WO2007090872A2 - Agents de liaison de la proteine-4 apparentee a frizzled (sfrp-4) - Google Patents

Agents de liaison de la proteine-4 apparentee a frizzled (sfrp-4) Download PDF

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WO2007090872A2
WO2007090872A2 PCT/EP2007/051229 EP2007051229W WO2007090872A2 WO 2007090872 A2 WO2007090872 A2 WO 2007090872A2 EP 2007051229 W EP2007051229 W EP 2007051229W WO 2007090872 A2 WO2007090872 A2 WO 2007090872A2
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sfrp
polypeptide
antibody
disease
cancer
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PCT/EP2007/051229
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WO2007090872A3 (fr
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Annette Zaar
Rainer Hillenbrand
Alessandra Vitaliti
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Novartis Ag
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Priority to JP2008553767A priority Critical patent/JP2009526020A/ja
Priority to AU2007213655A priority patent/AU2007213655A1/en
Priority to CA002637775A priority patent/CA2637775A1/fr
Priority to EP07712189A priority patent/EP1991582A2/fr
Priority to BRPI0710442-1A priority patent/BRPI0710442A2/pt
Publication of WO2007090872A2 publication Critical patent/WO2007090872A2/fr
Publication of WO2007090872A3 publication Critical patent/WO2007090872A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • This invention relates generally to immunoglobulins, antibodies and fragments thereof.
  • the present invention relates to the preparation and use of antibodies to secreted frizzled related protein type 4 (SFRP-4)-binding agents.
  • SFRP-4 frizzled related protein type 4
  • the Wnt gene family encodes proteins that serve key roles in differentiation and development. Wnt proteins interact with seven transmembrane receptors of the Frizzled family and activate a signalling pathway leading to the nucleus. Dennis et al, J. Cell Sci., 112(21): 3815-20 (1999). A primary biochemical effect of Wnt- 1 signalling is the stabilization of cytoplasmic (beta)-catenin which, in association with transcription factors of the Lef/tcf family, regulates gene expression.
  • SFRP secreted frizzled-related proteins
  • SFRP-4 is a soluble extracellular glycoprotein that antagonizes the signalling function of the Wnt/frizzled pathway by binding to either Wnts or frizzled receptors.
  • SFRP-4 is expressed in brain, kidney, lung, ovary, prostate, mammary gland and endometrium and shows complex functions with respect to cell survival. Jones et al., Bioessays 24: 811-20 (2002).
  • SFRP-4 has been suggested to be involved in bone metabolism (Fujita et al., Geriat. and Gerontol. Intern.
  • SFRP-4 has been considered as a potential biomarker (i.e., biological marker) in the field of cancer, diseases of hormonally- regulated reproductive tissues and neurodegeneration. Accordingly, there is a need in the art for additional agents useful to target (e.g., bind) and/or modulate SFRP-4 polypeptide.
  • the invention provides an agent useful to target and/or modulate SFRP-4 polypeptide.
  • the invention provides an antibody composition that binds immunospecifically to a SFRP-4 polypeptide selected from the group consisting of: SEQ ID NO: 3; SEQ ID NO: 4; and SEQ ID NO: 5; and binds immunospecifically to a polypeptide of SEQ ID NO: 2.
  • the antibody may be a polyclonal antibody; a monoclonal antibody; a chimaeric antibody; a humanized antibody, and an antibody-related polypeptide.
  • the invention also provides methods of making and using the antibody of the invention.
  • the invention therefore provides a pharmaceutical composition containing the antibody of the invention in a pharmaceutically-acceptable carrier.
  • the invention provides an isolated nucleic acid encoding an antibody or fragment thereof that binds immunospecifically to an SFRP-4 polypeptide.
  • the invention also provides a vector and a host cell containing the nucleic acid of the invention.
  • the invention further provides a method of treating or preventing a secreted frizzled related polypeptide type 4-associated disorder.
  • the disorder may be brain cancer; breast cancer; prostate cancer; uterine cancer; cancer of the spleen; pancreatic cancer; colon cancer; rectal cancer; cancer of the small intestine; stomach cancer; oesophageal cancer; apoptosis resulting from ischemia, heart failure; myocardial infarction; stroke; neurodegenerative disorder; Huntington's Disease; peripheral demyelinating disease; Multiple Sclerosis; Alzheimer's Disease; Amyotrophic Lateral Sclerosis; Parkinson ' s Disease; trauma; coronary heart disease; inflammation; or inflammatory bowel disease.
  • the invention provides a method of modulating the expression or activity of a polypeptide by administering the antibody of the invention.
  • the invention provides for the use of an antibody composition for the manufacture of a medicament for the treatment of a secreted frizzled related polypeptide type 4-associated disorder.
  • FIG. 1 shows Western blot analysis of His-tagged SFRP-4 Polypeptide.
  • Panel A shows immunoreactive His-tagged SFRP-4 polypeptide detected in 10 ⁇ l culture supernatant after 48 h (left lane) and 144 h (right lane) of culture.
  • Panel B shows the effect of deglycoslyation treatment of His-tagged SFRP-4-containing culture supernatant (48 h sample).
  • Lane 1 SFRP-4 immunoreactive protein observed in His-tagged SFRP-4-containing culture supernatant (48 h sample); Lane 2: SFRP-4 immunoreactive protein observed in mock-treated His-tagged SFRP-4-containing culture supernatant (48 h sample); Lane 3: SFRP-4 immunoreactive protein observed in PNGase treated His-tagged SFRP-4-containing culture supernatant (48 h sample).
  • FIG. 2 is a graph showing an SFRP-4 ELISA standard curve.
  • FIG. 3 is a picture of a High Throughput Western Blot with a chart showing the size of the spots in the tissues.
  • the tissues assayed are as follows: 1 , placenta; 2, empty lane; 3, adipose; 4, bladder; 5, brain; 6, breast: 7, cerebellum; 8, cervix; 9, colon; 10, diaphragm; 11, duodenum; 12, oesophagus; 13, gall bladder; 14, heart; 15, ileum; 16, jejunum; 17, kidney; 18, liver; 19, lung; 20, ovary; 21, pancreas; 22, placenta; 23, rectum; 24, skeletal muscle; 25, skin: 26, spleen; 27, stomach; 28, testis; 29, thymus; 30, thyroid; 31, tonsil; 32, uterus.
  • SFRP-4 secreted frizzled related protein-4" encompasses naturally occurring SFRP-4, as well as synthetic or recombinant SFRP-4. Further, the term “secreted frizzled related protein-4" encompasses allelic variants, species variants, splice variants and conserved amino acid substitution variants. Expression of SFRP-4 gene expression gives rise to SFRP-4 splice variants in mammals. Yam et al. Gene PMID: 16005582 (Epub ahead of print, July 7, 2005). The term also encompasses full length SFRP-4 as well as SFRP-4 fragments. SFRP-4 includes, but is not limited to, human SFRP-4 and murine SFRP-4.
  • the administration of an agent or drug to a subject or subject includes self-administration and the administration by another. It is also to be appreciated that the various modes of treatment or prevention of medical conditions as described are intended to mean “substantial”, which includes total but also less than total treatment or prevention, and wherein some biologically or medically-relevant result is achieved.
  • amino acid includes naturally-occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally-occurring amino acids.
  • Naturally-occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally-occurring amino acid, i.e., an ⁇ -carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g.
  • amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally- occurring amino acid. Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB
  • antibody means a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen, e.g., SFRP-4 polypeptide.
  • an antigen e.g., SFRP-4 polypeptide.
  • Use of the term antibody is meant to include whole antibodies, including single-chain whole antibodies, and antigen-binding fragments thereof.
  • antibody-related polypeptide means antigen- binding antibody fragments, including single-chain antibodies, which can comprise the variable regions alone, or in combination, with all or part of the following polypeptide elements: hinge region, CHi, CH 2 , and CH 3 domains of an antibody molecule. Also included in the invention are any combinations of variable regions and hinge region, CHj, CH 2 , and CH 3 domains.
  • Antibody-related molecules useful as binding agents of the invention include, e.g., but are not limited to, Fab, Fab' and F(ab') 2 , Fd, single-chain Fvs (scFv), single-chain antibodies, disulphide-linked Fvs (sdFv) and fragments comprising either a V L or V H domain.
  • Examples include: (i) a Fab fragment, a monovalent fragment consisting of the V L5 V H , C L and CHj domains; (ii) a F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulphide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and CHi domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a dAb fragment (Ward el ah, Nature 341 : 544-546, 1989), which consists of a
  • V H domain V H domain
  • CDR complementarity determining region
  • biological sample means sample material derived from or contacted by living cells.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.
  • CDR-grafted antibody means an antibody in which at least one CDR of an "acceptor” antibody is replaced by a CDR "graft” from a “donor” antibody possessing desirable antigen specificity.
  • chimaeric antibody means an antibody in which the Fc constant region of a monoclonal antibody from one species (e.g., a mouse Fc constant region) is replaced, using recombinant DNA techniques, with an Fc constant region from an antibody of another species (e.g., a human Fc constant region). See generally, Robinson et ⁇ l.,
  • the term ''comparison window' means a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600 amino acids or nucleotides, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • the term "consensus FR” means a framework (FR) antibody region in a consensus immunoglobulin sequence. The FR regions of an antibody do not contact the antigen.
  • the term "consensus sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). That is, in a family of proteins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence.
  • the term "contacted" when applied to a cell refers to the process by which an SFRP-4-binding agent of the present invention (e.g., antibody, antibody composition, cytotoxic agent or moiety, gene, protein, and/or antisense sequence) is delivered to a target cell or is placed in direct proximity with the target cell.
  • an SFRP-4-binding agent of the present invention e.g., antibody, antibody composition, cytotoxic agent or moiety, gene, protein, and/or antisense sequence
  • This delivery can be in vitro or in vivo and can involve the use of a recombinant vector system.
  • cytotoxic moiety means a moiety that inhibits cell growth or promotes cell death when proximate to or absorbed by a cell.
  • the cytotoxic moiety can be, by way of non-limiting example, a chemotherapeutic agent, a photoactivated toxin or a radioactive agent.
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (V L ) in the same polypeptide chain (V H V L ).
  • VH heavy-chain variable domain
  • V L light-chain variable domain
  • the domains are forced to pair with the complementary domains of another chain and create two antigen binding sites.
  • Diabodies are described more fully in, e.g., EP 404,097; WO 93/11161; and Hollinger et al, Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).
  • effector cell means an immune cell which is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response.
  • exemplary immune cells include a cell of a myeloid or lymphoid origin, e.g., lymphocytes (e.g., B cells and T cells including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils. Effector cells express specific Fc receptors and carry out specific immune functions.
  • lymphocytes e.g., B cells and T cells including cytolytic T cells (CTLs)
  • CTLs cytolytic T cells
  • killer cells e.g., natural killer cells
  • macrophages e.g., monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils
  • An effector cell can induce antibody-dependent cell- mediated cytotoxicity (ADCC), e.g., a neutrophil capable of inducing ADCC.
  • ADCC antibody-dependent cell- mediated cytotoxicity
  • monocytes, macrophages, neutrophils, eosinophils, and lymphocytes which express Fc ⁇ R are involved in specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens.
  • An effector cell can also phagocytose a target antigen, target cell, metastatic cancer cell, or microorganism.
  • epipe means a protein determinant capable of specific binding to an antibody.
  • Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconforniational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • the term "effective amount" of a composition is a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g. , an amount which results in the prevention of or a decrease in the symptoms associated with a disease that is being treated, e.g., the diseases associated with target polypeptide listed herein.
  • compositions of the invention administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • the compositions of the present invention can also be administered in combination with each other, or with one or more additional therapeutic compounds.
  • expression * includes but is not limited to one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.
  • a "fusion polypeptide” comprises an SFRP-4 polypeptide operatively- liiiked to a polypeptide having an amino acid sequence corresponding to a polypeptide that is not substantially homologous to the SFRP-4 polypeptide, e.g., a polypeptide that is different from the SFRP-4 polypeptide and that is derived from the same or a different organism.
  • the term '"gene means a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.
  • genotype means an unphased 5" to 3' sequence of nucleotide pairs found at one or more polymorphic or mutant sites in a locus on a pair of homologous chromosomes in an individual.
  • genotype includes a full- genotype and/or a sub-genotype.
  • human sequence antibody includes antibodies having variable and constant regions (if present) derived from human germline immunoglobulin sequences.
  • the human sequence antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g. , mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • Such antibodies can be generated in non-human transgenic animals, e.g., as described in PCT Publication Nos. WO 01/14424 and WO 00/37504.
  • human sequence antibody is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (e.g., humanized antibodies).
  • nucleic acids or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e..
  • sequences that have deletions and/or additions, as well as those that have substitutions can account for gaps and the like.
  • identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.
  • an "isolated” or “purified” polypeptide or biologically-active portion thereof is substantially free of cellular material or other contaminating polypeptides from the cell or tissue source from which the SFRP-4-binding agent is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • an isolated SFRP-4-binding agent which is an anti-SFRP-4 antibody would be free of materials that would interfere with diagnostic or therapeutic uses of the agent.
  • Such interfering materials may include enzymes, hormones and other proteinaceous and nonproteinaceous solutes.
  • intact antibody means an antibody that has at least two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulphide bonds.
  • the term "immune response” refers to the concerted action of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of cancerous cells, metastatic tumour cells, malignant melanoma, invading pathogens, cells or tissues infected with pathogens, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
  • the terms "immunologically cross-reactive” and “immunologically reactive " ' are used interchangeably to mean an antigen which is specifically reactive with an antibody which was generated using the same (“immunologically reactive") or different (“immunologically cross-reactive") antigen.
  • the antigen is SFRP-4, a variant or subsequence thereof.
  • immunologically reactive conditions' * means conditions which allow an antibody, generated to a particular epitope of an antigen, to bind to that epitope to a detectably greater degree than the antibody binds to substantially all other epitopes, generally at least two times above background binding, preferably at least five times above background.
  • Immunologically reactive conditions are dependent upon the format of the antibody binding reaction and typically are those utilized in immunoassay protocols. See, Harlow & Lane, Antibodies, A Laboratory Manual (Cold Spring Harbor Publications, New York, 1988) for a description of immunoassay formats and conditions.
  • lymphocyte means any of the mononuclear, nonphagocytic leukocytes, found in the blood, lymph, and lymphoid tissues, e.g., B and T lymphocytes.
  • lymphoid tissues e.g., B and T lymphocytes.
  • medical condition includes, but is not limited to, any condition or disease manifested as one or more physical and/or psychological symptoms for which treatment and/or prevention is desirable, and includes previously and newly identified diseases and other disorders.
