobulin V | signal sequence (Loh et al., Cell 33: 85-93, 1983). Particularly preferred signal sequences are the SUC2 signal sequence (Carlson et al., Mol. Cell. Bil. 3: 439-447, 1983) and the PDGF receptor signal sequence. The signal sequence may be used singly or in conjunction with a sequence encoding the third domain of the "Barrier" (described pending, disclosed to the public U.S. Patent Application No. 104,316, which is incorporated herein by reference in its entirety). The third domain of the "Barrier" may be placed in the correct reading frame 12 'for the DNA sequence of interest, or 5' for the DNA sequence and in the correct reading frame having both the sequence signal sequence and the DNA sequence which have interest.

In one embodiment of the present invention, a sequence encoding a dimerization protein is joined with a sequence encoding a peptide dimer. As shown herein, in the present invention, such an arrangement is used to promote the association of the peptide or receptor to form a biologically active molecule upon secretion. Suitable dimerization proteins include the repressible acid phosphatase of S. cerevisiae (Mizunaga et al., J. Biochem. (Tokyo) 103: 321-326, 1988), type 1 killer prepotoxin of S. cerevisiae (Sturley et al., EMBO J. 5: 3381-3390, 1986), alpha-galactosidase melibiase from S. carlsbergensis (Sumner-Smith et al., Gene 36: 333-340, 1985), and omithine decarboxylase from Neurospora crassa (Digangi et al., J. Biol. Chem. 262: 7889-7893, 1987). In this regard, sequences encoding a hinge region of a heavy immunoglobulin chain (Takahashi et al., Cell 29: 671-679, 1982) are preferred, the S. cerevisiae SUC2 gene (Carlson et al., Mol Cell. Biol. 3: 439-447, 1983), immunoglobulin sequences and portions thereof. In one embodiment of the invention, 20 immunoglobulin gene sequences are used to drive the association of non-immunoglobulin peptides. Constant regions of immunoglobulins include distinct in domains that show similarity in their three-dimensional conformation. These distinct domains correspond to exons of the immunoglobulin heavy chain constant domain domain (Hood et al., In Immunology, The Benjamin / Cummings Publishing Company, Inc., Menlo Park, CA;

Honjo et al., Cell 18: 559-568, 1979, and Takahashi et al., Cell 29: 671-679, 1982). As the dimerization protein, it is particularly preferred to use a constant region of the light immunoglobulin chain in combination with at least one constant region domain of the heavy immunoglobulin chain, which domain is joined by an immunoglobulin hinge region. Constant domain domains in the heavy immunoglobulin chain include C | _ | 1, Cj-j2, 13, Ογ1, Ογ2, Cv3, Cy4, and μ. A particularly preferred constant domain domain of the heavy immunoglobulin chain is C1-1. Host cells for use in the practice of the present invention include mammalian cells and fungal cells. Fungal cells, including species of yeast (e.g., Saccharomyces spp., Schizosaccharomyces spp.) Or filamentous fungi (e.g., Aspergillus spp., Neurospora spp.), Can be used as host cells in the present invention. Strains of the yeast Saccharomyces cerevisiae are particularly preferred.

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Suitable yeast vectors for use in the present invention include YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76: 1035-1039, 1978), YEp13 (Broach et al., Gene 8: 121-133, 1979). , pJDB249 and pJDB219 (Beggs, Nature 275: 104-108, 1978) and derivatives thereof. Such vectors will generally comprise a selectable marker, which may be any of a variety of genes exhibiting a dominant phenotype for which a phenotypic assay exists so that transformants can be selected. Preferred selectable markers are those markers that complement host cell cell atrophy, provide antibiotic resistance or enable a cell to use specific carbon sources, and they include LEU2 (Broach et al., Ibid.), URA3 (Botstein et al., Gene 8: 17, 1979), HIS3 (Struhl et al., Ibid.) Or POT1 (Kawasaki and Bell, EP 171,142). Other suitable selectable markers include the CAT gene which confers yeast cells chloramphenicol resistance, or the lacZ gene which results in blue colonies when active β-galactosidase is expressed.

Preferred promoters for use in yeast include promoters of glycolytic yeast genes (Hitzeman et al., J. Biol. Chem. 255: 12073-12080, 1980; Alberog Kawasaki, J. Mol. Appl. Genet. 1: 419-434, 1982; Kawasaki, U.S. Pat.

4,599,311) or alcohol dehydrogenase genes (Young et al., In Genetic Engineering of Microorganisms for Chemicals, Hollaender et al., (Ed.), P. 355, Pie, New York, 1982; Ammer, Meth. Enzymol. 101: 192-201, 1983). In this regard, particularly preferred promoters are the TP11 promoter (Kawasaki, U.S. Patent No. 4,599,311, 1986) and the ADH2-4c promoter (Russell et al., Nature 304: 652-654, 1983, and Irani and Kilgore, which are disclosed in U.S. Patent Application Nos. 29,867 and 183,130, incorporated herein by reference. Expression units may also comprise a transcription terminator. A preferred transcription terminator is the TPI1 terminator (Alber and Kawasaki, ibid.).

In addition to yeast, proteins of the present invention can be expressed in filamentous fungi, e.g. strains of the fungus Aspergillus (McKnight and Upshall, which are disclosed in pending U.S. Patent Applications Nos. 820,519 and 942,494, corresponding to published European Patent Application EP 272,277, which is incorporated herein by reference). Examples of useful promoters include those promoters derived from glycolytic genes in Aspergillus nidulans such as the ADH3 promoter (McKnight et al., EMBO J. 4: 2093-2099, 1985) and the tpiA promoter. An example of a suitable terminator is the ADH3 terminator (McKnight et al., Ibid.).

The expression units utilizing such components are cloned into vectors which can be inserted into the chromosomal DNA of Aspergillus.

Methods for transforming fungi are well known in the literature, and they are e.g. have been described by Beggs (ibid.), Hinnen et al. (Proc. Natl. Acad. Sci.

USA 75: 1929-1933, 1978), Yelton et al. (Proc. Natl. Acad. Sci. USA 81: 1740-1747, 1984) and Russell (Nature 301: 167-169, 1983). The host cell geotype will generally comprise a genetic defect complemented by the selectable marker present on the expression vector. One skilled in the art can select a particular host and selectable marker.

In a preferred embodiment, a yeast host cell containing a genetic defect is used in a gene necessary for the asparagine-linked glycoprotein cosylation of glycoproteins. Preferably, the yeast host cell contains a genetic defect in the MNN9 gene (described in pending U.S. Patent Application No. 116,095, incorporated herein by reference in its entirety). Most preferably, the yeast host cell contains an interrupt 5 of the MNN9 gene. Yeast host cells containing such defects can be produced using standard methods of mutagenization and selection. Ballou et al. (J. Biol. Chem. 255: 5986-5991, 1980) have described the isolation of mannoprotein biosynthetic mutants that are defective in genes affecting asparagine-linked glycosylation. Briefly, mutagenized yeast cells were screened using fluorescent antibodies directed against the outer mannose chains present on the wild-type yeast. Mutant cells that did not bind antibody were further characterized and were found to be defective in the addition of asparagine-linked oligosaccharide units. To optimize the production of heterologous proteins, it is preferred that the host strain carry a mutation, such as the yeast pep4 mutation (Jones, Genetics 85: 23-33, 1977), which results in decreased proteolytic activity.

In addition to fungal cells, cultured mammalian cells can be used in the present invention. Preferred mammalian cell lines include the cell lines COS-20 1 (ATCC CRL 1650), BHK, p363.Ag.8.653 (ATCC CRL 1580), FO (ATCC

CRL 1646) and 293 (ATCC CRL 1573; Graham et al., J. Gen. Virol. 36: 59-72, 1977). A preferred BHK cell line is the tk13 ts BHK cell line (Waechter and Baserga, Proc. Natl. Acad. Sci. USA 79: 1106-1110, 1982). A particularly preferred cell line is SP2 / 0-Ag14 (ATCC CRL Further, in the present invention, a variety of other cell lines can be used, including rat Hep I (ATCC CRL 1600), rat Hep II (ATCC CRL 1548), TCMK (ATCC CCL 139), human lung cells (ATCC CCL 75.1). ), human hepatoma cells (ATCC HTB-52), Hep G2 cells (ATCC HB 8065), mouse liver cells (ATCC CC 29.1) and DUKX cells (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77: 30 4216 -4220, 1980).

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Mammalian expression vectors for use in the practice of the present invention will comprise a promoter capable of directing the transcription of a cloned gene or cDNA. Preferred promoters include viral promoters and cellular promoters. Preferred viral promoters include the major late promoter of adenovirus 2 (Kaufman and Sharp, Mol. Cell. Biol. 2: 1304-1319, 1982) and the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1: 854 -864, 1981). Preferred cellular promoters include the mouse metal lothionein I promoter (Palmiter et al., Science 222: 809-814, 1983) and the mouse V1 promoter (Grant et al., Nuc. Acids Res. 15: 5496, 1987). A particularly preferred promoter is the mouse Vj_j promoter (Loh et al., Ibid.). Such expression vectors may also contain a set of RNA splicing sites, located downstream of the promoter, and upstream of the DNA sequence encoding the peptide or protein of interest. Preferred RNA splicing sites can be obtained from adenovirus genes and / or immunoglobulin genes. Also in the expression vectors is a polyadenylation signal located downstream of the insertion site. Polyadenylation signals include the early or late polyadenylation signal from SV40 (Kaufman and Sharp, ibid.), The polyadenylation signal from the E1B region of adenovirus 5, and the human growth hormone terminator (DeNoto et al., Nuc. Acids Res. 9: 20 3719-3730, 1981). . A particularly preferred polyadenylation signal is the V 1 gene terminator (Loh et al., Ibid.). The expression vectors may comprise a non-coding viral leader sequence, such as the adenovirus 2 triplicate leader sequence located between the promoter and the RNA splicing sites. Preferred vectors may also include amplifier sequences such as the SV40-25 amplifier and the mouse μ amplifier. Expression vectors may also include sequences encoding adenovirus VA RNAs.

Cloned DNA sequences can be introduced into cultured mammalian cells by e.g. calcium phosphate mediated transfection (Wigeret al., Cell 14: 725,1978; Corsaro 30 and Pearson, Somatic Cell Genetics 7: 603, 1981; Graham and Van der Eb, 17 DK 176057 B1

Virology 52: 456, 1973). Other methods may also be used to introduce cloned DNA sequences into mammalian cells, such as electroperforation (Neumann et al., EMBO J. 1: 841-845, 1982).

In order to identify cells that have integrated the cloned DNA, a gene providing a selectable phenotype (a selectable marker) is usually introduced into the cells along with the gene or cDNA of interest. Preferred selectable markers include genes that confer resistance to drugs such as neomycin, hygromycin and methotrexate. The selectable marker can be a selectable marker which can be amplified. A preferred amplifiable, selectable marker is the DHFR gene. A particularly preferred marker is the DHFRr cDNA (Simonsen and Levinson, Proc. Natl.

Acad. Sci. USA 80: 2495-2499, 1983). Thilly has provided a list of selectable markers (Mammalian Cell Technology, Butterworth Publishers, 15 Stoneham, MA) and one skilled in the art will be able to select the selectable markers.

Selectable markers can be introduced into the cell on a separate plasmid at the same time as the gene of interest, or they can be introduced on the same plasmid. If the selectable marker and gene of interest are located on the same plasmid, it may be under the control of different promoters or the same promoter, with the latter scheme providing a di-cistronic message. Constructions of this type are known in the art (e.g., Levinson and Simonsen, U.S. Patent No. 4,713,339). It may also be advantageous to add additional DNA, known as "carrier DNA", to the mixture introduced into the cells.

Transfected mammalian cells are allowed to grow for a period of time, typically 1 to 2 days, for initial expression of the DNA sequence (s) of interest. A drug selection is then performed to select growth 30 of cells expressing the selectable marker in a stable manner. For cells that have been transfected with an amplifiable selectable marker, the drug concentration can be increased in a stepwise manner to select for increased copy number of the cloned sequences, thereby increasing expression levels.

