US 7041801 B1
The present invention relates to a member of a novel gene family referred to as developmentally-regulated endothelial cell locus-1 (del-1). In particularly, the invention related to del-1 nucleotide sequences, Del-1 amino acid sequences, methods of expressing a functional gene product, and methods of using the gene and gene product. Since del-1 is expressed in endothelial cells and certain cancer cells, it may be useful as an endothelial cell and tumor marker. In addition, anti-Del-1 antibodies for immunological assessment of Del-1 protein expression as well as for inhibiting or stimulating Del-1 function are provided.
1. An isolated, purified antibody which binds to a polypeptide of a mammalian Del-1, wherein said mammalian Del-1 comprises an amino acid sequence as shown in a SEQ ID NO selected from the group consisting of: SEQ ID NO:10; SEQ ID NO:29; SEQ ID NO:30 and SEQ ID NO:31.
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This is a divisional application of U.S. patent application Ser. No. 08/659,235, filed Jun. 5, 1996, now U.S. Pat. No. 5,877,281, issued on Mar. 2, 1999, which is a continuation-in-part of U.S. patent application Ser. No. 08/480,229, filed Jun. 7, 1995, now U.S. Pat. No. 5,874,562, issued on Feb. 23, 1999, each of which is incorporated herein in its entirety.
This invention was made, in part, with government support under HD 25580 awarded by the National Institutes of Health. The government may have certain rights in the invention.
The present invention relates to a member of a novel gene family referred to as developmentally-regulated endothelial cell locus-1 (del-1). In particular, the invention relates to Del-1 nucleotide sequences, Del-1 amino acid sequences, methods of expressing a functional gene product, antibodies specific for the gene product, and methods of using the gene and gene product. Since Del-1 is expressed in endothelial cells and certain cancer cells, it may be useful as an endothelial cell and tumor marker. In addition, the ability of Del-1 protein to inhibit vascular formation provides for its use as an anti-angiogenic agent.
2.1 Endothelial Cell Biology and Blood Vessel Development
The endothelium occupies a pivotal position at the interface between the circulating humoral and cellular elements of the blood, and the solid tissues which constitute the various organs. In this unique position, endothelial cells regulate a large number of critical processes. Such processes include leukocyte adherence and transit through the blood vessel wall, local control of blood vessel tone, modulation of the immune response, the balance between thrombosis and thrombolysis, and new blood vessel development (Bevilacqua et al., 1993, J. Clin. Invest. 91: 379–387; Folkman et al., 1987, Science 235: 442–447; Folkman et al., 1992, J. Biol. Chem. 267: 10931–10934; Gimbrone, 1986, Churchill Livingstone, London; Issekutz, 1992, Curr. Opin. Immunol. 4:287–293; Janssens et al., 1992, J. Biol. Chem. 267:14519–14522; Lamas et al. 1992, Proc. Natl. Acad. Sci. U.S.A. 89:6348–6352, Luscher et al. 1992, Hypertension 19:117–130; Williams et al., 1992, Am. Rev. Respir. Dis. 146:S45–S50; Yanagisawa, et al., 1988, Nature 332:411–415).
Endothelial cell dysfunction has been postulated as a central feature of vascular diseases such as hypertension and atherosclerosis. In this context, the ability of the endothelium to synthesize smooth muscle cell mitogens and factors which control smooth muscle contraction has received much attention (Janssens et al., 1992, J. Biol. Chem. 267:14519–14522; Lamas et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:6348–6352; Luscher et al., 1992, Hypertension 19:117–130; Raines et al., 1993, Br. Heart J. 69:S30–S37; Yanagisawa et al., 1988, Nature 332:411–415). The endothelial cell has also become the focus of attention in the study of diseases which are not primarily vascular in nature. Diverse disease processes such as adult respiratory distress syndrome, septic shock, solid tumor formation, tumor cell metastasis, rheumatoid arthritis, and transplant rejection are now understood to be related to normal or aberrant function of the endothelial cell. A rapidly increasing number of pharmacologic agents are being developed whose primary therapeutic action will be to alter endothelial cell function. In addition, recent attention on gene therapy has focused on the endothelial cell (Nabel et al., 1991, J. Am. Coll. Cardiol. 17:189B–194B). Transfer of genes into the endothelial cell may afford a therapeutic strategy for vascular disease, or the endothelium may serve simply as a convenient cellular factory for a missing blood borne factor. Hence, information regarding fundamental processes in the endothelial cell will aid the understanding of disease processes and allow more effective therapeutic strategies.
Studies from a number of laboratories have characterized the ability of the endothelial cell to dramatically alter basic activities in response to cytokines such as tumor necrosis factor (TNF)-alpha. TNF-alpha stimulation induces significant alterations in the production of vasoactive compounds such as nitric oxide and endothelin, increases surface stickiness toward various types of leukocytes, and modulates the expression of both pro- and anti-coagulant factors (Cotran et al., 1990, J. Am. Soc. Nephrol. 1:225–235; Mantovani et al., 1992, FASEB J. 6:2591–2599). In turn, endothelial cells have been shown to be an important source for the production of cytokines and hormones, including interleukin 1, 6 and 8 (Gimbrone et al., 1989, Science 246:1601–1603; Locksley et al. 1987, J. Immunol. 139:1891–1895; Loppnow et al., 1989, Lymphokine. Res. 8:293–299; Warner et al., 1987, J. Immunol. 139:1911–1917).
The ability of endothelial cells to produce granulocyte, granulocyte-macrophage, and macrophage colony stimulating factors has led to speculation that endothelial cells are an important facet of hematopoietic development (Broudy et al., 1987, J. Immunol. 139:464–468; Seelentag et al., 1987, EMBO J. 6:2261–2265). Early studies have provided the foundation for the cloning of a large number of “endothelial cell-specific” genes. Some of these include ICAM-1, ICAM-2, VCAM-1, ELAM-1, endothelin-1, constitutive endothelial cell nitric oxide synthetase, thrombomodulin, and the thrombin receptor (Bevilacqua et al., 1989, Science 243:1160–1165; Jackman et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:8834–8838; Janssens et al., 1992, J. Biol. Chem. 267:14519–14522; Lamas et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:6348–6352; Osborn et al., 1989, Cell 59:1203–1211; Staunton et al., 1–969, Nature 339:61–64, Staunton et al., 1988, Cell 52:925–933; Vu et al, 1991, Cell 64:1057–1068; Yanagisawa et al., 1988, Nature 332:411–415).
All blood vessels begin their existence as a capillary, composed of only endothelial cells. Much of the molecular research investigating the role of endothelial cells in blood vessel development has focused on this process in the adult organism, in association with pathological conditions. In these situations, new blood vessels are formed by budding and branching of existing vessels. This process, which depends on endothelial cell division, has been termed angiogenesis. Research on this process has focused primarily on small proteins which are growth factors for endothelial cells (Folkman et al., 1987, Science 235:442–447; Folkman et al., 1992, J. Biol. Chem. 267:10931–10934). Sensitive bioassays for angiogenesis have allowed the characterization of a number of angiogenic factors, from both diseased and normal tissues. Members of the fibroblast growth factor (FGF) family, platelet-derived endothelial cell growth factor, and vascular endothelial cell growth factor (vascular permeability factor), are a few of the angiogenic factors which have been characterized (Folkman et al., 1987, Science 235:442–447; Folkman et al., 1992, J. Biol. Chern. 267:10931–10934; Ishikawa et al., 1989, Nature 338:557–562; Keck et al., 1989, Science 246:1309–1312; Leung et al., 1989, Science 246:1306–1309).
Such information has provided some insight into the study of blood vessel development in the embryo. Studies linking vascular development to an angiogenic factor have resulted in the work with vascular endothelial cell growth factor (VEGF). VEGF expression has been correlated in a temporal and spatial fashion with blood vessel development in the embryo (Breier et al., 1992, Development 114:521–532). A high affinity VEGF receptor, flk-1, has been shown to be expressed on the earliest endothelial cells in a parallel fashion (Millauer et al., 1993, Cell 72:835–846).
Blood vessels form by a combination of two primary processes. Some blood vessel growth depends on angiogenesis, in a process very similar to that associated with pathological conditions in the adult. For instance, the central nervous system depends solely on angiogenesis for development of its vascular supply (Noden, 1989, Am. Rev. Respir. Dis. 140:1097–1103; Risau et al., 1988, EMBO J. 7:959–962). A second process, vasculogenesis, depends on the incorporation of migratory individual endothelial cells (angioblasts) into the developing blood vessel. These angioblasts appear to be components of almost all mesoderm, and are able to migrate in an invasive fashion throughout the embryo (Coffin et al., 1991, Anat. Rec. 231:383–395; Noden, 1989, Am. Rev. Respir. Dis. 140:1097–1103; Noden 1991, Development 111:867–876). The precise origin of this cell, and the characteristics of its differentiation have not been defined.
Understanding of the molecular basis of endothelial cell differentiation in blood vessel development may allow manipulation of blood vessel growth for therapeutic benefit. The ability to suppress blood vessel growth may also provide therapeutic strategies for diseases such as solid tumors and diabetic retinopathy. On the other hand, diseases such as coronary artery disease may be treated through pharmacologic induction of directed blood vessel growth, through increasing collateral circulation in the coronary vascular bed. Both vascular diseases such as atherosclerosis and hypertension and nonvascular diseases which depend on the endothelial cell will benefit from a better understanding of endothelial cells.
