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WO2008013928A2 - Traitement du cancer par administration de gène interféron en association avec un inhibiteur tgf-beta - Google Patents

Traitement du cancer par administration de gène interféron en association avec un inhibiteur tgf-beta Download PDF

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Publication number
WO2008013928A2
WO2008013928A2 PCT/US2007/016880 US2007016880W WO2008013928A2 WO 2008013928 A2 WO2008013928 A2 WO 2008013928A2 US 2007016880 W US2007016880 W US 2007016880W WO 2008013928 A2 WO2008013928 A2 WO 2008013928A2
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WIPO (PCT)
Prior art keywords
pyridin
methyl
pyrimidin
administration
imidazo
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Ceased
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PCT/US2007/016880
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WO2008013928A3 (fr
Inventor
Michael Parr
Leona Ling
Steven Albelda
Samuel Kim
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Biogen Inc
University of Pennsylvania Penn
Biogen MA Inc
Original Assignee
University of Pennsylvania Penn
Biogen Idec Inc
Biogen Idec MA Inc
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Publication of WO2008013928A2 publication Critical patent/WO2008013928A2/fr
Publication of WO2008013928A3 publication Critical patent/WO2008013928A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • This invention relates cancer treatment. Specifically, the invention relates to methods for improved cancer therapy comprising administering to a subject a vector, e.g., a viral vector comprising a nucleic acid encoding an interferon polypeptide, e.g., interferon beta, and an inhibitor of TGF- ⁇ .
  • a vector e.g., a viral vector comprising a nucleic acid encoding an interferon polypeptide, e.g., interferon beta, and an inhibitor of TGF- ⁇ .
  • Interferons are proteins having a variety of biological activities, some of which are antiviral, immunomodulating and antiproliferative. They are relatively small, species-specific, single chain polypeptides, produced by mammalian cells in response to exposure to a variety of inducers such as viruses, polypeptides, mitogens and the like. Interferons protect animal tissues and cells against viral attack and are an important host defense mechanism. In most cases, interferons provide better protection to tissues and cells of the kind from which they have been produced than to other types of tissues and cells, indicating that human-derived interferon could be more efficacious in treating human diseases than interferons from other species.
  • interferon- alpha [ ⁇ ] interferon- alpha [ ⁇ ]
  • fibroblast interferon-beta [ ⁇ ]
  • immune interferon-gamma [ ⁇ ]
  • Interferons have potential in the treatment of a large number of human cancers since these molecules have anti-cancer activity which acts at multiple levels.
  • interferon proteins can directly inhibit the proliferation of human tumor cells.
  • the anti-proliferative activity is also synergistic with a variety of approved chemotherapeutic agents such as cis-platin, 5FU and taxol.
  • the immunomodulatory activity of interferon proteins can lead to the induction of an anti-tumor immune response. This response includes activation of NK cells, stimulation of macrophage activity and induction of MHC class I surface expression leading to the induction of anti-tumor cytotoxic T lymphocyte activity.
  • TFN- ⁇ protein may have antiangiogenic activity.
  • Angiogenesis new blood vessel formation, is critical for the growth of solid tumors.
  • Evidence indicates that IFN- ⁇ may inhibit angiogenesis by inhibiting the expression of pro-angiogenic factors such as bFGF and VEGF.
  • interferon proteins may inhibit tumor invasiveness by affecting the expression of enzymes such as collagenase and elastase which are important in tissue remodeling.
  • Treatment of cancer via the delivery of interferon gene products via is an alternative to parenteral administration of interferon polypeptides.
  • Interferon delivery, e.g., IFN- ⁇ delivery, via viral vectors has proven to be effective treatment for various cancers. See, e.g., U.S. Patent No. 6,696,423, and U.S. Patent Application Publication No.
  • Gene product delivery vectors e.g. , viral vectors, including replication-defective viral vectors, are being used as delivery vehicles for a wide range of transgenes in pre-clinical and clinical studies for many pathological indications, including treatment of various cancers.
  • Administration of viral vectors can occur through systemic or local, e.g., intratumoral, routes.
  • routes of administration including intravenous, intraperitoneal, and subcutaneous administration can result in transduction of cells followed by expression of a viral vector-encoded transgene and detectable circulating levels of a secreted transgene product.
  • TGF- ⁇ Transforming Growth Factor- ⁇
  • BMPs bone morphogenetic proteins
  • GDFs growth and differentiation factors
  • MIS mullerian inhibiting substance
  • TGF- ⁇ isoform is synthesized as a precursor protein that is cleaved intracellularly into a C-terminal region (latency associated peptide (LAP) ) and an N-terminal region known as mature or active TGF- ⁇ .
  • LAP latency associated peptide
  • TGF- ⁇ binds at high affinity to the type II receptor (TGF- ⁇ RII), a constitutively active serine/threonine kinase.
  • TGF- ⁇ RII type II receptor
  • the ligand-bound type II receptor phosphorylates the TGF- ⁇ type I receptor (Alk5) in a glycine/serine rich domain, which allows the type I receptor to recruit and phosphorylate downstream signaling molecules, Smad2 or Smad3. See, e.g. , Huse, M. et al., MoI. Cell. 8: 671-682 (2001).
  • Phosphorylated Smad2 or Smad3 can then complex with Smad4, and the entire hetero- Smad complex translocates to the nucleus and regulates transcription of various TGF- ⁇ - responsive genes. See, e. g.,Massagu ⁇ , J. Ann. Rev. Biochem. Med. 67: 773 (1998).
  • TGF- ⁇ activity is found to manifest in patients with progressive cancers. Studies have shown that in late stages of various cancers, both the tumor cells and the stromal cells within the tumors generally overexpress TGF- ⁇ . This leads to stimulation of angiogenesis and cell motility, suppression of the immune system, and increased interaction of tumor cells with the extracellular matrix. See, e.g., Hojo, M. et al., Nature 397: 530-534 (1999). As a result, the tumor cells become more invasive and metastasize to distant organs. See, e.g. , Maehara, Y. et al, J. Clin.Oncol. 17: 607-614 (1999) and Picon, A. et al.
  • agents which inhibit TGF- ⁇ signaling pathways are useful for treating and/or preventing various cancer, including carcinomas of the lung, breast, liver, biliary tract, gastrointestinal tract, head and neck, pancreas, prostate, cervix as well as multiple myeloma, melanoma, glioma, and glioblastomas.
  • the present invention provides novel cancer treatments in which interferon gene delivery is combined with administration of one or more inhibitors of TGF- ⁇ .
  • This invention provides an improved method of cancer therapy, e.g., reduction of tumor size, elimination of tumors, prevention of tumor growth, prevention of metastases, prolonged survival and the like, comprising administering to a subject a vector comprising a nucleic acid encoding an interferon gene product, e.g., an interferon ⁇ protein or biologically active fragment, variant, or derivative thereof, in combination with a TGF- ⁇ inhibitor.
  • an interferon gene product e.g., an interferon ⁇ protein or biologically active fragment, variant, or derivative thereof
  • the vector is a viral vector, for example, an adenovirus vector.
  • the TGF- ⁇ inhibitor may be administered prior to administering the viral vector.
  • the TGF- ⁇ inhibitor may be administered 24 hours or less prior to administering the viral vector, 1 hour or less prior to administering the viral vector, or five minutes or less prior to administering the viral vector.
  • the TGF- ⁇ inhibitor may be administered concurrently with the vector.
  • the subject is a rodent.
  • the subject is a primate.
  • the primate is a human.
  • the vector is administered by a route selected from the group consisting of oral administration; nasal administration; parenteral administration; transdermal administration; topical administration; intraocular administration; intrabronchial administration; intraperitoneal administration; intravenous administration; subcutaneous administration; intramuscular administration; direct injection into cells; direct injection into tissues; direct injection into organs; direct injection into tumors; and a combination of two or more of these routes of administration.
  • the TGF- ⁇ inhibitor is administered by a route selected from the group consisting of oral administration; nasal administration; parenteral administration; transdermal administration; topical administration; intraocular administration; intrabronchial administration; intraperitoneal administration; intravenous administration; subcutaneous administration; intramuscular administration; direct injection into cells; direct injection into tissues; direct injection into organs; direct injection into tumors; and a combination of two or more of these routes of administration.
  • the viral vector is an adenovirus vector.
  • the viral vector is a replication-defective viral vector.
  • the interferon polypeptide is interferon- ⁇ , or a biologically or therapeutically active variant, fragment, or derivative thereof.
  • the interferon- ⁇ is human interferon- ⁇ .
  • kits for delivering a viral vector encoding an interferon polypeptide comprising a vector encoding an interferon polypeptide and a TGF- ⁇ inhibitor.
  • the vector is a viral vector.
  • the viral vector is an adenoviral vector.
  • the viral vector is a replication-defective viral vector.
  • the interferon polypeptide is interferon- ⁇ , or a biologically or therapeutically active variant, fragment, or derivative thereof.
  • the interferon- ⁇ is human interferon- ⁇ .
  • Figure 1 Effect of Ad INF B and SM 16 Combination in LKR tumors.
  • Figure 2 Effect of Ad INF and SM 16 combo on AB12 tumors.
  • Figure 3 Effect of Ad INF+SM16 on survival in LKR orthotopic lung cancer model.
  • the term “a” or “an” entity refers to one or more of that entity.
  • the terms “a” or “an”, “one or more,” and “at least one” can be used interchangeably.
  • the term “administration” refers to systemic and/or local administration.
  • systemic administration refers to non-localized administration such that an administered substance may affect several organs or tissues throughout the body or such that an administered substance may traverse several organs or tissues throughout the body in reaching a target site.
  • administration of a gene delivery vector into a subject's circulation may result in expression of a therapeutic product from an administered vector in more than one tissue or organ, or may result in expression of a therapeutic product from an administered vector at a specific site, e.g., due to natural tropism or operable linkage of tissue- specific promoter elements.
  • systemic administration including those forms of administration encompassed by parenteral administration such as intravenous, intramuscular, intraperitoneal, and subcutaneous administration.
  • systemic administration can be used to elicit a systemic effect associated with treatment of a local or systemic disease or condition.
  • a systemic effect may be desirable for a local disease or condition, for example, to prevent spread of said disease or condition.
  • the term "local administration" refers to administration at or near a specific site.
  • local administration is associated with treatment of a disease or condition where a local effect is desired ⁇ e.g. administration to the lung for the treatment of lung cancer).
  • a local effect may be desired in association with either local or systemic diseases or conditions.
  • a local effect may be desired in association with a systemic disease or condition to treat a local aspect of a systemic disease or condition.
  • the term “comprise” and variations such as “comprises” or “comprising” indicate the inclusion of any recited integer or group of integers but not the exclusion of any other integer or group of integers in the specified method, structure, or composition.
  • the term “consists of and variations such as “consist of or “consisting of indicate the inclusion of any recited integer or group of integers but that no additional integer or group of integers may be added to the specified method, structure, or composition.
  • polypeptide is intended to encompass a singular "polypeptide” as well as plural “polypeptides” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds).
  • polypeptide refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product.
  • peptides, dipeptides, tripeptides, oligopeptides, "protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide,” and the term “polypeptide” may be used instead of or interchangeably with any of these terms.
  • polypeptide is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids.
  • Polypeptides as described herein may include fragment, variant, or derivative molecules thereof without limitation.
  • fragment when referring to a polypeptide include any polypeptide which retains at least some biological or therapeutic activity.
  • Polypeptide fragments may include proteolytic fragments, deletion fragments, and fragments which more easily reach the site of action when delivered to an animal.
  • Polypeptide fragments further include any portion of the polypeptide which comprises an antigenic or immunogenic epitope of the native polypeptide, including linear as well as three-dimensional epitopes.
  • Polypeptide fragments may comprise variant regions, including fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions.
  • Variants may occur naturally, such as an allelic variant.
  • allelic variant By an "allelic variant” is intended alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes ⁇ , Lewin, B., ed., John Wiley & Sons, New York (1985). Non- naturally occurring variants may be produced using art-known mutagenesis techniques.
  • Polypeptide fragments of the invention may comprise conservative or non-conservative amino acid substitutions, deletions or additions. Variant polypeptides may also be referred to herein as "polypeptide analogs.” Polypeptide fragments of the present invention may also include derivative molecules. As used herein a "derivative" of a polypeptide or a polypeptide fragment refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Also included as “derivatives" are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids.
  • 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.
  • fusion protein means a protein comprising a first polypeptide linearly connected, via peptide bonds, to a second, polypeptide.
  • the first polypeptide and the second polypeptide may be identical or different, and they may be directly connected, or connected via a peptide linker.
  • the terms "linked,” “fused” or “fusion” are used interchangeably. These terms refer to the joining together of two more elements or components, by any means including chemical conjugation or recombinant means.
  • An “in-frame fusion” refers to the joining of two or more open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct reading frame of the original ORFs.
  • the resulting recombinant fusion protein is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature.) Although the reading frame is thus made continuous throughout the fused segments, the segments may be physically or spatially separated by, for example, in-frame linker sequence.
  • a "linker" sequence is a series of one or more amino acids separating two polypeptide coding regions in a fusion protein.
  • a “linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminal direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide.
  • polynucleotide is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g. , messenger RNA (mRNA) or plasmid DNA (pDNA).
  • mRNA messenger RNA
  • pDNA plasmid DNA
  • a polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)).
  • a polynucleotide can contain the nucleotide sequence of the full length cDNA sequence, including the untranslated 5' and 3' sequences, the coding sequences, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence.
  • the polynucleotide can be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single- stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • the polynucleotides can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. Polynucleotides may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons.
  • nucleic acid refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.
  • isolated nucleic acid or isolated polynucleotide refers to a nucleic acid molecule, DNA or RNA, which has been removed from its native environment.
  • an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present invention.
  • Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically.
  • polynucleotide or a nucleic acid may be or may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.
  • a "coding region” is a portion of nucleic acid which consists of codons translated into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. Two or more coding regions of the present invention can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors.
  • any vector may contain a single coding region, or may comprise two or more coding regions.
  • a vector, polynucleotide, or nucleic acid of the invention may encode heterologous coding regions, either fused or unfused to a nucleic acid encoding a polypeptide or polypeptide fragment of the present invention.
  • Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.
  • the polynucleotide or nucleic acid is DNA.
  • a polynucleotide comprising a nucleic acid which encodes a polypeptide normally includes a promoter and/or other transcription or translation control elements operably associated with one or more coding regions.
  • An operable association is when a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s).
  • Two DNA fragments are "operably associated" if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed.
  • a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid.
  • the promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells.
  • transcription control elements besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell- specific transcription.
  • Suitable promoters and other transcription control regions are disclosed herein.
  • a variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus).
  • transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone, and rabbit ⁇ -globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters ⁇ e.g., promoters inducible by interferons or interleukins).
  • translation control elements include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picomaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).
  • a polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA).
  • mRNA messenger RNA
  • Polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention.
  • proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated.
  • polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or "full length" polypeptide to produce a secreted or "mature” form of the polypeptide.
  • the native signal peptide is used or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it.
  • a heterologous mammalian signal peptide, or a functional derivative thereof may be used.
  • the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse ⁇ -glucuronidase.
  • the term "expression” as used herein refers to a process by which a gene produces a biochemical, for example, a RNA or polypeptide.
  • the process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes, without limitation, transcription of the gene into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), or any other RNA product, and the translation of such mRNA into polypeptide(s). If the final desired product is biochemical, expression includes the creation of that biochemical and any precursors.
  • mRNA messenger RNA
  • tRNA transfer RNA
  • shRNA small hairpin RNA
  • siRNA small interfering RNA
  • small molecule As used herein, the terms "small molecule,” “small molecule inhibitor,” and analogous terms include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e,. including heteroorganic and organometallic compounds) having a molecular weight less than about 1,000 daltons, organic or inorganic compounds having a molecular weight less than about 500 daltons, organic or inorganic compounds having a molecular weight less than about 250 daltons, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
  • organic or inorganic compounds i.e,. including heteroorganic and organometallic compounds
  • the terms “treat” and “treatment” refers to therapeutic treatment, including prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of cancer.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of the extent of a disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
  • subject or “individual” or “animal” or “patient” or “vertebrate” or “mammal,” is meant any subject, e.g., a mammalian subject, for whom diagnosis, prognosis, or therapy is desired.
  • Mammalian subjects include, but are not limited to, humans; domestic animals; farm animals; zoo animals; sport animals; pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; ursids such as bears, food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on.
  • the mammal is a human subject.
  • a “therapeutically effective amount” or “therapeutic dose” refers to an amount effective, at dosages and for periods of time necessary, to achieve at least one desired therapeutic result.
  • a therapeutic result may be, e.g., lessening of symptoms, prolonged survival, improved mobility, and the like.
  • a therapeutic result need not be a "cure”.
  • the present invention provides combination therapies.
  • the "therapeutically effective amount" of any one of the agents may be lesser than if the agents were given individually.
  • the present invention may enable the dose of one or more of the agents to be reduced (dose sparing). This may lead to a reduction the likelihood/occurrence of the side effects, such as liver toxicity which may occur, e.g, upon delivery of high doses of adenoviral vectors.
  • dose sparing A reduction of side effects of a therapeutic agent or an increase in effectiveness of the agent during treatment of human or animal patients being treated is known as "dose sparing".
  • a prophylactically effective amount or “prophylactic dose” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, a prophylactically effective amount will be less than a therapeutically effective amount for treatment of an advanced stage of disease.
  • gene delivery refers to a procedure in therapy, e.g., treatment of a disease, is effected through the introduction and expression of genetic information into the affected organism.
  • Gene delivery includes "ex vivo gene delivery,” in which cells are removed from a subject and cultured in vitro.
  • ex vivo gene delivery a polynucleotide such as a functional gene is introduced into the cells in vitro, the modified cells are expanded in culture, and then reimplanted in the subject.
  • Gene delivery also includes "in vivo gene delivery,” in which target cells are not removed from the subject.
  • the transferred polynucleotide e.g., a gene encoding, upon expression, an interferon
  • the transferred polynucleotide is introduced into cells of the recipient organism in situ, that is, within the recipient.
  • gene delivery includes any condition amenable to gene delivery such as genetic diseases (i.e., a disease condition that is attributable to one or more gene defects), acquired pathologies (e.g., a pathological condition, e.g., an infectious disease, which is not attributable to an inborn defect), cancers, and nascent or early stage conditions.
  • tumor refers, in part, to any undesirable proliferation of cells, including malignant and non-malignant tumors, solid or fluid tumors, carcinomas, myelomas, sarcomas, leukemias, lymphomas, and other cancerous, neoplastic, or tumorigenic diseases.
  • tumorigenic diseases include malignant and non-malignant tumors, solid or fluid tumors, carcinomas, myelomas, sarcomas, leukemias, lymphomas, and other cancerous, neoplastic, or tumorigenic diseases.
  • a binding molecule comprises, consists essentially of, or consists of at least one binding domain which, either alone or in combination with one or more additional binding domains, specifically binds to a target gene product (such as a messenger RNA, a protein, an antigen or other binding partner), e.g., a TGF- ⁇ polypeptide, a TGF- ⁇ receptor polypeptide, or a fragment or variant thereof.
  • a binding molecule comprises, consists essentially of, or consists of at least two binding domains, for example, two, three, four, five, six, or more binding domains. Each binding domain may bind to a target molecule separately, or two or more binding domains may be required to bind to a given target, for example, a combination of an immunoglobulin heavy chain and an immunoglobulin light chain.
  • Binding molecules e.g., binding polypeptides, e.g., TGF- ⁇ - or TGF- ⁇ receptor-specific antibodies used in the methods disclosed herein may comprise, consist essentially of, or consist of two or more subunits thus forming multimers, e.g., dimers, trimers or tetramers.
  • certain binding molecules comprise a polypeptide dimer, typically, a heterodimer comprising two non-identical monomeric subunits.
  • Other binding molecules comprise tetramers, which can include two pairs of homodimers, e.g., two identical monomeric subunits, e.g., an antibody molecule consisting of two identical heavy chains and two identical light chains.
  • binding molecules e.g., binding polypeptides to be utilized in the methods disclosed herein comprise at least one amino acid sequence derived from an immunoglobulin.
  • a polypeptide or amino acid sequence "derived from" a designated protein refers to the origin of the polypeptide.
  • the polypeptide or amino acid sequence which is derived from a particular starting polypeptide or amino acid sequence has an amino acid sequence that is essentially identical to that of the starting sequence, or a portion thereof, wherein the portion consists of at least 10-20 amino acids, preferably at least 20-30 amino acids, more preferably at least 30-50 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the starting sequence.
  • a polypeptide or amino acid sequence derived from a designated protein may be similar, e.g., have a certain percent identity to the starting sequence, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the starting sequence, as described in more detail below.
  • binding polypeptides comprise, consist essentially of, or consist of an amino acid sequence derived from a human amino acid sequence.
  • binding polypeptides may comprise one or more contiguous amino acids derived from another mammalian species.
  • a primate heavy chain portion, hinge portion, or binding site may be included in the subject binding polypeptides.
  • one or more murine-derived amino acids may be present in a non-murine binding polypeptide, e.g., in an antigen binding site of a binding molecule.
  • preferred binding molecules to be used in the methods of the invention are not immunogenic in the animal to which the binding polypeptide is administered.
  • binding polypeptides for use in the methods disclosed herein may be modified such that they vary in amino acid sequence from the naturally occurring binding polypeptide from which they were derived. For example, nucleotide or amino acid substitutions leading to conservative substitutions or changes at "non-essential" amino acid residues may be made.
  • a binding polypeptide for use in the methods of the invention comprises an amino acid sequence or one or more moieties not normally associated with that binding polypeptide. Exemplary modifications are described in more detail below.
  • a binding polypeptide of the invention may comprise a flexible linker sequence, or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label).
  • a binding polypeptide for use in the methods of the invention may comprise, consist essentially of, or consist of a fusion protein.
  • Fusion proteins are chimeric molecules which comprise a binding domain with at least one target binding site, and at least one heterologous portion.
  • a "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence with which it is not naturally linked in nature.
  • the amino acid sequences may normally exist in separate proteins that are brought together in the fusion polypeptide or they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide.
  • a chimeric protein may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.
  • heterologous as applied to a polynucleotide or a polypeptide, means that the polynucleotide or polypeptide is derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared. For instance, a heterologous polynucleotide or antigen may be derived from a different species origin, different cell type, or the same type of cell of distinct individuals.
  • ligand binding domain or “ligand binding portion” as used herein refers to any native receptor (e.g., cell surface receptor) or any region or derivative thereof retaining at least a qualitative ligand binding ability, and preferably the biological activity of a corresponding native receptor.
  • receptor binding domain or "receptor binding portion” as used herein refers to any native ligand or any region or derivative thereof retaining at least a qualitative receptor binding ability, and preferably the biological activity of a corresponding native ligand.
  • TGF- ⁇ inhibitors for use in the methods disclosed herein are "antibody” or “immunoglobulin” molecules, or immunospecific fragments thereof, e.g., naturally occurring antibody or immunoglobulin molecules or engineered antibody molecules or fragments that bind antigen in a manner similar to antibody molecules.
  • Typical TGF- ⁇ inhibitor antibodies of the present invention are directed to either TGF- ⁇ - or TGF- ⁇ receptor, and block the interaction between TGF- ⁇ - or TGF- ⁇ receptor, thereby inhibiting TGF- ⁇ induced signaling.
  • the terms "antibody” and “immunoglobulin” are used interchangeably herein.
  • immunoglobulin molecules used in the methods of the invention are also described as "immunospecific” or “antigen-specific” or “antigen-binding” molecules and are used interchangeably to refer to antibody molecules and fragments thereof.
  • An antibody or immunoglobulin comprises at least the variable domain of a heavy chain, and normally comprises at least the variable domains of a heavy chain and a light chain.
  • Basic immunoglobulin structures in vertebrate systems are relatively well understood. See, e.g., Harlow et ah, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988), incorporated herein by reference.
