EP1287121A2 - Extracellular domain of ryk protein for regulating angiogenesis - Google Patents
Extracellular domain of ryk protein for regulating angiogenesisInfo
- Publication number
- EP1287121A2 EP1287121A2 EP01935233A EP01935233A EP1287121A2 EP 1287121 A2 EP1287121 A2 EP 1287121A2 EP 01935233 A EP01935233 A EP 01935233A EP 01935233 A EP01935233 A EP 01935233A EP 1287121 A2 EP1287121 A2 EP 1287121A2
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- European Patent Office
- Prior art keywords
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- protein
- ryk
- variant
- amino acid
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- This invention relates to a method of regulating angiogenesis using newly identified polypeptides or polynucleotides encoding such polypeptides. More particularly, the method comprises using the Ryk protein, or fragments derived therefrom, to regulate angiogenesis in human or animal tissue.
- Ryk is a member of the Receptor Tyrosine Kinase (RTK) family that has impaired catalytic activity and whose ligand(s) have not yet been identified.
- RTK Receptor Tyrosine Kinase
- the open reading frame of human Ryk encodes 607 amino acids (aa), has 2 potential transmembrane domains and exhibits closest homology in its catalytic (i.e., activation and nucleotide binding) domain to RTKs such as met (HGF/SF-R) and IGF-1R (Tamagnone et al., Oncogene 1993; 8:2009).
- the Ryk catalytic region shows closest homology with v-sea (39%) (Wang et al., Mol. Med. 1996; 2:189).
- Ryk differs from other RTK family members at a number of conserved residues in its catalytic domain (Katso et al., Mol. Cell. Biol. 1999; 19:6427).
- the amino acid substitutions in this domain account for Ryk's inability to undergo autophosphorylation or to phosphorylate substrates.
- ligand stimulation of a Ryk chimeric receptor results in activation of mitogen- activated protein kinase pathway (Katso et al., Mol. Cell. Biol. 1999; 19:6427).
- Ryk does not exhibit significant homology in its extracellular domain to other RTK families.
- Ryk has a relatively small extracellular domain. Variations of the amino acid/nucleotide sequence of Ryk has been described by several authors (See e.g., Tamagnone et al., Oncogene 1993; 8:2009 (the extracellular domain is described as having an amino acid sequence consisting of 191 amino acids) and Halford et al., J. Biol. Chem. 1999; 274:7379 (the extracellular domain is described as having the same amino acid sequence as described by Tamagnone; however, ten amino acids at the C terminus are missing making the extracellular domain 181 amino acids)).
- Ryk RNA is present at greatly increased levels in the basal layer of skin and tongue epithelia and the interviUous layer and some crypt bases of the intestine (Serfas et al., Oncogene 1998; 17:3435). Ryk is induced in epithelial cells seeking a final place in a differentiated tissue or during remodeling of the endometrium, and it has been hypothesized to be involved in cellular recognition (Serfas et al., Oncogene 1998; 17:3435).
- Human Ryk is overexpressed in borderline and malignant ovarian tumors (Katso et al., Cancer Res. 1999; 59:2265). In serous and clear cell tumor subtypes, increased expression is observed in epithelium, stroma, and blood vessels. In malignant tumors, the increased expression is predominantly confined to epithelium (Wang et al., Mol. Med. 1996; 2:189).
- the human Ryk cDNA has been isolated from a complementary DNA library of SKON-3, an epithelial ovarian cancer cell line, using PCR (Wang et al., Mol. Med. 1996; 2:189). There is minimal to absent expression of human Ryk on the surface epithelium of normal ovaries.
- RYK is the mammalian homologue of Drosophila Lio protein which has been determined to be involved in learning and memory and axon guidance (pathway selection) in the embryo & adult (Moreau-Fauvarque et al, Mech. Dev. 1998; 78:47).
- Lionette was identified in screens for embryonic nervous system axonal guidance defects called derailed.
- Lionette mutants are viable but present structural brain defects in the adult central complex where the central brain axons behave as if abnormally attracted by midbrain area. In derailed mutant embryos, a small set of intemeurons that expressed the gene failed to make correct pathway choices (Callahan et al. Nature 1995; 376:171).
- Ryk is expressed in CD3-, CD4-, and CD8- T cells, pre-T cells, thymic epithelial cells, and mature myeloid cells, but not myeloid precursors or B cell precursors (Simoneaux et al., J. Immunol. 1995; 154:1157).
- Ryk expression is observed in differentiated cells (Lin+) but not in precursor cells (Lin-), and it was hypothesized that Ryk expression may be regulated during hematopoietic development by lineage commitment and stage of maturation.
- Ryk is also expressed by a small subset of developing embryonic muscles and neighboring epidermal cells during muscle attachment site selection (Callahan et al., Development 1996; 122:2761). In derailed mutants, muscles often fail to attach at appropriate locations although their epidermal attachment cells appear unaffected.
- Angiogenesis the formation of new capillaries from preexisting blood vessels, is a multistep, highly orchestrated process involving vessel sprouting, endothelial cell migration, proliferation, tube differentiation, and survival.
- angiogenesis is essential for the growth and persistence of solid tumors and their metastases (Folkman et al. (1989) Nature 339:58-61; Hori et al. (1991) Cancer Research 51:6180-84; Kim et al. (1993) Nature 362:841-844; MiUauer et al. (1996) Cancer Research 56:1615-20).
- tumors upregulate their production of a variety of angiogenic factors, including the fibroblast growth factors (FGF and BFGF) (Kandel et al. (1991) Cell. 66:1095-104) and vascular endothelial cell growth factor/vascular permeability factor (VEGF/VPF).
- FGF and BFGF fibroblast growth factors
- VEGF/VPF vascular endothelial cell growth factor/vascular permeability factor
- angiostatin and thrombospondin Chombospondin (Chen et al. (1195) Cancer Research 55:4230-33; Good et al. (1990) Proc Natl Acad Sci U S A. 87:6624-28; O'Reilly et al. (1994) Cell 79:315-28).
- angiogenic phenotype is the result of a net balance between these positive and negative regulators of neovascularization (Good et al. (1990), supra; O'Reilly et al. (1994), supra; Parangi et al. (1996) Proc Natl Acad Sci U S A. 93:2002-07; Rastinejad et al. (1989) Cell 56:345-55).
- endogenous inhibitors of angiogenesis have been identified, although not all are associated with the presence of a tumor. These include platelet factor 4 (Gupta et al. (2000) Blood 95:147-55), interferon-alpha, interferon-inducible protein 10 (Angiolilloet al.
- Angiostatin is an approximately 38 kiloDalton (kDa) specific inhibitor of endothelial cell proliferation.
- Angiostatin is an internal fragment of plasminogen containing at least three of the five kringles of plasminogen.
- Angiostatin has been shown to reduce tumor weight and to inhibit metastasis in certain tumor models. (O'Reilly et al. (1994), supra).
