NZ246256A - Methods of production of purified, soluble of beta-pdgf (platelet derived growth factor) receptor fragments - Google Patents

Description

New Zeala Zo4 O 5 6 International No. PCT/US92/10430 TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION Priority dates: 3L- *2.' °i\ Internationa! filing date: v- *^"2.

Classification: c\2.P3- \ csz.

Publication date: 2 6 JAN 1996 Journal No.: II+.00 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of invention: METHODS FOR PRODUCTION OF PURIFIED SOLUBLE TYPE B AND TYPE A HUMAN PLATELET-DERIVED GROWTH FACTOR RECEPTOR FRAGMENTS Name, address and nationality of appiicant(s) as in international application form: COR THERAPEUTICS, INC. of 256 East Grand Avenue, Suite 80, South San Francisco, California 94080, United States of America. liFollalwrJ lov M) i A METHODS FOR PRODUCTION OF PURIFIED SOLUBLE TYPE B AND TYPE A HUMAN PLATELET-DERIVED GROWTH FACTOR RECEPTOR FRAGMENTS BACKGROUND OF THE INVENTION The present invention relates generally to methods for producing soluble platelet-derived growth factor receptor 10 fragments. More particularly, it provides cell lines and methods using those cell lines, allowing for highly efficient production of purified platelet-derived growth factor receptor soluble fragments exhibiting ligand binding functions.

Polypeptide growth factors are mitogens that act upon 15 cells by specifically binding to receptors located on the cell plasma membrane. The platelet-derived growth factor (PDGF) stimulates a diverse group of biochemical responses, e.g., changes in ion fluxes, activation of various kinases, alteration of cell shape, transcription of various genes, and 20 modulation of enzymatic activities associated with phospholipid metabolism. See, e.g., Bell et al. (1989) Circulation Research 65:1075-1065. The platelet-derived growth factor is a polypeptide factor which interacts with a membrane bound receptor, the platelet-derived growth factor receptor (PDGF-R). 25 The receptor has a binding site which binds the PDGF ligands.

Particular medical conditions result from abnormal receptor-ligand interactions. The specificity of binding allows the use of one binding partner to determine, qualitatively or quantitatively, the presence of the other, and 30 to detect abnormal interactions. These diagnostic reagents would be useful.

Receptors for platelet-derived growth factor have been expressed in various cells. See, e.g., Orchansky et al. (1988) J. Biol. Chem. 263:15159-15165; Duan et al. (1991) 35 Biol, chem. 266:413-418; Heidaran et al. (1990) J. Biol. Chem. 265:18741-18744; Claesson-Welsh et al. (1988) Mol. and Cell. Biol. 8:3476-3486; and Escobedo et al. (1988) J. Biol. Chem. 263:1482-1487.

WO 93/11223 PCT/US92/10430 246256 Although others have reported expressing PDGF receptors or fragments thereof in cells, the ligand binding fragments have all been of approximately intact extracellular regions of the receptor. In many instances, a smaller and soluble ligand binding segment would be useful. For example, a soluble ligand binding fragment may serve as an antagonist to modulate the effect of PDGF ligands. Antagonists which are soluble and smaller than the original receptor will be useful. The physiological bioavailability of small soluble antagonists 10 will be better than the natural intact receptor. The intact receptor is a membrane bound protein and would "typically not circulate in the blood. Soluble antagonist fragments, e.g., which are shorter than the native receptor binding site, will typically also be produced in greater quantities at lower cost. 15 Moreover, a smaller soluble peptide is more likely to be capable of reaching remote and circulation compromised regions of the body.

Thus, a need exists for a highly efficient means for producing a purified ligand binding region, and for soluble 20 molecules which have PDGF binding activities. Fragments smaller than the intact extracellular region of each receptor are desired. Economical and high efficiency production of PDGF ligand binding proteins, e.g., fragments containing critical ligand binding regions, is greatly desired. The present 25 invention provides these and other needs.

SUMMARY OF THE INVENTION The present invention provides an immortalized mammalian cell line which expresses a soluble peptide fragment 30 which binds a platelet-derived growth factor (PDGF) ligand, said fragment consisting essentially of from one to three contiguous domains of a PDGF ligand binding region derived from a platelet-derived growth factor receptor (PDGF-R) extracellular region sequence, said domains being selected from 35 the group consisting of Dl, D2 and D3. 246256 2a In a further aspect the present invention provides a pharmaceutical composition comprising a protein produced from a cell line of Claim 1 together with a pharmaceutically acceptable carrier or diluent. provides a method for preparing a soluble polypeptide exhibiting human platelet-derived growth factor receptor binding activity comprising the steps of: a) growing a cell line of Claim 1; and b) isolating said cell product from said cell line.