  • the term "modulator” includes inhibitors and activators.
  • Inhibitors are agents that, e.g. , bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of an SFRP-4 polypeptide, e.g., antagonists.
  • Activators are agents that, e.g., bind to, stimulate, increase, open, activate, facilitate, enhance activation, sensitize or up regulate the activity of an SFRP-4 polypeptide, e.g., agonists.
  • Modulators include agents that, e.g., alter the interaction of an SFRP-4 polypeptide with: proteins that bind activators or inhibitors, receptors, including proteins, peptides, lipids, carbohydrates, polysaccharides, or combinations of the above, e.g., lipoproteins, glycoproteins, and the like.
  • Modulators include genetically modified versions of a naturally-occurring SFRP-4 polypeptide, e.g., with altered activity, as well as naturally- occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like.
  • the term "monoclonal antibody”' means 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.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including, e.g., but not limited to, hybridoma, recombinant, and phage display technologies.
  • neutralizing antibody means an antibody molecule that is able to eliminate or significantly reduce at least one (1) biological function of an SFRP-4 polypeptide or an SFRP-4-like polypeptide.
  • nucleotide pair means the two nucleotides bound to each other between the two nucleotide strands.
  • pharmaceutically-acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, compatible with pharmaceutical administration.
  • polyclonal antibody means a preparation of antibodies derived from at least two different antibody producing cell lines. The use of this term includes preparations at least two antibodies that contain antibodies that specifically bind to different epitopes or regions of an antigen.
  • RNA or DNA means any RNA or DNA, which may be unmodified or modified RNA or DNA.
  • polypeptide As used herein, the terms "polypeptide,” “peptide,” and “protein” are used interchangeably herein to mean a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres.
  • Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and to longer chains, generally referred to as proteins.
  • Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
  • Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that 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.
  • the term "recombinant" when used with reference, e.g. , to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the material is derived from a cell so modified.
  • the terms ''single chain antibodies'" or “single chain Fv (scFv)” refer to an antibody fusion molecule of the two domains of the Fv fragment, V L and V H - Although the two domains of the Fv fragment, V L and V H , are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules. known as single chain Fv (scFv). See, e.g., Bird et al, Science 242: 423-426 (1988); and
  • small molecule means a composition that has a molecular weight of less than about 5 kDa and more preferably less than about 2 kDa.
  • Small molecules can be, e.g., nucleic acids, peptides, polypeptides, glycopeptides, peptidomimetics, carbohydrates, lipids, lipopolysaccharides, combinations of these, or other organic or inorganic molecules.
  • binding means the contact between SFRP-4- binding agent and an antigen with a binding affinity of at least 10 " M.
  • Preferred binding agents bind with affinities of at least about 10 "7 M, and preferably 10 '8 M to 10 "9 M, 10 '10 M,
  • stringent hybridization conditions means conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen,
  • stringent conditions are selected to be about 5-1O 0 C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength pH.
  • T m is the temperature (under defined ionic strength, pH. and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplary stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42 0 C, or, 5x SSC, 1% SDS, incubating at 65 0 C, with wash in 0.2x SSC, and 0.1% SDS at 65 0 C.
  • the term "subject” means that preferably the subject is a mammal, such as a human, but can also be an animal, e.g., domestic animals (e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horses and the like) and laboratory animals (e.g., monkey, rats, mice, rabbits, guinea pigs and the like).
  • domestic animals e.g., dogs, cats and the like
  • farm animals e.g., cows, sheep, pigs, horses and the like
  • laboratory animals e.g., monkey, rats, mice, rabbits, guinea pigs and the like.
  • target cell means any cell in a subject (e.g., a human or animal) that can be targeted by the SFRP-4-binding agent of the invention.
  • SFRP-4-binding agent e.g., SFRP-4 binding agent
  • the present invention provides SFRP-4 binding agents directed to SFRP-4 polypeptides, fragments and variants thereof.
  • SFRP-4 is a member of the SFRP family that contains a cysteine-rich domain homologous to the putative Wnt-binding site of Frizzled proteins.
  • the Wnt networks influence biological processes ranging from developmental cell fate, cell polarity and adhesion to tumourigenesis and apoptosis. Jones et al., Bioessays 24: 81 1-20 (2002).
  • SFRPs act as soluble modulators of Wnt signalling. Multiple protein products may be derived from mammalian expression of SFRP-4 gene by alternate splicing mechanism. Yam et al. Gene PMID: 16005582 (Epub ahead of print, July 7 (2005).
  • a nucleotide sequence encoding human SFRP-4 (NM_003014.2; SEQ ID NO: 1) is shown in TABLE l .
  • SEQ ID NO: 1 An amino acid sequence of a human SFRP-4 polypeptide (NP_OO3OO5.1 ; SEQ ID NO: 2) is shown in TABLE 2.
  • SFRP-4 binding agents of the invention it is an object of the invention to provide immunogenic compositions useful for the preparation of SFRP-4 binding agents of the invention.
  • amino acid sequence of human SFRP-4 polypeptide was analyzed using Biobench2 (Novartis) and the BLAST network service at the National Center for Biotechnology Information to identify SFRP-4 domains useful as targets for binding agents of the present invention (see EXAMPLE I). This analysis resulted in the identification of four SFRP-4 domains which are summarized below in TABLE 3.
  • Target domains may be prepared by any technique useful to produce polypeptides, e.g., chemical synthesis, molecular biological technique, proteolytic or chemical degradation of SFRP-4 polypeptide.
  • the SFRP-4 target domain is prepared by chemical synthesis. Methods for chemical synthesis of peptides are well known in the art.
  • the SFRP-4 target domain is modified to facilitate conjugation to another compound, e.g., ovalbumin, keyhole limpet haemocyanin. See generally below, Preparation of Polyclonal Antiser a and Immunogens .
  • a cysteine residue is incorporated into the C-terminus of the SFRP-4 target domain to facilitate conjugation of the SFRP-4 target domain to another compound.
  • Sequences comprising the target domains of the invention and useful for conjugation to another compound are detailed below in TABLE 4.
  • the immunogenic composition of the present invention comprises at least one (1) polypeptide selected from the group consisting of: SEQ ID NO: 3; SEQ ID NO: 4: SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; and SEQ ID NO: 10.
  • SFRP-4 is elaborated by a number of human and animal tumours, e.g., breast, prostate, uterine, spleen, colon, and other tissues.
  • SFRP-4 expression is a biological marker, therefore, of pathological conditions such as cancer (e.g., brain, breast, prostate, uterine, spleen, pancreas, gastrointestinal tract (e.g., colon, rectum, small intestine, stomach, oesophagus) apoptosis resulting from ischemia, (e.g., heart failure, myocardial infarction; stroke); neurodegenerative disease (e.g., Huntington's Disease, peripheral demyelinating disease, Multiple Sclerosis, Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Parkinson's Disease; trauma); coronary heart disease, inflammation (e.g., inflammatory bowel disease).
  • cancer e.g., brain, breast, prostate, uterine, spleen, pancreas, gastrointestinal tract (e
  • SFRP-4-binding agents directed to each of the target binding domains 1 through 4 are useful, alone or in combination, to detect SFRP-4 polypeptide (a.k.a., target polypeptide) in test sample (e.g., a biological sample) as well as to modulate SFRP-4-mediated function.
  • test sample e.g., a biological sample
  • various aspects of the present invention further relate to diagnostic/theranostic methods and kits that use the SFRP-4-binding agents of the invention to identify individuals predisposed to a medical condition or to classify individuals with regard to drug responsiveness, side effects, or optimal drug dose.
  • SFRP -4 may be used alone, or in combination with other biological markers, e.g., apoptosis or proliferation-related markers ⁇ e.g., c-myc, cyclin-Dl); Wnt- pathway markers (see, e.g., Etheredge et al, Stem Cells 22:849-860 (2004)); Erbl; or prostatic specific antigen.
  • apoptosis or proliferation-related markers e.g., c-myc, cyclin-Dl
  • Wnt- pathway markers see, e.g., Etheredge et al, Stem Cells 22:849-860 (2004)
  • Erbl or prostatic specific antigen.
  • the invention provides methods for the use of SFRP-4-binding agents to prevent or treat SFRP-4-mediated disorders as well as to screen and/or validate ligands, e.g., small molecules that bind, SFRP -4 polypeptide.
  • SFRP-4-binding agents of the present invention are useful for the prophylactic treatment, or therapeutic treatment of disorders manifested by changes, e.g., increase or decrease, in SFRP -4 polypeptide expression, e.g., cancer ⁇ e.g., brain, breast, prostate, uterine, spleen, pancreas, gastrointestinal tract ⁇ e.g., colon, rectum, small intestine, stomach, oesophagus) apoptosis resulting from ischemia ⁇ e.g., heart failure, myocardial infarction; stroke); neurodegenerative disease ⁇ e.g., Huntington's Disease, peripheral demyelinating disease, Multiple Sclerosis, Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Parkinson's Disease; trauma); coronary heart disease, inflammation ⁇ e.g., inflammatory bowel disease).
  • cancer e.g., brain, breast, prostate, uterine, spleen, pancreas, gastrointestinal tract ⁇ e
  • the present invention provides SFRP-4-binding agent compositions, a.k.a., the binding agent.
  • Binding agents of the present invention can be described or specified in terms of the epitopes or portions of a polypeptide of the present invention which are recognized or specifically bound by the binding agent, e.g., a region of SFRP-4 polypeptide that is located on the surface of the polypeptide ⁇ e.g., a hydrophilic region).
  • hydropathy plots showing regions of hydrophilicity and hydrophobicity can be generated by any method well known in the art, including, e.g., the Kyte-Doolittle or the Hopp- Woods methods, either with or without Fourier transformation (see, e.g., Hopp & Woods, Proc. Nat. Acad. ScL USA 78: 3824-3828 (1981); Kyte & Doolittle, J. MoI. Biol. 157: 105-142 (1982)).
  • the epitopes or polypeptide portions can be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues.
  • the present invention includes binding agents that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.
  • Binding agents of the present invention can also be described or specified in terms of their cross-reactivity. Binding agents that do not bind any other analog, ortholog, or homolog of the target polypeptide of the present invention are included. Binding agents that do not bind polypeptides with less than 95%, less than 90%. less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a target polypeptide of the present invention are also included in the present invention.
  • binding agents which only bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions. Binding agents of the present invention can also be described or specified in terms of their binding affinity.
  • Preferred binding affinities include those with a dissociation constant or K d less than 10 '6 M, 5 X 10 "6 M, 10 "7 M, 5 X 10 '7 M, 10 "8 M, 5 X 10- 8 M, 10 "9 M, 5 X 10- 9 M, 10 "10 M, 5 X 10 "10 M, 10 "u M, 5 X lO '11 M, 10 "12 M, 5 X 10 "12 M, 10- 13 M, 5 X 10 "13 M, 10 "14 M, 5 X lO "14 M, W 15 M, and 5 X 10 "15 M.
  • SFRP-4-binding agents within the scope of the present invention includes, e.g., but is not limited to, monoclonal, polyclonal, chimaeric. humanized, diabody, and human monoclonal and human polyclonal antibodies which specifically bind the target polypeptide, a homologue, derivative or a fragment thereof.
  • an "SFRP-4-like" polypeptide means a polypeptide that is different from the SFRP-4 polypeptide but which is immunologically reactive with an SFRP-4-binding agent of the invention.
  • An SFRP-4-like polypeptide may be derived from the same organism or a different organism as an SFRP-4 polypeptide.
  • An SFRP-4-like polypeptide may be encoded by the same gene or a different gene as SFRP-4 polypeptide.
  • the antibodies useful as binding agents of the present invention include, e.g., but are not limited to, IgG (including IgGj, IgG 2 , IgG 3 , and IgG 4 ), IgA (including IgAi and IgA 2 ), IgD, IgE, or IgM, and IgY.
  • the binding agent of the invention is an antibody-related polypeptide directed to an SFRP-4 polypeptide, homologue or derivative thereof.
  • the antigen-binding region of a binding agent e.g., anti-SFRP-4-binding region
  • the SFRP-4-binding agent is an anti-SFRP-4 polypeptide antibody, such as anti-SFRP-4 polypeptide monoclonal antibody, anti-SFRP-4 polypeptide chimaeric antibody, and anti-SFRP-4 polypeptide humanized antibody which have been modified by, e.g., deleting, adding, or substituting portions of the antibody.
  • an anti-SFRP-4 polypeptide antibody may be modified to increase half-life, e.g., serum half-life, stability or affinity of the antibody.
  • selection of antibodies that are specific to a particular domain of an SFRP-4 polypeptide is facilitated by generation of hybridomas that bind to the fragment of an SFRP-4 polypeptide possessing such a domain.
  • SFRP-4-binding agents which are antibodies that are specific for a desired domain within an SFRP-4 polypeptide, or a derivative, fragment, analog or homolog thereof, are also provided herein.
  • the present invention further includes antibodies which are anti-idiotypic to the binding agents of the present invention.
  • the binding agents of the present invention can be monospecific, bispecific, trispecific or of greater multispecificity.
  • Multispecific binding agents can be specific for different epitopes of an SFRP-4 polypeptide of the present invention or can be specific for both a polypeptide of the present invention as well as for heterologous compositions, such as a heterologous polypeptide or solid support material. See, e.g., WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al., J. Immunol. 147: 60- 69 (1991); U.S. Pat. Nos. 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648; 6,106,835; Kostelny et at, J. Immunol. 148: 1547-1553 (1992).
  • the binding agents of the invention can be from any animal origin including birds and mammals. Preferably, the binding agents are human, murine, rabbit, goat, guinea pig, camel, horse, or chicken.
  • the binding agents of the invention are suitable for administration to a subject where it is desirable, e.g., to modulate SFRP-4 function. Accordingly, it is a further object of the invention to provide for SFRP-4-binding agent compositions that are SFRP-4 modulators, e.g., functional antagonists or functional agonists of an SFRP-4 polypeptide. It is also an object of the invention to provide for SFRP-4-binding agent compositions that are partial antagonists and partial agonists of an SFRP-4 polypeptide. Likewise included are neutralizing anti-SFRP-4 antibodies which bind the SFRP-4 polypeptide.
  • the binding agent of the invention will be purified: (1) to greater than 95% by weight of antibody as determined by the Lowry method (Lowry et at., J. Biol. Chem. 193: 265. 1951) and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated binding agent includes the polypeptide in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an SFRP-4-binding agent, e.g., an isolated anti-SFRP-4 antibody, will be prepared by at least one purification step.
  • the invention further relates to structure-based methods useful in identifying, designing and producing compounds which act as modulators of SFRP-4 polypeptide.