  • immunoglobulin comprises five broad classes of polypeptides that can be distinguished biochemically. All five classes are clearly within the scope of the present invention, the following discussion will generally be directed to the IgG class of immunoglobulin molecules.
  • IgG a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000. The four chains are typically joined by disulfide bonds in a "Y" configuration wherein the light chains bracket the heavy chains starting at the mouth of the "Y” and continuing through the variable region.
  • Both the light and heavy chains are divided into regions of structural and functional homology.
  • the terms "constant” and “variable” are used functionally.
  • the variable domains of both the light (V L ) and heavy (V H ) chain portions determine antigen recognition and specificity.
  • the constant domains of the light chain (C L ) and the heavy chain (C H 1 , C H 2 or C H 3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like.
  • the numbering of the constant region domains increases as they become more distal from the antigen binding site or amino-terminus of the antibody.
  • the N-terminal portion is a variable region and at the C-terminal portion is a constant region; the C H 3 and C L domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.
  • Light chains are classified as either kappa or lambda (K, ⁇ ). Each heavy chain class may be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells.
  • heavy chains In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.
  • heavy chains are classified as gamma, mu, alpha, delta, or epsilon, ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ) with some subclasses among them ⁇ e.g., ⁇ l- ⁇ 4). It is the nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA IgG, or IgE, respectively.
  • the immunoglobulin subclasses isotypes) e.g., IgGi, IgGz.
  • IgG3, IgG-i, IgAi, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discemable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant invention.
  • variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the V L domain and V H domain of an antibody combine to form the variable region that defines a three dimensional antigen binding site. This quaternary antibody structure forms the antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three complementary determining regions (CDRs) on each of the V H and V L chains.
  • CDRs complementary determining regions
  • a complete immunoglobulin molecule may consist of heavy chains only, with no light chains.
  • each antigen binding domain is short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding domain as the antibody assumes its three dimensional configuration in an aqueous environment.
  • the remainder of the amino acids in the antigen binding domains referred to as "framework" regions, show less inter-molecular variability.
  • the framework regions largely adopt a ⁇ -sheet conformation and the CDRs form loops which connect, and in some cases form part of, the ⁇ -sheet structure.
  • framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.
  • the antigen binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope.
  • the amino acids comprising the CDRs and the framework regions, respectively can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined ⁇ see, "Sequences of Proteins of Immunological Interest," Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI.
  • V H H the heavy chain variable region
  • the main differences between camelid V H H variable regions and those derived from conventional antibodies (V H ) include (a) more hydrophobic amino acids in the light chain contact surface of V H as compared to the corresponding region in V H H, (b) a longer CDR3 in V H H, and (c) the frequent occurrence of a disulfide bond between CDRl and CDR3 in V H H.
  • an antigen binding molecule for use in the methods of the invention comprises at least one heavy or light chain CDR of an antibody molecule.
  • an antigen binding molecule for use in the methods of the invention comprises at least two CDRs from one or more antibody molecules.
  • an antigen binding molecule for use in the methods of the invention comprises at least three CDRs from one or more antibody molecules.
  • an antigen binding molecule for use in the methods of the invention comprises at least four CDRs from one or more antibody molecules.
  • an antigen binding molecule for use in the methods of the invention comprises at least five CDRs from one or more antibody molecules.
  • an antigen binding molecule for use in the methods of the invention comprises at least six CDRs from one or more antibody molecules.
  • Exemplary antibody molecules comprising at least one CDR that can be included in the subject antigen binding molecules are known in the art and exemplary molecules are described herein.
  • Antibodies or immunospecific fragments thereof for use in the methods of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab') 2 .
  • Immunoglobulin or antibody molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class ⁇ e.g., Igd, IgG 2 , IgG 3 , IgG 4 , IgAi and IgA 2 ) or subclass of immunoglobulin molecule.
  • Antibody fragments may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, C H 1, C H 2, and C H 3 domains of the heavy chain, or C L of the light chain. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, C H I, C H 2, C H 3, or C L domain.
  • Antibodies or immunospecific fragments thereof for use in the methods disclosed herein may be from any animal origin including birds and mammals.
  • the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies.
  • variable region may be condricthoid in origin ⁇ e.g., from sharks).
  • "human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and that do not express endogenous immunoglobulins, as described infra and, for example, in U.S. Pat. No. 5,939,598 by Kucherlapati et al.
  • heavy chain portion includes amino acid sequences derived from an immunoglobulin heavy chain.
  • a polypeptide comprising a heavy chain portion comprises at least one of: a C H 1 domain, a hinge ⁇ e.g., upper, middle, and/or lower hinge region) domain, a C H 2 domain, a C H 3 domain, or a variant or fragment thereof.
  • a heavy chain portion may comprise a polypeptide chain comprising a C H I domain; a polypeptide chain comprising a C H I domain, at least a portion of a hinge domain, and a C H 2 domain; a polypeptide chain comprising a C H l domain and a C H 3 domain; a polypeptide chain comprising a C H I domain, at least a portion of a hinge domain, and a C H 3 domain, or a polypeptide chain comprising a C H I domain, at least a portion of a hinge domain, a C H 2 domain, and a C H 3 domain.
  • the heavy chain portion may also include a polypeptide comprising a polypeptide chain comprising a C H 3 domain.
  • a binding polypeptide for use in the invention may lack at least a portion of a C H 2 domain ⁇ e.g., all or part of a C H 2 domain).
  • these domains ⁇ e.g., the heavy chain portions
  • these domains may be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molecule.
  • the heavy chain portions of one polypeptide chain of a multimer are identical to those on a second polypeptide chain of the multimer.
  • heavy chain portion-containing monomers for use in the methods of the invention are not identical.
  • each monomer may comprise a different target binding site, forming, for example, a bispecific antibody.
  • the heavy chain portions of a binding polypeptide for use in the methods disclosed herein may be derived from different immunoglobulin molecules.
  • a heavy chain portion of a polypeptide may comprise a C H I domain derived from an IgGj molecule and a hinge region derived from an IgG 3 molecule.
  • a heavy chain portion can comprise a hinge region derived, in part, from an IgGi molecule and, in part, from an IgG 3 molecule.
  • a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgGi molecule and, in part, from an IgG 4 molecule.
  • the term "light chain portion" includes amino acid sequences derived from an immunoglobulin light chain.
  • the light chain portion comprises at least one of a V L or C L domain.
  • An isolated nucleic acid molecule encoding a non-natural variant of a polypeptide derived from an immunoglobulin can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of the immunoglobulin such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more non-essential amino acid residues.
  • binding affinities include those with a dissociation constant or Kd less than 5 x 10 "2 M, 10 "2 M, 5 x 10 3 M, 10° M, 5 x 10 ⁇ M, 10 "4 M, 5 x 10 s M, 10 "5 M, 5 x 10 "6 M, 10 '6 M, 5 x 10 "7 M, 10 '7 M, 5 x 10 '8 M, 10 "8 M, 5 x 10 9 M, 10 "9 M, 5 x 10 "10 M, 10 10 M, 5 x 10 " “ M, l ⁇ “ M, 5 x lO "12 M, 10 "12 M, 5 x 10 "13 M, 10 "13 M, 5 x 10 '14 M, 10 "14 M, 5 x 10 15 M, or lO '15 M.
  • Antibodies or immunospecific fragments thereof for use in the methods disclosed herein act as TGF- ⁇ inhibitors as described herein.
  • an antibody for use in the methods of the present invention may function as an antagonist, blocking or inhibiting signal transduction mediated by a TGF- ⁇ polypeptide.
  • chimeric antibody will be held to mean any antibody wherein the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial or modified in accordance with the instant invention) is obtained from a second species.
  • the target binding region or site will be from a non-human source (e.g. mouse or primate) and the constant region is human.
  • engineered antibody refers to an antibody in which the variable domain in either the heavy and light chain or both is altered by at least partial replacement of one or more CDRs from an antibody of known specificity and, if necessary, by partial framework region replacement and sequence changing.
  • the CDRs may be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRs will be derived from an antibody of different class and preferably from an antibody from a different species.
  • An engineered antibody in which one or more "donor" CDRs from a non-human antibody of known specificity is grafted into a human heavy or light chain framework region is referred to herein as a "humanized antibody.” It may not be necessary to replace all of the CDRs with the complete CDRs from the donor variable region to transfer the antigen binding capacity of one variable domain to another. Rather, it may only be necessary to transfer those residues that are necessary to maintain the activity of the target binding site.
  • Certain embodiments of the present invention provide methods of cancer treatment comprising a combination therapy, where the therapy comprises administration of a TGF- ⁇ inhibitor and a vector comprising a isolated polynucleotide which encodes an interferon polypeptide or therapeutically or biologically active fragment, variant, or derivative thereof to a subject in need of treatment.
  • An "interferon” also referred to as "IFN" is a small, species-specific, single chain polypeptide, produced by mammalian cells in response to exposure to a variety of inducers such as viruses, polypeptides, mitogens and the like.
  • interferons are in recombinant form and recombinant DNA methods for producing proteins including the various interferons are known and are not intended to limit the invention in any way. See for example, U.S. Patents 4,399,216, 5,149,636, 5,179,017 (Axel et al) and 4,470,461 (Kaufman).
  • recombinant forms of interferon-alpha, beta, gamma and consensus interferon have been produced.
  • Forms of interferon may be expressed from cells containing polynucleotide sequences encoding variants such as cysteine-depleted mutants (e.g. , for interferon-beta) and methionine- depleted mutants.
  • Preferred interferon-encoding polynucleotides that may be used in the present methods of the invention are derived from the wild-type interferon gene sequences of various vertebrates, preferably mammals, and are obtained using methods that are well-known to those having ordinary skill in the art. See, for example: U. S Patent 5,641,656 (issued Jun. 24, 1997: DNA encoding avian type I interferon proprotein and mature avian type I interferon), U.S. Patent 5,605,688 (Feb. 25, 1997- recombinant dog and horse type I interferons); U.S. Patent 5,554,513 (Sep. 10, 1996; DNA sequence which codes for human interferon-beta2A); U.S.
  • Patent 4,695,543 (Sep. 22, 1987, human alpha- interferon Gx-I gene and U.S. Patent 4,456,748 (Jun. 26, 1984, DNA encoding sub-sequences of different, naturally, occurring leukocyte interferons).
  • Examples of naturally-occurring IFN polypeptides include, but are not limited to, IFN polypeptides from rodents, primates, and humans having the amino acid sequences listed in Table 1.
  • a vector for use in the methods of the present invention comprises an isolated polynucleotide which encodes at least one interferon polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence identical to a reference amino acid sequence, except for up to twenty amino acid substitutions, wherein the reference amino acid sequence is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 38 as shown in Table 1, or a combination or composite of two or more of said sequences.
  • a vector for use in the methods of the present invention comprises an isolated polynucleotide which encodes at least one interferon polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 38 as shown in Table 1, or a combination or composite of two or more of said sequences.
  • a vector for use in the methods of the present invention comprises an isolated polynucleotide which encodes at least one interferon polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 38 as shown in Table 1, or a combination or composite of two or more of said sequences.
  • vectors comprising IFN-encoding polynucleotides which encode fragments, variants, or derivatives of any of the interferon polypeptides disclosed herein, and which retain biological or therapeutic activity, are provided.
  • a reference amino acid sequence is meant the specified sequence without the introduction of any amino acid substitutions.
  • the “isolated polypeptide” of the invention comprises an amino acid sequence which is identical to the reference amino acid sequence.
  • Interferon polypeptides encoded by vectors described herein may have various alterations such as substitutions, insertions or deletions.
  • Exemplary amino acids that can be substituted in the polypeptide include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e
  • sequence identity between two polypeptides is determined by comparing the amino acid sequence of one polypeptide to the sequence of a second polypeptide.
  • whether any particular polypeptide is at least about 70%, 75%, 80%, 85%, 90% or 95% identical to another polypeptide can be determined using methods and computer programs/software known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711).
  • BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences.
  • the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference polypeptide sequence and that gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.
  • Mutant members of the interferon family of genes may be used in accordance with this invention. Mutations in the wild-type interferon polynucleotide sequence may be naturally-occuring variants, or are developed using conventional methods of directed mutagenesis, known to those of ordinary skill in the art. The term “mutant” is also meant to encompass genetic fusions so that the following interferon sequences, incorporated herein by reference, would all be considered “mutant” sequences: [0089] U.S. Patent 5,273,889 (Dec. 28, 1993, DNA construct comprising gamma-interferon gene linked to a sequence encoding an immunogenic leukotoxin); U.S. Patent 4,959,314 (Sep.
  • a vector comprising an isolated polynucleotide encoding a consensus interferon is also provided.
  • Consensus interferon is a nonnaturally occurring polypeptide, which predominantly includes those amino acid residues that are common to all naturally-occurring human interferon subtype sequences and which include, at one or more of those positions where there is no amino acid common to all subtypes, an amino acid which predominantly occurs at that position and in no event includes any amino acid residue which is not extant in that position in at least one naturally-occurring subtype.
  • Consensus interferon sequences encompass consensus sequences of any two or more of the interferons polypeptide sequences known in the art or described herein.
  • consensus interferons are disclosed in U.S. Patents 4,695,623 and 4,897,471 (Amgen). Polynucleotides encoding consensus interferon may be synthesized as described in these patents or by other standard methods. Consensus interferon polypeptides are preferably the products of expression of manufactured DNA sequences, transformed or transfected into hosts, as described herein. That is, consensus interferon is preferably recombinantly produced. Such materials may be purified by procedures well known in the art. [0091 J Moreover, isolated interferon-encoding polynucleotides as described herein and in the cited references can be altered to provide for functionally equivalent polynucleotides. A polynucleotide is "functionally equivalent" compared with those of the above sequences if it satisfies at least one of the following conditions:
  • the "functional equivalent” is a polynucleotide that hybridizes to any of the foregoing sequences under standard or stringent hybridization conditions and/or is degenerate to any of the foregoing sequences. Most preferably, it encodes a mutant interferon having a desired therapeutic or biological activity of a wild type interferon, e.g., antiproliferative or apoptotic activity;
  • the "functional equivalent” is a polynucleotide that encodes an amino acid sequence encoded by any of the polynucleotides of the foregoing interferon sequences.
  • the term "interferon” includes, but is not limited to, the agents listed above as well as their functional equivalents.
  • the term “functional equivalent” therefore refers to an interferon protein or a polynucleotide encoding the interferon protein that has the same or an improved beneficial effect on the mammalian recipient as the interferon of which is it deemed a functional equivalent.
  • a functionally equivalent protein can be produced by recombinant techniques, e.g., by expressing a "functionally equivalent DNA”.
  • the instant invention embraces interferon encoded by naturally-occurring DNAs, as well as by non-naturally-occurring DNAs which encode the same protein as encoded by the naturally-occurring DNA. Due to the degeneracy of the nucleotide coding sequences, other polynucleotides may be used to encode interferons. These include all,,, or portions of the above sequences which are altered by the substitution of different codons that encode the same amino acid residue within the sequence, thus producing a silent change. Such altered sequences are regarded as equivalents of these sequences.
  • Trp (F) is coded for by two codons, TTC or TTT
  • Tyr (Y) is coded for by TAC or TAT
  • His (H) is coded for by CAC or CAT.
  • Trp (W) is coded for by a single codon, TGG.
  • transformation or “transform” refers to any genetic modification of cells and includes both “transfection” and “transduction”.
  • transfection of cells refers to the acquisition by a cell of new genetic material by incorporation of added DNA.
  • transfection refers to the insertion of nucleic acid (e.g., DNA) into a cell using physical or chemical methods.
  • nucleic acid e.g., DNA
  • transfection techniques are known to those of ordinary skill in the art including: calcium phosphate DNA co-precipitation (Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Ed. E. J. Murray, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; and tungsten particle-facilitated microparticle bombardment (Johnston, S.
  • transduction of cells refers to the process of transferring nucleic acid, into a cell using a DNA or RNA virus.
  • One or more isolated polynucleotide sequences encoding one or more interferon proteins contained within the virus may be incorporated into the chromosome of the transduced cell.
  • a cell is transduced with a virus but the cell will not have the isolated polynucleotide incorporated into its chromosomes but will be capable of expressing interferon extrachromosomally within the cell.
  • the cells are transformed (i.e., genetically modified) ex vivo.
  • the cells are isolated from a mammal (preferably a human) and transformed (i.e., transduced or transfected in vitro) with a vector containing an isolated polynucleotide such as a recombinant gene operatively linked to one or more expression control sequences for expressing a recombinant secreted protein (e.g., an interferon).
  • the cells are then administered to a mammalian recipient for delivery of the protein in situ.
  • the mammalian recipient is a human and the cells to be modified are autologous cells, i.e., the cells are isolated from the mammalian recipient. The isolation and culture of cells in vitro has been reported.
  • the cells are transformed or otherwise genetically modified in vivo.
  • the cells from the mammalian recipient preferably a human
  • a vector containing isolated polynucleotide such as a recombinant gene operatively linked to one or more expression control sequences for expressing a secreted protein (e.g., recombinant interferon) and the protein is delivered in situ.
  • a secreted protein e.g., recombinant interferon
  • the isolated polynucleotides encoding the secreted protein (e.g., a cDNA encoding one or more therapeutic interferon proteins) is introduced into the cell ex vivo or in vivo by genetic transfer methods, such as transfection or transduction, to provide a genetically modified cell.
  • Various expression vectors e.g., vehicles for facilitating delivery of the isolated polynucleotide into a target cell
  • the expressed polypeptide like IFN, is a secreted protein, surrounding cells which do not contain the vector or polynucleotide are still affected.
  • the present methods typically do not require use of a selectable gene.
  • the expression vector may optionally include a selection gene, for example, a neomycin resistance gene, for facilitating selection of cells that have been transfected or transduced with the expression vector.
  • the cells are transfected with two or more expression vectors, at least one vector containing the gene(s) encoding the interferon(s), the other vector containing a selection gene.
  • a suitable promoter, enhancer, selection gene and/or signal sequence (described below) is deemed to be within the scope of one of ordinary skill in the art without undue experimentation.
  • a nucleic acid encoding an interferon protein or therapeutically or biologically active fragment thereof is provided in a vector that allows introduction of and expression of the interferon polypeptide in the subject to be treated.
  • the vector allows for encapsulation of the gene of the encoded therapeutic product into a particle.
  • a suitable particle is a viral particle, e.g., an adenovirus particle.
  • the nucleic acid is carried by a non-viral vector.
  • Vectors may include appropriate transcriptional and translational control signals operatively linked to the polynucleotide sequence for a particular therapeutic gene. Promoters and enhancers may also be used to control expression of therapeutic proteins or gene products. Promoter activation may be tissue specific or inducible by a metabolic product or administered substance.
  • Such promoters and enhancers include, but are not limited to, the native E2F promoter, the cytomegalovirus immediate-early promoter and enhancer (Karasuyama et al., J. Exp. Med. 169: 13 (1989)), the human beta-actin promoter (Gunning et al., Proc. Nat. Acad. Sci. USA 84: 4831 (1987)), the glucocorticoid- inducible promoter present in the mouse mammary tumor virus long terminal repeat (MMTV LTR) (Klessig et al., MoI. Cell. Biol.
  • MMTV LTR mouse mammary tumor virus long terminal repeat
  • MoLV LTR Moloney murine leukemia virus
  • SSV Rous sarcoma virus
  • HSV herpes simplex virus
  • the introduced genetic material includes an isolated polynucleotide encoding, e.g., an interferon polypeptide (usually in the form of a cDNA comprising the exons coding for the interferon) together with a promoter to control transcription of the new gene.
  • the promoter characteristically has a specific nucleotide sequence necessary to initiate transcription.
  • the genetic material could include intronic sequences which will be removed from the mature transcript by RNA splicing.
  • a polyadenylation signal should be present at the 3 1 end of the gene to be expressed.
  • the introduced genetic material also may include an appropriate secretion "signal" sequence for secreting the therapeutic gene product (e.g., an interferon) from the cell to the extracellular milieu.
  • the isolated genetic material further includes additional sequences (e.g., enhancers) required to obtain the desired gene transcription activity.
  • additional sequences e.g., enhancers
  • an “enhancer” is simply any non-translated DNA sequence which works contiguous with the coding sequence (in cis) to change the basal transcription level dictated by the promoter.
  • isolated IFN-encoding DNA of the invention is operably associated with a promoter to permit transcription.
  • Typical vectors of the present invention comprise an exogenous promoter element to control transcription of the inserted interferon gene.
  • exogenous promoters include both constitutive and inducible promoters.
  • constitutive promoters control the expression of proteins that regulate essential cell functions. As a result, a gene under the control of a constitutive promoter is expressed under all conditions of cell growth.
  • exemplary constitutive promoters include the promoters for the following genes which encode certain constitutive or "housekeeping" functions: hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolate reductase (DHFR) (Scharfmann et al., Proc. Natl. Acad. Sci.
  • viral promoters function constitutively in eucaryotic cells. These include: the early and late promoters of SV40 (See Bemoist and Chambon, Nature, 290:304 (1981)); the long terminal repeats (LTRs) of Moloney Leukemia Virus and other retroviruses (See Weiss et al. , RNA Tumor Viruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1985)); the thymidine kinase promoter of Herpes Simplex Virus (HSV) (See Wagner et al., Proc. Nat. Acad. Sci.
  • SV40 See Bemoist and Chambon, Nature, 290:304 (1981)
  • LTRs long terminal repeats
  • HSV Herpes Simplex Virus
  • IEl cytomegalovirus immediate-early
  • RSV Rous sarcoma virus
  • adenovirus major late promoter Yamada et al., Proc. Nat. Acad. Sci. USA, 82: 3567 (1985)
  • any of the above-referenced constitutive promoters can be used to control transcription of a gene insert.
  • tissue-specific promoters may be used.
  • tissue-specific promoters include liver-specific promoters of hepatitis B virus (Sandig et al., Gene Therapy 3: 1002-1009 (1996) and the albumin gene (Pinkert et al., Genes and Development, 1: 268-276 (1987); see also Guo et al.. Gene Therapy, 3: 802-810 (1996) for other liver-specific promoter.
  • promoters described in the literature which are only expressed in specific tumors.
  • PSA promoter prostate carcinoma
  • carcinoembryonic antigen promoter colon and lung carcinoma
  • ⁇ -casein promoter mimmary carcinoma
  • tyrosinase promoter melanoma
  • calcineurin Aa promoter glioma, neuroblastoma
  • c-sis promoter osteosarcoma
  • ⁇ -fetoprotein promoter hepatoma
  • inducible promoters Genes that are under the control of inducible promoters are expressed only, or to a greater degree, in the presence of an inducing agent, (e.g., transcription under control of the metallothionein promoter is greatly increased in presence of certain metal ions). See also the glucocorticoid-inducible promoter present in the mouse mammary tumor virus long terminal repeat (MMTV LTR) (Klessig et al, MoL. Cell. Biol., 4: 1354 (1984)).
  • Inducible promoters include responsive elements (REs) which stimulate transcription when their inducing factors are bound. For example, there are REs for serum factors, steroid hormones, retinoic acid and cyclic AMP. Promoters containing a particular RE can be chosen in order to obtain an inducible response and in some cases, the RE itself may be attached to a different promoter, thereby conferring inducibility to the recombinant gene.
  • an inducing agent e.g.
  • in situ expression by genetically modified cells of interferon protein encoded by an interferon gene under the control of the metallothionein promoter is enhanced by contacting the genetically modified cells with a solution containing the appropriate (e.g., inducing) metal ions in situ.