In a still further aspect the present invention wo 93/1,223 zTgIFS BRIEF DESCRIPTION OF THE FIGURES Fig. 1 illustrates a strategy for oligonucleotide directed in vitro deletion mutagenesis of soluble hPDGF-R extracellular domains. Many of these constructs will be soluble peptides, or can be modified to be such. The abbreviations used are: PR = PDGF-R; intact P = PDGF—R; extracellular region TM = transmembrane 10 K = kinase S = signal sequence Fig. 2 illustrates the structure of a plasmid derived from pcDL-Sa296 used for expressing various deletion polypeptides.

Fig. 3 illustrates the structure of a plasmid pBJA derived from pcDLa296. See Takabe et al. (1988) Mol. Cell. Biol. 8:466-472, which is incorporated herein by reference. 1. The pcDL—SRa296 is cut with Xhol. 2. A polylinker (Xhol-Xbal-Sfil-Notl-EcoRI-20 EcoRV-Hindlll-Clal-Sall) is inserted into the Xhol cut vector. 3. Sail is compatible with the Xhol site; and generates both a Sail and an Xhol site. 4. The SV4 0 16s splice junction is no longer 25 present.

DESCRIPTION OF THE PREFERRED EMBODIMENTS In accordance with the present invention, novel cells for producing soluble polypeptides which bind platelet-derived 30 growth factor (PDGF) ligands and compositions comprising such fragments are provided. The subject cells contain nucleic acid sequences which encode desired polypeptide fragments. The cells will express the nucleic acids, thereby producing large amounts of the fragments. In preferred embodiments, the 35 fragments are usually secreted into the medium and available for purification. The compositions are capable of specifically binding to platelet-derived growth factor (PDGF) ligands with affinity and specificity.

WO 93/11223 PCT/US92/10430 The polypeptide fragments will find use as diagnostic and therapeutic compositions. For example, they will be useful as antagonists of PDGF ligands, or as reagents for gualitative or quantitative assay of a PDGF ligand. The cells may also be used in methods for preparing large quantities of binding polypeptides, e.g., as sources of starting material for purification processes.

Analogues of the PDGF protein are referred to as PDGF ligands. These ligands typically are agonists of the PDGF-R. 10 The PDGF ligands will usually be proteinaceous, but may be other compounds which exhibit structural features important in interaction with the receptors.

I. Cell lines The cells of the present invention are typically capable of stable cultured production of the PDGF ligand binding polypeptide. The cells will usually be eukaryotic, e.g., from a mammal which provides cells that are easily manipulated. Rodent cells, e.g., mouse, hamster or rat cells, 20 are preferred embodiments. Cells which will process or modify the soluble proteins in manners analogous to human proteins will be preferred. Glycosylation and other protein modifications are particularly important.

In some embodiments, the cells and DNA constructs are 25 derived from a human cell line. Especially preferred cell lines include the pAl-5 line, which contains an expression vector for producing a type B PDGF-R polypeptide fragment, and the pAaRF line, which contains an expression vector for producing a type A PDGF-R polypeptide fragment. Each of these 30 cell lines efficiently express the appropriate polypeptide fragments.

Novel cell lines are provided herein for expressing fragments of the extracellular region of the platelet-derived growth factor receptor. These fragments exhibit unexpected 35 properties, in part, because it has been discovered that deletions of various portions of the extracellular region of the PDGF-R do not affect ligand binding. Particular segments of the extracellular region of the receptor have been WO 93/11223 PCT/US92/10430 identified which do not significantly contribute to ligand binding affinity or specificity. Deletion of these extraneous segments of the PDGF-R extracellular region increase, the solubility of the shortened polypeptide and decrease its immunogenicity. Moreover, higher efficiency production and lower cost provide significant commercial advantages.

The cell products will typically be soluble fragments of human PDGF receptor polypeptides. Type B or type A receptors fragments will be produced. By virtue of their human 10 origin, the fragments introduced into human subjects should exhibit low immunogenicity and function as more effective therapeutic reagents. Their homology to natural human proteins should decrease the likelihood of adverse reactions and side effects from therapeutic administration. 15 The ligand binding regions (LBRs) are defined, in part, by their effect on the affinity or specificity of binding to PDGF ligands. The natural, native full length PDGF-R binds its natural ligand with a Kd of about 0.2 mM. See, e.g., Duan et al. (1991) J. Biol. Chem. 266:413-418, which is hereby 20 incorporated herein by reference. A ligand binding region is a segment of the polypeptide whose presence significantly affects ligand binding, e.g., absolute affinity and specificity. Affinity will usually beaffected by a factor of at least about two, typically by a least a factor of about four, more 25 typically by at least a factor of about eight, and preferably by at least about a factor of twelve or more. Measures for specificity are more difficult to quantitate, but will typically be evaluated by comparison to comparative affinity to ligands exhibiting similar structural features. 30 Preparation of mammalian cells of the present invention can be accomplished by standard methods of transforming many different types of mammalian cells with appropriate expression vectors. See, e.g., Ausubel et al. (1987 and supplements) Current Protocols in Molecular Biolocrv. 35 Greene Publishing/Wiley-Interscience, New York, which is hereby incorporated herein by reference. Proper selection of a combination of cellular properties and expression vector WO 93/11223 PCT/US92/10430 24 properties can lead to improved methods of producing the desired platelet-derived growth factor receptor fragment.

