Nothing Special   »   [go: up one dir, main page]

WO2009157919A1 - Détection de erbb3 soluble, régulation et traitement du cancer - Google Patents

Détection de erbb3 soluble, régulation et traitement du cancer Download PDF

Info

Publication number
WO2009157919A1
WO2009157919A1 PCT/US2008/067910 US2008067910W WO2009157919A1 WO 2009157919 A1 WO2009157919 A1 WO 2009157919A1 US 2008067910 W US2008067910 W US 2008067910W WO 2009157919 A1 WO2009157919 A1 WO 2009157919A1
Authority
WO
WIPO (PCT)
Prior art keywords
serbb3
antibody
therapeutic composition
seq
cancer
Prior art date
Application number
PCT/US2008/067910
Other languages
English (en)
Inventor
Nita J. Maihle
Hakjoo Lee
Original Assignee
Tumor Biology Investment Group, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tumor Biology Investment Group, Inc. filed Critical Tumor Biology Investment Group, Inc.
Priority to PCT/US2008/067910 priority Critical patent/WO2009157919A1/fr
Publication of WO2009157919A1 publication Critical patent/WO2009157919A1/fr

Links

Classifications

    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators

Definitions

  • Embodiments of the present invention generally pertain to methods and therapeutics that relate to soluble ErbB3 proteins (sErbB3), such as p85-sErbB3, p45- sErbB3 and other isoforms of sErbB3.
  • sErbB3 soluble ErbB3 proteins
  • Embodiments of the present invention pertain to methods of treating cancer and cancer therapeutics comprising an sErbB3 or an sErbB3 agent regulating expression or activity of an sErbB3.
  • Additional embodiments of the present invention pertain to sErbB3 regulation of heregulin or at least one ErbB receptor activity and treatment of associated conditions.
  • Other embodiments relate to risk assessment and cancer prevention, screening, diagnosis, prognosis, theragnosis, monitoring of responsiveness to treatment, and monitoring of disease progression, recurrence, or metastasis of a cancer based on aberrant sErbB3 concentrations or localization in biological samples, for example serum or tissue.
  • the heregulins also called neuregulins, neu differentiation factor
  • NDF acetylcholine receptor inducing activity
  • GGFs glial growth factors
  • NRG-I neuroregulin-1
  • ErbB-mediated signal transduction exerted by heregulins has been implicated in the regulation of diverse biological events including Schwann cell differentiation, neural regulation of skeletal muscle differentiation, heart development, and proliferation and differentiation of normal and malignant breast epithelial cells (Alroy and Yarden 1997; Sundaresan, Penuel et al. 1999). Research has shown that breast carcinoma cells respond to heregulin by activating signal transduction pathways that result in cellular proliferation, differentiation, as well as morphogenesis. Carcinoma cells expressing heregulin are typically hormone-independent and associated with the ability for metastasis in experimental studies. [0007] ErbB3 is a transmembrane glycoprotein encoded by the c-erbB3 gene
  • the ErbB3 receptor belongs to the ErbB family which is composed of four growth factor receptor tyrosine ldnases, known as ErbBl/EGFR/HERl, ErbB2/Neu/HER2, ErbB4/HER4, as well as ErbB3/HER3.
  • ErbB3 and ErbB4 are receptors for heregulins, whereas ErbB2 is a coreceptor (Carraway and Burden 1995).
  • receptors are structurally related and include three functional domains: an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic tyrosine kinase domain (Plowman, Culouscou et al. 1993).
  • the extracellular domain can be further divided into four subdomains (I-IV), including two cysteine-rich regions (II and IV) and two flanking regions (I and III).
  • ErbB3 is unusual among these receptor tyrosine kinases in that its catalytic domain is defective.
  • ErbB3 is an important mediator of both heregulin and epidermal growth factor (EGF) responsiveness by way of its ability to heterodimerize with other ErbB family members, but especially with ErbB2.
  • Heregulin binding induces ErbB3 to associate with other members of the ErbB family to form heterodimeric receptor complexes.
  • ErbB3 then activates the kinase of its partner receptor which initiates a variety of c signaling cascades. ErbB3 also can relay information in response to diverse ligands, such as interferon and TNF-alpha, presumably through receptor cross-talk.
  • the ErbB3 receptor is important in regulating normal and aberrant cellular growth and differentiation, metastasis, and related pathologies.
  • Transgenic mice that have been engineered to overexpress heregulin in mammary glands have been reported to exhibit persistent terminal end buds and, over time, to develop mammary adenocarcinomas (Krane and Leder 1996).