  • the amount of interferon that is delivered in situ is regulated by controlling such factors as: (1) the nature of the promoter used to direct transcription of the inserted gene, (e.g., whether the promoter is constitutive or inducible, strong or weak or tissue specific); (2) the number of copies of the exogenous gene that are inserted into the cell; (3) the number of transduced/transfected cells that are administered (e.g., implanted) to the patient; (4) the size of an implant (e.g., graft or encapsulated expression system) in ex vivo methods; (5) the number of implants in ex vivo methods; (6) the number of cells transduced/transfected by in vivo administration; (7) the length of time the transduced/transfected cells or implants are left in place in both ex vivo and in vivo methods; and (8) the production rate of the interferon by the genetically modified cell.
  • the nature of the promoter used to direct transcription of the inserted gene e.g., whether the promoter is
  • Expression vectors compatible with mammalian host cells for use in gene delivery to tumor cells include, for example, plasmids; avian, murine and human retroviral vectors; adenovirus vectors; herpes viral vectors; parvoviruses; and non-replicative pox viruses.
  • replication-defective recombinant viruses can be generated in packaging cell lines that produce only replication-defective viruses. See Current Protocols in Molecular Biology: Sections 9.10-9.14 (Ausubel et al., eds.), Greene Publishing Associcates, 1989.
  • viral vectors for use in gene transfer systems include adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, baculoviral vectors, Epstein Barr viral vectors, papovaviral vectors, vaccinia viral vectors, and herpes simplex viral vectors.
  • adenoviral vectors include adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, baculoviral vectors, Epstein Barr viral vectors, papovaviral vectors, vaccinia viral vectors, and herpes simplex viral vectors.
  • Madzak et al J. Gen. Virol. 73: 1533-36 (1992): papovavirus SV40; Moss et al, Curr. Top. Microbiol. Immunol. 158: 25-38 (1992): vaccinia virus; Margulskee, Curr. Top. Microbiol. Immunol.
  • HSV herpes simplex virus
  • EBV Epstein-Barr virus
  • Miller Curr. Top. Microbiol. Immunol. 158: 1-24 (1992): retrovirus; Brandyopadhyay et al, MoI Cell. Biol.
  • recombinant viral vectors designed for use in gene delivery are used in the invention. See, e.g., Hu and Pathak, Pharmacol Rev. 52: 493-512 (2000); Somia and Verma, Nature Rev. 7: 91-99 (2000); van Beusechem et al, Gene Ther. 7: 1940-1946 (2000); Glorioso et al, Nature Med. 7: 33-40 (2001). Additionally, viral vectors may be administered in combination with transient immunosuppressive or immunomodulatory therapies. See, e.g., Jooss et al, Hum Gene Ther. 7: 1555-1566 (1996); Kay et al, Pro. Nat. Acad. ScL USA 94: 4686-4691.
  • a vector for delivering a nucleic acid is an adenovirus-based vector.
  • the adenovirus-based vector is an Ad-2 or Ad-5 based vector. See, e.g., Muzyczka, Curr. Top. Microbiol. Immunol 158: 97-123(1992); AIi et al, Gene Therapy 1: 367-384 (1994); and U.S. Pat. Nos. 4,797,368 and 5,399,346.
  • Adenoviruses can be modified to efficiently deliver a therapeutic or reporter transgene to a variety of cell types.
  • the general adenoviruses types 2 and 5 Ad2 and Ad5, respectively
  • Ad2 and Ad5 which cause respiratory disease in humans
  • DMD Duchenne Muscular Dystrophy
  • CF Cystic Fibrosis
  • Both Ad2 and Ad5 belong to a subclass of adenovirus that are not associated with human malignancies.
  • Adenovirus vectors are capable of providing high levels of transgene delivery to diverse cell types, regardless of the mitotic state of the cell.
  • 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 No. CRLl 573) 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, e.g., Watanabe, Atherosclerosis 36: 261-268 (1986); Tanzawa et al, FEBS Letters 775(1): 81-84 (1980); Golasten et al, New Engl. J. Med.
  • Some replication-deficient adenoviruses which have been developed for clinical trials contain deletions of the entire EIa region and part of the EIb region. These replication-defective viruses are grown in 293 cells containing a functional adenovirus EIa gene which provides a trans-acting EIa protein. El -deleted viruses are capable of replicating and producing infectious virus in certain cells ⁇ e.g., 293 cells), which provide EIa and EIb 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).
  • adenoviral vectors developed for clinical trials may be used in the invention. Examples include Ad vectors with recombinant fiber proteins for modified tropism ⁇ e.g., van Beusechem et al., Gene Ther. 7: 1940-1946 (2000)), protease pre-treated viral vectors (e.g., Kuriyama et al., Hum. Gene Ther. 11: 2219-2230 (2000)), E2a temperature sensitive mutant Ad vectors ⁇ e.g., Engelhardt et al., Hum. Gene Ther.
  • Ad vectors e.g., Armentano et al., J. Virol. 71: 2408-2416 (1997); Chen et al., Proc. Nat. Acad. Sci. USA 94: 1645-1650 (1997); Schied ⁇ r et al., Nature Genetics 18: 180-183 (1998)).
  • Adenoviruses have a broad host range, can infect quiescent or terminally differentiated cells such as neurons, and appear to be essentially non-oncogenic. Adenoviruses additionally do not appear to integrate into the host genome. Because they exist extrachromosomally, the risk of insertional mutagenesis is greatly reduced. See, e.g., AIi et al. 1994, 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.
  • Adeno-associated viruses AAV have also been used 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) are involved in replication, rescue and integration of the AAV genome.
  • the cap proteins (VPl, VP2 and VP3) 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.
  • the entire rep and cap domains can be excised and replaced with a therapeutic or reporter transgene. See, e.g., Carter, "The Growth Cycle of Adeno-Associated Virus,” in Handbook of Parvoviruses, vol. I, pp. 155-168, Tijssen, ed., CRC Press (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 encapsidated ssDNA, and shortly thereafter are delivered to the nucleus where the AAV DNA stably integrates into 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 herpesvirus or adenovirus which encodes helper functions that are required by AAV to aid in its excision from host chromatin (BJ. Carter, supra).
  • the infecting parental single-stranded (ss) DNA 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.
  • AAV have significant potential in gene delivery.
  • 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.
  • High level gene expression fom AAV in mice was shown to persist for at least 1.5 years. See Xiao, Li and Samulski Journal of Virology 70, 8089-8108 (1996).
  • Kessler et al. (Proc. Natl. Acad. ScL USA 93: 14082-14087 (1996)) showed high-level expression of the erythropoietin (Epo) gene following intramuscular injection of AAV in mice. Epo protein was demonstrated to be present in circulation and an increase in the red blood cell count was reported, indicative of therapeutic potential.
  • Other work by this group has used AAV expressing the HSV tk gene as a treatment for cancer. High level gene expression in solid tumors has been described.
  • baculovirus primarily derived from the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV)
  • AcMNPV Autographa californica multiple nuclear polyhedrosis virus
  • Recombinant baculovirus has several potential advantages for gene delivery.
  • viral serotypes e.g., the general adenovirus types 2 and 5 (Ad2 and Ad5, respectively) may be administered, possibly on an alternating dosage schedule where multiple treatments will be administered. Specific dosage regimens may be administered: over the course of several days, when an immune response against the viral vector is anticipated, or both.
  • Ad5-based viral vectors may be used on day 1
  • Ad2-based viral vectors may be used on day 2, or vice versa.
  • nucleic acids are additionally provided in replication-defective recombinant viruses or viral vectors. These can be generated in packaging cell lines that produce only replication-defective viruses. See, e.g., Sections 9.10-9.14, Ausubel et al.
  • a vector comprising a nucleic acid encoding a interferon polypeptide or a biologically or therapeutically active fragment, variant, or derivative thereof acts synergistically with an agent which inhibits TGF- ⁇ signaling activity.
  • the term “synergistic” or “synergistically” refers to the combined effect being greater than the sum of the individual effects.
  • the viral vector and TGF- ⁇ inhibitor may be formulated such that the individual components if dosed separately would comprise a sub-therapeutic dose.
  • subtherapeutic dose refers to an amount of an active agent that would not provide treatment, prevention of amelioration of the condition to be treated or prevented if dosed alone, without other active agents.
  • Interferon polynucleotides are administered to a cell via an expression vector.
  • the selection and optimization of a particular expression vector for expressing a specific interferon gene product in an isolated cell is accomplished by obtaining the interferon gene, preferably with one or more appropriate control regions (e.g., promoter); preparing a vector construct comprising the vector into which is inserted the interferon gene; transfecting or transducing cultured cells in vitro with the vector construct; and determining whether the interferon gene product is present in the cultured cells.
  • the effect of transfection with polynucleotides encoding interferons may be tested in vitro using any one of a number of readily available human tumor cell lines.
  • Such cell line include a human bladder carcinoma cell line, 5637 (ATCC HTB9), a human breast carcinoma cell line, MDA-MB-468 (ATCC HTB 132); a human prostate carcinoma cell line, E)U145 (ATCC HTB81); a human osteosarcoma cell line, SAOS2 (ATCC HTB85); a human fibrosarcoma metastatic to lung cancer cell line, Hs913T (ATCC HTB 152); a human cervical carcinoma cell line, HeLa (ATCC ECL 2).
  • Each of these cell lines may be transfected with the appropriate polynucleotides encoding interferons and the effect of transfection on cell growth and cellular morphology may be tested using procedures known in the art such as the Trypan blue exclusion assay to measure cell viability, cell counting to measure propagation over time and tritiated- thymidine incorporation to measure DNA replication.
  • interferon sequences can be identified by conventional methods such as nucleic acid hybridization using probes comprising sequences that are homologous/complementary to the inserted mutant interferon sequences. Interferon transcription can be measured by reverse transcriptase polymerase chain reaction. Alternatively, interferon protein is measured in the cell-conditioned medium by conventional antiviral assay or ELISA assay. In another approach, the 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
  • interferon-beta Ia a polynucleotide encoding interferon-beta Ia is inserted into a vector having a dominant selectable marker gene such as a neomycin phosphotransferase gene under separate control of an SV40 early promoter
  • the sequence can be identified by the presence of the marker gene function (Geneticin resistance).
  • Other methods of detecting the appropriate vector will be readily available to persons having ordinary skill in the art.
  • Interferon proteins have been shown to possess anti-oncogenic activity in many settings. For reviews, see Wadler and Schwartz, Cancer Research 50: 3473-3486 (1990); Martin-Odegard, DN&P, 4: 116-117 (1991); and Spiegel, Seminars in Oncology 15 (5): 41-45 (1988). Treatment with interferon-alpha and interferon-beta polypeptides, as well as delivery of IFN-encoding polynucleotides, have shown efficacy against several cancers. Interferon-encoding polynucleotide delivery, in addition to combining with administration of TGF- ⁇ inhibitors as described herein, could be done in conjunction with conventional surgery, radiation or chemotherapy.
  • gliomas account for 60-80% of all primary brain tumors in adults.
  • Human glioma cells can be implanted intracerebrally into immuno-deficient (nude) mice to provide a glioma model.
  • Interferon-beta protein treatment has been shown to increase survival in these mice.
  • a problem with some of the interferon-beta protein trials in glioma has been the high toxicity following parenteral administration (intravenous or intramuscular) of interferon-beta.
  • Melanoma is an excellent target for methods of the present invention.
  • the prognosis for metastatic malignant melanoma is poor.
  • the incidence of disease is increasing dramatically and conventional chemotherapies are ineffective.
  • Melanoma appears to be an immunogenic tumor type, in that the patient response may depend on the host immune response.
  • Both the antiproliferative and immunomodulatory activities of interferon-beta could be effective in this setting.
  • interferon-beta protein has direct antiproliferative effects on cultured malignant melanoma cells.
  • Hemangioma is a proliferation of capillary endothelium resulting in the accumulation of mast cells, fibroblasts and macrophages, and leads to tissue damage. Although usually harmless, hemangiomas can endanger vital organs and cause fatalities.
  • Interferon-alpha protein was shown to induce early regression of steroid-resistant hemangiomas in infants (Martin-Odegard, supra).
  • Interferon proteins have been shown to be effective in the treatment of leukemias, lymphomas and myelomas. The efficacy shown in these diseases is contrary to the general finding that, although efficacy of interferon proteins in in vitro cancer treatment is well-characterized, in vivo efficacy is far less common. Nevertheless, interferon-alpha is efficacious against hairy cell leukemia, chronic myeloid leukemia, cutaneous T cell lymphomas, Hodgkin's lymphoma and multiple myeloma in human clinical trials. Interferon-beta protein inhibits the growth of renal cell carcinoma cells in culture. DFN- ⁇ has already been approved for use in the treatment of renal cell carcinoma.
  • Colorectal cancer is a major cause of cancer-related deaths in the U.S.
  • IFN- ⁇ , IFN- ⁇ and IFN- ⁇ proteins on cultured human colon carcinoma cells. Colon carcinoma often generates metastases in the liver with dire consequences.
  • Adenovirus or other liver-tropic delivery systems could be used according to the methods provided herein to deliver an interferon encoding polynucleotide to the liver for treatment of these metastases.
  • Hepatocellular_carcinoma is an attractive target due to the high efficiency of liver delivery by adenovirus. It has been observed that IFN- ⁇ protein significantly inhibits the proliferation of human hepatoma cells in culture.
  • Interferon proteins have shown efficacy in the treatment of inoperable non-small cell lung carcinoma in some clinical trials, but not in others.
  • Two human lung cancer cell lines are found to be sensitive to growth inhibition by interferon-beta protein.
  • significant interferon-related toxicity was observed after intravenous interferon administration.
  • Local delivery of the interferon-beta gene to the lung could be efficacious without the toxicity observed following systemic protein delivery.
  • the inhibition in proliferation of small-cell lung carcinoma cells using interferon-beta protein has been observed.
  • An orthotopic model of lung cancer has been shown to be sensitive to Ad.INFbeta given intratracheally.
  • malignant mesotheliomas have been shown to be sensitive to gene delivery using interferon beta.
  • Vector can be delivered intrapleurally.
  • Interferon-alpha protein inhibits the growth of breast cancer xenografts in nude mice.
  • Interferon- beta may be efficacious against breast cancer due not only to its antiproliferative effects but also due to its induction of estrogen receptors and progesterone receptors in vivo to sensitize breast carcinomas to the anti-estrogen tamoxifen.
  • Ovarian cancer is a disease target. Therapy, in this case, could be done by installation of the LFN- ⁇ -encoding gene delivery vector into the peritoneum as this type of tumor tends to fill the peritoneal cavity.
  • interferon proteins and administration of IFN-encoding polynucleotides have demonstrated anti-oncogenic properties in a number of settings.
  • IFN- ⁇ and IFN- ⁇ proteins have been tested in conjunction with conventional chemotherapeutics and have shown synergy with these drugs in many indications including cervical cancer cells, laryngeal carcinoma cells, leukemia cells, renal cell carcinomas, colon adenocarcinoma and myeloma. It is also believed that interferons possess antiangiogenesis activity.
  • Some data indicate that a sustained level of IFN- ⁇ protein is necessary for the inhibition of angiogenesis. In that case, interferon-encoding polynucleotide delivery would be preferable to protein therapy in which the high levels of interferon protein fall off to low or undetectable levels quite rapidly.
  • the most commonly used rodent cancer model is the human tumor xenograft model in nude (nu/nii) mice.
  • the human cancer cells are propagated in culture and transfected or infected with a gene encoding interferon operably linked to the appropriate expression control sequences. These cells are then injected into a nude mouse.
  • the tumor cells are injected subcutaneously into the back of the mouse leading to the formation of a solid tumor mass.
  • the tumor cells could be injected orthotopically into the organ in which they would naturally appear (lung cancer cells would be injected into the lung; colon carcinoma cells into the colon, etc.). Tumor growth can then be followed by measuring the diameter of the tumor mass over time.
  • adenoviruses have also been used in the treatment of solid tumors in animal models and in early human clinical trials. Many of these studies used similar nude mouse/human xenograft models. Some examples of these modeling experiments are listed below, dayman et at. Cancer Research 55: 1-6 (1995) set up a model of human squamous cell carcinoma of the head and neck in nude mice. They found that adenovirus expressing wild type p53 prevented formation of these tumors.
  • Hirschowitz et al. Human Gene Therapy 6: 1055-1063 (1995) introduced human colon carcinoma cells into nude mice. After tumors are established, they injected these tumors directly with adenovirus expressing the E.
  • CD coli cytosine deaminase gene
  • 5FC 5-fluorocytosine
  • Cancer models also can be set up in immunocompetent mice and rats. These tumors can be established from syngeneic rodent tumor cells which are injected into the mice. Alternatively, the tumors can derive from endogenous cells. In these cases, the endogenous tumors could be due to treatment of the animal with a carcinogen or, alternatively, can form spontaneously due to the genetic background of the mouse (deficient in p53, for instance). Some examples follow.
  • Retrovirus vectors were the first vectors used in human gene delivery clinical trials.
  • One report which is relevant to the present patent application is that of Roth et al. Nature Medicine 2: 985-991
  • the TGF- ⁇ inhibitor is a TGF- ⁇ type I receptor kinase inhibitor, e.g. 4-[4- benzo[l,3]dioxol-5-yl-5-(6-methyl-pyridin-2-yl)-lH-imidazol-2-yl]-bicyclo[2.2.2]octane-l-carboxylic acid amide:
  • the TGF- ⁇ inhibitor is an antagonist of the TGF- ⁇ type I receptors, AIkF and/or Alk4, e.g. as disclosed in U.S. published application 20060063809 (U.S. Appln. 10/510,459) and having the Formula I:
  • R] is aryl, heteroaryl, aralkyl, or heteroaralkyl
  • each R a is independently alkyl, alkenyl, alkynyl, alkoxy, acyl, halo, hydroxy, amino, nitro, oxo, thioxo, cyano, guanadino, amidino, carboxy, sulfo, mercapto, alkylsulfanyl, alkylsulfinyl, alkylsulfonyl, aminocarbonyl, alkylcarbonylamino, alkylsulfonylamino, alkoxycarbonyl, alkylcarbonyloxy, urea, thiourea, sulfamoyl, sulfamide, carbamoyl, cycloalkyl, cycloalkyloxy, cycloalkylsulfanyl, heterocycloalkyl, heterocycloalkyloxy, heterocycloalkylsulfanyl, aryl, aryloxy, arylsulf
  • X is cycloalkyl or heterocycloalkyl
  • Y is a bond, -C(O)-, -C(O)-O-, -O-C(O)-, -S(O) p -O- ( -O-S(O) P -, -C(O)-N(R 1 ,)-, -N(R b )-C(O)-, -O-
  • R b and R 0 is independently hydrogen, hydroxy, alkyl, alkoxy, amino, aryl, aralkyl, heterocycloalkyl, heteroaryl, or heteroaralkyl; p is 1 or 2; and q is 1-4;
  • R 2 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl,
  • each of Ai and A 2 independently, is O, S, N, or NR b J provided that at least one OfA 1 and A 2 is N;
  • m is 0, 1, 2, or 3; provided that when m>2, two adjacent R a groups can join together to form a 4- to 8-membered optionally substituted cyclic moiety.
  • Non-limiting compounds of Formula I include 4-[4-(4-benzo[l ,3]dioxol-5-yl-5-pyridin-2-yl-lH- imidazol-2-yl)-piperidine- 1 -sulfonylj-benzoic acid; 2-[5 -benzo[ 1 ,3]dioxol-5-yl-2-( 1 - phenylmethanesulfonyl-piperidin-4-yl)-3H-imidazol-4-yl]-pyridine; 2- ⁇ 5-benzo[l,3]dioxol-5-yl-2-[l- (butane-1 -sulfonyl)-piperidin-4-yl]-3H-imidazol-4-yl ⁇ -pyridine; 2- ⁇ 5 -benzo[ 1 ,3]dioxol-5-yl-2-[ 1 -(3,5- dichloro-phenylme
  • each of X ⁇ n, Xn -2 , Xn- 3 , and X IM can be independently CR ⁇ "x or N, provided that only two of Xn -I , X ⁇ i- 2 , X ⁇ o. and Xn -4 can be N simultaneously.
  • Each of Y 1H and Yu -2 can be independently CR 11' * or N, provided that at least one of Yn_ ⁇ and Yn. 2 must be N.
  • the ring having Yn-i and Yn -2 ring atoms can be a pyrimidinyl or pyridyl.
  • Each R 11'1 can be independently alkyl, alkenyl, alkynyl, alkoxy, acyl, halo, hydroxy, amino, nitro, cyano, guanadino, amidino, carboxy, sulfo, mercapto, alkylsulfanyl, alkylsulfinyl, alkylsulfonyl, aminocarbonyl, alkylcarbonylamino, alkylsulfonylamino, alkoxycarbonyl, alkylcarbonyloxy, urea, thiourea, sulfamoyl, sulfamide, carbamoyl, cycloalkyl, cycloalkyloxy, cycloalkylsulfanyl, heterocycloalkyl, heterocycloalkyloxy, heterocycloalkylsulfanyl, aryl, aryloxy, arylsulfanyl, aroyl, heteroaryl,
  • Each R 11"2 can be independently alkyl, alkenyl, alkynyl, acyl, halo, hydroxy, -NH 2 , - NH(alkyl), -N(alkyl) 2 , -NH(cycloalkyl), -N(alkyl)(cycloalkyl), -NH(heterocycloalkyl), -
  • R II x and R Il y can be independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, acyl, halo, hydroxy, amino, nitro, cyano, guanadino, amidino, carboxy, sulfo, mercapto, alkylsulfanyl, alkylsulfinyl, alkylsulfonyl, cycloalkylcarbonyl, (cycloalkyl)alkylcarbonyl, aroyl, aralkylcarbonyl, heterocycloalkylcarbonyl, (heterocycloalkyl)acyl, heteroaroyl, (heteroaryl)acyl, aminocarbonyl, -M-
  • alkylcarbonylamino (amino)aminocarbonyl, alkylsulfonylaminocarbonyl, alkylsulfonylamino, cycloalkylcarbonylamino, cycloalkylsulfonylamino, (cycloalky ⁇ alkylcarbonylamino,
  • heterocycloalkytyalkylcarbonylamino (heterocycloalkyljalkylsulfonylamino, heteroarylcarbonylamino, heteroarylsulfonylamino, heteroaralkylcarbonylamino, heteroaralkylsulfonylamino, alkoxycarbonyl, alkylcarbonyloxy, urea, thiourea, sulfamoyl, sulfamide, carbamoyl, cycloalkyl, cycloalkyloxy, cycloalkylsulfanyl, (cycloalkyl)alkyl, (cycloalkyl)alkoxy, (cycloalky ⁇ alkylsulfanyl, heterocycloalkyl, heterocycloalkyloxy, heterocycloalkylsulfanyl, (heterocycloalkyl)alkyl, (heterocycloalkyl)alkoxy, (heterocyclo
  • two adjacent R 11"1 groups can optionally together to form a 4- to 8-membered optionally substituted cyclic moiety. That is, the 2-pyridyl ring can fuse with a 4- to 8-membered cyclic moiety to form a moiety such as 7H-[l]pyrindinyl, 6,7-dihydro-5H- [l]pyrindinyl, 5,6,7,8-tetrahyd ⁇ o-quinolinyl, 5,7-dihydro-furo[3,4-b]pyridinyl, or 3,4-dihydro-lH- thiopyrano[4,3-c]pyridinyl.