- Assays used for measuring effect on angiogenesis activity include comeal pocket assay (Invest. Ophtalmol. Vis. Sci. 1999, 40: 2498-505), chorioallantoic membrane angiogenesis assay (CAM) (Am. J. Pathol. 1983, l l l(3):282-7), and endothelial cell proliferation and migration assays (Biochem. Biophys. Res. Comm. 2000, 271:499-508).
- the Ryk protein possesses a novel biological activity in regulating angiogenesis. This activity has been demonstrated via in vitro and in vivo assays for detecting molecules having activity in modulating angiogenesis. The activity was measured using variant Ryk proteins.
- a "variant Ryk protein” is intended to include a Ryk protein which lacks the transmembrane portion of the protein (e.g., the extracellular domain the Ryk protein) or fragments derived therefrom, as described further below, where the variant Ryk protein exhibits activity in regulating angiogenesis.
- the instant invention encompasses the use of a variant Ryk protein for regulating or modulating angiogenesis.
- the current invention further encompasses the use of a variant Ryk protein for the treatment of a disease or clinical condition where angiogenesis is relevant to the causation or treatment of the disease or clinical condition, including but not limited to cancer, wound healing, diabetic retinopathies, macular degeneration, and cardiovascular diseases.
- Further uses of the protein include treatment of clinical conditions involving angiogenesis in the reproductive system, including regulation of placental vascularization or use as an abortifacient.
- the instant invention also encompasses pharmaceutical compositions containing a variant Ryk protein and the use of the pharmaceutical compositions for the treatment of the above-mentioned diseases or clinical conditions.
- novel mature polypeptides comprising the amino acid sequence shown in Figure 1 (SEQ ID NO:l), as well as biologically active and diagnostically or therapeutically useful fragments, analogues and derivatives thereof.
- novel mature polypeptides comprising the amino acid sequence shown in Figure 2 (SEQ ID NO:2), as well as biologically active and diagnostically or therapeutically useful fragments, analogues and derivatives thereof.
- novel mature polypeptides comprising the amino acid sequence shown in Figure 3 (SEQ ID NO:3), as well as biologically active and diagnostically or therapeutically useful fragments, analogues and derivatives thereof.
- antibodies to the polypeptides of the present invention especially antibodies which bind specifically to an epitope made up of the sequences shown in Figures 1-3 (SEQ ID NOS:l-3) or a sequence which shares at least a 60%, preferably at least a 70%, more preferably at least an 80%, still more preferably a 90%, or most preferably at least a 95% sequence identity over at least 20, preferably at least 30, more preferably at least 40, still more preferably at least 50, or most preferably at least 100 residues to the sequences shown in Figures 1-3 (SEQ ID NOS:l-3).
- nucleic acid molecules encoding the polypeptides of the present invention, including mRNAs, DNAs, cDNAs, genomic DNA, as well as antisense analogs thereof and biologically active and diagnostically or therapeutically useful fragments thereof.
- processes for producing such polypeptides by recombinant techniques through the use of recombinant vectors there are provided recombinant prokaryotic and/or eukaryotic host cells comprising a nucleic acid sequence encoding a polypeptide of the present invention.
- a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides, for therapeutic purposes involving the regulation of angiogenesis for example, the treatment of cancer, wound healing, diabetic retinopathies, macular degeneration, cardiovascular diseases, and clinical conditions involving angiogenesis in the reproductive system, including regulation of placental vascularization or use as an abortifacient.
- nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to a polynucleotide encoding a polypeptide of the present invention.
- diagnostic assays for detecting diseases or susceptibility to diseases related to mutations in a nucleic acid sequence of the present invention and for detecting over-expression or underexpression of the polypeptides encoded by such sequences.
- gene therapy is defined as the process of providing for the expression of nucleic acid sequences of exogenous origin in an individual for the treatment of a disease condition within that individual.
- Figure 1 shows the amino acid sequence of the extracellular domain of the Ryk protein as described by Tamagnone et al., as well as the corresponding nucleotide sequence.
- Figure 2 shows the amino acid sequence of the extracellular domain of the Ryk protein as described by Halford et al, J. Biol. Chem. 1999; 274:7379, as well as the corresponding nucleotide sequence.
- Figure 3 shows the amino acid sequence of a portion of the extracellular domain of the Ryk protein consisting of the Wnt inhibitory factor (WIF) domain, as well as the corresponding nucleotide sequence.
- WIF Wnt inhibitory factor
- FIGS 4A and 4B show the dose dependent inhibitory effect of Ryk-Fc on HUVEC capillary-like organization in MATRIGEL matrix. Results are expressed as percentage of control, which represents the capillary-like organization of untreated HUVEC in MATRIGEL matrix.
- Figure 5 shows the dose dependent inhibitory effect of Ryk-myc-His containing supernatant on HUVEC capillary-like organization in MATRIGEL matrix. Results are expressed as percentage of control, which represents the capillary-like organization of untreated HUVEC in MATRIGEL matrix.
- Figure 6 shows the dose dependent inhibitory effect of Ryk-Fc on HUVEC capillary-like organization in MATRIGEL matrix in the presence of exogenously added bFGF, VEGF or IL-8. Results are expressed as percentage of control, which represents the capillary-like organization of untreated HUVEC in MATRIGEL matrix.
- Figures 7A and 7B show the dose dependent inhibitory effect of WIF, WIF-Fc, NWIF- his, WIF-his and Ryk-his on HUVEC capillary-like organization in MATRIGEL matrix in the presence of exogenously added IL-8. Results are expressed as percentage of control, which represents the capillary-like organization of untreated HUNEC in MATRIGEL matrix.
- Figure 8 shows the specificity of the Ryk-Fc fusion protein using the single chain antibody generated against the Ryk-Fc fusion protein (scFv lb4).
- Figure 9 shows the effect of the expression of Ryk-Fc on the growth of B16 melanoma lung metastases in vivo in syngeneic mice.
- B16 cells were infected ex vivo with an adenovirus containing the Ryk-Fc gene and implanted in mice by tail vein injection. Results are expressed as number of lung metastases.
- Figure 10 shows the effect of the expression of Ryk-Fc on angiogenesis in vivo in the rat cornea. Hydron pellets containing the angiogenic agent, recombinant bFGF, and varying concentrations of Ryk-Fc were implanted into the rat cornea and angiogenesis was examined microscopically seven days later. Grades of angiogenesis were scored blindly by two individuals using points of reference.
- Figure 11 shows the purification of the anti-Ryk scFv antibody using Mono Q 5/5 ion- exchange chromatography.
- Figure 12 shows the purification anti-Ryk scFv 1B4 and endotoxin removal using Mono Q 5/5 ion-exchange chromatography.
- Figure 13 shows an SDS-PAGE of the anti-Ryk scFv Mono Q 5/5 purified antibody, highly purified from E. Coli.
- An aliquot of the Mono Q 5/5 elution (lane 1) and molecular size markers of the indicated size in kDa (lane 2) were resolved on a 4-12% bis-tris gel ( ⁇ ovex, Carlsbad, CA) and developed with coomassie stain.
