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WO2000069454A1 - Suppression de l'igfbp-2 endogene visant a inhiber le cancer - Google Patents

Suppression de l'igfbp-2 endogene visant a inhiber le cancer Download PDF

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WO2000069454A1
WO2000069454A1 PCT/US2000/013574 US0013574W WO0069454A1 WO 2000069454 A1 WO2000069454 A1 WO 2000069454A1 US 0013574 W US0013574 W US 0013574W WO 0069454 A1 WO0069454 A1 WO 0069454A1
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igfbp
cell
cells
cancer
tumor
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WO2000069454A8 (fr
WO2000069454A9 (fr
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Shila Chakrabarty
Pomila Singh
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University of Texas System
University of Texas at Austin
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University of Texas System
University of Texas at Austin
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    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1754—Insulin-like growth factor binding proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/30—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • 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
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4745—Insulin-like growth factor binding protein

Definitions

  • the present invention relates generally to the fields of biochemistry and cancer More particularly, it concerns Insulin-like Growth Factor Binding Protein 2 (IGFBP-2) and its relevance to cancer
  • Prostate cancer is the most common non-skin cancer diagnosed in men in the United States With about 184,000 new cases found, and about 39,200 deaths attributed to prostate cancer each year, it is second only to lung cancer as the most common cause of death from malignancy
  • the cause of prostate cancer is unknown, and the tumor often remains clinically silent until it has reached an advanced stage in men over 60 years of age
  • the methods currently used to diagnose prostate cancer such as physical exam, monitoring Prostate-Specific Antigen (PSA) levels, tissue biopsy and ultrasound and bone scans, are restricted in both sensitivity and specificity Since the survival of prostate cancer patients depends on early detection and treatment, novel strategies for early diagnosis and treatment of the disease must be developed
  • IGF Insulin-like Growth Factor
  • the Insulin-like Growth Factor (IGF) system is a multi-component network of molecules (Juul et al, 1995) involved in the regulation of cell growth in a variety of organs (Rajaram et al. 1997), including the prostate (Cohen et ai, 1994, Thrasher et al, 1996)
  • the IGF binding proteins (IGFBPs) are a family of high affinity binding proteins, which regulate IGF availability to the receptors, and are now being recognized as a new class of growth modulators (Cohen and Rosenfeld, 1994)
  • IGFBP-2 is elevated in the serum of patients with prostate cancer (Ho and Baxter, 1997, Kanety et al, 1993, Figueroa et al., 1998), but not in benign prostatic hyperplasia (BPH) (Ho and Baxter, 1997, Cohen et al, 1993) Elevation of IGFBP-2 is related to the stage of the tumor and serum prostate-specific anti
  • the present invention discloses a method of inhibiting an IGFBP-2 dependent cancer cell comprising reducing IGFBP-2 activity level with an IGFBP-2 modulator
  • Inhibiting an IGFBP-2 dependent cancer cell includes altering proliferation, metastasis, contact inhibition, soft agar growth, cell cycle regulation, tumor formation, tumor progression, differentiation, or tumor invasion
  • the IGFBP-2 modulator may decrease the amount of IGFBP-2, inhibit expression, transcription, translation, secretion, or inhibit the functional activity of IGFBP-2
  • the IGFBP-2 modulator may be an agonist or antagonist of IGFBP-2
  • the modulator of IGFBP-2 is a nucleic acid containing a promoter operably linked to a IGFBP-2 gene segment
  • the IGFBP-2 gene segment is positioned, in reverse orientation, under the control of a promoter that directs expression of an antisense product
  • the nucleic acid encodes a ribozyme specific for an RNA transcript of IGFBP-2 in a cell expressing an RNA transcript of IGFBP-2
  • the promoter that is operably linked to a gene segment that encodes IGFBP-2 functions in a mammalian cell
  • the nucleic acid segment containing a promoter operably linked to a IGFBP-2 gene segment may be located on a vector, such as a plasmid vector
  • the vector is a viral vector selected from the group consisting of adenovirus, retrovirus, herpes virus, adeno-associated virus, and vaccinia virus
  • the nucleic acid may be packaged in a virus particle
  • the promoter that is operably linked to a gene segment that encodes IGFBP-2 functions in a mammalian cell
  • the IGFBP-2 modulator is an antibody composition comprising an antibody that recognizes IGFBP-2
  • the antibody composition may comprise a monoclonal antibody, polyclonal antibodies, or a humanized antibody
  • the IGFBP-2 modulator may be at least one polypeptide that interacts with IGFBP-2, and may comprises IGF-I, IGF-II, IFGBP-2 receptor, or a fusion protein or fragment thereof
  • the IGFBP-2 modulator may also be a polypeptide involved in post-translational modification of IGFBP-2
  • the IGFBP-2 dependent cell may be in cell culture or in an animal The animal may be a mammal and may be a human
  • the IGFBP-2 dependent cell may be derived from the brain (e.g , glioblastoma, astrocytoma, oligodendroglioma, ependymomas), neurofibrosarcoma, meningia, lung, liver, spleen, kidney, lymph node, pancreas, small intestine, blood cells, colon or colorectal region, stomach, thyroid, breast, endomet ⁇ um, prostate, testes, ovary, skin, head and neck, esophagus, bone marrow, blood or other tissue
  • the cell is derived from the prostate
  • the IGFBP-2 modulator may be an antibody composition comprising an antibody that recognizes IGFBP-2
  • the antibody composition may comprises a monoclonal antibody, polyclonal antibodies, or a humanized antibody
  • the modulator may be a ribozyme or antisense sequence complementary to the IGFBP-2 gene
  • Treating a subject with cancer may result in killing a cancer cell, inhibiting the growth of a cancer cell, inducing apoptosis of a cancer cell, inhibiting metastatic potential of a cancer cell, reducing tumor burden, or inducing tumor regression
  • the cancer may be a cancer of the brain (e.g , glioblastoma, astrocytoma, oligodendroglioma, ependymomas), neurofibrosarcoma, meningia, lung, liver, spleen, kidney, lymph node, pancreas, small intestine, blood cells, colon or colorectal region, stomach, thyroid, breast, endometrium, prostate, testes, ovary, skin, head and neck, esophagus, bone marrow, blood or other tissue
  • the cancer is prostate cancer
  • the method of treating a subject with cancer may also comprise treating the subject with a second agent, wherein the second agent is a second modulator of IGFBP-2, a therapeutic polypeptide, nucleic acid encoding a therapeutic polypeptide, a chemotherapeutic agent, or a radiotherapeutic agent
  • Also disclosed is a method of screening a candidate substance for anti-tumor activity comprising the steps of contacting a first IGFBP-2 dependent cell with a candidate substance, and comparing one or more characteristics of the first cell in the presence of the candidate substance with one or more characteristics of a second
  • IGFBP-2 dependent cell in the absence of the candidate substance Characteristic that can be examined may be selected from the group consisting of IGFBP-2 expression, proliferation, metastasis, contact inhibition, soft agar growth, cell cycle regulation, tumor formation, tumor progression, and tumor invasion
  • the IGFBP-2 dependent cell may be contacted in vitro or in vivo
  • the methods of treating a cancer patient involve administering to the patient a composition that contains a peptide having at least 8 contiguous amino acids from the amino acid sequence of human IGFBP-2 (SEQ ID NO 2) in an amount effective to convey a therapeutic benefit on the patient
  • therapeutic benefit used throughout this application refers to anything that promotes or enhances the well- being of the subject with respect to the medical treatment of his condition, which includes treatment of cancer or other hyperproliferative diseases
  • a list of nonexhaustive examples of such benefit includes extension of the subject's life by any period of time, decrease or delay in the neoplastic development of the disease, decrease in hyperproliferation, reduction in tumor growth or size, delay of metastases, reduction in cancer cell or tumor cell proliferation rate, and a decrease in pain to the subject that can be attributed to the subject's condition
  • the therapeutic benefit conveyed on the patient may generally involve inhibition of
  • the prevenative and therapeutic methods involve a composition that includes an adjuvant in addition to an IGFBP-2 peptide or polypeptide
  • the adjuvant may or may not be linked to the peptide or polypeptide If the adjuvant is linked to the IGFBP-2 peptide or polypeptide, they may be covalently linked
  • the IGFBP-2 peptide may be of any length, though the peptide of SEQ ID NO 3 has been used to generate antibodies
  • a peptide that contains at least 8 contiguous amino acids from SEQ ID NO 3 may be employed in the methods of the present invention, as well as a peptide comprising SEQ ID NO 3
  • the therapeutic methods of the invention may be employed in combination with another anti-cancer treatment such as chemotherapy, radiotherapy, surgery, hormone therapy, or gene therapy, which may be administered before administration of the IGFBP-2 directed agent, which includes an IGFPB-2 peptide that confers a therapeutic benefit, as well as after or at the same time as the
  • the preventative methods of the invention rely upon the immune system to effect a response against IGFBP-2
  • An immune response includes a humoral as well as a cellular or cell-mediated response
  • a subject could be immunized with an IGFBP-2 peptide or polypeptide to generate a level of humoral and/or cellular immunity against IGFBP-2
  • Multiple immunizations or boosters may be given to a subject to maintain the immune response
  • compositions comprising a recombinant vector containing a promoter operably linked to a IGFBP-2 gene segment
  • the pharmaceutical composition may have the IGFBP-2 gene segment positioned, in reverse orientation, under the control of a promoter that directs expression of an antisense product
  • the pharmaceutical composition may be a recombinant vector expressing a ribozyme specific for an RNA transcript of IGFBP-2 in a cell expressing an RNA transcript of IGFBP-2
  • kits comprising a suitable container and at least a first antibody composition that recognizes IGFBP-2 or a kit comprising a suitable container and at least a recombinant vector containing a promoter operably linked to a nucleic acid segment that encodes IGFBP-2
  • FIG. IA, FIG. IB, FIG. IC, and FIG. ID Effect of IGFBP-2 and IGFBP-2 antibody (Ab) on the growth of LNCap and DU145 prostate cancer cells
  • LNCap and DU145 cells were treated with different doses of IGFBP-2 or IGFBP-2 Ab, with or without IGF-I (250 ng/ml), and growth was measured by counting the total number of cells (FIG IA - LNCap, FIG IB - DU145), or by an MTT assay (FIG IC - LNCap, FIG 1D - DU145)
  • the Y axis of panels FIG IC and FIG ID represent Optical Density at 560 nm In the sections designated by "Serum” and "Control,” the three bars represent growth of Control cells in 1%, 5% and 10% concentrations of fetal calf serum (FCS)
  • FCS fetal calf serum
  • FIG. 2 Tumorigenic potential of DU145 cells grown in the presence of
  • IGFBP-2 or IGFBP-2 Ab determined in a soft agar clonogenic assay The total number of colonies/well in wells seeded with IGFBP-2 Ab was less than 1-5% of the numbers in wells seeded with IGFBP-2
  • FIG. 3 Growth of AS-clones of LNCap cells in the presence of different concentrations of serum Growth was measured in terms of optical density (O D ) by an MTT assay Results are mean ⁇ SEM of 8 observations with the representative clones
  • FIG. 4 IGF-I protein levels in CM collected from Control, IGF and IGFBP-2 treated cultures of DU145 cells The levels of IGF increase 3-4 fold times following treatment with IGF However, the levels of IGF are undetectable following treatment with IGFBP-2 antibody
  • FIG. 5 Initiation of DU145-generated tumors in nude mice following inoculation with cells mixed with non-immune IgG (Control) and cells mixed with anti- IGFBP-2 IgG (Treated) The initiation of tumors was delayed significantly (p ⁇ 0 001) when cells are co-inoculated with anti-IGFBP-2
  • FIG. 6 Weight comparison of tumors generated in male athymic nude mice from wild type (WT) and anti-IGFBP-2 IgG mixed cells (Cells + IGFBP-2 antibody ("BP-2 Ab")) Tumors generated from WT cells are significantly (p ⁇ 0 001) larger than those generated from cells mixed with anti-IGFBP-2 IgG
  • FIG. 7 Effect of anti-IGFBP-2 IgG on the growth of DU145 prostatic tumors as measured by tumor weight Group 1 Inoculated with untreated cells (2 x 10 cells) on right, and 0 1 ml of 1 ml cell suspension of 20 x 10 6 cells treated with 30 ⁇ g anti-IGFBP-2 IgG on left Group II Inoculated with untreated cells (2 x lO 6 cells) on right, and 0 1 ml of 1 ml cell suspension of 20 x 10 6 cells treated with 60 ⁇ g anti-IGFBP-2 IgG on left Group III Inoculated with untreated cells (2 x 10 6 cells) on right, and 0 1 ml of 1 ml cell suspension of 20 x 10 6 cells treated with 120 ⁇ g anti-IGFBP-2 IgG on left
  • FIG. 8 Effect of anti-IGFBP-2 IgG on total tumor weight in control and treated groups of animals Animals of Group A were inoculated on the left side with 2 x 10 6 DU145 cells mixed with 12 ⁇ g of non-immune IgG, and with an equal concentration of untreated cells on the right side Group B animals were inoculated on both sides with untreated DU145 cells
  • the present invention concerns the role of IGFBP-2 in cancer Cancers that are dependent on IGFBP-2 would benefit from therapies targeting IGFBP-2
  • the invention discloses methods for inhibiting IGFBP-2 dependent cancers by modulating IGFBP-2 activity Modulation can be achieved by decreasing the amounts of IGFBP-2 protein or IGFBP-2 activity, as well as other polypeptides that interact with or modify IGFBP-2
  • Preferred modulators of IGFBP-2 include antibodies specific for IGFBP-2 and antisense or ribozymes specific for IGFBP-2 nucleic acid These modulators are useful for treating cancers derived from any tissue, with cancer of the prostate being preferred
  • Methods of treating a subject with cancer are disclosed as well as compositions of IGFBP-2 modulators and kits containing such modulators
  • the present invention is directed at methods for reducing IGFBP-2 activity in such a way as to affect an IGFBP-2 dependent cell
  • the effect on the IGFBP-2 dependent cell can be an alteration in proliferation, metastasis, contact inhibition, soft agar growth, cell cycle regulation, tumor formation, tumor progression, differentiation, or tumor invasion IGFBP-2 activity can be reduced m vitro or in vivo by a number of methods as detailed below
  • IGFBP-2 Inhibiting the synthesis is done in cells that are actively synthesizing IGFBP-2 IGFBP-2 IGFBP-2 may be synthesized by the IGFBP-2 dependent cell, where the IGFBP-2 would be acting in an autocrine manner on the same cell Alternatively, IGFBP-2 synthesis may be by a second cell distinct from the IGFBP-2 dependent cell in an endocrine manner Strategies for inhibiting the synthesis of IGFBP-2 can target transcription of the gene encoding IGFBP-2, target IGFBP-2 messenger RNA, target translation of the IGFBP-2 message, or target secretion of IGFBP-2
  • IGFBP-2 transcription The promoter for IGFBP-2 has been isolated and the inventors describe assays for determining the activity of this promoter in a given cell
  • Methods for inhibiting transcription include synthesis of antisense RNA complementary to the IGFBP-2 nucleic acid sequence Antisense RNA can interact with genomic DNN forming heteroduplexes that inhibit transcription of the D ⁇ A sequence
  • Alternative methods could include targeting transcription factors or other accessory factors involved in transcription
  • Methods for introducing genetic constructs into IGFBP-2 producing cells are described, as are methods for determining IGFBP-2 expression and screens for candidate compounds that affect IGFBP-2 transcription
  • IGFBP-2 messenger R ⁇ A Inhibition of IGFBP-2 synthesis could either be brought about by decreasing the amount of IGFBP-2 mR ⁇ A or inhibiting its translation into IGFBP-2 polypeptide
  • Methods for achieving this include producing antisense IGFBP-2 R ⁇ A that forms a duplex with IGFBP-2 mR ⁇ A or producing IGFBP-2 specific ribozymes
  • methods for introducing genetic constructs into IGFBP-2 producing cells are described, as are methods for determining IGFBP-2 expression and screens for candidate compounds that effect IGFBP-2 translation
  • IGFBP-2 is a secreted protein and methods for inhibiting its secretion would decrease IGFBP-2 activity
  • One general method of inhibiting IGFBP-2 secretion is based on producing polypetides that would interact with IGFBP-2 inside the cell and interfere with its secretion
  • polypeptides include antibodies with specificity to IGFBP-2, with single chain antibodies described as one approach
  • several polypeptides are known to interact with IGFBP-2, including IGF-I, IGF-II, and IGFBP receptors Cosynthesis of such polypeptides, or fragments or fusion proteins containing such polypeptides, could interfere with the secretion of IGFBP-2
  • Polypeptides such as those described above can also be used to bind and reduce
  • IGFBP-2 activity after it has been secreted from the cell synthesizing it.
