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WO2001016169A2 - COMPOSITIONS A BASE DE LIGAND 5 (RetL5) ET SES UTILISATIONS - Google Patents

COMPOSITIONS A BASE DE LIGAND 5 (RetL5) ET SES UTILISATIONS Download PDF

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Publication number
WO2001016169A2
WO2001016169A2 PCT/US2000/024111 US0024111W WO0116169A2 WO 2001016169 A2 WO2001016169 A2 WO 2001016169A2 US 0024111 W US0024111 W US 0024111W WO 0116169 A2 WO0116169 A2 WO 0116169A2
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WIPO (PCT)
Prior art keywords
ret
seq
retl5
polypeptide
ligand polypeptide
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PCT/US2000/024111
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English (en)
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WO2001016169A3 (fr
Inventor
Dane Worley
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Biogen, Inc.
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Priority to AU71039/00A priority Critical patent/AU7103900A/en
Publication of WO2001016169A2 publication Critical patent/WO2001016169A2/fr
Publication of WO2001016169A3 publication Critical patent/WO2001016169A3/fr

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • One of the goals of current research on cell signaling and receptor activation is to enable therapeutic modulation of processes involved in cell growth and survival. Such processes determine outcome in diverse medical conditions, including organ failure, fetal development, and tumor growth, among others. Each of these conditions is of worldwide clinical importance, and has limited efficacious treatment options. It is an object of the invention to provide compositions and - methods for promoting regeneration or survival of damaged tissue, as well as for treating disorders involving the aberrant growth and development of tissues. Tissue loss or end-stage organ failure affects millions of people worldwide each year and adds substantially to health care costs. Organ or tissue loss is usually treated by transplanting organs from donors, by surgical reconstruction, or with mechanical devices. Each of these remedies has shortcomings. Transplantation is limited by donor shortage, surgical reconstruction can create other long-term problems, and mechanical devices cannot perform all the functions of a single organ, and therefore cannot prevent progressive deterioration. Thus, a real medical need exists for new solutions to these problems.
  • Protein factors that affect the growth, differentiation and/or survival of cells may be useful in the treatment of disorders of organs which contain responsive cells.
  • Factors or ligands that interact with receptors of the receptor protein tyrosine kinase (RPTK) family are of particular interest in this regard. These receptors are involved in many cellular programs including cell growth and differentiation, and the genesis of many neoplasias. Thus the factors or ligands that interact with these receptors may prove useful in treating disorders of certain organs where the tissue has been damaged. Alternatively, it may be useful to block the interaction of these factors with their receptors in order to block tumor growth.
  • RPTK receptor protein tyrosine kinase
  • the Ret proto-oncogene encodes a receptor tyrosine kinase that is expressed during development in a variety of tissues, including the peripheral and central nervous systems and the kidney.
  • the abnormalities present in Ret null mice suggest that Ret is critical for the migration and innervation of enteric neurons to the hindgut, and for proliferation and branching of the ureteric bud epithelium during kidney development (Nature 367; 380-383 (1994)).
  • the search for a key component of the Ret signaling pathway, the Ret ligand, has been an area of intensive research.
  • This invention relates generally to novel Ret ligand 5 (RetL5) compositions and uses thereof.
  • the invention provides a purified and isolated DNA molecule coding for a novel Ret ligand, RetL5, having the nucleotide sequence of any RetL5, but specifically including murine RetL5 cDNA (SEQ ID NO:2).
  • the invention further provides a RetL5 protein, with an amino acid sequence comprising that of murine RetL5 (SEQ ID NO:3).
  • the invention includes a signal sequence in RetL5, preferably amino acids 1-21 of SEQ ID NO. 3 (which correspond to nucleotides 1- 63 of SEQ T NO. 2).
  • the invention in another embodiment, includes a first cysteine rich domain in RetL5, preferably comprising amino acids 22-112 of SEQ ID NO. 3 (which correspond to nucleotides 64-336 of SEQ ID NO. 2).
  • the invention also includes a second cysteine rich domain in RetL5, preferably comprising amino acids 1 13-218 of SEQ ID NO. 3 (which correspond to nucleotides 337-654 of SEQ ID NO. 2).
  • the invention includes a flexible hinge region in RetL5, preferably a first flexible hinge region comprising amino acids 219-247 of SEQ ID NO. 3 (which correspond to nucleotides 655-741 of SEQ ID NO. 2).
  • the invention includes a GPl cleavage/attachment site in RetL5, preferably comprising amino acids 248-250 of SEQ VD NO. 3 (which correspond to nucleotides 742-750 of SEQ ID NO. 2).
  • the invention includes an amino linked glycosylation site in RetL5, preferably comprising amino acids 184-186 of SEQ ID NO. 3 (which correspond to nucleotides 550-558 of SEQ ID NO. 2).
  • the invention incudes a RetL5 polypeptide having a domain structure including a signal sequence, two cysteine rich domains, a flexible hinge, a GPl cleavage/attachment site, and an N-linked glycosylation site.
  • the RetL5 polypeptide binds specifically to a GDNF family member polypeptide.
  • the domains of the present RetL5 polypeptide have the following approximate sites: signal sequence, 20 amino acids; cysteine rich domains, at least 45 amino acids up to at most about 110 amino acids; flexible hinge, at least 25 amino acids; GPl cleavage/attachment site, two amino acids; and an N-linked glycosylation site, two amino acids.
  • the invention includes an extracellular RetL5 polypeptide having a domain structure including two cysteine rich domains, a flexible hinge, a GPl cleavage/attachment site, and an N-linked glycosilation site.
  • the RetL5 polypeptide binds specifically to a GDNF family member polypeptide.
  • the domains of the present RetL5 polypepetide have the following approximate sites: cysteine rich domains, at least 45 amino acids up to at most about 110 amino acids; flexible hinge, at least 25 amino acids; GPl cleavage/attachment site, two amino acids; and an N-linked glycosylation site, two amino acids.
  • the invention includes a soluble RetL5 polypeptide having a domain structure including two cysteine rich domains, a flexible hinge and an N-linked glycosilation site.
  • the RetL5 polypeptide binds specifically to a GDNF family member polypeptide.
  • the domains of the present RetL5 polypepetide have the following approximate sites: cysteine rich domains, at least 45 amino acids up to at most about 110 amino acids; flexible hinge, at least 25 amino acids, two amino acids; N-linked glycosylation site, two amino acids.
  • a purified and isolated DNA molecule for use in securing expression in a prokaryotic or eukaryotic host cell of a polypeptide product has at least a part of the primary structural conformation and the biological activity of RetL5; a) the DNA may be a DNA molecule which comprises murine RetL5 cDNA or the complementary strand of murine RetL5 cDNA; b) DNA molecules which hybridize under stringent conditions to the DNA molecules defined in a) or unique fragments thereof; or c) DNA molecules which, but for the degeneracy of the genetic code, would hybridize to the DNA molecules defined in a) and b).
  • a purified and isolated DNA molecule coding for a polypeptide fragment or variant of a human RetL5 having the biological activity of a RetL5 is also within the invention.
  • the invention is an isolated nucleic acid comprising a nucleotide sequence complementary to or which hybridizes under conditions of medium stringency to high stringency to the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or the complement of any of the foregoing.
  • a complementary strand or complement (RNA or DNA) of a nucleic acid sequence encoding a RetL5 protein has a nucleic acid sequence which can form base pairs with the nucleic acid sequence encoding the RetL5 proteins.
  • the invention relates to nucleic acid sequences which hybridize to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 under conditions of moderate to high stringency.
  • Any of the recombinant DNA molecules of the invention if desired, can be operably linked to an expression control sequence.
  • the vector may encompass a DNA molecule encoding a RetL5 or a variant of a RetL5.
  • the invention includes prokaryotic or eukaryotic host cells stably transformed or transfected by a vector comprising a DNA molecule encoding a native or variant RetL5.
  • a purified and isolated human RetL5 substantially free of other human proteins is specifically within the invention, as is a process for the production of a polypeptide product having part or all of the primary structural conformation and the biological activity of a RetL5.
  • Such a process may include the steps of growing, under suitable culture conditions, prokaryotic or eukaryotic host cells transformed or transfected with any DNA molecule of the invention, in a manner allowing expression of such polypeptide product, and recovering a RetL5.
  • the polypeptide product of the expression in a procaryotic or eukaryotic host cell of a DNA is also included.
  • the invention also includes proteins and protein fragments, variants and derivatives, whether soluble or membrane bound.
  • the protein has an amino acid sequence which comprises murine RetL5, or is a variant thereof.
  • the protein is a fusion protein including a RetL5, fused to another molecule or molecular fragment, such as an immunoglobulin, toxin, imageable compound or radionuclide.
  • Other embodiments of the invention include specific monoclonal antibodies to a RetL5 of the invention. Such an antibody may be associated with a toxin, imageable compound or radionuclide.
  • the invention further includes a method of promoting growth of new tissue, or promoting survival of damaged tissue in a subject, including administering to the subject a therapeutically effective amount of a compound which interacts with cellular Ret and thereby induces autophosphorylation of Ret.