- Figure 14 shows the interaction of the anti-Ryk scFv antibody with Ryk-Fc.
- the BIAcore 2000 instrument was used to characterize the interaction of the scFv antibody with Ryk- Fc. Data was collected using at least 6 different concentrations of Ryk-Fc, injected in duplicate. All experiments were conducted at 25oC using parameters discussed herein.
- the variant Ryk proteins of the present invention comprise full length, wild type Ryk proteins which lack the transmembrane portion, thereby rendering the protein soluble.
- the variant Ryk proteins of the present invention comprise the extracellular domain ("ECD") of the full length, wild type Ryk protein, or a fragment the ECD which maintains the ability to modulate angiogenesis.
- ECD extracellular domain
- Slight variations of the amino acid sequence of the Ryk protein's extracellular domain have been described in the literature by at least two different groups (See e.g., Tamagnone et al., Oncogene 1993; 8:2009 (the extracellular domain is described as having an amino acid sequence consisting of 191 amino acids (SEQ ID NO:l) and Halford et al., J. Biol. Chem.
- the extracellular domain is described as having the same amino acid sequence as described by Tamagnone; however, ten amino acids at the C terminus are missing making the extracellular domain 181 amino acids (SEQ ID NO:2)); these references are incorporated herein in their entirety.
- the variant Ryk proteins of the present invention comprise the Wnt inhibitory factor (WIF) domain of the extracellular domain of the full length, wild type Ryk protein (SEQ ID NO:3), or a fragment thereof which maintains the ability to modulate angiogenesis.
- WIF Wnt inhibitory factor
- polypeptides of the present invention include those polypeptides having the deduced amino acid sequences given by SEQ ID NOS:l-3.
- the polypeptides of the present invention may include additional amino acid sequences appended to the N- or C-terminal of the peptides having the deduced amino acid sequences given by SEQ ID NOS:l-3.
- the polypeptides of the present invention may be recombinant polypeptides, natural polypeptides, or synthetic polypeptides, preferably recombinant polypeptides.
- protein is synonymous with "polypeptide.”
- the present invention further relates to polypeptides which share at least a 60%, preferably at least a 70%, more preferably at least an 80%, still more preferably a 90%, yet still more preferably a 95%, or most preferably at least a 98% sequence identity over at least 20, preferably at least 30, more preferably at least 40, still more preferably at least 50, yet still more preferably at least 100, or most preferably at least 150 residues with SEQ ID NOS:l-3.
- a "variant Ryk protein” is intended to also include polypeptides of the present invention as referred to in this paragraph.
- the polypeptide of the present invention is at least 20, preferably at least 30, more preferably at least 40, still more preferably at least 50, or most preferably at least 100 residues long.
- the peptide of the present invention is less than 600, preferably less than 550, more preferably less than 500, still more preferably less than 480 residues long.
- the peptide of the present invention is less than 250, preferably less than 185, more preferably less than 150, still more preferably less than 100 residues long, or most preferably less than 60 residues long.
- the invention also contemplates polypeptides which share at least a 60%, preferably at least a 70%, more preferably at least an 80%, still more preferably a 90%, or most preferably at least a 95% sequence identity over at least 20, preferably at least 30, more preferably at least 40, still more preferably at least 50, or most preferably at least 100 residues SEQ ID NOS:l-3.
- Polypeptides of the present invention exhibiting such a percent identity to sequences as above-described may be assayed for biological activity as described herein by using the MATRIGEL matrix assay, as described herein, or by using other assays directed to measuring activity in regulating angiogenesis, as are known in the art.
- the process of measuring for biological activity is within the scope of one skilled in the art given the disclosure herein.
- Modulating angiogenesis includes processes resulting in either upregulation or downregulation of angiogenic processes.
- Such a polypeptide as described above may be (i) one in which one or more of the amino acid residues are substituted (as compared to SEQ ID NOS:l-3) with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethyleneglycol), or (iv) one in which additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence or mature protein sequence beyond the Ryk extracellular domain, or (v) one in which one or more amino acids are deleted from or inserted into the sequence of the polypeptide.
- a polypeptide of the present invention may contain amino acids other than the 20 gene-encoded amino acids.
- the polypeptides may be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side- chains, and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications.
- Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslational natural processes or may be made by synthetic methods.
- Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
- polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
- isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
- a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
- Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
- Similarity refers to sequence conservation, or “homology”, between two or more peptides or two or more nucleic acid molecules, normally expressed in terms of percentages.
- a position in the compared sequences is occupied by the same base or amino acid (“residue"), then the molecules are identical at that position.
- a position in two compared peptide sequences is occupied by an amino acid with similar physical properties (a conservative substitution as determined by a given scoring matrix; similarity is thus dependent on the scoring matrix chosen)
- the percent identity or similarity can be maximized by aligning the compared sequences alongside each other, sliding them back and forth, and conservatively introducing gaps in the sequences where necessary.
- the percent identity is calculated by counting the number of identical aligning residues dividing by the total length of the aligned region, including gaps in both sequences, and multiplying by 100. Identity would thus be expressed as, e.g., "60% identity over 200 amino acids,” or “57% identity over 250 amino acids.” Alternatively, such sequences would be described as being “60% identical over 200 amino acids” or “57% identical over 250 amino acids,” respectively. Similarity is calculated by counting both identities and similarities in the above calculation. For example, the alignment below has 37.5% sequence identity over 56 amino acids ( (21 identities/56 residues)X100% ), where 56 is the total length of the aligned region. As used herein, "percent identity” and “percent sequence identity” are interchangeable.
- the same alignment below has 55.4% sequence similarity over 56 amino acids ( (31 similarities/56 residues)X100% ), where 56 is the total length of the aligned region.
- conservative substitutions are indicated by a plus sign and the total similarities is given by the sum of the identities and the conservative substitutions. (As noted above, determination of conservative substitutions is dependent on the scoring matrix chosen. The same alignment below may yield a different value for percent similarity using a different scoring matrix.)
- the invention further encompasses polynucleotides which code for the above- described polypeptides of the present invention.
- These polynucleotides may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
- the DNA may be double-stranded or single-stranded.
- the polynucleotides may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide (and, optionally, additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.
- the term "polynucleotide encoding a polypeptide” encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
- the present invention further relates to variants of the herein above-described polynucleotides.
- the variants of the polynucleotides may be naturally occurring allelic variants of the polynucleotides or non-naturally occurring variants of the polynucleotides.
- an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion, or addition of one or more nucleotides which does not substantially alter the function of the encoded polypeptides.
- the present invention includes polynucleotides encoding the same mature polypeptides as described in Example 1, below, as well as variants of such polynucleotides which variants include deletion variants, substitution variants, and addition or insertion- variants.
- the present invention also includes polynucleotides wherein the coding sequence for the mature polypeptides may be fused to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell.
- the polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide.
- the polynucleotides may also encode for a proprotein which is the mature protein plus additional amino acid residues.