  • the fused ring moiety can be optionally substituted with one or more substituents such as alkyl (including carboxyalkyl, hydroxyalkyl, and haloalkyl such as trifluoromethyl; see the definiton of "alkyl” below), alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkoxy, aryl, heteroaryl, aryloxy, heteroaryloxy, aralkyloxy, heteroarylalkoxy, aroyl, heteroaroyl, amino, nitro, carboxy, alkoxycarbonyl, alkylcarbonyloxy, aminocarbonyl, alkylcarbonylamino, cycloalkylcarbonylamino, cycloalkyl-alkylcarbonylamino, arylcarbonylamino, aralkylcarbonylamino, heterocycloalkyl-carbonylamino, heterocycloalkyl-carbonylamino, heterocycloalkyl
  • the 4- to 8-membered cyclic moiety formed by two adjacent R " groups can be optionally substituted with substituents such as alkyl (including carboxyalkyl, hydroxyalkyl, and haloalkyl such as trifluoromethyl; see the definiton of "alkyl” below), alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkoxy, aryl, heteroaryl, aryloxy, heteroaryloxy, aralkyloxy, heteroarylalkoxy, aroyl, heteroaroyl, amino, nitro, carboxy, alkoxycarbonyl, alkylcarbonyloxy, aminocarbonyl, alkylcarbonylamino, cycloalkylcarbonylamino, cycloalkyl-alkylcarbonylamino, arylcarbonylamino, aralkylcarbonylamino, heterocycloalkyl-carbonylamino, heterocycloalkyl-carbony
  • WO 04/022054 which is incorpoarated in its entrity by reference, describes synthetic methods for producing inhibitors of formula II.
  • Examples of compounds of formula II include, but are not limited to, 215) 4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[l ,5-a]pyridin-3-yl]-pyrimidin-2-ylamine; 216) 4-(2-pyridin-2-yl-pyrazolo[l,5-a]pyridin-3-yl)-pyrimidin-2-ylamine;
  • the antagonists have the structure shown in formula HI:
  • each ofXnn, Xm- 2> X ⁇ i-3. and Xm -4 can be independently CR 111 " or N, provided that only two of Xm -I , Xm- 2 , X 111 -3, and X m . 4 can be N simultaneously.
  • Each of Y UM and Ym -2 can be independently CR III y or N, provided that at least one of Ym., and Ym -2 must be N.
  • R 1 " 1 can be independently alkyl, alkenyl, alkynyl, alkoxy, acyl, halo, hydroxy, amino, nitro, cyano, guanadino, amidino, carboxy, sulfo, mercapto, alkylsulfanyl, alkylsulfinyl, alkylsulfonyl, aminocarbonyl, alkylcarbonylamino, alkylsulfonylamino, alkoxycarbonyl, alkylcarbonyloxy, urea, thiourea, sulfamoyl, sulfamide, carbamoyl, cycloalkyl, cycloalkyloxy, cycloalkylsulfanyl, heterocycloalkyl, heterocycloalkyloxy, heterocycloalkylsulfanyl, aryl, aryloxy, arylsulfanyl, aroyl, heteroaryl,
  • Each of R 1 " "2 can be independently alkyl, alkenyl, alkynyl, acyl, halo, hydroxy, -NH 2 , -NH(alkyl), -N(alkyl) 2 , -NHCcycloalkyl), -N(alkyl)(cyclocalkyl), - NH(heterocycloalkyl), -NH(heteroaryl), -NH-alkyl-heterocycloalkyl, -NH-alkyl-heteroaryl, - NH(aralkyl), cycloalkyl, (cycloalkyl)alkyl, aryl, aralkyl, aroyl, heterocycloalkyl, (heterocycloalkyl)alkyl, heteroaryl, heteroaralkyl, heteroaroyl, nitro, cyano, guanadino, amidino, carboxy, sulfo, mercapto, alk
  • R ul x and R ⁇ "y can be independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, acyl, halo, hydroxy, amino, nitro, cyano, guanadino, amidino, carboxy, sulfo, mercapto, alkylsulfanyl, alkylsulfinyl, alkylsulfonyl, cycloalkylcarbonyl, (cycloalkyl)alkylcarbonyl, aroyl, aralkylcarbonyl, heterocycloalkylcarbonyl, (heterocycloalkyl)acyl, heteroaroyl, (heteroaryl)acyl, aminocarbonyl, alkylcarbonylamino, (amino)aminocarbonyl, alkylsulfonylaminocarbonyl, alkylsulfonylamino, cycloalkylcarbonylamino, cycloal
  • heterocycloalkyOalkylcarbonylamino (heterocycloalkytyalkylsulfonylamino, heteroarylcarbonylamino, heteroarylsulfonylamino, heteroaralkylcarbonylamino, heteroaralkylsulfonylamino, alkoxycarbonyl, alkylcarbonyloxy, urea, thiourea, sulfamoyl, sulfamide, carbamoyl, cycloalkyl, cycloalkyloxy, cycloalkylsulfanyl, (cycloalkyl)alkyl, (cycloalkyl)alkoxy, (cycloalkyl)alkylsulfanyl, heterocycloalkyl, heterocycloalkyloxy, heterocycloalkylsulfanyl, (heterocycloalkyl)alkyl, (heterocycloalkyl)alkoxy, (heterocyclo
  • each OfXm -1 , Xm -2 , Xin-3, and Xm -4 can be independently CR 1 " '* or N, provided that only two of Xnn, X ⁇ i- 2 , X ⁇ n- 3 ) and Xm-4 can be N simultaneously.
  • Each of Ynn and Yin- 2 can be independently CR I1I y or N, provided that at least one of Ym-i and Ym -2 must be N.
  • R 1 " ' ' can be independently alkyl, alkenyl, alkynyl, alkoxy, acyl, halo, hydroxy, amino, nitro, cyano, guanadino, amidino, carboxy, sulfo, mercapto, alkylsulfanyl, alkylsulfinyl, alkylsulfonyl, aminocarbonyl, alkylcarbonylamino, alkylsulfonylamino, alkoxycarbonyl, alkylcarbonyloxy, urea, thiourea, sulfamoyl, sulfamide, carbamoyl, cycloalkyl, cycloalkyloxy, cycloalkylsulfanyl, heterocycloalkyl, heterocycloalkyloxy, heterocycloalkylsulfanyl, aryl, aryloxy, arylsulfanyl, aroyl, hetero
  • Each of R 1 " "2 can be independently alkyl, alkenyl, alkynyl, acyl, halo, hydroxy, -NH 2 , -NH(alkyl), -N(alkyl) 2 , -NHCcycloalkyl), -N(alkyl)(cyclocalkyl), - NHCheterocycloalkyl), -NH(heteroaryl), -NH-alkyl-heterocycloalkyl, -NH-alkyl-heteroaryl, -NH-alkyl-heteroaryl, -NH-alkyl-heteroaryl, -
  • R 1 " " and R III y can be independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, acyl, halo, hydroxy, amino, nitro, cyano, guanadino, amidino, carboxy, sulfo, mercapto, alkylsulfanyl, alkylsulfinyl, alkylsulfonyl, cycloalkylcarbonyl, (cycloalkyl)alkylcarbonyl, aroyl, aralkylcarbonyl, heterocycloalkylcarbonyl, (heterocycloalkyl)acyl, heteroaroyl, (heteroaryl)acyl, aminocarbonyl, alkylcarbonylamino, (amino)aminocarbonyl, alkylsulfonylaminocarbonyl, alkylsulfonylamino, cycloalkylcarbonylamino, cycloalkyl
  • heterocycloalkyl alkylcarbonylamino, (heterocycloalkyl)alkylsulfonylamino, heteroarylcarbonylamino, heteroarylsulfonylamino, heteroaralkylcarbonylamino, heteroaralkylsulfonylamino, alkoxycarbonyl, alkylcarbonyloxy, urea, thiourea, sulfamoyl, sulfamide, carbamoyl, cycloalkyl, cycloalkyloxy, cycloalkylsulfanyl, (cycloalkyl)alkyl, (cycloalkyl)alkoxy, (cycloalkyl)alkylsulfanyl, heterocycloalkyl, heterocycloalkyloxy, heterocycloalkylsulfanyl, (heterocycloalkyl)alkyl, (heterocycloalkyl)alkoxy, (heterocycl
  • Each R IV"a can be independently alkyl, alkenyl, alkynyl, alkoxy, acyl, halo, hydroxy, amino, nitro, oxo, thioxo, cyano, guanadino, amidino, carboxy, sulfo, mercapto, alkylsulfanyl, alkylsulfinyl, alkylsulfonyl, aminocarbonyl, alkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, alkylsulfonylamino, arylsulfonylarnino, heteroarylsulfonylamino, alkoxycarbonyl, alkylcarbonyloxy, urea, thiourea, sulfamoyl, sulfamide, carbamoyl, cycloalkyl, cycloalkyloxy, cycloalkylsulf
  • R IV 1 can be a bond, alkylene, alkenylene, alkynylene, or -(CH 2 ) r i-O-(CH 2 )r2-, wherein each of rl and r2 is independently 2 or 3.
  • R ⁇ v"2 can be cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, or a bond.
  • R IV 3 can be -C(O)-, -C(O)O-, -OC(O)-, -C(O)-N(R Iv b )-, -N(R 1 v b ) -C(O)-, -O-C(O)-N(R lv b )-, -
  • RTM "4 can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, heterocycloalkyl,
  • heterocycloalkyl alkyl
  • cycloalkenyl cycloalkenyl
  • cycloalkenyl heterocycloalkenyl
  • heterocycloalkenyl heterocycloalkenyl
  • heterocycloalkenyl heterocycloalkenyl
  • R ⁇ v'5 can be hydrogen, unsubstituted alkyl, halo-substituted alkyl, alkoxy, alkylsulfinyl, amino, alkenyl, alkynyl, cycloalkyl, cycloalkoxy, cycloalkylsulfinyl, heterocycloalkyl, heterocycloalkoxy, heterocycloalkylsulfinyl, aryl, aryloxy, arylsulfinyl, heteroaryl, heteroaryloxy, or heteroarylsulf ⁇ nyl.
  • Ring A can be an aromatic ⁇ ng containing 0-4 hetero ring atoms
  • nng B can be a 5- to 7-membered aromatic or nonaromatic ⁇ ng containing 0-4 hetero ⁇ ng atoms, provided that at least one of ⁇ ng A and ring B contains one or more hetero ring atoms
  • Ring A' can be an aromatic ring containing 0-4 hetero ring atoms
  • ring B' can be a 5- to 7-membered saturated or unsaturated ⁇ ng containing 0-4 hetero ⁇ ng atoms, provided that at least one of ring A' and ring B' contains one or more hetero ring atoms.
  • Each X 1 can be independently N or C.
  • R IV h and R ⁇ v ' can be independently alkyl, alkenyl, alkynyl, alkoxy, acyl, halo, hydroxy, ammo, nitro, oxo, thioxo, cyano, guanadino, amidino, carboxy, sulfo, mercapto, alkylsulfanyl, alkylsulfinyl, alkylsulfonyl, ammocarbonyl, alkylcarbonylamino, arylcarbonylamino, heteroarylcarbonyl- ammo, alkylsulfonylammo, arylsulfonylamino, heteroarylsulfonylammo, alkoxycarbonyl,
  • R IV 6 is 2-naphthy ⁇ dinyl, 4-quinohnyl, irmdazo[l,2-a]py ⁇ dyl, or benzimidazolyl
  • -R' ⁇ '-R' ⁇ .R' ⁇ .R' V - 4 IS not H, unsubstituted alkyl, -CH 2 -C(O)-N(H)-alkyl, -CH 2 -C(O)-N(alkyl) 2 , or benzyl
  • R V 1 can be heteroaryl
  • Each R v'a can be alkyl, alkenyl, alkynyl, alkoxy, acyl, halo, hydroxy, amino, nitro, oxo, thioxo, cyano, guanadino, amidino, carboxy, sulfo, mercapto, alkylsulfanyl, alkylsulf ⁇ nyl, alkylsulfonyl, aminocarbonyl, alkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, alkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, alkoxycarbonyl, alkylcarbonyloxy, urea, thiourea, sulfamoyl, sulfamide, carbamoyl, cycloalkyl, cycloalkyloxy, cycloalky
  • Xv can be cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or a bond.
  • Yv can be a bond, -C(O)-, -C(O)-O-, -O-C(O)-, -S(O) P -O-, -O-S(O) P -, -C(O)-N(R v b )-, -N(R V" 1 O-C(O)-, -O-C(O)-N(R v b )- > -N(R v b )-C(O)-O-, -C(O)-N(R ⁇ )-O-, -O-N(R v b )-C(O)-, -O-S(O)p-N(R * *)-, -N(R v b )- S(O) p -O-, -S(O) p -N(R v b )-O-, -O-N(R v b )-S(S(O
  • R v"b and R v-C independently, can be hydrogen, hydroxy, alkyl, alkoxy, amino, aryl, aralkyl, heterocycloalkyl, heteroaryl, or heteroaralkyl.
  • R v-2 can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, (cycloalkenyl)alkyl, aryl, aralkyl, arylalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, heterocycloalkenyl, (heterocycloalkenyl)alkyl, heteroaryl, heteroaralkyl, or (heteroaryl)alkenyl.
  • Each of A v l and A v 2 can be N or NR v b . It is to be understood that when A v l is NR v b , A v ⁇ 2 is N, and vice versa.
  • the variable, m can be 0, 1, 2, or 3.
  • the pyrimidinyl ring can be unsubstituted or substituted with 1 -3 R v"a groups. Note that when m > 2, two adjacent R v'a groups can optionally together to form a 4- to 8-membered optionally substituted cyclic moiety.
  • the pyrimidinyl ring can fuse with a cyclic moiety to form a moiety, that can be optionally substituted with one or more substituents such as alkyl (including carboxyalkyl, hydroxyalkyl, and haloalkyl such as trifluoromethyl), alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkoxy, aryl, heteroaryl, aryloxy, heteroaryloxy, aroyl, heteroaroyl, amino, nitro, carboxy, alkoxycarbonyl, alkylcarbonyloxy, aminocarbonyl, alkylcarbonylamino, cyano, halo, hydroxy, acyl, mercapto, alkylthio, sulfoxy, sulfamoyl, oxo, or carbamoyl.
  • substituents such as alkyl (including carboxyalkyl, hydroxyalkyl, and haloalkyl such as
  • R v"2 is hydrogen or alkyl
  • m is 1, 2, or 3
  • at least one R v"a is substituted at the 2-pyrimidinyl position (i.e., the position of the pyrimidinyl ring that is between the two nitrogen ring atoms).
  • Compounds of formula V may be prepared by a number of known methods from commercially available or known starting materials.
  • compounds of formula V are prepared according to Scheme described below. Specifically, in Scheme V-Ia, optionally substituted 2-methylpyrimidine (II) is deprotonated by LDA before reacting with R v' " -substituted carboxylic acid methoxy-methyl-amide (I) to form an R ⁇ '- ⁇ -methylpyrimidinyty-ketone (III), wherein R v l has been defined above.
  • the methoxy- methyl-amide can be prepared by reacting a corresponding acid chloride (i.e., R V' '-CO-C1) with N,O- dimethylhydroxylamine hydrochloride.
  • R V' '-CO-C1 a corresponding acid chloride
  • the R v'1 -(6-methylpyrimidinyl)-ketone (HI) can then be treated with sodium nitrite in acetic acid to afford an ⁇ -keto-oxime (IV), which can undergo a further reaction with an appropriate substituted (and optionally protected) aldehyde (VI) in the presence of ammonium acetate to yield a compound of formula V.
  • the above-described compounds of formula V can be prepared according to Scheme V-Ib below. Specifically, l,l-dimethoxy-propan-2-one can first react with dimethoxymethyl- dimethyl-amine at an elevated temperature to produce the intermediate 4-dimethylamino-l,l-dimethoxy- but-3-en-2-one, which can then react an R V a -substituted amidine to form an R v'a -substituted pyrimidine- 2-carbaldehyde (Ha).
  • This carbaldehyde (Ha) can then react with aniline and diphenyl phosphite to form a resulting N.P-acetal, which can further couple with an R v" ' -substituted aldehyde to produced an (R v" '- methyl)-pyrimidinyl-ketone (IHa). See, e.g., Journet et al., Tetrahedron Lett. 39:1717-1720 (1998).
  • a compound of formula V can be prepared by reacting intermediate (IV) or (FVa) with an aldehyde (V ⁇ ) to yield a further intermediate (VlIl), which can then react with compound (DC) to yield a compound of formula V.
  • V ⁇ aldehyde
  • VlIl further intermediate
  • moieties Y' and Y" are precursors of moiety Yv. See Scheme V-2 below.
  • desired substitutions at R v"a can be obtained by selecting, for example, the appropriate compound (Ha) intermediate.
  • moiety Xv in compound (VII) is a nitrogen-containing heterocycloalkyl (e.g., piperidine).
  • the nitrogen ring atom can be protected by a nitrogen protecting group (e.g., Cbz, Boc, or FMOC) before coupling to compound (IV) or (IVa) and deprotected afterwards (see first step of Scheme 3) to yield compound (Villa).
  • This compound can further react with various compounds (DC) to produce a compound of formula V. See second steps of Scheme V-3 below.
  • compound (VIII) or compound (Villa) can be a compound of formula V as well.
  • Examples of compounds of formula V include, but are not limited to, 469) 4-[4-benzo[l,3]dioxol- 5-yl-5-(2-methylsuifanyl-pyrimidin-4-yl)-lH-imidazol-2-yl]-benzamide; 470) 4-[4-benzo[l,3]dioxol-5-yl- 5-(2-methylsulfanyl-pyrimidin-4-yl)-lH-imidazol-2-yl]-benzonitrile; 471) 4-[5-(2-methanesulfonyl- pyrimidin-4-yl)-4-(6-methyl-pyridin-2-yl)-lH-imidazol-2-yl]-bicyclo[2.2.2]octane-l-carboxylic acid methyl ester; 472) 4-[5-(2-methoxy-pyrimidin-4-yl)-4-(6-methyl-pyridin-2-yl)
  • each R VI"a can be alkyl, alkenyl, alkynyl, alkoxy, acyl, halo, hydroxy, -NH 2 , -NH(unsubstituted alkyl), -N(unsubstituted alkyl) 2 , nitro, oxo, thioxo, cyano, guanadino, amidino, carboxy, sulfo, mercapto, alkylsulfanyl, alkylsulf ⁇ nyl, alkylsulfonyl, aminocarbonyl, alkylcarbonylamino, arylcarbonylamino, heteroarylcarbonyl amino, alkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, alkoxycarbonyl, alkylcarbonyloxy, urea, thiourea, sulf
  • R v ⁇ ' can be a bond, alkylene, alkenylene, alkynylene, or -(CH 2 ) r i-O-(CH 2 ) ⁇ -, wherein each of rl and r2, independently, is 2 or 3.
  • R VI"2 can be cycloalkylene, heterocycloalkylene, cycloalkenylene, heterocycloalkenylene, arylene, heteroarylene, or a bond.
  • R VI 3 can be -C(O)-, -C(O)-O-, -O-C(O)-, -S(O) P -O-, -O-S(O) P -, -C(O)-N(R VI b )-,
  • R VI b and R VI*C independently, can be hydrogen, hydroxy, alkyl, aryl, aralkyl, heterocycloalkyl, heteroaryl, or heteroaralkyl.
  • p can be 1 or 2 and q can be
  • R v ⁇ 4 can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, heterocycloalkyl,
  • heterocycloalkyl alkyl
  • cycloalkenyl cycloalkenyl
  • cycloalkenyl heterocycloalkenyl
  • heterocycloalkenyl heterocycloalkenyl
  • heterocycloalkenyl heterocycloalkenyl
  • R VI"5 can be hydrogen, unsubstituted alkyl, halo-substituted alkyl, alkoxy, alkylsulfinyl, amino, alkenyl, alkynyl, cycloalkoxy, cycloalkylsulfinyl, heterocycloalkoxy, heterocycloalkylsulfinyl, aryloxy, arylsulfinyl, heteroaryloxy, or heteroarylsulfinyl
  • R VI"6 can be a 5- to 6-membered monocyclic heterocyclyl or a 8- to 11-membered bicyclic heteroaryl, and optionally substituted with alkyl, alkenyl, alkynyl, alkoxy, acyl, halo, hydroxy, amino, nitro, oxo, thioxo, cyano, guanadino, amidmo, carboxy, sulfo, mercapto, alkylsulfanyl, alkylsulfinyl, alkylsulfonyl, aminocarbonyl, alkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, alkylsulfonylammo, arylsulfonylamino, heteroarylsulfonylammo, alkoxycarbonyl, alkylcarbonyloxy, urea, thiourea, sulfamoyl,
  • m can be 0-3, and when m > 2, two adjacent R a groups can optionally together to form a 4- to 8-membered optionally substituted cyclic moiety.
  • a pyrimidine of formula II which contains a 2-( ⁇ , ⁇ -unsaturated carbonyl) substituent can cyclize with hydrazine to form a pyrazole core ring to produce a 2-(pyrazol-3-yl)-py ⁇ midine intermediate (III).
  • the pyrimidine of formula II can be prepared by known methods (see, e.g., Jameson, D. and Guise, L. Tetrahedron Letters, 32(18): 1999-2002).
  • the intermediate (III) can be further substituted at the 4-position of the pyrazole core ring with a good leaving group such as halo (e.g., iodo or bromo) by reacting with a halogenation reagent (e.g., bromination reagent such as Br 2 or iodination reagent such as N-iodosuccinimide) to form a 2-(4-halo-pyrazol-3-yl)-pyrimidine (TV).
  • halo is represented by moiety X in Scheme VI-I .
  • the halo substituent forms an ideal platform for R v ⁇ '6 substitutions.
  • the iodo substituent can be converted into a boronic acid substituent (see compound (V) below), which can react with a R VI'6 -halide (VI) (e.g., an aryl halide or a heteroaryl halide) via Suzuki coupling reaction to form a compound of formula VI.
  • R VI'6 -halide e.g., an aryl halide or a heteroaryl halide
  • Other substitution reactions can also be employed to produce a wide range of compounds of formula VI (see, e.g., via a reaction between the protected iodinated compound (FVa) and phthalic anhydride to form a di-keto intermediate (VII), which can undergo a cyclization reaction with an R g -substituted hydrazine to form a compound (VI); for reference, see J. Med.
  • a compound of formula VI can be prepared according to Scheme VI-3 shown below.
  • a dimethoxymethyl-substituted pyrimidine of formula (Ha) can be prepared by reacting dimethylformamide dimethylacetal with l,l-dimethoxy-propan-2-one to form 4-dimethylamino- l,l-dimethoxy-but-3-en-2-one as an intermediate, which can further react with an R vl a -substituted acetamidine (i.e., R VI a -C(NH)-NH 2 ) to produce a compound of formula (Ha).
  • R VI a -C(NH)-NH 2 an R vl a -substituted acetamidine
  • the compound of formula (Ila) can then be deprotected in an acidic medium (e.g., aqueous HBr) and react with aniline and diphenylphosphite to form a compound of formula (lib), which can then react with an R VI 6 -substituted aldehyde to form a compound of formula (lie). Further reaction of a compound of formula (lie) with N,N-dimethylformamide dimethylacetal (DMFDMA), followed by hydrazine hydrate, yields a compound of formula VI.
  • DMFDMA N,N-dimethylformamide dimethylacetal
  • a compound of formula VI can also be prepared via a phenylacetyl pyrimidine compound (DC) as shown in Scheme VI-5 below. Specifically, a pyrimidine-carboxyaldehyde compound (VIII) is converted to the N 1 P acetal intermediate with aniline and diphenylphosphite. This acetal intermediate is then coupled to an aldehyde substituted with R v ⁇ "6 in basic condition (e.g., Cs 2 CO 3 ) to afford an enamine intermediate, which is hydrolyzed to a ketone intermediate (DC).
  • DC ketone intermediate
  • a compound of formula VI wherein the 1 -position of the pyrazole core ring is unsubstituted can undergo a conjugate addition reaction as shown in reaction (B) below.
  • the electrophile or acceptor in the addition reaction generally contains a double bond connecting to an electron-withdrawing group or a double bond conjugating to groups such as carbonyl, cyano, or nitro.
  • RVI-1 RVI-1.
  • R VI-2 R VI-3.
  • R VM VM
  • the -R VI 1 -R VI"2 -R VI 3 -R VM group can be further transformed into other functionalities as shown in Scheme VI-8 below.
  • a compound of formula VI wherein the -R VM -R VI'2 -R VI"3 -R VM group is cyanoalkyl can be reduced to aminoalkyl, which can be further converted to other functionalities such as heteroaralkyl, heterocycloalkylalkyl, and carboxylic acid.