Various methods are available for expressing defined proteins at high level. Amplification methods similar to those using dehydrofolate reductase (DHFR) can be applied. See, e.g., Kaufman et al. (1985) Mol. Cell. Biol. 5:1750-1759.

Other well known high expression techniques will also be applicable.

These cells are particularly selected for high level 10 expression of desired receptor fragments. Cell lines resulting from transformation with vectors encoding the desired peptides are provided. Different isolates of transformants will have different copy numbers and integration sites resulting in differential expression levels. Favorable cell strains will 15 have integrated DNA in positions and numbers providing particularly high receptor fragment expression.

With respect to the constructs, PaI through Pa9 refer to DNA constructs of the type B receptor inserted into cloning sites in the expression vectors. The PaI construct inserted 20 into the pBJ vector is designated pBJPA. The vector is transferred into a specific cell background and various clonal transformants are isolated and designated, e.g., pAi-i, pAl-2, etc. Each clonal isolate differs from others by sequence copy number and integration sites, with resultant variability in 25 expression levels. The pAl-5 is a particularly useful cell line embodiment expressing high levels of type B ligand binding regions.

The pAaRF cell line expresses the entire extracellular region of a human type A PDGF receptor. The 30 encoding DNA sequence was introduced into a pBJ-1. CHO cells were transformed with the resulting DNA construct and selected for both expression of the neo plasmid and for production of a type A receptor fragment.

II. Methods The present invention also provides methods for producing the described fragments. In particular, cell cultures are available to express the nucleic acids described. 24 7 Usually, the fragments are secreted thereby considerably simplifying purification of the receptor fragments. The cells need not be disrupted and cellular contamination is minimized. Thus, the cells will be separable from the secreted products by physical techniques while allowing recovery of the intact cells. See, e.g., Ausubel et al. (1987 and supplements) Current Protocols in Molecular Biology. Greene/Wiley-Interscience, New York, especially section 10:Vii. will be susceptible to recovery from the medium. Various techniques will be available for separating the soluble proteins in the media from the cells, e.g., filtration or centrifugation. Cell cultures attached to solid substrates will be easily separable from the medium by filtration or centrifugation, while suspension cultures of fragile cells will usually be subjected to centrifugation. used, e.g., chromatography, centrifugation, precipitations, electrophoresis, immunoaffinity methods, and other techniques well known to protein chemists and enzymologists. See, e.g., Deutscher et al. (1990) Protein Purification, in Methods in Enzvmolocrv: and Ausubel et al. (1987 and supplements) Current Protocols in Molecular Biology. affinity reagents, e.g., either PDGF-ligand affinity columns or immunoaffinity columns. A PDGF-ligand affinity column will be readily prepared using a cloned PDGF-ligand sequence or analog for isolation of the protein product, and attachment to a solid substrate. An immunoaffinity column will be readily prepared by attaching immunoglobulins prepared against PDGF-R peptides, either produced by the cells of the invention, or by other methods. binding affinities of 106 M"1, preferably 107 to 1010, or stronger, will typically be made by standard procedures as described, e.g., in Harlow and Lane, Antibodies: A Laboratory Manual. CSH Laboratory (1988); or Goding, Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press, Usually, the soluble proteins will be secreted, and Standard methods for protein purification will be Particularly useful purification reagents include PDGF-receptor specific monoclonal antibodies with WO 93/11223 PCT/US92/10430 • 8 2482! New York (1986), which are hereby incorporated herein by reference. Briefly, appropriate animals will be immunized. After the appropriate period of time, the spleens of such animals are excised and individual spleen cells fused, typically, to immortalized myeloma cells. The fusion products are subjected to appropriate selection conditions and clonally separated. The supernatants of each clone are tested for antibody specific for binding the desired region of the antigen.

Other suitable techniques involve in vitro exposure of lymphocytes to the antigenic polypeptides or alternatively to selection of libraries of antibodies in phage or similar vectors. See. Huse et al. (1989) "Generation of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage 15 Lambda," Science 246:1275-1281; and Ward et al. (1989) "Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli." Nature 341:544-546; each of which is hereby incorporated herein by reference. The polypeptides and antibodies of the present invention may be 20 used with or without modification.

The appropriate affinity reagent, e.g., the PDGF or antibody, will usually be attached to a solid substrate.

Typical substrates include chromatography matrices, e.g., CNBr-sepharose, glass beads, and plastics. Usually, the attachment 25 will be covalent, though non-covalent attachment methods may, under certain conditions, be sufficient.

Once the appropriate affinity reagent, or combination of affinity reagents is prepared, solutions containing the soluble fragments will be passed though the affinity reagent 30 for specific binding and elution. These steps may be interspersed with other purification or concentration steps. A particularly useful fragment purification method incorporates an immunoaffinity substrate comprising monoclonal antibodies. The receptor fragments are attached to immobilized antibodies 35 in the column and eluted at high pH. There may also be incorporated additional steps for specific removal of identified contaminating components, e.g., by affinity removal techniques.