- a mature protein having a prosequence is a proprotein and is an inactive form of the protein.
- the polynucleotides of the present invention may code for a mature protein or for a protein having a prosequence or for a protein having both a prosequence and a presequence (leader sequence).
- the polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention.
- the marker sequence may be, for example, a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
- the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein. Wilson et al., 1984, Cell 37: 767.
- the FLAG tag is based on the FLAG marker octapeptide (N- AspTyrLysAspAspAspAspLys-C) (SEQ ID NO: 13).
- the FLAG sequence is hydrophilic and the last 5 amino acids (AspAspAspAspLys) (subsequence of SEQ ID NO: 13) represent the target sequence of the protease enterokinase.
- gene means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). Fragments of a gene may be used as a hybridization probe for a cDNA library to isolate the full length gene and to isolate other genes which have a high sequence similarity to the gene or similar biological activity. Probes of this type typically have at least 20 bases and preferably have at least 30 bases and may contain, for example, 50 or more bases.
- the probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promoter regions, exons, and introns.
- An example of a screen comprises isolating the coding region of a gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
- the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 80% identity, more preferably at least a 90% identity, still more preferably at least a 95% identity, and most preferably at least 98% identity to a polynucleotide which encodes a polypeptide of the present invention, as well as fragments thereof, which fragments have at least 20 bases and preferably have at least 30 bases and more preferably have at least 50 bases, and to polypeptides encoded by such polynucleotides.
- a preferred embodiment is given by polynucleotides that have at least a 70% identity, preferably at least 80% identity, more preferably at least a 90% identity, still more preferably at least a 95% identity, and most preferably at least 98% identity to a polynucleotide which encodes the polypeptides having the sequences given by SEQ ID NOS:l-3.
- Such polynucleotides may be contained within larger nucleotide sequences, as in embodiments of the present invention given by SEQ ID NO.T7-20.
- the invention is directed to polynucleotides which are less than 1500, preferably less than 1000, more preferably less than 600, still more preferably less than 200, yet still more preferably less than 100, most preferably less than 35 bases long.
- the present invention also relates to vectors that include polynucleotides of the present invention as above described, host cells that are genetically engineered with vectors of the invention, and the production of polypeptides of the invention by recombinant techniques.
- Host cells may be genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector.
- the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
- the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the gene for the polypeptide of the present invention.
- the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
- the polynucleotide of the present invention may be employed for producing a polypeptide by recombinant techniques.
- the polynucleotide sequence may be included in any one of a variety of expression vehicles, in particular vectors or plasmids for expressing a polypeptide.
- vectors include chromosomal, non-chromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
- any other vector or plasmid may be used as long as they are replicable and viable in the host.
- the appropriate DNA sequence may be inserted into the vector by a variety of procedures. Such procedures and others are deemed to be within the scope of those skilled in the art.
- the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
- promoters there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda PL promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
- the expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator.
- the vector may also include appropriate sequences for amplifying expression.
- the expression vectors preferably contain a gene to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
- the vector containing the appropriate DNA sequence as herein above described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
- appropriate hosts there may be mentioned: bacterial cells, such as E.
- the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
- the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
- the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
- regulatory sequences including, for example, a promoter, operably linked to the sequence.
- suitable vectors and promoters are known to those of skill in the art and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, phagescript, psiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene), pTRC99A, pKK223-3, pKK233-3, pDR540, PRIT5 (Pharmacia).
- Eukaryotic pWLneo, pSV2cat, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, PSVL (Pharmacia).
- Promoter regions can be selected from any desired gene using CAT (chloramphenicol acetyl transferase) vectors or other vectors with selectable markers.
- CAT chloramphenicol acetyl transferase
- Two appropriate vectors are pKK232-8 and pCM7.
- Particular named bacterial promoters include laci, lacZ, T3, T7, gpt, lambda PR, PL and trp.
- Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
- the present invention also relates to host cells containing the above-described construct.
- the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
- Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation.
- the constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
- the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
- Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al. (Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989; the disclosure of which is hereby incorporated by reference).
- Enhancers are cis-acting elements of DNA, usually from about 10 to 300 bp, that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin (bp 100 to 270), a cytomegalovirus early promoter enhancer, a polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
- recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S.
- heterologous structural sequence is assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.
- the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
- Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation, initiation, and termination signals in operable reading phase with a functional promoter.
- the vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desirable, to provide amplification within the host.
- Suitable prokaryotic hosts for transformation include E.coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
- Useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).
- cloning vector pBR322 ATCC 37017
- Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wl.) These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
- the selected promoter may be de-repressed, if necessary, by appropriate means (e.g., temperature shift or chemical induction) and the cells may be cultured for an additional period.
- Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
- Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
- mammalian cell culture systems can also be employed to express recombinant protein.
- mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts (82) and other cell lines capable of expressing protein from a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
- Mammalian expression vectors will generally comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcription termination sequences, and 5' flanking nontranscribed sequences.
- DNA sequences derived from the SV40 viral genome for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required non-transcribed genetic elements.
- polypeptide of the present invention may be recovered and purified from recombinant cell cultures by methods used heretofore, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
- HPLC high performance liquid chromatography
- the polypeptide of the present invention may be a naturally purified product, or a product of chemical synthetic-procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated with mammalian or other eukaryotic carbohydrates or may be non-glycosylated. Polypeptides of the present invention may also include an initial methionine amino acid residue.
- Polypeptides of the present invention may be used in a process of gene therapy.
- gene therapy may be involved in the treatment of a disease or clinical condition which may include but is not limited to cancer, wound healing, diabetic retinopathies, macular degeneration, cardiovascular diseases, and clinical conditions involving angiogenesis in the reproductive system, including regulation of placental vascularization or use as an abortifacient.
- cells may be engineered with a polynucleotide (DNA or RNA) encoding for the polypeptide ex vivo, the engineered cells are then provided to a patient to be treated with the polypeptide.
- DNA or RNA polynucleotide
- Such methods are well-known in the art.
- cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding for the polypeptide of the present invention.
- Retrovirus-mediated DNA transfer See, e.g., Kay et al. (1993) Science, 262:117-19; Anderson (1992) Science 256:808-13.
- Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
- the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus.
- DNA viruses include adenoviruses (preferably Ad-2 or Ad-5 based vectors), herpes viruses (preferably herpes simplex virus based vectors), and parvoviruses (preferably "defective" or non-autonomous parvovirus based vectors, more preferably adeno-associated virus based vectors, most preferably AAV-2 based vectors).
- adenoviruses preferably Ad-2 or Ad-5 based vectors
- herpes viruses preferably herpes simplex virus based vectors
- parvoviruses preferably "defective" or non-autonomous parvovirus based vectors, more preferably adeno-associated virus based vectors, most preferably AAV-2 based vectors.