  • Substituents at the pyrimidinyl ring can also be converted into other functionalities.
  • R VI a a compound of formula VI wherein R VI"a is bromo
  • R VI"a a bromo-substituted compound of formula VI (Sigma-Aldrich, St. Louis, MO))
  • R VI"a a bromo-substituted compound of formula VI
  • R v ⁇ "6 moiety can be further converted into other functionalities as well.
  • substituents of the R v ⁇ "6 moiety can be further converted into other functionalities as well.
  • suitable protecting groups see, e.g., T. W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, Inc., New York (1981).
  • Examples of compounds of formula VI include, but are not limited to, 500) 4-(4-benzo[l,3]dioxol-5-yl- 1 H-pyrazol-3-yl)-2-methyl-pyrimidine, 500a) 6-[3-(2-methyl-pyrimidin-4-yl)-l H-pyrazol-4-yl]- [l,2,4]triazolo[l,5-a]pyridine, 501) 6-[3-(2-trifluoromethyl-pyrimidin-4-yl)-lH-pyrazol-4-yl]- [l,2,4]triazolo[l,5-a]pyridine, 502) 6-[3-(2-methyl-pyrimidin-4-yl)-lH-pyrazol-4-yl]-quinoxaline, 502a) 6-[3-(2-trifIuoromethyl-pyrimidin-4-yl)-lH-pyrazol-4-yl]-quinoxaline, 503) 6-[3-(2-cyclo
  • the TGF- ⁇ inhibitor is an inhibitor of the TGF- ⁇ signaling pathway, e.g. as disclosed in U.S. Pat. 6,906,089, having Formula VII:
  • Ri is naphthyl, or phenyl optionally substituted with one or more substituents selected from the group consisting of halo, -0-C 1-6 alkyl, -S-Ci -6 alkyl, Ci -6 alkyl, Ci -6 haloakyl, -O-(CH 2 ) n -Ph, -S- (CH 2 ),, -Ph, cyano, phenyl, and CO 2 R, wherein R is hydrogen or C 1-6 alkyl, and n is 0, 1, 2 or 3; or Ri is phenyl fused with an aromatic or non-aromatic cyclic ring of 5-7 members wherein said cyclic ring optionally contains up to three heteroatoms, independently selected from N, O and S; [0237] R 2 is H, C -6 alkyl, Ci -6 alkoxy, phenyl, NH(CH 2 ) n -Ph, NH-C -6 alkyl,
  • R 3 is CO 2 H, CONH 2 , CN, NO 2 , Ci -6 alkylthio, SO 2 -C 1-6 alkyl, C,. 6 alkoxy, SONH 2 , CONHOH, NH 2 , CHO, CH 2 OH, CH 2 NH 2 , or CO 2 R, wherein R is hydrogen or C, -6 alkyl.
  • Particular examples of compounds having Formula II include 4-(4-Benzo[l,3]dioxol-5-yl-5- pyridin-2-yl-lH-imidazol-2-yl)phenol; 4-(4-Benzo[l,3]dioxol-5-yl-5-pyridin-2-yl-lH-imidazol-2-yl)-N- methyl-benzamide; 4-(4-Benzo[l,3]dioxol-5-yl-5-pyridin-2-yl-lH-imidazol-2-yl)-N-methoxy-benzamide; 2- ⁇ 4-Benzo[l,3]dioxol-5-yl-2-[4-(2H-tetrazol-5-yl)-phenyl]-lH-imidazol-5-yl ⁇ -pyridine; [4-(4-(4-(4-(4-benzo[l,3]dioxol-5-yl-5-
  • the TGF- ⁇ inhibitor is an inhibitor of the TGF- ⁇ signaling pathway as described in International Application WO02/055077 and U.S. Pat. 6,602,877 having Formula VIII:
  • R 1 is optionally substituted heteroaryl
  • R 2 is optionally substituted aryl or optionally substituted heteroaryl
  • Particular examples of compounds of Formula HI include ⁇ 2-[5-(2-cyclopropylamino-pyrimidin- 4-yl)-4-(4-fluoro-phenyl)-lH-imidazol-2-yl]-5-methyl-[l,3]dioxan-5-yl ⁇ -mo ⁇ holin-4-yl-methanone, trans isomer; 2-[5-(2-Amino-pyrimidin-4-yl)-4-(4-fluoro-phenyl)-lH-imidazol-2-yl]-5-methy 1- [ 1 ,3]dioxan-5-yl ⁇ -mo ⁇ holin-4-yl-methanone, trans-isomer; ⁇ 2-[5-(2-Dimethylamino-pyrimidin-4-yl)-4- (4-fluoro-phenyl)-lH-imidazol-2-yl]-5-methyl-[l,3]dioxan-5-y
  • the TGF- ⁇ inhibitor is an antagonist of the TGF- ⁇ type I described in published U.S. Application 20060148835 (U.S. Appln. 10/537,711) having Formula DC:
  • R 3 is selected from the group consisting of H, carboxyl, alkyloxycarbonyl, 5'- (phenyloxadiazol-2'-yl), 5'-(pyridyl-4"-oxadizol-2'-yl), CONHR 9 , wherein R 9 is selected from the group consisting of C 2 -C 8 fatty acid, benzoxamido, isonicotinamido, un-substituted or mono- or multi- substituted phenyl wherein the substituent may be hydroxyl, Cj-C 8 alkoxyl, CF 3 , carboxyl, alkyloxycarbonyl, OCH 2 CO 2 H, NO 2 , halogen, SO 3 H, SO 2 NHR M , wherein R 11 is selected from the group consisting of hydrogen, amidino, 2"-thiazolyl, 3"-(5"-methylisooxazolyl), 2"-pyrimidinyl, 2"-(4",6"- di
  • Particular examples of compounds having Formula IV include 3-ethoxycarbonyl-6-chloro-7- hydroxy-8-nitro-coumarin, S-ethoxycarbonyl- ⁇ -ethyl ⁇ -hydroxy- ⁇ -nitro-couinarin, 3-ethoxycarbonyl-6- nit ⁇ o-7,8-dimethoxy-coumarin, 3-ethoxycarbonyl-6,8-dinitro-7-methoxy-coumarin, 3-ethoxycarbonyl-6,8- dinitro-7-hydroxy-coumarin, 3 -(3 '-hydroxy-4'-carboxy-phenylamidocarbonyl)-6-ethyl-7-methoxy- coumarin, 3-(3'-carboxy-4'-hydroxy-phenylamidocarbonyl)-6-ethyl-7-methoxy-coumarin, 3-(m- carboxyphenylamidocarbonyl)-7-methoxycoumarin, 3-(3'-hydroxy-4'-carboxyphenylamidocarbonyl)-7-7-me
  • an effective amount is the amount required to confer a therapeutic effect on the treated patient.
  • an effective amount can range, for example, from about 1 mg/kg to aboutl50 mg/kg (e.g. , from about 1 mg/kg to about 100 mg/kg).
  • Effective doses will also vary, as recognized by those skilled in the art, dependant on route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatments including use of other therapeutic agents and/or radiation therapy.
  • Compounds of formulas (I)-(DC) can be administered in any manner suitable for the administration of pharmaceutical compounds, including, but not limited to, pills, tablets, capsules, aerosols, suppositories, liquid formulations for ingestion or injection or for use as eye or ear drops, dietary supplements, and topical preparations.
  • the pharmaceutically acceptable compositions include aqueous solutions of the active agent, in an isotonic saline, 5% glucose or other well-known pharmaceutically acceptable excipient.
  • Solubilizing agents such as cyclodextrins, or other solubilizing agents well-known to those familiar with the art, can be utilized as pharmaceutical excipients for delivery of the therapeutic compounds.
  • the compositions can be administered orally, intranasally, transdermally, intradermally, vaginally, intraaurally, intraocularly, buccally, rectally, transmucosally, or via inhalation, implantation (e.g., surgically), or intravenous administration.
  • the compositions can be administered to an animal (e.g.
  • a mammal such as a human, non-human primate, horse, dog, cow, pig, sheep, goat, cat, mouse, rat, guinea pig, rabbit, hamster, gerbil, or ferret, or a bird, or a reptile, such as a lizard).
  • TGF- ⁇ inhibitors of the present invention are soluble transforming growth factor-beta receptor polypeptides and polypeptide fragments, which downregulate or neutralize TGF- ⁇ in vivo.
  • TGFRII Full-length TGFRII consists of a signal sequence, an extracellular domain, a transmembrane domain and a cytoplasmic domain. A protein kinase domain is also present within the cytoplasmic domain.
  • polypeptide sequence was reported as the human TGFRH polypeptide (isoform A) sequence and has the accession number NP OO 1020018 in Genbank.
  • Variants of human TGFRII include, but are not limited to, the polypeptides with the following mutations: M36V, V191I, L308P, T315M, Y336N, A355P, G357W, V387M, N435S, V447A, S449F,
  • YLTRHVISWE DLRKLGSSLA RGIAHLHSDH TPCGRPKMPI VHRDLKSSNI LVKNDLTCCL CDFGLSLRLD PTLSVDDLAN SGQVGTARYM APEVLESRMN LENVESFKQT DVYSMALVLW EMTSRCNAVG EVKDYEPPFG SKVREHPCVE SMKDSVLRDR GRPEIPSFWL NHQGIQIVCE TLTECWDHDP EARLTAQCVA ERFSELEHPE RLSGRSCSQE KIPEDGSLNT TK
  • Variants of mouse TGFRII include, but are not limited to, the polypeptides with the following mutations:G8A, D117N, A354R, NSGQ440 ⁇ 43KQRE, C486W, H506P and PE569-570MD.
  • Variants of rat TGFRH include, but are not limited to, the polypeptides with the following mutations:KN388-389RS, R403G, D405S and K477R.
  • the partial pig TGFRII polypeptide sequence is shown below as SEQ ID NO:44.
  • TGFRH domain designations used herein are defined as follows: Table 2.
  • Full-length TGFRI consists of a signal sequence, an extracellular domain, a transmembrane domain and a cytoplasmic domain.
  • a protein kinase domain and an ATP binding domain are also present within the cytoplasmic domain.
  • the alanine at amino acid 26 is a double alanine in the human TGFRI polypeptide.
  • Variants of human TGFRI include, but are not limited to, the polypeptides with the following mutations:T200I, M318R, D400G and R487P.
  • rat TGFRI polypeptide is shown below as SEQ ID NO:47 and SEQ ID NO:48. Both of these sequence have been designated as rat TGFRI in Ge ⁇ bank.
  • TGFRI domain designations used herein are defined as follows: Table 3.
  • TGFRHI Full-length TGFRHI consists of a signal sequence, an extracellular domain, a transmembrane domain and a cytoplasmic domain.
  • a zona pellucida (ZP) domain is also present within the extracellular domain.
  • Variants of human TGFRIII include, but are not limited to, the polypeptides with the following mutations:S14N, S15F, W163L, NA358-359N, A634T, G764R, RTAG70-73ALR or NR349-350IV.
  • the following polypeptide sequence was reported as the mouse TGFRIII polypeptide sequence and has the accession number NP_035708 in Genbank.
  • Full-Length Mouse TGFRm (SEQ ID NO:50):
  • Variants of mouse TGFRIII include, but are not limited to, the polypeptides with the following mutations:S533A, S544A, Y322I, P391 S, N712K.
  • Variants of mouse TGFRIII include, but are not limited to, the polypeptides with the following mutations:Ll 64V.
  • TGFRIII domain designations used herein are defined as follows:
  • Some embodiments of the invention provide an inhibitor of the TGF- ⁇ signaling pathway which comprises a soluble TGFRI, TGFRH or TGFRm polypeptide.
  • soluble TGFRI, TGFRD or TGFRIII polypeptides of the present invention include fragments, variants, or derivative thereof of a soluble TGFRI, TGFRII or TGFRIII polypeptide.
  • Tables 2-4 above describes the various domains of the TGFRI, TGFRH or TGFRDI polypeptide.
  • Soluble TGFRI, TGFRII or TGFRIII polypeptides of the invention generally comprise a portion or all of the extracellular domain of the polypeptides.
  • Soluble TGFRI, TGFRn or TGFRHI polypeptides generally lack the transmembrane domain and cytoplasmic domains.
  • the entire extracellular domain of TGFRI, TGFRH or TGFRi ⁇ may comprise additional or fewer amino acids on either the C-terminal or N-terminal end of the extracellular domain polypeptide.
  • Soluble human TGFRH polypeptides for use in the methods of the present invention include, but are not limited to, a soluble TGFRII polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence identical to a reference amino acid sequence, except for up to twenty amino acid substitutions, wherein said reference amino acid sequence is selected from the group consisting of amino acids a to 166 of SEQ ED NO: 39 or 40, amino acids 23 to b of SEQ ID NO: 39 or 40, and a to b of SEQ ID NO: 39 or 40,wherein a is any integer from 1 to 25, and b is any integer from 50 to 170, wherein said polypeptide inhibits transforming growth factor (TGF)-beta signaling.
  • TGF transforming growth factor
  • Soluble TGFRH polypeptides for use in the methods of the present invention include, but are not limited to, a soluble TGFRII polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence identical to a reference amino acid sequence, except for up to twenty amino acid substitutions, wherein said reference amino acid sequence is selected from the group consisting of amino acids a to 191 of SEQ BD NO: 39 or 40, amino acids 23 to b of SEQ ID NO: 39 or 40, and a to b of SEQ ED NO: 39 or 40, wherein a is any integer from 1 to 25, and b is any integer from 50 to 195, wherein said polypeptide inhibits transforming growth factor (TGF)-beta signaling.
  • TGF transforming growth factor
  • the soluble TGFRII polypeptide comprises amino acids 23 to 166 of SEQ ID NO: 39 or 40. In another embodiment, the soluble TGFRII polypeptide comprises amino acids 1 to 159 of SEQ ED NO: 39 or 40. In another embodiment, the soluble TGFRII polypeptide comprises amino acids 23 to 191 of SEQ ED NO: 39 or 40.
  • a reference amino acid sequence is meant the specified sequence without the introduction of any amino acid substitutions.
  • the “isolated polypeptide” of the invention comprises an amino acid sequence which is identical to the reference amino acid sequence.
  • Soluble human TGFRI polypeptides for use in the methods of the present invention include, but are not limited to, a soluble TGFRI polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence identical to a reference amino acid sequence, except for up to twenty amino acid substitutions, wherein said reference amino acid sequence is selected from the group consisting of amino acids a to 125 of SEQ ID NO: 45, amino acids 25 to b of SEQ ID NO: 45, and a to b of SEQ ID NO: 45, wherein a is any integer from 1 to 30, and b is any integer from 50 to 130, wherein said polypeptide inhibits transforming growth factor (TGF)-beta signaling.
  • TGF transforming growth factor
  • the soluble TGFRI polypeptide comprises amino acids 25 to 125 of SEQ ID NO: 45.
  • the soluble TGFRI polypeptide comprises amino acids 1 to 124 of SEQ ID NO: 45.
  • Soluble human TGFRIII polypeptides for use in the methods of the present invention include, but are not limited to, a soluble TGFRHI polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence identical to a reference amino acid sequence, except for up to twenty amino acid substitutions, wherein said reference amino acid sequence is selected from the group consisting of amino acids a to 787 of SEQ ID NO: 49, amino acids 21 to b of SEQ ID NO: 49, and a to b of SEQ ID NO: 49, wherein a is any integer from 1 to 454, and b is any integer from 500 to 790, wherein said polypeptide inhibits transforming growth factor (TGF)-beta signaling.
  • TGF transforming growth factor
  • the soluble TGFRHI polypeptide comprises amino acids 21 to 787 of SEQ ED NO: 49.
  • the soluble TGFRm polypeptide comprises amino acids 1 to 781 of SEQ ID NO: 49.
  • Soluble TGFRI, TGFRII or TGFRIII polypeptides described herein may have various alterations such as substitutions, insertions or deletions.
  • Exemplary amino acids that can be substituted in the polypeptide include amino acids with basic side chains (e.g.
  • lysine, arginine, histidine acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • soluble TGFRI, TGFRII or TGFRHI polypeptides at least 70%, 75%, 80%, 85%, 90%, or 95% identical to the polypeptides and reference polypeptides described herein are also contemplated.
  • sequence identity between two polypeptides is determined by comparing the amino acid sequence of one polypeptide to the sequence of a second polypeptide.
  • sequence identity can be determined using methods and computer programs/software known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711).
  • BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences.
  • the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference polypeptide sequence and that gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.
  • a soluble TGFRI, TGFRII or TGFRIII polypeptide or polypeptide fragment of the invention is typically administered directly as a preformed polypeptide.
  • Some embodiments of the invention involve the use of a soluble TGFRI, TGFRII or TGFRIII polypeptide fused to a heterologous polypeptide moiety to form a fusion protein.
  • fusion proteins can be used to accomplish various objectives, e.g., increased serum half-life, improved bioavailability, in vivo targeting to a specific organ or tissue type, improved recombinant expression efficiency, improved host cell secretion, ease of purification, and higher avidity.
  • the heterologous moiety can be inert or biologically active.
  • a chosen heterologous moiety can be preformed and chemically conjugated to the soluble TGFRI, TGFRII or TGFRIII polypeptide moiety of the invention. In most cases, a chosen heterologous moiety will function similarly, whether fused or conjugated to the soluble TGFRI, TGFRII or TGFRIII polypeptide moiety.
  • heterologous amino acid sequences can be joined to the soluble TGFRI, TGFRII or TGFRIII polypeptide moiety in the form of a fusion protein or as a chemical conjugate.
  • Soluble TGFRI, TGFRII or TGFRi ⁇ polypeptides for use in the treatment methods disclosed herein include derivatives that are modified, e.g., by the covalent attachment of any type of molecule such that covalent attachment does not prevent the soluble TGFRI, TGFRH or TGFRIII polypeptide from inhibiting TGF- ⁇ signalling.
  • the soluble TGFRI, TGFRII or TGFRi ⁇ polypeptides of the present invention may be modified e.g., by glycosylation, acetylation, pegylation, phosphylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
  • Soluble TGFRI, TGFRII or TGFRIII polypeptides for use in the treatment methods disclosed herein can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids.
  • Soluble TGFRI, TGFRTI or TGFREI polypeptides may be modified by natural processes, such as posttranslational 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 the soluble TGFRI, TGFRII or TGFRIII polypeptide including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini, or on moieties such as carbohydrates. 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 soluble TGFRI, TGFRII or TGFRHI polypeptide. Also, a given soluble TGFRI, TGFRII or TGFRIII polypeptide may contain many types of modifications. Soluble TGFRI, TGFRH or TGFRHI 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 soluble TGFRI, TGFRII or TGFRHI 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 phosphotidylinositol, 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
  • Polypeptides described herein may be cyclic. Cyclization of the polypeptides reduces the conformational freedom of linear peptides and results in a more structurally constrained molecule.
  • Many methods of peptide cyclization are known in the art. For example, "backbone to backbone” cyclization by the formation of an amide bond between the N-terminal and the C-terminal amino acid residues of the peptide.
  • the "backbone to backbone” cyclization method includes the formation of disulfide bridges between two ⁇ -thio amino acid residues ⁇ e.g.. cysteine, homocysteine).
  • Certain peptides of the present invention include modifications on the N- and C- terminus of the peptide to form a cyclic polypeptide. Such modifications include, but are not limited, to cysteine residues, acetylated cysteine residues, cysteine residues with a NH2 moiety and biotin. Other methods of peptide cyclization are described in Li & Roller, Curr. Top. Med. Chem. 3:325-341 (2002) and U.S Patent Publication No. U.S. 2005-0260626 Al, which are incorporated by reference herein in their entirety.
  • the heterologous polypeptide to which the soluble TGFRI, TGFRH or TGFRHI polypeptide is fused is useful theraeutically or is useful to target the soluble TGFRI, TGFRH or TGFRIII polypeptide.
  • Soluble TGFRI, TGFRH or TGFRIII fusion proteins can be used to accomplish various objectives, e.g., increased serum half-life, improved bioavailability, in vivo targeting to a specific organ or tissue type, improved recombinant expression efficiency, improved host cell secretion, ease of purification, and higher avidity.
  • the heterologous moiety can be inert or biologically active.
  • a chosen heterologous moiety can be preformed and chemically conjugated to the polypeptide. In most cases, a chosen heterologous moiety will function similarly, whether fused or conjugated to the soluble TGFRI, TGFRn or TGFRm polypeptide. Therefore, in the following discussion of heterologous amino acid sequences, unless otherwise noted, it is to be understood that the heterologous sequence can be joined to the soluble TGFRI, TGFRH or TGFRIII polypeptide in the form of a fusion protein or as a chemical conjugate.
  • Pharmacologically active polypeptides such as soluble TGFRI, TGFRII or TGFRHI polypeptides may exhibit rapid in vivo clearance, necessitating large doses to achieve therapeutically effective concentrations in the body.
  • polypeptides smaller than about 60 IcDa potentially undergo glomerular filtration, which sometimes leads to nephrotoxicity.
  • Various heterologous amino acid sequences e.g., polypeptide moieties or "carriers,” for increasing the in vivo stability, e.g., serum half-life, of therapeutic polypeptides are known.
  • serum albumins such as, e.g., bovine serum albumin (BSA) or human serum albumin (HSA).
  • HSA human serum albumin
  • HSA can be used to form a fusion protein or polypeptide conjugate that displays pharmacological activity by virtue of the soluble TGFRI, TGFRII or TGFRm polypeptide moiety while displaying significantly increased in vivo stability, e.g., 10-fold to 100-fold higher.
  • the C-terminus of the HSA can be fused to the N-te ⁇ ninus of the soluble TGFRI, TGFRII or TGFRIII polypeptide moiety.
  • soluble TGFRI, TGFRII or TGFRIII polypeptides for use in the methods of the present invention further comprise a targeting moiety.
  • Targeting moieties include a protein or a peptide which directs localization to a certain part of the body.
  • Some embodiments of the invention employ a soluble TGFRI, TGFRII or TGFRHI polypeptide moiety fused to a hinge and Fc region, i.e., the C-terminal portion of an Ig heavy chain constant region.
  • amino acids in the hinge region may be substituted with different amino acids.
  • Exemplary amino acid substitutions for the hinge region according to these embodiments include substitutions of individual cysteine residues in the hinge region with different amino acids. Any different amino acid may be substituted for a cysteine in the hinge region.
  • Amino acid substitutions for the amino acids of the polypeptides of the invention and the reference amino acid sequence can include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • Typical amino acids to substitute for cysteines in the reference amino acid include alanine, serine, threonine, in particular, serine and alanine. Making such substitutions through engineering of a polynucleotide encoding the polypeptide fragment is well within the routine expertise of one of ordinary skill in the art.
  • soluble TGFRI, TGFRII or TGFRIII polypeptide-Fc fusion include solubility, in vivo stability, and multivalency, e.g., dimerization.
  • the Fc region used can be an IgA, IgD, or IgG Fc region (hinge-CH2-CH3). Alternatively, it can be an IgE or IgM Fc region (hinge-CH2-CH3- CH4).
  • An IgG Fc region is generally used, e.g., an IgGl Fc region or IgG4 Fc region.
  • Fc fusion protein such as those described in Capon et al., U.S. Patent Nos. 5,428,130 and 5,565,335.
  • Fully intact, wild-type Fc regions display effector functions that normally are unnecessary and undesired in an Fc fusion protein used in the methods of the present invention. Therefore, certain binding sites typically are deleted from the Fc region during the construction of the secretion cassette. For example, since coexpression with the light chain is unnecessary, the binding site for the heavy chain binding protein, Bip (Hendershot et al, Immunol.
  • the IgGl Fc region is most often used.
  • the Fc region of the other subclasses of immunoglobulin gamma (gamma-2, gamma-3 and gamma-4) can be used in the secretion cassette.