  • the binding agents of the present invention can be used either alone or in combination with other compositions.
  • the SFRP-4-binding agents of the present invention can further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions.
  • SFRP-4-binding agents of the present invention can be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins.
  • the SFRP-4-binding agents of the present invention are anti- SFRP-4 antibodies or anti-SFRP-4 antibody-related polypeptides that are coupled or conjugated to one or more therapeutic or cytotoxic moieties to yield an SFRP-4-binding agent conjugate protein of the invention.
  • the SFRP-4-binding agent conjugate protein of the invention can be used to modify a given biological response or create a biological response (e.g., to recruit effector cells).
  • the therapeutic moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the therapeutic moiety can be a protein or polypeptide possessing a desired biological activity.
  • proteins can include, e.g., an enzymatically active toxin, or active fragment thereof, such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin; a protein such as tumour necrosis factor or interferon-alpha; or, biological response modifiers such as, e.g., lymphokines, interleukin-1 ("IL-I”), interleukin-2 ("IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM- CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • IL-I interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • GM- CSF granulocyte macro
  • a target polypeptide is chosen to which a binding agent (e.g., anti-SFRP-4 antibody) can be raised.
  • a binding agent e.g., anti-SFRP-4 antibody
  • Techniques for generating binding agents directed to target polypeptides are well known to those skilled in the art.
  • Binding agents e.g., anti-SFRP-4 antibodies
  • Binding agents include monoclonal and polyclonal antibodies, and antibody fragments such as Fab, Fab', F(ab') 2 , Fd, scFv, diabodies, antibody light chains, antibody heavy chains and/or antibody fragments.
  • Methods useful for the high yield production of antibody Fv-containing polypeptides, e.g., Fab' and F(ab")2 antibody fragments have been described. See U.S. Pat. No. 5,648,237.
  • Originating species is any species which was useful to generate the binding agent of the invention or library of binding agents, e.g., rat, mice, rabbit, chicken, monkey, human, and the like.
  • SFRP-4-binding agents are anti-SFRP-4 antibodies.
  • Phage or phagemid display technologies are useful techniques to derive the binding agents of the present invention.
  • Anti-SFRP-4 antibodies useful in the present invention are "human antibodies,” (e.g., antibodies isolated from a human) or "human sequence antibodies.” Human antibodies can be made by a variety of methods known in the art including phage display methods. See also, U.S. Pat. Nos.
  • the SFRP-4-binding agents of the invention preferably have a high immunoreactivity, that is, percentages of antibodies molecules that are correctly folded so that they can specifically bind their target antigen.
  • Expression of sequences encoding binding agents, e.g., antibodies of the invention, can be carried out in E. coli as described below. Such expression usually results in imrmmoreactivity of at least 80%, 90%, 95% or 99%.
  • Methods of generating antibodies or antibody fragments of the invention typically include immunizing a subject (generally a non-human subject such as a mouse or rabbit) with purified SFRP-4 polypeptide or with a cell expressing SFRP-4 polypeptide. Any immunogenic portion of SFRP-4 can be employed as the immunogen.
  • An appropriate immunogenic preparation can contain, e.g., recombinantly-expressed SFRP-4 polypeptide or a chemically-synthesized SFRP-4 polypeptide.
  • An isolated SFRP-4 polypeptide, or a portion or fragment thereof, can be used as an immunogen to generate SFRP-4-binding agent that binds to an SFRP-4 polypeptide, or a portion or fragment using standard techniques for polyclonal and monoclonal antibody preparation.
  • the full-length SFRP-4 polypeptide can be used or, alternatively, the invention provides for the use of SFRP-4 polypeptide fragments as immunogens.
  • the SFRP-4 polypeptide comprises at least 4 amino acid residues of the amino acid sequence shown in SEQ ID NO: 1, and encompasses an epitope of SFRP-4 polypeptide such that an antibody raised against the peptide forms a specific immune complex with SFRP-4 polypeptide.
  • the antigenic peptide comprises at least 5, 8, 10, 15, 20, or 30 amino acid residues. Longer antigenic peptides are sometimes preferable over shorter antigenic peptides, depending on use and according to methods well known to those skilled in the art.
  • the immunogen will be at least about 8 amino acyl residues in length, and preferably at least about 10 acyl residues in length. Multimers of a given epitope are sometimes more effective than a monomer.
  • the immunogenicity of SFRP-4 can be increased by fusion or conjugation to a hapten such as keyhole limpet haemocyanin (KLH) or ovalbumin (OVA).
  • KLH keyhole limpet haemocyanin
  • OVA ovalbumin
  • a hapten such as keyhole limpet haemocyanin (KLH) or ovalbumin (OVA).
  • KLH keyhole limpet haemocyanin
  • OVA ovalbumin
  • Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels ⁇ e.g., aluminium hydroxide), surface active substances ⁇ e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory compounds. These techniques are standard in the art. [85] Following appropriate immunization, an SFRP-4-binding agent, e.g., anti-SFRP-4 polyclonal antibody can be prepared from the subject's serum. If desired, the antibody molecules directed against an SFRP-4 polypeptide can be isolated from the mammal ⁇ e.g., from the blood) and further purified by well known techniques, such as polypeptide A chromatography to obtain the IgG fraction.
  • SFRP-4-binding agent
  • the binding agent is an anti-SFRP-4 monoclonal antibody.
  • the anti-SFRP-4 monoclonal antibody is a human anti-SFRP-4 monoclonal antibody.
  • any technique that provides for the production of antibody molecules by continuous cell line culture can be utilized. Such techniques include, but are not limited to, the hybridoma technique (see, e.g. , Kohler & Milstein, 1975.
  • Human monoclonal antibodies can be utilized in the practice of the invention and can be produced by using human hybridomas (see, e.g., Cote, et al, Proc Natl Acad Sci USA 80: 2026-2030 (1983)) or by transforming human B-cells with Epstein Barr Virus in vitro (see, e.g., Cole, et al, Monoclonal Antibodies and Cancer Therapy (Alan R. Liss, Inc., 1985) pp. 77-96).
  • hybridomas expressing anti-SFRP-4 monoclonal antibodies can be prepared by immunizing a subject and then isolating hybridomas from the subject's spleen using routine methods.
  • a selected monoclonal antibody with the desired properties can be used as expressed by the hybridoma, it can be bound to a molecule such as polyethylene glycol (PEG) to alter its properties, or a cDNA encoding it can be isolated, sequenced and manipulated in various ways.
  • PEG polyethylene glycol
  • Hybridoma techniques include those known in the art and taught in Harlow et ah, Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 349 (1988); Hammerling et ah, Monoclonal Antibodies And T-CeIl Hybridomas, 563-681 (1981). Other methods for producing hybridomas and monoclonal antibodies are well known to those of skill in the art.
  • Phage Display Technique As noted above, the binding agents of the present invention can be produced through the application of recombinant DNA and phage display technology. For example, binding agents of the invention, e.g., anti-SFRP-4 antibodies, can be prepared using various phage display methods known in the art.
  • phage display methods functional antibody domains are displayed on the surface of a phage particle which carries polynucleotide sequences encoding them.
  • Phage with a desired binding property are selected from a repertoire or combinatorial antibody library (e.g., human or murine) by selecting directly with antigen, typically antigen bound or captured to a solid surface or bead.
  • Phage used in these methods are typically filamentous phage including fd and M 13 with Fab, Fv or disulphide stabilized Fv antibody domains are recombinantly fused to either the phage gene III or gene VIII protein.
  • methods can be adapted for the construction of Fab expression libraries (see, e.g., Huse, et ah,.
  • phage display methods that can be used to make the binding agents of the present invention include those disclosed in Huston et ⁇ h, Proc. N ⁇ tl. Ac ⁇ d. Sci U.S.A., 85: 5879-5883, 1988; Chaudhary et ⁇ l. , Proc. N ⁇ tl. Ac ⁇ d. Sci U.S.A., 87: 1066-1070, 1990; Brinkman et ⁇ l.. J. Immunol.
  • 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.
  • techniques to recombinantly produce Fab, Fab' and F(ab') 2 fragments can also be employed using methods known in the art such as those disclosed in WO 92/22324; Mullinax el al, BioTechniques 12: 864-869, 1992; and Sawai et al, AJRI 34: 26-34, 1995; and Better et al, Science 240: 1041-1043, 1988.
  • hybrid antibodies or hybrid antibody fragments that are cloned into a display vector can be selected against the appropriate antigen in order to identify variants that maintained good binding activity, because the antibody or antibody fragment will be present on the surface of the phage or phagemid particle.
  • a display vector can be selected against the appropriate antigen in order to identify variants that maintained good binding activity, because the antibody or antibody fragment will be present on the surface of the phage or phagemid particle.
  • Other vector formats could be used for this process, such as cloning the antibody fragment library into a lytic phage vector (modified T7 or Lambda Zap systems) for selection and/or screening.
  • libraries of Nucleic Acids Encoding Sfrp-4-Binding Agents result in libraries of nucleic acid sequences encoding SFRP-4-binding agents, e.g., anti- SFRP-4 antibody chains, having specific affinity for an SFRP-4 polypeptide or an SFRP -4- like polypeptide.
  • the libraries of nucleic acids typically have at least 5, 10, 20, 50, 100, 1000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 different members. Usually, no single member constitutes more than 25% or 50% of the total sequences in the library.
  • library members encode SFRP-4-binding agents with specific affinity for the an SFRP-4 polypeptide or an SFRP-4-like polypeptide.
  • a pair of nucleic acid segments encoding heavy and light chains respectively is considered a library member.
  • the nucleic acid libraries can exist in free form, as components of any vector or transfected as a component of a vector into host cells. Methods useful to produce libraries of genes encoding antigen combining molecules or antibodies which does not require an in vivo procedure have been described by Wigler et al, U.S. Pat. Nos. 6,303,313; 6,479,243.
  • the binding agents of the present invention can be produced through the application of recombinant DNA technology.
  • Recombinant polynucleotide constructs encoding an SFRP-4-binding agent of the present invention typically include an expression control sequence operably linked to the coding sequences of anti-SFRP-4 antibody chains, including naturally-associated or heterologous promoter regions.
  • another aspect of the invention includes vectors containing one or more nucleic acid sequences encoding an SFRP-4-binding agent of the present invention.
  • the nucleic acid containing all or a portion of the nucleotide sequence encoding the SFRP-4-binding agent is inserted into an appropriate cloning vector, or an expression vector ⁇ i.e., a vector that contains the necessary elements for the transcription and translation of the inserted polypeptide coding sequence) by recombinant DNA techniques well known in the art and as detailed below. Methods for producing diverse populations of vectors have been described by Lerner et al. , U.S. Pat. No. 6,291,160; 6.680,192.
  • expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences encoding an SFRP-4-binding agent, and the collection and purification of the SFRP-4-binding agent, e.g., cross-reacting anti-SFRP-4 antibodies. See generally U.S. Application No. 20020199213.
  • Vectors can also encode signal peptide, e.g. , pectate lyase, useful to direct the secretion of extracellular antibody fragments. See U.S. Pat. No. 5,576,195.
  • the recombinant expression vectors of the invention comprise a nucleic acid encoding a compound with SFRP-4-binding properties in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression that is operatively-linked to the nucleic acid sequence to be expressed.
  • regulatory sequences are described, e.g., in Goeddel, Gene Expression Technology: Methods in Enzymology, 185 (Academic Press, San Diego, Calif., 1990).
  • a polynucleotide encoding an SFRP-4-binding agent of the invention is operably linked to an ara B promoter and expressible in a host cell. See U.S. Pat. 5,028,530.
  • the expression vectors of the invention can be introduced into host cells to thereby produce polypeptides or peptides, including fusion polypeptides, encoded by nucleic acids as described herein (e.g., SFRP-4-binding agents, etc.).
  • Another aspect of the invention pertains to SFRP-4-binding agent-expressing host cells, which contain a nucleic acid encoding one or more SFRP-4-binding agents.
  • the recombinant expression vectors of the invention can be designed for expression of an SFRP-4- binding agent in prokaryotic or eukaryotic cells.
  • SFRP-4-binding agent can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), fungal cells, e.g., yeast, yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology, 185 (Academic Press, San Diego, Calif., 1990).
  • the recombinant expression vector can be transcribed and translated in vitro, e.g. using T7 promoter regulatory sequences and T7 polymerase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith & Johnson, Gene 67: 31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, NJ.) that fuse glutathione S-transferase (GST), maltose E binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.
  • GST glutathione S-transferase
  • maltose E binding polypeptide or polypeptide A, respectively, to the target recombinant polypeptide.
  • E. coli expression vectors examples include pTrc (Amrann et al, (1988) Gene 69: 301-315) and pET 1 Id (Studier et al., Gene Expression Technology: Methods in Enzymology, 185 (Academic Press, San Diego, Calif., 1990) pp. 60- 89). Methods for targeted assembly of distinct active peptide or protein domains to yield multifunctional polypeptides via polypeptide fusion have been described by Pack et al, U.S. Pat. Nos. 6,294,353; 6,692,935. One strategy to maximize recombinant polypeptide expression, e.g., SFRP-4-binding agent, in E.
  • SFRP-4-binding agent e.g., SFRP-4-binding agent
  • coli is to express the polypeptide in host bacteria with an impaired capacity to proteolytically cleave the recombinant polypeptide. See, e.g., Gottesman, Gene Expression Technology: Methods in Enzymology, 185 (Academic Press, San Diego, Calif., 1990) pp. 1 19-128.
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the expression host, e.g., E. coli (see, e.g., Wada, et al, Nucl. Acids Res. 20: 211 1-2118, 1992).
  • the SFRP-4-binding agent expression vector is a yeast expression vector.
  • yeast Saccharomyces cerivisae examples include pYepSecl (Baldari, et al, 1987. EMBOJ. 6: 229-234), pMFa (Kurjan & Herskowitz, Cell 30: 933-943, 1982), pJRY88 (Schultz et al., Gene 54: 113-123, 1987), pYES2 (Invitrogen Corporation, San Diego, Calif), and picZ (InVitrogen Corp, San Diego, Calif).
  • SFRP-4-binding agent can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of polypeptides, e.g., SFRP-4-binding agent, in cultured insect cells include the pAc series (Smith, et al., MoI. Cell. Biol. 3: 2156-2165, 1983) and the pVL series (Lucklow & Summers, Virology 170: 31-39 (1989)).