- Adenoviruses have the advantage that they have a broad host range, can infect quiescent or terminally differentiated cells, such as neurons or hepatocytes, and appear essentially non-oncogenic. Adenoviruses do not appear to integrate into the host genome. Because they exist extrachromosomally, the risk of insertional mutagenesis is greatly reduced. Adeno-associated viruses exhibit similar advantages as adenoviral-based vectors. However, AAVs exhibit site-specific integration on human chromosome 19.
- gene therapy with DNA encoding a polypeptide of the present invention is provided to a patient in need thereof, concurrent with, or immediately after diagnosis.
- any suitable gene therapy vector containing DNA encoding a polypeptide of the present invention may be used in accordance with this embodiment.
- the techniques for constructing such a vector are known. See, e.g., Anderson (1998) Nature, 392 25-30; Verma (1998) Nature, 389 239- 42. Introduction of the vector to the target site may be accomplished using known techniques.
- the present invention also relates to a diagnostic assay for detecting levels of polypeptides of the present invention, e.g. in various tissues, since an over-expression of the proteins compared to normal control tissue samples may detect the presence of abnormal cellular proliferation, for example, a tumor.
- Assays used to detect levels of protein in a sample derived from a host are well-known to those of skill in the art and include radioimmunoassays, competitive-binding assays, Western Blot analysis, ELISA assays and "sandwich" type assays. Diagnostic assays may also include the detection of polynucleotides which code for the polypeptides of the present invention.
- the polypeptides of the present invention can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies.
- the present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
- Antibodies generated against the polypeptides of the present mvention are included in the present invention.
- "Antibody” as used herein includes intact immunoglobulin molecules (e.g., IgGl, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA), as well as fragments thereof, such as Fab, F(ab') 2 , scFv, and Fv, which are capable of specific binding to an epitope of a variant Ryk protein.
- antibodies that specifically bind to variant Ryk do not detect other proteins in immunochemical assays.
- preferable antibodies of this invention are human antibodies.
- human antibodies of the present invention specifically bind to human variant Ryk protein with a K d of about 0.1 nM to about 10 ⁇ M, about 2 nM to about 1 ⁇ M, about 2 nM to about 200 nM, about 2 nM to about 150 nM, or about 50 nM to about 100 nM. More preferred human antibodies specifically bind to human variant Ryk with a K d selected from the group consisting of about 2 nM, about 7 nM, about 10 nM, and about 11 nM. Preferred K d s range from about 1 nM, about 3 nM, about 9 nM, about 13 nM, about 14 nM, to about 15 nM.
- the K d of human antibody binding to variant Ryk can be assayed using any method known in the art, including technologies such as real-time Bimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal Chem. 63, 2338-2345, 1991, and Szabo et al, Curr. Opin. Struct. Biol. 5, 699-705, 1995).
- BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcoreTM). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
- Antibodies generated against the polypeptides of the present invention can be obtained in various manners. For example, antibodies by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide or as a diagnostic reagent.
- any technique which provides antibodies produced by continuous cell line cultures can be used. See generally Antibodies: A Laboratory Manual, Harlow and Lane, eds. (1988) Cold Spring Harbor Laboratory. Examples include the hybridoma technique (Kohler and Milstein (1975) Nature 256:495- 97), the trioma technique, the human B-cell hybridoma technique (Kozbor et al. (1983) Immunology Today, 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al. (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
- Human antibodies with the variant Ryk binding characteristics described above can be identified from the MorphoSys HuCAL library as follows. Human variant Ryk protein is coated on a microtiter plate and incubated with the MorphoSys HuCAL-Fab phage library (see Example 9, below). Those phage-linked Fabs not binding to variant Ryk can be washed away from the plate, leaving only phage which tightly bind to variant Ryk. The bound phage can be eluted by a change in pH and amplified by infection of E. coli hosts. This panning process can be repeated once or twice to enrich for a population of antibodies that tightly bind to variant Ryk. The Fabs from the enriched pool are then expressed, purified, and screened in an ELISA assay. The identified hits are then screened in the enzymatic assay described in Bickett et al, 1993, and Bodden et al, 1994.
- Human antibodies with the characteristics described above also can be purified from any cell that expresses the antibodies using methods known to the skilled artisan, and which are described above relating to the purification of a protein. Moreover, human antibodies can be produced using chemical methods to synthesize its amino acid sequence, again, using techniques well known to the skilled artisan and described above relating to the purification of a protein.
- Ryk-Fc fusion protein The cDNA corresponding to the extracellular domain of Ryk as described by Halford (SEQ ID NO:2) was fused at its 3' end to the cDNA encoding the Fc tag (SEQ ID NO:4) and at its 5' end to the cDNA encoding the signal peptide sequence (SEQ ID NO:5).
- the resulting fusion gene (polynucleotide sequence given in SEQ ID NO: 16) was cloned into a vector containing an hCMV promoter to drive gene expression and an ampicillin resistance gene for selection, and the protein was expressed in hEK293E cells (Invitrogen, Carlsbad, CA).
- the Ryk-Fc fusion protein having the sequence given by SEQ ID NO: 8 (lacking the signal peptide sequence, which is removed during processing by the cells during secretion) was purified from conditioned medium by a one step purification using a Protein-A-Sepharose affinity resin. Elution of the Ryk-Fc fusion protein from the affinity resin was performed with 0.2 M gly cine, pH 2.8. The eluate was immediately neutralized with saturated sodium bicarbonate to pH 7.5-8.0 and dialyzed into phosphate buffered saline with dialysis membrane (6,000 dalton cut-off) overnight at 5°C.
- Ryk-myc-His fusion protein This variant Ryk protein described by SEQ ID NO: 9 was prepared in an adeno viral expression system. A cDNA corresponding to the Ryk extracellular domain as described by Tamagnone (SEQ ID NO:l) was fused to cDNAs corresponding to a signal sequence peptide (SEQ ID NO: 5) and a myc-(6X)His sequence (SEQ ID NO:21). The resulting cDNA, described by SEQ ID NO:17, was cloned into the Bglll-Hindlll sites of a CMV shuttle. (He et al. (1998) Proc Natl Acad Sci USA..).
- This plasmid is known as RYK-myc-his6 pShuttleCMV.
- 293A cells Quantum Biotechnologies
- OPTI-MEM 1 reduced serum medium Life Technologies, Gaithersburg, MD
- LIPOFECTAMINE transfection solution was produced according to the supplier's information (Gibco BRL, Gaithersburg, MD).
- RYK-myc-his6-pShuttleCMV DNA was diluted in 0.25 ml OPTIMEM 1 reduced serum medium and mixed with 30 ⁇ l LIPOFECTAMINE transfection solution that was diluted in 0.25 ml OPTI-MEM 1 reduced serum medium. The mixture was added to the cells and incubated for 4.5 hours. DMEM+10%FBS was added and incubated over night. The next day the medium was changed to DMEM 10%FBS. After another 24 hours the medium was changed to OPTI-MEM 1 reduced serum medium. 293A cells which had not been transfected were also incubated in OPTI-MEM 1 reduced serum medium to serve as a control in subsequent assays. The supernatant was collected after 48 hours of incubation.