Host cells containing DNA constructs of the present invention are grown in a suitable growth medium. The growth medium is usually a medium which selects for cells containing the DNA construct. The term "appropriate growth medium" as used herein means a medium containing nutrients necessary for cell growth. Nutrients needed for cell growth may include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals and growth factors. Yeast cells are grown e.g. preferably in a chemically defined medium comprising a nitrogen source which is not an amino acid, inorganic salts, vitamins and supplements of essential amino acids. The pH of the medium is preferably maintained at a value greater than 2 and less than 8, preferably a pH of 6.5. Methods for maintaining a stable pH value include the use of buffers and constant pH control, preferably by the addition of sodium hydroxide. A preferred buffering agent is succinic acid. Yeast cells which have a defect in a gene necessary for asparagine-linked glycosylation are preferably cultured in a medium containing an osmotic stabilizer. A preferred osmotic stabilizer is sorbitol added to the medium at a concentration of from 0.1 M to 1.5 M, preferably 0.5 or 1.0 M. Cultured mammalian cells are generally cultured in commercially available serum-containing or serum-free media. Selection of a medium suitable for the particular cell line used can be carried out by one of ordinary skill in the art.

The culture medium from appropriately cultured, transformed or transfected host cells is separated from the cell material and the presence of sej in serrated peptide dimers, for example in the form of PDGF receptor analogs. A preferred method for detecting PDGF receptor analogs is e.g. to bind the receptors or portions of the receptors to a receptor-specific antibody. A specific preferred antibody is PR7212, a mouse monoclonal antibody against the PDGF receptor. Another particularly preferred antibody which can be used to detect substance P-labeled peptides and proteins is the commercially available monoclonal rat antibody to substance P which can be used to visualize peptides or proteins fused to the C-terminal amino acids. in substance P. Ligand binding assays can also be used to detect the presence of receptor analogs. In the case of PDGF receptor analogs, it is preferred to use fetal foreskin fibroblasts expressing PDGF receptors to compete with the PDGF receptor analogs 10 of the present invention for labeled PDGF or PDGF isoforms.

Assays to detect secreted, biologically active peptide dimers may include Western transfer, protein blot or colony filter. A Western transfer filter can be produced by using substantially the method described by Towbin et al. (Proc. Natl. Acad. Sci. USA 76: 4350-4354, 1979). Briefly, samples are subjected to electrophoresis in a sodium dodecyl sulfate polyacrylamide gel. The proteins are electrophoretically transferred to nitrocellulose paper. Protein blot filters can be prepared by filtering supernatant samples or concentrates through nitrocellulose filters using e.g. a 20 "minifold" (Schleicher and Schueli). Colony filters can be prepared by growing colonies on a nitrocellulose filter which has been placed on top of an appropriate growth medium. In this method, a solid medium is preferred. The cells are allowed to grow on the filters for at least 12 hours. The cells are removed from the filters by washing with a suitable buffer which does not remove the proteins bound to the filters. A preferred buffer comprises 25 mM Tris base, 19 mM glycine, pH 8.3.20% methanol.

The secreted peptide dimers present on the Western transfer filter, protein blot filter or colony filter can be visualized by specific antibody binding 30 using known methods. Towbin et al. (ibid.) describes e.g. visualization of proteins immobilized on nitrocellulose filters with a specific antibody followed by a labeled second antibody directed to the first antibody. Kits and reagents needed for visualization can be obtained commercially, e.g. from Vector Laboratories (Burlingame, CA) and from Sigma Chemical Company (St. Louis, MO).

5

Secreted, biologically active peptide dimers can be isolated from the medium from host cells grown under conditions that allow secretion of the biologically active peptide dimers. The cellular material is removed from the culture medium and the biologically active peptide dimers are isolated using known methods. Suitable isolation methods include precipitation and fractionation by a variety of chromatographic methods, including gel filtration, ion exchange chromatography and immunoaffinity chromatography. A particularly preferred purification method is immunoaffinity chromatography using an antibody directed to the peptide dimer. The antibody is preferably immobilized or attached to a solid support or substrate. A particularly preferred substrate is CNBr-activated Sepharose (Pharmacia, Piscataway, NJ). In this method, the medium is combined with the antibody / substrate under conditions that allow binding to take place. The complex can be washed to remove unbound material and the peptide is released or eluted using conditions which are unfavorable for complex formation. Particularly useful elution methods include changes in pH where the immobilized antibody has a high affinity for the ligand at a first pH and a decreased affinity at a second (higher or lower) pH; changes in the concentration of certain chaotropic agents or by the use of detergents.

25

The secreted biologically active PDGF receptor analog dimers of the present invention can be used in a variety of assays to detect the presence of native PDGF, PDGF isoforms or PDGF-like molecules. Typically, these assays will involve PDGF receptor analog dimers, which may be bound to a solid substrate such as polymeric microtiter plate wells, in combination with a biological sample under conditions that allow the formation of receptor / ligand complexes . The detection can be achieved by using a label attached to the receptor or by using a labeled antibody that reacts with the receptor. Alternatively, the labeled antibody may be an antibody that reacts with the ligand. A wide variety of labels can be used, such as radionuclides, fluorophores, enzymes and luminescent compounds. Complexes can also be detected visually, viz. in immunoprecipitation assays which do not require the use of a label.

Secreted PDGF receptor analog dimers of the present invention may also be labeled with a radioisotope or other imaging agent and used for in vivo diagnostic purposes. Preferred imaging radioisotope agents include iodine-125 and technitium-99, with technitium-99 being particularly preferred. Methods for producing protein isotope conjugates are well known and are described e.g. by Eckelman et al. (U.S. Patent No. 4,652,440), Parker et al. (WO 87/05030) and Wilber et al. (EP 203,764). Alternatively, the secreted PDGF receptor analog dimers can be bound to spin labeling enhancers and used for magnetic resonance imaging (MRI) imaging. Suitable spin labeling enhancers include stable, sterically inhibited, free radical compounds such as nitroxides. Methods for labeling ligands for 20 MRI imaging are e.g. described by Coffman et al. (U.S. Patent No. 4,656,026). For administration, the labeled PDGF receptor analog dimers are combined with a pharmaceutically acceptable carrier or diluent such as sterile saline or sterile water. Administration is preferably by bolus injection, preferably intravenously. These imaging agents are particularly useful in identifying sites with atherosclerotic plaques, PDGF-producing tumors, and receptor-bound PDGF.

The secreted PDGF receptor analog dimers of the present invention may also be used in diagnostic kits. Briefly, the dimers in question are preferably in a freeze-dried form or immobilized on the walls of a suitable container, either alone or in combination with additional antibodies capable of binding to a native PDGF or selected PDGF. isoforms or specific ligands. The antibodies which may be conjugated to a label or non-conjugated are generally included in the kit along with appropriate buffers such as phosphate, stabilizers, inert proteins 5 or the like. These materials are generally present in an amount of less than ca. 5% by weight based on the total amount of active receptor analogue and is usually present in an amount of at least approx. 0.001% by weight. It may also be desirable to include an inert excipient to dilute the active ingredients. Such an excipient may be present in an amount of from ca. 1 to 99% by weight of the total composition. Furthermore, the kit will typically comprise other standard reagents, instructions and, depending on the nature of the label involved, reagents needed to produce a detectable product. Where an antibody capable of binding to the receptor or receptor / ligand complex is used, this antibody will usually be present in a separate container. The antibody is typically conjugated with a label and formulated in an analogous manner to the formulations briefly described above. The diagnostic kits comprising the containers can be produced and packaged using conventional kit making procedures.

20

As noted above, the secreted PDGF receptor analog dimers of the present invention can be used in methods of purifying PDGF from a variety of samples. In a preferred method, the secreted PDGF receptor analog dimers are immobilized or attached to a substrate or carrier such as polymeric test tubes, beads, polysaccharide particles, polysaccharide-containing materials, polyacrylamide or other water-insoluble polymeric materials. Immobilization methods are well known in the art (Mosbach et al., U.S. Patent No. 4,415,665; Clarke et al., Meth. Enzymology 68: 436-442, 1979). A common immobilization method is CNBr-30 activation. Activated substrates are commercially available from a variety of suppliers, including Pharmacia (Piscataway, NJ), Pierce Chemical Co. (Rock-23 DK 176057 B1 ford, IL) and Bio-Rad Laboratories (Richmond, CA). A preferred substrate is CNBr-activated Sepharose (Pharmacia, Piscataway, NJ). The substrate / receptor analog complex will generally be in the form of a column. The sample is then combined with the immobilized dimerized receptor analog 5 under conditions that allow binding to take place. The immobilized receptor analog substrate is first equilibrated with a buffer solution having a composition in which the receptor analog has previously been shown to bind to its ligand. The sample is then placed on the substrate / receptor analog complex in the solution used for equilibration. When the complex is in the form of a column, it is preferred that the sample be passed through the column two or more times to allow full ligand binding to receptor analog. The complex is then washed with the same solution to elute unbound material. In addition, another washing solution can be used to minimize non-specific binding. The bound material can then be released or eluted using conditions which are unfavorable for complex formation. Particularly useful methods include changes in pH where the immobilized dimerized receptor has a high affinity for PDGF at a first pH and a decreased affinity at a second (higher or lower) pH; changes in concentration of certain chaotropic agents or with the use of detergents.

The secreted PDGF receptor analogs fused to dimerization proteins of the present invention can be used in pharmaceutical compositions for topical or intravenous use. The PDGF-25 receptor analogs fused to dimerization proteins are used in conjunction with physiologically acceptable carrier or diluent. Preferred carrier materials and thinners include saline and sterile water. Pharmaceutical compositions may also contain stabilizers and adjuvants.

The resulting aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, combining the lyophilized preparation with a sterile aqueous solution prior to administration.

24 DK 176057 B1

The following examples are given as an illustration of the invention and not as a limitation.

5 EXAMPLES

Enzymes, including restriction enzymes, DNA polymerase I (Klenow fragment), T4 DNA polymerase, T4 DNA ligase, and T4 polynucleotide kinase, were obtained from New England Biolabs (Beverly, MA), Bethesda Research La-10 boratories ( Gaithersburg, MD) and Boehringer-Mannheim Biochemicals (Indianapolis, IN) and were used as stated by the manufacturer or as described in Maniatis et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY, 1982).

Example 1

Cloning of the PDGF Receptor cDNA:

Complementary DNA (cDNA) libraries were prepared from poly (A) -20 RNA from diploid human dermal fibroblast cells produced by normal adult exploration, which was largely performed as described by Hagen et al. (Proc. Natl. Acad. Sci. USA 83: 2412 * 2416, 1986). Briefly, poly (A) RNA was primed with oligo (T) and cloned into Agt11 using Eco R1 linkers. The stochastically primed library was screened for the presence of human PDGF receptor cDNAs using three oligonucleotide probes complementary to sequences of the mouse PDGF receptor (Yarden et al., Nature 323: 226-232, 1986). . Ca. one million phages from the stochastically primed human fibroblast cell library were screened using oligonucleotides ZC904, ZC905 and ZC906 (Table 1). Eight 30 positive clones were identified and plaque purified. Two clones, designated RP41 and RP51, were selected for further analysis by restriction enzyme mapping and DNA sequence analysis. RP51 was found to contain 356 bp of the non-coding 5 'sequence, the ATG translation initiation codon, and 738 bp of the amino-terminal coding sequence. RP41 was found to overlap the clone RP51 and contained 2649 bp, which encodes amino acids 43-925 of the receptor protein.