  • Methods for using polypeptides that interact with IGFBP-2, including IGF-I, IGF-II, and IGFBP receptors, or fragments or fusion proteins containing such polypeptides are anticipated
  • antibodies specific for IGFBP-2 can be used to bind and inactivate IGFBP- 2
  • Such antibodies can be monoclonal or polyclonal as well as human or humanized antibodies
  • Polypeptides such as these that bind IGFBP-2 can lead to its inactivation, increased clearance rate or shorter half-life, all of which will decrease overall IGFBP-2 activity
  • a final method contemplated for reducing IGFBP-2 activity is to effect post- translational modifications that affect its activity
  • Post-translational modifications that can be targetted include phosphorylation, proteolysis and glycosylation, as well as polypeptides involved in these modifications, including kinases, phosphatases and proteases
  • IGFBP-2 is a major prostatic binding protein and is dramatically elevated in serum of all patients with prostate cancer, but not in non-cancerous benign prostatic hyperplasia; the degree of elevation is directly related to the severity of the cancer This suggests that IGFBP-2 may be involved in the development and/or progression of prostate cancer.
  • IGFs Insulin-like Growth Factors
  • IGF-I and IGF-II are mitogenic in vitro for a wide variety of cells in tissue culture
  • IGF-I insulin-like Growth Factors
  • Both IGFs stimulate in vitro the growth of various tissues and in particular they induce collagen synthesis
  • IGF-I mediates the growth promoting effect of growth hormone in chondrogenesis and bone formation and is therefore essential for normal growth of an organism This is demonstrated by the fact that pygmies and toy poodles are deficient in IGF-I, but have normal growth hormone level in their serum.
  • IGF-II is believed to play a key role in fetal development and nerve growth.
  • IGF-I and IGF-II also exhibit growth-stimulating functions on other tissues
  • Wound fibroblasts are known to produce IGFs that are effective in stimulating fibroblasts to grow and synthesize collagen, a structural protein normally required for wound healing Vascularization of the wound tissue also is induced. Further, it has also been found that IGFs have an erythropoietin-like activity in that they induce hematopoiesis
  • IGFs produced by certain cancer cells e.g., breast and kidney cancer cells
  • both IGFs show a spectrum of metabolic activities similar to those of insulin, in that they stimulate, in particular, the transport and metabolism of glucose
  • the biological effects of IGFs and insulin are mediated through their binding to specific receptors
  • both IGFs have the ability to bind to the insulin receptor with approximately 100-fold lower affinity than does insulin
  • IGFs have a concentration in blood approximately a hundred-fold higher than that of insulin Hypoglycemia is prevented by a regulatory mechanism that involves carrier proteins present in blood and able to form complexes with IGFs
  • IGFs circulate in the blood in the form of a complex that has no insulin-like activity Through their association with carrier protein (hereinafter referred to as IGF-binding proteins or IGFBPs), binding of IGFs to cell surface receptors is inhibited
  • IGF-binding proteins carrier protein
  • IGF binding proteins In the circulation, in other body fluids, and in media conditioned by cultured cells, the somatomedins (IGF-I and IGF-II) are bound to specific high-affinity carrier proteins that have been implicated as modulators of IGF actions
  • IGF binding proteins The history of IGF binding proteins (BPs) dates back to 1984 when the existence of specific somatomedin carrier proteins in serum was first shown (Hintz, 1984)
  • IGFBPs IGF binding proteins
  • IGFBPs are produced locally in all tissues to concentrate locally produced IGF-I near cells requiring the IGF-I, reducing the active role of IGF-I bound to BPs and IGF-I circulating in the blood (Isaksson et al , 1987) It has been reported, for example, that IGF-I is produced locally in bone by GH (Nilsson et al,
  • IGFBPs Intracellular growth factor proteins
  • IGFBPs Intracellular growth factor proteins
  • Several major functions have been proposed for IGFBPs in regulating the activities of IGFs These are 1) to act as transport proteins in plasma and to modulate the location and movement of bound IGFs, 2) to modulate the half-life and clearance rates of IGFs, 3) to provide a means of tissue- and cell-type specific localization of IGFs, and 4) to directly modulate interactions between IGFs and IGF receptors and the resulting effects of receptor binding
  • IGFBPs include direct effects of IGFBPs on cell function in the absence of IGFs and specific cellular receptors for IGFBPs distinct from IGF receptors
  • Amniotic fluid was the first source from which IGFBP- 1 was detected (Chochinov et al , 1977)
  • the protein has been purified also from tissue extract of fetal and maternal placenta and named placental protein (Kiostinen et al, 1986)
  • the mature protein contains 2
  • Carrier proteins of the IGFBP-2 class have been isolated from human fetal liver and rat and bovine cell lines (Binkert et al, 1989, Rosenfeld et al , 1990) In humans, the mature form contains 289 amino acids and has an apparent molecular mass of 31- 40 kD, depending on the stage of reduction on SDS-PAGE In humans high IGFBP-2 levels have been found in the cerebrospinal fluid The abundance of this protein in fetal tissue suggests that it has a role in regulating development IGFBP-2 preferentially binds IGF-II
  • SEQ ID NO 1 The nucleic acid sequence of the cDNA encoding human IGFBP-2 is shown as SEQ ID NO 1 and the deduced amino acid sequence of human IGFBP-2 is shown as SEQ ID NO 2
  • IGFBP-3 The majority of serum IGFs are bound to a BP composed of two parts forming a complex of molecular mass 125-150 kD IGFBP-3 is the IGF binding subunit ( ⁇ - subunit) in this complex (Baxter and Martin, 1989) It is an acid-stable glycoprotein appearing on SDS-PAGE as a major and minor band, corresponding to molecular weights of 53 kD and 47 kD, respectively
  • the other components in the complex are the acid-labile, non-IGF-binding subunit ( ⁇ -subunit) with a molecular mass of 84-86 kD (Baxter, WO 90/0569), and IGF-I or IGF-II ( ⁇ -subunit) Sequencing of the cloned cDNA for IGFBP-3 (previously known as IGFBP-53) predicts a molecular mass of 28 7 kD for the non-glycosylated protein and reveals that IGFBP-3 shares 33% sequence identity
  • IGFBP-4 has been isolated from cultured human osteoblast-like TE89 osteosarcoma cell conditioned media and sequenced (Mohan et al, 1989) A similar, if not identical, IGFBP was isolated from human prostatic carcinoma cells and sequenced
  • IGFBP-5 is a 252 amino acid protein that is highly conserved between species
  • IGFBP-5 insulin-like growth factor-I
  • IGFBP-5 stimulated all aspects of the myogenic response to IGF-L the later rise in myogenin mRNA, the elevation of creatine kinase activity, and the fusion of myoblasts into myotubes
  • IGFBP-5 inhibited both proliferation and differentiation (Ewton et al. 1998). This was seen in other studies, where IGFBP-5 inhibits muscle differentiation, implying a role for IGFBP-5 in regulating IGF action during myogenic development in vivo (James et al, 1996).
  • IGFBP-6 has only recently been isolated and cloned It is expressed during mouse development in a number of tissues, including brain (Putzer et al, 1998) The effects of IGFBP-6 on neuroblastoma cell growth has been studied (Grellier et al, 1998, Babajko et al, 1997) These findings suggest that IGFBP-6 contributes as an autocrine inhibitor in the regulation of growth by the IGF system in these neuroblastoma cells In other studies on IGFBP-6 actions, IGFBP-6 completely inhibited IGF-II- induced [ ⁇ jthymidine incorporation in MC3T3-E1 mouse osteoblast cells, while it had only minimal inhibitory effects on IGF-I-induced [ 3 H]thymidine incorporation This differential effect is associated with the fact that IGFBP-6 has greater affinity for IGF- II than IGF-I (Srinivasan et al. , 1996)
  • IGFBP-3 The levels of IGFBP in adult serum have been found to reflect the growth hormone (GH) status of individuals who are either GH-deficient or acromegalic Thus, high levels of IGFBP-3 correlate with high levels of GH (Martin and Baxter, 1985) Under normal conditions about 95-98% of the IGF-I in human plasma is bound to the IGFBPs. Studies on size-fractionated human serum, subjected to IGF-I RIA after extraction of each fraction to remove binding activity, have indicated that 72% of the endogenous peptide is associated with the 150-kD fraction and 25% with the 50-kD fraction (Daughaday et al , 1982)
  • IGFBP-2 The specific actions of IGFBP-2 and IGFs are unclear Purified IGFBP-2 has been shown to inhibit [ 3 H] thymidine incorporation into DNA in two different cell systems (Rechler and Nissley, 1990, Knauer and Smith, 1980) Inhibition of DNA synthesis was also seen using purified human IGFBP-2 in a human lung carcinoma line (Reeve et al, 1993) One explanation for the observed inhibitory actions is that IGFBP-2 inhibited binding of IGFs to IGF cell surface receptors (Ross et al, 1989) However, IGFBP-2 has been shown to be a weak potentiator of IGF action as well IGFBP-2 was shown to enhance the effect of IGF-I in microvascular endothelial cells in serum-free medium (Bar et al, 1989) Also, in aortic smooth muscle cells, IGFBP-2 was shown to potentiate the actions of IGF (Bourner et al, 1992)
  • IGFBP refers to a protein that binds IGFs in the circulation, in other body fluids, and in media conditioned by cultured cells, as defined in the Workshop on IGF Binding Proteins held in Vancouver, Canada in June 1989 discussed above and reported in Ballard et al (1989)
  • This term includes IGFBP- 1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6, and other as yet unidentified IGFBPs that have the characteristics common to all the known IGF binding proteins
  • the term includes animal equivalents to human IGFBPs as well as human IGFBPs, for example, the bovine, ovine, murine, porcine, and equine species It may be from any source, whether natural, synthetic, or recombinant, provided that it will bind to the appropriate binding domain of IGFs
  • IGFBP-2 dependent cancer cell refers to cells whose biologic activity is modulated by IGFBP-2
  • Specific biologic properties or cell functions contemplated in this application include altering proliferation, metastasis, contact inhibition, soft agar growth, cell cycle regulation, tumor formation, tumor progression, differentiation, or tumor invasion
  • Other described functions of IGFBP-2 may include modulating chemotaxis, chemokinesis, immune response, and amino acid and glucose uptake These responses may be mediated directly by IGFBP- 2 or by IGFBP-2 's modulation of IGF function as described above
  • Cells other than cancer that are possibly affected by IGFBP-2 include fibroblasts, smooth muscle cells, chondrocytes, osteoblasts, keratinocytes, skelatal muscle cells, neuronal cells, mammary epithelial cells, mesangial cells, thyroid follicular cells, erythroid progenitor cells, thymic epithelium, oocytes, granulosa
  • Another embodiment of the present invention is a monclonal antibody and more preferably a human monoclonal antibody immunoreactive with the peptide sequence designated herein as SEQ ID NO 2
  • antibodies can be used for inhibiting an IGFBP-2 dependent cancer cell by binding or modulating IGFBP-2
  • this antibody is useful for screening samples from human patients for the purpose of detecting IGFBP-2 present in the samples
  • the antibody also may be useful in the screening of expressed DNA segments or peptides and proteins for the discovery of related antigenic sequences
  • the antibody may be useful in passive immunotherapy for cancer All such uses of the said antibody and any antigens or epitopic sequences so discovered fall within the scope of the present invention
  • the present invention provides antibodies that bind with high specificity to the IGFBP-2 polypeptides provided herein
  • antibodies that bind to the protein products of the isolated nucleic acid sequences of SEQ ID NOT are provided.
  • antibodies generated against the full length proteins antibodies also may be generated in response to smaller constructs comprising epitopic core regions, including wild-type and mutant epitopes.
  • antibody is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE Generally, IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting
  • Monoclonal antibodies are recognized to have certain advantages, e.g. , reproducibility and large-scale production, and their use is generally preferred
  • the invention thus provides monoclonal antibodies of the human, murine, monkey, rat, hamster, rabbit and even chicken origin. Due to the ease of preparation and ready availability of reagents, murine monoclonal antibodies will often be preferred.
  • "humanized” antibodies are also contemplated, as are chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, bispecific antibodies, recombinant and engineered antibodies and fragments thereof
  • Methods for the development of antibodies that are "custom-tailored" to the patient's disease are likewise known and such custom-tailored antibodies are also contemplated
  • antibody is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab') 2 , single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like.
  • DABs single domain antibodies
  • Fv single domain antibodies
  • scFv single chain Fv
  • a polyclonal antibody is prepared by immunizing an animal with an immunogenic IGFBP-2 polypeptide composition in accordance with the present invention and collecting antisera from that immunized animal
  • a wide range of animal species can be used for the production of antisera.
  • the animal used for production of antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
  • a given composition may vary in its immunogenicity It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier.
  • exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA)
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin also can be used as carriers
  • Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine
  • immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants
  • Suitable molecule adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins or synthetic compositions
  • Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12, ⁇ -interferon,
  • MHC antigens may even be used
  • MHC antigens include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant
  • BRM biologic response modifiers
  • CCM Cimetidine
  • CYP Cyclophosphamide
  • cytokines such as ⁇ -interferon, IL-2, or IL-12 or genes encoding proteins involved in immune helper functions, such as B-7
  • the amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal) The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization
  • a second, booster injection also may be given The process of boosting and titering is repeated until a suitable titer is achieved When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate MAbs
  • the animal For production of rabbit polyclonal antibodies, the animal can be bled through an ear vein or alternatively by cardiac puncture The removed blood is allowed to coagulate and then centrifuged to separate serum components from whole cells and blood clots
  • the serum may be used as is for various applications or else the desired antibody fraction may be purified by well-known methods, such as affinity chromatography using another antibody, a peptide bound to a solid matrix, or by using, e.g. , protein A or protein G chromatography
  • MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U S Patent 4,196,265, incorporated herein by reference.
  • this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified IGFBP-2 polypeptide, peptide or domain, be it a wild-type or mutant composition
  • a selected immunogen composition e.g., a purified or partially purified IGFBP-2 polypeptide, peptide or domain
  • the immunizing composition is administered in a manner effective to stimulate antibody producing cells.
  • the methods for generating monoclonal antibodies generally begin along the same lines as those for preparing polyclonal antibodies Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells also is possible The use of rats may provide certain advantages (Goding, 1986, pp 60-61), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions
  • the animals are injected with antigen, generally as described above
  • the antigen may be coupled to carrier molecules such as keyhole limpet hemocyanin if necessary
  • the antigen would typically be mixed with adjuvant, such as Freund's complete or incomplete adjuvant Booster injections with the same antigen would occur at approximately two-week intervals
  • somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells) are selected for use in the MAb generating protocol
  • B cells B lymphocytes
  • These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible
  • a panel of animals will have been immunized and the spleen of an animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe
  • a spleen from an immunized mouse contains approximately 5 x 10 7 to 2 x 10 8 lymphocytes
  • the antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas)
  • any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, pp 65-66, 1986, Campbell, pp 75-83, 1984)
  • the immunized animal is a mouse
  • R210 RCY3, Y3-Ag 1 2 3, IR983F and 4B210, and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions
  • NS-1 myeloma cell line also termed P3-NS-l-Ag4-l
  • P3-NS-l-Ag4-l NS-1 myeloma cell line
  • mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line
  • Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2 1 proportion, though the proportion may vary from about 20 1 to about 1 1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes Fusion methods using Sendai virus have been described by Kohler and Milstein (1975, 1976), and those using polyethylene glycol (PEG), such as 37%) (v/v) PEG, by Gefter et al. (1977) The use of electrically induced fusion methods also is appropriate (Goding pp 71-74, 1986)
  • the selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media
  • agents are aminopterin, methotrexate, and azaserine Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis
  • aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides
  • HAT medium Where azaserine is used, the media is supplemented with hypoxanthine
  • the preferred selection medium is HAT Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium
  • the myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive
  • HPRT hypoxanthine phosphoribosyl transferase
  • the B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells
  • This culturing provides a population of hybridomas from which specific hybridomas are selected Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity
  • the assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like
  • the selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide MAbs
  • the cell lines may be exploited for MAb production in two basic ways
  • a sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion (e.g., a syngeneic mouse)
  • the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection
  • the injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid
  • the body fluids of the animal such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration
  • the individual cell lines could be cultured n vitro, where the MAbs are naturally secreted into the culture medium
  • MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography Fragments of the monoclonal antibodies of the invention can be obtained from the monoclonal antibodies so produced by methods which include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction Alternatively, monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer
  • a molecular cloning approach may be used to generate monoclonals
  • combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the spleen of the immunized animal, and phagemids expressing appropriate antibodies are selected by panning using cells expressing the antigen and control cells
  • the advantages of this approach over conventional hybridoma techniques are that approximately 10 4 times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination which further increases the chance of finding appropriate antibodies
  • Humanized monoclonal antibodies are antibodies of animal origin that have been modified using genetic engineering techniques to replace constant region and/or variable region framework sequences with human sequences, while retaining the original antigen specificity Such antibodies are commonly derived from rodent antibodies with specificity against human antigens Such antibodies are generally useful for in vivo therapeutic applications This strategy reduces the host response to the foreign antibody and allows selection of the human effector functions
  • humanized immunoglobulins having one or more complementarity determining regions (CDR's)
  • CDR's complementarity determining regions
  • the humanized immunoglobulins are substantially non-immunogenic in humans and retain substantially the same affinity as the donor immunoglobulin to the antigen, such as a protein or other compound containing an epitope
  • monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer, or by expression of full-length gene or of gene fragments in E. coh
  • peptides corresponding to one or more antigenic determinants of the IGFBP-2 polypeptides of the present invention also can be prepared so that an immune response against IGFBP- 2 is raised
  • vaccination with an IGFBP-2 peptide or polypeptide may generate an autoimmune response in an immunized animal such that autoantibodies that specifically recognize the animal's endogenous IGFBP-2 protein
  • This vaccination technology is shown in U S Patent Nos 6,027,727, 5,785,970, and 5,609,870, which are hereby incorporated by reference
  • Such peptides should generally be at least five or six amino acid residues in length and will preferably be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or about 30 amino acid residues in length, and may contain up to about 35-50 residues
  • these peptides may comprise IGFBP-2 amino acid sequence, such as 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, and 50 or more contiguous amino acids from SEQ ID NO 2
  • Synthetic peptides will generally be about 35 residues long, which is the approximate upper length limit of automated peptide synthesis machines, such as those available from Applied Biosystems (Foster City, CA) Longer peptides also may be prepared, e.g., by recombinant means
  • IGFBP-2 sequence disclosed herein in SEQ ID NO 2 A peptide with the sequence LFYNEQQEARGVHTQRMQ (SEQ ID NO 3) has been used to generate antibodies against human IGFBP-2
  • major antigenic determinants of a IGFBP-2 polypeptide may be identified by an empirical approach in which portions of the gene encoding the IGFBP-2 polypeptide are expressed in a recombinant host, and the resulting proteins tested for their ability to elicit an immune response
  • PCRTM can be used to prepare a range of peptides lacking successively longer fragments of the C-terminus of the protein The immunoactivity of each of these peptides is determined to identify those fragments or domains of the polypeptide that are immunodominant
  • Further studies in which only a small number of amino acids are removed at each iteration then allows the location of the antigenic determinants of the polypeptide to be more precisely determined
  • Another method for determining the major antigenic determinants of a polypeptide is the SPOTsTM system (Genosys Biotechnologies, Inc , The Woodlands, TX) In this method, overlapping peptides are synthesized on a cellulose membrane, which following synthesis and deprotection, is screened using a polyclonal or monoclonal antibody The antigenic determinants of the peptides which are initially identified can be further localized by performing subsequent syntheses of smaller peptides with larger overlaps, and by eventually replacing individual amino acids at each position along the immunoreactive peptide
  • polypeptides are prepared that contain at least the essential features of one or more antigenic determinants.
  • the peptides are then employed in the generation of antisera against the polypeptide Minigenes or gene fusions encoding these determinants also can be constructed and inserted into expression vectors by standard methods, for example, using PCRTM cloning methodology
  • immunogenic carrier protein such as hepatitis B surface antigen, keyhole limpet hemocyanin or bovine serum albumin, or other adjuvants discussed above (adjuvenated peptide)
  • Alum is an adjuvant that has proven sufficiently non-toxic for use in humans
  • Other immunopotentiating compounds are also contemplated for use with the compositions of the invention such as polysaccharides, including chitosan, which is described in U S Patent No 5,980,912, hereby incorporated by reference Multiple (more than one) IGFBP-2 epitopes may be crosslinked to one another (e.g., polymerized)
  • a nucleic acid sequence encoding an IGFBP-2 peptide or polypeptide may be combined with a nucleic acid sequence that heightens the immune response
  • Such fusion proteins may comprise part or all of a foreign (non-self)
  • Antibody titers effective to achieve a response against endogenous IGFBP-2 will vary with the species of the vaccinated animal, as well as with the sequence of the administered peptide However, effective titers may be readily determined, for example, by testing a panel of animals with varying doses of the specific antigen and measuring the induced titers of autoantibodies (or anti-self antibodies) by known techniques, such as ELISA assays, and then correlating the titers with IGFBP-2-related cancer characteristics, e.g., tumor growth or size
  • immuno response includes both cellular and humoral immune responses
  • B lymphocyte and T lymphocyte assays are well known, such as ELISAs, cytotoxic T lymphocyte (CTL) assays, such as chromium release assays, proliferation assays using peripheral blood lymphocytes (PBL), tetramer assays, and cytokine production assays
  • CTL cytotoxic T lymphocyte
  • PBL peripheral blood lymphocytes
  • tetramer assays cytokine production assays See Benjamini et al, 1991, hereby incorporated by reference
  • dimers or multimers of IGFBP-2 peptides, or an IGFBP-2 peptide and a non-self, non-IGFBP-2 peptide or adjuvant may be joined via a biologically- releasable bond, such as a selectively-cleavable linker or amino acid sequence
  • peptide linkers that include a cleavage site for an enzyme preferentially located or active within a tumor environment are contemplated
  • Exemplary forms of such peptide linkers are those that are cleaved by urokinase, plasmin, thrombin, Factor IXa, Factor Xa, or a metallaproteinase, such as collagenase, gelatinase, or stromelysin
  • the linkers and coupling agents described herein may be employed to join an anti-IGFBP-2 antibody with a therapeutic or preventative agent, such as a toxin
  • Amino acids such as selectively-cleavable linkers, synthetic linkers, or other amino acid sequences may be used to separate an IGFBP-2 peptide from another peptide or adjuvant, or anti-IGFBP-2 antibodies from a therapeutic compound
  • linkers While numerous types of disulfide-bond containing linkers are known that can successfully be employed to conjugate the toxin moiety with the targeting agent, certain linkers will generally be preferred over other linkers, based on differing pharmacologic characteristics and capabilities For example, linkers that contain a disulfide bond that is sterically "hindered” are to be preferred, due to their greater stability in vivo, thus preventing release of the toxin moiety prior to binding at the site of action Furthermore, while certain advantages in accordance with the invention will be realized through the use of any of a number of toxin moieties, the inventors have found that the use of ricin A chain, and even more preferably deglycosylated A chain, will provide particular benefits
  • Cross-linking reagents are used to form molecular bridges that tie together functional groups of two different molecules, e.g , a stablizing and coagulating agent
  • a stablizing and coagulating agent e.g., a stablizing and coagulating agent
  • hetero-bifiinctional cross-linkers can be used that eliminate unwanted homopolymer formation
  • An exemplary hetero-bifunctional cross-linker contains two reactive groups one reacting with primary amine group (e.g , N-hydroxy succinimide) and the other reacting with a thiol group (e.g , pyridyl disulfide, maleimides, halogens, etc )
  • primary amine group e.g , N-hydroxy succinimide
  • a thiol group e.g , pyridyl disulfide, maleimides, halogens, etc
  • the cross-linker may react with the lysine residue(s) of one protein (e.g , the selected antibody or fragment) and through the thiol reactive group, the cross-linker, already tied up to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the other protein (e.g , the selective agent)
  • a targeted peptide composition will generally have, or be derivatized to have, a functional group available for cross-linking purposes This requirement is not considered to be limiting in that a wide variety of groups can be used in this manner
  • groups can be used in this manner
  • primary or secondary amine groups, hydrazide or hydrazine groups, carboxyl alcohol, phosphate, or alkylating groups may be used for binding or cross-linking
  • the spacer arm between the two reactive groups of a cross-linkers may have various length and chemical compositions
  • a longer spacer arm allows a better flexibility of the conjugate components while some particular components in the bridge (e.g., benzene group) may lend extra stability to the reactive group or an increased resistance of the chemical link to the action of various aspects (e.g., disulfide bond resistant to reducing agents)
  • peptide spacers such as L-Leu-L-Ala-L-Leu- L-Ala, is also contemplated
  • SMPT cross-linking reagents for use in immunotoxins
  • SMPT is a bifunctional cross-linker containing a disulfide bond that is "sterically hindered" by an adjacent benzene ring and methyl groups
  • stearic hindrance of the disulfide bond serves a function of protecting the bond from attack by thiolate anions such as glutathione which can be present in tissues and blood, and thereby help in preventing decoupling of the conjugate prior to the delivery of the attached agent to the tumor site.
  • the SMPT agent may
  • the SMPT cross-linking reagent lends the ability to cross-link functional groups such as the SH of cysteine or primary amines (e.g , the epsilon amino group of lysine)
  • cross-link functional groups such as the SH of cysteine or primary amines (e.g , the epsilon amino group of lysine)
  • Another possible type of cross-linker includes the hetero-bifiinctional photoreactive phenylazides containing a cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido) ethyl- 1,3'- dithiopropionate
  • the N-hydroxy-succinimidyl group reacts with primary amino groups and the phenylazide (upon photolysis) reacts non-selectively with any amino acid residue.