  • the compound can be RetL5, a fragment of a full-length RetL5, or an antibody which binds to Ret.
  • the compound can be administered concurrently with a therapeutically effective amount of a second compound, such as GDNF, neurturin or a GDNF family member molecule.
  • tissues of interest for these methods can include any tissue, preferred tissues include renal tissue, neural tissue, heart, stomach, small intestine, spinal cord, or lung.
  • the RetL5 is a soluble RetL5.
  • the subject of the methods can be human.
  • Ret signal transduction between a first cell expressing a RetL5 and a second cell expressing Ret is inhibited by contacting the first cell with a soluble Ret ligand protein or with an antibody to the RetL5.
  • the soluble Ret ligand protein may be a fusion protein.
  • the invention also includes a method for targeting a toxin, imageable compound or radionuclide to a cell expressing Ret, encompassing contacting the cell with a RetL5 fusion protein conjugated to a toxin, imageable compound or radionuclide.
  • the RetL5 fusion protein comprises a soluble RetL5 polypeptide.
  • growth of a tumor cell which expresses Ret is suppressed, with a step of the method being contacting the cell with a fusion protein of a RetL5 and a toxin or radionuclide, conjugated to a toxin or radionuclide.
  • the cell can be within a subject, and the protein or the conjugated antibody is administered to the subject.
  • Also encompassed within the invention is a method for targeting a toxin, imageable compound or radionuclide to a cell expressing a RetL5, comprising contacting the cell with an anti-RetL5 antibody conjugated to a toxin, imageable compound or radionuclide.
  • Another embodiment includes the method of suppressing growth of a tumor cell which expresses a RetL5, comprising contacting the cell with a fusion protein of Ret and a toxin or radionuclide or with an anti- RetL5 antibody conjugated to a toxin or radionuclide; the cell can be within a subject, and the protein administered to the subject.
  • the RetL for any of the methods of the invention is RetL5, or a variant or unique fragment of RetL5.
  • One embodiment is a method of treating a subject with a disorder of Ret metabolism, comprising administering to the subject a vector comprising a DNA molecule encoding a RetL5, as well as a method of promoting growth of new tissue in a subject, comprising administering such a vector to the subject.
  • Another embodiment includes a method of promoting survival of damaged tissue in a subject, one step of the method being administering a therapeutically effective amount of a vector encoding a RetL5 to the subject.
  • FIGURE 1 is a murine RetL5 cDNA sequence predicted from DSW300 sequence by Visual Inspection Method (SEQ ID NO: 2)
  • FIGURE 2 is a murine RetL5 cDNA sequence predicted from DSW300 sequence by GENESCAN/GENE ALEX (SEQ ED NO: 4).
  • FIGURE 3 is a murine RetL5 amino acid sequence predicted from DSW300 by Visual Inspection Method (SEQ ID NO: 3).
  • the presumed signal sequence is underlined at the N-terminus of the protein and the presumed cleavage site/GPI attachment at the C-terminus is in bold and underlined.
  • FIGURE 4 is a murine RetL5 amino acid sequence predicted from DSW300 sequence by GENESCAN/GENE ALEX (SEQ ED NO: 5). The presumed signal sequence is underlined at the N-terminus of the protein.
  • FIGURE 5 is a predicted human RetL5 cDNA sequence (SEQ ID NO: 6).
  • FIGURE 6 is a predicted human RetL5 amino acid sequence (SEQ ID NO: 7). The presumed signal sequence is underlined at the N-terminus of the protein and a possible GPl cleavage/attachment site is in red at the C-terminus
  • FIGURE 7 is a murine RetL5 cDNA (SEQ ⁇ D NO: 8) alternatively spliced from the genomic sequence of SEQ ED NO: 1.
  • FIGURE 8 is a deduced murine RetL5 amino acid sequence (SEQ ED NO: 9) of SEQ ED NO: 8.
  • FIGURE 9 depicts murine RetL5-Fc fusion protein binding to the following GDNF family members.
  • GDNF Global cell line-Derived Neurotrophic Factor
  • NTN neurotrophic factor
  • NBN neuroblastin
  • PSP persephin
  • FIGURE 10 depicts fusion proteins of rat RetLl (rLl-Ig), human RetL2 (rL2-Ig), murine RetL3 (mL3-Ig) and murine RetL5 (mL5-Ig) binding to rat neublastin (rNBN).
  • FIGURE 11 depicts fusion proteins of rat RetLl (rLl-Ig), human RetL2 (rL2-Ig), murine RetL3 (mL3-Ig) and murine RetL5 (mL5-Ig) binding to neublastin and rat Ret AP (alkaline phosphatase).
  • SEQ ED NO: 1 mouse RetL5 genomic sequence.
  • SEQ ED NO: 2 mouse RetL5 cDNA predicted by Visual Inspection Method.
  • SEQ ED NO: 4 mouse RetL5 cDNA sequence predicted from DSW300 sequence by GENESCAN/GENE ALEX.
  • SEQ ED NO: 5 mouse RetL5 - RetL5 amino acid sequence predicted from
  • SEQ ED NO: 6 predicted human RetL5 cDNA.
  • SEQ ED NO: 7 predicted human RetL5 amino acid sequence.
  • SEQ ED NO: 8 murine RetL5 cDNA alternatively spliced from SEQ ED NO: 1.
  • RetL (also refe ⁇ ed to herein as “GFR ⁇ ” or "c- RetL”) means any protein which specifically interacts with the receptor protein Ret, (also referred to herein as “c-Ret") and which, when it interacts with Ret, triggers Ret dimerization and/or autophosphorylation of the tyrosine kinase domain of Ret.
  • the DNA sequences which code for RetL and for Ret are termed “RetL” and “Ret”, respectively.
  • a ligand optionally can be soluble, or present as a membrane-bound molecule on the same or on a different cell as the Ret molecule for which it is triggering autophosphorylation.
  • Ligands of the invention may require additional molecules to trigger autophosphorylation.
  • Ligands of the invention include co-receptors or accessory ligand cofactors.
  • Ligands of the invention further include anti-Ret mAbs which act as Ret agonists, triggering Ret dimerization and autophosphorylation.
  • the ligand can also be modified in various ways, such as incorporated as a portion of a fusion protein, or conjugated to a toxin or radionuclide.
  • alignment of sequences is meant the positioning of one sequence, either nucleotide or amino acid, with that of another, to allow a comparison of the sequence of relevant portions of one with that of the other.
  • An example of one method of this procedure is given in Needleman et al. (J. Mol. Biol. 48:443-453 (1970)). The method may be implemented conveniently by computer programs such as the Align program (DNAstar, Inc.).
  • homologous or functionally equivalent sequences include functionally equivalent arrangements of positionally conserved amino acid residues such as the cysteine residues, within the sequence.
  • “Functionally equivalent arrangements” can include amino acid insertions or deletions which alter the linear arrangement of conserved residues, but do not materially impair their relationship in the folded structure of the protein. Therefore, minor internal gaps and amino acid insertions in a candidate sequence are ignored for purposes of calculating the level of amino acid sequence homology or identity between the candidate sequence and a reference sequence (here, RetL5).
  • a reference sequence here, RetL5
  • One characteristic frequently used in establishing the homology of proteins is the similarity of the number and location of the cysteine residues between one protein and another.
  • unique fragment of a polypeptide sequence is meant a peptide consisting of at least 8 contiguous amino acids of said polypeptide sequence, preferably at least about 12 contiguous amino acids.
  • unique fragment of a nucleic acid sequence refers to at least 8 contiguous codons of a polypeptide encoding a sequence of a similar length of non-coding nucleic acid sequence.
  • cloning is meant the use of in vitro recombination techniques to insert a particular gene or other DNA sequence into a vector molecule.
  • in vitro recombination techniques to insert a particular gene or other DNA sequence into a vector molecule.
  • it is necessary to employ methods for generating DNA fragments, for joining the fragments to vector molecules, for introducing the composite DNA molecule into a host cell in which it can replicate, and for selecting the clone having the target gene from amongst the recipient host cells.
  • cDNA is meant complementary or copy DNA produced from an RNA template by the action of RNA-dependent DNA polymerase (reverse transcriptase).
  • a “cDNA clone” means a duplex DNA sequence complementary to an RNA molecule of interest, carried in a cloning vector.
  • cDNA library is meant a collection of recombinant DNA molecules containing cDNA inserts which together comprise a representation of the mRNA molecules present in an entire organism or tissue, depending on the source of the RNA templates.
  • a cDNA library may be prepared by methods known to those of skill, and described, for example, in Maniatis et al., Molecular Cloning: A Laboratory Manual, supra.
  • RNA is first isolated from the cells of an organism from whose genome it is desired to clone a particular gene. Preferred for the purposes of the present invention are mammalian, and particularly human, cell lines.
  • RNA may be isolated from a tumor cell, derived from an animal tumor, and preferably from a human tumor.