2.2 Epidermal Growth Factor-Like Domain
Epidermal growth factor (EGF) stimulates growth of a variety of cell types. EGF-like domains have been found in a large number of extracellular and membrane bound proteins (Anderson, 1990, Experientia 46(1): 2; and Doolittle, 1985, TIBS, June:233). These proteins include molecules that function as soluble secreted proteins, growth factors, transmembrane signal and receptor molecules, and components of the extracellular matrix (Lawler and Hynes, 1986, J. Cell Biol. 103:1635; Durkin et al., 1988, J. Cell Biol. 107:2749; Wu et al., 1990, Gene 86:275; Bisgrove and Raff, 1993, Develop. Biol. 157:526).
In many cases, multiple tandem repeats of a characteristic 40 amino acid long, 6 cysteine-containing sequence are observed (Anderson, 1990, Experientia 46(1): 2). EGF-like domains are homologous to the peptide growth factor EGF which consists of a single copy of the standard EGF domain. These domains have been highly conserved in evolution, being found in species as diverse as nematodes, Drosophila, sea urchins, and vertebrates.
The EGF molecule and the closely related transforming growth factor (TGF) alpha induce cell proliferation by binding to a tyrosine kinase receptor. It has been suggested that other EGF-like domains also function as ligands for receptor molecules (Engel, 1989, FEBS Lett. 251:1–7). Fundamentally, EGF repeats are protein structures that participate in specific protein-protein binding interactions.
The Drosophila Notch protein, the Nematode lin-12 and glp-1 proteins, and the closely related vertebrate homologs, Motch (mouse Notch), Xotch (Xenopus Notch), rat Notch, and TAN 1 (human Notch) are membrane bound receptor molecules that control the specification of cell fate for a variety of cell types early in embryogenesis (Rebay et al., 1991, Cell 67:687; Hutter and Schnabel, 1994, Development 120:2051; Del Amo et al. 1992, Development 115:737; Reaume et al. 1992 Develop. Biol. 154:377; and Ellisen et al., 1991, Cell 66:649). Specific EGF-like repeats in the Notch receptors are binding sites that attach to protein ligands leading to signal transduction (Rebay et al., 1991 Cell 67:687; Couso and Arias, 1994, Cell 79:259; Fortini and Artavanis-Tsakonas, 1994, Cell 79:273; Henderson et al., 1994, Development 120:2913). Extracellular matrix proteins such as thrombospondin, entactin, tenascin and laminin play key roles in morphogenesis by providing the physical scaffold to which cells attach to form and maintain tissue morphologies (Frazier, 1987, J. Cell. Biol. 105:625; Taraboletti et al., 1990, J. Cell. Biol. 111:765; Ekblom et al., 1994, Development 120:2003).
2.3 Discoidin I/Factor VIII-Like Domains
A homologous domain structure has been discovered in coagulation factors VIII and V (Kane and Davie, 1986, Proc. Natl. Acad. Sci. U.S.A. 83:6800). This domain is related to a more ancient structure first observed in the discoidin I protein produced by the cellular slime mold Dictyostelium discoideum. Discoidin I is a carbohydrate binding lectin secreted by Dictyostelium cells during the process of cellular aggregation and is involved in cell-substratum attachment and ordered cell migration (Springer et al., 1984, Cell 39:557).
Discoidin I/factor VIII-like domains have also been observed in a number of other proteins. For example, milk fat globule protein (BA46), milk fat globule membrane protein (MFG-E8), breast cell carcinoma discoidin domain receptor (DDR), and the Xenopus neuronal recognition molecule (A5) (Stubbs et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:8417; Larocca et al., 1991, Cancer Res. 51:4994; Johnson et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5677). The discoidin I/factor VIII-like domains of the vertebrate proteins are all distantly related to the Dictyostelium sequence but more closely related to each other.
Discoidin I/factor VIII-like domains are rich in positively charged basic amino acids and are believed to bind to negatively charged substrates such as anionic phospholipids or proteoglycans. Both of the milk fat globule proteins have been shown to associate closely with cell membranes and the coagulation factors VIII and V interact with specific platelet membrane proteins (Stubbs et al., 1990 Proc. Natl. Acad. Sci. U.S.A. 87:8417; Larocca et al., 1991, Cancer Res. 51:4994).
The present invention relates to a novel gene family referred to as del-1. In particular, it relates to del-1 nucleotide sequences, expression vectors containing the sequences, genetically-engineered host cells expressing del-1, Del-1 protein, Del-1 mutant polypeptides, methods of expressing del-1 and methods of using del-1 and its gene product in various normal and disease conditions such as cancer.
The invention is based, in part, upon Applicants' isolation of a murine DNA clone (SEQ ID NO: 9), del-1, and its homologous human counterpart, (SEQ ID NO: 11). Structural features of the Del-1 protein are deduced by homology comparisons with sequences in the Genbank and NBRF-PIR databases. The protein is a modular molecule composed of repeats of two different sequence motifs which are present in a number of distinct proteins. The two sequence motifs are known as the EGF-like domain (SEQ ID NO: 26) and the discoidin I/factor VIII-like domain (SEQ ID NOS: 1–8). These domains are defined by characteristic patterns of conserved amino acids distributed throughout the molecule at specific locations. While Del-1 shows certain sequence homology with other proteins, it is unique in both its primary sequence and its overall structure. In all cases in which EGF-like and discoidin I-like domains have been identified, both of these structures are always found in extracellular locations. Variant forms of Del-1 protein exist, and one form is shown herein to be an extracellular matrix protein and is associated with the cell surface. The expression pattern of del-1 further indicates that it is involved in endothelial cell function. In addition, a number of human tumor cells express del-1. Furthermore, host-derived blood vessels that traverse the tumor nodule also express del-1. The Del-1 protein inhibits vascular morphogenesis and binds to αVβ3 as its cellular receptor. Therefore, a wide variety of uses are encompassed by the present invention, including but not limited to, the use of Del-1 as a tumor marker for cancer diagnosis and treatment, the isolation of embryonic endothelial cells, the identification of Del-1 binding partners, and the stimulation or inhibition of endothelial cell growth and blood vessel formation.
The present invention relates to a novel family of genes herein referred to as del-1. Described below are methods for cloning members of this gene family, characteristics of a murine member and its human homolog, expression of recombinant gene products, and methods of using the gene and its gene product. Structurally, members of this gene family contain three EGF-like domains and two discoidin I/factor VIII-like domains.
The overall structure of the del-1 molecule is similar to the milk fat globule membrane protein (MFG-E8) (Stubbs et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:8417). MFG-E8 is highly expressed by a large portion of human breast tumors as well as by lactating mammary epithelial cells. It consists of two tandem EGF-like domains followed by two discoidin I/factor VIII-like domains. The function of MFG-E8 is not known but it has been shown to associate closely with cell membranes and has been investigated as a target for antibody-based tumor imaging techniques. The observed association of MFG-E8 with cell membranes indicates the potential use of antibodies against Del-1 to identify and sort endothelial cells from mixed cell populations, and to target tumor cells that express Del-1 for diagnosis and therapy.
The second EGF-like repeat of MFG-E8 contains the amino acid sequence arg-gly-asp (RGD) in the same position as the second EGF-like repeat of Del-1. The RGD sequence has been shown to be a cell binding site for fibronectin, discoidin I, nidogen/entactin, and tenascin (Anderson, 1990, Experientia 46:2). The binding of fibronectin to cell surface integrin molecules through the RGD sequence has been extensively studied (Main et al., 1992, Cell 71:671; Hynes, 1992, Cell 69:11). Integrins appear to be the major receptors by which cells attach to extracellular matrices. Substrate binding to integrins has been shown to initiate signal transduction leading to events such as tyrosine phosphorylation, cytoplasmic alkalinization, activation of secretion and differentiation (Hynes, 1992, Cell 69:11). The presence of the RGD sequence in Del-1 indicates that this portion of the molecule may bind cell surface integrins, possibly triggering certain developmental events. In particular, Del-1 is shown to bind to integrin αVβ3 on endothelial cells. In several cases, synthetic peptides containing the RGD sequence have been shown to compete with native protein for integrin binding and prevent the initiation of downstream events (Brooks et al., 1994, Cell 79:1157).
For clarity of discussion, the invention is described in the subsections below by way of example for the del-1 genes and their products in mice and in humans. However, the findings disclosed herein may be analogously applied to other members of the del-1 family in all species.
5.1 The DEL-1 Coding Sequence
The present invention relates to nucleic acid molecules and polypeptides of the del-1 gene family. In a specific embodiment by way of example in Section 6, infra, murine and human del-1 nucleic acid molecules were cloned, and their nucleotide and deduced amino acid sequences characterized. Both the nucleotide coding sequence and deduced amino acid sequence of del-1 are unique. In accordance with the invention, any nucleotide sequence which encodes the amino acid sequence of the del-1 gene product can be used to generate recombinant molecules which direct the expression of del-1 gene.
Enhancer trapping is a strategy which has been successfully employed in genetic analysis in Drosophila but is also applicable to higher organisms. This method identifies regulatory regions in genomic loci through their influence on reporter genes (Okane et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84:9123–9127). The reporter gene, as a transcriptional unit under the control of a weak constitutively expressed eukaryotic promoter, is introduced into a large number of organisms. The offsprings of these organisms are then screened by analysis of the pattern of reporter gene expression. Lines which show expression in the appropriate cells at the appropriate time are maintained for further study. This strategy has successfully identified a number of loci in Drosophila involved in complex developmental processes.