WO 93/11223 Q"71^92^!04^ Isolated soluble peptide fragments may be used for various purposes, e.g., as diagnostic reagents or for therapeutic reagents. The cells will be useful for transfer into an animal, e.g., into a mouse, or for culturing in an 5 appropriate implantation container, which will allow for diffusion of the product into the body of a host organism. Appropriate cell culturing apparatuses which can be implanted into a subject to secrete therapeutic cell products are described, e.g., in U.S. Pat. No. 4,806,355; 4,402,694; and 10 3,093,831.

EXPERIMENTAL In general, standard techniques of recombinant DNA technology are described in various publications, e.g., 15 Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory; Ausubel et al. (1987) Current Protocols in Molecular Biology, vols. 1 and 2 and supplements; and Wu and Grossman (eds.) (1987) Methods in Enzvmologv. Vol. 53 (Recombinant DNA Part D); each of which is incorporated 20 herein by reference.

I. Human Extracellular Region Equivalent techniques for construction, expression, and determination of the physiological effect of truncation or 25 deletion analogues of the soluble extracellular receptor fragments from the human receptor may be performed using the nucleic acid, polypeptide, and other reagents provided herein.

A. Type B Segments 30 Constructs of type B receptor polypeptides were made as follows: The 3.9 kb EcoRI-Hind III cDNA fragment of the human type B hPDGF-R was subcloned into the EcoRI-Hind III site of M13 Mpl8 to produce a vector Mpl8PR. For techniques, see 35 Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, N.Y., which is incorporated herein by reference. Verification of subcloning was performed by restriction enzyme digestion analysis and dideoxy chain termination sequencing, as described by Sanger et al. (1977) Proc. Nat'l Acad. Sci. USA 74:5463. Oligonucleotide directed in vitro mutagenesis was performed according to the method described by Kunkel et al. (1987) Methods in Enzvmol.. 154:3 67. The strategy for oligonucleotide directed in vitro deletion mutagenesis of MplSPR is outlined in Fig. 1. to create a nested set of soluble type B hPDGF receptor extracellular regions by deletion mutagenesis. Intact domains have been deleted. These domains are designated Domain 1 through Domain 5 (D1-D5), suitable for expression in an appropriate eukaryotic expression system. Exemplary mutagenic oligonucleotides are listed in Table l. These oligonucleotides are complementary to regions of the human PDGF receptor spanning various defined IgG-like domains. See, e.g., Williams (1989) Science 243:1564-1570, which is hereby incorporated herein by reference.

In brief, a series of oligonucleotides were designed PCT /US92/10430 11 24 TABLE 1 m HUMAN B-type PDGF-R MUTAGENESIS OLIGOMERS [note that these are complementary; given 3'-5'] pal 31-GTG TGA GGA ACG GGA AAT TCA TCG AAG GAC ATC CCC CGA-5» (SEQ ID 10 NO:1) a pa2 3'-GGA AGC TGG ATG TCT AGT TAA TCG AAG GAC ATC CCC CGA C-5 • (SEQ ID NO: 2) Pa3 3'-TAG TGG CAC CAA CTC TCG CCG ATC GAA GGA CAT CCC CCG AC-5 • (SEQ ID NO: 3) '0 Pa4 3'-ATG TCT GAG GTC CAC AGT AGG ATC GAA GGA CAT CCC CCG AC-51 (SEQ ID NO: 4) Pa5 "■ 3 • —GAG ATG TAG AAA CAC GGT CTA GGG ATC GAA GGA CAT CCC CCG AC-5' (SEQ ID NO:5) Pa 6 3 • —GTC TAG AGA GTC CCG GAC CAG TGG CAC CCG AAG GAG GGA TTA GTA-5 ' (SEQ C ID NO:6) Pa7 ^3' -GTC TAG AGA GTC CCG GAC CAG TAG TTG CAG AGA CAC TTG CGT CAC GTC-5 • (SEQ ID NO:7) Pa8 3 * —GTC TAG AGA GTC CCG GAC CAG ATG CAC GCC GAG GAC CCT CTC GAC-5 • (SEQ ID NO:8) • Pa9 3'-GTC TAG AGA GTC CCG GAC CAG CAG GCT CAC GAC CTC GAT TCA-5' (SEQ ID NO: 9) wo 93/11223 ^CT/L|92/W43fl (0 12 w £ j The resulting constructs are labeled as indicated in Table 2. The antisense strand was used for mutagenesis throughout. Mutagenesis of PaI, Pa2, Pa3, Pa4, and Pa5 utilized Mpl8PR as the template and mutagenesis of Pa6, Pa7, 5 Pa8, and Pa9 utilized Mpl8 PaI as the template. PaI, a 41 bp oligomer, introduced a TAG stop codon after Lysine49g (K4g9) of D5 and removed the transmembrane (TM) as well as entire intracellular kinase domain (K), producing an Mpl8 PaI (see Fig. 1).