  • a library may be prepared from, for example, a human adrenal tumor, but any tumor may be used.
  • DNA polymorphism refers to the condition in which individuals can have different nucleotide sequences at a particular site in the DNA of an organism. The differing sequences isolated from such individuals are thus “polymorphic variants.”
  • “Expression vector” includes vectors which are capable of expressing DNA sequences contained therein, i.e., the coding sequences are operably linked to other sequences capable of effecting their expression. It is implied, although not always explicitly stated, that these expression vectors must be replicable in the host organisms either as episomes or as an integral part of the chromosomal DNA.
  • a useful, but not a necessary, element of an effective expression vector is a marker encoding sequence, which is a sequence encoding a protein which results in a phenotypic property (e.g. tetracycline resistance) of the cells containing the protein which permits those cells to be readily identified.
  • expression vector is given a functional definition, and any DNA sequence which is capable of effecting expression of a specified contained DNA code is included in this term, as it is applied to the specified sequence.
  • Such vectors are frequently in the form of plasmids, so “plasmid” and “expression vector” are often used interchangeably.
  • the invention is intended to include such other forms of expression vectors, including phage, which serve equivalent functions and which may from time to time become known in the art.
  • GDNF-related molecule means any molecule which is a GDNF family member, i.e., which has at least 40% amino acid sequence similarity to either GDNF or neurturin. Preferred GDNF family members are capable of specifically binding to RetL5.
  • gene means a polynucleotide sequence encoding a peptide.
  • homogeneous is meant, when referring to a peptide or DNA sequence, that the primary molecular structure (i.e., the sequence of amino acids or nucleotides) of substantially all molecules present in the composition under consideration is identical.
  • oligonucleotide as used herein in referring to probes, oligomer fragments to be detected, oligomer controls, unlabeled blocking oligomers and primers for amplification of sequences is defined as a molecule comprised of more than three deoxyribonucleotides or ribonucleotides. Its exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide.
  • probe refers to a ligand of known qualities capable of selectively binding to a target antiligand.
  • probe refers to a strand of nucleic acid having a base sequence complementary to a target strand.
  • Recombinant host cells refers to cells which have been transformed with vectors constructed using recombinant DNA techniques. As defined herein, the antibody or modification thereof produced by a recombinant host cell is by virtue of this transformation, rather than in such lesser amounts, or more commonly, in such less than detectable amounts, as would be produced by the untransformed host.
  • the terms “restriction endonucleases” and “restriction enzymes” refer to bacterial enzymes each of which cut double-stranded DNA at or near a specific nucleotide sequence.
  • RFLP restriction fragment length polymorphism
  • a molecule is said to be "substantially similar” to another molecule if the sequence of amino acids in both molecules is substantially the same, and if both molecules possess a similar biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if one of the molecules contains additional amino acid residues not found in the other, or omits residues that are found in the other, or if the sequence of amino acid residues is not identical.
  • a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, etc.
  • the moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc. Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Perm. (1980).
  • vector is meant a DNA molecule, derived from a plasmid or bacteriophage, into which fragments of DNA may be inserted or cloned.
  • a vector will contain one or more unique restriction sites, and may be capable of autonomous replication in a defined host or vehicle organism such that the cloned sequence is reproducible.
  • substantially pure any protein of the present invention, or any gene encoding any such protein, which is essentially free of other proteins or genes, respectively, or of other contaminants with which it might normally be found in nature, and as such exists in a form not found in nature.
  • the invention includes cDNA coding for a novel Ret ligand, RetL5, including for example the nucleotide sequence of murine RetL5 cDNA.
  • the compounds of the invention include sequences which are derivatives of these sequences.
  • the invention also includes vectors, liposomes and other carrier vehicles which encompass one of these sequences or a derivative of one of these sequences.
  • the invention also includes proteins transcribed and translated from RetL5 cDNA, preferably, murine RetL5 cDNA, including but not limited to murine RetL5 and derivatives and variants.
  • the invention relates to the use of the nucleic acids and proteins of the present invention to design probes to isolate other proteins which have structural or functional properties of the RetL5 proteins of the invention.
  • the probes can be a variety of base pairs in length.
  • a nucleic acid probe can be between about 10 base pairs in length to about 150 base pairs in length.
  • the nucleic acid probe can be greater than about 150 base pairs in length. Exceptional methods are provided in Ausubel et al, "Current Protocols in Molecular Biology" J. Wiley (ed.) (1999), the entire teachings of which are herein incorporated by reference in their entirety.
  • oligonucleotide also referred to herein as nucleic acid
  • N's fewest ambiguous bases
  • T m 80° C (assuming 2°C for each A or T and 4° for each G or C).
  • the oligonucleotide should preferably be labeled with ⁇ - " P ATP (specific activity 6000 Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for labeling oligonucleotides. Other labeling techniques can also be used. Unincorporated label should preferably be removed by gel filtration chromatography or other established methods. The amount of radioactivity incorporated into the probe should be quantitated by measurement in a scintillation counter. Preferably, specific activity of the resulting probe should be approximately 4 x 10 dpm/pmole.
  • the bacterial culture containing the pool of full-length clones should preferably be thawed and 100 ⁇ l of the stock used to inoculate a sterile culture flask containing 25 ml of sterile L-broth containing ampicillin at 100 ⁇ g/ml.
  • the culture should preferably be grown to saturation at about 37°C, and the saturated culture should preferably be diluted in fresh L-broth.
  • Aliquots of these dilutions should preferably be plated to determine the dilution and volume which will yield approximately 5000 distinct and well-separated colonies on solid bacteriological media containing L-broth containing ampicillin at 100 ⁇ g/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at about 37°C. Other known methods of obtaining distinct, well-separated colonies can also be employed. Standard colony hybridization procedures should then be used to transfer the colonies to nitrocellulose filters and lyse, denature and bake them.
  • Highly stringent (also referred to herein as “high stringency”) conditions are those that are at least as stringent as, for example, lx SSC at about 65°C, or lx SSC and 50%o formamide at about 42°C.
  • Moderate stringency conditions are those that are at least as stringent as 4x SSC at about 65°C, or 4x SSC and 50% formamide at about 42°C.
  • Moderate or reduced stringency conditions are those that are at least as stringent as 4x SSC at about 50°C, or 6x SSC and 50% formamide at 40°C.
  • the filter is then preferably incubated at about 65°C for 1 hour with gentle agitation in 6X SSC (20x stock is 175.3 g NaCl/liter, 88.2 g Na citrate/liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS, 100 ⁇ g/ml of yeast RNA, and 10 mM EDTA (approximately 10 mL per 150 mm filter).
  • 6X SSC 20x stock is 175.3 g NaCl/liter, 88.2 g Na citrate/liter, adjusted to pH 7.0 with NaOH
  • SDS 100 ⁇ g/ml of yeast RNA
  • 10 mM EDTA approximately 10 mL per 150 mm filter.
  • the probe is then added to the hybridization mix at a concentration greater than or equal to 1 x 10 dpm/mL.
  • the filter is then preferably incubated at about 65°C with gentle agitation overnight.
  • the filter is then preferably washed in 500 mL of 2x SSC/0.5% SDS at room temperature without agitation, preferably followed by 500 mL of 2x SSC/0.1% SDS at room temperature with gentle shaking for 15 minutes. A third wash with O.lx SSC/0.5% SDS at about 65°C for 30 minutes to 1 hour is optional.
  • the filter is then preferably dried and subjected to autoradiography for sufficient time to visualize the positives on the X-ray film. Other known hybridization methods can also be employed.
  • the positive colonies are then picked, grown in culture, and plasmid DNA isolated using standard procedures. The clones can then be verified by restriction analysis, hybridization analysis, or DNA sequencing.
  • the invention relates to nucleic acid sequences (e.g., DNA, RNA) that hybridize to nucleic acids of RetL5 ligands.
  • nucleic acids which hybridize to SEQ ED NO: 1, SEQ ED NO: 2, SEQ ED NO: 4 or SEQ ED NO: 6 under high, moderate or reduced stringency conditions as described above.
  • the invention relates to a complement of nucleic acid of RetL5.
  • it relates to complements of SEQ ED NO: 1, SEQ ED NO: 2, SEQ ED NO: 5 or SEQ ED NO: 5.
  • the invention relates to an RNA counterpart of the
  • DNA nucleic acid of RetL5. relates to RNA counterparts of SEQ ED NO: 1, SEQ ED NO: 2, SEQ ED NO: 4 or SEQ ED NO: 6.
  • Soluble variants lack at least a portion of the intramembrane section of the native RetL5.
  • the soluble variant lacks the phosphatidylinositol glycan linkage ("GPl linkage") of the native RetL5.
  • Soluble variants include fusion proteins which encompass derivatives of RetL5 that lack a phosphatidylinositol motif.
  • Variants can differ from naturally occurring RetL5 in amino acid sequence or in ways that do not involve sequence, or both.
  • Variants in amino acid sequence are produced when one or more amino acids in naturally occurring RetL5 is substituted with a different natural amino acid, an amino acid derivative or non-native amino acid.