Enhancer trap experiments have been employed in mice to a limited extent (Allen et al., 1988, Nature 333:852–855). A number of such experiments were through fortuitous integration of a reporter gene into a locus of interest (Kothary et al., 1988, Nature 335:435–437). Using this method coupled with genomic and cDNA cloning, the murine del-1 locus associated with the transgene was identified. A genomic library is generated from the transgenic mouse, and a probe from the transgene used to isolate clones containing the transgene and sequences flanking the integration site. Characterization of the regulatory region is accomplished by employing flanking sequences in functional assays, via transfection experiments with an appropriate cell culture line, or via further transgenic experiments (Bhat et al., 1988, Mol. Cell. Biol. 8:3251–3259).
For analysis of the transcription unit, it is necessary to identify a region of flanking sequence which contains a portion of exon. This has been accomplished by blindly using flanking genomic sequences as probes in northern blots or zoo blots (Soinen et al., 1992, Mechanisms of Development 39:111–123). DNA fragments thus identified to contain exon sequence are employed as probes for cDNA cloning. Similar cloning experiments have been conducted to characterize loci inactivated by insertional mutagenesis associated with transgene integration. These experiments indicate that deletions of large regions of genomic DNA may accompany transgene integration, and that complexity of the transcription unit may greatly complicate this type of analysis (Karls et al., 1992, Mol. Cell. Biol. 12:3644–3652; Woychik et al., 1990, Nature 346:850–853).
Subsequent analysis of the del-1 sequence has revealed both EGF-like and discoidin I/factor VIII-like domains. The shared homology between del-1 and other known molecules is discussed in Section 6.2, infra. However, this molecule also contains regions of previously unreported unique nucleotide sequences. Northern blot hybridization analysis indicates that del-1 mRNA is highly expressed in fetal cells. In addition, the del-1 sequence is expressed in certain tumor cells.
In order to clone the full length cDNA sequence from any species encoding the entire del-1 cDNA or to clone variant forms of the molecule, labeled DNA probes made from nucleic acid fragments corresponding to any murine and human of the partial cDNA disclosed herein may be used to screen a cDNA library. More specifically, oligonucleotides corresponding to either the 5′ or 3′ terminus of the cDNA sequence may be used to obtain longer nucleotide sequences. Briefly, the library may be plated out to yield a maximum of 30,000 pfu for each 150 mm plate. Approximately 40 plates may be screened. The plates are incubated at 37° C. until the plaques reach a diameter of 0.25 mm or are just beginning to make contact with one another (3–8 hours). Nylon filters are placed onto the soft top agarose and after 60 seconds, the filters are peeled off and floated on a DNA denaturing solution consisting of 0.4N sodium hydroxide. The filters are then immersed in neutralizing solution consisting of 1M Tris HCl, pH 7.5, before being allowed to air dry. The filters are prehybridized in casein hybridization buffer containing 10% dextran sulfate, 0.5M NaCl, 50 mM Tris HCl, pH 7.5, 0.1% sodium pyrophosphate, 1% casein, 1% SDS, and denatured salmon sperm DNA at 0.5 mg/ml for 6 hours at 60° C. The radiolabelled probe is then denatured by heating to 95° C. for 2 minutes and then added to the prehybridization solution containing the filters. The filters are hybridized at 60° C. for 16 hours. The filters are then washed in 1× wash mix (10× wash mix contains 3M NaCl, 0.6M Tris base, and 0.02M EDTA) twice for 5 minutes each at room temperature, then in 1× wash mix containing 1% SDS at 60° C. for 30 minutes, and finally in 0.3× wash mix containing 0.1% SDS at 60° C. for 30 minutes. The filters are then air dried and exposed to x-ray film for autoradiography. After developing, film is aligned with the filters to select a positive plaque. If a single, isolated positive plaque cannot be obtained, the agar plug containing the plaques will be removed and placed in lambda dilution buffer containing 0.1M NaCl, 0.01M magnesium sulfate, 0.035M Tris HCl, pH 7.5, 0.01% gelatin. The phage well may then be replated and rescreened to obtain single, well isolated positive plaques. Positive plaques may be isolated and the cDNA clones sequenced using primers based on the known cDNA sequence. This step may be repeated until a full length cDNA is obtained.
It may be necessary to screen multiple cDNA libraries from different tissues to obtain a full length cDNA. In the event that it is difficult to identify cDNA clones encoding the complete 5′ terminal coding region, an often encountered situation in cDNA cloning, the RACE (Rapid Amplification of cDNA Ends) technique may be used. RACE is a proven PCR-based strategy for amplifying the 5′ end of incomplete cDNAs. 5′-RACE-Ready cDNA synthesized from human fetal liver containing a unique anchor sequence is commercially available (Clontech). To obtain the 5′ end of the cDNA, PCR is carried out on 5′-RACE-Ready cDNA using the provided anchor primer and the 3′ primer. A secondary PCR reaction is then carried out using the anchored primer and a nested 3′ primer according to the manufacturer's instructions. Once obtained, the full length cDNA sequence may be translated into amino acid sequence and examined for certain landmarks such as a continuous open reading frame flanked by translation initiation and termination sites, EGF-like domain, discoidin I-like domain, a potential signal sequence and transmembrane domain, and finally overall structural similarity to the del-1 genes disclosed herein.
5.2 Expression of DEL-1 Sequence
In accordance with the invention, a del-1 polynucleotide sequence which encodes the Del-1 protein, mutant polypeptides, peptide fragments of Del-1, Del-1 fusion proteins or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of Del-1 protein, Del-1 peptide fragments, fusion proteins or a functional equivalent thereof, in appropriate host cells. Such del-1 polynucleotide sequences, as well as other polynucleotides which selectively hybridize to at least a part of such del-1 polynucleotides or their complements, may also be used in nucleic acid hybridization assays, Southern and Northern blot analyses, etc.
Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence, may be used in the practice of the invention for the cloning and expression of the Del-1 protein. Such DNA sequences include those which are capable of hybridizing to the murine and/or human del-1 sequences under stringent conditions. The phrase “stringent conditions” as used herein refers to those hybridizing conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C.; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M Sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS.
Altered DNA sequences which may be used in accordance with the invention include deletions, additions or substitutions of different nucleotide residues resulting in a sequence that encodes the same or a functionally equivalent gene product. The gene product itself may contain deletions, additions or substitutions of amino acid residues within a Del-1 sequence, which result in a silent change thus producing a functionally equivalent Del-1 protein. Such amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine, histidine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include the following: glycine, asparagine, glutamine, serine, threonine, tyrosine; and amino acids with nonpolar head groups include alanine, valine, isoleucine, leucine, phenylalanine, proline, methionine, tryptophan.
The DNA sequences of the invention may be engineered in order to alter a del-1 coding sequence for a variety of ends, including but not limited to, alterations which modify processing and expression of the gene product. For example, mutations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, phosphorylation, etc.
Based on the domain organization of the Del-1 protein, a large number of Del-1 mutant polypeptides can be constructed by rearranging the nucleotide sequences that encode the Del-1 domains. Since the EGF-like domains of Del-1 are known to be involved in protein binding, Del-1 may directly bind to other cell surface receptors or extracellular matrix proteins via these domains, thereby controlling cell fate determination or differentiation in a manner similar to Notch and Notch ligands. Additionally, the RGD sequence in the second EGF-like domain is known to bind to certain integrins, thus Del-1 may regulate cell adhesiveness, migration, differentiation and viability via this sequence. The discoidin I-like domains of Del-1 are involved in a separate type of cell binding activity. In accordance with the observed properties of Factors V and VIII, Del-1 may directly bind proteoglycans in the extracellular matrix or on the cell surface via those domains. Therefore, the combination of various domains of full-length Del-1 permits the molecule to perform diverse types of binding. For example, the major form of Del-1 may be able to cluster integrin receptors by way of both EGF-like and discoidin I-like domains. In contrast, smaller fragments of Del-1 or its minor form would bind integrins without the ability to induce receptor clustering, and thus induce alternative signals to cells.
In view off the foregoing, the Del-1 mutant polypeptides can be generated and their functional activities compared. In addition to the minor form, Del-1 mutants may be constructed to contain only the EGF-like or discoidin I-like domains. Additionally, smaller polypeptides can be made from constructs that contain any one of the EGF-like and discoidin I-like domains.
In another embodiment of the invention, a del-1 or a modified del-1 sequence may be ligated to a heterologous sequence to encode a fusion protein. For example, for screening of peptide libraries for molecules that bind Del-1, it may be useful to encode a chimeric Del-1 protein expressing a heterologous epitope that is recognized by a commercially available antibody. A fusion protein may also be engineered to contain a cleavage site located between a Del-1 sequence and the heterologous protein sequence, so that the Del-1 may be cleaved away from the heterologous moiety.
In an alternate embodiment of the invention, the coding sequence of Del-1 could be synthesized in whole or in part, using chemical methods well known in the art. See, for example, Caruthers et al., 1980, Nuc. Acids Res. Symp. Ser. 7:215–233; Crea and Horn, 180, Nuc. Acids Res. 9(10): 2331; Matteucci and Caruthers, 1980, Tetrahedron Letter 21:719, and Chow and Kempe, 1981, Nuc. Acids Res. 9(12):2807–2817. Alternatively, the protein itself could be produced using chemical methods to synthesize a Del-1 amino acid sequence in whole or in part. For example, peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography. (e.g., see Creighton, 1983, Proteins Structures and Molecular Principles, W.H. Freeman and Co., N.Y. pp. 50–60). The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, 1983, Proteins, Structures and Molecular Principles, W.H. Freeman and Co., N.Y., pp. 34–49).
