Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates generally to methods for producing soluble platelet-derived growth factor receptor 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 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 inases, alteration of cell shape, transcription of various genes, and 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) .
• 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 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) ___. Biol. Chem. 266:413-418; Heiong 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.
• 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 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. Moreover, a smaller soluble peptide is more likely to be capable of reaching remote and circulation compromised regions of the body.
• the present invention provides cell lines and methods for high efficiency production of soluble peptides which bind platelet-derived growth factor (PDGF) .
• Cell lines are described which secrete fragments of an extracellular region of a PDGF receptor (PDGF-R) , or related peptides which exhibit the activity of PDGF ligand binding. Many of these fragments result from deletion of segments of the extracellular region which do not contribute to ligand binding affinity or specificity.
• Methods for using these cell lines to produce PDGF-binding polypeptides, e.g., from human receptors, are also provided. BRIEF DESCRIPTION OF THE FIGURES Fig.
• 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:
• FIG. 2 illustrates the structure of a plas id derived from pcDL-S ⁇ 296 used for expressing various deletion polypeptides.
• Fig. 3 illustrates the structure of a plasmid pBJ ⁇ derived from pcDL ⁇ 296. See Takabe et al. (1988) Mol. Cell. Biol. 8:466-472, which is incorporated herein by reference.
• a polylinker (XhoI-Xbal-Sfil-Notl-EcoRI- EcoRV-Hindlll-Clal-Sall) is inserted into the Xhol cut vector.
• Sail is compatible with the Xhol site; and generates both a Sail and an Xhol site.
• novel cells for producing soluble polypeptides which bind platelet-derived 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.
• the 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.
• the polypeptide fragments will find use as diagnostic and therapeutic compositions.
• 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.
• PDGF ligands Analogues of the PDGF protein are referred to as PDGF ligands. These ligands typically are agonists of the PDGF-R.
• the PDGF ligands will usually be proteinaceous, but may be other compounds which exhibit structural features important in interaction with the receptors.
• 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, 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.
• the cells and DNA constructs are derived from a human cell line.
• Especially preferred cell lines include the p ⁇ l-5 line, which contains an expression vector for producing a type B PDGF-R polypeptide fragment, and the p ⁇ Q.RF line, which contains an expression vector for producing a type A PDGF-R polypeptide fragment.
• p ⁇ l-5 line which contains an expression vector for producing a type B PDGF-R polypeptide fragment
• the p ⁇ Q.RF line which contains an expression vector for producing a type A PDGF-R polypeptide fragment.
• Novel cell lines are provided herein for expressing fragments of the extracellular region of the platelet-derived growth factor receptor. These fragments exhibit unexpected 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 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.
• 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.
• 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.
• 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 be affected by a factor of at least about two, typically by a least a factor of about four, more 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. 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.
• DHFR dehydrofolate reductase
• These cells are particularly selected for high level 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 have integrated DNA in positions and numbers providing particularly high receptor fragment expression.
• P ⁇ l through P ⁇ 9 refer to DNA constructs of the type B receptor inserted into cloning sites in the expression vectors.
• the P ⁇ I construct inserted into the pBJ vector is designated pBJP ⁇ .
• the vector is transferred into a specific cell background and various clonal transformants are isolated and designated, e.g., p ⁇ l-1, p ⁇ l-2, etc.
• Each clonal isolate differs from others by sequence copy number and integration sites, with resultant variability in expression levels.
• the p ⁇ l-5 is a particularly useful cell line embodiment expressing high levels of type B ligand binding regions.
• the p ⁇ RF cell line expresses the entire extracellular region of a human type A PDGF receptor.
• the 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.
• the present invention also provides methods for" producing the described fragments.
• cell cultures are available to express the nucleic acids described.
• the fragments are secreted thereby considerably simplifying purification of the receptor fragments.
• the cells need not be disrupted and cellular contamination is minimized.
• 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.
• the soluble proteins will be secreted, and 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.
• Standard methods for protein purification will be used, e.g., chromatography, centrifugation, precipitations, electrophoresis, immunoaffinity methods, and other techniques well known to protein chemists and enzy ologists. See, e.g., Deutscher et al. (1990) Protein Purification, in Methods in Enzvmology; and Ausubel et al. (1987 and supplements) Current Protocols in Molecular Biology.
• Particularly useful purification reagents include affinity reagents, e.g., either PDGF-ligand affinity columns or immunoaffinity columns.
• 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.
• PDGF-receptor specific monoclonal antibodies with binding affinities of 10 5 M "1 , preferably 10 7 to 10 10 , 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, 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 Lambda," Science 246:1275-1281; and Ward et al.
• the appropriate affinity reagent e.g., the PDGF or antibody
• a solid substrate include chromatography matrices , e.g., CNBr- sepharose, glass beads, and plastics.
• the attachment will be covalent, though non-covalent attachment methods may, under certain conditions, be sufficient.