  • Particularly preferred variants include naturally occurring RetL5, or biologically active fragments of naturally occurring RetL5, whose sequences differ from the wild type sequence by one or more conservative amino acid substitutions, which typically have minimal influence on the secondary structure and hydrophobic nature of the protein or peptide.
  • Variants may also have sequences which differ by one or more non-conservative amino acid substitutions, deletions or insertions which do not abolish the RetL5 biological activity.
  • Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics such as substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • variants within the invention are those with modifications which increase peptide stability. Such variants may contain, for example, one or more non- peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are: variants that include residues other than naturally occurring L-amino acids, such as D-amino acids or non-naturally occurring or synthetic amino acids such as beta or gamma amino acids and cyclic variants. Incorporation of D- instead of L-amino acids into the polypeptide may increase its resistance to proteases. See, e.g., U.S. Patent 5,219,990.
  • the peptides of this invention may also be modified by various changes such as insertions, deletions and substitutions, either conservative or nonconservative where such changes might provide for certain advantages in their use. Splice variants are specifically included in the invention.
  • a RetL5 polypeptide or fragment is biologically active if it exhibits a biological activity of naturally occurring RetL5.
  • Such biological activities can include the ability to specifically bind the extracellular portion of Ret, with an affinity that is at least 50% of, and preferably at least equal to, the affinity of naturally occurring RetL5 for the extracellular portion of Ret.
  • RetL5 biological activity can include binding to a GDNF family member molecule (e.g., GDNF, neurturin, persephen, neublastin) with an affinity that is at least 50% of the affinity of naturally occurring RetL5 for binding to the GDNF family member molecule.
  • RetL5 polypeptide Another biological activity is the ability to bind to an antibody which is directed at an epitope which is present on naturally occurring RetL5.
  • Biologically active fragments of the RetL5 polypeptide can be predicted using computer programs known to those of skill in the art, including for example, the computer program P-SORT.
  • variants with amino acid substitutions which are less conservative can also result in desired derivatives, e.g., by causing changes in charge, conformation and other biological properties.
  • substitutions would include for example, substitution of hydrophilic residue for a hydrophobic residue, substitution of a cysteine or proline for another residue, substitution of a residue having a small side chain for a residue having a bulky side chain or substitution of a residue having a net positive charge for a residue having a net negative charge.
  • the derivatives may be readily assayed according to the methods disclosed herein to determine the presence or absence of the desired characteristics.
  • substitutions that may be expected to induce changes in the functional properties of Ret polypeptides are those in which: (i) a hydrophilic residue, e.g., serine or threonine, is substituted by a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, or alanine; (ii) a cysteine residue is substituted for (or by) any other residue; (iii) a residue having an electropositive side chain, e.g., lysine, arginine or histidine, is substituted for (or by) a residue having an electronegative charge, e.g., glutamic acid or aspartic acid; or (iv) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having such a side chain, e.g., glycine.
  • a hydrophilic residue e.g., serine or
  • Variants within the scope of the invention include proteins and peptides with amino acid sequences having at least sixty percent homology with murine RetL5 (SEQ ED NO:3). More preferably the sequence homology is at least eighty, at least ninety percent, or at least ninety-five percent. For the purposes of determining homology the minimum length of comparison sequences will generally be at least 8 amino acid residues, usually at least 12 amino acid residues.
  • Variants of the compounds of the invention also includes any protein which 1) has an amino acid sequence which is at least forty percent homologous to a RetL5 protein of the invention, and also which 2) after being placed in an optimal alignment with the RetL5 sequence, has at least 80% of its cysteine residues aligned with cysteines in the RetL5 protein of the invention.
  • Non-sequence modifications may include, for example, in vivo or in vitro chemical derivatization of portions of naturally occurring RetL5, as well as changes in acetylation, methylation, phosphorylation, carboxylation or glycosylation.
  • agents which specifically bind to a protein of the invention include Ig fusion proteins and antibodies (including single chain, double chain, Fab fragments, and others, whether native, humanized, primatized, or chimeric). Additional descriptions of these categories of agents are in PCT application WO95/16709, the disclosure of which is herein incorporated by reference.
  • Native and variant RetL5, anti-RetL5 antibodies, and fusion proteins of Ret and of RetL5 may have therapeutic utility in situations where it is desirable to block or to activate the Ret signaling pathway, to stimulate renal and/or neuronal cell growth or survival in disease situations where these cells are lost or damaged, or to suppress growth of or to kill undesirable cells such as tumor cells that express Ret or a RetL5.
  • compounds of the invention that bind to Ret, inducing dimerization and/or autophosphorylation of Ret, are useful for stimulating growth of or limiting damage to Ret-expressing tissues.
  • the compounds of the invention are useful for stimulating renal tissue growth and/or survival, supporting renal function, and in minimizing damage to renal tissue after various insults.
  • Particular conditions which may be beneficially treated with the compounds of the invention include acute renal failure, acute nephritis, chronic renal failure, nephrotic syndrome, renal tubule defects, kidney transplants, toxic injury, hypoxic injury, and trauma.
  • Renal tubule defects include those of either hereditary or acquired nature, such as polycystic renal disease, medullary cystic disease, and medullary sponge kidney. This list is not limited, and may include many other renal disorders (see, e.g., Harrison's Principles of Internal Medicine, 13th ed., 1994, which is herein incorporated by reference.)
  • genes and proteins of the invention may be used to treat conditions where neural growth and regeneration is desirable. This would include any conditions involving disorders of neural degeneration, such as
  • disorders of the cranial nerves and of the spinal cord including disorders involving traumatic, inflammatory, congenital or vascular etiologies, are specifically included, as are disorders affecting the autonomic nervous system. Also included are developmental neural disorders such as mental retardation, autism, fetal alcohol syndrome, Down's syndrome, and cerebral palsy.
  • the compounds of the invention may also be used to treat syndromes involving the peripheral nervous system, particularly peripheral neuropathies. These disorders include those caused by any of the factors previously listed, and specifically include Lyme disease, HEV-associated neuropathies, polymyositis, muscular dystrophy, and myasthenia gravis.
  • Anti-RetL5 antibodies and Ret fusion proteins of the invention which specifically bind to the protein of murine RetL5 or fragments of thereof, are useful in several methods.
  • the compounds may be used therapeutically to inhibit or block Ret receptor signaling, such as for blocking growth of tumors which depend on activation of Ret signaling for growth.
  • These agents may also be fused to detectable markers, such as fluoroscopically or radiographically opaque substances, and administered to a subject to allow imaging of tissues which express a RetL5.
  • the agents may also be bound to substances, such as horseradish peroxidase, which can be used as immunocytochemical stains to allow visualization of areas of RetL5- positive cells on histological sections.
  • a specific antibody could be used alone in this manner, and sites where it is bound can be visualized in a sandwich assay using an anti-immuno globulin antibody which is itself bound to a detectable marker.
  • Specific antibodies to any RetL5 are also useful in immunoassays to quantify the substance for which a given antibody has specificity.
  • Specific antibodies to a RetL5 may also be bound to solid supports, such as beads or dishes, and used to remove the ligand from a solution, either for use in purifying the protein or in clearing it from the solution. Each of these techniques is routine to those of skill in the immunological arts.
  • RetL5 may act as an antagonist by interacting with Ret in a manner which blocks activation of Ret by a GDNF family member/Ret Ligand complex.
  • This theory is based on the observation that RetL5 could interact with a GDNF family protein and this complex would not be able to interact with Ret. RetL5 would therefore bind to/tie up GDNF family proteins and prevent their proper interactions with other RetLigands.
  • RetL5 is a truncated RetLigand that may be lacking the domain that would bind a GDNF family member yet still maintain the amino acids necessary to interact with.
  • Other methods of the invention include modulating Ret-RetL5 signaling by contacting Ret with an anti-Ret monoclonal antibody.
  • the effect of such a mAb-Ret contact can be to either block or to stimulate activation of the Ret signaling pathway, depending on the characteristics of the interaction of each particular mAb with Ret.
  • Certain mAbs interact with Ret as agonists, with the agonist mAb-Ret binding triggering the dimerization and autophosphorylation of Ret.
  • Other mAbs act as Ret antagonists.
  • the interaction of Ret with an antagonist mAb prevents Ret signaling activation by other RetL5, or by complexes comprising RetL5, which would otherwise activate the Ret signaling pathway.
  • a RetL5 and/or antibodies to Ret or to a Ret fusion protein can be used to allow imaging of tissues which express Ret, or in the imrnunohisto logical or preparative methods described above for antibodies to a RetL5.
  • Fusion proteins encompassing a RetL5 and/or anti-Ret antibodies can be used to specifically target medical therapies against cancers and tumors which express Ret.
  • Such tumors might include the several different tumor phenotypes which have been associated with mutations in Ret (N. Engl. J. Med. 335:943-951, 1996; Nature 367: 319-320, 1996; Trends Gen. 12:138-144, 1996).