TABLE 2 HUMAN TYPE B PDGF-R EXPRESSION CONSTRUCTS Soluble Membrane Bound pBJPR pBJPAl PBJPA2 PBJPA3 PBJPa4 pBJPA5 pBJPA6 PBJPa7 pBJPA8 pBJPA9 The human PDGF receptor constructs were subsequently subcloned into the EcoRI-Hind III site of pBJl a derivation of pCDL-SRa296, as described in Takabe et al. (1988) Molec. Cell Biol. 8:466, and co-transfected with pSV2NE0, as described by Southern and Berg (1982) J. Mol. AppI. Gen.. 1: 327, into Chinese hamster ovary cells (CHO). See. Figs. 2 and 3.

Function of the purified or semipurified constructs was demonstrated as follows: A sample of 0.3 3 nM PDGF BB ligand is preincubated 10 for 1 hr at 4°C under the following conditions: 1. a polyclonal antibody to human PDGF (this antibody recognizes human PDGF AA, PDGF BB and PDGF AB); 2. 18 nM (60 fold molar excess to PDGF BB) human type B PDGF receptor; 3. phosphate buffered saline solution that the receptor and antibody are in; or WO 93/11223 - rs-f/US92/10430 «- zVi 4. no additions but the ligand itself.

In a duplicate set of experiments, 0.33 nM PDGF AA is incubated with three of the above preincubation conditions, e.g., 2, 3, and 4 above. The human type B PDGF receptor does not appreciably recognize PDGF AA but this ligand will still activate cell-associated human type A PDGF receptor from NIH3T3 cells and so is a control for human type B PDGF receptor specificity and PDGF BB-dependent activation versus nonspecific general cellular effect, e.g., cytotoxicity. 10 The preincubated materials were in a final volume of 0.5 ml. They were placed in one well each of a six well tissue culture dish containing a confluent layer of serum starved (quiescent) NIH3T3 cells which were chilled to 4°C. The cells and incubation mixtures were agitated, e.g., rocked, at 4°C for 15 2 h. They were then washed twice with 4°C phosphate buffered saline. Forty (i 1 of 125 mM Tris(hydroxymethyl)amino methane (Tris), pH 6.8, 20% (v/v) glycerol, 2% (w/v) sodium dodecyl sulfate (SDS), 2% (v/v) 2-mercaptoethanol, and 0.001% bromphenol blue, (known as SDS sample buffer), was added per 20 microtiter well followed by 4 0 /xl of 100 mM Tris, pH 8.0, 3 0 mM sodium pyrosphosphate, 50 mM sodium fluoride, 5 mM ethylenediaminetetraacetic acid (EDTA), 5 mM ethylenebis(oxyethylenenitrilio)tetraacetic acid, 1% (w/v) SDS, 100 mM dithiothreitol, 2 mM phenylmethylsulfonylfluoride 25 (PMSF) , and 200 fiM. sodium vanadate was added to the cells. The cells were solubilized and 40 /xl additional SDS sample buffer was added to the solubilizate. This material was boiled 5 minutes and loaded onto a single gel sample well of a 7.5% sodium dodecyl sulfate polyacrylamide gel. Cellular proteins 30 were separated by electrophoresis.

The separated proteins were transferred to nitrocellulose by electrotransfer and the resulting "Western blot" was incubated with 3 changes of 0.5% (w/v) sodium chloride, 5 mg/ml bovine serum albumin, 50 mM Tris, pH 7.5, 35 (designated blocking buffer) for 20 minutes each at room temperature. A 1/1000 dilution of PY20 (a commercially available monoclonal antibody to phosphotyrosine [ICN]) in blocking buffer was incubated with the blot overnight at 4°C.

WO 93/11223 PCT/US92/10430 14 24 8 25 The blot was washed 3 times for 20 minutes each at room temperature in blocking buffer. The blot was incubated with 4 /xCi/40 ml of 125I-Protein A [Amersham] in blocking buffer for 1 hour at room temperature and washed 3 times for 2 0 minutes each at room temperature in blocking buffer. The blot was exposed to X-ray film for 48 h with one intensifying screen at -70°C and developed with standard reagents.