• affinity reagent or combination of affinity reagents
• solutions containing the soluble fragments will be passed though the affinity reagent 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 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. 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 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 3,093,831.
• Type B Segments Constructs of type B receptor polypeptides were made as follows:
• oligonucleotides were designed 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 1. 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.
• the resulting constructs are labeled as indicated in Table 2.
• the antisense strand was used for mutagenesis throughout. Mutagenesis of P ⁇ I, P ⁇ 2, P ⁇ 3, P ⁇ 4, and P ⁇ 5 utilized Mpl ⁇ PR as the template and mutagenesis of P ⁇ 6, P ⁇ 7, P ⁇ 8, and P ⁇ 9 utilized Mpl8 P ⁇ I as the template.
• P ⁇ I, a 41 bp oligomer introduced a TAG stop codon after Lysine 4g9 (K 499 ) of D5 and removed the transmembrane (TM) as well as entire intracellular kinase domain (K) , producing an Mpl8 P ⁇ I (see Fig. 1) .
• the human PDGF receptor constructs were subsequently subcloned into the EcoRI-Hind III site of pBJl a derivation of pCDL-SR ⁇ 296, as described in Takabe et al. (1988) Molec. Cell Biol. 8:466, and co-transfected with pSV2NEO, as described by Southern and Berg (1982) J. Mol. Appl. Gen.. l: 327, into Chinese hamster ovary cells (CHO) . See. Figs. 2 and 3.
• a sample of 0.33 nM PDGF BB ligand is preincubated for 1 hr at 4°C under the following conditions:
• a polyclonal antibody to human PDGF (this antibody recognizes human PDGF AA, PDGF BB and PDGF AB) ;
• PDGF AA 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 non ⁇ specific general cellular effect, e.g., cytotoxicity.
• the preincubated materials were in a final volume of
• the cells were solubilized and 40 ⁇ l 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 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, (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. The blot was washed 3 times for 20 minutes each at room temperature in blocking buffer.
• the blot was incubated with 4 ⁇ Ci/40 ml of 125 I-Protein A [Amersham] in blocking buffer for 1 hour at room temperature and washed 3 times for 20 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.
• 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.
• Polystyrene microtiter plates (Immulon, Dynatech Laboratories) were coated with the extracellular region fragment of the type B human PDGF receptor (described above) by incubating approximately 10-100 ng of this purified protein per well in 100 ⁇ l 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 MEM ⁇ ) at a concentration of 0.2 - 1 ⁇ g/ml, with a total protein concentration of 150 - 300 ⁇ g/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 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.
• the wells were then drained, rinsed once with 200 ⁇ l 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 ⁇ l 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 ⁇ l each).
• 90 ⁇ l of the 0.3% gelatin solution was put in each well (wells used to test nonspecific binding received just 80 ⁇ l and then 10 ⁇ l of 0.01 mg/ml non-labeled PDGF in the 0.3% gelatin solution).
• PDGF BB (A gen) was iodinated at 4°C to 52,000 CPM/ng with di-iodo Bolton-Hunter reagent (Amersham) and approximately 40,000 CPM was added per well in 10 ⁇ l, containing 0.024% BSA, 0.4% gelatin, 20 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 ⁇ l each with 0.3% gelatin, 25 mM Hepes, 100 mM NaCl, pH 7.4.
• the bound radioactivity remaining was solubilized from the wells in 200 ⁇ l 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 125 I-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 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.
• iodinated PDGF-BB the specific binding of less than 10 pg of ligand can be detected in an assay volume of 100 ⁇ g/well.
• the binding of 125 I-PDGF BB to immobilized receptor is saturable and of high affinity.
• the Kd by Scatchard analysis was about 1 nM with 1.8 x l ⁇ 10 sites per well.
• Type B human PDGF-R fragment was prepared from cells 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 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.
• 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 is hereby incorporated herein by reference. Either polyclonal or monoclonal antibodies will be prepared.
• mice 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 with about 100 ⁇ g of the appropriate purified receptor fragment. Each mouse was boosted with about 100 ⁇ 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 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) .
• ELISA enzyme-linked immunosorbent assay
• 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 10 6 cells were injected into a mouse in 1 ml. Antibodies from ascites fluid were purified by standard methods.
• Clone IC705 is a preferred monoclonal antibody with type B receptor fragment binding specificity
• clone 1H-2H8 is a preferred monoclonal antibody with type A receptor fragment binding specificity.
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• CAGCCCCCTA CAGGAAGCTA GCCGCTCTCA ACCACGGTGA T 41
• MOLECULE TYPE DNA (genomic)
• CAGCCCCCTA CAGGAAGCTA GGATGACACC TGGAGTCTGT A 41
• MOLECULE TYPE DNA (genomic)
• GAGCCCCCTA CAGGAAGCTA GGGATCTGGC ACAAAGATGT AGAG 44
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)

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• 1992
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