  • Therapeutic interventions against neoplasias which express a RetL5 utilize fusion proteins which incorporate Ret and/or an anti-RetL5 antibody.
  • the anti-Ret antibody or anti-RetL5 antibody may be effective by itself through antibody-dependent and complement- dependent cytolysis mediated by the Fc domain.
  • Such hybrid ligands and antibodies can be made more effective as cancer therapeutics by using them as delivery vehicles for antineoplastic drugs, toxins and cytocidal radionuclides, such as yttrium 90.
  • Cytotoxic effector cells may be targeted to tumor cells using heteroconjugate antibodies, where an antibody specific for either Ret or for a RetL5 expressed by a tumor is covalently coupled to an antibody directed against a surface protein on cytotoxic effector cells, such as NK cells or CTLs.
  • an anti-Ret antibody or RetL5 therapy is to conjugate the toxic A chain of ricin or a modified full-length form of ricin (which can no longer bind cells) to a RetL5 or to an antibody directed against the Ret polypeptide expressed on the surface of malignant cells.
  • a toxin is conjugated to Ret or to an anti-RetL5 antibody to selectively target and kill RetL5- positive cells, such as a tumor expressing a RetL5. Success with an analogous approach is reported in Grossbard et al., Blood 79:5761 (1992).
  • Other toxins are equally useful, as known to those of skill in the art. Such toxins include, but are not limited to, pseudomonas exotoxin, diphtheria toxin, and saporin.
  • Radioisotope labeled RetL5 or anti-Ret antibodies will preferentially target radioactivity to tumor sites in cells expressing Ret, sparing normal tissues.
  • the radiation emitted from a radiolabeled antibody bound to a tumor cell may also kill nearby malignant tumor cells that do not express Ret.
  • a variety of radionuclides may be used. Isotopes that emit ⁇ particles (for example, 131 I) have been successful when employed with monoclonal antibodies against CD20 present on B-cell lymphomas (Kaminski et al., N. Engl. J. Med. 329: 459 (1993); Press et al, N.
  • Radionuclides emitting ⁇ particles generate radioactive emissions that are tumoricidal over distances spanning several cell diameters, permitting the eradication of antigen negative cells and diminishing the consequences of nonhomogenous deposition of antibody or ligand in tumors.
  • Radionuclides emitting particles may also be employed.
  • the low dose rate irradiation generated by radionuclide labeled RetL5 or anti-Ret antibodies may be more therapeutically effective than the instantaneous irradiation delivered externally in conventional radiation therapy.
  • Low dose rate irradiation can induce apoptosis (programmed cell death) in certain cell lines (Macklis et al., Radiat. Res. 130: 220 (1992); Maklis et al, Radiopharm. 5: 339 (1992).
  • the compounds of the invention are administered in therapeutically-effective amounts, which means an amount of a compound which produces a medically desirable result or exerts an influence on the particular condition being treated.
  • subject used herein is taken to mean any mammal to which Ret ligand or gene may be administered.
  • Subjects specifically intended for treatment with the method of the invention include humans, as well as nonhuman primates, sheep, horses, cattle, goats, pigs, dogs, cats, rabbits, guinea pigs, hamsters, gerbils, rats and mice, as well as the organs, tumors, and cells derived or originating from these hosts.
  • the RetL5 genes of the invention are introduced into damaged tissue to stimulate production of a RetL5 by the transfected cells, to promote cell growth and/or survival of cells that express Ret.
  • a RetL5 gene may be introduced into a renal or neural target tissue of choice.
  • a RetL5 would then be stably expressed and stimulate Ret receptor-positive cells to grow, divide, differentiate, and/or potentiate cell survival.
  • RetL5 genes may be introduced into a target cell using a variety of well-known methods that use either viral or non-viral based strategies.
  • Non-viral methods include electroporation, membrane fusion with liposomes, high velocity bombardment with DNA-coated microprojectiles, incubation with calcium-phosphate-DNA precipitate, DEAE-dextran mediated transfection, and direct micro-injection into single cells.
  • a RetL5 gene may be introduced into a cell by calcium phosphate coprecipitation (Pillicer et al., Science, 209: 1414-1422 (1980); mechanical microinjection and/or particle acceleration (Anderson et al., Proc. Natl. Acad. Sci.
  • liposome based DNA transfer e.g., LEPOFECTIN-mediated transfection- Fefgner et al., Proc. Nat. Acad. Sci., USA, 84: 471-477, 1987; Gao and Huang, Biochim. Biophys. Res. Comm., 179: 280-285, 1991; DEAE Dextran-mediated transfection; electroporation (U.S.
  • Patent 4,956,288) or polylysine-based methods in which DNA is conjugated to deliver DNA preferentially to liver hepatocytes (Wolff et al., Science, 247: 465-468, 1990; Curiel et al., Human Gene Therapy 3: 147-154, 1992).
  • Target cells may be transfected with the genes of the invention by direct gene transfer. See, e.g., Wolff et al., "Direct Gene Transfer Into Moose Muscle In Vivo", Science 247:1465-68, 1990. In many cases, vector-mediated transfection will be desirable. Any of the methods known in the art for the insertion of polynucleotide sequences into a vector may be used.
  • Promoter activation may be tissue specific or inducible by a metabolic product or administered substance.
  • promoters/enhancers include, but are not limited to, the native RetL promoter, the cytomegalovirus immediate-early promoter/enhancer (Karasuyama et al., J. Exp. Med.. 169: 13 (1989)); the human beta-actin promoter (Gunning et al, Proc. Nat.
  • MMTV LTR mouse mammary tumor virus long terminal repeat
  • MoLV LTR Moloney murine leukemia virus
  • SSV Rous sarcoma virus
  • HSV Rous sarcoma virus
  • the RetL5 gene may also be introduced by specific viral vectors for use in gene transfer systems which are now well established. See for example: Madzak et al., J. Gen . Virol.. 73: 1533-36, 1992 (papovavirus SV40); Berkner et al., Curr. Top. Microbiol. Immunol. 158: 39-61, 1992 (adenovirus); Hofmann et al, Proc. Natl. Acad. Sci. 92: 10099-10103, 1995 (baculovirus); Moss et al., Curr. Top. Microbiol. Immunol.. 158: 25-38, 1992 (vaccinia virus); Muzyczka, Curr. Top. Microbiol. Immunol..
  • Preferred vectors are DNA viruses that include adenoviruses (preferably Ad- 2 or Ad-5 based vectors), baculovirus, he ⁇ es viruses (preferably he ⁇ es 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
  • baculovirus preferably he ⁇ es viruses (preferably he ⁇ es 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 are eukaryotic DNA viruses that can be modified to efficiently deliver a therapeutic or reporter transgene to a variety of cell types.
  • Ad2 and Ad5 The general adenoviruses types 2 and 5 (Ad2 and Ad5, respectively), which cause respiratory disease in humans, are currently being developed for gene therapy of Duchenne Muscular Dystrophy (DMD)and Cystic Fibrosis (CF). Both Ad2 and Ad5 belong to a subclass of adenovirus that are not associated with human malignancies.
  • Adenovirus vectors are capable of providing extremely high levels of transgene delivery to virtually all cell types, regardless of the mitotic state.
  • High titers (10 13 plaque forming units/ml) of recombinant virus can be easily generated in 293 cells (an adenovirus-transformed, complementation human embryonic kidney cell line: ATCC CRL1573) and cryo-stored for extended periods without appreciable losses.
  • the efficacy of this system in delivering a therapeutic transgene in vivo that complements a genetic imbalance has been demonstrated in animal models of various disorders. See Y. Watanabe, Atherosclerosis. 36: 261-268 (1986); K. Tanzawa et al, FEBS Letters.
  • Human adenoviruses are comprised of a linear, approximately 36 kb double- stranded DNA genome, which is divided into 100 map units (m.u.), each of which is 360 bp in length.
  • the DNA contains short inverted terminal repeats (ITR) at each end of the genome that are required for viral DNA replication.
  • ITR inverted terminal repeats
  • the gene products are organized into early (El through E4) and late (LI through L5) regions, based on expression before or after the initiation of viral DNA synthesis. See, e.g., Horwitz, Virology, 2d edit., ed. B.N. Fields, Raven Press Ltd., New York (1990).
  • the first-generation recombinant, replication-deficient adenoviruses which have been developed for gene therapy of DMD and other inherited disorders contain deletions of the entire Ela and part of the Elb regions.
  • This replication-defective virus is grown in 293 cells containing a functional adenovirus Ela gene which provides a transacting Ela protein.
  • El -deleted viruses are capable of replicating and producing infectious virus in the 293 cells, which provide Ela and Elb region gene products in trans.
  • the resulting virus is capable of infecting many cell types and can express the introduced gene (providing it carries its own promoter), but cannot replicate in a cell that does not carry the El region DNA unless the cell is infected at a very high multiplicity of infection.