TABLE 1

Oligonucleotide Sequences 1 ° ZC582 5'AAT TCC CGG G 3 'ZC583 5'GAT CCC CGG G 3' ZC904 5 'CAT GGG CAC GTA AT C TAT AGA TTC ATC CTT GCT CAT ATC CAT GTA 3'

ZC905 5 'TCT TGC CAG GGC ACC TGG GAC ATC TGT TCC CAC ATG

ACC GG 3 'ZC906 5' AAG CTG TCC TCT GCT TCA GCC AGA GGT CCT GGG CAG CC 3 'ZC1380 5' CAT GGT GGA ATT CCT GCT GAT 3 '

20 ZC1453 5 'AAT TCA TTA TGT TGT TGC AAG CCT TCT TGT TCC TGC

TAG CTG GTT TCG CTG TTA A 3 'ZC1454 5' GAT CTT AAC AGC G AA ACC AGC TAG CAG G AA CAA GAA GGC TT G CAA CAA CAT AAT G 3 'ZC1478 5' ATC GCG AGC ATG CAG ATC TGA 3 '

25 ZC1479 5 'AGC TTC AGA TCT GCA TGC TCG CGA T

ZC1776 5 'AGC TGA GCG CAA ATG TTG TGT CGA GTG CCC ACC GTG CCC AGC TTA GAA TTC T 3' ZC1777 5 'CTA GAG AAT TGT AAG CTG GGC AAC GTG GGC ACT CGA CAC AAC ATT TGC GCT G 3' 26 DK 176057 B1 ZC1846 5 'GAT CGG CCA CTG TCG GTG CGC TGC ACG CTG CGC AAC GCT GTG GGC CAG GAC ACG CAG GAG GTC ATC GTG GTG CCA CAC TCC TTG CCC TTT AAG CA 3'

ZC1847 5 'AGC TTG CTT AAA GGG C AA GGA GTG TGG CAC CAC GAT 5 GAC CTC CTG CGT GTC CTG GCC CAC AGC GTT GCG CAG

CGT GCA GCG CAC CGA CAG TGG CC 3 'ZC1886 5' CCA GTG CCA AGC TTG TCT AGA CTT ACC TTT AAA GGG CAAGAAG 3 'ZC1892 5' AGC TTG AGC GT 3 '10 ZC1893 5' CTA GAC GCT CA 3 'ZC1894 5' AGC TTC CAG TTC TTC GGC CTC ATG TCA GTT CTT CGG CCT CAT GTG AT 3 'ZC1895 5' CTA GAT CAC ATG AGG CCG AAG AAC TGA CAT GAG GCC G AA G AA CTG GA 3 '15 The 3-end of the cDNA was were not isolated by the first cloning and were later isolated by screening 6 x 10 5 phages of the oligo (dT) -primed cDNA library with a 630 bp Sst Eco Eco R1 fragment derived from the 3-end of the clone RP41. An isolate, designated OT91, was further analyzed by restriction enzyme mapping and DNA sequencing. This clone was found to comprise the 3-end of the receptor coding region and 1986 bp of the untranslated 3-sequence.

Clones RP51, RP41 and OT91 were ligated together to produce a full-length cDNA encoding the entire PDGF receptor. RP41 was digested with Acc I and Barn H1 to isolate a 2.12 kb fragment. RP51 was digested with Eco RI and Acc I to isolate a 982 bp fragment. The 2.12 kb RP41 fragment and the 982 bp RP51 fragment were joined by a tripartite ligation with pUC13, which had been linearized by digestion with Eco RI and Barn H1. The resulting plasmid was designated 51/41. Plasmid 51/41 was digested with Eco RI and Bam H1 to isolate a 27 kb fragment containing the partial PDGF receptor cDNA. OT91 was digested with Barn HI and Xba I to isolate the 1.4 kb fragment containing the 3 portion of the PDGF receptor cDNA. The Eco RI-Bam HI-51/41 fragment, the Bam HI-Xba I-OT91 fragment, was joined in a tripartite ligation with Eco RI-Xba 1 digested pUC13. The resulting plasmid was designated pR-RX1 (Figure 2).

Example 2 Construction of a SUC2 Signal Sequence PDGF Receptor Fusion:

To direct the PDGF receptor into the yeast secretion pathway, the PDGF receptor cDNA was joined to a sequence encoding the SUC2 signal sequence from Saccharomyces cerevisiae. Oligonucleotides ZC1453 and ZC1454 (Table 1) were designed to form an adapter encoding the SUC2 secretion peptide flanked by an Eco R1 sticky 5 'end and a Bgl II sticky 3 end. ZC1453 and ZC1454 were annealed under conditions described by Maniatis et al. (Ibid.). Plasmid pR-RX1 was digested with Bgl II and Sst II to isolate a 1.7 kb fragment, as if * 20 captured the PDGF receptor coding sequence from amino acids 28 to 596. Plasmid pR-RX1 was also cut with Sst II and Hind III to isolate a 1.7 kb fragment that included the coding sequence from amino acid 597 to the translation termination codon and 124 bp of the untranslated 3 DNA.

The two 1.7 kb pR-RX1 fragments and the ZC1453 / ZC1454 adapter were joined with pUC19, which had been linearized by digestion with Eco RI and Hind III. The resulting plasmid, comprising the SUC2 signal sequence fused in reading frame with the PDGF receptor cDNA, was designated pBTL10 (Figure 2).

Example 3 28 DK 176057 B1

Construction of pCBS22: The BAR1 gene product, "Barrier", is an exported protein that has been found to have three domains. When used in conjunction with a signal sequence (von 5 Heinje, Eur. J. Biochem. 133: 17-21, 1983; J. Mol. Biol. 184: 99-105, 1985;

Nuc. Acids Res. 14: 4683-4690, 1986), it has been found that the third domain of the "Barrier" protein assists in the secretion of proteins to the medium (MacKay et al., U.S. Patent Application No. 104,316).

The third domain of the BAR1 gene was joined to a sequence encoding the C-terminal portion of substance P (subP; Munro and Pelham, EMBO J. 3: 3087-3093,1984). The presence of substance P-amino acids at the terminal end of the fusion protein allowed the protein to be detected using commercially available antibodies against substance P.

Plasmid pZV9 (deposited as a transformant in E. coli strain RR1, ATCC deposit # 53283), comprising the entire BAR1 coding region and its associated flanking regions, was cut with Sal I and Barn H1 to isolate a 1.3 kb long BAR1 fragment. This fragment was subcloned into pUC13, which had been cut with Sal I and Barn HI, to generate the plasmid designated pZV17. Plasmid pZV17 was digested with Eco RI to remove the outermost 0.5 kb 3-end of the BAR1 coding region. The vector BAR1 fragment was ligated to generate the plasmid designated pJH66 (Figure 3). Plasmid pJH66 was linearized with Eco RI and provided with blunt ends with DNA polymerase I (Klenow fragment). China-25 seed-treated Barn HI linkers (5 ′ CCG GAT CCG G 3 ′) were added, and excess linkers were removed by digestion with Barn HI before ligation. The resulting plasmid was designated pSW8 (Figure 3).

Plasmid pSW81, which included the TP11 promoter, the BAR1 coding region fused to the coding region of the C-terminal portion of substance P (Munro and Pelham, EMBO J. 3: 3087-3093, 1984) and TPI1. the temninator, 29 DK 176057 B1 was derived from pSW8. Plasmid pSW8 was cut with Sal I and Bam H1 to isolate an 824 bp fragment encoding amino acids 252 to 516 in BAR1. Plasmid pPM2, which contains the synthetic oligonucleotide sequence encoding the dimer form of the C-terminal portion of substance P (subP) in 5 M13mp8, was obtained from Hugh Pelham (MRC Laboratory of Molecular Biology, Cambridge, England). Plasmid pPM2 was linearized by digestion with Bam HI and Sal I and ligated with the 824 bp BAR1 fragment from pSW8. The resulting plasmid, pSW14, was digested with Sal I and Sma I to isolate an 871 bp BAR1 substance P fragment. Plasmid pSV16, which comprised a fragment of BAR1-encoding amino acids from 1 to 250, was cut with Xba I and Sal I to isolate a 767 bp BAR1 fragment. This fragment was ligated to the 871 bp BAR1 substance P fragment in a three-part ligation with pUC18 cut with Xba I and Sma I. The resulting plasmid, designated pSW15, was digested with Xba I and Sma I for isolation. of a 1.64 kb BAR1 substance P fragment. The ADH1 promoter was obtained from pRL029. Plasmid pRL029, comprising the ADH1 promoter and the 5 'region of BAR1 encoding amino acids 1 to 33, in pUC18, was digested with Sph I and Xba I to isolate a 0.42 kb ADH1 promoter fragment. . The TPI1 terminator (Alber and Kawasaki, ibid.) Was obtained as a linearized fragment containing the TPI1 terminator and pUC18 with a Klenow-filled Xba I end and a Sph I end. This fragment was ligated with the 0.42 kb ADH1 promoter fragment and the 1.64 kb BAR1 substance P fragment in a tripartite ligation to generate the plasmid pSW22.

The ADH1 promoter and the coding region of BAR1 from the translation initiation code ATG to the Eco RV site in pSW22 were removed by digestion with Hind III and Eco RV. The 3.9 kb vector fragment, which included 401 bp between the Eco RV and Eco R1 sites of the BAR1 gene fused to the subP and the TPI1 terminator, was isolated by gel electrophoresis. Oligonucleotide ZC1478 (Table 1) was kinase treated and annealed with oligonucleotide ZC1479 (Table 1) using conditions described by Maniatis et al.

DK 176057 B1 (ibid.). The annealed oligonucleotides formed an adapter which included a Hind III adhesive end and a polylinker encoding the Bgl II, Sph I, Nru I and Eco RV restriction sites. The ZC1479 / ZC1478 adapter was ligated to the gel-purified pSW22 fragment. The resulting plasmid was designated 5 pCBS22 (Figure 3).

Example 4

Construction of pBTL13: 10

To enhance the secretion of the receptor and to facilitate the identification of the secreted protein, a sequence encoding the third domain of BAR1 fused to the C-terminal amino acids of substance P was fused in reading frame with the 5 '1240 bp of the PDGF receptor. Plasmid pBTLIO 15 (Example 2) was digested with Sph I and Sst I to isolate the 4 kb fragment comprising the SUC2 signal sequence, part of the PDGF receptor cDNA, and the pUC19 vector sequences. Plasmid pCBS22 was digested with Sph I and Sst I to isolate the 1.2 kb fragment which contained the BAR1 subP fusion and the TPI1 terminator. These two fragments were ligated and the resulting plasmid was designated pBTL13 (Figure 4).

Example 5

Construction of an expression vector encoding the entire PDGF receptor: 25

The entire PDGF receptor was directed into the secretory pathway by fusing a SUC2 signal sequence to the 5-end of the PDGF receptor coding sequence. This fusion was placed behind the TP11 promoter and inserted into the vector YEp13 for expression in yeast.

The B1 TP11 promoter was obtained from the plasmid pTPIC10 (Alber and Kawasaki, J.

Moth. Appl. Genet. 1: 410-434, 1982) and plasmid pFATPOT (Kawasaki and Bell, EP 171,142; ATCC 20699). Plasmid pTPIC10 was cut at the unique Kpn I site, the TP11 coding region was removed with Bal-31-5 exonuclease, and an Eco R1 linker (sequence: GGA ATT CC) was added to the 3 'end of the promoter. Digestion with Bgl II and Eco RI yielded a TPI1 promoter fragment with Bgl II and Eco R1 sticky ends. This fragment was then joined with plasmid YRp7 '(Stinchcomb et al., Nature 282: 39-43, 1979), which had been cut with Bgl II and Eco RI (partial). The resulting plasmid, TE32, was digested with Eco RI (partial) and Barn HI to remove part of the tetracycline resistance gene. The linearized plasmid was then restored to a circle by the addition of an Eco Ri-Barn HI linker to generate the plasmid TEA32. Plasmid TEA32 was digested with Bgl II and Eco RI, and the 900 bp partial TP11 promoter fragment was gel purified. Plasmid plC19H (Marsh et al., Gene 32: 481-486, 1984) was cut with Bgl II and Eco RI and the vector fragment was gel purified. The TPI1 promoter fragment was then ligated to the linearized plC19H and the mixture was used to transform E. coli RR1. Plasmid DNA was prepared and screened for the presence of a ca. 900 bp long Bgl II Eco 20 R1 fragment. A correct plasmid was selected and designated pICTPIP.