  • non-hindered linkers also can be employed in accordance herewith
  • Other useful cross-linkers include SATN SPDP and 2-iminothiolane
  • the peptide generally will be purified to separate the conjugate from unconjugated targeting agents or coagulants and from other contaminants
  • a large a number of purification techniques are available for use in providing conjugates of a sufficient degree of purity to render them clinically useful Purification methods based upon size separation, such as gel filtration, gel permeation or high performance liquid chromatography, will generally be of most use Other chromatographic techniques, such as Blue-Sepharose separation, may also be used
  • an IGFBP-2 polypeptide, peptide, or antibody may be modified at the protein level Included within the scope of the invention are IgA protein fragments or other derivatives or analogs that are differentially modified during or after translation, for example by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, and proteolytic cleavage Any number of chemical modifications may be carried out by known techniques, including but not limited to specific chemical cleavage by cyanogen bromide, tryps
  • Important aspects of the present invention concern isolated DNA segments and recombinant vectors expressing IGFBP-2 antisense or sense, and the creation and use of recombinant host cells through the application of DNA technology, including such sequences encoding one or more IGFBP-2 polypeptides, or functional equivalents thereof
  • the present invention concerns DNA segments, isolatable from mammalian and human cells, that are free from total genomic DNA and are capable of expressing IGFBP-2 polypeptide in a recombinant host cell when incorporated into the recombinant host cell
  • DNA segment refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species Therefore, a DNA segment encoding IGFBP-2 refers to a DNA segment that contains coding sequences of IGFBP-2, yet is isolated away from, or purified free from, total mammalian or human genomic DNA Included within the term "DNA segment”, are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like Similarly, a DNA segment comprising an isolated or purified IGFBP-2 gene refers to a DNA segment including any of the IGFBP-2 gene coding sequences and, in certain aspects, regulatory sequences, isolated substantially away from other naturally occurring genes or protein encoding sequences In this respect, the term “gene” is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit As will be understood by those in the art, this functional term includes both genomic sequence
  • IGFBP-2 isolated substantially away from other coding sequences
  • the gene of interest in this case any IGFBP-2 gene, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNN such as large chromosomal fragments or other functional genes or cD ⁇ A coding regions
  • the invention concerns isolated D ⁇ A segments and recombinant vectors incorporating D ⁇ A sequences that encode an IGFBP-2 polypeptide that includes within its amino acid sequence a contiguous amino acid sequence of human IGFBP-2 polypeptide, or functional equivalents thereof
  • DNA segment or vector encodes an IGFBP-2 polypeptide, or is intended for use in expressing the IGFBP-2 polypeptide
  • the most preferred sequences are those that are essentially as set forth in the contiguous sequence of SEQ ID NO 1
  • sequence of an IGFBP-2 polypeptide will substantially correspond to a contiguous portion of that shown in SEQ ID NO 2, and have relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids shown in SEQ ID NO 2
  • biologically functional equivalent is well understood in the art and is further defined in detail herein
  • sequences that have between about 70% and about 80%, or more preferably, between about 81%> and about 90%, or even more preferably, between about 91% and about 99%, of amino acids that are identical or functionally equivalent to the amino acids of SEQ ID NO 2 will be sequences that are "essentially as set forth in SEQ ID NO.2 "
  • the invention concerns isolated DNA segments and recombinant vectors that include within their sequence a contiguous nucleic acid sequence from that shown in SEQ ID NO 1
  • This definition is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a contiguous portion of that shown in SEQ ID NOT and has relatively few codons that are not identical, or functionally equivalent, to the codons of SEQ LD NO.1
  • the term "functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids See Table 2 below, which lists the codons preferred for use in humans, with the codons listed in decreasing order of preference from left to right in the table (Wada et al, 1990) Codon preferences for other organisms also are well known to those of skill in the art (Wada et al , 1990, included herein in its entirety by reference
  • amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned
  • terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e , introns, which are known to occur within genes
  • sequences that have between about 70% and about 79%, or more preferably, between about 80% and about 89%, or even more preferably, between about 90% and about 99% of nucleotides that are identical to the nucleotides shown in the sequences of SEQ ID NO 1 will be sequences that are "essentially as set forth in SEQ ID NO 1 " Sequences that are essentially the same as those set forth in SEQ ID NO 1 also may be functionally defined as sequences that are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID NO 1 under relatively stringent conditions Suitable relatively stringent hybridization conditions will be well known to those of skill in the art
  • nucleic acid sequences that are “complementary” are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules
  • complementary sequences means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ ID NO 1 under relatively stringent conditions
  • nucleic acid segments of the present invention may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol
  • nucleic acid fragments may be prepared that include a short contiguous stretch identical to or complementary to the sequence shown in SEQ ID NO 1, such as about 14 nucleotides, and that are up to about 10,000 or about 5,000 base pairs in length, with segments of about 3,000 being preferred in certain cases DNA segments with total lengths of about 1,000, about 500, about 200, about 100 and about 50 base pairs in length (including all intermediate lengths) are also contemplated to be useful
  • intermediate lengths means any length between the quoted ranges, such as 14, 15, 16, 17, 18, 19, 20, etc , 21, 22, 23, etc , 30, 31, 32, etc , 50, 51, 52, 53, etc , 100, 101, 102, 103, etc , 150, 151, 152, 153, etc , including all integers through the 200-500, 500-1,000, 1,000-2,000, 2,000-3,000, 3,000-5,000, 5,000-10,000 ranges, up to and including sequences of about 12,001, 12,002, 13,001, 13,002 and the like
  • the various probes and primers designed around the nucleotide sequences of the present invention may be of any length
  • an algorithm defining all primers can be proposed n to n + y
  • the probes correspond to bases 1 to 10, 2 to 11, 3 to 12 and so on
  • the probes correspond to bases 1 to 15, 2 to 16, 3 to 17 and so on
  • the probes correspond to bases 1 to 20, 2 to 21, 3 to 22 and so on
  • Recombinant vectors and isolated DNA segments may therefore variously include the IGFBP-2 coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include IGFBP-2 -coding regions or may encode biologically functional equivalent proteins or peptides that have variant amino acids sequences
  • DNA segments of the present invention encompass biologically functional equivalent IGFBP-2 proteins and peptides Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged Changes designed by man may be introduced through the application of site-directed mutagenesis techniques, e.g , to introduce improvements to the antigenicity of the protein
  • fusion proteins and peptides e.g , where the
  • IGFBP-2 coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes (e.g , proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively)
  • DNA segments encoding relatively small peptides such as, for example, peptides of from about 15 to about 50 amino acids in length, and more preferably, of from about 15 to about 30 amino acids in length, and also larger polypeptides up to and including proteins corresponding to the full-length sequences set forth in SEQ ID NO.2, or to specific fragments of SEQ ID NO 1 that correspond to differences as compared to the published sequence for IGFBP-2
  • Nucleic acids used as a template for amplification are isolated from cells contained in the biological sample, according to standard methodologies (Sambrook et al, 1989)
  • the nucleic acid may be genomic DNA or fractionated or whole cell RNA Where RNA is used, it may be desired to convert the RNA to a complementary DNA
  • the RNA is whole cell RNA and is used directly as the template for amplification
  • primer as defined herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process
  • primers are oligonucleotides from ten to twenty base pairs in length, but longer sequences can be employed
  • Primers may be provided in double-stranded or single-stranded form, although the single-stranded form is preferred
  • the nucleic acid primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis
  • Multiple rounds of amplification also referred to as “cycles,” are conducted until a sufficient amount of amplification product is produced
  • the amplification product is detected
  • the detection may be performed by visual means
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals
  • PCRTM polymerase chain reaction
  • the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated
  • a reverse transcriptase PCRTM amplification procedure may be performed in order to quantify the amount of mRNA amplified
  • Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al, 1989
  • Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases These methods are described in WO 90/07641, filed December 21, 1990, incorporated herein by reference
  • Polymerase chain reaction methodologies are well known in the art
  • LCR ligase chain reaction
  • Qbeta Replicase described in PCT Application No PCT/US87/00880, incorporated herein by reference, also may be used as still another amplification method in the present invention
  • SDA Strand Displacement Amplification
  • RCR Repair Chain Reaction
  • modified primers are used in a PCR-like, template- and enzyme-dependent synthesis
  • the primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme)
  • a capture moiety e.g., biotin
  • a detector moiety e.g., enzyme
  • an excess of labeled probes are added to a sample In the presence of the target sequence, the probe binds and is cleaved catalytically After cleavage, the target sequence is released intact to be bound by excess probe Cleavage of the labeled probe signals the presence of the target sequence
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR Gingeras et al, PCT Application WO 88/10315, incorporated herein by reference Davey et al, EPA No 329 822 (incorporated herein by reference in its entirety) disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA”), ssDNN and double-stranded D ⁇ A (dsD ⁇ A), which may be used in accordance with the present invention
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • dsD ⁇ A double-stranded D ⁇ A
  • amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods See Sambrook et al, 1989
  • chromatographic techniques may be employed to effect separation
  • adsorption, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas chromatography
  • Amplification products must be visualized in order to confirm amplification of the marker sequences
  • One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light
  • the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation
  • a labeled nucleic acid probe is brought into contact with the amplified marker sequence
  • the probe preferably is conjugated to a chromophore but may be radiolabeled
  • the probe is conjugated to a binding partner, such as an antibody or biotin, and the other member of the binding pair carries a detectable moiety
  • detection is by Southern blotting and hybridization with a labeled probe
  • the techniques involved in Southern blotting are well known to those of skill in the art and can be found in many standard books on molecular protocols See Sambrook et al, 1989 Briefly, amplification products are separated by gel electrophoresis The gel is then contacted with a membrane, such as nitrocellulose or nylon, permitting transfer of the nucleic acid and non-covalent binding Subsequently, the membrane is incubated with a chromophore-conjugated probe that is capable of hybridizing with a target amplification product Detection is by exposure of the membrane to x-ray film or ion-emitting detection devices
  • expression vector or construct means any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed and subsequently translated
  • promoter refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene
  • operatively positioned means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene
  • the promoter may be in the form of the promoter that is naturally associated with any of the IGFBP-2 genes, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCR technology, in connection with the compositions disclosed herein (PCR technology is disclosed in U S Patent 4,683,202 and U S Patent 4,683, 195, each incorporated herein by reference)
  • a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with an IGFBP-2 gene in its natural environment
  • promoters may include promoters normally associated with other genes, and/or promoters isolated from any other bacterial, viral, eukaryotic, or mammalian cell
  • promoter that effectively directs the expression of the DNA segment in the cell type, organism, or even animal, chosen for expression
  • the use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al (1989), incorporated herein by reference
  • the promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides
  • the promoter preferentially expresses or is specific for a tissue or cell type, such as a cell type that expresses IGFBP-2 It is contemplated that such cell types may be from, but not limited to, prostate and breast tissues, and may be altered or cancerous
  • At least one module in a promoter functions to position the start site for RNA synthesis
  • the best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation
  • promoter elements regulate the frequency of transcriptional initiation Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another
  • the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription
  • a promoter that is capable of expressing the nucleic acid in the targeted cell
  • a promoter might include either a human or viral promoter
  • the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter and the Rous sarcoma virus long terminal repeat can be used to obtain high-level expression of transgenes
  • CMV cytomegalovirus
  • SV40 early promoter the Rous sarcoma virus long terminal repeat
  • the use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a transgene is contemplated as well, provided that the levels of expression are sufficient for a given purpose Tables 3 and 4 below list several enhancers, promoters and inducible elements which may be employed, in the context of the present
  • Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA This ability to act over a large distance had little precedent in classic studies of prokaryotic transcriptional regulation Subsequent work showed that regions of DNA with enhancer activity are organized much like promoters That is, they are composed of many individual elements, each of which serves as a binding region for one or more transcriptional proteins.
  • enhancers are operational
  • An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements
  • a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities
  • Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization
  • Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct
  • Tumor Necrosis Factor FMA Hensel et ⁇ /., 1989 Thyroid Stimulating Thyroid Hormone Chatterjee et al, 1989 Hormone a Gene
  • IGFBP-2 polypeptides once a suitable clone or clones have been obtained, whether they be cDNA based or genomic, one may proceed to prepare an expression system
  • the engineering of DNA segment(s) for expression in a prokaryotic or eukaryotic system may be performed by techniques generally known to those of skill in recombinant expression It is believed that virtually any expression system may be employed in the expression of the proteins of the present invention
  • cDNA and genomic sequences are suitable for eukaryotic expression, as the host cell will generally process the genomic transcripts to yield functional mRNA for translation into protein Generally speaking, it may be more convenient to employ as the recombinant gene a cDNA version of the gene It is believed that the use of a cDNA version will provide advantages in that the size of the gene will generally be much smaller and more readily employed to transfect the targeted cell than will a genomic gene, which will typically be up to an order of magnitude larger than the cDNA gene However, the inventors do not exclude the possibility of employing a genomic version of a particular gene where desired
  • polyadenylation signal to effect proper polyadenylation of the transcript
  • the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed
  • Preferred embodiments include the SV40 polyadenylation signal and the bovine growth hormone polyadenylation signal, convenient and known to function well in various target cells
  • a terminator an element of the expression cassette. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences
  • a specific initiation signal also may be required for efficient translation of coding sequences
  • These signals include the ATG initiation codon and adjacent sequences
  • Exogenous translational control signals, including the ATG initiation codon, may need to be provided
  • One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals It is well known that the initiation codon must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert
  • the exogenous translational control signals and initiation codons can be either natural or synthetic.
  • engineered and recombinant cells are intended to refer to a cell into which an exogenous DNA segment or gene, such as a cDNA or gene encoding an IGFBP-2 polypeptide has been introduced Therefore, engineered cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced exogenous DNA segment or gene Engineered cells are thus cells having a gene or genes introduced through the hand of man Recombinant cells include those having an introduced cDNA or genomic gene, and also include genes positioned adjacent to a promoter not naturally associated with the particular introduced gene
  • an expression vector that comprises a IGFBP-2-encoding nucleic acid under the control of one or more promoters
  • the "upstream” promoter stimulates transcription of the DNA and promotes expression of the encoded recombinant protein This is the meaning of "recombinant expression” in this context
  • A. ANTISENSE Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences By complementary, it is meant that polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G.C) and adenine paired with either thymine (A T) in the case of DNN or adenine paired with uracil (A U) in the case of R ⁇ A Inclusion of less common bases such as inosine, 5- methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing
  • Antisense polynucleotides when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability
  • Antisense RNA constructs, or DNA encoding such antisense RNAs may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject
  • Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene It is contemplated that the most effective antisense constructs may include regions complementary to intron exon splice junctions Thus, antisense constructs with complementarity to regions within 50-200 bases of an intron-exon splice junction may be used It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof The amount of exonic material included will vary depending on the particular exon and intron sequences used One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.
  • complementary or “antisense” means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches Other sequences with lower degrees of homology also are contemplated.
  • an antisense construct which has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme) could be designed These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions
  • genomic DNA may be combined with cDNA or synthetic sequences to generate specific constructs
  • a genomic clone will need to be used
  • the cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence
  • IGFBP-2-specific ribozymes The use of IGFBP-2-specific ribozymes is claimed in the present application. The following information is provided in order to compliment the earlier section and to assist those of skill in the art in this endeavor.