  • Adenoviruses have the advantage that they have a broad host range, can infect quiescent or terminally differentiated cells such as neurons, and appear essentially non-oncogenic. Adenoviruses do not appear to integrate in to the host genome. Because they exist extrachromasomally, the risk of insertional mutagenesis is greatly reduced. Ali et al., supra, at 373. Recombinant adenoviruses (rAdV) produce very high titers, the viral particles are moderately stable, expression levels are high, and a wide range of cells can be infected. Their natural host cells are airway epithelium, so they are useful for therapy of lung cancers.
  • Baculovirus-mediated transfer has several advantages. Baculoviral gene transfer can occur in replicating and nonreplicating cells, and can occur in renal cells, as well as in hepatocytes, neural cells, spleen, skin, and muscle. Baculovirus is non-replicating and nonpathogenic in mammalian cells. Humans lack pre-existing antibodies to recombinant baculovirus which could block infection. In addition, baculovirus is capable of inco ⁇ orating and transducing very large DNA inserts.
  • Adeno-associated viruses have also been employed as vectors for somatic gene therapy.
  • AAV is a small, single-stranded (ss) DNA virus with a simple genomic organization (4.7 kb) that makes it an ideal substrate for genetic engineering.
  • Two open reading frames encode a series of rep and cap polypeptides.
  • Rep polypeptides rep78, rep68, rep 62 and rep 40
  • the cap proteins form the virion capsid.
  • Flanking the rep and cap open reading frames at the 5' and 3' ends are 145 bp inverted terminal repeats (ITRs), the first 125 bp of which are capable of forming Y- or T-shaped duplex structures.
  • ITRs inverted terminal repeats
  • the entire rep and cap domains can be excised and replaced with a therapeutic or reporter transgene. See BJ. Carter, in Handbook of Parvoviruses, ed., P. Tijsser, CRC Press, pp. 155-168 (1990). It has been shown that the ITRs represent the minimal sequence required for replication, rescue, packaging, and integration of the AAV genome.
  • the AAV life cycle is biphasic, composed of both latent and lytic episodes.
  • AAV virions enter a cell as an encapsilated ssDNA, and shortly thereafter are delivered to the nucleus where the AAV DNA stably integrates in to a host chromosome without the apparent need for host cell division.
  • the integrated AAV genome remains latent but capable of being activated and rescued.
  • the lytic phase of the life cycle begins when a cell harboring an AAV provirus is challenged with a secondary infection by a he ⁇ esvirus or adenovirus which encodes helper functions that are recruited by AAV to aid in its excision from host chromatin (B.J. Carter, supra).
  • the infecting parental ssDNA is expanded to duplex replicating form (RF) DNAs in a rep dependent manner.
  • the rescued AAV genomes are packaged into preformed protein capsids (icosahedral symmetry approximately 20 nm in diameter) and released as infectious virions that have packaged either + or - ssDNA genomes following cell lysis.
  • Adeno-associated viruses AAV have significant potential in gene therapy.
  • the viral particles are very stable and recombinant AAVs (rAAV) have "drug-like" characteristics in that rAAV can be purified by pelleting or by CsCl gradient banding. They are heat stable and can be lyophilized to a powder and rehydrated to full activity.
  • Their DNA stably integrates into host chromosomes so expression is long-term. Their host range is broad and AAV causes no known disease so that the recombinant vectors are non-toxic.
  • sequences of interest can be identified by conventional methods such as nucleic acid hybridization using probes comprising sequences that are homologous/complementary to the inserted gene sequences of the vector.
  • sequence(s) may be identified by the presence or absence of a "marker" gene function (e.g, thymidine kinase activity, antibiotic resistance, and the like) caused by introduction of the expression vector into the target cell.
  • a "marker" gene function e.g, thymidine kinase activity, antibiotic resistance, and the like
  • the compounds of the invention may be administered in any manner which is medically acceptable. This may include injections, by parenteral routes such as intravenous, intravascular, intraarterial, subcutaneous, intramuscular, intratumor, intraperitoneal, intraventricular, intraepidural, or others as well as oral, nasal, ophthalmic, rectal, or topical. Sustained release administration is also specifically included in the invention, by such means as depot injections or erodible implants. Localized delivery is particularly contemplated, by such means as delivery via a catheter to one or more arteries, such as the renal artery or a vessel supplying a localized tumor.
  • pharmaceutically acceptable carrier means one or more organic or inorganic ingredients, natural or synthetic, with which the mutant proto-oncogene or mutant oncoprotein is combined to facilitate its application.
  • a suitable carrier includes sterile saline although other aqueous and non-aqueous isotonic sterile solutions and sterile suspensions known to be pharmaceutically acceptable are known to those of ordinary skill in the art.
  • carrier encompasses liposomes and the HEV-1 tat protein (See Chen et al., Anal. Biochem. 227: 168-175, 1995) as well as any plasmid and viral expression vectors.
  • an “effective amount” refers to that amount which is capable of ameliorating or delaying progression of the diseased, degenerative or damaged condition.
  • An effective amount can be determined on an individual basis and will be based, in part, on consideration of the symptoms to be treated and results sought. An effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.
  • the liposome system may be any variety of unilamellar vesicles, multilamellar vesicles, or stable plurilamellar vesicles, and may be prepared and administered according to methods well known to those of skill in the art, for example in accordance with the teachings of United States Patents 5,169,637, 4,762,915, 5,000,958 or 5,185,154.
  • treatment of human acute renal failure with liposome-encapsulated RetL5 may be performed in vivo by introducing a RetL5 into cells in need of such treatment using liposomes.
  • the liposomes can be delivered via catheter to the renal artery.
  • the recombinant RetL5 protein is purified, for example, from CHO cells by immunoaffinity chromatography or any other convenient method, then mixed with liposomes and inco ⁇ orated into them at high efficiency.
  • the encapsulated protein may be tested in vitro for any effect on stimulating cell growth.
  • This invention also contemplates that the novel polypeptide of this invention may be administered to an animal via liposome delivery system in order to enhance their stability and/or immunogenicity.
  • liposomes Delivery of the novel polypeptides via liposomes may be particularly advantageous because the liposome may be internalized by phagocytic cells in the treated animal. Such cells, upon ingesting the liposomal membrane and subsequently present the polypeptides to the immune system in conjunction with other molecules required to elicit a strong immune response.
  • any of the novel RetL5 polypeptides of this invention may be used in the form of a pharmaceutically acceptable salt.
  • Suitable acids and bases which are capable of forming salts with the polypeptides of the present invention are well known to those of skill in the art, and include inorganic and organic acids and bases.
  • EXAMPLE 1 Functional Screening Assay for RetL5 Genomic DNA as well as a full length RetL5 cDNA (generated by overlap- extension PCR) is cloned into an expression vector for protein production in E293 cells.
  • RetL5 peptide polyclonal antibodies are produced in rabbits using techniques known to those of skill in the art and are used to identify the expressed RetL5 proteins.
  • a truncated Ret Ligand2, (RetL2c) is constructed for comparative pu ⁇ oses. The isolated RetL5 proteins are assayed by ELISA to identify interactions with known members of the GDNF family of proteins.
  • RetL5 is capable of interacting with cRET in the presence of any of the GDNF family members
  • E293 cells are transfected with the RetL5 cDNA clones and are assayed for a Ret-Fc fusion protein binding to the cells by a FACS (Flourescently Activated Cell Sorting) assay.
  • RetL5 protein can also be used in a RET ternary complex ELIS A, wherein a GDNF family protein and Ret-AP (alkaline hosphatase) fusion protein are mixed in solution with a RetL5-Fc fusion protein.
  • the protein mixture can then be captured on an antibody (anti Fc) coated ELISA well and the readout for the assay can be through the AP tag of the Ret-AP fusion protein.
  • a positive signal indicates that a ternary complex is formed between the GDNF family protein, RetL5 fusion protein, and the Ret fusion protein.
  • a human mouse somatic cell hybrid cell line which contains human chromosome 20 in a murine A9 cell background was obtained.
  • mRNA and cDNA libraries may be made from this cell line.
  • the RNA and the cDNA can be assayed for the presence of known human genes surrounding the locus for RetL5. If they are present it should be assumed that RetL5 is also present.
  • the A9/human chromosome 20 cDNA library is screened with a murine RetL5 probe to obtain clones which cross-hybridize.
  • a radioactive probe generated by random priming a portion of the murine RetL5 coding region (Feinberg, A.P. and et al, Anal.
  • the RetL5 probe can also be used to screen other human cDNA libraries.
  • the choice of cDNA libraries may be determined from results obtained by hybridizing the probe to commercially available Northern blots of human organs and cell lines.
  • the peptide sequence of murine RetL3, as disclosed in WO97/44356, inco ⁇ orated by reference herein, is used to search the GenBank database with the program BLAST in order to identify related proteins (i.e. isologs).
  • BLAST or Basic Local Alignment Search Tool, uses the method of Altschul et al. (J. Mol. Biol. 215: 403-410, 1990) to search for similarities between a query sequence and all the sequences in the sequence database.
  • the query sequence and the database to be searched can be either peptide or nucleotide in any combination.