- HUVEC MATRIGEL matrix assay An in vitro assay identified as the HUVEC MATRIGEL matrix assay. This assay mimics endothelial cell capillary organization and is a standard in vitro assay used to evaluate angiogenic mechanisms.
- HUVEC Human umbilical cord endothelial cells
- the HUVEC complete medium contained F12K medium with 2 mM L-glutamine, 100 ug/ml Heparin, 50 ug/ml endothelial cell growth supplement (ECGS), and 10% fetal bovine serum (FBS).
- ECGS 50 ug/ml endothelial cell growth supplement
- FBS fetal bovine serum
- Murine lung endothelial cells MLuEC were seeded at 5 x 10 4 cells per well in a complete medium containing DMEM with 2mM L-glutamine, 1% Pen/Strep, and 10% FBS.
- MATRIGEL basement membrane matrix (Becton Dickinson, Franklin Lakes, NJ) was prepared using pre-cooled pipettes, tips, plates and tubes during handling of the matrix. The matrix was thawed at 4°C overnight on ice, used to coat a 24-well plate (Costar, VWR, West Chester, PA) at 0.3 ml/well, and then polymerized at 37°C for 2 hours. Test samples were added in 0.5 ml of complete medium per well, and either HUVEC or MluEC cells were added in 0.5 ml of medium per well, so the total volume of medium per well was 1.0 ml.
- capillary-like structures refers to the organizational structures that result from the behavior of endothelial cells in vitro on a MATRIGEL Matrix.
- capillary-like structures can also include organized cells in vivo or in vitro leading up to and participating in angiogenesis which results in the cells in association with each other and forming capillaries.
- Ryk-Fc was added at different concentrations to HUVEC grown in culture on MATRIGEL matrix. Twenty-four hours later cells were fixed and evaluated for capillary-like organization. Measurement of the capillary-like organization in each well allows a quantitative analysis of the biological effect of tested compounds.
- IL-8- TVR has the two amino acids Thr- Val substituted for the two amino acids Glu-Leu, respectively, found at positions 3 and 4 of the wild-type IL-8 protein amino acid sequence.
- buffer alone
- endotoxin at concentrations of 0.1 Eu/mL and 1 Eu/ml was tested to determine if endotoxin could be responsible for the inhibitory effect.
- concentration of endotoxin in the Ryk-Fc treated wells varied from 0.7 x 10 "8 Eu/ml to 0.7 Eu/ml for the 2.5 fM and 250 nM Ryk-Fc concentration, respectively. The results show that endotoxin alone cannot be responsible for the inhibitory effect (data not shown).
- Ryk-myc-His was made in 293 cells transfected with a plasmid construct containing the Ryk-myc-His gene as described above in Example 1.
- the Ryk-myc-His fusion protein- containing supernatant was added at various dilutions to HUVEC grown in culture on MATRIGEL matrix. Twenty-four hours later cells were fixed and evaluated for capillarylike organization.
- Ryk-Fc fusion protein in combination with known angiogenic factors including bFGF, VEGF and IL-8 was tested at different concentrations in HUVEC grown in culture on MATRIGEL matrix. Twenty-four hours later cells were fixed and evaluated for capillary-like organization.
- HUVEC capillary-like organization was tested alone and shown to have no effect on HUVEC capillary-like organization. This result suggests that angiogenic stimuli necessary for HUNEC capillary-like organization are already present either in the MATRIGEL Matrix or in the medium.
- the specificity of the Ryk-Fc fusion protein was also evaluated in vitro using the HUVEC MATRIGEL matrix assay. Briefly, a single chain antibody was generated against the Ryk-Fc fusion protein (scFvlb4). Details concerning the generation of this antibody are described in the examples below. In this experiment, however, HUVEC were cultured with 2.5nM Ryk-Fc fusion protein and increasing concentrations of scFvlb4. Twenty-four hours later cells were fixed and evaluated for capillary-like organization. Results from a representative experiment are presented in Figure 8. In this experiment , scFvBB served as a negative control. ScFvBB is a single chain antibody that was generated against human placental bikunin using methods described in examples below.
- B16-F10 cells were infected ex vivo with non- replicative adenoviral particles containing the Ryk-Fc gene (ad-Ryk-Fc) for 24 hours and then implanted in C57BL/6 mice (Charles River Laboratories, Wilmington, Mass.) intravenously tlirough the tail vein.
- ad-Ryk-Fc non- replicative adenoviral particles containing the Ryk-Fc gene
- tumor burden is evaluated by counting lung metastases.
- the controls included mice receiving untreated B16-F10 cells or B16-F10 cells that had been infected ex vivo with non-replicative adenoviral particles containing the IL-2 gene (ad-IL-2) or the VEGF gene (ad- VEGF).
- ad-IL-2 non-replicative adenoviral particles containing the IL-2 gene
- ad- VEGF ad- VEGF gene
- the expression of IL-2 was shown to have an inhibitory effect on lung metastases whereas the expression of VEGF was shown to have a stimulatory effect.
- the results presented in Figure 9 show that B16-F10 cells infected with ad-Ryk-Fc gave rise to lower metastasis number than non-infected B16-F10 cells.
- the expression of IL-2 also inhibited tumor growth whereas the expression of VEGF stimulated tumor growth.
- variant Ryk protein was tested in vivo in the rat cornea model.
- recombinant bFGF is dissolved in a hydron pellet that allows for sustained release of bFGF.
- Hydron pellets are comprised of 1% Sucrose Octasulfate Aluminum Complex, 12%) Hydron Polymer Type NCC in 95% alcohol. All ingredients are mixed at room temperature overnight. Then, bFGF is added to the suspension under sterile conditions. Test materials with possible anti-angiogenic activity, such as the Ryk-Fc fusion protein, are included in the hydron pellet with bFGF.
- each pellet would normally contain 400 ng of bFGF and varying amounts of test materials.
- each pellet contained 400 ng of bFGF and Ryk-Fc fusion protein was included at doses ranging from 80 ng to 400 ng. Pellets will form at room temperate within 2 hours.
- pellets The implantation of pellets is performed on rats that are under anesthesia. Under aseptic conditions, a micropocket (2mm x 2mm) is produced surgically near the center of the cornea with a pliable iris spatula. The hydron pellets are pre-wetted with saline, implanted and dissolved in rat corneas. Rats are sacrificed 7 days later. Visualization of cornea angiogenesis is enhanced by heart perfusion with Indian Ink. Rat corneas are isolated, fixed in formalin overnight, and mounted onto slides. The level of angiogenesis is examined microscopically. The extent of angiogenesis is scored double-blindly: 0 for no angiogenesis; 1 for modest angiogenesis; 2 for significant angiogenesis and 3 for extensive angiogenesis.