In order to express a biologically active Del-1, the nucleotide sequence coding for Del-1, or a functional equivalent, is inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. The del-1 gene products as well as host cells or cell lines transfected or transformed with recombinant del-1 expression vectors can be used for a variety of purposes. These include but are not limited to generating antibodies (i.e., monoclonal or polyclonal) that competitively inhibit activity of Del-1 protein and neutralize its activity; and antibodies that mimic the activity of Del-1 binding partners such as a receptor. Anti-Del-1 antibodies may be used in detecting and quantifying expression of Del-1 levels in cells and tissues such as endothelial cells and certain tumor cells, as well as isolating Del-1 positive cells.
5.3 Expression Systems
Methods which are well known to those skilled in the art can be used to construct expression vectors containing the del-1 coding sequence and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, for example, the techniques described in Sambrook et al., 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y.
A variety of host-expression vector systems may be utilized to express the del-1 coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the del-1 coding sequence; yeast transformed with recombinant yeast expression vectors containing the del-1 coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the del-1 coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the del-1 coding sequence; or animal cell systems. The expression elements of these systems vary in their strength and specificities. Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used in the expression vector. For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter; cytomegalovirus promoter) and the like may be used; when cloning in insect cell systems, promoters such as the baculovirus polyhedrin promoter may be used; when cloning in plant cell systems, promoters derived from the genome of plant cells (e.g., heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll α/β binding protein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; the coat protein promoter of TMV) may be used; when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used; when generating cell lines that contain multiple copies of the del-1 DNA, SV40-, BPV- and EBV-based vectors may be used with an appropriate selectable marker.
In bacterial systems a number of expression vectors may be advantageously selected depending upon the use intended for the del-1 expressed. For example, when large quantities of del-1 are to be produced for the generation of antibodies or to screen peptide libraries, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include but are not limited to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the del-1 coding sequence may be ligated into the vector in frame with the lacZ coding region so that a hybrid AS-lacZ protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic acids Res. 13:3101–3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 264:5503–5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety. In particular, murine del-1 major and minor coding sequences have been inserted in pET28a (Novagen Inc.) which contains a T7 promoter, and pMALC2 (New England Biolabs). These vectors encode fusion proteins which can be readily purified.
In yeast, a number of vectors containing constitutive or inducible promoters may be used. For a review see, Current Protocols in Molecular Biology, Vol. 2, 1988, Ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant et al., 1987, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 1987, Acad. Press, N.Y., Vol. 153, pp. 516–544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3; and Bitter, 1987, Heterologous Gene Expression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel, Acad. Press, N.Y., Vol. 152, pp. 673–684; and The Molecular Biology of the Yeast Saccharomyces, 1982, Eds. Strathern et al., Cold Spring Harbor Press, Vols. I and II.
In cases where plant expression vectors are used, the expression of the del-1 coding sequence may be driven by any of a number of promoters. For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al. 1984, Nature 310:511–514), or the coat protein promoter of TMV (Takamatsu et al., 1987, EMBO J. 6:307–311) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al., 1984, EMBO J. 3:1671–1680; Broglie et al., 1984, Science 224:838–843); or heat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley et al., 1986, Mol. Cell. Biol. 6:559–565) may be used. These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, etc. For reviews of such techniques see, for example, Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421–463; and Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7–9.
An alternative expression system which could be used to express del-1 is an insect system. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The del-1 coding sequence may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of the del-1 coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. (e.g., see Smith et al., 1983, J. Viol. 46:584; Smith, U.S. Pat. No. 4,215,051). A commercially available baculovirus expression vector pFastBac 1 (Gibco BRL, Inc.) has been constructed to contain the murine del-1 coding sequence.
In mammalian host cells, a number of viral based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the del-1 coding sequence may be ligated to an adenovirus transcripton/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing del-1 in infected hosts. (e.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655–3659). Alternatively, the vaccinia 7.5K promoter may be used. (See, e.g., Mackett et al., 1982, Proc. Natl. Acad. Sci. USA 79:7415–7419; Mackett et al., 1984, J. Virol. 49:857–864, Panicali et al., 1982, Proc. Natl. Acad. Sci. USA 79:4927–4931).
Additionally, both the murine del-1 and human coding sequences have been inserted in a mammalian expression vector, pcDNA3 (Invitrogen, Inc.), which is under the control of the cytomegalovirus promoter. Regulatable expression vectors such as the tetracycline inducible vectors may also be used to express the coding sequences in a controlled fashion.
Specific initiation signals may also be required for efficient translation of inserted del-1 coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where the entire del-1 gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of the del-1 coding sequence is inserted, exogenous translational control signals, including the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the del-1 coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:516–544).
In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. The presence of several consensus N-glycosylation sites in the del-1 extracellular domain support the possibility that proper modification may be important for Del-1 function. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, W138, yolk sac cells, etc.
For long term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the del-1 may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with the del-1 DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of foreign DNA, engineered cells may be allowed to grow for 1–2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the Del-1 protein on the cell surface. Such engineered cell lines are particularly useful in screening for molecules or drugs that affect del-1 function.
A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowry, et al., 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al., 1980, Proc. Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981), Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147) genes. Recently, additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, 1988, Proc. Natl. Acad. Sci. USA 85:8047) and ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L., 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.).
5.4 Identification of Cells that Express DEL-1
The host cells which contain the coding sequence and which express a biologically active del-1 gene product or fragments thereof may be identified by at least four general approaches; (a) DNA-DNA or DNA-RNA hybridization, (b) the presence or absence of “marker” gene functions; (c) assessing the level of transcription as measured by the expression of del-1 mRNA transcripts in the host cell; and (d) detection of the gene product as measured by immunoassay or by its biological activity. Prior to the identification of gene expression, the host cells may be first mutagenized in an effort to increase the level of expression of del-1, especially in cell lines that produce low amounts of del-1.
In the first approach, the presence of the del-1 coding sequence inserted in the expression vector can be detected by DNA-DNA or DNA-RNA hybridization using probes comprising nucleotide sequences that are homologous to the del-1 coding sequence, respectively, or portions or derivatives thereof.
In the second approach, the recombinant expression vector/host system can be identified and selected based upon the presence or absence of certain “marker” gene functions (e.g., thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.). For example, the del-1 coding sequence is inserted within a marker gene sequence of the vector, recombinants containing the del-1 coding sequence can be identified by the absence of the marker gene function. Alternatively, a marker gene can be placed in tandem with the del-1 sequence under the control of the same or different promoter used to control the expression of the del-1 coding sequence. Expression of the marker in response to induction or selection indicates expression of the del-1 coding sequence.
In the third approach, transcriptional activity for the del-1 coding region can be assessed by hybridization assays. For example, RNA can be isolated and analyzed by Northern blot using a probe homologous to the del-1 coding sequence or particular portions thereof. Alternatively, total nucleic acids of the host cell may be extracted and assayed for hybridization to such probes. Additionally, RT-PCR may be used to detect low-levels of gene expression.
In the fourth approach, the expression of the Del-1 protein product can be assessed immunologically, for example by Western blots, immunoassays such as radioimmunoprecipitation, enzyme-linked immunoassays and the like. This can be achieved by using an anti-Del-1 antibody and a Del-1 binding partner such as αVβ3. Alternatively, the biologic activities of Del-1 can be determined by assaying its ability to inhibit vascular morphogenesis of endothelial cells.
5.5 Uses of DEL-1 Engineered Cell Lines
In an embodiment of the invention, the Del-1 protein and/or cell lines that express Del-1 may be used to screen for antibodies, peptides, small molecules natural and synthetic compounds or other cell bound or soluble molecules that bind to the Del-1 protein. For example, anti-Del-1 antibodies may be used to inhibit or stimulate Del-1 function. Alternatively, screening of peptide libraries with recombinantly expressed soluble Del-1 protein or cell lines expressing Del-1 protein may be useful for identification of therapeutic molecules that function by inhibiting or stimulating the biological activity of Del-1. The uses of the Del-1 protein and engineered cell lines, described in the subsections below, may be employed equally well for other members of the del-1 gene family in various species.
In an embodiment of the invention, engineered cell lines which express most of the del-1 coding region or a portion of it fused to another molecule such as the immunoglobulin constant region (Hollenbaugh and Aruffo, 1992, Current Protocols in Immunology, Unit 10.19, Aruffo et al., 1990, Cell 61:1303) may be utilized to produce a soluble molecule to screen and identify its binding partners. The soluble protein or fusion protein may be used to identify such a molecule in binding assays, affinity chromatography, immunoprecipitation, Western blot, and the like. Alternatively, portions of del-1 may be fused to the coding sequence of the EGF receptor transmembrane and cytoplasmic regions. Assuming that Del-1 can function as a cell-bound receptor, this approach provides for the use of the EGF receptor signal transduction pathway as a means for detecting molecules that bind to Del-1 in a manner capable of triggering an intracellular signal. On the other hand, Del-1 may be used as a soluble factor in binding to cell lines that express specific known receptors such as integrins. Synthetic compounds, natural products, and other sources of potentially biologically active materials can be screened in assays that are well known in the art.