  • a nucleic acid encoding an SFRP-4-binding agent of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include, e.g., but are not limited to, pCDM8 (Seed,. Nature 329: 840, 1987) and pMT2PC (Kaufman et al, EMBOJ. 6: 187-195. 1987).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type ⁇ e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al.,. Genes Dev. 1 : 268-277 (1987)), — ⁇ n —
  • lymphoid-specific promoters Calame & Eaton, Adv. Immunol. 43: 235-275 (1988)
  • promoters of T cell receptors Winoto & Baltimore, EMBO J. 8: 729-733 (1989)
  • immunoglobulins Bonerji, et al, Cell 33: 729-740 (1983); Queen & Baltimore, Cell 33: 741-748 (1983)
  • neuron-specific promoters e.g., the neurofilament promoter; Byrne & Ruddle, Proc. Natl. Acad. Sci.
  • pancreas-specific promoters Eslund et al, Science 230: 912-916 (1985)
  • mammary gland-specific promoters ⁇ e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166.
  • Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel & Gruss, Science 249: 374-379 (1990)) and the ⁇ -fetoprotein promoter (Campes & Tilghman,. Genes Dev. 3: 537-546 (1989)).
  • a host cell can be any prokaryotic or eukaryotic cell.
  • an SFRP-4-binding agent can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells. Mammalian cells are a preferred host for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes To Clones, (VCH Publishers, NY, 1987).
  • nucleic acid libraries can be expressed to generate polyclonal libraries of SFRP-4-binding agents, e.g., anti-SFRP-4-binding antibodies, having specific affinity for an SFRP-4 polypeptide or an SFRP-4-like polypeptide.
  • the composition of such libraries is determined from the composition of the nucleotide libraries.
  • such libraries typically have at least 5, 10, 20, 50, 100, 1000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 members with different amino acid composition. Usually, no single member constitutes more than 25% or 50% of the total polypeptides in the library.
  • the percentage of anti-SFRP-4 antibody chains in an anti-SFRP-4 antibody chain library having specific affinity for an SFRP-4 polypeptide or an SFRP-4-like polypeptide is typically lower than the percentage of corresponding nucleic acids encoding the anti-SFRP-4 antibody chains. The difference is due to the fact that not all polypeptides fold into a structure appropriate for binding despite having the appropriate primary amino acid sequence to support appropriate folding. In some libraries, at least 25%, 50%, 75%, 90%, 95%, 99% or 99.9% of anti-SFRP-4 antibody chains have specific affinity for the target molecules. Again, in libraries of multi-chain antibodies, each anti-SFRP-4 antibody (such as a Fab or intact antibody) is considered a library member.
  • the different anti-SFRP-4 antibody chains differ from each other in terms of fine binding specificity and affinity for the target.
  • Some such libraries comprise members binding to different epitopes on the same antigen, e.g., SFRP-4 polypeptide.
  • Such libraries may comprise at least two members that bind to the same antigen without competing with each other.
  • Polyclonal libraries of human anti-SFRP-4 antibodies resulting from the above methods are distinguished from natural populations of human antibodies both by the high percentages of high affinity binders in the present libraries, and in that the present libraries typically do not show the same diversity of antibodies present in natural populations.
  • Libraries of reduced diversity can be derived form nonhuman transgenic animals that provide the source materials not including all human immunoglobulin genes.
  • some polyclonal antibody libraries are free of anti-SFRP-4 antibodies having lambda light chains.
  • Some anti-SFRP-4 polyclonal antibody libraries of the invention have antibody heavy chains encoded by fewer than 10, 20, 30 or 40 V H genes.
  • Some polyclonal anti-SFRP-4 antibody libraries of the invention have antibody light chains encoded by fewer than 10, 20, 30 or 40 V L genes.
  • the binding agent of the invention is a single chain anti-SFRP-4 antibody.
  • techniques can be adapted for the production of single-chain antibodies specific to an SFRP-4 polypeptide (see, e.g., U.S. Pat. No. 4,946,778). Examples of techniques which can be used to produce single-chain Fvs and antibodies of the invention include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et ah, Methods in Enzymology, 203: 46-88, 1991; Shu, L. et al, Proc. Natl Acad.
  • the binding agent of the invention is a chimaeric anti-SFRP-4 antibody.
  • the binding agent of the invention is a humanized anti-SFRP-4 antibody.
  • the donor and acceptor antibodies are monoclonal antibodies from different species.
  • the acceptor antibody is a human antibody (to minimize its antigenicity in a human), in which case the resulting CDR-grafted antibody is termed a "humanized" antibody.
  • Recombinant anti-SFRP-4 antibodies such as chimaeric and humanized monoclonal antibodies, comprising both human and non-human portions, can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • chimaeric, humanized, or human anti-SFRP-4 antibodies it is preferable to use chimaeric, humanized, or human anti-SFRP-4 antibodies.
  • Such chimaeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art. Such useful methods include, e.g., but are not limited to, methods described in International Application No. PCT/US86/02269; U.S. Pat. No.
  • antibodies can be humanized using a variety of techniques including CDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. No. 5,530,101: 5,585,089; 5,859,205; 6,248,516; EP460167), veneering or resurfacing (EP 0 592 106; EP 0 519 596; Padlan E.
  • a cDNA encoding a murine anti-SFRP-4 monoclonal antibody is digested with a restriction enzyme selected specifically to remove the sequence encoding the Fc constant region, and the equivalent portion of a cDNA encoding a human Fc constant region is substituted (see Robinson el ah, PCT/US86/02269; Akira et ah, European Patent Application 184,187; Taniguchi, European Patent Application 171,496; Morrison et ah, European Patent Application 173,494; Neuberger et ah, WO 86/01533; Cabilly et a U.S. Patent No.
  • the binding agent of the invention is an anti- SFRP-4 CDR antibody.
  • the donor and acceptor antibodies used to generate the anti-SFRP-4 CDR antibody are monoclonal antibodies from different species; typically the acceptor antibody is a human antibody (to minimize its antigenicity in a human), in which case the resulting CDR-grafted antibody is termed a "humanized" antibody.
  • the graft may be of a single CDR (or even a portion of a single CDR) within a single V H or V L of the acceptor antibody, or can be of multiple CDRs (or portions thereof) within one or both of the V H and V L .
  • all three CDRs in all variable domains of the acceptor antibody will be replaced with the corresponding donor CDRs, though one need replace only as many as necessary to permit adequate binding of the resulting CDR-grafted antibody to MetAp3.
  • Methods for generating CDR-grafted and humanized antibodies are taught by Queen et ah U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,693,761; U.S. Pat. No.
  • either or both the heavy and light chain variable regions are produced by grafting the CDRs from the originating species into the hybrid framework regions.
  • Assembly of hybrid antibodies or hybrid antibody fragments having hybrid variable chain regions with regard to either of the above aspects can be accomplished using conventional methods known to those skilled in the art.
  • DNA sequences encoding the hybrid variable domains described herein i.e., frameworks based on the target species and CDRs from the originating species
  • the nucleic acid encoding CDR regions can also be isolated from the originating species antibodies using suitable restriction enzymes and ligated into the target species framework by ligating with suitable ligation enzymes.
  • the framework regions of the variable chains of the originating species antibody can be changed by site-directed mutagenesis.
  • the resulting FR fits a human consensus FR for that position, or is at least 90% or more identical to such a consensus FR, doing so may not increase the antigenicity of the resulting modified anti-SFRP-4 CDR antibody significantly compared to the same antibody with a fully human FR.
  • recombinant anti- SFRP-4 antibodies, SFRP-4 polypeptides or SFRP-4-binding agent are rendered non-immunogenic, or less immunogenic, to a given species by identifying in their amino acid sequences one or more potential epitopes for T-cells of the given species and modifying the amino acid sequence to eliminate at least one of the T-cell epitopes. This eliminates or reduces the immunogenicity of the polypeptide or protein when exposed to the immune system of the given species.
  • Monoclonal antibodies and other immunoglobulin-like molecules can particularly benefit from being de-immunized in this way - for example, mouse-derived immunoglobulins can be de-immunized for human therapeutic use.
  • Methods for de-immunizing a polypeptide or protein in the art See, e.g., Carr, et al. US Pat. Application 20030153043; and De Groot, et al, AIDS Res. and Human Retroviruses 13: 539- 541 (1997): Schafer, et al, Vaccine 16: 1880-1884 (1998); De Groot, et al, Dev. Biol.
  • Antibody fragments that contain the idiotypes to an SFRP-4 polypeptide can be produced by techniques known in the art including, but not limited to: (i) a F(ab') 2 fragment produced by pepsin digestion of an antibody molecule; (ii) a Fab fragment generated by reducing the disulphide bridges of an F(ab') 2 fragment; (iii) a Fab fragment generated by the treatment of the antibody molecule with papain and a reducing compound; and (iv) Fv fragments. See, e.g., Greenspan et al, FASEB J. 7: 437-444, 1993 and Nissinoff, J Immunol. 147: 2429-2438, 1991.
  • antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to ligand can be used to generate anti-idiotypes that "mimic" the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize target polypeptide and/or its ligand.
  • anti-idiotypes or Fab fragments of such antiidiotypes can be used in therapeutic regimens to neutralize target polypeptide ligand.
  • anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby modulate, e.g., increase or decrease target polypeptide biological activity relative to the level of biological activity observed in the absence of anti-idotypic antibodies.
  • the binding agent of the invention is a fusion protein.
  • the addition of peptide moieties to facilitate handling of polypeptides is familiar and routine techniques in the art.
  • the SFRP-4-binding agent of the invention can be fused to marker sequences, such as a peptide which facilitates purification of the fused polypeptide.
  • the marker amino acid sequence is a hexa-histidine peptide, such as lhe tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al, Proc. Natl. Acad.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • Another peptide tag useful for purification corresponds to an epitope derived from the influenza haemagglutinin protein. Wilson et al, Cell 37: 767. 1984.
  • any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention. Also, the fusion protein can show an increased half-life in vivo.
  • Fusion proteins having disulphide-linked dimeric structures can be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone.
  • EP-A-O 464 533 discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof.
  • the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, e.g., improved pharmacokinetic properties. See EP-A 0232 262.
  • the Fc portion can hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
  • human proteins such as hIL-5
  • Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. Bennett et al., J. Molecular Recognition 8: 52-58, 1995; K. Johanson et al., J. Biol. Chem., 270: 9459-9471, 1995.
  • SFRP-4-binding agents of the invention are prepared using genomic DNA or ESTs encoding candidate binding agents as part of fusion proteins which form inclusion bodies upon expression in host cells. Methods useful to prepare genomic DNA or ESTs encoding candidate binding agents as part of fusion proteins which form inclusion bodies upon expression in host cells have been described. See U.S. Pat. No. 6,653,068; U. S. S.N. 20040157291. For example, the inclusion bodies are useful to generate binding partners, e.g., SFRP-4-binding agents, which bind specifically to the target (poly)peptide. [1 18] SFRP-4-Binding Agent Conjugate Protein.
  • the SFRP-4 -binding agent of the present invention are anti-SFRP-4 antibodies coupled or conjugated to one or more therapeutic or cytotoxic moieties to yield an SFRP-4- binding agent conjugate protein of the invention.
  • the SFRP-4-binding agents of the invention of this invention are useful as SFRP-4-binding agent-cytotoxin conjugate molecules, as exemplified by the administration for treatment of neoplastic disease.
  • a variety of bifunctional or polyfunctional reagents both homo- and hetero-functional (such as those described in the catalogue of the Pierce Chemical Co., Rockford, IL.), can be employed as a linker group. Coupling can be affected, e.g., through amino groups, carboxyl groups, sulphhydryl groups or oxidized carbohydrate residues (see, e.g., U.S. Pat. No. 4,671,958).
  • a moiety can be coupled to the SFRP-4-binding agents of the invention, e.g., through an oxidized carbohydrate group at a glycosylation site, as described in U.S. Pat. Nos. 5,057,313 and 5.156,840.
  • Yet another alternative method of coupling an SFRP-4-binding agent to a moiety is by the use of a non-covalent binding pair, such as streptavidin/biotin, or avidin/biotin.
  • one member of the pair is covalently coupled to the SFRP-4-binding agent and the other member of the binding pair is covalently coupled to the moiety.
  • Cleavable linkers where a cytotoxic or therapeutic moiety is more potent when free from the SFRP-4-binding agent portion of the immunoconjugates of the present invention, it can be desirable to use a linker group which is cleavable during or upon internalization into a cell, or which is gradually cleavable over time in the extracellular environment.
  • linker groups which are cleavable during or upon internalization into a cell, or which is gradually cleavable over time in the extracellular environment.
  • a number of different cleavable linker groups have been described. Examples of the intracellular release of a cytotoxic moiety from these linker groups include, e.g., but is not limited to, cleavage by reduction of a disulphide bond ⁇ e.g., U.S. Pat. No.
  • the SFRP-4-binding agent of the invention is coupled to more than one therapeutic, cytotoxic and/or imaging moiety.
  • an SFRP-4-binding agent can be made useful as a contrasting agent for several visualization techniques, or a therapeutic antibody can be labelled for tracking by a visualization technique.
  • multiple molecules of a cytotoxic moiety are coupled to one SFRP-4-binding agent.
  • the SFRP-4-binding agent of the invention is coupled to a mixture of at least two moieties selected from the group consisting of: a cytotoxic moiety; therapeutic moiety; and labelling/imaging moiety. That is, more than one type of moiety can be coupled to one SFRP-4-binding agent.
  • a therapeutic moiety such as a polynucleotide or antisense sequence
  • a chemotoxic or radiotoxic moiety can be conjugated to SFRP-4-binding agent in conjunction with a chemotoxic or radiotoxic moiety, to increase the effectiveness of the chemo- or radiotoxic therapy, as well as lowering the required dosage necessary to obtain the desired therapeutic effect.
  • immunoconjugates with more than one moiety can be prepared in a variety of ways.
  • more than one moiety can be coupled directly to an SFRP-4-binding agent, or linkers that provide multiple sites for attachment (e.g., dendrimers) can be used.
  • a carrier with the capacity to hold more than one cytotoxic moiety can be used.
  • an SFRP-4-binding agent can bear the moieties in a variety of ways, including covalent bonding either directly or via a linker group, and non-covalent associations,
  • the SFRP-4-binding coupled protein can be combined with encapsulation carriers. This is especially useful in chemotoxic therapeutic embodiments, as they can allow the therapeutic compositions to gradually release an SFRP-4-binding agent chemotoxic moiety over time while concentrating it in the vicinity of the target cells.
  • SFRP-4-Binding Agent Conjugated with Radionuclides In one embodiment, the SFRP-4-binding agent of the present invention is coupled with a cytotoxic moiety which is a radionuclide.
  • Preferred radionuclides for use as cytotoxic moieties of the invention are radionuclides which are suitable for pharmacological administration.