  • EXAMPLE 5 Identification and characterization of genomic clones for RetL5 An antisense oligonucleotide was generated from the AU035938 EST sequence: KD2-819 (AGC GCT CCA GGG GCT CCA GGC AAG AG (SEQ ED NO: 10); corresponding to the complement of nucleotides 69-95). lxlO 6 plaque forming units from the Stratagene murine 129 SvJ lambda FIXE genomic library (cat# 946313) were screened in duplicate on OptitranTM filters.
  • the filters were hybridized with 32 P-labeled oligonucleotide KD2-819 in 300 mis plaque screen buffer (50mM Tris pH 7.5, 1M NaCl, 0.1% sodium pyrophosphate, 0.2% Polyvinylpryrolidine and 0.2%o Ficoll) containing 10% Dextran sulfate and lOO ⁇ g/ml tRNA and 100 pmol 32 P-labeled oligonucleotide at 65Covernight.
  • the filters were washed twice with 2X SSC/1%SDS and exposed to film. Four duplicate positives were purified. DNA from each of these clones was analyzed by restriction enzyme digestion followed by agarose gel electrophoresis and Southern blotting.
  • the Southern filter was hybridized to KD2-819 to confirm the inserts hybridized to the probe.
  • several restriction enzyme fragments from the insert of DSW300 were subcloned into the plasmid vector pBluescript ⁇ SK(+) (Stratagene) for sequence analysis.
  • DSW320 is a Notl/Hind H fragment spanning nucleotides 1-2551
  • DSW318 is a HindH/HindEH fragment spanning nucleotides 2552-5612
  • DSW319 is a HindH/Hind ⁇ i fragment spanning nucleotides 5613-5889
  • DSW316 is a HindE VBamHI fragment spanning nucleotides 5890-10948
  • DSW304 is a BamHI/Notl fragment spanning nucleotides 10949-15914.
  • DSW318, DSW319, DSW316, and DSW304 were entirely sequenced and the sequences obtained were 100%) identical to the corresponding portion of DSW300.
  • EXAMPLE 6 Protein coding region prediction The sequence of DSW300 was used to query all the public databases using
  • BLAST This search indicated the presence of a novel member of the disintegrin family (ADAM) at the 5' end of DSW300 as well as sequence homology to GFR ⁇ family (RetL family). Based on homology to the Ret Ligand gene family, exons 4-8 for a novel family member were identified. Through visual alignment of DSW300 with the coding regions of RetLl, 2, 3 and 4, five exons can be identified and a putative coding region lacking an initiation codon and at least a portion of the signal sequence can be inferred from nucleotides 12443-12806 of SEQ ED NO. 1 (exonA) (the reading frame begins at nucleotide 12445 of SEQ ED NO.
  • Exon 1 contains a putative secretion signal with the initiating MET beginning at NT 11113 of SEQ ED NO. 1.
  • the 5' end of exonl is not predicted and the 3' end of exonl is at NT 11158 of SEQ ED NO. 1;
  • exon2 is predicted at nt 12443-12810 of SEQ ED NO. 1;
  • RetL5 shares with the other members of the RetLigand family a hydrophobic N-terminus indicative of a signal sequence and a hydrophobic C-terminus indicative of a phosphatidylinositol glycan linkage motif. Although these methods vary in the protein -coding region predictions, in all predictions the putative protein domain structure of RetL5 differs from other family members in that domain 1 and the first flexible hinge region encoded by exons 2 and 3 of other family members is deleted.
  • EXAMPLE 7 Murine RefL5-Alternative Splice Variant Murine Genomic DNA containing the entire murine RetL5 gene (DSW304- BamHI/Notl) was cloned into the expression vector CH269 for protein production in E293 cells. mRNA was isolated from these cells and subjected to RT-PCR to identify the RetL5 transcript. These cDNAs were then subcloned into the sequencing vector TOPO-TA(Invitrogen) for sequence analysis.
  • Murine RetL5-GPI2 cDNA Sequence See Figure 7 (SEQ ED NO. 8)
  • the cDNA for the GPl splice variant of murine RetL4(5), (SEQ ED NO: 8) would be transiently transfected into E293 cells. Post transfection 48 hours the cells would be removed from the flask with EDTA and then washed with PBS. The cells are incubated with rat Ret-human Ig +/- GDNF family member (persephin; PSP) for 1.5 hours at RT. The cells are washed and are incubated with PE- conjugated anti-human Fc for 20 min at room temperature and read in the FACSCAN.
  • PSP rat Ret-human Ig +/- GDNF family member
  • chromosome assignments can be erroneous where specific tagged sequences (STSs) turn out to encompass moderately repetitive elements that reside on more than one chromosome; thus, the mapping data in public databases can be wrong or result from a chimeric BAC, derived from more than one chromosome.
  • STSs specific tagged sequences
  • Murine RetL5 Binding Data Murine RetL5 can interact with NBN.
  • the murine RetL5 used is a RetL5 fusion (amino acids 1-247) with the Fc portion of human IgG (Sanicola, et al, PNAS 94:6238-6243 (1997)).
  • the fusion protein interacts with the recently decribed GDNF family member neublastin, NBN. ( Figures 9 and 10).
  • Oligonucleotide sequence used in constructing the RetL5 fusion protein were as follows:
  • kd3-071 CAGCCGCCTG TGCCGGCCCC GTCTCCTTGC (SEQ ED NO: 11)
  • kd3-072 GCAAGGAGAC GGGGCCGGCA CAGGCGGCTG (SEQ ED NO: 12)
  • kd3-073 ACGCAGGCCT CATAGGCACC GTGGTCACCC (SEQ ED NO: 13)
  • kd3-074 GGGTGACCAC GGTGCCTATG AGGCCTGCGT (SEQ ED NO: 14)
  • kd3-075 GGAACCCCTG CTTGGATGGT GCCATACAAG
  • kd3-076 CTTGTATGGC ACCATCCAAG CAGGGGTTCC
  • kd3-077 GGACCAGACT GCTGGGCAAG GCACGAGTGG
  • kd3-079 ATAGTTTAGC GGCCGCTCAG AGCAGGGCCT GGAGAG (SEQ ED NO: 18) kd3-079: ATAGTTTAGC GGCCGCTCAG AGCAGGGCCT GG
  • Reaction A oligos: kd3-l 17, kd3-l 19; template: DSW304-2 Reaction 4: oligos: kd3-l 18, kd3-072; template: DSW304-2 Reaction 5: oligos: kd3-071, kd3-074; template: DSW304-2 Reaction 6: oligos: kd3-073, kd3-076; template: DSW304-2 Reaction 7: oligos: kd3-075, kd3-080; template: DSW304-2 Reaction 8: oligos: kd3-077, kd3-079; template: DSW304-2 Reaction A/4: oligos: kd3-l 17, kd3-072; template: gel-purified products from Reaction A and Reaction 4 Reaction 5/6: oligos: kd3-071, kd3-076; template: gel-purified products from Reaction 5 and Reaction 6
  • Reaction 7/8 oligos: kd3-075, kd3-079; template: gel-purified products from Reaction 7 and Reaction 8
  • Reaction 5/8 oligos: kd3-071, kd3-079; template: gel-purified products from Reaction 5/6 and Reaction 7/8
  • Reaction A/8 oligos: kd3-l 17, kd3-079; template: gel-purified products from Reaction A/4 and Reaction 5/8
  • Reaction A/8 yielded the cDNA of murine RetL5 according to the visual inspection prediction.
  • the cDNA of murine RetL5 was digested with Notl and inserted into the Notl site of the CH269 vector in a two-way ligation to produce construct DSW321.
  • the DSW322 (murine RetL5-human Fc fusion) construct was produced by amplifying DSW321 by PCR with oligos kd3-l 17 and kd3-121 and digested with Notl/Sall.
  • the Notl /Sail murine RetL5 fragment was ligated to the Sail /Notl human Fc fragment from SAB144 (Sanicola, et al, PNAS 94:6238-6243 (1997)) and inserted into the Notl site of the CH269 vector in a three-way ligation to produce construct DSW322.
  • the murine RetL5 -human Fc fusion protein was expressed in EBNA293 cells by transient transfection.
  • Murine RetL5-human Fc fusion protein conditioned media was purified over a protein A Sepharose column, pooled, and then purified over a Superdex 200 column.
  • Protein A-purified material was concentrated to 500 ⁇ l using MiUipore 10k cutoff Ultrafree-4 centrifugal filter device (UFV4BGC25). The entire sample was applied that to a gel filtration column. The column was a prepacked Pharmacia 1 X 30 cm Superdex 200 column (17-1088-01).
  • the protein was eluted with a 45 minute isocratic elution (25 mM HEPES, 150 mM NaCl, pH 7.5) at 0.5 mL/min on the BioCad. 500 uL fractions were collected and 10 uL of the fractions 11-30 applied to a 10-20% reducing SDS-PAGE gel.