- phage preparation 10 n -10 12 tu
- 100 ⁇ L of the phage preparation 10 n -10 12 tu
- 100 ⁇ l 5%MPBS-0.1% Tween 20 containing 312.5 ⁇ g/ml IL4R-Fc as blocking protein
- the coated wells were washed 2X with 400 ⁇ l PBS.
- Pre-blocked phage mix 200 ⁇ L was transferred into each coated well, the plate sealed, and incubated for 30 min at RT on the microtiter plate shaker followed by another 30 min standing at RT.
- the phage solution was removed and the wells were washed as follows: 1st round of selection: 3x 400 ⁇ l PBST ( PBS containing 0.05% Tween 20) quick - 2x 400 ⁇ l PBST for 5 min on a shaker at RT; 3x 400 ⁇ l PBS quick - 2x 400 ⁇ l PBS for 5 min on a shaker at RT
- Bound phage were eluted by incubation for 10 min with 200 ⁇ l 100 mM triethylamine. The eluate was rapidly neutralized by transferring to a tube containing 100 ⁇ l 1M Tris-HCl pH 7.0. The remaining phage in the selection well were rescued by addition of 200 ⁇ l of E. coli TGI cells at OD 600nm of 0.8. At the same time 4.5 ml of an E. coli TGI culture with an OD 600nm of 0.8 were added to the neutralized phage eluate and both this tube and the microtiter plate were incubated for 45 min at 37°C without shaking. The two samples of infected E.
- coli TGI were combined and centrifuged for 2 min at 5000 g. The supernatant was removed, and the pellet was resuspended and plated on a 150mm LB/Cm/Glu (2xTY medium, 34 ⁇ g/ml chloramphenicol, 1% glucose) agar plate, which was incubated overnight at 30°C.
- the bacteria were scraped from the agar plate with 2 ml 2xTY/Cm/Glu containing 15%) glycerol.
- Ten ml 2xTY/Cm/Glu medium (in 50 ml plastic tubes) were inoculated with 86 ⁇ l bacteria and incubated for 45 min at 37°C in a shaking at 250 rpm.
- Five mL were transferred to culture tube, 50 ⁇ l helper phage were added, and the tube was incubated in a water bath for 30 min at 37°C without shaking followed by 30 min at 37°C in a shaker at 250 rpm.
- the bacteria were harvested by centrifugation at 4500 g for 5 min at 4°C.
- the pellet was resuspended in 25 ml 2xTY/ Cm/50 mg/mL Kan/0.1 mM IPTG- without glucose, then incubated at 30°C, shaking at 250 rpm overnight.
- the bacteria were pelleted by centrifugation, and the phage were recovered from the supernatant by precipitation with 5 ml P ⁇ G/NaCl.
- the phage pellet was resuspended in 1 ml sterile PBS, cleared by centrifugation, filtered through a 0.8 ⁇ m syringe filter and stored at 4°C.
- the second round of selection was carried out as described above, using this phage preparation instead of the HuCAL library primary phage preparation. After the second round of selection, the bacteria were scraped from two agar plates with 2 mL 2xTY/Cm/Glu containing 15% glycerol. 500 microliters of scraping from each plate was used directly for a miniprep.
- DNA was prepared by a standard miniprep method and digested with EcoRI and Xbal, The scFv band obtained from the pool of selected of HuCAL TAG-CAL clones was ligated by standard methods into the expression vector pMx7_FH. The ligation reaction was transformed into E. coli JM83 and plated on LB/Cm/Glu agar plates.
- Single colonies from the transformation were picked and innoculated both into a grid pattern on a 150 mm LB/Cm/l%Glu agar plate and into a 96-well microtiter plates containing 100 ⁇ l 2xTY/Cm/l%Glu medium.
- the plate was sealed with gas-permeable tape and incubated O/N at 37°C with shaking
- approximately 5 ⁇ l per well of the master plate were transferred to the corresponding well of a new culture plate containing 100 ⁇ l 2xTY/Cm/0.1%Glu.
- the culture plate was incubated at 37°C with shaking for 4 hours, then induced by addition of 100 ⁇ l/well of 2xTY/Cm containing ImM IPTG.
- the plate was incubated for an additional 4 h at 30°C with shaking.
- the plate was centrifuged to pellet the bacteria, the supernatants were removed, and 125/well ⁇ l ice-cold BBS (100 mM boric acid, 150 mM NaCl, 2 mM ⁇ DTA, pH 8.0) was added.
- the plate was sealed and incubated overnight at 4°C with shaking.
- the bacteria were pelleted by centrifugation, and 50 ⁇ L of the supernatants were used in the following scFv ⁇ LISA.
- a Maxisorp microtiter plate was coated with 100 ⁇ l Ryk-Fc in PBS (5 ⁇ g/ml) per well overnight at 4°C. The antigen solution was removed and the plate blocked for 2 h with 400 ⁇ l/well of 5%MPBS shaking on a microtiter plate shaker at RT. The plates were rinsed once with 400 ⁇ l per well TBST (50 mM Tris HC1, pH 7.4, 15 mM NaCl, 0.5% Tween-20), and 50 ⁇ l 5%MTBST (TBST containing 5% non-fat milk) were distributed to each well.
- TBST 50 mM Tris HC1, pH 7.4, 15 mM NaCl, 0.5% Tween-20
- the supernatants from the micro-expression plate above were transferred to the corresponding wells of the to the antigen-coated ⁇ LISA plate, which was incubated for 1.5 h at RT on a microtiter plate shaker.
- the plates were washed 5x quickly with TBST + 1 mM CaCl 2 -
- a mixture of monoclonal mouse anti-FLAG-tag antibodies Ml (Sigma F-3040) and M2 (Sigma F-3165) each diluted 1 :10000 in TBST + 1 mM CaCl 2 was added to the plate (100 ⁇ L/well), which was then incubated for 1 h at RT on a microtiter plate shaker.
- the plate was washed 5x quickly with TBST + 1 mM CaCl 2 , and 100 ⁇ l anti-mouse IgG-HRP conjugate (Sigma A-6782) diluted 1.TO 000 in TBST + 1 mM CaCl 2 was added to each well, followed by incubation for 1 h at RT on a microtiter plate shaker.
- the plate was washed 5x quickly with TBST + 1 mM CaCl 2 , and 100 ⁇ l/well Peroxidase substrate BM blue soluble (Roche) was added followed by incubation for 30 min at RT.
- the ELISA signals were read at 370 nm.
- ELISA-positive clones were innoculated from the grid agar plate into 1.2 mL 2XYT/Cm/l%Glu in a deep-well microtiter plate, and incubated 24 h at 37°C with shaking.
- DNA was prepared using the QiaRobot, and sequenced using the primers HuCAL for (TACCGTTGCTCTTCACCCC) and HuCAL rev (TTTTTCACTTCACAGGTC). The DNA sequences were compared by standard alignment methods to identify unique scFv sequences, and to determine the framework sequences of each unique clone.
- E. coli JM83 containing the HuCAL scFv was plated on a LB/Cm/Glu agar plate, and incubated overnight at 37°C.