  • Chemotoxic moieties In one embodiment, the SFRP-4-binding agent of the present invention is coupled with a chemotoxic moiety.
  • Preferred chemotoxic agents useful in the present invention include, but are not limited to, small-molecule drugs such as methotrexate, and pyrimidine and purine analogs.
  • the SFRP-4-binding agent of the present invention is coupled with a protein toxin moiety.
  • Preferred toxin proteins for use as cytotoxic moieties of the invention include, e.g., but are not limited to, Actinomycetes or Streptomyces antibiotics, duocarmycin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin didne, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Preferred toxin proteins for use as cytotoxic moieties further include ricin, abrin, diphtheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, pokeweed antiviral protein, and other toxin proteins known in the medicinal biochemistry arts.
  • these toxin agents can elicit undesirable immune responses in the subject, especially if injected intravascularly, it is preferred that they be encapsulated in a carrier for coupling to the SFRP-4-binding agents of the invention, e.g., anti-SFRP-4 antibody and antibody-related polypeptides of the invention.
  • the SFRP-4-binding agent of the present invention is coupled with an en2ymatically active toxin.
  • the enzymatically active toxin can be of bacterial or plant origin, or an enzymatically active fragment ("A chain * ') of such a toxin.
  • Enzymatically active toxins and fragments thereof useful in the present invention are diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin.
  • diphtheria A chain nonbinding active fragments of diphtheria toxin
  • exotoxin A chain from Pseudomonas aeruginosa
  • ricin A chain abrin A chain
  • modeccin A chain alpha-sarcin
  • Conjugates of the SFRP-4-binding agent of the present invention with cytotoxic moieties are made using a variety of bifunctional protein coupling agents.
  • reagents are SPDP, IT, bifunctional derivatives of imidoesters such a dimethyl adipimidate HCl, active esters such as disuccinimidyl suberate, aldehydes such as glutaraldehyde, bis-azido compounds such as bis (p-azidobenzoyl) hexanediamine, bis-diazonium derivatives such as bis-(p- diazoniumbenzoyl)-ethylenediamine, diisocyanates such as tolylene 2,6-diisocyanate, and bis- active fluorine compounds such as l,5-difluoro-2,4-dinitrobenzene.
  • the lysing portion of a toxin can be joined to the Fab fragment of antibodies, e.g., SFRP-4-binding agent..
  • Therapeutic Moieties In one embodiment, the SFRP-4-binding agent of the present invention is coupled with a therapeutic moiety. Techniques for conjugating such therapeutic moiety to an SFRP-4-binding agent of the present invention are well known. See, e.g., Arnon et al, ''Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc.
  • the SFRP-4-binding agent of the present invention is coupled with a label moiety, i.e., a detectable group.
  • a label moiety i.e., a detectable group.
  • the particular label or detectable group conjugated to the SFRP-4-binding agent of the invention is not a critical aspect of the invention, so long as it does not significantly interfere with the specific binding of the SFRP-4-binding agent of the present invention to an SFRP-4 polypeptide or an SFRP-4-like polypeptide.
  • the detectable group can be any material having a detectable physical or chemical property.
  • detectable labels have been well -developed in the field of immunoassays and imaging, in general, most any label useful in such methods can be applied to the present invention.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include magnetic beads (e.g., DynabeadsTM), fluorescent dyes ⁇ e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3 H, 14 C, 35 S, 125 1, 121 1, 112 In, "mTc), other imaging agents such as microbubbles (for ultrasound imaging), 18 F.
  • magnetic beads e.g., DynabeadsTM
  • fluorescent dyes ⁇ e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like
  • radiolabels e.g., 3 H, 14 C, 35 S, 125 1, 121 1, 112 In, "mTc
  • other imaging agents such as microbubbles (for ultrasound imaging), 18 F.
  • Non-radioactive labels are often attached by indirect means.
  • a ligand molecule e.g., biotin
  • an anti- ligand e.g., streptavidin
  • a signal system such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • a number of ligands and anti-ligands can be used. Where a ligand has a natural anti-ligand, e.g., biotin, thyroxine, and Cortisol, it can be used in conjunction with the labelled, naturally-occurring anti-ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody, e.g., anti-SFRP-4 antibody.
  • the molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore.
  • Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases.
  • Fluorescent compounds useful as labelling moieties include, but are not limited to, e.g., fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and the like.
  • Chemiluminescent compounds useful as labelling moieties include, but are not limited to, e.g., luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol.
  • Means of detecting labels are well known to those of skill in the art.
  • means for detection include a scintillation counter or photographic film as in autoradiography.
  • the label is a fluorescent label, it can be detected by exciting the fiuorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence can be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
  • CCDs charge coupled devices
  • enzymatic labels can be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
  • simple colorimetric labels can be detected simply by observing the colour associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the colour of the bead.
  • agglutination assays can be used to detect the presence of the target antibodies, e.g., anti- SFRP-4 antibodies.
  • antigen-coated particles are agglutinated by samples comprising the target antibodies.
  • none of the components need be labelled and the presence of the target antibody is detected by simple visual inspection.
  • Formulations of Pharmaceutical Compositions The SFRP -4-binding agent of the present invention can be incorporated into pharmaceutical compositions suitable for administration.
  • the pharmaceutical compositions generally comprise at least one (1) SFRP-4- binding agent and a pharmaceutically-acceptable carrier in a form suitable for administration to a subject.
  • Pharmaceutically-acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions for administering the antibody compositions (see, e.g., Remington's Pharmaceutical Sciences, 18 th ed., Mack Publishing Co., Easton, Pennsylvania, 1990).
  • the pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
  • GMP Good Manufacturing Practice
  • compositions, carriers, diluents and reagents are used interchangeably and represent that the materials are capable of administration to or upon a subject without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.
  • pharmaceutically-acceptable excipient means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
  • “Pharmaceutically-acceptable salts and esters” means salts and esters that are pharmaceutically-acceptable and have the desired pharmacological properties. A person of ordinary skill in the art, would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compositions of the present invention.
  • Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used.
  • the use of such media and compounds for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or compound is incompatible with the SFRP-4-binding agent, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • the SFRP-4-binding agent is prepared with carriers that will protect the SFRP-4-binding agent against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically-acceptable carriers. These can be prepared according to methods known to those skilled in the art, e.g. , as described in U.S. Pat. No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of binding agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the binding agent and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such SFRP-4-binding agent for the treatment of a subject.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, e.g., intravenous injection, local administration (see, e.g., U.S. Pat. No. 5,328,470) or by stereotactic injection (see, e.g., Chen, et ah, 1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
  • the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • Methods for identifying and/or screening the binding agents of the invention include any immunologically-mediated techniques known within the art. Components of an immune response can be detected in vitro by various methods that are well known to those of ordinary skill in the art.
  • cytotoxic T lymphocytes can be incubated with radioactively labelled target cells and the lysis of these target cells detected by the release of radioactivity
  • helper T lymphocytes can be incubated with antigens and antigen presenting cells and the synthesis and secretion of cytokines measured by standard methods (Windhagen A; et ah, Immunity, 2: 373-80, 1995)
  • antigen presenting cells can be incubated with whole protein antigen and the presentation of that antigen on MHC detected by either T lymphocyte activation assays or biophysical methods (Harding et ah, Proc. Natl. Acad.
  • mast cells can be incubated with reagents that cross-link their Fc-epsilon receptors and histamine release measured by enzyme immunoassay (Siraganian et ah, TIPS, 4: 432-437, 1983): and (5) enzyme-linked immunosorbent assay (ELISA).
  • enzyme immunoassay Siraganian et ah, TIPS, 4: 432-437, 1983
  • ELISA enzyme-linked immunosorbent assay
  • products of an immune response in either a model organism (e.g. , mouse) or a human subject can also be detected by various methods that are well known to those of ordinary skill in the art.
  • a model organism e.g. , mouse
  • a human subject can also be detected by various methods that are well known to those of ordinary skill in the art.
  • the production of antibodies in response to vaccination can be readily detected by standard methods currently used in clinical laboratories, e.g., an ELISA
  • the migration of immune cells to sites of inflammation can be detected by scratching the surface of skin and placing a sterile container to capture the migrating cells over scratch site (Peters et al, Blood, 72: 1310-5, 1988)
  • the proliferation of peripheral blood mononuclear cells in response to mitogens or mixed lymphocyte reaction can be measured using 3 H-thymidine
  • the phagocytic capacity of granulocytes, macrophages, and other phagocytes in PBMCs can be measured by placing PMBCs in well
  • SFRP-4-binding agents of the invention are selected using display of candidate binding agents on the surface of replicable genetic packages. See. e.g., U.S. Pat. Nos. 5.514,548; 5,837,500; 5,871,907; 5,885,793; 5,969,108; 6,225,447; 6,291,650; 6,492,160; EP 585 287; EP 605522; EP 616640; EP 1024191; EP 589 877; EP 774 511; EP 844 306.
  • Methods useful for producing/selecting a filamentous bacteriophage particle containing a phagemid genome encoding for a binding molecule with a desired specificity has been described. See, e.g., EP 774 51 1 ; US 5871907; US 5969108; US 6225447; US 6291650; US 6492160.
  • SFRP-4-binding agents of the invention are selected using display of candidate binding agents on the surface of a yeast host cell. Methods useful for the isolation of scFv polypeptides by yeast surface display have been described by Kieke et al, Protein Eng. 1997 Nov; 10(11): 1303-10.
  • SFRP-4-binding agents of the invention are selected using ribosome display. Methods useful for identifying ligands in peptide libraries using ribosome display have been described by Mattheakis et al, Proc. Natl. Acad. Sci. USA 91: 9022-26. 1994; and Hanes et al, Proc. Natl. Acad. ScL USA 94: 4937-42, 1997. [145] In one embodiment, SFRP-4-binding agents of the invention are selected using tRNA display of candidate binding agents. Methods useful for in vitro selection of ligands using tRNA display have been described by Merryman et al, Chem. Biol., 9: 741-46, 2002.
  • SFRP-4-binding agents of the invention are selected using RNA display.
  • Methods useful for selecting peptides and proteins using RNA display libraries have been described by Roberts et al Proc. Natl Acad. ScL USA, 94: 12297-302, 1997; and Nemoto et al, FEBSLeIt., 414: 405-8, 1997.
  • Methods useful for selecting peptides and proteins using unnatural RNA display libraries have been described by Frankel et ah, Curr. Opin. Struct. Biol, 13: 506-12, 2003.
  • SFRP-4-binding agents of the invention are expressed in the periplasm of gram negative bacteria and mixed with labelled SFRP -4 polypeptide. See WO 02/34886. In clones expressing recombinant polypeptides with affinity for the SFRP-4 polypeptide, the concentration of the labelled SFRP-4 bound to the binding agents is increased and allows the cells to be isolated from the rest of the library as described in Harvey el ah, Proc. Natl Acad. Sci. 22: 9193-98 2004 and U.S. Pat. No. 2004/0058403.
  • SFRP-4-binding agent After selection of a desired SFRP-4-binding agent, it is contemplated that it can be produced in large volume by any technique known to those skilled in the art, e.g., prokaryotic or eukaryotic cell expression and the like.
  • SFRP-4-binding agents which are, e.g., but not limited to, anti-SFRP-4 hybrid antibodies or fragments thereof can be produced by using conventional techniques to construct an expression vector that encodes an antibody heavy chain in which the CDRs and, if necessary, a minimal portion of the variable region framework, that are required to retain original species antibody binding specificity (as engineered according to the techniques described herein) are derived from the originating species antibody and the remainder of the antibody is derived from a target species immunoglobulin which can be manipulated as described herein, thereby producing a vector for the expression of a hybrid antibody heavy chain.
  • an SFRP-4 binding assay refers to an assay format wherein an SFRP-4 polypeptide and an SFRP-4-binding agent are mixed under conditions suitable for binding between the SFRP-4 polypeptide and the SFRP-4- binding agent and assessing the amount of binding between the SFRP-4 polypeptide and the SFRP-4-binding agent.
  • the amount of binding is compared with a suitable control, which can be the amount of binding in the absence of the SFRP-4 polypeptide, the amount of the binding in the presence of non-specific immunoglobulin composition, or both.
  • the amount of binding can be assessed by any suitable method.
  • Binding assay methods include, e.g., ELISA, radioreceptor binding assays, scintillation proximity assays, cell surface receptor binding assays, fluorescence energy transfer assays, liquid chromatography, membrane filtration assays, and the like.
  • Biophysical assays for the direct measurement of SFRP-4 binding to SFRP-4-binding agents are, e.g., nuclear magnetic resonance, fluorescence, fluorescence Al
  • SFRP-4-binding agent polarization, surface plasmon resonance (BIACOR chips) and the like. Specific binding is determined by standard assays known in the art, e.g., radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectroscopy and the like. If the specific binding of a candidate SFRP-4-binding agent is at least 1 percent greater than the binding observed in the absence of the candidate SFRP-4-binding agent, the candidate SFRP-4-binding agent is useful as an SFRP-4-binding agent of the invention.
  • Co-crystals of the SFRP-4 polypeptide and SFRP-4-binding agents are also provided by the present invention as a method of determining molecular interactions. Conditions suitable for binding between the SFRP-4-binding agent and an SFRP-4 will depend on the compound and its ligand and can be readily determined by one of ordinary skill in the art. [151] Measurement of SFRP-4-Binding Agent Biological Activity.
  • the SFRP-4-binding agents of the present invention e.g., anti-SFRP-4 antibodies and anti-SFRP-4 antibody-related polypeptides, can be specified as agonists or antagonists for biological activities comprising specific activities disclosed herein.
  • SFRP-4 agonists and antagonists which are SFRP-4-binding agents can be made using methods known in the art. See e.g., WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al, Blood 92: 1981-1988, 1998; Chen et al, Cancer Res., 58: 3668-3678, 1998; Harrop et al, J. Immunol. 161 : 1786-1794, 1998; Zhu et al, Cancer Res., 58: 3209-3214, 1998; Yoon et al, J. Immunol, 160: 3170-3179, 1998; Prat et al., J.
  • the biological activity, namely the agonist or antagonist properties of SFRP-4-binding agents can be characterized using any conventional in vivo and in vitro assays that have been developed to measure the biological activity of an SFRP-4 polypeptide.
  • the binding agents of the invention are useful in methods known in the art relating to the localization and/or quantitation of an SFRP-4 polypeptide (e.g., for use in measuring levels of the SFRP-4 polypeptide within appropriate physiological samples, for use in diagnostic methods, for use in imaging the polypeptide, and the like).