Random peptide libraries consisting of all possible combinations of amino acids attached to a solid phase support may be used to identify peptides that are able to bind to the ligand binding site of a given receptor or other functional domains of a receptor such as kinase domains (Lam, K. S. et al., 1991, Nature 354:82–84). The screening of peptide libraries may have therapeutic value in the discovery of pharmaceutical agents that stimulate or inhibit the biological activity of receptors through their interactions with the given receptor.
Identification of molecules that are able to bind to the Del-1 protein may be accomplished by screening a peptide library with recombinant soluble Del-1 protein. Methods for expression and purification of Del-1 are described in Section 5.2, supra, and may be used to express recombinant full length del-1 or fragments of del-1 depending on the functional domains of interest. For example, the EGF-like and discoidin I/factor VIII domains of del-1 may be separately expressed and used to screen peptide libraries.
To identify and isolate the peptide/solid phase support that interacts and forms a complex with Del-1, it is necessary to label or “tag” the Del-1 molecule. The Del-1 protein may be conjugated to enzymes such as alkaline phosphatase or horseradish peroxidase or to other reagents such as fluorescent labels which may include fluorescein isothiocyanate (FITC), phycoerythrin (PE) or rhodamine. Conjugation of any given label to Del-1 may be performed using techniques that are well known in the art. Alternatively, del-1 expression vectors may be engineered to express a chimeric Del-1 protein containing an epitope for which a commercially available antibody exists. The epitope specific antibody may be tagged using methods well known in the art including labeling with enzymes, fluorescent dyes or colored or magnetic beads.
The “tagged” Del-1 conjugate is incubated with the random peptide library for 30 minutes to one hour at 22° C. to allow complex formation between Del-1 and peptide species within the library. The library is then washed to remove any unbound protein. If Del-1 has been conjugated to alkaline phosphatase or horseradish peroxidase the whole library is poured into a petri dish containing substrates for either alkaline phosphatase or peroxidase, for example, 5-bromo-4-chloro-3-indoyl phosphate (BCIP) or 3,3′,4,4″-diaminobenzidine (DAB), respectively. After incubating for several minutes, the peptide/solid phase-Del-1 complex changes color, and can be easily identified and isolated physically under a dissecting microscope with a micromanipulator. If a fluorescent tagged Del-1 molecule has been used, complexes may be isolated by fluorescence activated sorting. If a chimeric Del-1 protein expressing a heterologous epitope has been used, detection of the peptide/Del-1 complex may be accomplished by using a labeled epitope specific antibody. Once isolated, the identity of the peptide attached to the solid phase support may be determined by peptide sequencing.
In addition to using soluble Del-1 molecules, in another embodiment, it is possible to detect peptides that bind to cell surface receptors using intact cells. The use of intact cells is preferred for use with receptors that are multi-subunits or labile or with receptors that require the lipid domain of the cell membrane to be functional. Methods for generating cell lines expressing del-1 are described in Section 5.3. The cells used in this technique may be either live or fixed cells. The cells may be incubated with the random peptide library and bind to certain peptides in the library to form a “rosette” between the target cells and the relevant solid phase support/peptide. The rosette can thereafter be isolated by differential centrifugation or removed physically under a dissecting microscope.
As an alternative to whole cell assays for membrane bound receptors or receptors that require the lipid domain of the cell membrane to be functional, the receptor molecules can be reconstituted into liposomes where label or “tag” can be attached.
Various procedures known in the art may be used for the production of antibodies to epitopes of the natural and recombinantly produced Del-1 protein. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library. Neutralizing antibodies, i.e., those which compete for the ligand binding site of the Del-1 protein are especially preferred for diagnostics and therapeutics.
Monoclonal antibodies that bind Del-1 may be radioactively labeled allowing one to follow their location and distribution in the body after injection. Radioisotope tagged antibodies may be used as a non-invasive diagnostic tool for imaging de novo cells of tumors and metastases.
Immunotoxins may also be designed which target cytotoxic agents to specific sites in the body. For example, high affinity Del-1 specific monoclonal antibodies may be covalently complexed to bacterial or plant toxins, such as diphtheria toxin, ricin. A general method of preparation of antibody/hybrid molecules may involve use of thiol-crosslinking reagents such as SPDP, which attack the primary amino groups on the antibody and by disulfide exchange, attach the toxin to the antibody. The hybrid antibodies may be used to specifically eliminate Del-1 expressing tumor cells.
For the production of antibodies, various host animals may be immunized by injection with the recombinant or naturally purified Del-1 protein, fusion protein or peptides, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.
Monoclonal antibodies to Del-1 may be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein, (Nature, 1975, 256:495–497), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today, 4:72; Cote et al., 1983, Proc. Natl. Acad. Sci., 80:2026–2030) and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal and Cancer Therapy, Alan R. Liss, Inc., pp. 77–96). In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci., 81:6851–6855; Neuberger et al., 1984, Nature, 312:604–608; Takeda et al., 1985, Nature, 314:452–454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce Del-1 specific single chain antibodies.
Hybridomas may be screened using enzyme-linked immunosorbent assays (ELISA) in order to detect cultures secreting antibodies specific for refolded recombinant Del-1. Cultures may also be screened by ELISA to identify those cultures secreting antibodies specific for mammalian-produced Del-1. Confirmation of antibody specificity may be obtained by western blot using the same antigens. Subsequent ELISA testing may use recombinant Del-1 fragments to identify the specific portion of the Del-1 molecule with which a monoclonal antibody binds. Additional testing may be used to identify monoclonal antibodies with desired functional characteristics such as staining of histological sections, immunoprecipitation of Del-1, or neutralization of Del-1 activity. Determination of the monoclonal antibody isotype may be accomplished by ELISA, thus providing additional information concerning purification or function.
Antibody fragments which contain specific binding sites of Del-1 may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab′)2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab′)2 fragments. Alternatively, Fab expression libraries may be constructed (Huse et al., 1989, Science, 246:1275–1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity to Del-1. Anti-Del-1 antibodies may be used to isolate Del-1-expressing cells or eliminate such cells from a cell mixture.
5.6 Uses of DEL-1 Polypeptide.
A del-1 polynucleotide may be used for diagnostic and/or therapeutic purposes. For diagnostic purposes, a del-1 polynucleotide may be used to detect del-1 gene expression or aberrant del-1 gene expression in disease states. Included in the scope of the invention are oligonucleotide sequences, that include antisense RNA and DNA molecules and ribozymes, that function to inhibit translation of del-1.
5.6.1 Diagnostic Used of a DEL-1 Polypeptide
A del-1 polynucleotide may have a number of uses for the diagnosis of diseases resulting from aberrant expression of del-1. For example, the del-1 DNA sequence may be used in hybridization assays of biopsies or autopsies to diagnose abnormalities of del-1 expression; e.g., Southern or Northern analysis, including in situ hybridization assays. Such techniques are well known in the art, and are in fact the basis of many commercially available diagnostic kits.
5.6.2 Therapeutic Uses of a DEL-1 Polypeptide
A del-1 polynucleotide may be useful in the treatment of various abnormal conditions. By introducing gene sequences into cells, gene therapy can be used to treat conditions in which the cells do not proliferate or differentiate normally due to underexpression of normal del-1 or expression of abnormal/inactive del-1. In some instances, the polynucleotide encoding a del-1 is intended to replace or act in the place of a functionally deficient endogenous gene. Alternatively, abnormal conditions characterized by overproliferation can be treated using the gene therapy techniques described below.
Abnormal cellular proliferation is an important component of a variety of disease states. Recombinant gene therapy vectors, such as viral vectors, may be engineered to express variant, signaling incompetent forms of Del-1 which may be used to inhibit the activity of the natural occurring endogenous Del-1. A signaling incompetent form may be, for example, a truncated form of the protein that is lacking all or part of its signal transduction domain. Such a truncated form may participate in normal binding to a substrate but lack signal transduction activity. Thus recombinant gene therapy vectors may be used therapeutically for treatment of diseases resulting from aberrant expression or activity of a Del-1. Accordingly, the invention provides a method of inhibiting the effects of signal transduction by an endogenous Del-1 protein in a cell comprising delivering a DNA molecule encoding a signaling incompetent form of the Del-1 protein to the cell so that the signaling incompetent Del-1 protein is produced in the cell and competes with the endogenous Del-1 protein for access to molecules in the Del-1 protein signaling pathway which activate or are activated by the endogenous Del-1 protein.
Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of recombinant Del-1 into the targeted cell population. Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors containing a del-1 polynucleotide sequence. See, for example, the techniques described in Maniatis et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y. Alternatively, recombinant Del-1 molecules can be reconstituted into liposomes for delivery to target cells.
Oligonucleotide sequences, that include anti-sense RNA and DNA molecules and ribozymes that function to inhibit the translation of a del-1 mRNA are within the scope of the invention. Anti-sense RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation. In regard to antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site, e.g., between −10 and +10 regions of a del-1 nucleotide sequence, are preferred.
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Within the scope of the invention are engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of del-1 RNA sequences.
Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.
Both anti-sense RNA and DNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
Various modifications to the DNA molecules may be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribo- or deoxy-nucleotides to the 5′ and/or 3′ ends of the molecule or the use of phosphorothioate or 2′ O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.
Methods for introducing polynucleotides into such cells or tissue include methods for in vitro introduction of polynucleotides such as the insertion of naked polynucleotide, i.e., by injection into tissue, the introduction of a del-1 polynucleotide in a cell ex vivo, i.e., for use in autologous cell therapy, the use of a vector such as a virus, retrovirus, phage or plasmid, etc. or techniques such as electroporation which may be used in vivo or ex vivo.