B. Type A Sequence Similar manipulations may be performed using either mutagenic oligonucleotides or PCR primers based upon the sequences presented in Table 3. Appropriate oligonucleotides are used to construct type A constructs, as listed in Table 4, which can be functionally tested by various formats as described in the type B assays detailed above. 24 TABLE 3 PROPOSED HUMAN A-type PDGF-R MUTAGENESIS OLIGOMERS [note that these are complementary; given 3'-5'] PaIOI 3'-CGA GGG TGG GAC GCA AGA CTT ATT GAC CGC CTA AGC TCC CC-51 (SEQ ID NO:10) PA102 3'-CTT GAC AAT TGA GTT CAA GGA ATT GAC CGC CTA AGC TCC CC-51 (SEQ ID NO:11) PA103 3'-TAA AGA CAG GTA CTC TTT CCA ATT GAC CGC CTA AGC TCC CC-5' (SEQ ID NO:12) PA104 31—ATA CGA AAT TTT CGT TGT AGT ATT GAC CGC CTA AGC TCC CC-51 (SEQ ID NO:13) PA105 3'-TAA ATG TAG ATA CAC GGT CTG GGT ATT GAC CGC CTA AGC TCC CC-51 (SEQ ID NO:14) PA106 3' -TCG GAT TAG GAG ACG GTC GAA CTA CAT CGG AAA CAT GGA GAT CCT-5' (SEQ ID NO:15) PA107 3'-TCG GAT TAG GAG ACG GTC GAA CTC GAC CTA GAT CTT TAC CTT CGA GAA-5 ' (SEQ ID NO: 16) PA108 3'-TCG GAT TAG GAG ACG GTC GAA AAG TAA. CTT TAG TTT GGG TGG AAG-5 1 (SEQ ID NO:17) PA109 '3'-TCG GAT TAG GAG ACG GTC GAA AGT AGG TAA GAC CTG AAC CAG-5' (SEQ ID NO:18) WO 93/11223 PCT/US92/10430 w i6 c y c J Table 4 SUGGESTED HUMAN TYPE A PDGF-R EXPRESSION CONSTRUCTS type A Soluble Membrane Bound pARSR pARSAl pARSA2 PARSA3 PARSA4 PARSA5 pARSA6 pARSA7 PARSA8 pARSA9 C. PDGF Plate Assay Polystyrene microtiter plates (Immulon, Dynatech Laboratories) were coated with the extracellular region fragment of the type B human PDGF receptor (described above) by 5 incubating approximately 10-100 ng of this purified protein per well in 100 Ml of 25 mM Tris, 75 mM NaCl, pH 7.75 for 12 to 18 h at 4°C.

The protein was expressed in transfected CHO cells and collected in serum-free media (Gibco MEMa) at a 10 concentration of 0.2 - 1 fzg/ml, with a total protein concentration of 150 - 300 fig/ml. The human PDGF type B receptor extracellular region fragment was concentrated and partially purified by passing the media over wheat germ-agglutinin-sepharose at 4°C (at 48 ml/h) in the presence of 1 15 mM PMSF. After extensive washing, the protein was eluted in 0.3 M N-acetyl-glucosamine, 25 mM Hepes, 100 mM NaCl, 1 mM PMSF, pH 7.4. This fraction was then applied to Sephacryl S—200 HR (Pharmacia) equilibrated in 0.15 M ammonium bicarbonate pH 7.9. The fractions containing receptor (3-10 20 ng//xl) were detected by SDS-PAGE and Western blotting with a polyclonal rabbit antibody, made by standard methods, against a Domain 1 (Dl) segment from the receptor external region. These fractions (3-10 ng/^1) were used to coat the microtiter wells as described above. The wells were then drained, rinsed once WO 93/11223 PCT/US92/10430 • » 24 6 25 with 200 jil each of 0.5% gelatin (Bio-Rad, EIA grade), 25 mM Hepes, 100 mM NaCl, pH 7.4, and incubated for 1-2 h at 24°C with 150 nl of this same solution. The wells were drained and rinsed twice with 0.3% gelatin, 25 mM Hepes, 100 mM NaCl, pH 7.4 (150 fil each) . 90 /il of the 0.3% gelatin solution was put in each well (wells used to test nonspecific binding received just 80 /il and then 10 nl of 0.01 mg/ml non-labeled PDGF in the 0.3% gelatin solution). PDGF BB (Amgen) was iodinated at 4°C to 52,000 CPM/ng with di-iodo Bolton-Hunter reagent (Amersham) 10 and approximately 40,000 CPM was added per well in 10 fx 1, containing 0.024% BSA, 0.4% gelatin, 2 0 mM Hepes, 80 mM NaCl, 70 mM acetic acid, pH 7.4. The plate was incubated for 2-3 h at 24°C, after which wells were washed three times with 150 /il each with 0.3% gelatin, 25 mM Hepes, 100 mM NaCl, pH 7.4. The 15 bound radioactivity remaining was solubilized from the wells in 200 /il 1% SDS, 0.5% BSA, and counted in a gamma-counter. The nonspecific binding was determined in the presence of a 150-fold excess of unlabeled PDGF BB (Amgen) and was about 7% of the total bound 1251-PDGF.

Similar assays will be possible using type A receptor fragments. However, the type A receptor fragments are more sensitive to the presence of other proteins than the type B fragments, and appear to require a different well coating reagent from the gelatin. Hemoglobin is substituted for 25 gelatin in the buffers at 1 part per 1000.