  • GFL GDNF Family Ligand
  • a Nunc-Immunoplate MaxiSo ⁇ Surface plate were coated with 250 ng/ml GDNF family member (GFL) in 50 mM sodium bicarbonate/ carbonate, pH 9.6 overnight at 4° C. The plate is then blocked with 1% BSA in TBS + Tween (TBST) for 1 hour RT. The block (TBST) was removed and fresh TBST added to each well. Equal molar stock solutions of GFR ⁇ -human Ig fusions (2 nM) were prepared (Sanicola, et al, PNAS 94:6238-6243 (1997)) and serially diluted. Plate were incubated at room temperature.
  • the plates were washed three times with TBST and incubated with an anti-human-HRP conjugate for 1 hour at RT.
  • the plates were washed again and developed with developing solution (12.5 ml 0.1 M sodium acetate trihydrate + 0.1 M citric acid (monohydrate), 125 ul 42 mM TMB in DMSO, 1.95 ul 30% hydrogen peroxide).
  • the reaction is allowed to continue for 5 minutes and stopped with an equal volume of 2 N H 2 S0 4 .
  • the ELISA plate is then read in at 450 nm and the results plotted in Deltagraph.
  • GFR 2c (RL2c)
  • encodes a protein with a similar domain structure as RetL5 in that domain 1 and the first flexible hinge region are deleted.

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Abstract

Cette invention concerne des compositions de Ret Ligand 5 (RetL5) et leurs utilisations. Elle porte sur des séquences isolées d'acides nucléiques et d'acides aminés pour ligands Ret. Les ligands Ret pour lesquels codent les séquences d'acides nucléiques isolées comportent une séquence signal N-terminal hydrophobe, une séquence C-terminal hydrophobe et un motif de couplage phosphatidylinositol glycane. L'invention concerne également des vecteurs et des cellules hôtes renfermant des ligands Ret codés par les séquences isolées d'acides nucléiques.
PCT/US2000/024111 1999-09-01 2000-09-01 COMPOSITIONS A BASE DE LIGAND 5 (RetL5) ET SES UTILISATIONS WO2001016169A2 (fr)

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US10023570B2 (en) 2015-07-16 2018-07-17 Array Biopharma Inc. Substituted pyrazolo[1,5-A]pyridine compounds as RET kinase inhibitors
WO2018136661A1 (fr) 2017-01-18 2018-07-26 Andrews Steven W Composés de pyrazolo[1,5-a]pyrazine substitués utilisés en tant qu'inhibiteurs de la kinase ret
WO2018136663A1 (fr) 2017-01-18 2018-07-26 Array Biopharma, Inc. Inhibiteurs de ret
US10112942B2 (en) 2016-10-10 2018-10-30 Array Biopharma Inc. Substituted pyrazolo[1,5-A]pyridine compounds as RET kinase inhibitors
US10144734B2 (en) 2016-10-10 2018-12-04 Array Biopharma Inc. Substituted pyrazolo[1,5-A]pyridine compounds as RET kinase inhibitors
WO2019075114A1 (fr) 2017-10-10 2019-04-18 Mark Reynolds Formulations comprenant du 6-(2-hydroxy-2-méthylpropoxy)-4-(6-(6-((6-méthoxypyridin-3-yl)méthyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile
WO2019075108A1 (fr) 2017-10-10 2019-04-18 Metcalf Andrew T Formes cristallines
WO2019143994A1 (fr) 2018-01-18 2019-07-25 Array Biopharma Inc. Composés de pyrazolyl[4,3-c]pyridine substitués utilisés en tant qu'inhibiteurs de la kinase ret
WO2019143977A1 (fr) 2018-01-18 2019-07-25 Array Biopharma Inc. Composés de pyrrolo[2,3-d]pyrimidines substitués utilisés en tant qu'inhibiteurs de la kinase ret
WO2020055672A1 (fr) 2018-09-10 2020-03-19 Array Biopharma Inc. Composés hétérocycliques condensés comme inhibiteurs de kinases ret
US10647730B2 (en) 2010-05-20 2020-05-12 Array Biopharma Inc. Macrocyclic compounds as TRK kinase inhibitors
US10966985B2 (en) 2017-03-16 2021-04-06 Array Biopharma Inc. Macrocyclic compounds as ROS1 kinase inhibitors
US11524963B2 (en) 2018-01-18 2022-12-13 Array Biopharma Inc. Substituted pyrazolo[3,4-d]pyrimidines as RET kinase inhibitors

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US10174028B2 (en) 2015-07-16 2019-01-08 Array Biopharma Inc. Substituted pyrazolo[1,5-A]pyridine compounds as RET kinase inhibitors
US10138243B2 (en) 2015-07-16 2018-11-27 Array Biopharma Inc. Substituted pyrazolo[1,5-a]pyridine compounds as RET kinase inhibitors
US10174027B2 (en) 2015-07-16 2019-01-08 Array Biopharma Inc. Substituted pyrazolo[1,5-a]pyridine compounds as RET kinase inhibitors
US10023570B2 (en) 2015-07-16 2018-07-17 Array Biopharma Inc. Substituted pyrazolo[1,5-A]pyridine compounds as RET kinase inhibitors
US10144734B2 (en) 2016-10-10 2018-12-04 Array Biopharma Inc. Substituted pyrazolo[1,5-A]pyridine compounds as RET kinase inhibitors
US10441581B2 (en) 2016-10-10 2019-10-15 Array Biopharma Inc. Substituted pyrazolo[1,5-A]pyridine compounds as RET kinase inhibitors
US10172845B2 (en) 2016-10-10 2019-01-08 Array Biopharma Inc. Substituted pyrazolo[1,5-A]pyridine compounds as RET kinase inhibitors
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US10172851B2 (en) 2016-10-10 2019-01-08 Array Biopharma Inc. Substituted pyrazolo[1,5-A]pyridine compounds as RET kinase inhibitors
US10112942B2 (en) 2016-10-10 2018-10-30 Array Biopharma Inc. Substituted pyrazolo[1,5-A]pyridine compounds as RET kinase inhibitors
US10137124B2 (en) 2016-10-10 2018-11-27 Array Biopharma Inc. Substituted pyrazolo[1,5-a]pyridine compounds as RET kinase inhibitors
US11648243B2 (en) 2016-10-10 2023-05-16 Array Biopharma Inc. Substituted pyrazolo[1,5-A]pyridine compounds as RET kinase inhibitors
US10953005B1 (en) 2016-10-10 2021-03-23 Array Biopharma Inc. Substituted pyrazolo[1,5-a]pyridine compounds as RET kinase inhibitors
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US10555944B2 (en) 2016-10-10 2020-02-11 Eli Lilly And Company Substituted pyrazolo[1,5-A]pyridine compounds as RET kinase inhibitors
WO2018136663A1 (fr) 2017-01-18 2018-07-26 Array Biopharma, Inc. Inhibiteurs de ret
US11168090B2 (en) 2017-01-18 2021-11-09 Array Biopharma Inc. Substituted pyrazolo[1,5-a]pyrazines as RET kinase inhibitors
WO2018136661A1 (fr) 2017-01-18 2018-07-26 Andrews Steven W Composés de pyrazolo[1,5-a]pyrazine substitués utilisés en tant qu'inhibiteurs de la kinase ret
US10966985B2 (en) 2017-03-16 2021-04-06 Array Biopharma Inc. Macrocyclic compounds as ROS1 kinase inhibitors
WO2019075108A1 (fr) 2017-10-10 2019-04-18 Metcalf Andrew T Formes cristallines
WO2019075114A1 (fr) 2017-10-10 2019-04-18 Mark Reynolds Formulations comprenant du 6-(2-hydroxy-2-méthylpropoxy)-4-(6-(6-((6-méthoxypyridin-3-yl)méthyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile
WO2019143977A1 (fr) 2018-01-18 2019-07-25 Array Biopharma Inc. Composés de pyrrolo[2,3-d]pyrimidines substitués utilisés en tant qu'inhibiteurs de la kinase ret
WO2019143994A1 (fr) 2018-01-18 2019-07-25 Array Biopharma Inc. Composés de pyrazolyl[4,3-c]pyridine substitués utilisés en tant qu'inhibiteurs de la kinase ret
US11472802B2 (en) 2018-01-18 2022-10-18 Array Biopharma Inc. Substituted pyrazolyl[4,3-c]pyridine compounds as RET kinase inhibitors
US11524963B2 (en) 2018-01-18 2022-12-13 Array Biopharma Inc. Substituted pyrazolo[3,4-d]pyrimidines as RET kinase inhibitors
US11603374B2 (en) 2018-01-18 2023-03-14 Array Biopharma Inc. Substituted pyrrolo[2,3-d]pyrimidines compounds as ret kinase inhibitors
WO2020055672A1 (fr) 2018-09-10 2020-03-19 Array Biopharma Inc. Composés hétérocycliques condensés comme inhibiteurs de kinases ret
US11964988B2 (en) 2018-09-10 2024-04-23 Array Biopharma Inc. Fused heterocyclic compounds as RET kinase inhibitors

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