  • Ribozymes are RNA-protein complexes that cleave nucleic acids in a site- specific fashion Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cech, 1987, Gerlack et al, 1987, Forster and Symons, 1987) For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cech et al, 1981, Michel and Westhof, 1990, Reinhold- Hurek and Shub, 1992) This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction
  • IGS internal guide sequence
  • Ribozyme catalysis has primarily been observed as part of sequence specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989, Cech et al, 1981)
  • U S Patent 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes
  • sequence-specific ribozyme- mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al, 1991, Sarver et al, 1990, Sioud et al, 1992)
  • ribozymes elicited genetic changes in some cell lines to which they were applied, the altered genes included the oncogenes H-ras, c-fos and genes of HIV Most of this work involved the modification of a target mRNN based on a specific mutant codon that is cleaved by a specific ribozyme
  • the altered genes
  • RNA cleavage activity examples include sequences from the Group I self splicing introns including tobacco ringspot virus (Prody et al, 1986), avocado sunblotch viroid (Palukaitis et al, 1979 and Symons, 1981), and Lucerne transient streak virus (Forster and Symons, 1987) Sequences from these and related viruses are referred to as hammerhead ribozymes based on a predicted folded secondary structure
  • ribozymes include sequences from RNase P with RNA cleavage activity (Yuan et al, 1992, Yuan and Altman, 1994), hairpin ribozyme structures (Berzal-Herranz et al, 1992, Chowrira et al, 1993) and hepatitis ⁇ virus based ribozymes (Perrotta and Been, 1992)
  • the general design and optimization of ribozyme directed RNA cleavage activity has been discussed in detail (Haseloff and Gerlach, 1988, Symons, 1992, Chowrira, et al, 1994, and Thompson, et al, 1995)
  • the other variable on ribozyme design is the selection of a cleavage site on a given target RNA Ribozymes are targeted to a given sequence by virtue of annealing to a site by complimentary base pair interactions Two stretches of homology are required for this targeting These stretches of homologous sequences flank the catalytic ribozyme structure defined above Each stretch of homologous sequence can vary in length from 7 to 15 nucleotides The only requirement for defining the homologous sequences is that, on the target RNA, they are separated by a specific sequence which is the cleavage site
  • the cleavage site is a dinucleotide sequence on the target RNA, uracil (U) followed by either an adenine, cytosine or uracil (NC or U, Perriman, et al, 1992, Thompson, et al, 1995)
  • the frequency of this dinucleotide occurring in any given R ⁇ A is statistically 3 out of 16 Therefore,
  • Another therapeutic embodiment of the present invention contemplates the use of single-chain antibodies to block the activity of IGFBP-2 in cells, in particular cancer cells
  • Single-chain antibodies can be synthesized by a cell, targeted to particular cellular compartments, and used to interfere in a highly specific manner with cell growth and metabolism (Richardson and Marasco, 1995) Recently, single-chain antibodies were utilized for the phenotypic knockout of growth-factor receptors, the functional inactivation of p2 Iras, and the inhibition of HIV- 1 replication
  • Intracellular antibodies offer a simple and effective alternative to other forms of gene inactivation, as well as demonstrate a clear potential as reagents for cancer therapy and for the control of infectious diseases
  • Single-chain antigen-binding proteins also represent potentially unique molecules for targeted delivery of drugs, toxins, or radionuclides to a tumor site, and show increased accessibility to tumor cells in vivo (Yokoda et al ,
  • Single-chain antibodies that bind IGFBP-2 can be introduced into a cell to functionally inactivate the IGFBP-2
  • a single-chain antibody is created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule
  • Single-chain antibody variable fragments in which the C-terminus of one variable domain is tethered to the N-terminus of the other via a 15 to 25 amino acid peptide or linker, have been developed without significantly disrupting antigen binding or specificity of the binding (Bedzyk et al , 1990, Chaudhary et al , 1990) These Fvs lack the constant regions (Fc) present in the heavy and light chains of the native antibody
  • single-chain antibody therapy can be combined with chemotherapeutic or radiotherapeutic intervention
  • chemotherapeutic or radiotherapeutic intervention The discussion of combined therapy with traditional chemotherapy or radiotherapy employed herein is incorporated into this section by reference
  • the IGFBP-2 genes are incorporated into an adenoviral infectious particle to mediate gene transfer to a cell
  • Additional expression constructs encoding other therapeutic agents as described herein may also be transferred via viral transduction using infectious viral particles, for example, by transformation with an adenovirus vector of the present invention as described herein below
  • retroviral or bovine papilloma virus may be employed, both of which permit permanent transformation of a host cell with a gene(s) of interest
  • viral infection of cells is used in order to deliver therapeutically significant genes to a cell
  • the virus simply will be exposed to the appropriate host cell under physiologic conditions, permitting uptake of the virus
  • adenovirus is exemplified, the present methods may be advantageously employed with other viral vectors, as discussed below
  • Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized DNA genome, ease of manipulation, high titer, wide target-cell range, and high infectivity
  • the roughly 36 kB viral genome is bounded by 100-200 base pair (bp) inverted terminal repeats (ITR), in which are contained as-acting elements necessary for viral DNA replication and packaging
  • ITR inverted terminal repeats
  • the El region encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes
  • the expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication These proteins are involved in DNA replication, late gene expression, and host cell shut off (Renan, 1990)
  • the products of the late genes (LI, L2, L3, L4 and L5), including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP)
  • MLP located at 16 8 map units
  • TL tripartite leader
  • Plasmids containing ITR's can replicate in the presence of a non-defective adenovirus (Hay et al, 1984) Therefore, inclusion of these elements in an adenoviral vector should permit replication
  • the packaging signal for viral encapsidation is localized between 194-385 bp (0 5-1 1 map units) at the left end of the viral genome (Hearing et al, 1987)
  • This signal mimics the protein recognition site in bacteriophage ⁇ DNA where a specific sequence close to the left end, but outside the cohesive end sequence, mediates the binding to proteins that are required for insertion of the DNA into the head structure
  • El substitution vectors of Ad have demonstrated that a 450 bp (0-1 25 map units) fragment at the left end of the viral genome could direct packaging in 293 cells (Levrero et al, 1991)
  • adenoviral genome can be incorporated into the genome of mammalian cells and the genes encoded thereby expressed These cell lines are capable of supporting the replication of an adenoviral vector that is deficient in the adenoviral function encoded by the cell line.
  • helping vectors e.g., wild-type virus or conditionally defective mutants
  • Replication-deficient adenoviral vectors can be complemented, in trans, by helper virus This observation alone does not permit isolation of the replication- deficient vectors, however, since the presence of helper virus, needed to provide replicative functions, would contaminate any preparation Thus, an additional element was needed that would add specificity to the replication and/or packaging of the replication-deficient vector That element, as provided for in the present invention, derives from the packaging function of adenovirus
  • helper viruses that are packaged with varying efficiencies
  • the mutations are point mutations or deletions
  • helper viruses with low efficiency packaging are grown in helper cells
  • the virus is packaged, albeit at reduced rates compared to wild- type virus, thereby permitting propagation of the helper
  • the wild-type packaging signals are recognized preferentially over the mutated versions
  • the virus containing the wild-type signals are packaged selectively when compared to the helpers If the preference is great enough, stocks approaching homogeneity should be achieved
  • Retrovirus are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990)
  • the resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins
  • the integration results in the retention of the viral gene sequences in the recipient cell and its descendants
  • the retroviral genome contains three genes - gag, pol and env - that code for capsid proteins, polymerase enzyme, and envelope components, respectively
  • a sequence found upstream from the gag gene, termed ⁇ functions as a signal for packaging of the genome into virions
  • Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome These contain strong promoter and enhancer sequences and also are required for integration in the host cell genome (Coffin, 1990)
  • a nucleic acid encoding a promoter is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective
  • a packaging cell line containing the gag, pol and env genes but without the LTR and ⁇ components is constructed (Mann et al, 1983)
  • a recombinant plasmid containing a human cDNN together with the retroviral LTR and ⁇ sequences is introduced into this cell line (by calcium phosphate precipitation for example), the ⁇ sequence allows the R ⁇ A transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988, Temin, 1986, Mann et al, 1983).
  • Retroviral vectors are able to infect a broad variety of cell types
  • integration and stable expression of many types of retroviruses require the division of host cells (Paskind et al, 1975).
  • AAV utilizes a linear, single-stranded DNA of about 4700 base pairs Inverted terminal repeats flank the genome
  • Two genes are present within the genome, giving rise to a number of distinct gene products
  • the first, the cap gene produces three different virion proteins (VP), designated VP- 1 , VP-2 and VP-3
  • the second, the rep gene encodes four non-structural proteins (NS)
  • VP virion proteins
  • NS non-structural proteins
  • the three promoters in AAV are designated by their location, in map units, in the genome These are, from left to right, p 5, pi 9 and p40 Transcription gives rise to six transcripts, two initiated at each of three promoters, with one of each pair being spliced
  • the splice site, derived from map units 42-46, is the same for each transcript
  • the four non-structural proteins apparently are derived from the longer of the transcripts, and three virion proteins all arise from the smallest transcript
  • AAV is not associated with any pathologic state in humans Interestingly, for efficient replication, AAV requires "helping" functions from viruses such as herpes simplex virus I and II, cytomegalovirus, pseudorabies virus and, of course, adenovirus
  • helpers The best characterized of the helpers is adenovirus, and many "early" functions for this virus have been shown to assist with AAV replication
  • Low level expression of AAV rep proteins is believed to hold AAV structural expression in check, and helper virus infection is thought to remove this block
  • the terminal repeats of the AAV vector can be obtained by restriction endonuclease digestion of AAV or a plasmid such as p201, which contains a modified AAV genome (Samulski et al 1987), or by other methods known to the skilled artisan, including but not limited to chemical or enzymatic synthesis of the terminal repeats based upon the published sequence of AAV
  • the ordinarily skilled artisan can determine, by well-known methods such as deletion analysis, the minimum sequence or part of the AAV ITRs which is required to allow function, i.e., stable and site-specific integration
  • the ordinarily skilled artisan also can determine which minor modifications of the sequence can be tolerated while maintaining the ability of the terminal repeats to direct stable, site-specific integration
  • AAV-based vectors have proven to be safe and effective vehicles for gene delivery in vitro, and these vectors are being developed and tested in pre-clinical and clinical stages for a wide range of applications in potential gene therapy, both ex vivo and in vivo (Flotte and Carter, 1995 , Chatterjee et al, 1995, Ferrari et al, 1996, Fisher et al, 1996, Flotte et al, 1993, Goodman et al, 1994, Kaplitt et al, 1994, 1996, Kessler et al, 1996, Koeberl et al, 1997, Mizukami et al, 1996, Xiao et al, 1996)
  • AAV-mediated efficient gene transfer and expression in the lung has led to clinical trials for the treatment of cystic fibrosis (Flotte and Carter, 1995, Flotte et al, 1993)
  • the prospects for treatment of muscular dystrophy by AAV-mediated gene delivery of the dystrophin gene to skeletal muscle, of Parkinson's disease by tyrosine hydroxylase gene delivery to the brain, of hemophilia B by Factor IX gene delivery to the liver, and potentially of myocardial infarction by vascular endothelial growth factor gene to the heart appear promising since AAV-mediated transgene expression in these organs has recently been shown to be highly efficient (Fisher et al, 1996, Flotte et al, 1993, Kaplitt et al, 1994, 1996, Koeberl et al, 1997, McCown et al, 1996, Ping et al, 1996, Xiao et al, 1996)
  • viral vectors may be employed as expression constructs in the present invention
  • Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988,
  • Virus, and herpesviruses may be employed These viruses offer several features for use in gene transfer into various mammalian cells B. Non-viral Transfer
  • DNA constructs of the present invention are generally delivered to a cell, in certain situations, the nucleic acid to be transferred is non-infectious, and can be transferred using non-viral methods
  • Non-viral methods for the transfer of expression constructs into cultured mammalian cells include calcium phosphate precipitation (Graham and Van Der Eb, 1973, Chen and Okayama, 1987, Rippe et al, 1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al, 1986, Potter et al, 1984), direct microinjection (Hariand and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982, Fraley et al, 1979), cell sonication (Fechheimer et al, 1987), gene bombardment using high velocity microprojectiles (Yang et al, 1990), and receptor-mediated transfection (Wu and Wu, 1987, Wu and Wu, 1988)
  • the nucleic acid encoding the therapeutic gene may be positioned and expressed at different sites
  • the nucleic acid encoding the therapeutic gene may be stably integrated into the genome of the cell This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation)
  • the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA
  • Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed
  • the expression construct may be entrapped in a liposome
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium
  • Muhilamellar liposomes have multiple lipid layers separated by aqueous medium They form spontaneously when phospholipids are suspended in an excess of aqueous solution
  • the lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991)
  • the addition of DNA to cationic liposomes causes a topological transition from liposomes to optically birefringent liquid-crystalline condensed globules (Radler et al, 1997)
  • These DNA-lipid complexes are potential non-viral vectors for use in gene therapy
  • Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful Using the ⁇ -lactamase gene, Wong et al. (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa, and hepatoma cells Nicolau et al. (1987) accomplished successful liposome-mediated gene transfer in rats after intravenous injection Also included are various commercial approaches involving "lipofection" technology
  • the liposome may be complexed with a hemagglutinating virus (HVJ) This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al, 1989)
  • HVJ hemagglutinating virus
  • the liposome may be complexed or employed in conjunction with nuclear nonhistone chromosomal proteins (HMG-1) (Kato et al, 1991)
  • HMG-1 nuclear nonhistone chromosomal proteins
  • the liposome may be complexed or employed in conjunction with both HNJ and HMG-1 In that such expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present invention
  • receptor-mediated delivery vehicles which can be employed to deliver a nucleic acid encoding a therapeutic gene into cells
  • receptor-mediated delivery vehicles These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993)
  • Receptor-mediated gene targeting vehicles generally consist of two components a cell receptor-specific ligand and a DNA-binding agent Several ligands have been used for receptor-mediated gene transfer The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferrin (Wagner et al, 1990)
  • ASOR asialoorosomucoid
  • transferrin transferrin
  • a synthetic neoglycoprotein which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle (Ferkol et al, 1993, Perales et
  • the delivery vehicle may comprise a ligand and a liposome
  • a ligand and a liposome For example, Nicolau et al. (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes
  • a nucleic acid encoding a therapeutic gene also may be specifically delivered into a cell type such as prostate, epithelial or tumor cells, by any number of receptor-ligand systems with or without liposomes
  • the human prostate-specific antigen (Watt et al, 1986) may be used as the receptor for mediated delivery of a nucleic acid in prostate tissue
  • the expression construct may simply consist of naked recombinant DNA or plasmids Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane This is applicable particularly for transfer in vitro, however, it may be applied for m vivo use as well Dubensky et al.
  • DNA encoding a CAM may also be transferred in a similar manner in vivo and express CAM
  • Another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment This method depends on the ability to accelerate DNA coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al, 1987) Several devices for accelerating small particles have been developed One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al, 1990)
  • the microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads
  • cancers include but are not limited to cancers of the brain (e.g., glioblastoma, astrocytoma, oligodendroglioma, ependymomas), neurofibrosarcoma, meningia, lung, liver, spleen, kidney, lymph node, pancreas, small intestine, blood cells, colon or colorectal region, stomach, thyroid, breast, endometrium, prostate, testes, ovary, skin, head and neck, esophagus, bone marrow, blood or other tissue
  • human monoclonal antibodies are employed in passive immunotherapy, as they produce few or no side effects in the patient
  • their application is somewhat limited by their scarcity and have so far only been administered intralesionally
  • Human monoclonal antibodies to ganglioside antigens have been administered intralesionally to patients suffering from cutaneous recurrent melanoma (Irie & Morton, 1986) Regression was observed in six out of ten patients, following, daily or weekly, intralesional injections
  • moderate success was achieved from intralesional injections of two human monoclonal antibodies (Irie et al, 1989)
  • Treatment protocols also may include administration of lymphokines or other immune enhancers as described by Bajorin et al. (1988) The development of human monoclonal antibodies is described in further detail elsewhere in the specification
  • an antigenic peptide, polypeptide or protein, or an autologous or allogenic tumor cell composition or "vaccine” is administered, generally with a distinct bacterial adjuvant (Ravindranath & Morton, 1991, Morton & Ravindranath, 1996, Morton et al, 1992, Mitchell et al, 1990, Mitchell et al, 1993)
  • a distinct bacterial adjuvant Rostranath & Morton, 1991, Morton & Ravindranath, 1996, Morton et al, 1992, Mitchell et al, 1990, Mitchell et al, 1993
  • melanoma immunotherapy those patients who elicit high IgM response often survive better than those who elicit no or low IgM antibodies (Morton et al, 1992) IgM antibodies are often transient antibodies and the exception to the rule appears to be anti-ganglioside or anticarbohydrate antibodies
  • the patient's circulating lymphocytes, or tumor infiltrated lymphocytes are isolated in vitro, activated by lymphokines such as IL-2 or transduced with genes for tumor necrosis, and readministered (Rosenberg et al, 1988,
  • activated lymphocytes will most preferably be the patient's own cells that were earlier isolated from a blood or tumor sample and activated (or "expanded") in vitro.