The present assays require less than 500 ng/well of receptor soluble form, which was expressed in transfected CHO cells, and partially purified by affinity and gel chromatography. Using iodinated PDGF-BB, the specific binding 30 of less than 10 pg of ligand can be detected in an assay volume of 100 ng/well. At 4°C, the binding of 125I-PDGF BB to immobilized receptor is saturable and of high affinity. The Kd by Scatchard analysis was about 1 nM with 1.8 x 1010 sites per well. The nonspecific binding, determined in the presence of a 35 100-fold excess of cold PDGF BB, was usually only about 5-10% of the total binding. The binding was also specific for the isoform of the ligand, insofar as excess cold PDGF AA did not inhibit 125I-PDGF BB binding. Furthermore, the external region # 18 of the type B PDGF receptor in solution competes with its immobilized form for binding iodinated PDGF BB (IC50 = 5nM) . The 125I-PDGF BB bound after 4 h at 4°C is only slowly dissociable in binding buffer (t1/,2 > 6 h) , but is completely 5 displaced by the addition of a 150-fold excess of unlabeled PDGF BB (t1/2 < l h).

D. Purification of hPDGF-R fragments Type B human PDGF-R fragment was prepared from cells 10 disclosed herein. Secreted fragments were purified by wheat germ agglutinin affinity chromatography and by S-200 sizing chromatography. Purity was evaluated by SDS-PAGE and estimated to be about 70% pure.

Type A human PDGF-R fragment was also prepared from 15 cells disclosed herein. Secreted fragments were purified by ion exchange chromatography, wheat germ agglutinin affinity chromatography, and S200 sizing chromatography. Purity was evaluated by SDS-PAGE and estimated to be about 70% pure.

E. Preparation of Antibodies Antibodies are prepared using standard techniques. See, e.g., Harlow and Lane (1990) Antibodies: A Laboratory Manual. Cold Spring Harbor Press, New York; and Goding (1986) Monoclonal Antibodies. Principles and Practice, each of which 25 is hereby incorporated herein by reference. Either polyclonal or monoclonal antibodies will be prepared.

Two Balb/C mice were immunized for preparation of monoclonal antibodies specific for each of type B and type A receptor fragments. Each mouse was injected intraperitoneally 30 with about 100 ng of the appropriate purified receptor fragment. Each mouse was boosted with about 100 y.g of the protein. The mouse was sacrificed and its spleen recovered. The spleen was disrupted and cells fused to myeloma line P3X using polyethylene glycol (PEG). Cell fusion products were 35 distributed into microtiter plates at limiting dilutions to clonally isolate antibody producing hybridomas. Antibody producing clones were identified by an enzyme-linked immunosorbent assay (ELISA).

WO 93/11223 PCT/US92/I0430 19 Selected antibody producing clones were grown to sufficient numbers and introduced into a mouse peritoneum to produce ascites fluid. Typically about 1-1.5 x 106 cells were injected into a mouse in 1 ml. Antibodies from ascites fluid were purified by standard methods.

Resulting antibodies were characterized for their binding specificity and binding affinity. Clone IC705 is a preferred monoclonal antibody with type B receptor fragment binding specificity; and clone 1H-2H8 is a preferred monoclonal 10 antibody with type A receptor fragment binding specificity.

F. Preparation of Fragments using Cells and Antibodies Appropriate receptor fragments will be easily prepared using the cells disclosed herein. As an example, 15 PaI—5 cells were grown in hollow fiber bioreactors in a 5 liter culture. Supernatant media was removed by centrifugation and remaining cells were removed by filtration. The media was then subjected to ultrafiltration and passed over a 50 cm monoclonal antibody-Tresyl agarose immunoaffinity column. The column was 20 washed and the bound receptor fragments eluted by high pH. The purified soluble receptor fragment was at least about 97% pure with a yield of about 50 mg from 5 liters of cell supernatant.

These studies were made possible by the availability of growth factor preparations substantially devoid of 25 contamination with other growth factors and by the use of a receptor expression system in which all of the measured PDGF responses could be attributed to this single transfected receptor cDNA.

All publications and patent applications herein are 30 incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and 35 modifications can be made thereto without departing from the spirit or scope of the appended claims.

WO 93/11223 PCT/US92/10430 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Wolf, David Tomlinson, James E. (ii) TITLE OF INVENTION: Methods for Production of Purified Soluble Type B and Type A Human Platelet-Derived Growth Factor Receptor Fragments (iii) NUMBER OF SEQUENCES: 18 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: William M. Smith (B) STREET: One Market Plaza, Steuart Tower, Suite 2 000 (C) CITY: San Francisco (D) STATE: California (E) COUNTRY: USA (F) ZIP: 94105 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (Vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: PCT (B) FILING DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 07/801,794 (B) FILING DATE: 02-DEC-1991 (Viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Smith, William M.