  • SFRP-4-binding agents that contain the antibody derived binding domain are useful as pharmacologically-active compositions. Binding agents of the invention are useful to isolate an SFRP-4 polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An SFRP-4-binding agent of the invention can facilitate the purification of a natural immunoreactive SFRP-4 polypeptide or an immunoreacitve SFRP -4- like polypeptide from a biological sample, e.g., cells as well as a recombinantly-produced immunoreactive SFRP-4 polypeptide or SFRP-4-like polypeptide expressed in a host system.
  • a SFRP-4-binding agent can be used to detect an immunoreactive SFRP-4 polypeptide or an immunoreactive SFRP-4-like polypeptide (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the immunoreactive polypeptide.
  • the SFRP-4-binding agents of the invention can be used diagnostically to monitor immunoreactive SFRP-4 polypeptide levels and/or immunoreactive SFRP-4-like polypeptide levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen.
  • the detection can be facilitated by coupling (i.e., physically linking) the SFRP-4-binding agent of the invention to a detectable substance.
  • An exemplary method for detecting the presence or absence of an SFRP-4 polypeptide or an SFRP-4-like polypeptide in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with an SFRP-4-binding agent of the invention capable of detecting an SFRP-4 polypeptide or an SFRP-4-like polypeptide such that the presence of an SFRP-4 polypeptide or an SFRP-4-like polypeptide is detected in the biological sample.
  • an SFRP-4-binding agent is an antibody raised against SEQ ID NO: 1, capable of binding to an SFRP-4 polypeptide or an SFRP-4-like polypeptide, preferably an antibody with a detectable label.
  • the term "labelled", with regard to the binding agent is intended to encompass direct labelling of the binding agent by coupling (i.e., physically linking) a detectable substance to the binding agent, as well as indirect labelling of the binding agent by reactivity with another compound that is directly labelled.
  • indirect labelling include detection of a primary antibody using a fluorescently-labelled secondary antibody and end-labelling of a DNA probe with biotin such that it can be detected with fluorescently- labelled streptavidin.
  • the detection method of the invention can be used to detect an SFRP-4 polypeptide or an SFRP-4-like polypeptide in a biological sample in vitro as well as in vivo.
  • In vitro techniques for detection of an SFRP-4 polypeptide or an SFRP-4-like polypeptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence.
  • ELISAs enzyme linked immunosorbent assays
  • Western blots Western blots
  • immunoprecipitations immunoprecipitations
  • immunofluorescence immunofluorescence
  • in vivo techniques for detection of an SFRP-4 polypeptide or an SFRP-4-like polypeptide include introducing into a subject a labelled SFRP-4-binding agent, e.g., anti-SFRP-4 antibody.
  • the antibody can be labelled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the biological sample contains poly
  • An SFRP-4-binding agent of the present invention can be used to assay SFRP-4 protein levels or SFRP-4-like protein levels in a biological sample using antibody-based techniques.
  • protein expression in tissues can be studied with classical immunohistological methods. Jalkanen, M. et al., J. Cell. Biol. 101 : 976-985, 1985; Jalkanen, M. et al.J. Cell. Biol. 105: 3087-3096, 1987.
  • Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • Suitable antibody assay labels include enzyme labels, such as, glucose oxidase, and radioisotopes or other radioactive agent, such as iodine ( 123 1, 121 I), carbon ( 14 C), sulphur ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium (“mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • enzyme labels such as, glucose oxidase, and radioisotopes or other radioactive agent, such as iodine ( 123 1, 121 I), carbon ( 14 C), sulphur ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium (“mTc)
  • fluorescent labels such as fluorescein and rhodamine, and biotin.
  • secreted SFRP-4 protein levels or SFRP-4-like protein levels can also be detected in vivo by imaging.
  • SFRP-4-binding agents e.g., anti-SFRP-4 antibody labels or markers for in vivo imaging of SFRP-4 protein levels or SFRP-4-like protein levels include those detectable by X-radiography, NMR or ESR.
  • suitable labels include radioisotopes such as barium or caesium, which emit detectable radiation but are not overtly harmful to the subject.
  • Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which can be incorporated into the SFRP-4-binding agent by labelling of nutrients for the relevant clone expressing SFRP-4-binding agent.
  • the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images.
  • the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of "mTc.
  • the labelled SFRP-4-binding agent will then preferentially accumulate at the location of cells which contain the specific target polypeptide.
  • in vivo tumour imaging is described in S. W. Burchiel et a!., Tumor Imaging: The Radiochemical Detection of Cancer 13 (1982).
  • the invention provides a diagnostic method of a medical condition e.g., an SFRP -4 associated disorder, which involves (a) assaying the expression of a polypeptide by measuring binding of an SFRP-4-binding agent of the present invention in cells or body fluid of an individual; (b) comparing the level of gene expression with a standard SFRP-4 polypeptide expression level, whereby an increase or decrease in the assayed SFRP-4 polypeptide expression level compared to the standard expression level is indicative of a medical condition.
  • a diagnostic method of a medical condition e.g., an SFRP -4 associated disorder, which involves (a) assaying the expression of a polypeptide by measuring binding of an SFRP-4-binding agent of the present invention in cells or body fluid of an individual; (b) comparing the level of gene expression with a standard SFRP-4 polypeptide expression level, whereby an increase or decrease in the assayed SFRP-4 polypeptide expression level compared to the standard expression level is indicative of a
  • the SFRP-4-binding compositions of the invention are useful in diagnostic methods.
  • the present invention provides methods using the binding agents of the invention useful in the diagnosis of SFRP-4-related medical conditions in a subject.
  • Binding agents of the invention may be selected such that they have any level of epitope binding specificity and very high binding affinity to the SFRP-4 polypeptide.
  • SFRP-4-binding agents of the invention useful in diagnostic assays usually have binding affinities of at least 10 8 , 10 9 , l ⁇ '°, 10 11 or 10 12 M "1 .
  • SFRP-4-binding agents used as diagnostic reagents have a sufficient kinetic on-rate to reach equilibrium under standard conditions in at least 12 hours, preferably at least five (5) hours and more preferably at least one (1) hour.
  • Some methods of the invention employ polyclonal preparations of anti-SFRP-4 antibodies and anti-SFRP-4 antibody related polypeptide compositions of the invention as diagnostic reagents, and other methods employ monoclonal isolates.
  • the use of polyclonal mixtures has a number of advantages compared to compositions made of one monoclonal anti-SFRP-4 antibody.
  • polyclonal anti-SFRP-4 antibodies or other polypeptides can generate a stronger signal (for diagnostics) than a monoclonal that binds to a single site on the SFRP-4 polypeptide or the SFRP-4-like polypeptide.
  • a polyclonal preparation can bind to numerous variants of a prototypical target sequence (e.g., allelic variants, species variants, strain variants, drug-induced escape variants) whereas a monoclonal antibody can bind only to the prototypical sequence or a narrower range of variants thereto.
  • monoclonal anti-SFRP-4 antibodies are advantageous for detecting a single antigen in the presence or potential presence of closely related antigens.
  • the preparation typically contains an assortment of SFRP-4-binding agents, e.g., antibodies, with different epitope specificities to the target polypeptide.
  • SFRP-4-binding agents e.g., antibodies
  • SFRP-4-binding agents which are human antibodies can be used as diagnostic reagents for any kind of sample, they are most useful as diagnostic reagents for human biological samples.
  • SFRP-4-binding agents can be used to detect a given SFRP-4 polypeptide in a variety of standard assay formats. Such formats include immunoprecipitation, Western blotting, ELISA, radioimmunoassay, and immunometric assays. See Harlow & Lane, Antibodies, A Laboratory Manual (Cold Spring Harbor Publications, New York, 1988); U.S. Pat. Nos.
  • Bio samples can be obtained from any tissue or body fluid of a subject.
  • Immunometric or sandwich assays are a preferred format for the diagnostic methods of the present invention. See U.S. Pat. No. 4,376,110, 4,486,530, 5,914,241, and 5,965,375.
  • Such assays use one SFRP-4-binding agent, e.g., anti-SFRP-4 antibody, or population of anti- SFRP-4 antibodies immobilized to a solid phase, and another anti-SFRP-4 antibody, or a population of anti-SFRP-4 antibodies.
  • the solution anti-SFRP-4 antibody or population of anti-SFRP-4 antibodies is labelled. If an antibody population is used, the population typically contains antibodies binding to different epitope specificities within the target polypeptide. Accordingly, the same population can be used for both solid phase and solution antibody.
  • first and second-SFRP-4 monoclonal antibodies having different binding specificities are used for the solid and solution phase.
  • Solid phase and solution antibodies can be contacted with target antigen in either order or simultaneously. If the solid phase antibody is contacted first, the assay is referred to as being a forward assay. Conversely, if the solution antibody is contacted first, the assay is referred to as being a reverse assay. If target is contacted with both antibodies simultaneously, the assay is referred to as a simultaneous assay. After contacting the SFRP-4 polypeptide with an anti-SFRP-4 antibody, a sample is incubated for a period that usually varies from about 10 min to about 24 hr and is usually about 1 hr.
  • a wash step is then performed to remove components of the sample not specifically bound to the anti-SFRP-4 antibody being used as a diagnostic reagent.
  • a wash can be performed after either or both binding steps.
  • binding is quantified, typically by detecting label linked to the solid phase through binding of labelled solution antibody.
  • a calibration curve is prepared from samples containing known concentrations of target antigen. Concentrations of an SFRP-4 polypeptide in samples being tested are then read by interpolation from the calibration curve. Analyte can be measured either from the amount of labelled solution antibody bound at equilibrium or by kinetic measurements of bound labelled solution antibody at a series of time points before equilibrium is reached. The slope of such a curve is a measure of the concentration of an SFRP-4 polypeptide in a sample
  • Suitable supports for use in the above methods include, e.g., nitrocellulose membranes, nylon membranes, and derivatized nylon membranes, and also particles, such as agarose, a dextran-based gel, dipsticks, particulates, microspheres, magnetic particles, test tubes, microtiter wells, SEPHADEXTM. (Amersham Pharmacia Biotech, Piscataway N.J., and the like. Immobilization can be by absorption or by covalent attachment.
  • anti- SFRP-4 antibodies can be joined to a linker molecule, such as biotin for attachment to a surface bound linker, such as avidin.
  • the invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to treat prophylactically a subject. Accordingly, one aspect of the invention relates to diagnostic assays for determining SFRP-4 polypeptide expression in a biological sample (e.g., blood, serum, cells, tissue) in order to determine whether subject is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant SFRP-4 polypeptide expression.
  • a biological sample e.g., blood, serum, cells, tissue
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with SFRP-4 polypeptide expression or activity. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with an SFRP-4 polypeptide. Furthermore, the methods of the present invention can also be used to assess whether an individual expresses an SFRP-4 or a polymorphic form of the SFRP-4 in instances where an SFRP-4-binding agent of the present invention has greater affinity for the SFRP-4 polypeptide for its polymorphic form (or vice versa).
  • the levels of certain polypeptides in a particular tissue (or in the blood) of a subject may be indicative of the toxicity, efficacy, rate of clearance or rate of metabolism of a given drug when administered to the subject.
  • the methods described herein can also be used to determine the levels of such polypeptides in subjects to aid in predicting the response of such subjects to these drugs.
  • Another aspect of the invention provides methods for determining SFRP-4 expression in an individual to thereby select appropriate therapeutic or prophylactic compounds for that individual (referred to herein as "pharmacogenomics").
  • Pharmacogenomics allows for the selection of compounds (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular compound.)
  • an SFRP-4-binding agent of the invention to an SFRP-4 polypeptide or an SFRP-4-like polypeptide, e.g., can be utilized to identify a subject having or at risk of developing a disorder associated with SFRP-4 polypeptide or SFRP-4-like polypeptide expression or activity, as described above.
  • the prognostic assays can be utilized to identify a subject having or at risk for developing the disease or disorder.
  • the invention provides a method for identifying a disease or disorder associated with aberrant SFRP-4 polypeptide or SFRP-4-like polypeptide expression or activity in which a test sample is obtained from a subject and SFRP-4-binding agent binding is detected, wherein the presence of an alteration of SFRP-4-polypeptide or SFRP-4 like polypeptide is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant SFRP-4 polypeptide or SFRP-4-like polypeptide expression or activity.
  • a test sample refers to a biological sample obtained from a subject of interest.
  • the prognostic assays described herein can be used to determine whether a subject can be administered a compound (e.g., an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant SFRP-4 polypeptide or SFRP-4-like polypeptide expression or activity.
  • a compound e.g., an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, or other drug candidate
  • such methods can be used to determine whether a subject can be effectively treated with a compound for an SFRP-4 polypeptide or an SFRP-4-like polypeptide-associated disorder.
  • the invention provides methods for determining whether a subject can be effectively treated with a compound for a disorder associated with aberrant SFRP-4 polypeptide or SFRP-4-like polypeptide expression or activity in which a test sample is obtained and an SFRP-4 polypeptide or an SFRP-4-like polypeptide is detected using SFRP-4-binding agent ⁇ e.g., wherein the presence of an SFRP-4 polypeptide or an SFRP-4-like polypeptide is diagnostic for a subject that can be administered the compound to treat a disorder associated with aberrant SFRP-4 polypeptide or SFRP-4-like polypeptide expression or activity).
  • the level of the SFRP-4 polypeptide or the SFRP-4-like polypeptide in a blood or tissue sample obtained from a subject is determined and compared with the level found in a blood sample or a sample from the same tissue type obtained from an individual who is free of the disease.
  • An overabundance (or underabundance) of the SFRP-4 polypeptide or the SFRP-4-like polypeptide in the sample obtained from the subject suspected of having the SFRP-4 polypeptide or the SFRP-4-like polypeptide-associated disease compared with the sample obtained from the healthy subject is indicative of the SFRP-4 polypeptide or the SFRP-4-like polypeptide-associated disease in the subject being tested. Further testing may be required to make a positive diagnosis.
  • the method of detecting an SFRP-4 polypeptide or an SFRP-4-like polypeptide in a sample can be used as a method of prognosis, e.g., to evaluate the likelihood that the subject will respond to the therapy or treatment.
  • the level of the relevant prognostic polypeptide in a suitable tissue or blood sample from the subject is determined and compared with a suitable control, e.g., the level in subjects with the same disease but who have responded favourably to the treatment.
  • the degree to which the prognostic polypeptide is overexpressed (or underexpressed) in the sample compared with the control may be predictive of likelihood that the subject will not respond favourably to the treatment or therapy.
  • predictive polypeptides There are a number of diseases in which the degree of overexpression (or underexpression) of certain target polypeptides, referred to herein as "predictive polypeptides", is known to be indicative of whether a subject will develop a disease.