The TP11 promoter was then subcloned to place appropriate restriction sites at its ends. Plasmid plC7 (Marsh et al., Ibid.) Was digested with Eco RI, the fragment ends blunted with DNA polymerase 25 I (Klenow fragment), and the linear DNA restored to a circle using T4 DNA ligase . The resulting plasmid was used to transform E. coli RR1. Plasmid DNA was prepared from the transformants and screened for the loss of the Eco R1 site. A plasmid with the correct restriction pattern was designated plC7RI *. Plasmid plC7RI * was digested with 30 Hind III and Nar I, and the 2500 bp fragment was gel purified. The partial TP11 promoter fragment (about 900 bp) was removed from pICTPIP using Nar I and Sph I and was gel purified. The remaining portion of the TPI1 promoter was obtained from plasmid pFATPOT by digesting the plasmid with Sph I and Hind III, and a 1750 bp fragment which comprised a portion of the TP11 promoter fragment from pICTPIP, and the fragment from pFATPOT was subsequently digested. combined in a tripartite ligation to generate pMVR1 (Figure 2).

The TP11 promoter was then joined to the SUC2-PDGF receptor fusion. Plasmid pBTL10 (Example 2) was digested with Eco RI and Hind III to isolate a 3.4 kb fragment which comprised the SUC2-10 signal sequence and the entire PDGF receptor coding region. Plasmid pMVR1 was digested with Bgl II and Eco RI to isolate a 0.9 kb TPI1 promoter fragment. The TP11 promoter fragment and fragment derived from pBTL10 were joined to YEp13, which had been linearized by digestion with Bam HI and Hind III, in a tripartite ligation. The resulting plasmid was designated pBTL12 (Figure 2).

Example 6

Construction of an expression vector encoding the extracellular 5'-20 portion of the PDGF receptor:

The extracellular portion of the PDGF receptor was directed into the secretory pathway by fusing the coding sequence with the SUC2 signal sequence. This fusion was placed in an expression vector behind the TP11 promoter. Plasmid pBTL10 (Example 2) was digested with Eco RI and Sph I to isolate the ca. 1.3 kb fragment containing the SUC2 signal sequence and the sequence encoding the extracellular domain of the PDGF receptor. Plasmid pMVR1 (Example 5) was digested with Bgl II and Eco RI to isolate a 0.9 kb TP11 promoter fragment. The TPI1-30 promoter fragment was joined with the fragment derived from pBTL10 and YEp13, which had been linearized by digestion with Bam H1 and 331 176057 B1.

Sph I, in a tripartite ligation. The resulting plasmid was designated pBTL11 (Figure 2).

Example 7 5

Construction of yeast expression vectors pBTL14 and pBTL15 and the expression of PDGF receptor-BAR1 subP fusions: A. Construction of pBTL14 10 The SUC2-PDGF-R fusion was joined to the third domain of BAR1 to enhance the secretion of the receptor and expression was cloned into a derivative of YEp13 designated pJH50. YEp13 was modified to destroy the Sal I site near the LEU2 gene. This was achieved by a 15 partial digestion of YEp13 with Sal I followed by complete digestion with Xho I. The 2.0 kb Xho l-Sal I fragment comprising the LEU2 gene and the 8.0 kb long , linear YEp13 vector fragments were isolated and ligated together. E. coli strain RR1 was transformed with the ligation mixture. DNA was prepared from the transformants and analyzed by digestion with Sal I and Xho I. A clone was isolated which showed a single Sal I site and an inactive Xho I site, indicating that the LEU2 fragment had been inserted with the opposite orientation to the parent plasmid YEp13. The plasmid was designated pJH50.

Reference is made to Figure 4, where plasmid pBTL12 (Example 5) was digested with Sal I and Pst I to isolate a 2.15 kb fragment which contained 270 bp of the YEp13 vector sequence, the TP11 promoter, the SUC2 signal sequence and 927 bp of PDGF receptor cDNA. Plasmid pBTL13 (Example 4) was digested with Pst I and Hind III to isolate a 1.48 kb fragment which comprised 30,313 bp of the PDGF receptor cDNA, the BAR1 subP fusion and the TPI1 terminator.

The fragments derived from pBTL12 and pBTL13 were joined to a tripartite ligation with 34 DK 176057 B1 pJH50, which had been linearized by digestion with Hind III and Sal I. The resulting plasmid was designated pBTL14.

B. Construction of pBTL15 5

Refer to Figure 5, where a yeast expression vector was constructed which included the TP11 promoter, the SUC2 signal sequence, 1.45 kb of the PDGF receptor cDNA sequence fused to the BAR1 subP fusion and the TPI1 terminator. Plasmid pBTL12 (Example 5) was digested with Sal I and Fsp I 10 to isolate a 2.7 kb fragment which included the TP11 promoter, the SUC2 signal sequence, the PDGF-R coding sequence and 270 bp of the YEp13 vector sequence. . Plasmid pBTL13 (Example 4) was digested with Nru I and Hind III to isolate a 1.4 kb fragment comprising the BAR1 subP fusion, the TPI1 terminator and 209 bp of the 3'-PDGF receptor-15 cDNA. sequence. The fragments derived from pBTL12 and pBTL13 were joined in a tripartite ligation with pJH50 which had been linearized by digestion with Hind III and Sal I. The resulting plasmid was designated pBTL15.

20 C. Expression of PDGF-R subP Fusions from pBTL14 and pBTL15 Yeast Expression Vectors pBTL14 and pBTL15 were transformed into Sac-charomyces cerevisiae strains ZY100 (MATa Ieu2-3,112 ade2-101 suc-Δ9 gal2 pep4 :: TPI1p-CAT ZY400 (MATa Ieu2-3,112 ade2-101 suc2- Δ9 25 gal2 pep :: TPIp-CAT Amnn9 :: URA3). Transformations were performed using largely the method described by Beggs (ibid.). Transformants were selected for their ability to grow on -LEUDS (Table 2).

TABLE 2 35 DK 176057 B1 -LeuThrT rp amino acid mixture: 4 g adenine 3 g L-arginine 5 5 g L-aspartic acid 2 g L-histidine free base 6 g L-isoleucine 4 g L-lysine monohydrochloride 2 g L methionine 10 6 g L-phenylalanine 5 g L-serine 5 g L-tyrosine 4 g uracil 6 g L-valine 15

All the ingredients are mixed and ground with a mortar and bump until the mixture is comminuted.

LEUDS: 20 20 g glucose 6.7 g gemino base without amino acids (DlFCO Laboratories Detroit, Ml) 0.6 g -LeuThrTrp amino acid mixture 182.2 g sorbitol 25 18 g Agar

All ingredients are mixed in distilled water. Distilled water is added to a final volume of 1 liter. Autoclave for 15 minutes. After autoclaving, 150 mg of L-threonine and 40 mg of L-tryptophan are added. The medium is poured into plates 30 and allowed to solidify.

36 DK 176057 B1 -LEUDS + sodium succinate, pH 6.5: 20 g yeast nitrogen base 0.6 g -LeuTrpThr amino acid mixture 5 182.2 g sorbitol 11.8 g succinic acid

All ingredients are mixed in distilled water to a final volume of 1 liter. The pH of the solution is adjusted to pH 6.5. Autoclave for 15 minutes. After 10 autoclaving, 150 mg of L-threonine and 40 mg of L-tryptophan are added.

Fermentation medium: 7 g / l yeast nitrogen base without amino acids or ammonium sulfate (DIFCO Laboratories) 0.6 g / l ammonium sulfate 0.5 M sorbitol 0.39 g / l adenine sulfate 0.01% polypropylene glycol 20

All ingredients are mixed in distilled water. Autoclave for 15 minutes.

80 ml of 50% glucose is added for each liter of medium.

Supersynthetic -LEUD, pH 6.5 (liquid or solid medium): 6.7 g of yeast nitrogen base without amino acids or ammonium sulfate (DIFCO) 6 g of ammonium sulfate 160 g of adenine 0.6 g of -LeuThrTrp amino acid mixture 30 20 g of glucose 11.8 g of succinic acid 37 DK 176057 B1

All ingredients are mixed and distilled water is added to a final volume of 800 ml. The pH of the solution is adjusted to pH 6.4. Autoclave for 15 minutes. To the solid medium is added 18 g of agar before autoclaving, after which 5 are autoclaved and the medium is poured into plates.

Supersynthetic -LEUDS, pH 6.4 (liquid or solid medium):

The same formula is used as for Super Synthetic -LEUD, pH 6.4, but before 10 autoclaving, 182.2 g of sorbitol is added.

YEPD: 20 g glucose 15 g g Bacto peptone (DIFCO Laboratories) 10 g Bacto yeast extract (DIFCO Laboratories) 18 g agar 4 ml 1% adenine 8 ml 1% L-leucine 20

All the ingredients are mixed in distilled water and brought to a final volume of 1 liter. Autoclave for 25 minutes and pour the media into plates.

The transformants were assayed for binding to a monoclonal antibody against the PDGF receptor (PR7212) or an antibody to substance P by protein blot analysis. ZY100 (pBTL14) and ZY100 (pBTL15) transformants were grown overnight at 30 ° C in 5 ml of Supersynthetic -LEUD, pH 6.4 (Table 2). ZY400 (pBTL14) and ZY400 (pBTL15) transformants were grown overnight at 30 ° C in 5 ml of Supersynthetic -LEUDS, pH 6.4 (Table 2). The cultures were pelleted by centrifugation, and the supernatants were analyzed by protein blot analysis using methods described in Example 13. Analysis results obtained with PR7212 are shown in Table 3.

TABLE 3 5

Results of a protein blot probed with PR7212:

Transformant: 10 ZY100 (pBTL14) + ZY400 (pBTL14) ++ ZY100 (pBTL15) + j ZY400 (pBTL15) + j! Example 8

Construction of a SUC2-PDGF-R fusion comprising the complete extracellular domain of PDGF-R: 20 A. Construction of PBTL22

The PDGF-R coding sequence present in pBTL10 was modified to remove the coding region 3 'of the extracellular PDGF-R domain. As shown in Figure 6, plasmid pBTL10 was digested with Sph I and Barn 25 HI and with Sph I and Sst II to isolate a 4.77 kb fragment and a 466 bp fragment, respectively. The 466 bp fragment was then digested with Sau 3A to isolate a 0.17 kb fragment. The 0.17 kb fragment and the 4.77 kb fragment were then joined by ligation.

The resulting plasmid was designated pBTL21.

30 39 DK 176057 B1

Plasmid pBTL21, which contained a Barn HI site that had been regenerated by the ligation of the Barn HI and Sau 3A sites, was digested with Hind III and Barn HI to isolate a 4.2 kb fragment. Synthetic oligonucleotides ZC1846 (Table 1) and ZC1847 (Table 1) were designed to form an adapter encoding PDGF-R from the Sau 3A site after bp 1856 (Figure 1) to the end of the extracellular domain at 1958 bp (Figure 1) and having a Barn H1 sticky 5 end which destroys the Barn H1 site and a Hind III sticky 3 end. The oligonucleotides ZC1846 and ZC1847 were canceled under conditions described by Maniatis et al. (Ibid.). The 4.2 kb pBTL21-10 fragment and the ZC1846 / ZC1847 adapter were joined by ligation. The resulting plasmid, designated pBTL22, comprises the SUC2 signal sequence fused in correct reading frame with the extracellular domain of PDGF-R in the vector pUC19 (Figure 6).

15 B. Construction of PBTL28

A translation stop codon was inserted into the reading frame immediately after the coding region of PDGF-R in pBTL22 using oligonucleotides ZC1892 (Table 1) and ZC1893 (Table 1). These oligonucleotides were designed to form an adapter that encodes a stop codon in reading frame with the PDGF-R coding sequence from pBTL22 flanked by a Hind III-sticky 5 'end and an Xba 1 sticky 3' end. Plasmid pBTL22 was digested with Eco RI and Hind III to isolate a 1.6 kb SUC2 PDGF-R fragment. Plasmid pMVR1 was digested with Eco RI and Xba I to isolate a 3.68 kb fragment containing the TP11 promoter, the plC7Rr vector sequences and the TPI1 terminator. Oligonucleotides ZC1892 and ZC1893 were canceled to form a Hind III-Xba I adapter. The 1.6 kb SUC2 PDGF-R fragment, the 3.86 kb pMVR1 fragment, and the ZC1892 / ZC1893 adapter were joined by a tripartite ligation. The resulting plasmid was designated pBTL27.