5.7 Uses of DEL-1 Protein
Analysis of β-gal expression in transgenic mice in which β-gal gene expression is controlled by the del-1 enhancer indicates that the del-1 gene is activated in endothelial cells undergoing vasculogenesis. Vasculogenesis refers to the development of blood vessels de novo from embryonic precursor cells. The related process of angiogenesis is the process through which existing blood vessels arise by outgrowth from preexisting ones. Vasculogenesis is limited to the embryo while angiogenesis continues throughout life as a wound healing response or to increase oxygenation of chronically stressed tissues (Pardanaud et al., 1989 Development 105:473; Granger 1994, Cell and Mol. Biol. Res. 40:81).
It is likely that Del-1 functions during embryonic vasculogenesis and in angiogenesis. For therapeutic use, it is essential that Del-1, portions of Del-1 or antibodies that block Del-1, may interact with angiogenic cells since it is stimulation or inhibition of these cells that is clinically relevant. Manipulation of Del-1 function may have significant effects on angiogenesis if Del-1 normally participates in this process.
The working examples in Sections 9 and 10 demonstrate that Del-1 exhibits an inhibitory effect on angiogenesis, which may be mediated by its interaction with αVβ3-expressing endothelial cells. Del-1 protein or recombinant proteins consisting of portions of Del-1 may function to suppress angiogenesis or induce endothelial cell apoptosis. This function could be clinically useful to prevent neovascularization of tissues such as tumor nodules. It has been demonstrated that inhibition of angiogenesis is useful in preventing tumor metastases (Fidler and Ellis, 1994, Cell 79:185). Recently, O'Reilly et al. (1994, Cell 79:315) reported that a novel angiogenesis inhibitor isolated from tumor-bearing mice, angiostatin, specifically inhibited endothelial cell proliferation. In vivo, angiostatin was a potent inhibitor of neovascularization and growth of tumor metastases. In a related report, Brooks et al. (1994, Cell 79:115) showed that integrin antagonists promoted tumor regression by inducing apoptosis of angiogenic blood vessels. These integrin antagonists included cyclic peptides containing an RGD amino acid sequence. Since Del-1 contains an RGD sequence, the use of this portion of the Del-1 molecule may have similar effects.
Manipulation of the discoidin I/factor VIII-like domains of Del-1 may also be used to inhibit angiogenesis. Apolipoprotein E (ApoE) has been shown to inhibit basic fibroblast growth factor (bFGF)-stimulated proliferation of endothelial cells in vitro (Vogel et al., 1994, J. Cell. Biochem. 54:299). This effect could also be produced with synthetic peptides based on a portion of the ApoE sequence. These results could be due to direct competition of ApoE with growth factors for binding to heparin sulfate proteoglycans, or through disruption by ApoE of cell-matrix interactions. It has been proposed that discoidin I/factor VIII-like domains such as those in Del-1 bind to proteoglycans. In addition, Del-1 is similar in structure to a number of extracellular matrix proteins. Thus, Del-1 may manipulated to effect the activity of growth factors such as bFGF or to alter interactions between endothelial cells and the extracellular matrix.
The anti-angiogenic activity of Del-1 may be used to treat abnormal conditions that result from angiogenesis. These conditions include, but are not limited to, cancer, diabetic retinopathy, rheumatoid arthritis and endometriosis. Additionally, the removal or inhibition of Del-1 in situations where it naturally inhibits blood vessel formation may be used to promote angiogenesis. These conditions include, but are not limited to, cardiac ischemia, thrombotic stroke, wound healing and peripheral vascular disease. Furthermore, Del-1 may be used to stimulate bone formation.
6.1. Materials and Methods
6.1.1. Generation of Transgenic Mice
The SLM275 transgenic mouse line was generated in a C57BL6×DBA/F1 background, and the transgenic animals had been crossed back against similar B6D2F1 animals for maintenance of the line and the generation of embryos. This transgene had been maintained in the heterozygous state, and these heterozygous mice had normal breeding capacity. However, preliminary experiments indicated that these animals were not viable in the homozygous state.
6.1.2. Molecular Cloning of DEL-1
A genomic library was constructed from high molecular weight DNA isolated from the kidney of an SLM275 transgenic animal. This DNA was subjected to partial digestion with Sau3A to obtain an average size of 20 kb, subjected to a partial fill-in reaction, and then cloned into a similarly treated lambdaphage vector (lambdaFix, Stratagene). The library constructed in this fashion had a base of approximately 2 million clones. These clones were amplified and the library stored at −70° C. A 200 basepair (bp) probe derived from the SV40 polyadenylation signal of the transgene was used as a probe and allowed the isolation of 12 lambdaphage clones. Six of these clones were randomly chosen for further investigation. These clones were mapped, and restriction fragments which did not contain transgene sequence identified. The clones were divided into two groups on the basis of common non-transgenic fragments. One such fragment from the first group of phage allowed specific hybridization to genomic blots and provided evidence that it was derived from a region adjacent to the integration site. Genomic DNA from a non-transgenic mouse of the same genetic background (B6D2 μl) was compared to that of an SLM275 transgene animal by hybridization to this probe. Rearranged bands representing fragments disrupted by transgene integration were seen in the SLM275 lanes with both EcoR1 and BamH1 digests. The flanking sequence probe was employed to screen a commercially available lambdaFixII genomic library constructed from the 129SV mouse strain (Stratagene).
A murine cDNA fragment was used as a probe to identify cDNA clones of its human homolog. The probe corresponded to nucleotides 1249 through 1566 in the murine del-1 major sequence. Human cDNA clones were isolated from a human fetal lung cDNA library (Clontech, Inc.) following standard procedures.
A transgenic mouse line was created through a fortuitous enhancer trap event. The original studies were designed to map the cell-specific and developmental-specific regulatory regions of the mouse SPARC promoter, 2.2 kilobases (kb) of the SPARC 5′ flanking sequence were placed upstream of the E. coli lacZ (beta-galactosidase or β-gal) reporter gene. The mouse SPARC gene is normally expressed in a wide variety of adult and embryonic cells which synthesize a specific extracellular matrix (Nomura et al., 1989, J. Biol. Chem. 264:12201–12207). However, one of the founder mouse lines showed a highly restricted pattern of expression quite distinct from the native SPARC gene. Expression of the lacZ reporter in this particular line of mice referred to as SLM275 was seen very early in cells of the endothelial lineage. Whole mount lacZ staining was employed for initial studies, and these embryos were subsequently sectioned and examined by light microscopy. The first cells to stain were endothelial cells forming the endocardium, the outflow tract, and the developing intervertebral vessels. Staining appeared to be predominantly restricted to endothelial cells associated with forming major blood vessels. Expression began to decline after 11.5 days pc.
The genomic region targeted by this transgene is herein referred to as del-1. Initial cloning experiments were aimed at isolating genomic sequences flanking the transgene integration site. A number of lambdaphage clones were isolated and mapped (
Exon trapping was used to evaluate genomic fragments for the presence of exons. This approach utilized a vector with a constitutive promoter driving transcription through a DNA fragment containing a splice donor site and a splice acceptor site. Between these splicing signals was a common cloning site where the genomic DNA fragment to be evaluated was cloned. Exons within this fragment would be spliced into the transcript when the construct was transfected into eukaryotic cells, such as COS cells. The transcript containing the trapped exon sequence was rescued from the COS cells by reverse transcriptase polymerase chain reaction (RT-PCR). PCR amplified DNA was cloned and evaluated.
A 160 bp exon was trapped from a fragment of genomic DNA located approximately 10 kb from the “left” integration site. Nucleotide sequence of the trapped exon was employed to screen various nucleic acid databanks through the BLAST routine at the NCBI, revealing no other gene with significant nucleic acid homology. The deduced amino acid sequence of the single open reading frame was subsequently employed in databank searches. These revealed that the protein domain encoded in the trapped exon was similar in part to domains in a number of proteins, including Factor V, Factor VIII and discoidin I (
The DNA fragment encoding the trapped exon was employed as a probe in a Southern blot experiment and shown to hybridize with regions of the del-1 locus outside of the region that was employed in the exon trap construct. Given this finding, cDNA cloning was pursued by using the exon trap probe to screen an 11.5 day embryonic mouse cDNA library. Clones were plaque purified, and inserts subcloned into plasmid for further analysis. Nucleotide sequence analysis showed that two of the embryonic cDNA clones contained the sequence of the trapped exon. Sequence from the clones was used to expand the deduced amino acid sequence of the discoidin I-like domain (
A human cDNA was isolated from a human fetal lung cDNA lambda phage library purchased from Clontech Inc. (
DNA sequence analysis of the human del-1 revealed an open reading frame of 1,446 base pairs predicted to encode a 481 amino acid protein with a molecular weight of, 53,797. The mouse cDNA encodes a 480 amino acid protein. Homology comparisons with DNA and protein databases indicated that the Del-1 protein was composed of three EGF-like protein domains, followed by two discoidin I/factor VIII-like domains (
A physiologic function for the Del-1 protein is implicated by the activities which have been demonstrated for EGF-like and discoidin I/factor VIII-like domains in other proteins. EGF-like domains have been shown to participate in protein-protein binding interactions, while the discoidin I-like domains of factor VIII are believed to mediate binding to cell membranes through association with negatively charged phospholipids. Thus, the Del-1 protein may generate a signal for endothelial cell determination or differentiation by binding to the membranes of precursor cells and interacting with an EGF-like domain receptor protein.