  • the IgGl Fc region of immunoglobulin gamma- 1 is generally used in the secretion cassette and includes at least part of the hinge region, the CH2 region, and the CH3 region.
  • the Fc region of immunoglobulin gamma- 1 is a CH2-deleted-Fc, which includes part of the hinge region and the CH3 region, but not the CH2 region.
  • a CH2-deleted-Fc has been described by Gillies et al., Hum.
  • the Fc region of one of IgA, IgD, IgE, or IgM is used.
  • Soluble TGFRI, TGFRTI or TGFRHI-polypeptide-moiety-Fc fusion proteins can be constructed in several different configurations. In one configuration the C-terminus of the soluble TGFRI, TGFRII or TGFRIII polypeptide moiety is fused directly to the N-terminus of the Fc hinge moiety.
  • a short polypeptide e.g., 2-10 amino acids, is incorporated into the fusion between the N-terminus of the soluble TGFRI, TGFRTI or TGFRIII polypeptide moiety and the C-terminus of the Fc moiety.
  • the short polypeptide is incorporated into the fusion between the C-terminus of the soluble TGFRI, TGFRII or TGFRIII polypeptide moiety and the N-terminus of the Fc moiety.
  • Such a linker provides conformational flexibility, which may improve biological activity in some circumstances.
  • the soluble TGFRI, TGFRn or TGFRm-polypeptide-moiety-Fc fusion will dimerize, thus forming a divalent molecule.
  • a homogeneous population of monomeric Fc fusions will yield monospecific, bivalent dimers.
  • a mixture of two monomeric Fc fusions each having a different specificity will yield bispecific, bivalent dimers.
  • any of a number of cross-linkers that contain a corresponding amino-reactive group and thiol- reactive group can be used to link a soluble TGFRI, TGFRQ or TGFRm polypeptide or polypeptide fragment of the invention to serum albumin.
  • suitable linkers include amine reactive cross- linkers that insert a thiol-reactive maleimide, e.g., SMCC, AMAS, BMPS, MBS, EMCS, SMPB, SMPH, KMUS, and GMBS.
  • Other suitable linkers insert a thiol-reactive haloacetate group, e.g., SBAP, SIA, SIAB.
  • Linkers that provide a protected or non-protected thiol for reaction with sulfhydryl groups to product a reducible linkage include SPDP, SMPT, SATA, and SATP. Such reagents are commercially available (e.g. , Pierce Chemical Company, Rockford, IL).
  • TGFRI, TGFRII or TGFRIII polypeptide or polypeptide fragment of the invention does not have to involve the N-terminus of a soluble TGFRI, TGFRII or TGFRIII polypeptide or polypeptide fragment of the invention or the thiol moiety on serum albumin.
  • TGFRI, TGFRII or TGFRIII -polypeptide-albumin fusions can be obtained using genetic engineering techniques, wherein the TGFRI, TGFRH or TGFRHI polypeptide moiety is fused to the serum albumin gene at its N-terminus, C-terminus, or both.
  • Soluble TGFRI, TGFRII or TGFRIII polypeptides of the invention can be fused to a polypeptide tag.
  • polypeptide tag is intended to mean any sequence of amino acids that can be attached to, connected to, or linked to a soluble TGFRI, TGFRII or TGFRm polypeptide and that can be used to identify, purify, concentrate or isolate the soluble TGFRI, TGFRII or TGFRm polypeptide.
  • the attachment of the polypeptide tag to the soluble TGFRI, TGFRII or TGFRm polypeptide may occur, e.g., by constructing a nucleic acid molecule that comprises: (a) a nucleic acid sequence that encodes the polypeptide tag, and (b) a nucleic acid sequence that encodes a soluble TGFRI, TGFRQ or TGFRIII polypeptide.
  • Exemplary polypeptide tags include, e.g. , amino acid sequences that are capable of being post-translationally modified, e.g., amino acid sequences that are biotinylated.
  • polypeptide tags include, e.g., amino acid sequences that are capable of being recognized and/or bound by an antibody (or fragment thereof) or other specific binding reagent.
  • Polypeptide tags that are capable of being recognized by an antibody (or fragment thereof) or other specific binding reagent include, e.g., those that are known in the art as "epitope tags.”
  • An epitope tag may be a natural or an artificial epitope tag. Natural and artificial epitope tags are known in the art, including, e.g., artificial epitopes such as FLAG, Strep, or poly-histidine peptides.
  • FLAG peptides include the sequence Asp-Tyr-Lys-Asp-Asp- Asp-Asp-Lys (SEQ DD NO: 53) or Asp-Tyr-Lys-Asp-Glu-Asp-Asp-Lys (SEQ Q) NO: 54) (Einhauer, A. and Jungbauer, A., J. Biochem. Biophys. Methods 49:1-3:455-465 (2001)).
  • the Strep epitope has the sequence Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (SEQ ID NO: 55).
  • VSV-G epitope can also be used and has the sequence Tyr-Thr-Asp-Ile-Glu-Met-Asn-Arg-Leu-Gly-Lys (SEQ ID NO: 56).
  • Another artificial epitope is a poly-His sequence having six histidine residues (His-His-His-His-His-His (SEQ ED NO: 57).
  • Naturally-occurring epitopes include the influenza virus hemagglutinin (HA) sequence Tyr-Pro- Tyr-Asp-Val-Pro-Asp-Tyr-Ala- ⁇ e-Glu-Gly-Arg (SEQ ID NO: 58) recognized by the monoclonal antibody 12CA5 (Murray et al., Anal. Biochem.
  • HA hemagglutinin
  • the soluble TGFRI, TGFRII or TGFRIII polypeptide and the polypeptide tag may be connected via a linking amino acid sequence.
  • a "linking amino acid sequence” may be an amino acid sequence that is capable of being recognized and/or cleaved by one or more proteases. Amino acid sequences that can be recognized and/or cleaved by one or more proteases are known in the art.
  • Exemplary amino acid sequences are those that are recognized by the following proteases: factor Vila, factor DCa, factor Xa, APC, t-PA, u-PA, trypsin, chymotrypsin, enterokinase, pepsin, cathepsin B,H,L,S,D, cathepsin G, renin, angiotensin converting enzyme, matrix metalloproteases (collagenases, stromelysins, gelatinases), macrophage elastase, Cir, and Cis.
  • the amino acid sequences that are recognized by the aforementioned proteases are known in the art. Exemplary sequences recognized by certain proteases can be found, e.g., in U.S. Patent No. 5,811,252.
  • a soluble TGFRI, TGFRII or TGFRIII polypeptide fusion construct is used to enhance the production of a soluble TGFRI, TGFRII or TGFRIII polypeptide moiety in bacteria.
  • a bacterial protein normally expressed and/or secreted at a high level is employed as the N-terminal fusion partner of a soluble TGFRI, TGFRII or TGFRm polypeptide or polypeptide fragment of the invention. See, e.g., Smith et al., Gene 67:31 (1988); Hopp et al., Biotechnology 6:1204 (1988); La Value et al., Biotechnology /7:187 (1993).
  • a soluble TGFRI, TGFRU or TGFRIH polypeptide moiety By fusing a soluble TGFRI, TGFRU or TGFRIH polypeptide moiety at the amino and carboxy termini of a suitable fusion partner, bivalent or tetravalent forms of a soluble TGFRI, TGFRII or TGFRIII polypeptide or polypeptide fragment of the invention can be obtained.
  • a soluble TGFRI, TGFRn or TGFRi ⁇ polypeptide moiety can be fused to the amino and carboxy termini of an Ig moiety to produce a bivalent monomeric polypeptide containing two soluble TGFRI, TGFRII or TGFRIII polypeptide moieties.
  • a tetravalent form of a soluble TGFRI, TGFRII or TGFRIH polypeptide is obtained.
  • Such multivalent forms can be used to achieve increased binding affinity for the target.
  • Multivalent forms of a soluble TGFRI, TGFRII or TGFRIH polypeptide or polypeptide fragment of the invention also can be obtained by placing soluble TGFRI, TGFRII or TGFRIH polypeptide moieties in tandem to form concatamers, which can be employed alone or fused to a fusion partner such as Ig or HSA. 5.
  • Conjugated Polymers (other than polypeptides)
  • Some embodiments of the invention involve a soluble TGFRI, TGFRII or TGFRIII polypeptide or polypeptide fragment of the invention wherein one or more polymers are conjugated (covalently linked) to the soluble TGFRI, TGFRII or TGFRi ⁇ polypeptide.
  • polymers suitable for such conjugation include polypeptides (discussed above), sugar polymers and polyalkylene glycol chains.
  • a polymer is conjugated to the soluble TGFRI, TGFRH or TGFRIII polypeptide or polypeptide fragment of the invention for the purpose of improving one or more of the following: solubility, stability, or bioavailability.
  • the class of polymer generally used for conjugation to a soluble TGFRI, TGFRII or TGFRm polypeptide or polypeptide fragment of the invention is a polyalkylene glycol.
  • Polyethylene glycol (PEG) is most frequently used.
  • PEG moieties e.g., 1, 2, 3, 4 or 5 PEG polymers, can be conjugated to each soluble TGFRI, TGFRII or TGFRIII polypeptide to increase serum half life, as compared to the soluble TGFRI, TGFRII or TGFRIII polypeptide alone.
  • PEG moieties are non-antigenic and essentially biologically inert.
  • PEG moieties used in the practice of the invention may be branched or unbranched.
  • the number of PEG moieties attached to the soluble TGFRI, TGFRII or TGFRm polypeptide and the molecular weight of the individual PEG chains can vary. In general, the higher the molecular weight of the polymer, the fewer polymer chains attached to the polypeptide. Usually, the total polymer mass attached to a soluble TGFRI, TGFRH or TGFRIII polypeptide or polypeptide fragment is from 20 kDa to 40 kDa. Thus, if one polymer chain is attached, the molecular weight of the chain is generally 20-40 kDa. If two chains are attached, the molecular weight of each chain is generally 10-20 kDa. If three chains are attached, the molecular weight is generally 7-14 kDa.
  • the polymer e.g., PEG
  • the polymer can be linked to the soluble TGFRI, TGFRII or TGFRIII polypeptide through any suitable, exposed reactive group on the polypeptide.
  • the exposed reactive group(s) can be, e.g. , an N-terminal amino group or the epsilon amino group of an internal lysine residue, or both.
  • An activated polymer can react and covalently link at any free amino group on the soluble TGFRI, TGFRII or TGFRi ⁇ polypeptide.
  • Free carboxylic groups suitably activated carbonyl groups, hydroxyl, guanidyl, imidazole, oxidized carbohydrate moieties and mercapto groups of the soluble TGFRI, TGFRII or TGFRIII polypeptide (if available) also can be used as reactive groups for polymer attachment.
  • a conjugation reaction from about 1.0 to about 10 moles of activated polymer per mole of polypeptide, depending on polypeptide concentration, is typically employed. Usually, the ratio chosen represents a balance between maximizing the reaction while minimizing side reactions (often nonspecific) that can impair the desired pharmacological activity of the soluble TGFRI, TGFRII or TGFRIII polypeptide moiety.
  • the soluble TGFRI, TGFRII or TGFRIII polypeptide is retained, and most preferably nearly 100% is retained.
  • the polymer can be conjugated to the soluble TGFRI, TGFRII or TGFRIII polypeptide using conventional chemistry.
  • a polyalkylene glycol moiety can be coupled to a lysine epsilon amino group of the soluble TGFRI, TGFRII or TGFRHI polypeptide.
  • Linkage to the lysine side chain can be performed with an N-hydroxylsuccinimide (NHS) active ester such as PEG succinimidyl succinate (SS-PEG) and succinimidyl propionate (SPA-PEG).
  • Suitable polyalkylene glycol moieties include, e.g., carboxymethyl-NHS and norleucine-NHS, SC.
  • Additional amine-reactive PEG linkers can be substituted for the succinimidyl moiety. These include, e.g., isothiocyanates, nitrophenylcarbonates (PNP), epoxides, benzotriazole carbonates, SC-PEG, tresylate, aldehyde, epoxide, carbonylimidazole and PNP carbonate. Conditions are usually optimized to maximize the selectivity and extent of reaction. Such optimization of reaction conditions is within ordinary skill in the art.
  • PEGylation can be carried out by any of the PEGylation reactions known in the art. See, e.g., Focus on Growth Factors, 3: 4-10, 1992 and European patent applications EP 0 154 316 and EP 0 401 384. PEGylation may be carried out using an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer).
  • PEGylation by acylation generally involves reacting an active ester derivative of polyethylene glycol. Any reactive PEG molecule can be employed in the PEGylation. PEG esterified to N- hydroxysuccinimide (NHS) is a frequently used activated PEG ester.
  • acylation includes without limitation the following types of linkages between the therapeutic protein and a water-soluble polymer such as PEG: amide, carbamate, urethane, and the like. See, e.g., Bioconjugate Chem. 5: 133- 140, 1994. Reaction parameters are generally selected to avoid temperature, solvent, and pH conditions that would damage or inactivate the soluble TGFRI, TGFRH or TGFRIII polypeptide.
  • the connecting linkage is an amide and typically at least 95% of the resulting product is mono-, di- or tri-PEGylated. However, some species with higher degrees of PEGylation may be formed in amounts depending on the specific reaction conditions used.
  • purified PEGylated species are separated from the mixture, particularly unreacted species, by conventional purification methods, including, e.g., dialysis, salting-out, ultrafiltration, ion-exchange chromatography, gel filtration chromatography, hydrophobic exchange chromatography, and electrophoresis.
  • PEGylation by alkylation generally involves reacting a terminal aldehyde derivative of PEG with a soluble TGFRI, TGFRII or TGFRm polypeptide or polypeptide fragment of the invention in the presence of a reducing agent.
  • a reducing agent e.g. one can manipulate the reaction conditions to favor PEGylation substantially only at the N-terminal amino group of the soluble TGFRI, TGFRU or TGFRHI polypeptide, e.g. a mono-PEGylated protein.
  • the PEG groups are typically attached to the protein via a -CH2-NH- group. With particular reference to the -CH2- group, this type of linkage is known as an "alkyl" linkage.
  • the polymer molecules used in both the acylation and alkylation approaches are selected from among water-soluble polymers.
  • the polymer selected is typically modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization may be controlled as provided for in the present methods.
  • An exemplary reactive PEG aldehyde is polyethylene glycol propionaldehyde, which is water stable, or mono Cl-ClO alkoxy or aryloxy derivatives thereof (see, e.g., Harris et al., U.S. Pat. No. 5,252,714).
  • the polymer may be branched or unbranched.
  • the polymer(s) selected typically have a single reactive ester group.
  • the polymer(s) selected typically have a single reactive aldehyde group.
  • the water-soluble polymer will not be selected from naturally occurring glycosyl residues, because these are usually made more conveniently by mammalian recombinant expression systems.
  • Methods for preparing a PEGylated soluble TGFRI, TGFRH or TGFRHI polypeptide of the invention generally includes the steps of (a) reacting a soluble TGFRI, TGFRII or TGFRIII polypeptide or polypeptide fragment of the invention with polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions whereby the molecule becomes attached to one or more PEG groups, and (b) obtaining the reaction product(s).
  • polyethylene glycol such as a reactive ester or aldehyde derivative of PEG
  • the optimal reaction conditions for the acylation reactions will be determined case-by-case based on known parameters and the desired result. For example, a larger the ratio of PEG to protein, generally leads to a greater the percentage of poly- PEGylated product.
  • Reductive alkylation to produce a substantially homogeneous population of mono- polymer/soluble TGFRI, TGFRII or TGFRIII polypeptide generally includes the steps of: (a) reacting a soluble TGFRI, TGFRII or TGFRIII polypeptide or polypeptide fragment of the invention with a reactive PEG molecule under reductive alkylation conditions, at a pH suitable to permit selective modification of the N-terminal amino group of soluble TGFRI, TGFRII or TGFRIII; and (b) obtaining the reaction product(s).
  • the reductive alkylation reaction conditions are those that permit the selective attachment of the water-soluble polymer moiety to the N-terminus of a soluble TGFRI, TGFRII or TGFRHI polypeptide or polypeptide fragment of the invention.
  • Such reaction conditions generally provide for pKa differences between the lysine side chain amino groups and the N-terminal amino group.
  • the pH is generally in the range of 3-9, typically 3-6.
  • Soluble TGFRI, TGFRII or TGFRIII polypeptides of the invention can include a tag, e.g., a moiety that can be subsequently released by proteolysis.
  • the lysine moiety can be selectively modified by first reacting a His-tag modified with a low-molecular-weight linker such as Traut's reagent
  • polypeptide will then contain a free SH group that can be selectively modified with a PEG containing a thiol-reactive head group such as a maleimide group, a vinylsulfone group, a haloacetate group, or a free or protected SH.
  • a thiol-reactive head group such as a maleimide group, a vinylsulfone group, a haloacetate group, or a free or protected SH.
  • Traut's reagent can be replaced with any linker that will set up a specific site for PEG attachment.
  • Traut's reagent can be replaced with SPDP, SMPT, SATA, or SATP (Pierce Chemical
  • amine-reactive linker that inserts a maleimide (for example SMCC, AMAS, BMPS, MBS, EMCS, SMPB, SMPH, KMUS, or GMBS), a haloacetate group (SBAP, SIA, SIAB), or a vinylsulfone group and react the resulting product with a PEG that contains a free SH.
  • a maleimide for example SMCC, AMAS, BMPS, MBS, EMCS, SMPB, SMPH, KMUS, or GMBS
  • SBAP haloacetate group
  • SIAB SIA, SIAB
  • the polyalkylene glycol moiety is coupled to a cysteine group of the soluble TGFRI, TGFRII or TGFRIU polypeptide. Coupling can be effected using, e.g., a maleimide group, a vinylsulfone group, a haloacetate group, or a thiol group.
  • the soluble TGFRI, TGFRII or TGFRIII polypeptide is conjugated to the polyethylene-glycol moiety through a labile bond.
  • the labile bond can be cleaved in, e.g., biochemical hydrolysis, proteolysis, or sulfhydryl cleavage.
  • the bond can be cleaved under in. vivo
  • the reactions may take place by any suitable method used for reacting biologically active materials with inert polymers, generally at about pH 5-8, e.g., pH 5, 6, 7, or 8, if the reactive groups are on the alpha amino group at the N-terminus.
  • the process involves preparing an activated polymer and thereafter reacting the protein with the activated polymer to produce the soluble protein suitable for formulation.
  • Binding Molecules e.g., Antibodies Which Interrupt TGF- ⁇ Signaling
  • a TGF- ⁇ inhibitor for use in the methods of the invention is an antibody molecule, or immunospecific fragment thereof which interrupts the TGF- ⁇ signaling pathway, by, for example, binding to TGF- ⁇ - or to a TGF- ⁇ receptor.
  • Antibodies which specifically bind to a TGF- ⁇ - or to a TGF- ⁇ receptor, and which interrupt the TGF- ⁇ signaling pathway have been identified and characterized.
  • antibodies which specifically bind to TGF- ⁇ - or to TGF- ⁇ receptor may be produced by a person of ordinary skill in the art using routine experimentation and well-known methods as well as methods described elsewhere herein.
  • a "fragment thereof in reference to an antibody refers to an immunospecific fragment, e.g., an antigen-specific fragment.
  • a binding molecule e.g., an antibody of the invention is a bispecific binding molecule, binding polypeptide, or antibody, e.g., a bispecific antibody, minibody, domain deleted antibody, or fusion protein having binding specificity for more than one epitope, e.g., more than one antigen or more than one epitope on the same antigen.
  • a bispecific binding molecule, binding polypeptide, or antibody has at least one binding domain specific for at least one epitope on a target polypeptide disclosed herein, e.g., TGF- ⁇ - or a TGF- ⁇ receptor.
  • a bispecific binding molecule, binding polypeptide, or antibody has at least one binding domain specific for an epitope on a target polypeptide and at least one target binding domain specific for a drug or toxin.
  • a bispecific binding molecule, binding polypeptide, or antibody has at least one binding domain specific for an epitope on a target polypeptide disclosed herein, and at least one binding domain specific for a prodrug.
  • a bispecific binding molecule, binding polypeptide, or antibody may be a tetravalent antibody that has two target binding domains specific for an epitope of a target polypeptide disclosed herein and two target binding domains specific for a second target.
  • a tetravalent bispecific binding molecule, binding polypeptide, or antibody may be bivalent for each specificity.
  • Antibody binding molecules for use in the treatment methods of the present invention can comprise a constant region which mediates one or more effector functions. For example, binding of the Cl component of complement to an antibody constant region may activate the complement system. Activation of complement is important in the opsonisation and lysis of cell pathogens.
  • FcR Fc receptor
  • IgG gamma receptors
  • IgE epsilon receptors
  • IgA alpha receptors
  • IgM mi receptors
  • ADCC antibody-dependent cellrmediated cytotoxicity
  • methods of treating hyperproliferative diseases according to the present invention comprise administration of an antibody, or immunospecific fragment thereof, in which at least a fraction of one or more of the constant region domains has been deleted or otherwise altered so as to provide desired biochemical characteristics such as reduced effector functions, the ability to non- covalently dimerize, increased ability to localize at the site of a tumor, reduced serum half-life, or increased serum half-life when compared with a whole, unaltered antibody of approximately the same immunogenicity.
  • certain antibodies for use in the diagnostic and treatment methods described herein are domain deleted antibodies which comprise a polypeptide chain similar to an immunoglobulin heavy chain, but which lack at least a portion of one or more heavy chain domains.
  • one entire domain of the constant region of the modified antibody will be deleted, for example, all or part of the C H 2 domain will be deleted.
  • the Fc portion may be mutated to decrease effector function using techniques known in the art.
  • the deletion or inactivation (through point mutations or other means) of a constant region domain may reduce Fc receptor binding of the circulating modified antibody thereby increasing tumor localization.
  • constant region modifications consistent with the instant invention moderate complement binding and thus reduce the serum half life and nonspecific association of a conjugated cytotoxin.
  • modifications of the constant region may be used to modify disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or antibody flexibility.
  • the resulting physiological profile, bioavailability and other biochemical effects of the modifications such as tumor localization, biodistribution and serum half-life, may easily be measured and quantified using well know immunological techniques without undue experimentation.
  • Modified forms of antibodies or immunospecific fragments thereof for use in the methods disclosed herein can be made from whole precursor or parent antibodies using techniques known in the art. Exemplary techniques are discussed in more detail herein.
  • both the variable and constant regions of TGF- ⁇ - or TGF- ⁇ receptor- specific antibodies or immunospecific fragments thereof for use in the treatment methods disclosed herein are fully human.
  • Fully human antibodies can be made using techniques that are known in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled. Exemplary techniques that can be used to make such antibodies are described in US patents: 6,150,584; 6,458,592; 6,420,140. Other techniques are known in the art. Fully human anti bodies can likewise be produced by various display technologies, e.g., phage display or other viral display systems, as described in more detail elsewhere herein.
  • Binding molecules e.g., binding polypeptides, e.g., TGF- ⁇ - or TGF- ⁇ receptor-specific antibodies or immunospecific fragments thereof for use in the treatment methods disclosed herein can be made or manufactured using techniques that are known in the art.
  • antibody molecules or fragments thereof are "recombinantly produced," i.e., are produced using recombinant DNA technology. Exemplary techniques for making antibody molecules or fragments thereof are discussed in more detail elsewhere herein.
  • Binding molecules e.g., binding polypeptides, e.g., TGF- ⁇ - or TGF- ⁇ receptor-specific antibodies or immunospecific fragments thereof for use in the treatment methods disclosed herein include derivatives that are modified, e.g., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from specifically binding to its cognate epitope.
  • the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
  • a binding molecule e.g., a binding polypeptide, e.g., a TGF- ⁇ - or TGF- ⁇ receptor-specific antibody or immunospecific fragment thereof for use in the treatment methods disclosed herein will not elicit a deleterious immune response in the animal to be treated, e.g., in a human.