40 DK 176057 B1

The expression unit present in pBTL27 was inserted into the yeast expression vector pJH50 by first digesting pJH50 with Barn HI and Sal I to isolate a 10.3 kb vector fragment. Plasmid pBTL27 was digested with Bgl II and Eco RI and with Xho I and Eco RI to isolate a 0.9 kb TPI1-5 promoter fragment and a 1.65 kb fragment, respectively. The 10.3 kb pJH50 vector fragment, the 0.9 kb TP11 promoter fragment, and the 1.65 kb fragment were joined by a tripartite ligation. The resulting plasmid was designated pBTL28.

10 C. Construction of plasmid pBTL3Q

The PDGF-R coding sequence present in plasmid pBTL22 was modified to encode the twelve C-terminal amino acids of substance P and a stop codon in the reading frame. Plasmid pBTL22 was digested with Eco RI and Hind 15 III to isolate a 1.6 kb SUC2 PDGF-R fragment. Plasmid pMVR1 was digested with Eco RI and Xba I to isolate a 3.68 kb fragment which included the TP11 promoter, pLC7RI * and the TPI1 terminator. Synthetic oligonucleotides ZC1894 (Table 1) and ZC1895 (Table 1) were canceled to form an adapter comprising the codons of the twelve C-terminal amino acids of substance P followed by a stop codon in reading frame and flanked at the 5 'end. with a Hind III sticky end and at the 3 'end with an Xba 1 sticky end. The ZC1894 / ZC1895 adapter, the 1.6 kb SUC2 PDGF-R fragment, and the pMVR1 fragment were joined by a tripartite ligation. The resulting plasmid, designated pBTL29, was digested with Eco RI and Xho I to isolate a 1.69 kb SUC2-PDGF-R subP-TPI1 terminator fragment. Plasmid pBTL27 was digested with Bgl II and Eco RI to isolate a 0.9 kb TP11 promoter fragment. Plasmid pJH50 was digested with Barn HI and Sal I to isolate a 10.3 kb vector fragment. The 1.69 kb pBTL29 fragment, the 0.9 kb TPH promoter fragment, and the 10.3 kb 30 vector fragment were joined in a tripartite ligation. The resulting plasmid was designated pBTL30.

41 DK 176057 B1

Example 9

Construction and Expression of a SUC2-PDGF-R-IgG Hinge Expression Vector:

An expression unit was constructed which included the TPI1 promoter, the SUC2 signal sequence, the extracellular PDGF-R domain, an immunoglobulin hinge region, and the TPI1 terminator. Plasmid pBTL2210 was digested with Eco I and Hind III to isolate a 1.56 kb fragment. Plasmid pMVR1 was digested with Eco RI and Xba I to isolate a 3.7 kb fragment which included the TP11 promoter, the plC7RI * vector sequences and the TPI1 temninator. Oligonucleotides ZC1776 (Table 1) and ZC1777 (Table 1) were designed to form, when canceled, an adapter encoding an immunoglobulin hinge region with a Hind III adhesive 5 end and an Xba 1 adhesive 3 end . Oligonucleotides ZC1776 and ZC1777 were canceled under conditions described by Maniatis et al. (Ibid.). The 1.56 kb pBTL22 fragment, the 3.7 kb fragment, and the ZC1776 / ZC1777 adapter were joined by a tripartite ligation, resulting in plasmid 20 pBTL24.

The pBTL24 expression unit comprising the TP11 promoter, the SUC2 signal sequence, the extracellular PDGF-R domain sequence, the hinge region sequence, and the TPI1 terminator were inserted into pJH50.

Plasmid pBTL24 was digested with Xho I and Hind III to isolate a 2.4 kb expression unit. Plasmid pJH50 was digested with Hind III and Sal I to isolate a 9.95 kb fragment. The 2.4 kb pBTL24 fragment and the 9.95 kb pJH50 vector fragment were joined by ligation. The resulting plasmid was designated pBTL25.

30 in 42 DK 176057 B1

Saccharomyces cerevisiae strain ZY400 was transformed with plasmid pBTL25 using substantially the method described by Beggs (ibid.). Transformants were selected for their ability to grow on -LEUDS (Table 2). The transformants were tested for their ability to bind the monoclonal antibody to PDGF-R PR7212 using the colony assay described in Example 13. Plasmid pBTL25 transformants were transferred onto nitrocellulose filters which had been wetted and applied to solid YEPD. medium. Antibody PR7212 was bound to the PDGF-R-IgG hinge fusion secreted by ZY400 (pBTL25) transformants.

10

Example 10

Construction and expression of a SUC2 signal sequence extracellular PDGF-R domain SUC2 fusion: 15

As shown in Figure 6, an expression unit comprising the TPI1 promoter, the SUC2 signal sequence, the extracellular PDGF-R domain sequence and the SUC2 coding sequence was constructed as follows. Plasmid pBTL22 was digested with Eco RI and Hind III to isolate a 20 1.6 kb SUC2 PDGF-R fragment. Plasmid pMVR1 was digested with Bgl II and Eco RI to isolate a 0.9 kb TP11 promoter fragment. The SUC2 coding region was obtained from pJH40. Plasmid pJH40 was constructed by inserting the 2.0 kb Hind III-Hind IIII-SUC2 fragment from pRB58 (Carlson et al., Cell 28: 145-154, 1982) into the Hind III site of pUC19 followed by destruction. of the Hind III site 3 'for the coding region. Plasmid pJH40 was digested with Hind III and Sal I to isolate the 2.0 kb SUC2 coding sequence. Plasmid pJH50 was digested with Sal I and Barn HI to isolate a 10.3 kb vector fragment. The 0.9 kb Bgl II Eco RI-TPI1 promoter fragment, the 1.6 kb Eco RI Hind III-SUC2-PDGF-R, the 2.0 30 kb Hind III-Sal I-SUC2 fragment and the 10.3 kb Bam HI-Sal I-r —— - - --- - —— DK 176057 B1 43 in vector fragment was joined by a four-part ligation. The resulting plasmid was designated pBTL26 (Figure 6).

Saccharomyces cerevisiae strain ZY400 was transformed with plasmid 5 pBTL26, largely using the method described by Beggs (ibid.). Transformants were selected for their ability to grow on -LEUDS (Table 2). ZY400 transformants (ZY400 (pBTL26)) were analyzed by protein blot (Example 13), colony blot (Example 13), and competition analysis.

10

Protein blot analyzes were performed on ZY400 (pBTL26) transformants and ZY400 (pJH50) transformants (control) which had been grown in bottles. Two hundred and fifty μΙ of a 5 ml night culture of ZY400 (pBTL26) in -LEUDS + sodium succinate, pH 6.5 (Table 2), was inoculated into 50 ml of -LEUDS + sodium 15 succinate, pH 6.5. The cultures were incubated for 35 hours in a shaker in air at 30 ° C. The culture supernatants were harvested by centrifugation. The culture supernatants were analyzed as described in Example 13 and found to bind PR7212 antibody.

Colony analyzes on ZY400 (pBTL26) transformants were performed. ZY400 (pBTL26) transformants were stained on wetted nitrocellulose filters mounted on a YEPD plate. This colonial assay, performed as described in Example 13.A., showed that ZY400 (pBTL26) antibodies bound PR7212 antibodies.

25

Competing binding assays on ZY400 (pBTL26) and ZY400 (pJH50) transformants were performed. The transformants were grown in 2 L fermentation medium (Table 2) in a New Brunswick Bioflo2 (New Brunswick, Philadelphia, PA) with continuous pH control at pH 6.4. The pH of the cultures was adjusted to 7.5 immediately before harvest. The culture supernatants were concentrated in an Amicon concentrator (Amicon, San Francisco, CA) using an Amicon 10 µm spiral filter element. The concentrated supernatants were further concentrated with "Amicon Centriprep 10s". Fifteen | ml of the concentrated supernatant samples was added to "Centriprep," eme, and "The centrifuges were centrifuged in a Beckman GRP centrifuge (Beckman 5 Instruments, Inc., Carlsbad, CA) at a setting of 5 for a total of 60 minutes. The concentrates were removed from The "centriprep" and was analyzed by the competition analysis.

!

The competing binding assay measured the amount of 125 µpQp remaining to be bound to fetal foreskin fibroblast cells after preincubation with the concentrate containing the PDGF-R-SUC2 fusion protein. The concentrate was serially diluted in binding medium (Table 4). The dilutions were mixed with 0.5 ng of iodinated PDGF-AA, PDGF-BB or PDGF-AB and the mixtures were incubated for two hours at room temperature. To each sample mixture 15, 300 µg of unlabelled PDGF-BB was added. The sample mixtures were added to 24-well plates containing confluent fetal foreskin fibroblast cells (AG1523, available from the Human Genetic Mutant Cell Repository,

Camden, NJ). The cells were incubated with the mixture for 4 hours at 4 ° C. The supernatants were aspirated from the wells and the wells were rinsed three times with phosphate buffered saline maintained at 4 ° C (PBS; Sigma, St.

Louis, Mo.). To each well, 500 μΙ PBS + 1% NP-40 was added and the plates were shaken on a plate shaker for 5 minutes. The cells were harvested and the amount of iodinated PDGF determined. The results of the concurrent binding assay showed that the PDGF-R-SUC2 fusion protein was able to competitively bind all isoforms of PDGF.

TABLE 4

Binding medium: 30 45 DK 176057 B1 500 ml Ham's F-12 medium 12 ml 1 MHEPES, pH 7.4 5 ml 100 x PSN (Penicillin / Streptomycin / Neomycin, Gibco) 1 g rabbit serum albumin 5

Western Transfer Buffer: 25 mM Tris, pH 8.3 19 mM Glycine, pH 8.3 10 20% Methanol

Western Buffer A: 50 ml 1 M Tris, pH 7.4 20 ml 0.25 mM EDTA, pH 7.0 5 mM 0% NP-40

37.5 ml of 4 M NaCl

2.5 g of gelatin 20 Tris, EDTA, NP-40 and NaCl were diluted to a final volume of 1 Ilter with distilled water. To 300 ml of this solution was added gelatin and the solution was heated in a microwave oven until the gelatin was in solution. The gelatin solution was added to the remainder of the first solution and stirred at 4 ° C until cooled. The buffer was stored at 4 ° C.

25

Western Buffer B: 50 ml 1 M Tris, pH 7.4 20 ml 0.25 M EDTA, pH 7.0 5 ml 10% NP-40

58.4 g of NaCl

2.5 g of gelatin 4 g of N-lauroylsarcosine

Tris, EDTA, NP-40 and NaCl were mixed and diluted to a final volume of 1.5 liters. To 300 ml of this solution was added gelatin and heated in a microwave oven until the gelatin was in solution. The gelatin solution was added to the original solution and N-lauroylsarcosine was added. The final mixture was stirred at 4 ° C until the solids were completely dissolved. This buffer was stored at 4 ° C.

10 2 x loading buffer:

36 ml 0.5 M Tris-HCl, pH 6.8 16 ml glycerol 16 ml 20% SDS

4 ml 0.5 bromophenol blue in 0.5 M Tris-HCl, pH 6.8

All ingredients are mixed. Immediately before use, 100 μΙ of β-mercaptoethanol is added to each 900 μΙ of color mixture.

20

Example 11

Construction and Expression of PDGF Receptor Analogs from BHK Cells: 25 A. Construction of pBTL1 14 and pBTL1 15

The portions of the extracellular PDGF receptor domain present in pBTL14 and pBTL15 were placed in a mammalian expression vector. Plasmidial pBTL14 and pBTL15 were digested with Eco RI to isolate the 1695 bp 30 and 1905 bp SUC2 signal PDGF-R-BAR1 fragments, respectively. The 1695 bp fragment and the 1905 bp fragment were each ligated 47 Z172257 B1 to Zem229R, which had been linearized by digestion with Eco RI. The mammalian expression vector Zem229R is a pUC18-based expression vector containing a unique Eco RI site for insertion of foreign DNA between the mouse metallothionein-1 promoter and the SV40 transcription terminator.