  • This form of immunotherapy has produced several cases of regression of melanoma and renal carcinoma, but the percentage of responders were few compared to those who did not respond.
  • the present invention provides methods for the treatment of various malignancies involving IGFBP-2 directed therapy, which includes compounds or compositions that affect IGFBP-2.
  • Treatment methods will involve treating an individual with an effective amount of a viral particle, as described above, containing a therapeutic gene of interest.
  • Other treatment methods involve treating an individual with an effective amount of an antibody.
  • treatment may involve administering an IGFBP-2 peptide or polypeptide that elicits an immune response, which effects similar results as administration of an anti-IGFBP-2 antibody.
  • An effective amount is described, generally, as that amount sufficient to detectably and repeatedly ameliorate, reduce, minimize or limit the extent of a disease or its symptoms.
  • Target cancer cells include cancers of the lung, brain, prostate, kidney, liver, ovary, breast, skin, stomach, esophagus, head and neck, testicles, colon, cervix, lymphatic system and blood. Of particular interest are cancers of the prostate.
  • the IGFBP-2 directed therapy may be combined with surgery or compositions comprising other agents effective in the treatment of cancer.
  • the IGFBP-2 directed therapy may be combined with other anti-cancer therapies or agents, such as surgery, immunotherapy, chemotherapy, radiotherapy, gene therapy, or hormone therapy.
  • compositions would be provided in a combined amount effective to kill or inhibit proliferation of a cancer cell
  • This process may involve contacting the cells with the IGFBP-2 directed therapy and the agent(s) or factor(s) at the same time
  • This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the IGFBP-2 directed therapy and the other includes the second agent
  • the IGFBP-2 directed therapy may precede or follow the other agent treatment by intervals ranging from minutes to weeks
  • the other agent and IGFBP-2 directed therapy are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and IGFBP-2 directed therapy would still be able to exert an advantageously combined effect on the cell.
  • Cancer therapies can include a variety of combination therapies with both chemical and radiation based treatments
  • Combination chemotherapies include, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, taxol, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate or any analog or derivative variant thereof
  • Radioisotopes Other factors that cause DNA damage and have been used extensively include what are commonly known as ⁇ -rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation It is most likely that all of these factors effect a broad range of damage on DNN on the precursors of D ⁇ A, on the replication and repair of D ⁇ A, and on the assembly and maintenance of chromosomes Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells
  • Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies.
  • Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed Tumor resection refers to physical removal of at least part of a tumor
  • treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs' surgery) It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue
  • the secondary treatment is gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time as the IGFBP-2 directed agent
  • Genes encoding proteins that induce cellular proliferation, that inhibit cell proliferation, or that regulate apoptosis are especially useful in the context of the present invention
  • Genes encoding proteins that induce cellular proliferation include erbN erbB, fos, jun, myc, src, ras, abl, fins, neu, her, myb, and rel
  • anti-sense mR ⁇ A directed to a particular inducer of cellular proliferation is used to prevent expression of the inducer of cellular proliferation
  • Genes that encode inhibitors of cell proliferation include genes known as tumor suppressor genes Examples are p53, pi 6, Rb, CCAM, APC, DCC, ⁇ F-1, NF-2, and WT-1 Many proteins involved in apoptosis are in the Bel family Some of these act like Bcl-2 (
  • IGFBP- 1, -3, -4, or -5 may be used in gene therapy methods in combination with an IGFBP-2 directed therapy
  • the level of IGFBP-3 may be modulated in conjunction with the administration of an IGFBP-2 directed therapy
  • the level of proteins implicated in the regulation of IGFBPs such as prostate-specific antigen (PSA) may also be altered in a combination treatment
  • PSA prostate-specific antigen
  • levels of PSA may be reduced by any of the methods described herein, such as by antisense of antibody modulation, or a vaccine against PSA may be used See U S Patent No 5,925,362, which is hereby incorporated by reference
  • genes involved in IGFBP modes of action for example, IGF-II-R, IGFs, IGF-I-R may also be similarly employed
  • Hormonal therapy may also be used in conjunction with the present invention or in combination with any other cancer therapy previously described
  • hormones may be employed in the treatment of certain cancers such as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen (e.g., anti-androgen or anti-estrogen agents)
  • This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases
  • hyperthermia is a procedure in which a patient's tissue is exposed to high temperatures (up to 106°F)
  • External or internal heating devices may be involved in the application of local, regional, or whole-body hyperthermia
  • Local hyperthermia involves the application of heat to a small area, such as a tumor Heat may be generated externally with high-frequency waves targeting a tumor from a device outside the body Internal heat may involve a sterile probe , including thin, heated wires or hollow tubes filled with warm water, implanted microwave antennae, or radiofrequency electrodes
  • a patient's organ or a limb is heated for regional therapy, which is accomplished using devices that produce high energy, such as magnets.
  • devices that produce high energy such as magnets.
  • some of the patient's blood may be removed and heated before being perfused into an area that will be internally heated
  • Whole-body heating may also be implemented in cases where cancer has spread throughout the body
  • Warm-water blankets, hot wax, inductive coils, and thermal chambers may be used for this purpose
  • IGFB-2 directed agent is "A” and the second anti-cancer therapy is "B"
  • IGFBP-2 directed therapy of the present invention will follow general protocols for the administration of chemotherapeutics, taking into account the toxicity, if any, of the therapies It is expected that the treatment cycles would be repeated as necessary It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described IGFBP-2 directed therapies
  • compositions according to the present invention will be via any common route so long as the target tissue is available via that route This includes oral, nasal, buccal, rectal, vaginal or topical Topical administration would be particularly advantageous for treatment of skin cancers Alternatively, administration will be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.
  • ex v vo therapies also are contemplated.
  • Ex vivo therapies involve the removal, from a patient, of target cells The cells are treated outside the patient's body and then returned
  • One example of ex vivo therapy would involve a variation of autologous bone marrow transplant
  • ABMT fails because some cancer cells are present in the withdrawn bone marrow, and return of the bone marrow to the treated patient results in repopulation of the patient with cancer cells
  • the withdrawn bone marrow cells could be treated while outside the patient with an viral particle that targets and kills the cancer cell
  • the treatments may include various "unit doses " Unit dose is defined as containing a predetermined-quantity of the therapeutic composition calculated to produce the desired responses in association with its administration, i.e , the appropriate route and treatment regimen
  • the quantity to be administered, and the particular route and formulation are within the skill of those in the clinical arts Also of importance is the subject to be
  • One of the preferred embodiments of the present invention involves the use of viral vectors to deliver therapeutic genes to cancer cells
  • the cancer may be directly injection a tumor with the viral vector
  • the tumor may be infused or perfused with the vector using any suitable delivery vehicle Local or regional administration, with respect to the tumor, also is contemplated
  • systemic administration may be performed Continuous administration also may be applied where appropriate, for example, where a tumor is excised and the tumor bed is treated to eliminate residual, microscopic disease Delivery via syringe or catherization is preferred
  • Such continuous perfusion may take place for a period from about 1-2 hours, to about 2-6 hours, to about 6-12 hours, to about 12-24 hours, to about 1-2 days, to about 1-2 wk or longer following the initiation of treatment
  • the dose of the therapeutic composition via continuous perfusion will be equivalent to that given by a single or multiple injections, adjusted over a period of time during which the perfusion occurs
  • the volume to be administered will be about 4-10 ml (preferably 10 ml), while for tumors of ⁇ 4 cm, a volume of about 1-3 ml will be used
  • Multiple injections delivered as single dose comprise about 0 1 to about 0 5 ml volumes
  • the viral particles may advantageously be contacted by administering multiple injections to the tumor, spaced at approximately 1 cm intervals
  • Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months These treatments may be of varying dosages as well
  • the tumor being treated may not, at least initially, be resectable Treatments with therapeutic viral constructs may increase the resectability of the tumor due to shrinkage at the margins or by elimination of certain particularly invasive portions Following treatments, resection may be possible Additional viral treatments subsequent to resection will serve to eliminate microscopic residual disease at the tumor site
  • a typical course of treatment, for a primary tumor or a post-excision tumor bed, will involve multiple doses Typical primary tumor treatment involves a 6 dose application over a two-week period
  • the two-week regimen may be repeated one, two, three, four, five, six or more times
  • the need to complete the planned dosing may be re-evaluated
  • compositions of the present invention comprise an effective amount of the IGFBP-2 directed therapeutic dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium
  • pharmaceutically acceptable carrier refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art.
  • compositions should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
  • the biological material should be extensively dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle, where appropriate.
  • the active compounds will then generally be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub- cutaneous, intralesional, or even intraperitoneal routes.
  • parenteral administration e.g., formulated for injection via the intravenous, intramuscular, sub- cutaneous, intralesional, or even intraperitoneal routes.
  • the preparation of an aqueous composition that contains an RBP agent as an active component or ingredient will be known to those of skill in the art in light of the present disclosure.
  • such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms
  • An IGFBP-2 directed agent of the present invention can be formulated into a composition in a neutral or salt form
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like
  • the technology of U S Patents 4,608,251, 4,601,903, 4,599,231, 4,599,230, 4,596,792, and 4,578,770 each incorporated herein by reference, may be used
  • the carrier also can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils
  • a coating such as lecithin
  • surfactants The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like
  • isotonic agents for example, sugars or sodium chloride
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof
  • the preparation of more, or highly, concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like also can be employed
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580)
  • Some variation in dosage will necessarily occur depending on the condition of the subject being treated The person responsible for administration will, in any event, determine the appropriate dose for the individual subject
  • other pharmaceutically acceptable forms include, e.g , tablets or other solids for oral administration, liposomal formulations, time release capsules, and any other form currently used, including cremes
  • Nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained Thus, the aqueous nasal solutions usually are isotonic and slightly buffered to maintain a pH of 5 5 to 6 5
  • antimicrobial preservatives similar to those used in ophthalmic preparations, and appropriate drug stabilizers, if required, may be included in the formulation
  • Various commercial nasal preparations are known and include, for example, antibiotics and antihistamines and are used for asthma prophylaxis
  • suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum, vagina or the urethra After insertion, suppositories soften, melt or dissolve in the cavity fluids
  • traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides, such suppositories may be formed from mixtures containing the active ingredient in the range of 0 5% to 10%, preferably l%-2%
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders
  • oral pharmaceutical compositions will comprise an inert diluent or assimilable edible ca ⁇ ier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet
  • the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like
  • Such compositions and preparations should contain at least 0 1% of active compound The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 75% of the weight of the
  • the tablets, troches, pills, capsules and the like may also contain the following a binder, as gum tragacanth, acacia, cornstarch, or gelatin, excipients, such as dicalcium phosphate, a disintegrating agent, such as corn starch, potato starch, alginic acid and the like, a lubricant, such as magnesium stearate, and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring
  • a liquid carrier Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit For instance, tablets, pills, or capsules may be coated with shellac, sugar or both
  • a syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and
  • kits can contain antibody compositions that recognize IGFBP-2, recombinant vectors containing a promoter operably linked to a nucleic acid segment that encodes IGFBP- 2, or any other modulators as described or anticipated in this application All the essential materials and reagents required for inhibiting an IGFBP-2 dependent tumor cell may be assembled together in a kit
  • the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being particularly preferred
  • a chemotherapeutic agent may be formulated into a single or separate pharmaceutically acceptable syringeable composition
  • the container means may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the formulation may be applied to an infected area of the body, such as the lungs, injected into an animal, or even applied to and mixed with the other components of the kit
  • kits of the invention may also be provided in dried or lyophilized forms When reagents or components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent It is envisioned that the solvent also may be provided in another container means
  • the kits of the invention may also include an instruction sheet defining administration of the gene therapy and/or the chemotherapeutic drug
  • kits of the present invention also will typically include a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained Irrespective of the number or type of containers, the kits of the invention also may comprise, or be packaged with, an instrument for assisting with the injection/administration or placement of the ultimate complex composition within the body of an animal
  • an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle
  • the present invention also contemplates the use of IGFBP-2 dependent cells, IGFBP-2 and active fragments, and nucleic acids coding thereof, in the screening of compounds for activity in inhibiting an IGFBP-2 dependent cell
  • Assays may make use of a variety of different formats and may depend on the kind of "activity" for which the screen is being conducted
  • Contemplated functional "read-outs" include binding to a compound, inhibition of binding to a substrate, ligand, receptor or other binding partner by a compound, phosphatase activity, anti-phosphatase activity, phosphorylation of IGFBP-2, dephosphorylation of IGFBP-2, inhibition or stimulation of cell-to-cell signaling, growth, metastasis, cell division, cell migration, soft agar colony formation, contact inhibition, invasiveness, angiogenesis, apoptosis, tumor progression or other malignant phenotype
  • the invention is to be applied for the screening of compounds that bind to the IGFBP-2 peptide or polypeptide
  • the polypeptide or fragment may be either free in solution, fixed to a support, expressed in or on the surface of a cell Either the polypeptide or the compound may be labeled, thereby permitting determining of binding
  • the assay may measure the inhibition of binding of IGFBP-2 to a natural or artificial substrate or binding partner
  • Competitive binding assays can be performed in which one of the agents (IGFBP-2, binding partner or compound) is labeled
  • the polypeptide will be the labeled species
  • One may measure the amount of free label versus bound label to determine binding or inhibition of binding
  • the assay may measure the inhibition of IGFBP-2 transcription, translation or secretion
  • these assays will generally be cell-based assays where detection of the secreted IGFBP-2 is monitored A decrease in IGFBP-2 in the presence of a candidate substance compared to the levels of IGFBP-2 in the absence of a candidate substance would be screened for.