  • binding molecules e.g., binding polypeptides, e.g., TGF- ⁇ - or TGF- ⁇ receptor-specific antibodies or immunospecific fragments thereof for use in the treatment methods disclosed herein be modified to reduce their immunogenicity using art-recognized techniques.
  • antibodies can be humanized, primatized, deimmunized, or chimeric antibodies can be made. These types of antibodies are derived from a non-human antibody, typically a murine or primate antibody, that retains or substantially retains the antigen-binding properties of the parent antibody, but which is less immunogenic in humans.
  • CDRs complementarity determining regions
  • De-immunization can also be used to decrease the immunogenicity of an antibody.
  • the term "de-immunization” includes alteration of an antibody to modify T cell epitopes (see, e.g., WO9852976A1, WO0034317A2).
  • V H and V L sequences from the starting antibody are analyzed and a human T cell epitope "map" from each V region showing the location of epitopes in relation to complementarity-determining regions (CDRs) and other key residues within the sequence.
  • Individual T cell epitopes from the T cell epitope map are analyzed in order to identify alternative amino acid substitutions with a low risk of altering activity of the final antibody.
  • a range of alternative V H and V L sequences are designed comprising combinations of amino acid substitutions and these sequences are subsequently incorporated into a range of binding polypeptides, e.g., TGF- ⁇ - or TGF- ⁇ receptor-specific antibodies or immunospecific fragments thereof for use in the treatment methods disclosed herein, which are then tested for function.
  • variant antibodies typically, between 12 and 24 variant antibodies are generated and tested. Complete heavy and light chain genes comprising modified V and human C regions are then cloned into expression vectors and the subsequent plasmids introduced into cell lines for the production of whole antibody. The antibodies are then compared in appropriate biochemical and biological assays, and the optimal variant is identified.
  • administration is to an animal, e.g., a human, in need of treatment for cancer or other hyperproliferative disorder.
  • a binding molecule e.g., a -I l l-
  • binding polypeptide e.g., a TGF- ⁇ - or TGF- ⁇ receptor-specific antibody or immunospecific fragment thereof may be administered, along with a vector comprising a polynucleotide which encodes an interferon polypeptide or biologically active fragment thereof, to a human patient diagnosed with a tumor, other cancerous lesion, or other hyperproliferative disorder, a human patient who has been treated for cancer and is in remission, but is in need of further chronic treatment to prevent recurrence or spread of cancer, a human who exhibits early warning signs for a certain cancer or hyperproliferative disorder and is a candidate for preventative treatment, or preventatively to a human who is genetically predisposed to contract a certain cancer.
  • a vector comprising a polynucleotide which encodes an interferon polypeptide or biologically active fragment thereof
  • in vitro assays to demonstrate the therapeutic utility of binding molecule described herein include the effect of a binding molecule on a cell line or a patient tissue sample.
  • the effect of the binding molecule on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, apoptosis assays and cell lysis assays.
  • in vitro assays which can be used to determine whether administration of a specific binding molecule is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.
  • Antibodies or fragments thereof for use as therapeutic binding molecules may be generated by any suitable method known in the art.
  • Polyclonal antibodies to an antigen of interest can be produced by various procedures well known in the art.
  • a TGF- ⁇ - or TGF- ⁇ receptor can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen.
  • adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et ai, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988); Hammerling et al, in: Monoclonal Antibodies and T-CeIl Hybridomas Elsevier, N.Y., 563-681 (1981) (said references incorporated by reference in their entireties).
  • the term "monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Thus, the term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma and recombinant and phage display technology.
  • antibodies are raised in mammals by multiple subcutaneous or intraperitoneal injections of the relevant antigen (e.g., purified TGF- ⁇ - or TGF- ⁇ receptor or cells or cellular extracts comprising such antigens) and an adjuvant.
  • This immunization typically elicits an immune response that comprises production of antigen-reactive antibodies from activated splenocytes or lymphocytes.
  • the resulting antibodies may be harvested from the serum of the animal to provide polyclonal preparations, it is often desirable to isolate individual lymphocytes from the spleen, lymph nodes or peripheral blood to provide homogenous preparations of monoclonal antibodies (MAbs).
  • the lymphocytes are obtained from the spleen.
  • the relatively short-lived, or mortal, lymphocytes from a mammal which has been injected with antigen are fused with an immortal tumor cell line (e.g. a myeloma cell line), thus, producing hybrid cells or "hybridomas" which are both immortal and capable of producing the genetically coded antibody of the B cell.
  • an immortal tumor cell line e.g. a myeloma cell line
  • hybrid cells or "hybridomas" which are both immortal and capable of producing the genetically coded antibody of the B cell.
  • the resulting hybrids are segregated into single genetic strains by selection, dilution, and regrowth with each individual strain comprising specific genes for the formation of a single antibody. They produce antibodies which are homogeneous against a desired antigen and, in reference to their pure genetic parentage, are termed "monoclonal.”
  • Hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • reagents, cell lines and media for the formation, selection and growth of hybridomas are commercially available from a number of sources and standardized protocols are well established.
  • culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies against the desired antigen.
  • the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by in vitro assays such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • in vitro assays such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the monoclonal antibodies secreted by the subclones may be separated from culture medium, ascites fluid or serum by conventional purification procedures such as, for example, protein-A, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.
  • the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a desired target polypeptide, e.g., TGF- ⁇ - or TGF- ⁇ receptor.
  • a desired target polypeptide e.g., TGF- ⁇ - or TGF- ⁇ receptor.
  • Antibody fragments that recognize specific epitopes may be generated by known techniques.
  • Fab and F(ab')2 fragments may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • F(ab')2 fragments contain the variable region, the light chain constant region and the C H I domain of the heavy chain.
  • DNA encoding antibodies or antibody fragments may also be derived from antibody phage libraries.
  • phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
  • Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • Phage used in these methods are typically filamentous phage including fd and Ml 3 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.
  • Exemplary methods are set forth, for example, in EP 368 684 Bl; U.S. patent. 5,969,108, Hoogenboom, H.R. and Chames, Immunol. Today 27:371 (2000); Nagy et al. Nat. Med. 5:801 (2002); Huie et al, Proc. Natl. Acad. Sci. USA 98:2682 (2001); Lui et al., J. MoI. Biol.
  • Ribosomal display can be used to replace bacteriophage as the display platform (see, e.g., Hanes et al., Nat. Biotechnol. 75: 1287 (2000); Wilson et al., Proc. Natl. Acad. Sci. USA 98:3750 (2001); or Irving et al,, J.
  • cell surface libraries can be screened for antibodies (Boder et al., Proc. Natl. Acad. Sci. USA 97: 10701 (2000); Daugherty et al., J. Immunol. Methods 243:211 (2000)).
  • Such procedures provide alternatives to traditional hybridoma techniques for the isolation and subsequent cloning of monoclonal antibodies.
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • DNA sequences encoding V H and V L regions are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of lymphoid tissues) or synthetic cDNA libraries.
  • the DNA encoding the V H and V L regions are joined together by an scFv linker by PCR and cloned into a phagemid vector (e.g., p CANTAB 6 or pComb 3 HSS).
  • the vector is electroporated in E. coli and the E. coli is infected with helper phage.
  • Phage used in these methods are typically filamentous phage including fd and M13 and the V H or V 1 . regions are usually recombinantly fused to either the phage gene III or gene VIEL Phage expressing an antigen binding domain that binds to an antigen of interest (e.g., a TGF- ⁇ - or TGF- ⁇ receptor polypeptide or a fragment thereof) can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • an antigen of interest e.g., a TGF- ⁇ - or TGF- ⁇ receptor polypeptide or a fragment thereof
  • phage display methods that can be used to make the antibodies include those disclosed in Brinkman et al., J. Immunol. Methods 752:41-50 (1995); Ames et al., J. Immunol. Methods 184: 177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 757:9-18 (1997); Burton et al., Advances in Immunology 57: 191-280 (1994); PCT Application No.
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria.
  • Fab, Fab 1 and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 72 ⁇ :864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240: 1041 -1043 (1988) (said references incorporated by reference in their entireties).
  • a chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.
  • Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, Science 229: 1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods 725:191-202 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entireties.
  • Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non- human species and framework regions from a human immunoglobulin molecule.
  • CDRs complementarity determining regions
  • framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
  • These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No.
  • Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et ah, Protein Engineering 7 ⁇ :805-814 (1994); Roguska.
  • Completely human antibodies are particularly desirable for therapeutic treatment of human patients.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.
  • Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
  • the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells.
  • the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes.
  • the mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production.
  • the modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice.
  • the chimeric mice are then bred to produce homozygous offspring that express human antibodies.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a desired target polypeptide.
  • Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B-cell differentiation, and subsequently undergo class switching and somatic mutation.
  • Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection.”
  • a selected non-human monoclonal antibody e.g., a mouse antibody
  • a completely human antibody recognizing the same epitope e.g., a mouse antibody
  • DNA encoding desired monoclonal antibodies may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the isolated and subcloned hybridoma cells serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells or myeloma cells that do not otherwise produce immunoglobulins.
  • prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells or myeloma cells that do not otherwise produce immunoglobulins.
  • the isolated DNA (which may be synthetic as described herein) may be used to clone constant and variable region sequences for the manufacture antibodies as described in Newman et al., U.S. Pat. No. 5,658,570, filed January 25, 1995, which is incorporated by reference herein.
  • RNA from the selected cells conversion to cDNA, and amplification by PCR using Ig specific primers. Suitable primers for this purpose are also described in U.S. Pat. No. 5,658,570.
  • transformed cells expressing the desired antibody may be grown up in relatively large quantities to provide clinical and commercial supplies of the immunoglobulin.
  • the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability.
  • CDRs complementarity determining regions
  • one or more of the CDRs may be inserted within framework regions, e.g. , into human framework regions to humanize a non-human antibody.
  • the framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. MoI. Biol.
  • the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds to at least one epitope of a desired polypeptide, e.g., TGF- ⁇ - or TGF- ⁇ receptor.
  • a desired polypeptide e.g., TGF- ⁇ - or TGF- ⁇ receptor.
  • one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen.
  • such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds.
  • Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region, e.g., humanized antibodies.
  • lymphocytes can be selected by micromanipulation and the variable genes isolated.
  • peripheral blood mononuclear cells can be isolated from an immunized mammal and cultured for about 7 days in vitro. The cultures can be screened for specific IgGs that meet the screening criteria. Cells from positive wells can be isolated.
  • Individual Ig-producing B cells can be isolated by FACS or by identifying them in a complement-mediated hemolytic plaque assay.
  • Ig- producing B cells can be micromanipulated into a tube and the V H and V L genes can be amplified using, e.g., RT-PCR.
  • the V H and V L genes can be cloned into an antibody expression vector and transfected into cells (e.g., eukaryotic or prokaryotic cells) for expression.
  • antibody-producing cell lines may be selected and cultured using techniques well known to the skilled artisan. Such techniques are described in a variety of laboratory manuals and primary publications. In this respect, techniques suitable for use in the invention as described below are described in Current Protocols in Immunology, Coligan et al., Eds., Green Publishing Associates and Wiley- Interscience, John Wiley and Sons, New York (1991) which is herein incorporated by reference in its entirety, including supplements.
  • Antibodies for use in the diagnostic and therapeutic methods disclosed herein can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques as described herein.
  • RNA may be isolated from the original hybridoma cells or from other transformed cells by standard techniques, such as guanidinium isothiocyanate extraction and precipitation followed by centrifugation or chromatography. Where desirable, mRNA may be isolated from total RNA by standard techniques such as chromatography on oligo dT cellulose. Suitable techniques are familiar in the art.
  • cDNAs that encode the light and the heavy chains of the antibody may be made, either simultaneously or separately, using reverse transcriptase and DNA polymerase in accordance with well known methods.
  • PCR may be initiated by consensus constant region primers or by more specific primers based on the published heavy and light chain DNA and amino acid sequences.
  • PCR also may be used to isolate DNA clones encoding the antibody light and heavy chains. In this case the libraries may be screened by consensus primers or larger homologous probes, such as mouse constant region probes.
  • modified antibodies for use in the methods disclosed herein are minibodies.
  • Minibodies can be made using methods described in the art (see, e.g., see e.g., US patent 5,837,821 or WO 94/09817Al).
  • the present invention also provides the use of antibodies that comprise, consist essentially of, or consist of, variants (including derivatives) of antibody molecules (e.g., the V H regions and/or V L regions) described herein, which antibodies or fragments thereof immunospecifically bind to a TGF- ⁇ - or or to a TGF- ⁇ receptor polypeptide or fragment or variant thereof.
  • Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a binding molecule, including, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis which result in amino acid substitutions.
  • the variants encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the reference V H region, V H CDR1, V H CDR2, V H CDR3, V L region, V L CDR1, V L CDR2, or V L CDR3.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge.
  • Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains ( e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e
  • mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity (e.g., the ability to bind a TGF- ⁇ - or TGF- ⁇ receptor polypeptide).
  • mutations only in framework regions or only in CDR regions of an antibody molecule.
  • Introduced mutations may be silent or neutral missense mutations, i.e., have no, or little, effect on an antibody's ability to bind antigen. These types of mutations may be useful to optimize codon usage, or improve a hybridoma's antibody production.
  • non-neutral missense mutations may alter an antibody's ability to bind antigen. The location of most silent and neutral missense mutations is likely to be in the framework regions, while the location of most non-neutral missense mutations is likely to be in CDR, though this is not an absolute requirement.
  • the encoded protein may routinely be expressed and the functional and/or biological activity of the encoded protein, (e.g., ability to immunospecifically bind at least one epitope of a TGF- ⁇ - or TGF- ⁇ receptor polypeptide) can be determined using techniques described herein or by routinely modifying techniques known in the art.
  • Recombinant expression of a TGF- ⁇ - or TGF- ⁇ receptor-specific antibody, a soluble TGF- ⁇ receptor, or a fragment, derivative or analog thereof may be carried out via construction of an expression vector containing a polynucleotide that encodes the antibody or soluble receptor.
  • a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, a soluble receptor, or portion thereof has been obtained, the vector for the production of the molecule is typically produced by recombinant DNA technology using techniques well known in the art.
  • methods for preparing a protein by expressing a polynucleotide containing an antibody or soluble receptor encoding polynucleotide sequence are described herein.
  • the invention provides replicable vectors comprising a nucleotide sequence encoding at least the antigen-binding domains of an antibody molecule or a soluble receptor molecule of the invention, operably linked to a promoter.
  • the expression vector is transferred to a host cell by conventional techniques and the transformed, transduced, or transfected cells are then cultured by conventional techniques to produce an antibody or use in the methods described herein.
  • the invention includes host cells containing a polynucleotide encoding an antibody or soluble TGF- ⁇ receptor of the invention, or a heavy or light chain thereof, operably linked to a promoter.
  • vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule.
  • a variety of host-expression vector systems may be utilized to express TGF- ⁇ - or TGF- ⁇ receptor antibody or soluble TGF- ⁇ receptor molecules for use in the methods described herein.
  • Such host- expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed, transduced, or transfected with the appropriate nucleotide coding sequences, express a TGF- ⁇ - or TGF- ⁇ receptor antibody or soluble TGF- ⁇ receptor of the invention in situ.
  • These include but are not limited to microorganisms such as bacteria ( . e.g., E. coli, B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing coding sequences
  • yeast e.g., Saccharomyces, Pichi ⁇
  • insect cell systems infected with recombinant virus expression vectors e.g.
  • baculovirus containing coding sequences
  • plant cell systems infected with recombinant virus expression vectors e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV
  • recombinant plasmid expression vectors e.g., Ti plasmid
  • mammalian cell systems e.g., COS, CHO, BLK, 293, 3T3 cells harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
  • mammalian cells such as Chinese hamster ovary cells (CHO)
  • CHO Chinese hamster ovary cells
  • a vector such as the major intermediate early gene promoter element from human cytomegalovirus
  • TGF- ⁇ - or TGF- ⁇ receptor antibody or soluble TGF- ⁇ receptor a vector that promotes TGF- ⁇ receptor for TGF- ⁇ receptor.
  • a number of expression vectors may be advantageously selected depending upon the use intended for the molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of a TGF- ⁇ - or TGF- ⁇ receptor antibody or soluble TGF- ⁇ receptor molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is typically used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • mammalian host cells a number of viral-based expression systems may be utilized.
  • the TGF- ⁇ - or TGF- ⁇ receptor antibody or soluble TGF- ⁇ receptor coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non- essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts, (e.g., see Logan & Shenk, Proc. Natl. Acad.
  • Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 753:51-544 (1987)).
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • Such mammalian host cells include but are not limited to CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
  • TGF- ⁇ - or TGF- ⁇ receptor antibody or soluble TGF- ⁇ receptor molecule may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which stably express the TGF- ⁇ - or TGF- ⁇ receptor antibody or soluble TGF- ⁇ receptor molecule.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 77:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. ScL USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 1980) genes can be employed in tk-, hgprt- or aprt- cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. ScL USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. ScL USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad.
  • TGF- ⁇ - or TGF- ⁇ receptor antibody or soluble TGF- ⁇ receptor molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Academic Press, New York, Vol. 3. (1987)).
  • vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Academic Press, New York, Vol. 3. (1987)).
  • a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene.
  • the amplified region is associated with the TGF- ⁇ - or TGF- ⁇ receptor antibody or soluble TGF- ⁇ receptor molecule gene, production of the antibody will also increase (Crouse et al., MoI. Cell. Biol. 3:257 (1983)).
  • a host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
  • the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • a single vector may be used which encodes both heavy and light chain polypeptides.
  • the light chain is advantageously placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. ScL USA 77:2197 (1980)).
  • the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
  • TGF- ⁇ - or TGF- ⁇ receptor antibody or soluble TGF- ⁇ receptor molecule of the invention may be purified by any method known in the art for purification of a recombinant proteins, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • centrifugation e.g., centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • the invention includes at least one pharmaceutical composition comprising a vector, e.g. , a viral vector, e.g., an adenovirus vector, where the vector comprises a polynucleotide encoding an interferon polypeptide, e.g., an is interferon- ⁇ polypeptide, or a biologically or therapeutically active variant, fragment, or derivative thereof; a TGF- ⁇ inhibitor as described elsewhere herein, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is provided in a pharmaceutically effective amount.
  • pharmaceutically or pharmaceutically effective amount means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by a researcher or clinician. As described elsewhere herein, in the combination therapies of the present invention, each individual ingredient may be in an amount, which by itself, might not be “pharmaceutically effective.”
  • compositions form the active ingredients of a pharmaceutical composition, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier” materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like.
  • carrier suitable pharmaceutical diluents, excipients or carriers
  • the gene delivery vector which encodes an interferon polypeptide or biologically or therapeutically active fragment, variant, or derivative thereof may not be administered in the same carrier, or by the same route as the TGF- ⁇ inhibitor.
  • the compositions typically will include an effective amount of active compound or the pharmaceutically acceptable salt thereof,-and in addition, and may also include any carrier materials as are customarily used in the pharmaceutical sciences.
  • compositions may be in solid, semi-solid or liquid dosage form, such as, for example, injectables, tablets, suppositories, pills, time-release capsules, powders, liquids, suspensions, or the like, for example, in unit dosages.
  • Administration of the active compounds and salts described herein can be via any of the accepted modes of administration for therapeutic agents. These methods include systemic or local administration such as oral, nasal, parenteral, transdermal, subcutaneous, or topical administration modes.
  • systemic or local administration such as oral, nasal, parenteral, transdermal, subcutaneous, or topical administration modes.
  • the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
  • suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture.
  • Suitable binders include starch, magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, polyethylene glycol, waxes and the like.
  • Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol and the like.
  • Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum starches, alginic acid or its sodium salt, or effervescent mixtures, and the like.
  • Diluents include, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine.
  • the compounds of the invention can also be administered in such oral dosage forms as timed release and sustained release tablets or capsules, pills, powders, granules, elixers, tinctures, suspensions, syrups and emulsions.
  • Liquid, particularly injectable compositions can, for example, be prepared by dissolving, dispersing, etc.
  • the active compound is dissolved in or mixed with ,a pharmaceutically pure solvent such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form the injectable solution or suspension.
  • a pharmaceutically pure solvent such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like
  • solid forms suitable for dissolving in liquid prior to injection can be formulated.
  • Injectable compositions are, for example, aqueous isotonic solutions or suspensions.
  • the compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers.
  • the compounds of the present invention can be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
  • injectables can be prepared in conventional forms, either as liquid solutions or suspensions.
  • Parenteral injectable administration is generally used for subcutaneous, intramuscular or intravenous injections and infusions. Additionally, one approach for parenteral administration employs the implantation of a slow-release or sustained-released systems, which assures that a constant level of dosage is maintained, according to U.S. Pat. No. 3,710,795, incorporated herein by reference in its entirety.
  • certain compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art.
  • the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
  • other topical preparations include creams, ointments, lotions, aerosol sprays and gels, wherein the concentration of active ingredient would range from 0.1% to 15%, w/w or w/v.
  • excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like may be used.
  • the active compound defined above may be also formulated as suppositories using for example, polyalkylene glycols, for example, propylene glycol, as the carrier.
  • suppositories are advantageously prepared from fatty emulsions or suspensions.
  • Compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, containing cholesterol, stearylamine or phosphatidylchohnes.
  • a film of lipid components is hydrated with an aqueous solution of drug to a form lipid layer encapsulating the drug, as desc ⁇ bed in U.S. Pat. No. 5,262,564.
  • Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled.
  • the compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers
  • soluble polymers can include polyvinyl-pyrrohdone, pyran copolymer, polyhydroxypropyl-methacrylamide-phenol, polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues
  • the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example., polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
  • the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying ' agents, pH buffering agents, and other substances such as, for example, sodium acetate, t ⁇ ethanolamine oleate, etc
  • the dosage regimen utilizing the compounds is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient, the seventy of the condition to be treated, the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed
  • An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition
  • Compounds of the present invention may be administered in a single dose.
  • compounds of the invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily.
  • compounds of the invention may be administered over the course of several days or weeks Dosing regimens for administration of therapeutics are well known to persons skilled in the art
  • administration of the TGF- ⁇ inhibitor is commenced p ⁇ or to administering the interferon-encodmg vector.
  • administration of the TGF- ⁇ inhibitor may be initiated 1, 2, 3, or 4 days p ⁇ or to administering the interferon-encodmg vector, 24 hours or less p ⁇ or to administe ⁇ ng the interferon-encodmg vector, 6, 5, 4, 3, 2, or 1 hour p ⁇ or to admimstenng the lnterferon- encoding vector, one hour or less p ⁇ or to administe ⁇ ng the interferon-encodmg vector, or 30, 20, 10, or five minutes or less p ⁇ or to administe ⁇ ng the interferon-encodmg vector.
  • Administration of the TGF- ⁇ inhibitor can be a single dose, or administration may be m several doses or in a continuous fashion up to, du ⁇ ng, and concurrently with administration of the mterferon-encoding vector
  • administration of the TGF- ⁇ inhibitor may commence concurrently with administration of the vector.
  • administration of the interferon-encodmg vector may commence prior to administration of the TGF- ⁇ inhibitor. Dosing regimens and the sequence of administration may be easily determined by a person of ordinary skill in the art.
  • Any of the above pharmaceutical compositions may contain 0.1-99%, 1-70%, or 1-50% of the active compounds of the invention as active ingredients.
  • the compounds of the present invention may be administered with another therapeutic agent, as one or more pharmaceutical compositions.
  • the other therapeutic agent may be administered prior to, concurrently with or after the administration of the compounds of the present invention.
  • the other therapeutic agent may be, for example, a therapeutic agent known in the art for that particular indication.
  • An effective amount is the amount which is required to confer a therapeutic effect on the treated subject, e.g. a patient.
  • an effective amount can range from about 1 mg/kg to about 150 mg/kg (e.g., from about 1 mg/kg to about 100 mg/kg).