Zem229R also contains an expression unit of the early SV40 promoter, the mouse dihydrofolate reductase gene, and the SV40 transcription terminator.

The ligation mixtures were transformed into E. coli strain RR1. Plasmid DNA was prepared and the plasmids were subjected to restriction enzyme analysis. A plasmid with the 1695 pb pBTL14 fragment inserted into Zem229R in the correct orientation was designated pBTL114 (Figure 9). A plasmid with the 1905 bp pBTL15 fragment inserted into Zem229R in the correct orientation was designated pBTL115 (Figure 9).

B. Expression of oBTL114 and pBTL115 from tk ~ ts13 BHK cells:

Plasmids pBTL114 and pBTL115 were each transfected into tk "ts13 cells using calcium phosphate precipitation (roughly as described by Graham and van der Eb, J. Gen. Virol. 36: 59-72, 1977). The transfected cells were grown in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf serum, 1 x PSN antibiotic mixture (Gibco 600-5640), 2.0 mM L-glutamine, cells were selected in 250 nM methotrexate (MTX) for 14 days , and the resulting colonies were screened by immunofilter analysis (McCracken and Brown, Biotechniques, 82-87, March / April, 1984).

Plates were rinsed with PBS or medium without serum (DMEM plus 1 x PSN antibiotic mixture). Teflon nets (Spectrum Medical Industries, Los Angeles, CA) were then placed over the cells. Nitrocellulose filters were wetted with PBS or medium without serum, as appropriate, and placed over the mesh. After six hours of incubation at 37 ° C, the filters were removed and placed in Western Buffer A (Table 4) overnight at room temperature. The filters were then developed with the antibody PR7212 and the procedure described in Example 13. The filters showed that conditioned media from pBTL114-transfected and pBTL115-transfected BHK cells bound the PR7212-5 antibody, indicating the presence of secreted PDGF-R.

Example 12

Construction and Expression of PDGF Receptor Analogs from Cultured Mouse Myeloma Cells: A. Construction of the plCuPRE8 µ-Promoter and the µ-heavy immunoglobulin chain amplifier was subcloned into plC19H to provide a unique Hind III site 3 'for the promoter. Plasmid ρμ (Grosschedl and Baltimore, Cell 41: 885-897, 1985) was digested with Sal I and Eco RI to isolate a 3.1 kb fragment containing the μ promoter. Plasmid plC19H was linearized by digestion with Eco RI and Xho I. The μ promoter fragment and the linearized plC19H-20 vector fragment were joined by ligation. The resulting plasmid, designated plCp3, was digested with Ava II to isolate a 700 bp μ-promoter fragment. The 700 bp fragment was provided with blunt ends by treatment with DNA polymerase I (Klenow fragment) and deoxy nucleotide triphosphates. Plasmid plC19H was linearized by digestion with Xho I, and the sticky ends were filled by treatment with DNA polymerase I (Klenow fragment) and deoxynucleotide triphosphates. The blunt-ended Ava II fragment was ligated with the blunt-ended, linearized plC19H, and E. coli JM83 was transformed with the ligation mixture. Plasmid DNA was prepared from the transformants and analyzed by restriction digestion. A plasmid with a Bgl II site 5 'for the promoter was designated p1CPPR1 (-). Plasmid plCpPR1 (-) was digested with Hind III and Bgl II to isolate a 700 bp μ-promoter fragment. Plasmid plC19RI was linearized by digestion with Hind III and Barn H1. The 700 bp promoter fragment was joined to the linearized plC19RI by ligation. The resulting plasmid, designated plCpPR7, comprised the 5 μ promoter with a unique Sma I site 5 'for the promoter and a unique Hind Misted 3' for the promoter.

The μ-heavy immunoglobulin chain enhancer was inserted into the unique Sma I site to generate plasmid plCpPRE8. Plasmid pJ4 (obtained from 10 F. Blattner, Univ. Wisconsin, Madison, Wisconsin), which comprises the 1.5 kb Hind III-Eco RI-β amplifier fragment in the vector pAT153 (Amersham, Arlington Heights, IL), was digested with Hind III and Eco RI to isolate the 1.5 kb amplifier fragment. The sticky ends of the enhancer fragment were filled by treatment with T4 DNA polymerase and deoxynucleotide triphosphates. The blunt-ended fragment and plCpPR7, which had been linearized by digestion with Sma I, were joined by ligation. E. coli RR1 was transformed with the ligation mixture. Plasmid DNA was prepared from the transformants and the plasmids were analyzed by restriction digestion. A plasmid that included the μ amplifier in the correct orientation relative to the μ promoter was designated plCpPRE8 (Figure 7).

B. Construction of pSDL114

The DNA sequence encoding the extracellular domain of the PDGF-25 receptor was joined to the DNA sequence encoding the constant region of the light human immunoglobulin chain. The extracellular PDGF domain was obtained from mpBTL22, which included the Eco Ri-Hind I II fragment of pBTL22 (Example 8.A.) cloned into M13mp18 cut with Eco RI and Hind III. Single-stranded DNA was prepared from a 30 mpBTL22 phage clone, and the DNA was subjected to in vitro mutagenization using the oligonucleotide ZC1886 (Table 1) and the method described by Kunkel (Proc. Natl. Acad. Sci. USA 82: 488-492, 1985).

A phage clone comprising the mutagenized PDGF-R with a donor splice site (5 'splice site) at the 3' end of the extracellular PDGF-R domain was designated pBTLR-HX (Figure 7).

5

The native PDGF-R signal sequence was obtained from pPR5. Plasmid pPR5, which comprises 738 bp 5 coding sequence with an Eco RI site immediately 5 'of the translation initiation codon, was constructed by in vitro mutagenization of the PDGF-R cDNA fragment from RP51 (Example 1). RP51 DNA in replicative fprm was digested with Eco RI to isolate a 1.09 kb PDGF-R fragment. The PDGF-R fragment was cloned into the Eco R1 site of M13mp18. Single stranded template DNA was prepared from a phage clone containing the PDGF-R fragment in the correct orientation. The template DNA was subjected to in vitro mutagenization using oligonucleotide ZC1380 (Table 15 1) and the method described by Zoller and Smith (Meth. Enzymol. 100: 468-500, 1983). The mutagenization resulted in the placement of an Eco R1 site immediately 5 'of the translation initiation codon. Mutagenized phage clones were analyzed by dideoxy sequence analysis. A phage clone containing the ZC1380 mutation was selected and DNA in replicative form (Rf) 20 was prepared from the phage clone. The Rf DNA was digested with Eco RI and Acc I to isolate a 0.63 kb fragment. Plasmid pR-RXI (Example 1) was digested with Acc I and Eco RI to isolate a 3.7 kb fragment. The 0.63 kb fragment and the 3.7 kb fragment were joined by ligation, resulting in plasmid pPR5 (Figure 7).

25

As shown in Figure 7, the PDGF-R signal peptide was obtained from plasmid pPR5 as a 1.4 kb Eco RI-SpH 1 fragment. Replicative DNA from phage clone pBTLR-HX was digested with Sph I and Hind III to isolate a ca. 0.25 kb PDGF-R fragment. Plasmid pUC19 was linearized by digestion with 30 Eco RI and Hind III. The 1.4 kb Eco RI-Sph 1 PDGF-R fragment, the 0.25 kb Sph 1-Hind I II fragment from pBTLR-HX and pUC19 cut with Eco RI

51 DK 176057 B1 and Hind III were joined by a tripartite ligation. The resulting plasmid, pSDL110, was digested with Eco RI and Hind III to isolate a 1.65 kb PDGF-R fragment. Plasmid plCHuCk3 9.11 was used as the source of the kappagen gene (Figure 7). Plasmid plCHuCk3.9.11 comprises a 1.1 kb Sph 5 I-Hinf I fragment of a human capsule which has been treated with DNA polymerase I (Klenow fragment) to fill the Hine II sticky end and the subclone in pUC19 cut with Sph I and Hine II. Plasmid plCHuCk3.9.11 was digested with Hind III and Eco RI to isolate the 1.1 kb constant region of the human kappagen gene. Plasmid plC19H was linearized by digestion with Eco RI. The 1.65 kb PDGF-R fragment, the 1.1 kb fragment of the lightweight constant chain constant region, and the linearized pLC19H were joined by a tripartite ligation. The resulting plasmid, pSDL112, was digested with Barn HI and Cla I to isolate a 2.75 kb fragment. Plasmid ppPRE8 was linearized with Bgl II and Cla I.

The 2.75 kb fragment and the linearized ppPRE8 were joined by ligation. The resulting plasmid was designated pSDL114 (Figure 7).

Plasmid pSDL114 was linearized by digestion with Cla I and transfected into SP2 / 0-Ag14 (ATCC CRL 1581) by electroperforation using substantially the procedure described by Neumann et al. (EMBO J. 1: 841-845, 1982). Transfected cells were selected in growth medium containing methotrexate.

Media from drug resistant clones were examined for the presence of 25 secreted PDGF receptor analogs by immunoprecipitation. Ca. one million drug resistant transfected cells were grown in DMEM medium without cysteine + 2% fetal calf serum for 18 hours at 37 ° C in the presence of 50 µCi 3i> S-cysteine. per media was harvested from the labeled cells and 250 μΙ of the media used was examined for binding to the PDGF-30 receptor antibody PR7212. PR7212, diluted in PBS, was added to the media at a final concentration of 2.5 µg per ml. 250 µl of used media. Five µg of rabbit anti-mouse Ig diluted in PBS was added to the PR7212 / media mixtures. The immunocomplexes were precipitated by the addition of 50 µl of 10% fixed Staph A (weight / volume in PBS). The immunocomplexes were analyzed on 58% SDS-polyacrylamide gels followed by overnight autoradiography at - 70 ° C. The results of the immunoprecipitation showed that the PDGF receptor analog secreted by the transfected cells was bound by the antibody to the PDGF receptor.

10 C. Cotransfection with PSDL114 of a heavy immunoglobulin chain

Plasmid pSDL114 was linearized by digestion with Cla I and used for cotransfection together with pG1-Neo, which encodes a neomycin resistance gene and a complete gene for the heavy immunoglobulin chain. The heavy immunoglobulin chain gene in pG1-Neo comprises a mouse / human chimeric immunoglobulin gene (Figure 9). The mouse immunoglobulin heavy chain gene was isolated from a lambda genomic DNA library constructed from a mouse hybridoma cell line, NR-ML-05, using oligonucleotide probes designed to extend over the VH / D / JH junction sites. The 20 heavy human immunoglobulin chain gene was isolated from a human genomic library and oligonucleotide probes designed to extend over the hinge exon. The mouse immunoglobulin heavy chain gene was isolated as a 5.3 kb Sst 1-Hind III fragment from the original phage clone, and the human gamma-1-C gene was obtained from the original phage clone as a 2.0 kb Hind III Eco RI fragment. The chimeric gamma-1-C gene was created by joining the VH and CH fragments via the common Hind I II site and incorporating them with the E. coli neomycin resistance gene in pLC19H to generate pG1M-Neo.

30 SP2 / 0-Ag14 cells were transfected with linearized pSDL114 and pG1M-Neo by electroperforation. The transfected cells were selected in growth medium containing methotrexate and neomycin. Media from drug resistant clones were studied for their ability to bind PDGF in a competitive binding assay.

5 The competitive binding assay measured the amount of 12 µl PDGF remaining to bind to dermal human fibroblast cells after preincubation with the used media from pSDL114-pG1 neo-transfected cells. The media were serially diluted in binding medium (Table 4). The dilutions were mixed with 0.5 ng of iodinated PDGF-BB and the mixtures were incubated for two hours at room temperature. To each sample mixture, 300 µg of unlabelled PDGF-BB was added. The sample mixtures were added to 24-well plates containing confluent dermal human fibroblast cells. The cells were incubated with the mixture for 4 hours at 4 ° C. The supernatants were aspirated from the wells and the wells were rinsed three times with phosphate buffered saline, maintained at 4 ° C (PBS; Sigma, St. Louis, Mo.). To each well, 500 µl of PBS + 1% NP-40 was added and the plates were shaken on a plate shaker table for five minutes. The cells were harvested and the amount of iodinated PDGF determined. The results of the competitive binding assay showed that the protein produced from pSDL114-pG1 neo-transfected cells was capable of competitively binding PDGF-BB.