- a 10 ml culture in 2xTY/Cm/l%Glu medium was innoculated from a single colony and incubated overnight at 30°C in a shaker.
- the bacterial culture was cooled on ice to reach RT and IPTG was added to a final concentration of 0.5mM.
- the scFv was expressed at 22°C overnight, shaking at 180 rpm, to an OD 600nm of 0.5.
- the bacteria were harvested by centrifugation at 5 000 g for 20 min at 4°C.
- the pellet was resuspended in 100 ml pre-cooled running buffer (100 mM Tris, 1 mM EDTA pH 8.0) and disrupted using a MicroFluidics cell disrupter. Bacterial debris was removed by centrifugation at 20 000 g for 30 min at 4°C.
- the supernatant was filtered through a 0.2 ⁇ m filter (low protein binding) and applied to a Poros metal-chelator column from Perseptive Biosystems.
- the scFv was eluted with 10 column volumes of elution buffer (running buffer + 250 mM imidazole). The eluted fractions were dialyzed into PBS by standard methods, aliquotted and stored at -20°C.
- the cells were pelleted by centrifugation (2660 x g) and the pellet was resuspended in 20 mM sodium phosphate, pH 7.4, 0.5 M NaCl, 10 mM imidazole containing 1 ml of a protease inhibitor cocktail (Sigma) (Buffer A) and lysed using a microfluidizer (Microfluidics, Inc) at a pressure of 15,000 psi. The supernatant was centrifuged at 10,640 xg, filtered and applied to a 1 ml Poros MC (Perseptive Biosystems) metal-chelator column that had been charged with Ni+ as per manufacturer's instruction.
- a protease inhibitor cocktail Sigma
- microfluidizer Microfluidics, Inc
- the column was washed with buffer A, and protein was eluted with a step gradient of 30, 60, 90 and 250 mM imidazole in buffer A.
- the 1B4 antibody peak was dialyzed in buffer containing 10 mM Hepes, pH 7.5, and 10 mM NaCl (Buffer B) for 4 hrs at 4°C, filtered, and injected onto a Mono Q 5/5 ion-exchange column that was equilibrated in Buffer B using a flow rate of 1 ml/min. After washing with Buffer B to remove unbound material, protein was eluted using a 0 to 0.5 M NaCl gradient in Buffer B.
- the peak containing the anti-Ryk 1B4 antibody was collected, tested for endotoxin using a QCL-1000 test kit under manufacturer suggestions (Biowhittaker, Walkersville, MD). Because the endotoxin level in this peak was significant (1600 EU/ml), the Mono Q column was cleansed from endotoxin contamination and antibody was reapplied by first diluting the peak 1:5 with Buffer B and re-injected. The resulted peak was reassayed for endotoxin.
- Running buffer was BIACORE HBS-EP (lOmM HEPES, 150mM NaCl, 3mM EDTA, 0.005% Surfactant P20, pH 7.4).
- the anti-Ryk scFv 1B4 antibody was amine coupled to a CM5 sensor chip by first activating the surface with a 1:1 mixture of l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC) (1M) and N- hydroxysuccinimide (.25M) at 5 ⁇ l/min.
- EDC l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride
- the antibody was diluted to 1.8 ⁇ g/ml in 10 mM sodium acetate, pH 4.5, and injected until 250 response units (RU) were obtained. Following attachment, the remaining active sites on the sensor surface were blocked with 35 ⁇ l of 1 M ethanolamine, pH 8.5, at 5 ⁇ l/min.
- Various concentrations of Ryk-Fc (30 to 242 nM) in running buffer were injected over the surface (60 ⁇ l injection/ 30 ⁇ l/min flow rate) to follow Ryk-Fc-antibody association.
- Ryk-Fc-antibody was followed immediately after each association reaction by a two minute injection of running buffer.
- BIAcore chip surface regeneration was accomplished by injecting 50 ⁇ l of 10 mM gly cine pH 2.5 at a flow rate of 100 ⁇ l/min.
- the anti-Ryk scFv 1B4 antibody was purified from E.coli lysates by sequential chelating and ion-exchange chromatography. The majority of the antibody eluted from the Poros MC chelating column at 250 mM immidizole. The anti-Ryk scFv 1B4 antibody also eluted in the earlier washes but these peaks were not combined with the high imidazole elution. A total of 11.7 A280 nm units was detected in the final pool. The first Mono Q column elution contained multiple peaks, the first 2 of which were judged to be the scFv 1B4 antibody ( Figure 11).
- the BIAcore 2000 instrument is a powerful tool in the analysis of protein-protein interactions. Using appropriate coupling conditions, accurate association and dissociation constants for the binding of antibody to antigen can be determined.
- Figure 14 shows the sensorgrams generated for the binding of various concentrations of Ryk-Fc to a fixed concentration of immobilized anti-Ryk scFv 1B4 antibody. The kinetic constants calculated from these plots are listed in Table 1 below. A KD value of 13.3 nM was calculated for this interaction, demonstrating a high affinity between the two binding partners.
- the wild type Ryk protein or other variant Ryk proteins as described herein may be relevant as therapeutic agents to any disease where angiogenesis is involved, including but not limited to cancer, wound healing, diabetic retinopathies, macular degeneration, and cardiovascular diseases.
- the variant Ryk proteins described herein may further be used in the treatment of clinical conditions involving angiogenesis in the reproductive system, including regulation of placental vascularization, regulation of pregnancy, or use as an abortifacient.
- the polypeptides of the present invention may find use in diagnostic applications, as may the polynucleotides which code for the polypeptides of the present invention, and as may antibodies to the polypeptides of the present invention.
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US56878300A | 2000-05-10 | 2000-05-10 | |
US568783 | 2000-05-10 | ||
PCT/US2001/015043 WO2001085789A2 (en) | 2000-05-10 | 2001-05-09 | Extracellular domain of ryk protein for regulating angiogenesis |
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JP (1) | JP2004527206A (ja) |
AR (1) | AR028424A1 (ja) |
AU (1) | AU2001261343A1 (ja) |
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JO3283B1 (ar) | 2011-04-26 | 2018-09-16 | Sanofi Sa | تركيب يتضمن أفليبيرسيبت, حمض فولينيك, 5- فلورويوراسيل (5- Fu) وإرينوسيتان (FOLFIRI) |
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AU4606196A (en) * | 1994-12-23 | 1996-07-19 | Ludwig Institute For Cancer Research | Assay, receptor proteins and ligands |
ATE321856T1 (de) * | 1998-06-11 | 2006-04-15 | Humane rezeptortyrosinkinase | |
CA2348835A1 (en) * | 1998-10-28 | 2000-05-04 | Cornell Research Foundation, Inc. | Methods for regulating angiogenesis and vascular integrity using trk receptor ligands |
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SV2002000442A (es) | 2002-07-03 |
DOP2001000164A (es) | 2002-05-15 |
WO2001085789A2 (en) | 2001-11-15 |
CA2408349A1 (en) | 2001-11-15 |
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