Key structural features of the open reading frame of human Del-1 include:
Further cloning and analysis of both the human and murine del-1 genes revealed additional variant forms. For example, a human splicing variant (Z20 clone) was obtained in which 30 bp (i.e. 10 amino acids) (SEQ ID: 30 # 66-#75) between the first and second EGF-like domains of the major form (SEQ NO ID: 30) of del-1 had been removed (
7.1. Materials and Methods
7.1.1. Whole Mount Staining of Transgenic Mouse Embryos
Male transgenic animals of second or third generation had been crossed with 8–10 week B6D2F1 females, and embryos harvested at 7.5, 8.5, 9.5, 10.5, and 13.5 days. Timing was based on the convention that noon of the day of plugging was 0.5 day post-coitum (pc). Embryos were harvested, dissected free of decidua and membranes, fixed in 2% glutaraldehyde, and stained as a whole mount in a standard X-gal indicator solution according to standard protocols. An exception was that embryos older than 11.5 days were bisected which allowed better penetration of the fixative and staining solution. Stained tissues were identified in whole mount embryos by examination at 7–70× with an Olympus SZH10 stereomicroscope, and photographed under darkfield illumination. Embryos 7.5, 8.5, 9.5, and 13.5 days pc were embedded in paraffin, sectioned, counterstained with nuclear fast red and examined under brightfield with a Zeiss Axioplan microscope.
7.1.2 Northern Blot Analysis
In order to study the expression of: the del-1 gene, Northern blots containing RNA obtained from a variety of human and mouse tissues (Clontech, Palo Alto, Calif.) were hybridized with a radiolabeled DNA probe as shown in
7.1.3 Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated using standard laboratory procedures (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, NY). Approximately 1 μg of total RNA was reverse transcribed and the cDNA was amplified by PCR (Perkin Elmer, Norwalk, Conn.). The PCR amplification conditions were: 94° C. for 30 sec, 60° C. for 30 sec, 72° C. for 30 sec for a total of 40 cycles. The amplified products were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. The amplimers were:
Expression of del-1 in various human and mouse tissues and cell lines was investigated by whole mount staining, Northern blot analysis and RT-PCR. Results of experiments are summarized in the subsections below.
7.2.1 Expression Analysis by Histochemistry
When the earliest time point was investigated by whole mount and histochemical staining in transgenic mice at day 7.5 pc, expression of the lacZ reporter gene was shown in cells forming the extra embryonic mesoderm (
At day 8.5, lacZ staining was seen in cells in the blood islands of the yolk sac. Interestingly, staining was not detected in mature endothelial cells lining the blood island, but rather in round cells found in clumps within the blood island (
LacZ staining of embryos at day 13.5 of development was evaluated in the whole mount, and in sections made from paraffin embedded embryos. By this time, there was only patchy staining of endothelial cells in large vessels such as the aorta, whereas smaller vessels had virtually no staining (
The aforementioned observations indicate that the protein encoded by the transcription unit in the del-1 locus is involved in early developmental processes in the cardiovascular system. This gene is not only a lineage marker, since it is expressed in restricted groups of endothelial cells in a temporally regulated fashion. The restricted expression seen at later stages indicates a connection with the origin of these endothelial cells, the mechanism of blood vessel formation, or the context-derived phenotype of these cells. Cells of the primordial endocardium express this marker, indicating a role in cardiogenesis. Most striking is the pattern of expression in the developing valvular apparatus of the heart. Competent endothelial cells in the forming septum and valves have been shown to undergo an epithelial-mesenchymal transformation. This transformation appears to be due, at least-in part, to an inductive signal, such as transforming growth factor beta 3, which is released by the myocardium (Potts et al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88:1516–1520; Sinning et al., 1992, Anat. Rec. 232:285–292). Reporter gene expression in the SLM275 mouse marked the competent cells of the endocardium which would respond to this signal, and expression appeared to persist for some time after the transformation (
7.2.2. Expression Analysis by Northern Blot
Expression of del-1 in various fetal and adult tissues was examined by Northern blot analysis (Tables 1 and 2). A portion of the mouse cDNA clone (0.3 kb Sac I probe) was used as a probe on six poly A RNA filters purchased from Clontech Inc. Human fetal tissues which were undergoing vasculogenesis were positive (Table 2). An organ blot generated with RNA from a 15.5 day mouse embryo indicated expression in highly vascular organs such as kidney, lung, nervous system and head. Also, the time course of expression in whole mouse embryos was consistent with the β-gal staining results observed in transgenic mice (Table 3). In general, adult mouse tissues were negative, or only weakly positive (Table 4). Mouse cDNA clones isolated from a brain cDNA library appeared to be identical to the embryonic del-1. Two human cancer cell lines tested were weakly positive (Table 5). The results of Northern blot analysis were basically consistent with the pattern for a gene which was specifically active during endothelial cell development.
7.2.3 Expression Analysis by RT-PCR
RNA from mouse yolk sac (day 8 through day 12) and mouse fetal liver (day 13 through day 19) were tested for del-1 expression by RT-PCR. All tested samples were positive, consistent with the Northern blot analysis and results from β-gal staining in transgenic mice (Table 6). Several mouse yolk sac-derived cell lines were also tested by RT-PCR for expression of del-1. For comparison, several other cell lines and total d15 mouse fetal liver RNA samples were tested. All samples shown in Table 7 except ECV304 (a human endothelial cell line) were of mouse origin. The yolk sac-derived cell lines grown in long-term culture were not expressing del-1 at a detectable level. These cell cultures were not forming endothelial cell-like structures under these conditions. In contrast, an endothelial tumor line, EOMA, expressed high levels of del-1.
A number of human tumors implanted in nude mice and cultured in vitro were shown to express del-1 by RT-PCR. For example, Table 8 shows the expression of del-1 in human osteosarcoma cell line 143B in vivo and in vitro. EOMA was used as a positive control. CD34, flk-1 and tie-2 are known markers for endothelial cells. When human and mouse del-1 specific PCR primers were used, both human (tumor) and murine (host) del-1 expression was detected. In addition, a variety of human tumor cell lines expressed del-1 in culture (Table 9). These results indicate that Del-1 may be used as a tumor marker in certain cancers diagnostically and therapeutically. In addition, host expression of del-1 is also up-regulated, possibly due to angiogenesis in tumor sites.
8.1. Materials and Methods
8.1.1. Antibody Production
A partial del-1 cDNA encoding amino acids 353 to 489 of the murine gene was cloned into pMALC2 (New England Biolabs) to generate a maltose binding protein-partial Del-1 fusion protein. The del-1 sequence included in this construct encodes a portion of the carboxyl terminal discoidin-like domain. Recombinant fusion protein was expressed and purified over an amylose affinity matrix according to the manufacturer's recommendations. Protein was emulsified into Freund's complete adjuvant, and injected as multiple subcutaneous injections into two New Zealand White rabbits. Boosting and harvesting of immune serum was performed according to established methodology (Harlow and Lane, 1988, Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory). Immune serum obtained after the second boost was subjected to affinity purification. First, the anti-serum was precleared over a Sepharose column coupled to total bacterial lysate. Subsequently, the antiserum was purified over an affinity column made from recombinant fusion protein coupled to Sepharose. The specificity of the antiserum, was evaluated first with western blots containing proteins from bacteria expressing the recombinant fusion protein before and after cleavage with factor Xa, or the maltose binding protein alone. Whole bacterial lysates from cells induced with IPTG were run on polyacrylamide gels, transferred to nitrocellulose, and probed with the affinity-purified antiserum. While crude antiserum labeled bands corresponding to maltose binding protein and the Del-1 portion of the fusion protein, affinity-purified antiserum specifically labeled the Del-1 component of the fusion protein.
8.1.2 Western Blot
For western blots of eukaryotic proteins, cells were harvested by lysis in a standard lysis buffer or Laemmli loading buffer. Cell culture supernatant was collected and concentrated by centrifugation in a CENTRICON filter, and extracellular matrix harvested by first removing cells with 1 mM EDTA in PBS, and then scraping the cell culture dish with a small volume of Laemmli buffer at 90° C.
Immunohistochemistry was performed on sections prepared from Bouin's fixed, paraffin-embedded, staged mouse embryos according to well established methodology (Hogan et al., 1994, Manipulating the Mouse Embryo, Cold Spring Harbor Press; Quertermous et al., 1994 Proc. Natl. Acad. Sci. U.S.A. 91:7066). The affinity-purified Del-1 antiserum was employed at a dilution of 1:500 to 1:1000, and the specificity of staining verified by competition with recombinant protein. Staining of cartilage was amplified by pre-treating the section with dilute trypsin solution.
8.1.4 Transfection of Yolk Sac Cells
A eukaryotic expression vector was constructed by cloning the entire open reading frame of the major del-1 transcript into ohbAPr-3-neo (Gunning et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84:4831). This construct was transfected into yolk sac cells with Lipofectamine (Gibco BRL), and clones selected in the presence of 1000 μg/ml of G418. Clones were evaluated for del-1 expression by northern and western blotting, and a group of clones with varying amounts of Del-1 protein were selected for further study. To serve as negative controls, a group of clones were randomly selected from a transfection with the empty phbAPr-3-neo vector.