(B) REGISTRATION NUMBER: 30,223 (C) REFERENCE/DOCKET NUMBER: 12418-15 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 415-326-2400 (B) TELEFAX: 415-326-2422 # 21 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l AGCCCCCTAC AGGAAGCTAC TTAAAGGGCA AGGAGTGTG 39 m 22 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 AGCCCCCTAC AGGAAGCTAA TTGATCTGTA GGTCGAAGG 39 # 23 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CAGCCCCCTA CAGGAAGCTA GCCGCTCTCA ACCACGGTGA 41 m 24 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CAGCCCCCTA CAGGAAGCTA GGATGACACC TGGAGTCTGT A 41 WO 93/11223 # (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: GAGCCCCCTA CAGGAAGCTA GGGATCTGGC ACAAAGATGT AGAG 44 WO 93/11223 PCT/US92/10430 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: ATGATTAGGG AGGAAGCCCA CGGTGACCAG GCCCTGAGAG ATCTG 45 27 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CTGCACTGCG TTCACAGAGA CGTTGATGAC CAGGCCCTGA GAGATCTG 48 28 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CAGCTCTCCC AGGAGCCGCA CGTAGACCAG GCCCTGAGAG ATCTG 45 29 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: ACTTAGCTCC AGCACTCGGA CGACCAGGCC CTGAGAGATC TG 42 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: CCCCTCGAAT CCGCCAGTTA TTCAGAACGC AGGGTGGGAG C 41 31 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11 CCCCTCGAAT CCGCCAGTTA AGGAACTTGA GTTAACAGTT 41 m 32 (2) INFOPMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CCCCTCGAAT CCGCCAGTTA ACCTTTCTCA TGGACAGAAA T 41 33 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: CCCCTCGAAT CCGCCAGTTA TGATGTTGCT TTTAAAGCAT A 41 WO 93/11223 PCT/US92/10430 • „ 24 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CCCCTCGAAT CCGCCAGTTA TGGGTCTGGC ACATAGATGT AAAT 44 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: TCCTAGAGGT ACAAAGGCTA CATCAAGCTG GCAGAGGATT AGGCT 45 WO 93/11223 PCT/US92/10430 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 49 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: AAGAGCTTCC ATTTCTAGAT CCGACTCCAA GCTGGCAGAG GATTAGGCT 49 WO 93/11223 PCT/US92/10430 m 37 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GAAGGTGGGT TTGATTTCAA TGAAAAGCTC GCAGAGGATT AGGCT 45 WO 93/11223 PCT/US92/10430 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCR8PTION: SEQ ID NO:18: GACCAAGTCC AGAATGGATG AAAGCTCGCA GAGGATTAGG CT 42

Claims (19)

. 246256 39

1. An immortalized mammalian cell line which expresses a soluble peptide fragment which binds a platelet-derived growth factor (PDGF) ligand, said fragment consisting essentially of from one to three contiguous domains of a PDGF ligand binding region derived from a platelet-derived growth factor receptor (PDGF-R) extracellular region sequence, said domains being selected from the group consisting of Dl, D2 and D3.

2. A cell line of Claim 1, wherein said fragment is at least substantially 50 amino acids long.

3. A cell line of Claim 1, wherein said fragment has a dissociation constant to a human PDGF of at least substantially 0.2 mM.

4. A cell line of Claim 1, wherein said PDGF and PDGF-R are human.

5. A cell line of Claim 1, wherein said line is a Chinese hamster ovary cell line.

6. A cell line of Claim 1, wherein said extracellular region sequence is a type B or type A human PDGF-R sequence.

7. A cell line of Claim 1, wherein said fragment has both type B and type A PDGF-R sequences.

8. A cell line of Claim 1, wherein said cell comprises a plasmid selected from the group consisting of: a) PaI; b) Pa2 ; c) Pa3; d) PaIOI ; e) Pa102; and 246256 40 f) PaI03 .

9. A cell line of Claim 1, wherein said cell line further comprises an exogenous enzyme which post-translationally modifies said fragment.

10. A cell line of Claim 9, wherein said enzyme is a glycosylation enzyme.

11. A pharmaceutical composition comprising a protein produced from a cell line of Claim 1 together with a pharmaceutically acceptable carrier or diluent.

12. A method for preparing a soluble polypeptide exhibiting human platelet-derived growth factor receptor binding activity comprising the steps of: a) growing a cell line of Claim 1; and b) isolating said cell product from said cell line.

13. A method of Claim 12, wherein said cell product is secreted into cell growth medium.

14. A method of Claim 12, wherein the cell product is isolated by affinity chromatography from said medium.

15. A method of Claim 12, wherein the affinity chromatography comprises a monoclonal antibody immuno-affinity reagent.

16. An immortalized mammalian cell line as defined in claim 1 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.

17. A pharmaceutical composition as claJ claim 11 substantially as herein described with refer 246256 41 any example thereof and with or without reference to the accompanying drawings.

18. A method as defined in claim 12 for preparing a soluble polypeptide exhibiting human-platelet-derived growth factor receptor binding activity substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.

19. A polypeptide produced in accordance with the method of any one of claims 12 to 15. By the authorised agents A. J Park & Son