  • Purified IGFBP-2 can be coated directly onto plates for use in the aforementioned drug screening techniques
  • non-neutralizing antibodies to the polypeptide can be used to immobilize the polypeptide to a solid phase
  • fusion proteins containing a reactive region may be used to link the IGFBP-2 active region to a solid phase
  • IGFBP-2 Various cell lines containing wild-type or natural or engineered mutations in IGFBP-2 can be used to study various functional attributes of IGFBP-2 and how a candidate compound affects these attributes
  • Methods for engineering mutations are described elsewhere in this document, as are naturally-occurring alleles of IGFBP-2
  • the compound would be formulated appropriately, given its biochemical nature, and contacted with a target cell
  • culture may be required
  • the cell may then be examined by virtue of a number of different physiologic assays
  • molecular analysis may be performed in which the function of IGFBP-2, or related pathways, may be explored This may involve assays such as those for protein expression, enzyme function, substrate utilization, phosphorylation states of various molecules including IGFBP-2, cAMP levels, mRNA expression (including differential display of whole cell or polyA RNA) and others
  • the present invention also encompasses the use of various animal models
  • the identity seen between human and rat IGFBP-2 provides an excellent opportunity to examine the function of IGFBP-2 in a whole animal system where it is normally expressed
  • mutant cells lines that fail to express or overexpress normal IGFBP-2
  • These models may employ the orthotopic or systemic administration of tumor cells to mimic primary and/or metastatic cancers
  • one may induce cancers in animals by providing agents known to be responsible for certain events associated with malignant transformation and/or tumor progression
  • transgenic animals discussed below
  • that lack a wild-type IGFBP-2 may be utilized as models for cancer development and treatment
  • Treatment of animals with test compounds will involve the administration of the compound, in an appropriate form, to the animal Administration will be by any route that could be utilized for clinical or non-clinical purposes, including but not limited to oral, nasal, buccal, rectal, vaginal or topical Alternatively, administration may be by intratracheal instillation, bronchial instillation, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection Specifically contemplated are systemic intravenous injection, regional administration via blood or lymph supply and intratumoral injection
  • Determining the effectiveness of a compound in vivo may involve a variety of different criteria such criteria include, but are not limited to, survival, reduction of tumor burden or mass, arrest or slowing of tumor progression, elimination of tumors, inhibition or prevention of metastasis, increased activity level, improvement in immune effector function and improved food intake
  • drugs which have improved IGFBP-2 activity or which act as stimulators, inhibitors, agonists, antagonists or IGFBP-2 or molecules affected by IGFBP-2 function
  • IGFBP-2 genes and proteins of the present invention are also encompassed within the present invention.
  • amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies, binding sites on substrate molecules or receptors, DNA binding sites, or such like Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a protein with like (agonistic) properties It is thus contemplated by the inventors that various changes may be made in the sequence of IGFBP-2 proteins or polypeptides, or underlying DNN without appreciable loss of their biological utility or activity.
  • biologically functional equivalent protein or peptide or gene is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule and still result in a molecule with an acceptable level of equivalent biological activity
  • Biologically functional equivalent peptides are thus defined herein as those peptides in which certain, not most or all, of the amino acids may be substituted
  • residues are shown to be particularly important to the biological or structural properties of a protein or peptide, e.g., residues in binding regions or active sites, such residues may not generally be exchanged This is an important consideration in the present invention, where changes in the IGFBP-2 should be carefully considered and subsequently tested to ensure maintenance of biological function, where maintenance of biological function is desired In this manner, functional equivalents are defined herein as those peptides which maintain a substantial amount of their native biological activity
  • Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like
  • An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues, that alanine, glycine and serine are all a similar size, and that phenylalanine, tryptophan and tyrosine all have a generally similar shape Therefore, based upon these considerations, arginine, lysine and histidine, alanine, glycine and serine, and phenylalanine, tryptophan and tyrosine, are defined herein as biologically functional equivalents To effect more quantitative changes, the hydropathic index of amino acids may be considered Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are isoleucine (+4 5
  • IGFBP-2 peptidyl compounds described herein the inventors also contemplate that other sterically similar compounds may be formulated to mimic the key portions of the peptide structure or to interact specifically with, for example, substrates or receptor compounds Such compounds, which may be termed peptidomimetics, may be used in the same manner as the peptides of the invention and hence are also functional equivalents
  • Rat IGFBP-2 is purchased from Research Diagnostics, Inc (Flanders, NJ), and rat IGFBP-2 antibody is custom-made for the experiments by Genemed Synthesis Inc (San Francisco, CA)
  • a polyclonal antibody against human IGFBP-2 was generated using a peptide of the following sequence LFYNEQQEARGVHTQRMQ (SEQ ID NO 3)
  • MTT assay 6,000 cells are seeded/well in 96-well plates and grown overnight in 10%) FCS Cells are maintained under serum-free conditions for 24 h, and then stimulated for 48 h with various concentrations of IGF, IGFBP-2 and antibody to IGFBP-2 The final cell number is determined with the help of MTT dye (Sigma M- 2128) (Singh et al, 1994) The plates are read immediately at 540 nm on a scanning multi-well spectrophotometer (Model Vmax Molecular Devices, Melno Park, CA) Optical absorbance reading from test wells is corrected for basal control absorbance values from cell-free wells containing culture media only Clonogenic assay: Cells are seeded at equal concentrations (8,000 cells/well) in 6-well culture plates in 0 3% agar in growth medium containing IGF, IGFBP-2 and antibody to IGFBP-2 (MacPherson and Montagnier, 1964) The colonies are stained with methylene blue, and total number of colonies (> 1 mm
  • RNA Extraction Total RNA is extracted from cells by a single-step phenol and guanidinium thiocyanate extraction method, using RNA Stat 60 (Tel-Test, Inc., Friendswood, TX) Total RNA is quantitated by Spectrophotometric readings at wavelengths of 260/270/280
  • cDNA is prepared from 1 microgram of total RNN using AMV reverse transcriptase (Promega) Ten percent of the cD ⁇ A prepared is used for amplification by PCR, (30-32 PCR cycles), using appropriate primers
  • the PCR reaction is performed in 50 microliters buffer containing 2 microliters cD ⁇ N 20 pmol of each primer, 250 uM d ⁇ TPs, and 0 3 units Taq polymerase The reactions are denatured at 94°C for 30 sec, annealed at 60°C for 45 sec, and elongated at 72°C for 1 min/cycle Twenty microliters of mixture from each reaction is then electrophoresed on 1% agarose and visualized with ethidium bromide staining
  • RNA is electrophoresed through formaldehyde-agarose gels, transferred to nylon membranes, and cross-linked either with ultraviolet light or baking.
  • Partial length complementary probes are used for analysis of mRNA
  • the cDNA probes are labeled by nick translation, using commercial kits from Promega (Madison, WI), and then hybridized to membranes using standard conditions Membranes are washed, briefly air-dried, exposed to X-ray film for an appropriate amount of time for the signal to develop, and the autoradiograms analyzed B.
  • RNA samples prepared from LNCap and DU145 prostate cancer cell lines were analyzed for expression of IGFBP-2 mRNA transcripts by RT-PCR A DNA fragment consistent with the predicted size (442 bp) was present from the cells that were analyzed, demonstrating that message for IGFBP-2 is present in these cell lines
  • FIG IA shows the effects of either 125, 250 or 500 ng/ml IGFBP-2 or 125, 250 or 500 ng/ml IGFBP-2 antibodies on the growth of LNCap cells as compared to control cells grown grown in 1%, 5% or 10% FCS
  • FIG IB shows the effects of these same treatments on DU145 cells While IGFBP-2 stimulated the growth of both cell lines, antibodies to IGFBP-2 inhibitted the growth of both cell lines
  • FIG IC and FIG ID demonstrate the effects of IGFBP-2 or antibodies to IGFBP-2 in the presence or absence of IGF-I (250 ng/ml)
  • An MTT assay was used to measure relative cell numbers Again, antibodies to IGFBP-2 inhibited the growth of both LNCap and DU145 cells In addition, this inhibition was still observed in the presence of exogenous IGF-1
  • a clonogenic assay was used to test the growth of DU145 cells treated with different doses of IGFBP-2 Ab All cultures were maintained in 10%> serum and treatments were administered for 5 weeks Colonies were stained with 1% methylene blue, and colonies were counted and their relative size determined While IGFBP-2 treatment (250 ng/ml) resulted in larger colonies compared to control cultures, culturing cells in increasing concentrations of antibodies to IGFBP-2 (125, 250 and 500 ng/ml) resulted in smaller and fewer colonies compared to control cultures Tumorigenic potential of DU145 cells grown in the presence of IGFBP-2 or IGFBP-2 Ab was determined in a soft agar clonogenic assay (MacPherson and Montagnier, 1964) Results of colony number are shown in FIG 2 Cells were cultured in either 1, 5 or 10% serum alone (Serum samples, FIG 2 ) or serum free with 125, 250, or 500 ng/ml IGF-1, IGFBP-2 or antibodies to IGFBP-2 The total number
  • RT-PCR analysis was used to determine the relative levels of IGF-I mRNA in LNCap cells treated with IGFBP-2 Ab RNA samples from LNCap cells growing in culture for 3 days in the presence of varying concentrations of IGFBP-2 Ab (125, 250 or 500 ng/ml) were subjected to RT-PCR for 32 cycles, using the primers specific for hIGF-I cDNA Ethidium bromide staining of the reaction products was used to evaluate the relative density of IGF-1 PCR products from the three control cultures compared to the experimental cultures The relative density of IGF-I message following IGFBP-2 Ab treatment was decreased approximately 50-100%>, compared with the relative density of IGF-1 message in control samples
  • Prostate cancer cells are treated with either IGFBP-2 or IGFBP-2 Ab and the resultant changes in the expression of IGF system components (viz IGFs, IGF-IR, and IGFBP-2) is examined Expression of prostate cancer specific Prostate Tumor Inducing- 1 (PTI-1) gene is also examined.
  • the inventors are measuring IGF-I RNA and PTI-1 RNA levels using RT-PCR in response to IGFBP-2 Ab treatment They are also examining levels of IGF-II and IGF-IR by RT-PCR and Northern hybridization/RPN as well as receptor binding assays
  • the inventors examined the effect of IGFBP-2 on the growth of prostate cancer cell lines in vitro, and investigated the probable mechanism by which IGFBP-2 exerts its growth-modulatory effects
  • IGFBP- 2 strongly stimulates the growth of androgen-sensitive (L ⁇ Cap) and androgen- independent (DU145) prostatic cancer cell lines Growth of the cells is significantly inhibited by IGFBP-2 Antibody (Ab)
  • IGFBP-2 Antibody (Ab) The probable mechanism by which IGFBP-2 Ab inhibited growth was by down-regulating mRNA levels of IGF-I and Prostate Tumor Inducing- 1 (PTI-1) genes Since PTI-1 is a marker for prostate cancer (Shen et al , 1995, Sun et al , 1997), down-regulation of mRNA levels of PTI-1 indicated cancer suppression
  • IGFBP-2 Circulatory levels of IGFBP-2 have been reported to correlate directly with the stage and severity of cancer in prostate cancer patients Thus, circulatory levels of IGFBP-2 may be a biomarker for progression of prostate cancer
  • the role of IGFBP-2 in the initiation and progression of prostate cancer is not known to date
  • the present application demonstrates that IGFBP-2 strongly stimulated the growth of prostate cancer cell lines grown in tissue culture dishes, while suppression of IGFBP-2 significantly reversed these growth effects
  • Treatment with IGFBP-2 Ab resulted in reduced expression of IGF-I, which is a growth stimulating factor for prostate cells
  • IGF-I which is a growth stimulating factor for prostate cells
  • growth of prostate cancer cells can be potentiated by IGFBP-2, and significantly, retarded by suppression of endogenous IGFBP-2
  • IGFBP-2 promotes prostate cancer in the elderly by stimulating IGF-I Normally, the serum levels of IGF-I progressively decline due to aging Abnormal stimulation of IGF-I by high circulatory levels of IGFBP-2 could
  • the inventors used stably transfected clones of aggressive (DU145) and non- aggressive (LNCap) prostate cancer cell line that are over-expressing either antisense (AS) or sense (S) IGFBP-2 Growth of cells transfected with the empty vector will serve as control (C)
  • the growth potential and clonogenic potential of stably transfected S, AS and C clones of LNCap and DU145 prostate cancer cells will be measured in vitro
  • the inventors expect that S LNCap and DU145 cells will show increased clonogenic and growth potential, compared to that of the C clones, while the clonogenic potential of AS LNCap and DU145 cells will be significantly reduced
  • the inventors will measure the concentrations of the IGF factors and the PTI-1 gene products at the mRNA and protein levels in the transfected cells
  • the inventors are using the mammalian expression vector, pCEP4 (Invitrogen), for the studies
  • This vector uses the Epstein-Barr virus origin of replication, and has Epstein-Barr virus-encoded nuclear antigen- 1 (EBNA-1), which allows it to replicate extra-chromosomally in the nuclei of eukaryotic cells It has a hygromycin B gene, which allows for selection in eukaryotic cells, and a pBR322 origin of replication, which allows growth and maintenance in Escherichia coli cells It uses the cytomegalovirus promoter to drive expression of the insert in a constitutive manner
  • Plasmids containing either a partial length cDNA (503 bps, for constructing AS plasmids), or a full-length cDNA (for constructing S plasmids) of hIGFBP-2 are constructed using hIGFBP-2 cDNA clones as described (Shimasaki and Ling, 1991)
  • the appropriate cDNA fragment of hIGFBP-2 is cloned in the sense (S) or anti-sense (AS) direction in pCEP4 vectors
  • the directional cloning of the cDNA insert is confirmed by restriction mapping and DNA sequencing (Wood et al, 1993) Using lipofectamine, DU145 and LNCap cells are transfected with the AS and S pCEP4 vectors, while Control (C) cells are transfected with only the pCEP4 vectors (no insert) Cells are allowed to recover for 2 days in non-selective medium Hygromycin (Sigma Chemical Co ) is then added at the optimal
  • the inventors are analyzing the growth of non-transfected and transfected (S, AS and C) cells either by measuring the total cell number at the end of the experimental time point or using a semiautomatic tetrazolium-based clonogenic (MTT) assay as described in Example 1
  • MTT semiautomatic tetrazolium-based clonogenic