  • the method of detecting an SFRP-4 polypeptide or an SFRP-4-like polypeptide in a sample can be used as a method of predicting whether a subject will develop a disease.
  • the level of the relevant predictive polypeptide in a suitable tissue or blood sample from a subject at risk of developing the disease is determined and compared with a suitable control, e.g., the level in subjects who are not at risk of developing the disease.
  • a suitable control e.g., the level in subjects who are not at risk of developing the disease.
  • the degree to which the predictive polypeptide is overexpressed (or underexpressed) in the sample compared with the control may be predictive of likelihood that the subject will develop the disease. The greater the overexpression (or underexpression) relative to the control, the more likely the subject will development the disease.
  • the methods described herein can be performed, e.g. , by utilizing pre-packaged diagnostic kits comprising at least one probe reagent, e.g., SFRP-4-binding agent described herein, which can be conveniently used, e.g., in clinical settings to diagnose subjects exhibiting symptoms or family history of a disease or illness involving an SFRP-4 polypeptide or an SFRP-4-like polypeptide.
  • probe reagent e.g., SFRP-4-binding agent described herein
  • any cell type or tissue in which an SFRP-4 polypeptide or an SFRP-4-like polypeptide is expressed can be utilized in the prognostic assays described herein.
  • SFRP-4-binding agents of the present invention are useful to prevent or treat disease.
  • the invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant SFRP-4-binding agent expression or activity.
  • the present invention provides methods for the associated prevention and/or treatment of an SFRP-4-related medical condition, e.g., an EFRP-4-disorder, in a subject comprising administering an effective amount of SFRP-4-binding agent to a subject in need thereof.
  • a subject can be administered an SFRP-4-binding agent composition of the present invention in an effort to replace absent or decreased levels of the SFRP-4 polypeptide (e.g., insulin), to supplement absent or decreased levels of a different polypeptide (e.g., anti-SFRP-4 antibody), to inhibit the activity of a polypeptide (e.g., an oncogene), to activate the activity of an SFRP-4 polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth).
  • a polypeptide e.g., insulin
  • a different polypeptide e.g., anti-SFRP-4 antibody
  • an oncogene e.g., an oncogene
  • an SFRP-4 polypeptide e.g., an oncogene
  • the SFRP-4-binding agents of the present invention are useful in potential prophylactic and therapeutic applications implicated in a variety of disorders in a subject including, but not limited to: those involving development, differentiation, and activation of bone cells; in diseases or pathologies of cells in blood circulation such as red blood cells and platelets; various immunological disorders and/or pathologies; pulmonary diseases and disorders; autoimmune and inflammatory diseases; cardiovascular diseases; metabolic diseases; reproductive diseases, renal diseases, diabetes, brain trauma, cancer growth and metastasis (e.g., cancer of the brain, breast, prostate, uterine, spleen, pancreas, gastrointestinal tract (e.g., colon, rectum, small intestine, stomach, oesophagus); viral infections, cancer therapy, periodontal disease; tissue regeneration (e.g., nerve and bone); acute lymphoblastic leukaemia; gliomas; neurologic diseases; neurodegenerative disorders; haematopoietic disorders; apoptosis resulting from isch
  • neurodegenerative disease e.g., Huntington ' s Disease, peripheral demyelinating disease, Multiple Sclerosis, Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Parkinson's Disease; trauma
  • coronary heart disease e.g., inflammatory bowel disease
  • the SFRP-4-binding agents e.g., anti-SFRP-4 antibodies of the present invention are administered to the subject in effective amounts (i.e., amounts that have desired therapeutic effect). They will normally be administered parenterally.
  • the dose and dosage regimen will depend upon the degree of the SFRP-4- related disease or disorder, the characteristics of the particular SFRP-4-binding agent used, e.g., its therapeutic index, the subject, and the subject's history.
  • the SFRP-4- binding agent is administered continuously over a period of 1-2 weeks, intravenously to treat cells in the vasculature and subcutaneously and intraperitoneally to treat regional lymph nodes.
  • the administration is made during the course of adjunct therapy such as combined cycles of radiation, chemotherapeutic treatment, or administration of tumour necrosis factor, interferon or other cytoprotective or immunomodulatory agent.
  • adjunct therapy such as combined cycles of radiation, chemotherapeutic treatment, or administration of tumour necrosis factor, interferon or other cytoprotective or immunomodulatory agent.
  • the SFRP-4-binding agent will be formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically-acceptable parenteral vehicle.
  • Such vehicles are inherently nontoxic, and non-therapeutic.
  • IgG molecules can be preferred for certain applications, however IgG molecules by being smaller can be more able than IgM molecules to localize to certain types of infected cells.
  • complement activation in vivo leads to a variety of biological effects, including the induction of an inflammatory response and the activation of macrophages. Unanue & Benecerraf, Textbook of Immunology, 2nd Edition (Williams & Wilkins 1984) p. 218.
  • the increased vasodilation accompanying inflammation can increase the ability of various agents to localize in infected cells. Therefore, SFRP-4-antibody combinations of the type specified by this invention can be used therapeutically in many ways.
  • antigens e.g., purified SFRP-4 polypeptides, fragments or analogs thereof, (Hakomori, Ann. Rev. Immunol. 2: 103, 1984) or anti-idiotypic antibodies (Nepom et ah, Proc. Natl. Acad. ScL USA 81 : 2864, 1985; Koprowski et al, Proc. Natl. Acad. ScI USA 81: 216, 1984) relating to such antigens could be used to induce an active immune response in human subjects.
  • a response includes the formation of antibodies capable of activating human complement for a desirable biological effect, e.g., target cell destruction.
  • an SFRP-4 polypeptide can be treated with an SFRP-4-binding agent-based therapeutic compounds that antagonize (i.e., reduce or inhibit) activity, which can be administered in a therapeutic or prophylactic manner.
  • Therapeutic compounds that can be utilized include, but are not limited to: (i) an aforementioned SFRP-4-binding agent; and (ii) nucleic acids encoding an SFRP-4-binding agent.
  • SFRP-4-binding agent -based therapeutic compounds that increase (i.e., are agonists to) SFRP-4 activity.
  • Therapeutics that upregulate activity can be administered in a therapeutic or prophylactic manner.
  • Therapeutics that can be utilized include, but are not limited to, SFRP-4-binding agent that increases bioavailability.
  • Increased or decreased levels can be readily detected by quantifying an SFRP-4- binding agent-induced peptides and/or RNA, by obtaining a subject's tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed SFRP-4 polypeptide (or mRNAs of an aforementioned polypeptide).
  • tissue sample e.g., from biopsy tissue
  • Methods that are well known within the art include, but are not limited to, immunoassays (e.g., by
  • SFRP-4 is associated with numerous diseases and disorder, all of which could be affected by the development of an SFRP-4 binding agent.
  • SFRP-4-binding agents of the present invention are useful for the prophylactic treatment, or therapeutic treatment of disorders manifested by changes, e.g., increase or decrease, in SFRP-4 polypeptide expression, e.g., cancer (e.g., brain, breast, prostate, uterine, spleen, pancreas, gastrointestinal tract (e.g., colon, rectum, small intestine, stomach, oesophagus); apoptosis resulting from ischemia, (e.g., heart failure, myocardial infarction; stroke); neurodegenerative disease .(e.g., Huntington's Disease, peripheral demyelinating disease, Multiple Sclerosis, Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Parkinson's Disease; trauma); coronary heart disease, inflammation (e.g., inflammatory bowel disease).
  • cancer e.g., brain, breast, prostate, uterine, spleen, pancreas, gastrointestinal tract (e.g.,
  • the SFRP-4 binding agents of the present invention are useful in the detection, prevention or therapeutic treatment of breast cancer.
  • Wong and co-workers examined the role of SFRP and its relationship to the Wnt-signalling pathway in breast cancer.
  • In situ hybridization and immunohistochemical analyses of SFRP, WnM, APC, beta-catenin, and its target genes c-myc and cyclin Dl were conducted in 70 specimens of invasive ductal carcinomas of the human breast.
  • SFRP mRNA was down-regulated in 62 and elevated in eight tumour specimens, compared with adjacent normal tissues. In the course of tumour progression, however, SFRP mRNA steadily increased in both tumour and the adjacent tissues.
  • the SFRP-4 binding agents of the present invention are useful in the detection, prevention or therapeutic treatment of overload induced heart failure, myocardial hypertrophy, and apoptosis in subjects with apoptosis susceptible myocyte phenotype.
  • Schumann and co- workers compared myocardial mRNA expression of SFRPs and the level of soluble beta- catenin in tissue samples from nonfailing and failing hearts. Schumann, et al. (2002) Cardiovascular Res 45: 720-728, 2002.
  • the mRNA levels of proapoptotic sFRPs 3 and 4 but not of sFRP 1 and 2 were elevated in failing ventricles compared to donor hearts.
  • sFRPs 3 and 4 were expressed in cardiomyocytes and their expression correlated with the mRNA expression of the proapoptotic Fas/Fas-antagonist ratio, but inversely with the mRNA levels of the antiapoptotic bcl-xL.
  • the results support the hypothesis that in failing human myocardium the Wnt/beta-catenin pathway is attenuated by enhanced expression of two endogenous Wnt-antagonists. This may contribute to an apoptosis susceptible phenotype of overloaded human myocardium.
  • the SFRP-4 binding agents of the present invention are useful in the detection, prevention or therapeutic treatment of uterine cancer, e.g., endometrial cancer.
  • Hrzenjak and co-workers have observed an inverse correlation of secreted frizzled-related protein 4 and beta-catenin expression in endometrial stromal sarcomas. Hrzenjak et ah, J Pathol. 204(1): 19-27, 2004.
  • cDNA array analysis more than 300 genes deregulated in endometrial stromal sarcoma were selected and sequenced. Among the most significantly deregulated genes were those of SFRPs, in particular SFRP4.
  • SFRP4 may therefore be a putative tumour suppressor involved in deregulation of the Wnt pathway and in the pathogenesis of ESS and UES.
  • the SFRP-4 binding agents of the present invention are useful in the detection, prevention or therapeutic treatment of uterine cancer, e.g., endometrial cancer and breast cancer.
  • Abuh-Jawdeh and co-workers used the differential display technique to identify genes that are differentially expressed in human endometrial carcinoma compared with normal endometrium, and cloned SFRP.
  • SFRP was determined to be expressed by mesenchymal cells but not by epithelial cells.
  • SFRP SFRP-4 binding agents of the present invention are useful in the detection, prevention or therapeutic treatment of prostatic cancer.
  • Horvath and co-workers have demonstrated that membranous expression of secreted frizzled-related protein 4 predicts for good prognosis in localized prostate cancer and inhibits PC3 cellular proliferation in vitro. Horvath et al, Clin. Cancer Res. 10: 615-25, 2004. Because SFRP-4 mRNA is overexpressed in prostate cancers (PCs), the aim of Horvath' s study was to define the pattern of SFRP-4 protein expression in normal and malignant human prostate tissue and to determine whether changes in expression were associated with disease progression and prognosis, as well as to define the phenotype of SFRP-4-overexpression in an in vitro model of PC.
  • PCs prostate cancers
  • the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant SFRP-4 expression or activity, by administering to the subject an SFRP-4-binding agent that modulates an SFRP-4 polypeptide expression or at least one SFRP-4 polypeptide activity.
  • Subjects at risk for a disease that is caused or contributed to by aberrant an SFRP-4 polypeptide expression or activity can be identified by, e.g., any or a combination of diagnostic or prognostic assays as described herein.
  • compositions or medicaments of SFRP-4-binding agents are administered to a subject susceptible to, or otherwise at risk of a disease or condition (i.e.. an immune disease) in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the outset of the disease, including biochemical, histologic and/or behavioural symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease.
  • Administration of a prophylactic SFRP-4-binding agent can occur prior to the manifestation of symptoms characteristic of the aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • an SFRP-4-binding agent which acts as an SFRP-4 agonist or an SFRP -4 antagonist can be used for treating the subject.
  • the appropriate compound can be determined based on screening assays described herein.
  • compositions or medicaments are administered to a subject suspected of, or already suffering from such a disease in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease (biochemical, histologic and/or behavioural), including its complications and intermediate pathological phenotypes in development of the disease.
  • An amount adequate to accomplish therapeutic or prophylactic treatment is defined as a therapeutically- or prophylactically- effective dose.
  • a compound that modulates an SFRP-4 polypeptide activity is described herein, and may include, e.g., a nucleic acid encoding an SFRP-4-binding agent or an SFRP-4-binding agent polypeptide.
  • the SFRP-4-binding agent stimulates one or more SFRP-4 polypeptide activity.
  • Examples of such stimulatory compounds include an SFRP-4- binding agent and a nucleic acid molecule(z.e., polynucleotide) encoding an SFRP-4-binding agent that has been introduced into the cell.
  • the SFRP-4-binding agent inhibits one or more SFRP-4 polypeptide activity.
  • modulatory methods can be performed in vitro (e.g., by culturing the cell with the SFRP-4-binding agent) or, alternatively, in vivo (e.g., by administering the SFRP-4-binding agent to a subject).
  • the invention provides methods of treating an individual afflicted with an SFRP-4-associated disease or disorder characterized by aberrant expression or activity of an SFRP-4 polypeptide or nucleic acid molecules encoding SFRP-4 polypeptide.
  • the method involves administering an SFRP-4-binding agent (e.g., a compound identified by a screening assay described herein), or combination an SFRP-4-binding agents that modulates (e.g., up-regulates or down-regulates) SFRP-4 expression or activity.
  • an SFRP-4-binding agent e.g., a compound identified by a screening assay described herein
  • the method involves administering an SFRP-4-binding agent or nucleic acid molecule encoding SFRP-4- binding agent as therapy to compensate for reduced or aberrant SFRP-4 expression or activity. Stimulation of SFRP-4 activity is desirable in situations in which an SFRP-4 polypeptide is abnormally downregulated.
  • in vitro or in vivo assays are performed to determine the effect of a specific SFRP-4-binding agent-based therapeutic and whether its administration is indicated for treatment of the affected tissue in a subject.
  • in vitro assays can be performed with representative cells of the types involved in the subject's disorder, to determine if a given SFRP-4-binding agent- based therapeutic exerts the desired effect upon the cell types.
  • SFRP-4-binding agents for use in therapy can be tested in suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects. Similarly, for in vivo testing, any of the animal model system known in the art can be used prior to administration to human subjects.
  • compositions include a combination of multiple ⁇ e.g., two or more) SFRP-4-binding agents of the invention.
  • each of the SFRP-4-binding agents thereof of the composition is a monoclonal antibody or a human sequence antibody that binds to a distinct, pre-selected epitope of an SFRP-4 polypeptide.