D, Construction of pSDL113 i

As shown in Figure 8, the DNA sequence encoding the extracellular domain of the PDGF receptor was joined to the DNA sequence encoding a constant region of the heavy human immunoglobulin chain, which region is joined to a hinge sequence. . Plasmid pSDL110 was digested with Eco RI and Hind III to isolate a 1.65 kb PDGF-R fragment. Plasmid pICHuy 1M was used as the source of the constant region of the heavy chain and hinge region. Plasmid pICHuy 1M comprises 54 ca. 6 kb Hind III-Xho-1 fragment of a human gamma-1-C gene subcloned into pUC19 which had been linearized by digestion with Hind III and Sal I. Plasmid pICHuy 1M was digested with Hind III and Eco RI for isolating a 6 kb fragment encoding the constant region of the heavy human chain. Plasmid plC19H was linearized by digestion with Eco RI. The 1.65 kb PDGF-R fragment, the 6 kb heavy chain constant region fragment, and the linearized pLC19H were joined by a tripartite ligation. The resulting plasmid, pSDL111, was digested with Barn H1 to isolate the 7.7 kb fragment. Plasmid ppPRE8 was linearized with Bgl II and treated with calf intestinal phosphatase to prevent self-ligation. The 7.7 kb fragment and the linearized ppPRE8 were joined by ligation. A plasmid containing the insert in correct orientation was designated pSDL113 (Figure 8).

E. Cotransfection with PSDL113 and plCO05V | .HuC |, -Neo

Plasmid pSDL113 was linearized by digestion with Cla I and used for cotransfection together with ρΙΟΦδν ^ ΗυΟ ^ -Νβο, which encodes a neomycin resistance gene and a mouse / human chimeric immunoglobulin gene. The light immunoglobulin chain mouse gene was isolated from a lambda genomic DNA library constructed from a mouse hybridoma cell line, NR-ML-05 using oligonucleotide probes designed to extend over the VL / JL junction site. The human immunoglobulin light chain gene was isolated from a human genomic library and an oligonucleotide probe encoding a published human kappa C gene (Hieter et al., Cell 22: 197-207, 1980). The mouse immunoglobulin heavy chain gene was subcloned from the original mouse genomic phage clone into p1C19R as a 3 kb Xba 1-Hinc II fragment. The human kappa C gene was subcloned from the original human genomic phage clone in pUC19 as a 2.0 kb Hind III-Eco 30 R1 fragment. The chimeric kappagen gene was generated by joining the mouse kappagen gene and human kappagen gene via the common Sph I site and incorporating them together with the E. coli neomycin resistance gene in plC19H to generate plC <t> 5V | <HuC | <-Neo .

SP2 / 0-Ag14 cells were transfected with linearized 5 pSDL113 and pICOSV ^ HuC ^ -Neo by electroperforation. The transfected cells were selected in growth medium containing methotrexate and neomycin. Media from drug resistant clones were studied for their ability to bind PDGF in a competitive binding assay.

10 F. Cotransfection with PSDL113 and PSDL114

Plasmids pSDL113 and pSDL114 were linearized by digestion with Cla I. The linearized plasmids p416, which comprises a DHFR expression unit, are cotransfected by electroperforation. Transfected cells were selected in growth medium containing methotrexate. Media from drug resistant colonies were examined for ligand binding.

Example 13: Methods of Analysis: A. Preparation of Nitrocellulose Filters for Colonial Elves

Transformed cell colonies were examined for secretion of PDGF-25 receptor analogs by first culturing the cells on nitrocellulose filters which were placed on top of a solid growth medium. Nitrocellulose filters (Schleicher and Schuell, Keene, NH) were placed on top of a solid growth medium and allowed to become completely moist. Test colonies were stained on the wetted filters and grown at 30 ° C for approx. 40 hours. The filters were then removed from the solid medium and the cells were removed by four consecutive Westem transfer buffer rinses (Table 4). The nitrocellulose filters were soaked in Western buffer A (Table 4) for one hour at room temperature on a shaker table with two buffer shifts. Secreted PDGF-R analogs were visualized on the filters as described below.

5 B. Preparation of Protein Blot Filters

A nitrocellulose filter was soaked in Western Buffer A (Table 4) without the gelatin and placed in a "Minifold" (Schleicher and Schuel, Keene, NH).

Five ml of culture supernatant was added without dilution to the "Minifold" wells and the liquid was allowed to pass through the nitrocellulose filter by gravity. The nitrocellulose filter was removed from the "Minifold" apparatus and soaked in Western buffer A (Table 4). one hour on a shaking table at room temperature. The buffer was changed three times during the one hour incubation.

15 C. Preparation of Westem Blot Filters

The transformed cells were analyzed by Western blot, which was largely performed as described by Towbin et al. (Proc. Natl. Acad. Sci. USA 76: 20 4350-4354, 1979) and Gordon et al. (U.S. Patent No. 4,452,901). Culture supernatants from appropriately grown transformed cells were diluted with three volumes of 95% ethanol. The ethanol mixtures were incubated overnight at -70 ° C. The precipitates were centrifuged out of solution in an SS-24 rotor at 18,000 rpm. 20 minutes. The supernatants 25 were poured off and the precipitated cells were resuspended in 200 µl dl-O.

To each sample, 200 μΙ 2 x loading buffer (Table 4) was added and the samples incubated in a boiling water bath for 5 minutes.

The samples were electrophoresed in a 15% sodium dodecyl sulfate polyacrylamide gel under non-reducing conditions. The proteins were electrophoretically transferred to nitrocellulose paper under conditions described by Towbin et al. (Ibid.). The nitrocellulose filter was then incubated in Western Buffer A (Table 4) for 75 minutes at room temperature on a rocker table.

D. Processing the filters for visualization with PR7212 5

Filters treated as described above were tested for proteins bound by a PDGF receptor-specific monoclonal antibody, designated PR7212. The filters were removed from Western buffer A (Table 4) and placed in sealed plastic bags containing a 10 ml solution containing 10 10 µg / ml monoclonal PR7212 antibody diluted in Western buffer A. The filters were incubated on a rocker table overnight at 4 ° C or for an hour at room temperature. Excess antibody was removed by three 15 minutes of washing with Western buffer A on a shaking table at room temperature.

To the filters, 10 µl of biotin-conjugated equine antibody antibody (Vector Laboratories, Burlingame, CA) was added in 20 ml of Western buffer A. The filters were again incubated for one hour at room temperature on a rocker table and unbound conjugated antibody was removed. with three 15 minutes wash with Western buffer A.

The filters were preincubated for one hour at room temperature with a solution containing 50 μΙ of Vectastain Reagent A (Vector Laboratories) in 10 ml of Western Buffer A, which had been allowed to incubate at room temperature for 30 minutes before use. The filters were washed with a quick wash with distilled water followed by three 15 minutes of washing with Western buffer B (Table 4) at 25 room temperature. The Western Buffer B washes were followed by a distilled water wash.

During the above washing step, the substrate reagent was prepared. Sixty mg of horseradish peroxidase reagent (Bio-Rad, Richmond, CA) was dissolved in 20 ml of HPLC grade methanol. 90 ml of distilled water was added to the dissolved peroxidase followed by 2.5 ml of 2 M Tris, pH 7.4, and 3.8 ml of 4 M NaCl.

DK 176057 B1 58

Immediately before use, 100 μΙ of 30% H2O2 was added. · The washed filters were incubated with 75 ml of substrate and incubated at room temperature for 10 minutes under vigorous shaking. After 10 minutes of incubation, the buffer was changed and the filters were incubated for a further 10 minutes. The filters were then washed in distilled water for one hour at room temperature. Positive was scored as the samples that showed staining.

E. Treatment of filters for visualization with an antibody against substance P

Filters treated as described above were tested with an antibody to substance P. The filters were removed from Western buffer A and rinsed with Western transfer buffer, followed by a 5 minute wash in phosphate buffered saline (PBS, Sigma, St. Louis, MO). The filters were incubated with a 10 ml solution containing 0.5 M. 1-Ethyl-3,3-dimethylaminopropylcarbodiimide (Sigma) in 1.0 M NH 4 Cl for 40 minutes at room temperature. After incubation, the filters were washed three times in PBS for 5 min. wash. The filters were blocked with 2% dry milk diluted in PBS.

The filters were then incubated with a monoclonal rat antibody against Substance P (Accurate Chemical & Scientific Corp., Westbury, NY). Ten μΙ antibody was diluted in 10 ml antibody solution (PBS containing 20% fetal calf serum and 0.5% Tween-20). The filters were incubated at room temperature for one hour. Unbound antibody was removed with four 5 minute washes with PBS.

The filters were then incubated with a biotin-conjugated rabbit anti-rat peroxidase antibody (Cappel Laboratories, Melvem, PA). The conjugated antibody was diluted 1: 1000 in 10 ml antibody solution for 2 hours at room temperature. Excess conjugated antibody was removed by four 5 minute washes with PBS.

30 59 DK 176057 B1

The filters were preincubated for 30 minutes at room temperature with a solution containing 50 µl of "Vectastain Reagent A" (Vector Laboratories) and 50 µl of "Vectastain Reagent B" (Vector Laboratories) in 10 ml of antibody solution which had been allowed to incubate in 30 minutes before use. Excess Vec-5 tastain reagents were removed by four 5 minute washes with PBS.

During the above washing step, the substrate reagent was prepared. 60 mg of horseradish peroxidase reagent (Bio-Rad) was dissolved in 25 ml of HPLC grade methanol. Immediately before use, approx. 100 ml PBS and 200 μΙ 10 H2O2. The filters were incubated with the substrate reagent for 10 to 20 minutes.

The substrate was removed by vigorous washing with distilled water.

While the above invention has been described in some detail by way of illustration and examples for a clearer understanding, it is clear that certain modifications and modifications may be made within the scope of the appended claims.

Claims (11)