  • Receptor Binding Assay for studying the expression of IGF-IR S, AS and C clones of LNCap and DU145 cells are subcultured in 160 mm flasks and grown to subconfluency in culture medium containing 10% FCS Cells in culture are washed with Hank's balanced salt solution (HBSS, Gibco, Grand Island, NY) containing 0 1%> bovine serum albumin and 25 mmol/L HEPES (Sigma) Cells are scraped with a rubber policeman, collected in conical tissue culture polystyrene tubes, centrifuged and resuspended in HBSS at a concentration of 2-3 X 10 6 cells/ml Binding assays are performed by incubating 0 5 ml aliquots of suspended cells with 0 01 nmol/L 125 I-IGF-I (Amersham, Arlington Heights, IL) in the presence or absence of radio-inert IGF-I, at 16°C for 6 hours, at pH 8 in a shaking
  • CM Serum-free CM was collected from L ⁇ Cap cells following 7 days of culture
  • the CM samples were concentrated and loaded in each lane (equivalent to 10 cells) and analyzed by Western immunoblot analysis (WTB) with specific anti- IGFBP-2 antibody
  • WTB Western immunoblot analysis
  • Samples from several different clones were analyzed IGFBP-2 (BP2) was used as a positive control for the samples
  • the molecular mass of the bands (35,000 kDa) was matched with that obtained from recombinant IGFBP-2 Comparisons of IGFBP-2 signals from the different clones was used to assess the degree of overexpression under downregulation of IGFBP-2
  • the inventors examine the effect of IGFBP-2 and IGFBP-2 Ab treatment on the rate of spontaneous development of prostate cancer in male retired breeders of the Copenhagen strain of rats, in which spontaneous prostatic tumors are known to develop at 22 months age (Dunning, 1963)
  • IGFBP-2 immunoreactivity is measured in 50 microliter serum samples of Copenhagen rats, using rat IGFBP-2 kit (Diagnostics Systems Laboratories, Inc , Webster, TX) Immunoprecipitates obtained in the double antibody coated RIA tubes of the kit are pelleted after 4 hrs of incubation at room temperature, and radioactivity is measured in a gamma counter
  • the prostate glands are dissected out and fixed in Bouin's fixative Following routine histological procedures the glands are embedded in paraffin and 5 micron sections are stained with hematoxylin and eosin, mounted with permount and observed under the microscope for microanatomical changes, as described by Noble (1982)
  • IGFBP-2 is detected immunocytochemically on 5 micron paraffin sections, following a standardized procedure for immunocytochemistry (Chatterjee et al, 1996, Fang et al, 1998), and by using 3,3'-diaminobenzidine (DAB) as the chromogen Sections are counterstained with hematoxylin, dehydrated in alcohol and mounted in Permount for observation under the light microscope Control sections are processed in parallel with the omission of primary antibodies A rough estimate of percentage of cells positive for IGFBP-2 and an estimate of the intensity of IGFBP-2 staining is obtained by video image analysis of the cells using a video image analysis system (Nikon Inc , Torrence, CA)
  • RNA Extraction Frozen prostate glands obtained from rats of different age groups, and tumors generated in nude mice (Example 4) are homogenized, and total
  • cDNA is prepared from 1 microgram of total RNN using AMV reverse transcriptase (Promega) 10 % of the cD ⁇ A prepared is used for amplification by PCR, (30-32 PCR cycles), using appropriate primers
  • the PCR reaction is performed in 50 microliters buffer containing 2 microliters cD ⁇ N 20 pmol of each primer, 250 uM d ⁇ TPs, and 0 3 units Taq polymerase
  • the reactions are denatured at 94°C for 30 sec, annealed at 60°C for 45 sec, and elongated at 72°C for 1 min/cycle Twenty microliters of mixture from each reaction is then electrophoresed on 1% agarose and visualized with ethidium bromide staining
  • RNA is electrophoresed through formaldehyde-agarose gels, transferred to nylon membranes, and cross-linked either with ultraviolet light or baking Partial length complementary probes (cDNA) are used for analysis of mRNA levels for IGFs, IGF-IR or IGFBP-2
  • cDNA probes are labeled by nick translation, using commercial kits from Promega (Madison, WI), and then hybridized to membranes using standard conditions Membranes are washed, briefly air-dried, exposed to X-ray film for an appropriate amount of time for the signal to develop, and the autoradiograms analyzed
  • the inventors are examining whether spontaneous prostatic tumors develop in male retired breeders (4,12 and 20 months old) of Copenhagen rats is effected when the rats are administered IGFBP-2 or IGFBP-2 antibody Baseline levels of serum IGFBP-2 are measured in animals of the experimental age groups A total of eighty four rats are grouped as follows Group I (Control) -12 rats, Group II (IGFBP-2 treated) - 36 rats, and Group III (IGFBP-2 Ab treated) - 36 rats Osmotic pumps are used to deliver IGFBP-2 and/or IGFBP-2 Ab Loaded Alzet minipumps with flow regulators (flow rate 2 5 1/ hr) deliver either normal physiological saline (0 9%>) (Group I), IGFBP-2 (Group II) or IGFBP-2 antibody (Group III), are implanted into subcutaneous pockets on the dorsal surface of the experimental animals The daily doses of IGFBP-2 received by animals in Group II is pre-calculated at 50% (for 12 animals), 100% (for 12 animals) and 150% (for 12
  • the Alzet minipumps to be used can effectively function for 42 days, they will be replaced every 40 th day
  • the inventors are generating polyclonal antibodies against rat IGFBP-2, using the help of a commercial outfit (Genemed Synthesis, Inc , San Francisco, CA)
  • the crude serum will be tested for the presence of rat IGFBP-2 Ab, using IGFBP-2 ELISA assays, and the titre and specificity of the Ab will be determined
  • the efficacy of the Ab will be tested m vitro on the growth potency of LNCap cells to ensure that they produce effects similar to that measured with the commercially available antibody (Genemed Synthesis, Inc , San Francisco, CA)
  • IGFBP-2 treatment of Copenhagen rats should result in inducing pre- neoplastic/neoplastic changes in the prostate of rats at a younger age, compared to that in untreated control rats
  • IGFBP-2 Ab treatment should delay the onset of the spontaneous changes that are usually measured in aged Copenhagen rats (Dunning, 1963) Circulatory levels of IGFBP-2 are significantly elevated in prostate cancer patients (Ho and Baxter, 1997, Kanety et al, 1993, Figueroa et al, 1998) Since prostate cancer is associated with old age, elevated levels of IGFBP-2 may be involved in the development of malignancy in the prostate of older men If IGFBP-2 promotes growth of cancer cells, then IGFBP-2 Ab may prevent malignant transformations in the prostate, and act like tumor suppressors by retarding the rate of tumor development This application demonstrates that IGF-I and IGFBP-2 may be mediating growth effects in tumors, it is quite conceivable that IGFBP-2 Ab may negate tumorigenesis in much the same way as inhibitors/antibodies of
  • the inventors are testing the tumorigenic potential of aggressive (DU145) or non-aggressive (LNCap) prostate cancer cells following modulation of levels of IGFBP-2 within the cells
  • Stably transfected DU145 and LNCap clones that over- express either the S or the AS human IGFBP-2 mRNA (Example 2) are inoculated subcutaneously on contra-lateral sides of nude (athymic) male mice (10 animals per group) Cells will be inoculated at equal concentrations, approximately 1-10 x 10 6 cells for DU145, and 5-10 x 10 6 cells for LNCap cells in 0 2 ml Hank's balanced salt solution (HBSS), on each side At this concentration, DU145 cells form approximately 0 5 g tumors in about 60 days in 3 month old male athymic nude mice In the case of LNCap cells, inoculation with even 10 x 10 7 cells generally does not result in developing palpable tumors till 90 days of in vivo growth in nude mice At term,
  • the inventors expect to measure palpable tumors in mice inoculated with the S
  • LNCap clones but not C LNCap clones, within 60-90 days of in vivo growth
  • An increase in the tumorigenic potential of IGFBP-2 over-expressing DU145 is also expected
  • the AS DU145 clones, in which IGFBP-2 expression is suppressed will show delay in tumor formation in nude mice, in comparison to C DU145 clones
  • LNCap cells inoculated directly within the prostates can grow into tumors in the orthotopic environment (Gleave et al, 1992)
  • the inventors will test tumorigenic growth of S LNCap cells to that of C LNCap cells in an orthotopic environment
  • the inventors will generate recombinant adenoviruses and retroviruses expressing sense (S) and anti-sense (AS) IGFBP-2 and deliver these to the prostate via surgical procedures, or intra-uretherally Retroviral or adenoviral vectors expressing S or AS IGFBP-2 will be constructed (Singh et al , 1999)
  • the inventors can then deliver the S and AS expressing IGFBP-2 virus to the prostatic cells in situ, by using an optimal titer of the infectious viral particles (approximately 10 6 pfu/ml for retroviruses, and approximately 10 8 ot 10° pfu/ml of adenoviruses) Delivery of the viral particles will include 1) intra-peritoneal (i p ) injections (in the lower abdomen), which is likely to be least toxic, but may lack efficiency, 2) orthotopic injection into the prostatic mass bilaterally, using surgical procedures, 3) intra-uretheral injections at the site of the prostate with intra-urether
  • IGF-I, IGF-IR and IGF-II promoters are cloned into promoterless luciferase reporter vectors to construct the promoter-luciferase vectors While the promoters driving the expression of hIGF-I and hIGF-lR are well defined, at least 4 promoters can be potentially used to drive the expression of IGF-II (Singh et al, 1998) IGF-II promoters P3 and P4 are highly active in many cancers, while PI and P2 are relatively silent The inventors are confirming the activity of the four P1-P4 IGF-II promoters in the DU 145 and LNCap cells to use the appropriate promoter(s) for the present studies The promoter- luciferase vectors are transfected to LNCap and DU145 cells All cells are co- transfected with ⁇ -gal expression vectors to correct for transfection efficiency After transfection, cells are maintained in serum-free media
  • Luciferase and ⁇ -Gal assays Cells are washed with PBS and lysed using the reporter lysis buffer (Promega) as per the protocols provided by the company Cellular debris is pelleted and the supernatant saved for analysis of luciferase and ⁇ -Gal activities Luciferase activity is measured with the Luciferase Assay system (Promega), using 20 microliters of cell extract and 100 microliters luciferase assay buffer, at room temperature Luciferase activity is measured within 15 sec of adding the substrate, lucife ⁇ n, with a Turner TD-20e Luminometer (Turner Designs, Sunnyvale, CA) The ⁇ -Gal assay is performed with the ⁇ -Galactosidase Enzyme Assay System (Promega) as per the protocols provided by the company Ninety-six well plates are used for this assay 30 microliters of cell extracts will be mixed with 20 microliters of lx reporter buffer and 50 microliters
  • mice Twenty-one male athymic nude mice were divided into Control (8 animals) and
  • IgG in 0 1 ml HBSS on the dorsal surface The IgG was added to the cells immediately before injection Animals of the treated group were injected with an equivalent number of cells mixed with anti-IGFBP-2 IgG at the same concentration, (i.e 12 ⁇ g of anti-IGFBP-2 IgG per 2 x 10 6 cells)
  • the mean weight of the tumors in the two groups are presented in FIG 6
  • the mean tumor weight of tumors generated from Control DU145 cells was 1 04 + 0 16 grams, while that of cells treated with anti-IGFBP-2 IgG at the time of inoculation was 0.28 + 0 08 grams
  • Statistical analysis of the tumor weight following student's t-test revealed that the weight of the tumors generated from wild type DU145 cells was significantly greater than those generated from anti-IGFBP-2 treated DU145 cells
  • mice Twenty-one male athymic nude mice were divided into three groups Sets of 20 x 10 6 DU145 aggressive prostate cancer cells were mixed with 30, 60 or 120 ⁇ g (i.e., 10, 20 or 40 ⁇ l from 3 mg/ml stock) of anti-IGFBP-2 IgG Respective groups of animals were subcutaneously inoculated on the left side of the upper arm with 2 x 10 6 cells in 0 1 ml PBS The contra-lateral side was inoculated with an equal concentration of untreated DU145 cells, and served as the control Animals were examined for tumor development after ten days of inoculation When tumors attained a size of 1 cm across, 300 ng of IgG was administered twice a week into the tumors on the left side of tumor-bearing animals All treatments were continued for one month. At term, the tumors were carefully dissected out, weighed, and examined for gross anatomical features.
  • Group 1 Inoculated with untreated cells (2 x 10 6 cells) on right; and 0.1 ml of 1 ml cell suspension of 20 x 10 6 cells treated with 30 ⁇ g anti-IGFBP-2 IgG on left
  • Group II Inoculated with untreated cells (2 x 10 6 cells) on right, and 0.1 ml of 1 ml cell suspension of 20 x 10 6 cells treated with 60 ⁇ g anti-IGFBP-2 IgG on left.
  • Group III Inoculated with untreated cells (2 x 10 6 cells) on right; and 0 1 ml of 1 ml cell suspension of 20 x 10 6 cells treated with 120 ⁇ g anti-IGFBP-2 IgG on left
  • Group IV Inoculated with untreated cells (2 x 10 6 cells) on right, and 0 1 ml of 1 ml cell suspension of 20 x 10 6 cells treated with 60 ⁇ g of non-immune IgG on left
  • Tumors were first evident after 16 days on the right side of animals of Groups I-III There was an apparent delay of about 6 days in the appearance of the tumor on the left side of the animals, where cells were mixed with various concentrations of anti- IGFBP-2 IgG The size of the tumors on the left side (cells mixed with anti-IGFBP-2 IgG) was significantly smaller than tumors on the right side (untreated cells) in the experimental groups of animals (FIG. 7)
  • mice were divided into two groups (Groups A and B) Animals of Group A and B were inoculated on both sides with 2 x 10 6 DU145 cells Tumors were evident by day 16 in both groups of animals At this time, tumors in animals of Group A were treated with 300 ng of anti-IGFBP-2 IgG twice a week for one month, while tumors of Group B were administered 0 1 ml of saline The experiment was terminated after one month At autopsy, the tumors were carefully removed and weighed.
  • compositions and/or methods disclosed and claimed herein can significantly retard the growth of prostatic tumors in nude mice
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims
  • IGFBP insulin-like growth factor binding protein
  • Nicolas and Rubenstein, n Vectors. A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt (eds ), Stoneham Butterworth, pp 493-513,
  • IGF insulin-like growth factor

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Abstract

L'invention concerne le rôle de l'IGFBP-2 dans le cancer. Le traitement des cancers qui dépendent de l'IGFBP-2 bénéficieraient de thérapies ciblant l'IGFBP-2. L'invention concerne des procédés d'inhibition des cancers dépendant de l'IGFBP-2 qui reposent sur la modulation de l'activité de l'IGFBP-2. On peut obtenir la modulation en diminuant les quantités de protéine IGFBP-2 ou d'activité IGFBP-2, ainsi que d'autres polypeptides interagissant avec l'IGFBP-2 ou le modifiant. Parmi les modulateurs préférés de l'IGFBP-2 figurent les anticorps spécifiques à l'IGFBP-2 et les antisens ou ribozymes spécifiques à l'acide nucléique IGFBP-2. Ce type de modulateur est utile pour traiter les cancers dérivés d'un tissu quelconque, et de préférence le cancer de la prostate. L'invention concerne en outre des procédés de traitement d'un sujet atteint du cancer ainsi que des compositions de modulateurs d'IGFBP-2 et des kits renfermant de tels modulateurs.
PCT/US2000/013574 1999-05-17 2000-05-17 Suppression de l'igfbp-2 endogene visant a inhiber le cancer Ceased WO2000069454A1 (fr)

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Cited By (14)

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FR2821624A1 (fr) * 2001-03-01 2002-09-06 Sod Conseils Rech Applic Nouveau polynucleotide utilisable pour moduler la proliferation des cellules cancereuses
WO2002070700A1 (fr) * 2001-03-01 2002-09-12 Societe De Conseils De Recherches Et D'applications Scientifiques (S.C.R.A.S.) Polynucleotide utilisable pour moduler la proliferation des cellules cancereuses
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