The major murine del-1 coding sequence was inserted into a eukaryotic expression vector and transfected into Del-1-non-expressing yolk sac cells (Wei et al., 1995, Stem Cell 13:541). Pooled transfectants with an empty expression vector or the del-1 construct were selected in G418. Lysates, cell culture supernatants and extracellular matrix were prepared from transfected cells, and reacted with an affinity-purified rabbit antiserum in Western blots. The polyclonal antiserum was generated to recombinant Del-1 fusion protein expressed in bacteria.
Several stably transfected yolk sac cell clones with the del-1 gene were selected (
In order to test the expression of del-1 in tumor cells by immunohistochemistry, human glioma cells were implanted in nude mice. The tumor was isolated, sectioned and stained with the aforementioned antibody followed by an anti-rabbit antibody conjugated with horse radish peroxidase and developed with Sigma Fast Red substitute.
9.1 Materials and Methods
9.1.1. Angiogenesis Assays
In vitro angiogenesis assays on “MATRIGEL” (Biocoat, Becton Dickinson) were conducted in 24 well plates coated with 50 μl of “MATRIGEL”. del-1 transfectants and control transfectants were plated at a density of 5×104 cells/well (low density) or 2×105 cells/well (high density), and observed for seven days.
For the assay evaluating morphogenetic potential of wild type yolk sac cells on del-1 conditioned matrix, the matrix was generated by growing 106 del-1 transfectants in 6 cm dishes for 7 days. A control matrix was generated by growing control transfectants under identical conditions. Transfected cells were removed with 0.5 M EDTA and extensive washing, and 106 wild type yolk sac cells were plated on the matrix produced by the del-1 or the control transfectants. Cells were cultured and observed for seven days.
For the in vitro angiogenesis sprouting assay, del-1 and control transfectants were trypsinized, and 106 cells cultured in a 15 ml conical tubes for 48 hours. Cell cultures were then transferred into a bacterial petri dish, and cultured for 4–7 days. Under these conditions, cell aggregates were formed. Several aggregates were collected for del-1 and control transfectants, and these were transferred to 24 well plates coated with “MATRIGEL”. Sprouting angiogenesis was evaluated at 24 and 43 hours.
The yolk sac cell line, YS-B, was chosen as the parental cell for del-1 tansfection because it had characteristics of embryonic endothelial cells, did not express del-1, was clonal and long lived in culture (
The del-1 transfected yolk sac clones and mock-transfected yolk sac lines were compared for their ability to form branching vascular-like structures on “MATRIGEL”. After 24 hours on “MATRIGEL”, the negative control transfectants had established an intricate network typical for these cells (
Since Del-1 protein is deposited in the extracellular matrix, one del-1 expressing clone, L10, was used to generate a cell culture matrix containing Del-1 protein. Matrix generated by negative control clones should differ only by the absence of Del-1. Transfected and control lines were cultured for 7 days, and then gently removed from the culture dish by extensive washing with 1 mM EDTA. By visual inspection, only a rare cell was not removed with this technique. Non-transfected native yolk sac cells were then plated on the Del-1-containing and the control matrices, and scored for their ability co assemble into a network. The yolk sac cells required several days at high density to undergo morphogenesis, and the network was lace-like in appearance. Cells grown on the matrix produced by negative control transfectants were able to produce the network (
Next, an in vitro angiogenesis sprouting assay was employed with the transfected yolk sac lines. This assay has been employed to evaluate angiogenic potential (Pepper et al. 1991, J. Cell. Physiol. 146:170). Transfected cells were allowed to stand overnight in a conical tube to allow them to aggregate, and the cell mass was then placed on “MATRIGEL”. The ability of the del-1 expressing cells to migrate onto the “MATRIGEL” and assemble into branching structures was compared to control cells. Within 24 hours, the control cells formed a series of branching projections, while the cells expressing del-1 remained virtually confined to the cellular aggregate (
Hence, Del-1 inhibits vascular morphogenesis and may be used to regulate endothelial cell differentiation.
10.1 Materials and Methods
10.1.1. Recombinant DEL-1 Purification and Refolding
Recombinant murine Del-1 protein (major form) was prepared using an E. coli expression system and protein refolding technique. E. coli cells with the del-1 containing pET28a vector (Novagen Inc.) were grown and induced following the protocol recommended by the manufacturer. Approximately 50 to 100 mg of crude recombinant Del-1 were routinely produced from 1 L of bacterial culture in the form of insoluble cytoplasmic inclusion bodies. Inclusion bodies were isolated by sonication of the E. coli cells, centrifugation and collection of the pellet fraction. Inclusion bodies from 500 ml of culture were then washed three times with 50 ml of 2M Urea, 0.025 M Tris-Cl (pH 8.0), 0.025% Triton X100. This procedure yielded a crude, insoluble, Del-1 product of >80% purity.
Recombinant Del-1 was dissolved by suspending the pellet from 500 ml of culture in 2.5 ml of 8M Urea, 100 mM DTT, 0.1 M Tris-Cl (pH 8.0), 0.05% Triton X100, followed by incubation at room temperature for 1 hr. Insoluble material remaining was removed by centrifugation and the soluble supernatant fraction was diluted 10 fold to 25 ml with 8M Urea, 100 mM Tris-Cl (pH 8.0), 0.05% Triton X100. Protein concentration was then measured by Bradford protein determination assay.
Soluble, reduced Del-1 was refolded by diluting to a final concentration of 0.01 mg/ml into refolding buffer: 100 mM Tris-Cl (pH 8.0), 100 mM (NH4)2SO4, 2 mM reduced glutathione, 0.5 mM oxidized glutathione, 0.05% sodium azide, 0.025 mg/ml PMSF. Refolding was performed by incubating this reaction mix at 4° C. for one week. Refolded Del-1 was then concentrated using an Amicon spiral concentrator and the soluble material remaining was collected.
The recombinant Del-1 product produced from the pET28a expression vector is a fusion protein with both N-terminal and C-terminal polyhistidine tags. This product was purified using the Novagen His tag resin purification system, following the protocol recommended by the supplier.
Refolded murine recombinant Del-1 was soluble and stable when stored at 4° C. in Tris-Cl buffer with 100 mM (NH4)2SO4 at concentrations of less than or equal to 100 mg/ml.
10.1.2. Cell Adhesion Assays
Human umbilical vein endothelial cells (HUVEC) (Clonetics Inc., San Diego, Calif.) were grown as indicated by the supplier in endothelial growth media supplemented with 10 ng/ml human recombinant epidermal growth factor, 1 μg/ml hydrocortisone, 50 μg/ml gentamicin, 12 μg/ml bovine brain extract and 2% FBS. Cells were grown at: 37° C./5% CO2 to 70% confluency before use in the binding assay. Non-tissue culture treated 96 well plates were coated with appropriate levels of target protein (1–20 μg of either murine recombinant Del-1, vitronectin, or BSA) diluted in calcium and magnesium free PBS for 24 hrs at 4° C. The plates were washed with PBS and blocked for 30 min with a solution of heat treated (95° C. for 5 min) PBS containing 3% BSA. HUVEC cells were harvested by trypsinization and resuspended in an adhesion buffer (Hanks balanced salt solution pH 7.4 containing 10 mM Hepes, 2.2 mM MgCl2, 0.2 mM MnCl2 and 1% BSA). Cells (104/100 μl) were added to each well in the presence or absence of the indicated antagonists or controls at varying concentrations. Antagonists included anti-human αVβ3 (clone LM609, Chemicon Inc.), RGE peptides (the inactive control GRGESP) or RGD the stable antagonist GPenGRGDSPCA or GRGDdSP all from Gibco). Cells were incubated at 37° C./5% CO2 for 60–90 min and wells were washed until no cells remained in the BSA control. To count remaining cells, 100 μl of endothelial media was added to each well. Cells number was determined by the Promega Cell titer AQ as indicated by the manufacturer.
Recombinant Del-1 protein and del-1 transfectants bound HUVEC. In order to identify a cellular receptor on HUVEC for Del-1, various peptides and antibodies were used to inhibit the interactions between Del-1 and HUVEC in cell adhesion assays.
αVβ3 is an integrin expressed by certain cell types and is associated with bFGF-induced angiogenic endothelial cells. Agents that bind to this integrin induce apoptosis of angiogenic endothelial cells. Since Del-1 binds to this integrin, it may be used to induce apoptosis during angiogenesis in tumor sites to reduce tumor growth.
DNA from P1 clone 10043 was labeled with digoxigenin dUTP by nick translation. The labeled probe was combined with sheared human DNA and hybridized to normal metaphase chromosomes derived from PHA stimulated peripheral blood lymphocytes in a solution containing 50% formamide, 10% dextran sulfate and 2×SSC. Specific hybridization signals were detected by incubating the hybridized slides in fluoresceinated antidigoxigenin antibodies followed by counterstaining with DAPI. The initial experiment resulted in specific labeling of the long arm of a group B chromosome.
A second experiment was conducted in which a probe that had previously been mapped to 5q34, and confirmed by cohybridization with a probe from the cri du chat locus which is known to localize to 5p15, was cohybridized with clone 10043. This experiment resulted in the specific labeling of the mid and distal long arm of chromosome 5 (
12. Deposit of Microorganisms
The following organisms were deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852.
Strain Designation Accession No.
The present invention is not to be limited in scope by the exemplified embodiments or deposited organisms which are intended as illustrations of single aspect of the invention, and any clones, DNA or amino acid sequences which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. It is also to be understood that all base pair sizes given for nucleotides are approximate and are used for purposes of description.
All publications cited herein are incorporated by reference in their entirety.