A method of producing a secreted biologically active peptide dimer, characterized in that a DNA construct is introduced into a host cell capable of directing the expression and secretion of a biologically active peptide dimer, which DNA construct contains a transcription promoter operably linked to at least one secretion signal sequence that is followed downstream and in proper reading frame by a DNA sequence encoding a peptide which requires dimerization for biological activity and fused to a dimerization protein, that the host cell is grown in a suitable growth medium under conditions which allow dimerization and secretion of the peptide dimer, and that the peptide dimer is isolated from the host cell. 15 Process according to claim 1, characterized in that the host cell is a yeast cell which carries a defect in a gene whose product is necessary for the addition of outer chain oligosaccharide units to glycoproteins. Method according to claim 1, characterized in that the host cell is a yeast cell which contains a genetic defect in the MNN9 gene or an interruption in the MNN9 gene. Method according to claim 1, characterized in that the secretion signal sequence is selected from the group consisting of the MFa1 pre-pro sequence, the PH05 signal sequence, the BAR1 signal sequence, the SUC2 signal sequence, the PDGF receptor signal sequence and the mouse immunoglobulin Vj_ sequence. . DK 176057 B1 The method of claim 1, characterized in that the dimerization protein comprises at least a portion of a protein selected from the group consisting of a light immunoglobulin chain, a heavy immunoglobulin chain and yeast invertase, wherein this moiety associates as a dimer on a covalent or non-covalent moiety. covalent manner. The method according to claim 1, characterized in that the dimerization protein is selected from the group consisting of a hinge region of a heavy immunoglobulin chain and yeast invertase. The method of claim 1, characterized in that the peptide requiring dimerization for biological activity is selected from the group consisting of a polypeptide comprising the amino acid sequence shown in Figure 1 from iso-leucine # 29 to lysine no. 531, and a polypeptide comprising the amino acid sequence shown in Figure 1 from isoleucine # 29 to methionine # 441. Method according to claim 1, characterized in that the host cell is a yeast cell or a cultured mammalian cell. 20 DK 176057 Bi »oo ouou oo o υ r- moh γ- o ί · ο η Η οο ο in <Η ο ιη ι— Ο Η U οι Ο (Νι <ο Ο Ο Ο Η Ο Ο Η Ο < Ο Ο Ο <. <, Rj · Ο co Ο οι Ο ID Ο <Η Ο <Ο Ε-ι ιη {Η Η U νο <o Ε π ε <ο in <fi ιηυσ ο ο ο <u ε- <u <υ ο ο <Ο ο <ου Ο ξ-ι OU OO U * J Ο> Uhl UCU φ Ο ΓΟΟ οο r- ο <τ <c <HU (Νι <in ο Ο Ο Ο Ε-ι U ι UOU Ο-ι> <Ε-ι Eh UU Ο Ο <<OW ο 0> U (¾ ο ο <ο <<-ι υ <uinu cn u ου r> o UU Ο Ο Ο σ \ O -3 · 0 ο Ε-ι in «C <Ο Ε-Ι Ο <ο Ο Ο -« tOO ιη Η m Ο W ο <υ ο <* £ Ο U Ο OriJoCoUOE-iOO <<U <ιη Ο ο Ο σ% Ο ou ο C ΟΟ »<Εη ΟΟ» Ο Η U · Η <CJ »£ ΟΟ υ ο o <υ υ η ο ο <Ο Εη <Ε-» ο ο U <<<<C ο Ο ^ Ε-ι CO << W Eh W. Υ o υ υ <U Ε-ι Ε-ι Ε-Ι U ιο O ΟΟ · ίΟ co ο Ο Ε-ι Ο 0 C0 Ο «ί-ι σι O ν Η ο <u ε-ι ο ο υ < ττ <μ -ΰ-ε-'ΐΛ Ο Εη οο Ο U Ο Εη ΟΟ Ο U ο -r υ Η οο Ο ιη Εη (Μ Ε-ι Ε-ι <Επ Ο U HU «Η <(Μ Ο οΟ ο »-} ο ο ο> υ« U Ο Ο Επ Ε- · Ο Ε- · Ε- · Ο Ο Ε- <<Ε-ι <UE-iU <KCU Ο <<Εη U Ο Ο ^ 0 < ΟΡ <OW υ ο ε-ι ο ο Ε- · Ο <Ο Γ ~ <η <£ ιη Ο σι Ο Ε- <<Ε-ι Ο-ι Ο ο <co Ε-ι ο ο OA - «Row xrOJ ιη Ε-ι 3: ς.-) ΟΟ ΟΟ ΟΕ-ι OU OO„ οΕ-ι ΟΟ r'-U - <r <HU Ο Ο Ο Ο „fc? U Η γΙΒ <Νθ οο £ η ε-ι Ε-ι Η Γ Ί <U Ο <<Χ Οι4 0> 0> W- / Ο Ο-Ε ο ο · <U Ο Ε-ι << Ο Ε -ι Μ Ο OUUUOU Ε-ι Επ Ε-ι Γ_, UUU Ε-Ι U 0 * <UiJ UiJ ο> bid <Ε- · Ε- · U Ε-ι UU <<Ο C0 Ε-Ι CM. <ιο Ο ΟΟ Ο ο ο Εη Γ- «<oU oo ou ou ο u ο Ε- <ο ο n · Εη ^ ^ ο W in u Ο. οι <σ \ Ε-ι ou ου οθ U UrKE-injUoC Ο Ο <Ε-ι υ ο υ. <ο υ εη ο υ υ ε- · υ ε-ι ε- · up * υ u λ ο <υ ο ε-ι ε-ι ο Ε-ι Ε-ι Ε-ι Ο Ο-ι Ο ο <uooehoe-i Ε-ι ο υ ο υ u ο ο »-3 ο ^ οο ε-ι w Ε Ι ο σ σ H H H fi fi fi fi fi fi fi fi fi H H E E E E E E E E E E E E E ΗΟ ο Ε-ι οιο Ο Εη <U Εη Ο Ο Ο Ο <υ uc υ eh Eri ο υ υ ε- · ο Ε-ι υ * ί s ο ^ op «tun eh Ο <U Erl Ο Ο h Eh Eh OO <O UOCE-iOO Eh OOUU Ε-1 Ο <<E - 3 <E- * f- · U DK 176057 B1 σι in η μ σ> co o cm *} · vo ov Η «Η H pH Η« Η OO hJ · C CO O CM E- · Ό CJ O Eh OI cm <co cm Eh co U n C σ> Eh * r O (vo CZ Ό o O r- O> C Eh co OX co Eh ft cn C ft U Eh ucouu UOO Eh Eh o CC Eh O «C EH CM OQ OO OO OOOCC EH O Eh OOCOOO UO Eh CO U ft OW C Eh U ft OO rH <tnU σι Eh n O t ^ OHC in Eh rHU VO < C, rH C t "U CM H CO CO fn vo <Eh vo o Q r ~ OQ r · 'C Eh æ o> to O σ σ \ CMU eh CUOOU Eh, CU Eh Eh CU UO CZ U ft UO 0> UK C EH OUO Eh O Eh U ·, Eh <Eh Eh Eh CU 0> UK 0> Eh ft UO OQ C Eh NO OUO Eh htU coO csøh Ό C Ho o in U rH Eh vo Eh rH <r> C cm < . vo C co vo <Eh C 'Eh ft n- C M co U O coEh> h σι C Z U U U U C U U Sf \ U O Eh E-. u UO SJJ Eh CO Eh U cm EHft Uft Uft EhU l · ·· U EH ucuuu U— Eh Eh C £ h C Eh Eh Enft Eh ft £ h> h UO OQ 0> CM On UU mu in C cr> C n Eh r ^ U σ \ U nr C o Eh in C OU vo CJ rH C i · in <Eh vo Eh> hr ^ UO ι> · OW co C Eh ω U ft cn Eh> h UCOOCOU _, OCOC Eh Eh O f = l OO O «Uft OW 0> UO CCO rfc? EH CCUCCCCJCOCUOUO OQ OO CZ OC Uft OC Cft * 1 -vr O co O cm CJ vo E-π OU tt Eh co U Γ Ύ. co c nU σι O vc OO in Eh OC 1 == 1 in U α vo C Eh fold t ^ OQ co EU coO> cn OQUUO Eh CUO u CCC u CC OC OQ OW CZ Uft OQ OW OC Eh Eh Eh O Eh C Eh CU Eh O Eh CZUQ OQ Uft CM OO Enft mu σι o nu r ^ U hu in C σι ur ~ U cm O co U n Eh cn UC σι Eh in OC vo o O vo C Eh f'UQ r> C Eh co CZ co CMO - Eh OUUO Eh Eh UC Eh Eh CO CS C Eh OW Enft CH CZ OO CUOUOO Eh C Eh Eh OCCU OW uJ u Q oo ow ow e- * co I · DK 176057 B1 in co rH σ \ c ^ in co H «-HeoincocoocMM · CM CM CM cm cm co ro m I CO O <MU CO O Ofi M · O 00 U CM O CO O σι r £ in B oo co u rH o voo cm o r- <σι * t'Z. 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OOOUUOO <H <U <UU <2: O> OW HW UX Udo Udo PJ U fold O <TfU CO U ro o OO h <co <0 now H cm U to <m H Tf o Q Tf «< ice in o <in U ni io <to 10UO OW Η Η H rH i — I rH HH <uooouo <OH <CUC ^ xu Pi o> ua ow up <ow UOOUHOH SjJ IH U <4 Η OHO i HW * ί H < Ui 0> <W rtIS UK 1__o O o- U HO in O cn <C r »U r- <ΓΓ oo m U HH O <HH Γ 'O CN u O · * · Tf HS Tf eh cn in O> mow vo UW vo o O r ^ UW H t— («—C c — l« -) «—l <—l 1 OOU Η OOH i. Η H O U <O O UL O W O W U Pi 0 <OW O «HU --1 U U. Η H O U O • - 'H U H <H O O Co o> o <o> oa UW OO <Di J' Tf Eh OOU cn O vo o oo Tf H CO <<. i_i cn u Tf h oh in <hh o o rH * c · t-1 o C H Tf <m in U W mow «ο O> o o X Γ '<! X Γ— Η H rl r ~ IH ri rH fc ± = 3 U <H <H u U * ϊ O <OOH UCU ow «<HUX UPi HU UW ououuuu UO Η Η HO <uh oo ow hw o> upi oa 1 m <cn H r »o r- U hh mo cn <co un U cn <TfU o U m H oo n UP« Tf cj <Tf o W mo <«ou P« ιοθ> γ- <X Η Η Η H rH H oa <ou <o <OUOHUO Hi * <Ui HWU Pi 0> # <H HU UOOOHOUUU Η H <<U u <F-tj o> <x <zo <yoci DK 176057 B1 in r> m cn mom 00 cg ΓΟ m C- CTv iH • g · in in in in in in 10 ro O cg <10 U OO Tt O oo U c \ j O io Eh o C vocj H Ec Γ'Η CMC <r> o oo Eh . co O W co O W cn O> σι O W OOO O O Q M Eh S H 1 Μ Η Η H OICNCNCN OOOOOEhOO <0 <Eh0000 OW OW OW <M E-C S Eh CO <Eh O C. 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Eh W «Cm <m 0> OW cg O 100 OO u · O to O n O id O 00 Γ '« C cg Eh oo O co O co O -g · cn <in <t ^ ow 00 O> co < Eh σι Eh to cn <Eh oow o Eh> h riOCX H rH rH Η H CO (N (S oooououo << 0 <EH <EH <C OW OW HO OK OW EH i * <M OQ <EhO <OEhOO <Eh O O Eh O <O OW Eh W OW OW 0> OW Eh> h OW cn O rO ho in o cn on <r ^ O mo OO Η <Γ- * Eh n Eh r * Eh n O co <U | n Eh W co OW co O> σι O> σι O> OOW OOW MOW MMHMHrgcgcg Eh OOOOO Eh OO <O Eh Eh «£ <Eh <CO OCX Eh W 0> OW <« OK OW OOOOOOOO <<Eh Eh O <O << 2; OW OQ <£ i — 1 o <OO OW DK 176057 B1 σ r- ^ in ro <H σι cm -m · \ o co o H n vo vo vo vo vo t "r ~ OO xr U eo O (MU o O o E-ι -a · U * r Eh cn U m · Eh o «<in Eh hu io <cm <S cm O << no>> c · H ^ in o <in o Q (M CM CM CM CM CM CM O <O o O Eh o O 2 Eh <Eh 2_ OC Eh UOO> OW Eh A <Z <W. UOU Eh UOOU rt! * £. OOO <, O «<2« iz <C EH C Eh <w OW.-C O in O o \ u ro tJ t "O hu in ϋ ro <co * £ n Eh σ \ Eh tti <o <C in ΰ cm U O MOW n U hl o <H if OK m Eh> i in o O CM CM CM CM CM CM CM Eh Eh U U rtl U U O O <Eh 2 Eh O EhW <W Ο X << M 2 X U W <Eh. Eh OO Eh OOO <OO <<EH UX <W UA Eh> h 2 χ OW EhW CM U FOLD OO Tf u 00 O CM O l £> O cm O Γ "O co O co Eh ro O σι O tt U cm <w cm UX co OO η <H mOK o <in Eh w CM CM CM CM CM CM CM <H, OU EH UUUO Eh U Eh U <O Eh OWO <OW UA UX <C0 o> U <U <OU Eh OU <O Eh O * £ l // OO <EH UX OO OW UA ux ro Eh r ^ U rH Eh in C cn O ro O t ^ U ju- rlC iDU cm O (tO <MC co U ro O 11 cm UX <M EH CO co <C CO ro OO * r Eh X - ^ r UA in <co CM ΓΜ CM CM CM CM CM Eh <. O <UUUU 2 Eh 2 <OOII o <2x <s 2x OOU «UA O EH UUOUO t = | U EH EH U Eh o eh <Eh UW <H <Eh 0> U «UW 7. 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