  • Effective doses of the SFRP-4-binding agents of the present invention e.g., anti- SFRP-4 antibodies or anti-SFRP-4 antibody cytotoxin conjugates, for the treatment of SFRP-4-related conditions and diseases described herein vary depending upon many different factors, including means of administration, target site, physiological state of the subject, whether the subject is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic.
  • the subject is a human but nonhuman mammals including transgenic mammals can also be treated. Treatment dosages need to be titrated to optimize safety and efficacy.
  • an effective amount of the compositions of the present invention ranges from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day.
  • the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day.
  • the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg per day, of the host body weight.
  • dosages can be 1 mg/kg body weight or 10 mg/kg body weight per day or within the range of 1-10 mg/kg per day.
  • An exemplary treatment regime entails administration once per every two weeks or once a month or once every 3 to 6 months.
  • two or more SFRP-4 binding agents with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated.
  • SFRP-4-binding agent e.g., anti-SFRP-4 antibody is usually 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 antibody in the subject.
  • dosage is adjusted to achieve a plasma SFRP-4-binding agent, e.g., anti-SFRP-4 antibody concentration, of 1-1000 ⁇ g/ml and in some methods 25-300 ⁇ g/ml.
  • SFRP-4-binding agent e.g., anti- SFRP-4 antibody
  • Dosage and frequency vary depending on the half-life of the SFRP-4-binding agent in the subject.
  • human anti-SFRP-4 antibodies show the longest half life, followed by humanized anti-SFRP-4 antibodies, chimaeric anti-SFRP-4 antibodies, and nonhuman anti-SFRP-4 antibodies.
  • the dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some subjects continue to receive treatment for the rest of their lives.
  • a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patent can be administered a prophylactic regime.
  • Doses for nucleic acids encoding SFRP-4 immunogens range from about 10 ng to 1 g, 100 ng to 100 mg, 1 ⁇ g to 10 mg, or 30- 300 ⁇ g DNA per subject.
  • Doses for infectious viral vectors vary from 10-100, or more, virions per dose.
  • Toxicity Preferably, an effective dose of the SFRP-4-binding agents described herein will provide therapeutic benefit without causing substantial toxicity to the subject.
  • Toxicity of the SFRP-4-binding agent described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. , by determining the LD 50 (the dose lethal to 50% of the population) or the LD 100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human.
  • kits comprising the SFRP-4-binding agent compositions ⁇ e.g., antibody cytotoxin conjugates, monoclonal antibodies, human sequence antibodies, human antibodies, multispecific and bispecific molecules) of the invention and instructions for use.
  • kits are useful for detecting the presence of an SFRP-4 polypeptide or an SFRP-4-like polypeptide in a biological sample.
  • the kit can comprise: a labelled SFRP-4-binding agent capable of detecting an SFRP-4 polypeptide or an SFRP-4-like polypeptide in a biological sample; means for determining the amount of the SFRP-4 polypeptide or the SFRP-4-like polypeptide in the sample; and means for comparing the amount of the SFRP-4 polypeptide or the SFRP-4-like polypeptide in the sample with a standard.
  • the compound or compound can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect the SFRP-4 polypeptide or the SFRP-4-like polypeptide.
  • primers were used to amplify the nucleic acid sequence encoding human SFRP-4 polypeptide: sense primer 5 'CCCAAGCTTGCAGTGCCATGTTCCTCTCCATCCS' (SEQ ID NO: 11); anti-sense primer
  • the amplicon was gel purified (Qiagen Inc., Valencia, CA; Product No. 28704) and ligated into pCR-XL-TOPO according to the manufacturer's instructions (Invitrogen, Carlsbad, CA; Product No.45-0008). Sequence fidelity was confirmed by Solvias AG (Basel, Switzerland). The TOPO-construct was amplified in the host strain E. coli Top 10 (Invitrogen, Carlsbad, CA; Product No.44-0301).
  • the insert was excised with HindIII and Xbal, purified and ligated into pRS5a (origin: Novartis) using T4 DNA ligase (Roche Pharmaceuticals, Inc., Nutley, NJ; Product No. 481220).
  • the pRS5a construct was termed k 'pSFRP4fullHis" (PlasNova entry no.: NPL006815).
  • the pSFRP4fullHis construct encoded the natural Kozac and leader sequence, the complete coding sequence (CDS) and a His-tag directly attached to the last amino acid of the human SFRP-4 polypeptide. DNA and amino acid sequences were analyzed using the BLAST network service at the National Center for Biotechnology Information.
  • HEK293 Ebna cells were seeded in 6- well-clusters (10 6 cells/well) under standard growth conditions and incubated overnight; at 95 % confluence, cells in each well were transfected with 2 ⁇ g pSFRP4fullHis or pRS5a control using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen, Carlsbad, CA; Product No. 11668-027). Each well contained 2.5 ml medium. For transient production, cells were transferred after 48 h in FCS free DMEM (Invitrogen-Gibco, Carlsbad, CA; Product No. 31966-021).
  • Control transfections were performed with pRK-LacZ-1 encoding ⁇ -galactosidase (Novartis Pharma AG). The enzyme activity was visualized after 48 h on cellular level by staining with colourigenic substrate (Stratagene, La Jolla, Calif; Product No. 200384-2).
  • SFRP-4 production Hek293 Ebna cells were transfected with pRK-LacZ-1, pRS5a or pSFRP4fullHis. Transfection efficiency was approximately 60 % as estimated by in situ ⁇ -galactosidase staining of pRK-LacZ-1 control. Cells transfected with pSFRP4fullHis showed altered growth behaviour. They formed aggregates and died from 72 h onwards after transfer into FCS free medium. Only a few stable cell lines were obtained after 8-10 weeks of selection. In contrast, controls containing the vector alone showed normal growth behaviour.
  • N-Glycosidase F also known as PNGase F
  • PNGase F is an amidase that cleaves between the innermost GIcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins (Maley et al., Anal. Biochem., 180, 195-204, 1989).
  • PNGase F treatment of SFRP-4-containing culture supernatant converted the 55 kDa band SFRP-4 immunoreactive polypeptide to a polypeptide which migrated as a distinct band at 40 kDa (FIG. 1, panel B, lane 1 and lane 2 versus lane 3). This experiment showed that the fragment formation occurring over time was due to partial glycoslyation and not to proteolytic degradation.
  • the eluate was concentrated to 300 ⁇ l using a 0.5 ml Microcon YM-30 (Millipore Corporation, Bedford, MA; Product No. 42409).
  • the protein concentration was determined with the BCA method (Pierce Chemical Co., Rockford, IL, Product No. 23235).
  • the purity of the protein was determined on a NuPAGE 4-12 % Bis-Tris gel (Invitrogen. Carlsbad, CA, Product No. NP0323BOX) using Laemmli Sample Buffer with 10 % beta-mercaptoethanol (Bio-Rad, Hercules, CA, Product No. 161-0737), NuPAGE running buffer (Bio-Rad, Carlsbad, CA; Product No.
  • SFRP-4 peptide preparation for polyclonal antibody generation.
  • the amino acid sequence of SFRP-4 was analyzed using Biobench2 (Novartis) and the BLAST network service at the National Center for Biotechnology Information.
  • Two peptides of human SFRP-4, corresponding to amino acid residues 292-305 and amino acid 305-319, were selected a immunogens based on based on their potential antigenicity, hydrophobicity, surface probability, glycosylation probability and specificity.
  • the peptides chosen were identical in human and cynomolgus monkeys ⁇ Macaca fascicularis) SFRP -4.
  • Anti-SFRP-4 antibody production was analyzed by direct ELISA using standard methods. Briefly, the OVA-conjugated peptides EP040755 and EP040756 were coated separately onto MaxisorbTM (Nunc) at a concentration of 1 ⁇ g/mL. Plates were washed and blocked with SuperblockTM (Pierce) blocking medium. Serum samples and affinity-purified antibodies at various dilutions were added and incubated for 3 h at room temperature. Bound rabbit antibodies were detected by HRP-conjugated goat-anti- rabbit IgG Fc antibodies (Jackson ImmunoResearch, West Grove, Perm.; Product No.
  • Antibody characterization Antibody quality was analyzed by direct ELISA using standard methods. Recombinant SFRP-4 was coated onto MaxisorbTM (Nunc) at concentrations from 0-300 ng/mL. Plates were washed and blocked with SuperblockTM (Pierce) blocking medium. Serum samples and affinity-purified antibodies at various dilutions were added and incubated for 3 hours at room temperature.
  • Bound rabbit antibodies were detected by HRP-conjugated goat-anti-rabbit IgG Fc antibodies (Jackson ImmunoResearch, West Grove, PA; Product No. 111-035-046) and colourigenic substrate (Roche Diagnostics (a.k.a., Roche Molecular Systems, Inc.), Almeda, CA; Product No. 1484281). Signal intensity was detected with a Molecular Devices Thermomax microplate reader and data analyzed using Softmax Pro 3.1.
  • Anti-EP040755 was additionally evaluated by Western blot (WB) using a HRP- conjugated goat-anti-rabbit IgG Fc antibody (Jackson ImmunoResearch, West Grove, Pennsylvania; Product No. 111-035-046) and chemiluminogenic substrate (Hoffmann-La Roche Inc., Nutley, New Jersey; Product No. 2015196) for detection.
  • WB Western blot
  • chemiluminogenic substrate Hoffmann-La Roche Inc., Nutley, New Jersey; Product No. 2015196
  • Polyclonal antibody generation The ability of rabbit-antisera and affinity purified anti-EP040755 and anti-EP040756 to detect the respective OVA-coupled peptide in a direct ELISA was compared to preimmune sera. Antisera of both rabbits detected both antigens and recombinant SFRP-4 polypeptide.
  • Affinity purified antibodies detected their respective antigen. However, only purified anti-EP040755 detected also recombinant SFRP-4 polypeptide. The antibody (1 ⁇ g/ml) detected 8 ng/ml recombinant SFRP-4 polypeptide. Based on the known serum/plasma concentration of 5.5-80 ng/ml SFRP-4 polypeptide in man, this antibody was considered to be sensitive enough to measure SFRP-4 in monkey plasma. The affinity purified anti-EP040755 was used to set up an anti-SFRP-4 ELISA in monkey plasma. In Western blot analysis the detection limit of SFRP-4 using anti-EP040755 was 75 ng absolute amount.
  • the objective of the present study was to construct an SFRP-4 ELISA assay useful to detect SFRP-4 immunoreactive polypeptide in biological sample. Specifically, an SFRP-4-
  • ELISA assay was developed and used to detect SFRP-4 immunoreactive polypeptide in the serum of female ovariectomized cynomolgus monkeys (Macaca fascicularis) over a 4 week period.
  • SFRP-4 Quantification of SFRP-4 in plasma by ELISA.
  • SFRP-4 was determined using a specific sandwich ELISA assay.
  • the sandwich ELISA for SFRP-4 utilized two specific polyclonal antibodies for this protein, an affinity purified rabbit antibody to SFRP-4 peptide
  • Cs were prepared in a pool of equal portions of the cynomolgus plasmas, lot number: CmoPla002, CmoPla042, CmoPla043, CmoPla044, CmoPla45.
  • SFRP-4-rec. protein Origin In-house production at MAD (CH) Coating antibody Goat-anti-SFRP-4, R&D Systems No. Al 827 Detection antibody Rabbit-anti-SFRP-4 (origin: Eurogentec), affinity purified pAb to SFRP-4 peptide EP040755
  • Acceptance criteria Mean accuracy: 80 % to 120 % (70 % -130 % at the LLOQ and
  • the assay is specific for SFRP-4
  • High Throughput Western Blot The High Throughput Western Blot - 31 Human Adult Normal Tissues (Cat. No. W8234480-B) was performed according to the manufacturers instructions (BioCAT GmbH, Heidelberg, Germany).As primary antibody 2 ⁇ g/mL affinity purified rabbit anti-SFRP4 EP040755 (see EXAMPLE I) was used. As detection antibody 20 ng/niL goat anti-rabbit IgG (H+L)-HRP (Pierce cat no. 34075) was used. See FIG. 3. [227] High throughput Western blot with 31 different individual human tissues showed the highest SFRP-4 protein level in heart. This is useful for the detection of SFRP-4 as a disease biomarker (diagnosis, prognostic, disease staging, drug effect) for heart infarction, myopathy or coronary heart disease
  • High throughput Western blot showed high levels of SFRP-4 in colon, rectum, duodenum, and less but still over average expression in ileum, jejunum, oesophagus. This is useful for the detection of SFRP-4 as a disease biomarker (diagnosis, prognostic, disease staging, and drug effect) for colorectal cancer and other cancers of the digestion tract and as marker for the grade of inflammatory diseases of the digestion tract.
  • High throughput Western blot showed over average levels of SFRP-4 in spleen, and pancreas. This is useful for the detection of SFRP-4 as a disease biomarker (diagnosis, prognostic, disease staging, and drug effect) for spleen or pancreas cancer.
  • High throughput Western blot showed over average levels of SFRP-4 in brain. This is useful for the detection of SFRP-4 as a disease biomarker (diagnosis, prognostic, disease staging, and drug effect) for neuro-degenerative diseases (e.g. Huntington, ALS, Alzheimer's disease, MS, Parkinson, peripheral demyelinating disease) and brain tumours.
  • SFRP-4 a disease biomarker
  • neuro-degenerative diseases e.g. Huntington, ALS, Alzheimer's disease, MS, Parkinson, peripheral demyelinating disease

Abstract

La présente invention concerne de manière générale la préparation d'agents de liaison du SFRP-4 et leur utilisation. L'invention porte en particulier sur la préparation d'anticorps anti-SFRP-4 et de leur utilisation pour détecter le SFRP-4 et moduler les fonctions induites par le SFRP-4. Les compositions et les procédés de l'invention peuvent être utilisés dans divers tissus en vue de détecter des troubles manifestés par une modification de l'expression du polypeptide SFRP-4 et également de traiter et prévenir de tels troubles associés au polypeptide SFRP-4 chez un sujet nécessitant un tel traitement.
PCT/EP2007/051229 2006-02-09 2007-02-08 Agents de liaison de la proteine-4 apparentee a frizzled (sfrp-4) WO2007090872A2 (fr)

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AU2007213655A AU2007213655A1 (en) 2006-02-09 2007-02-08 Antibodies against secreted fri zzled related protein-4 (SFRP-4 )
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BRPI0710442-1A BRPI0710442A2 (pt) 2006-02-09 2007-02-08 agentes de ligação de proteìna-4 secretada relacionada com frizzled(sfrp-4)

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