  • Effective doses will also vary, as recognized by those skilled in the art, dependent on route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatments including use of other therapeutic agents and/or radiation therapy.
  • the small molecule TGF ⁇ inhibitors can be administered by any method that permits the delivery of the compounds in accordance with the therapeutic methods described herein.
  • the small molecule TGF ⁇ inhibitors can be administered via pills, tablets, capsules, aerosols, suppositories, liquid formulations for ingestion or injection or for use as eye or ear drops, dietary supplements, and topical preparations.
  • a pharmaceutically acceptable composition includes the aqueous solution of the small molecule TGF- ⁇ inhibitor, in an isotonic saline, 5% glucose or another well-known pharmaceutically acceptable excipient.
  • Solubilizing agents such as cyclodextrins, or other solubilizing agents well-known to those familiar with the art, can be utilized as pharmaceutical excipients for delivery of the therapeutic compounds.
  • the compositions can be administered orally, parenterally, intranasally, transdermally, intradermally, vaginally, intraaurally, intraocularly, buccally, rectally, transmucosally, or via inhalation, or intravenous administration.
  • parenteral as used here includes subcutaneous, intravenous, intramuscular, and intra-arterial injections with a variety of infusion techniques. Intra-arterial and intravenous injection as used herein includes administration through catheters. Oral administration is generally preferred.
  • compositions containing the active ingredient may be in any form suitable for the intended method of administration.
  • tablets, pellets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs may be prepared.
  • Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents including sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a palatable preparation.
  • Tablets and pellets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipient which are suitable for manufacture of tablets are acceptable.
  • excipients may be, for example, inert diluents, such as calcium or sodium carbonate, lactose, calcium or sodium phosphate; granulating and disintegrating agents, such as maize starch, or alginic acid; binding agents, such as starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc.
  • Tablets and pellets may be uncoated or may be coated by known techniques including microencapsulation to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.
  • Formulations for oral use may be also presented as hard gelatin capsules where the active ingredient is mixed with an inert solid diluent, for example calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.
  • an inert solid diluent for example calcium phosphate or kaolin
  • an oil medium such as peanut oil, liquid paraffin or olive oil.
  • Aqueous suspensions of the compounds contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide ⁇ e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate).
  • a suspending agent such as sodium
  • the aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxy- benzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose or saccharin.
  • preservatives such as ethyl or n-propyl p-hydroxy- benzoate
  • coloring agents such as ethyl or n-propyl p-hydroxy- benzoate
  • flavoring agents such as sucrose or saccharin.
  • sweetening agents such as sucrose or saccharin.
  • Oil suspensions may be formulated by suspending the active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oral suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents, such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation.
  • These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.
  • Dispersible powders, pellets, and granules of the compounds suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives.
  • a dispersing or wetting agent e.g., sodium EDTA
  • suspending agent e.g., sodium EDTA
  • preservatives e.g., sodium bicarbonate, sodium bicarbonate
  • the pharmaceutical compositions may also be in the form of oil-in-water emulsions.
  • the oily phase may be a vegetable oil, such as olive oil or arachis oil, a mineral oil, such as liquid paraffin, or a mixture of these.
  • Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate.
  • the emulsion may also contain sweetening and flavoring agents.
  • Syrups and elixirs may be formulated with sweetening agents, such as glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a flavoring or a coloring agent.
  • the pharmaceutical compositions may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butane-diol or prepared as a lyophilized powder.
  • a non-toxic parenterally acceptable diluent or solvent such as a solution in 1,3-butane-diol or prepared as a lyophilized powder.
  • sterile fixed oils may conventionally be employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid may likewise be used in the preparation of injectables.
  • compositions can be administered to an animal (e.g., a mammal such as a human, non-human primate, horse, dog, cow, pig, sheep, goat, cat, mouse, rat, guinea pig, rabbit, hamster, gerbil, ferret, lizard, reptile, or bird).
  • an animal e.g., a mammal such as a human, non-human primate, horse, dog, cow, pig, sheep, goat, cat, mouse, rat, guinea pig, rabbit, hamster, gerbil, ferret, lizard, reptile, or bird.
  • formulations suitable for oral administration may be presented as discrete units such as capsules, cachets, pellets, or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be administered as a bolus, electuary or paste.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free flowing form such as a powder or granules, optionally mixed with a binder (e.g., povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. , sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) surface active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach. This is particularly advantageous when such compounds are susceptible to acid hydrolysis.
  • compositions comprising the compounds can be administered by controlled- or delayed-release means.
  • Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts.
  • the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to treat or control the condition in a minimum amount of time.
  • Controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction m local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions.
  • Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like.
  • controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels.
  • controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under dosing a drug (e.g., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug.
  • Controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.
  • a variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the compositions. Examples include, but are not limited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548;.5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 Bl; each of which is incorporated herein by reference.
  • dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS ® (Alza Corporation, Mountain View, Calif. USA)), multilayer coatings, microparticles, liposomes, or microspheres or a combination thereof to provide the desired release profile in varying proportions.
  • ion exchange materials can be used to prepare immobilized forms of the compositions and thus effect controlled delivery of the drug. Examples of specific anion exchangers include, but are not limited to, DUOLITE A568 and DUOLITE AP 143 (Rohm & Haas, Spring House, Pa. USA).
  • One embodiment oencompasses a unit dosage form which comprises a TGF ⁇ inhibitor or a pharmaceutically acceptable salt, or a polymorph, solvate, hydrate, dehydrate, co-crystal, anhydrous, or amorphous form thereof, and one or more pharmaceutically acceptable excipients or diluents, wherein the pharmaceutical composition or dosage form is formulated for controlled-release.
  • Specific dosage forms utilize an osmotic drug delivery system.
  • OROS Alza Corporation, Mountain View, Calif. USA
  • This technology can readily be adapted for the delivery of TGF ⁇ inhibitors.
  • Various aspects of the technology are disclosed in U.S. Pat. Nos. 6,375,978 Bl ; 6,368,626 B 1; 6,342,249 Bl; 6,333,050 B2; 6,287,295 Bl; 6,283,953 Bl; 6,270,787 Bl; 6,245,357 Bl; and 6,132,420; each of which is incorporated herein by reference.
  • OROS that can be used to administer compounds and compositions
  • OROS Push-Pull Delayed Push-Pull
  • Multi-Layer Push-Pull Multi-Layer Push-Pull
  • Push-Stick Systems All of which are well known.
  • Additional OROS systems that can be used for the controlled oral delivery include OROS-CT and L- OROS. Id.; see also, Delivery Times, vol. IL issue II (Alza Corporation).
  • OROS oral dosage forms are made by compressing a drug powder (e.g., a composition) into a hard tablet, coating the tablet with cellulose derivatives to form a semi-permeable membrane, and then drilling an orifice in the coating (e.g., with a laser).
  • a drug powder e.g., a composition
  • the advantage of such dosage forms is that the delivery rate of the drug is not influenced by physiological or experimental conditions. Even a drug with a pH-dependent solubility can be delivered at a constant rate regardless of the pH of the delivery medium. But because these advantages are provided by a build-up of osmotic pressure within the dosage form after administration, conventional OROS drug delivery systems cannot be used to effectively deliver drugs with low water solubility.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • the unit dosage formulations are those containing a daily dose or unit, daily sub- dose, or an appropriate fraction thereof, of a drug.
  • TGF- ⁇ - or TGF- ⁇ receptor-specific antibody e.g., a TGF- ⁇ - or TGF- ⁇ receptor-specific antibody, a soluble TGF- ⁇ receptor, or a fragment, derivative or analog thereof
  • the route of administration of the TGF- ⁇ - or TGF- ⁇ receptor-specific antibody, a soluble TGF- ⁇ receptor, or a fragment, derivative or analog thereof may be, for example, oral, parenteral, by inhalation or topical.
  • parenteral as used herein includes, e.g., intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. While all these forms of administration are clearly contemplated as being within the scope of the invention, a form for administration would be a solution for injection, in particular for intravenous or intraarterial injection or drip.
  • a suitable pharmaceutical composition for injection may comprise a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. human albumin), etc.
  • a TGF- ⁇ - or TGF- ⁇ receptor-specific antibody, a soluble TGF- ⁇ receptor, or a fragment, derivative or analog thereof can be delivered directly to the site of the adverse cellular population thereby increasing the exposure of the diseased tissue to the therapeutic agent.
  • Preparations for parenteral administration includes sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • pharmaceutically acceptable carriers include, but are not limited to, 0.01-0. IM and preferably 0.05M phosphate buffer or 0.8% saline.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the composition must be sterile and should be fluid to the extent that easy sy ⁇ ngabihty exists. It should be stable under the conditions of manufacture and storage and will preferably be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., 16th ed. (1980).
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like.
  • isotonic agents for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • sterile injectable solutions can be prepared by incorporating an active compound (e.g., a TGF- ⁇ - or TGF- ⁇ receptor-specific antibody, a soluble TGF- ⁇ receptor, or a fragment, derivative or analog thereof, by itself or in combination with other active agents) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization.
  • an active compound e.g., a TGF- ⁇ - or TGF- ⁇ receptor-specific antibody, a soluble TGF- ⁇ receptor, or a fragment, derivative or analog thereof, by itself or in combination with other active agents
  • dispe ⁇ sions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying, which yields a powder of an active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the preparations for injections are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art. Further, the preparations may be packaged and sold in the form of a kit such as those described in co-pending U.S.S.N. 09/259,337 (US-2002-0102208 Al), which is incorporated herein by reference in its entirety.
  • Such articles of manufacture will preferably have labels or package inserts indicating that the associated compositions are useful for treating a subject suffering from, or predisposed to autoimmune or neoplastic disorders.
  • compositions of the present invention for treatment of hyperproliferative disorders as described herein vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic.
  • the patient is a human but non-human mammals including transgenic mammals can also be treated.
  • Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.
  • the dosage can range, e.g., from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, lmg/kg, 2 mg/kg, etc.), of the host body weight.
  • dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg, preferably at least 1 mg/kg.
  • Doses intermediate in the above ranges are also intended to be within the scope of the invention.
  • Subjects can be administered such doses daily, on alternative days, weekly or according to any other schedule determined by empirical analysis.
  • An exemplary treatment entails administration in multiple dosages over a prolonged period, for example, of at least six months. Additional exemplary treatment regimes entail administration once per every two weeks or once a month or once every 3 to 6 months.
  • Exemplary dosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kg weekly.
  • two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated.
  • a TGF- ⁇ - or TGF- ⁇ receptor-specific antibody, a soluble TGF- ⁇ receptor, or a fragment, derivative or analog thereof for use in the diagnostic and treatment methods disclosed herein can be administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of target polypeptide or target molecule in the patient. In some methods, dosage is adjusted to achieve a plasma polypeptide concentration of 1-1000 ⁇ g/ml and in some methods 25-300 ⁇ g/ml. Alternatively, binding molecules can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient.
  • the half-life of a binding molecule can also be prolonged via fusion to a stable polypeptide or moeity, e.g., albumin or PEG.
  • a stable polypeptide or moeity e.g., albumin or PEG.
  • humanized antibodies show the longest half-life, followed by chimeric antibodies and nonhuman antibodies.
  • the binding molecules of the invention can be administered in unconjugated form,
  • a TGF- ⁇ - or TGF- ⁇ receptor-specific antibody, a soluble TGF- ⁇ receptor, or a fragment, derivative or analog thereof can be administered multiple times in conjugated form.
  • the binding molecules of the invention can be administered in unconjugated form, then in conjugated form, or vise versa.
  • compositions comprising antibodies or a cocktail thereof are administered to a patient not already in the disease state or in a pre-disease state to enhance the patient's resistance. Such an amount is defined to be a "prophylactic effective dose.”
  • prophylactic effective dose the precise amounts again depend upon the patient's state of health and general immunity, but generally range from 0.1 to 25 mg per dose, especially 0.5 to 2.5 mg per dose.
  • a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives.
  • a relatively high dosage e.g., from about 1 to 400 mg/kg of binding molecule, e.g., antibody per dose, with dosages of from 5 to 25 mg being more commonly used for radioimmunoconjugates and higher doses for cytotoxin-drug conjugated molecules
  • the patent can be administered a prophylactic regime.
  • a TGF- ⁇ - or TGF- ⁇ receptor-specific antibody, a soluble TGF- ⁇ receptor, or a fragment, derivative or analog thereof can optionally be administered in combination with other agents that are effective in treating the disorder or condition in need of treatment (e.g. , prophylactic or therapeutic).
  • an interferon-encoding gene delivery vector in combination with a TGF- ⁇ inhibitor of the invention may be used in a combined therapeutic regimen with chemotherapeutic agents.
  • Such therapeutic regimens may comprise the sequential, simultaneous, concurrent or coextensive administration of the disclosed antibodies or other binding molecules and one or more chemotherapeutic agents.
  • Particularly preferred embodiments of this aspect of the invention will comprise the administration of a radiolabeled binding polypeptide.
  • the administration or application of the various components of the combined therapeutic regimen may be timed to enhance the overall effectiveness of the treatment.
  • chemotherapeutic agents could be administered in standard, well known courses of treatment followed within a few weeks by the therapy described herein.
  • the therapy described herein could be administered intravenously followed by tumor localized external beam radiation.
  • binding molecules may be administered concurrently with one or more selected chemotherapeutic agents in a single office visit.
  • a skilled artisan e.g. an experienced oncologist
  • chemotherapeutic agents that are compatible with the instant invention include alkylating agents, vinca alkaloids (e.g., vincristine and vinblastine), procarbazine, methotrexate and prednisone.
  • the four-drug combination MOPP (mechlethamine (nitrogen mustard), vincristine (Oncovin), procarbazine and prednisone) is very effective in treating various types of lymphoma and comprises a preferred embodiment of the present invention.
  • ABVD e.g., adriamycin, bleomycin, vinblastine and dacarbazine
  • ChIVPP chlorambucil, vinblastine, procarbazine and prednisone
  • CABS lastine, doxorubicin, bleomycin and streptozotocin
  • MOPP plus ABVD MOPP plus ABV (doxorubicin, bleomycin and vinblastine) or BCVPP (carmustine, cyclophosphamide, vinblastine, procarbazine and prednisone) combinations
  • Additional regimens that are useful in the context of the present invention include use of folate inhibitors, such as methotrexate, Alimta, or raletrexed, nucleoside inhibitors such as gemcitabine, single alkylating agents such as cyclophosphamide or chlorambucil, or combinations such as CVP (cyclophosphamide, vincristine and prednisone), CHOP (CVP and doxorubicin), C-MOPP (cyclophosphamide, vincristine, prednisone and procarbazine), CAP-BOP (CHOP plus procarbazine and bleomycin), m-BACOD (CHOP plus methotrexate, bleomycin and leucovorin), ProMACE-MOPP (prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide and leucovorin plus Standard MOPP), ProMACE-Cyta
  • CHOP has also been combined with bleomycin, methotrexate, procarbazine, nitrogen mustard, cytosine arabinoside and etoposide.
  • Other compatible chemotherapeutic agents include, but are not limited to, 2- chlorodeoxyadenosine (2-CDA), 2'-deoxycoformycin and fludarabine.
  • Salvage therapies employ drugs such as cytosine arabinoside, cisplatin, etoposide and ifosfamide given alone or in combination.
  • IMVP- 16 ifosfamide, methotrexate and etoposide
  • MIME methyl-gag, ifosfamide, methotrexate and etoposide
  • DHAP dexamethasone, high dose cytarabine and cisplatin
  • ESHAP etoposide, methylpredisolone, HD cytarabine, cisplatin
  • CEPP(B) cyclophosphamide, etoposide, procarbazine, prednisone and bleomycin
  • CAMP lomustine, mitoxantrone, cytarabine and prednisone
  • chemotherapeutic agent used in combination with the therapies disclosed herein may vary by subject or may be administered according to what is known in the art. See for example, Bruce A Chabner et ai, Antineoplastic Agents, in Goodman & Gilman's The Pharmacological Basis of Therapeutics 1233-1287 ((Joel G. Hardman et al, eds., 9 th ed. (1996).
  • kits comprising, for example a polynucleotide encoding an interferon polypeptide or a biologically or therapeutically active fragment, variant, or derivative thereof, and a TGF- ⁇ inhibitor, e.g., a small molecule inhibitor, a TGF- ⁇ - or TGF- ⁇ receptor antibody, a soluble TGF- ⁇ receptor, or a fragment, variant, derivative, or analog thereof that interrupts, inhibits blocks or causes a reduction in TGF- ⁇ signalling; and an instruction manual for use.
  • a TGF- ⁇ inhibitor e.g., a small molecule inhibitor, a TGF- ⁇ - or TGF- ⁇ receptor antibody, a soluble TGF- ⁇ receptor, or a fragment, variant, derivative, or analog thereof that interrupts, inhibits blocks or causes a reduction in TGF- ⁇ signalling
  • TGF- ⁇ inhibitor e.g., a small molecule inhibitor, a TGF- ⁇ - or TGF- ⁇ receptor antibody, a soluble TGF- ⁇ receptor,
  • the polynucleotide can be a viral polynucleotide, e.g., an adenovirus polynucleotide, and can be provided as part of a viral particle, e.g., if desired.
  • Kits of the present invention may have the various components packaged in various ways, e.g., for simultaneous administration or for sequential administration.
  • mice were inoculated on the flanks with AB 12 (mesothelioma) and LKR (lung cancer) cells respectively, and tumors were allowed to grow to large size.
  • Inhibition of TGF- ⁇ signaling in vivo was determined by measuring tumor phospho-Smad2 following the treatment of tumor-bearing mice with a single dose (0.45 mg/kg i.p.) of the small molecule TGF- ⁇ type I receptor kinase inhibitor, 4-[4- benzo[l,3]dioxol-5-yl-5-(6-methyl-pyridin-2-yl)-lH-imidazol-2-yl]-bicyclo[2.2.2]octane-l-carboxylic acid amide:
  • SM16 SM16
  • the changes in the tumor microenvironment were studied by harvesting tumors from SM16-treated mice and measuring mRNA expression levels for various cytokines/chemokines using real-time reverse transcription PCR.
  • the effect of combining oral SM16 with Ad-INF ⁇ therapy was studied by treating the tumor-bearing mice with the SM16 in chow and injecting Ad-INF ⁇ i.p. and measuring the tumor volume periodically.
  • the El and E3 deleted adenovirus H5.110CMVhIFN- ⁇ encodes human IFN- ⁇ ("AdhlFN- ⁇ "), which is driven by the cytomegalovirus (CMV) early promoter.
  • CMV cytomegalovirus
  • the virus preparations were highly purified by two rounds of cesium chloride banding and particle titers were determined as previously described. See, e.g. , Nyberg-Hoffman, et al., Nat. Med. 3: 808-811 (1997); Chardonnet and Dales, Virology 40: 462-477 (1970).
  • SM 16 inhibits TGF- ⁇ signaling by abolishing Smad2 phosphorylation in AB 12 tumors in vivo
  • AB 12 tumors were injected with SM16 (20 mg/kg) i.p., and tumors were harvested 1 and 3 hours post injection. Immunoblotting was then performed on the tumor extracts. AB 12 tumors showed constitutively elevated phospho-Smad2 levels. However, a single i.p. bolus of 20mg/kg SM16 was able to abolish phospho-Smad 2 in vivo for up to 3 hours (data not shown).
  • SMl 6 changes the tumor microenvironment by increasing the mRNA levels of immunostimulatory cytokines/chemokines
  • LKR tumors from control and SM16-treated mice were harvested 3 days after SM16 treatment given by chow at 0.45 mg/kg chow. RNA was isolated, and real-time reverse transcription PCR was performed. Tumors from the SM16-treated mice showed increased expression levels of mRNA for immunostimulatory cytokines/chemokines (Table 5). For instance, there was a 22 fold increase in the IL- 12 expression compared to the control tumor. There were modest elevations in message levels for IP-IO, RANTES, TNF-alpha, MIG, INF-gamma (Table 5), while the message for the immunosuppressive enzyme Arginase decreased by 45% (Table 6).
  • TGF- ⁇ signaling with either TGF- ⁇ antibody or SM16 causes tumor growth retardation in AB
  • mice were inoculated with AB 12 and LKR tumors, respectively, on day 0. Once the tumors reached the average size of 200mm 3 , the mice were started on SMl 6 chow [0.45 mg/kg of chow] or anti-TGF- ⁇ antibody (5mg/kg i.p. q 3 days) The tumors were measured every 3-4 days. The data indicated that both SM16 and anti-TGF- ⁇ antibody were effective in retarding tumor growth in both AB 12 and LKR tumors.
  • mice with LKR tumors there were approximately 7 and 3.5 fold decrease in the tumor burden in the SM16 (.45 mg/kg) treated mice and anti-TGF- ⁇ treated mice compared to the control mice respectively.
  • the growth of AB 12 tumors were significantly retarded by various oral doses of SM16 (see Fig. 2) and anti-TGF- ⁇ antibody as well (not shown).
  • the El and E3 deleted adenovirus H5.110CMVhIFN- ⁇ encodes human IFN- ⁇ ("AdhlFN- ⁇ "), which is driven by the cytomegalovirus (CMV) early promoter.
  • CMV cytomegalovirus
  • the virus preparations were highly purified by two rounds of cesium chloride banding and particle titers were determined as previously described. See, e.g., Nyberg-Hoffman, et at., Nat. Med. 3: 808-811 (1997); Chardonnet and Dales, Virology 40: 462-477 (1970).
  • mice were inoculated on the flanks with one million AB 12 (mesothelioma) and LKR (lung cancer) cells ,and tumors were allowed to grow to large size.
  • the mice were started on SM16 chow (0.45 mg/kg of chow) 22-25 days after cell injection when the tumors were about 400 mm 3 .
  • the mice were treated with Ad INF ⁇ (10 9 pfu per mouse, one injection, intratumorally.).
  • Both LKR and AB 12 tumor-bearing mice treated with combination SMl 6 and Ad-INF ⁇ showed statistically significant decreases in tumor volume compared to the mice treated with SM16 or Ad-INF ⁇ alone ( Figures 1 and 2).

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Abstract

L'invention concerne des procédés de traitement du cancer comprenant l'administration à un sujet nécessitant le traitement, une multithérapie comprenant (a) l'administration d'un inhibiteur TGF-β et (b) un vecteur comprenant un polynucléotide isolé qui code un polypeptide interféron.
PCT/US2007/016880 2006-07-28 2007-07-27 Traitement du cancer par administration de gène interféron en association avec un inhibiteur tgf-beta Ceased WO2008013928A2 (fr)

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US9233956B2 (en) 2008-05-06 2016-01-12 Novartis Ag Benzene sulfonamide thiazole and oxazole compounds
WO2010089443A2 (fr) 2009-02-05 2010-08-12 Digna Biotech,S.L. FORMULATIONS PHARMACEUTIQUES DE PEPTIDES INHIBITEURS DE TGF- β1
CN102307598A (zh) * 2009-02-05 2012-01-04 迪格纳生物技术公司 TGF-β1抑制肽的药物制剂
CN103119066A (zh) * 2010-08-30 2013-05-22 独立行政法人理化学研究所 具有抑制TGF-β受体活化的活性的化合物、该化合物的筛选方法、以及用于预防或治疗由丙型肝炎病毒引起的疾病的组合物
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US8951521B2 (en) 2010-08-30 2015-02-10 Riken Compounds having activity of suppressing activation of TGF-β receptor, method for screening of the compounds, and composition for preventing or treating disease caused by hepatitis C virus
CN103119066B (zh) * 2010-08-30 2016-04-20 独立行政法人理化学研究所 具有抑制TGF-β受体活化的活性的化合物、该化合物的筛选方法、以及用于预防或治疗由丙型肝炎病毒引起的疾病的组合物
WO2022165313A1 (fr) 2021-02-01 2022-08-04 Regenxbio Inc. Thérapie génique de céroïdes-lipofuscinoses neuronales

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