  • ErbB3 expression studies on tumor tissues and on cancer cell lines show frequent co-expression of both ErbB2 and ErbB3 receptors (Alimandi, Heiong et al. 1995; Meyer and Birchmeier 1995; Robinson, He et al. 1996; Siegel, Ryan et al. 1999).
  • mammary tumors formed in transgenic mice that harbor an activated form of ErbB2 and metastasize to the lungs are associated with elevated amounts of tyrosine-phosphorylated ErbB2/Neu and ErbB3 (Siegel, Ryan et al. 1999).
  • Many transformed cell lines used for experimental studies are either estrogen-dependent (MCF-7 and T47D, the low ErbB2 expressers) or estrogen-independent (SKBR3, high ErbB2 expressers). However, these cell lines do not exhibit metastatic phenotypes. When MCF-7 cells are transfected to overexpress ErbB2, MCF-7 cells gain an estrogen-independent phenotype, however, they never metastasize.
  • p85-sErbB3 is encoded by a 2.1 kb alternate c-erbB3 transcript (cDNA clone R31F) that is translated into a 543 aa protein composed of subdomains I through III and the first third of subdomain IV of the ErbB3 extracellular domain, and a unique 24 amino acid carboxy-terminal sequence.
  • p45- sErbB3 is encoded by a 1.7 kb alternate c-erbB3 transcript (cDNA clone R2F) that is translated into a 312 aa protein composed of subdomains I, II, and a portion of subdomain III of the extracellular domain of ErbB-3 followed by two unique glycine residues.
  • p50-sErbB3 is encoded by a 1.6 kb alternate c-erbB3 transcript (cDNA clone RlF) that is translated into a 381 aa protein composed of subdomains I, II, and a portion of subdomain III of the extracellular domain of ErbB-3 followed by 30 unique amino acids.
  • p75-sErbB3 is encoded by a 2.3 kb alternate c-erbB3 transcript (cDNA clone R35F) that is translated into a 515 aa protein composed of subdomains I through III, and has a unique 41 amino acid carboxy-terminal sequence (Fig. l)(Lee and Maihle 1998).
  • a recombinant dominant-negative ErbB3 mutant with a deleted cytoplasmic domain but which retains its transmembrane domain can inhibit full- length ErbB2 and ErbB3 receptor phosphorylation and signal transduction (Ram, Schelling et al. 2000).
  • expression of a naturally occurring sEGFR/ErbBl inhibits TGF ⁇ dependent cellular transformation (Flickinger, MaMe et al. 1992).
  • An aberrant soluble EGFR/sErbBl secreted by the A431 human carcinoma cell line has been reported to inhibit the kinase activity of full-length EGFR in a ligand-independent manner (Basu, Raghunath et al. 1989).
  • herstatin a naturally occurring soluble ErbB2 isoform which inhibits ErbB2 receptor phosphorylation and signaling, appears to function by blocking ErbB2 dimerization (Doherty, Bond et al. 1999). In no case do these soluble ErbB isoforms function as antagonists of ErbB receptor signaling through competitive, high affinity ligand binding.
  • Soluble ErbB3 proteins for example p85-sErbB3 and p45-sErbB3, are unique among other naturally occurring soluble ErbB isoforms in that they bind specifically to heregulin with high affinity. Consequently, sErbB3 inhibits heregulin binding to cell surface ErbB receptors and heregulin-induced activation of the ErbB receptors and their downstream effectors.
  • sErbB3, such as p85-sErbB3 and p45- sErbB3 can be used as therapeutic reagents for heregulin-regulated malignancies such as mammary, prostate, ovary, and lung tumors.
  • these soluble receptor isoforms may also be important for nonheregulin-associated conditions.
  • Embodiments of the present invention pertain to several novel isolated and purified nucleic acid sequences which encode soluble isoforms of ErbB3 (sErbB3).
  • embodiments of this aspect of the invention include nucleic acid sequences which specifically encode a soluble form of ErbB3 whose amino acid sequence comprises the sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
  • the related nucleic acid sequence embodiments comprise SEQ ID NO: 1 and SEQ ID NO: 3, respectively.
  • sErbB3 agent for expression of sErbB3 or regulation of sErbB3 function or expression (either transcription or translation).
  • an sErbB3 agent include without limitation a polypeptide, a nucleic acid sequence (RNA or DNA, sense or antisense), an sErbB3 antibody, an expression vector, a small molecule, or a polypeptide agonist or antagonist.
  • Various embodiments of the present invention pertain to methods for treating cancer comprising an sErbB3 agent.
  • Other embodiments relate to sErbBS agents as cancer therapeutics.
  • Additional embodiments of the present invention relate to isoforms of sErbB3 that bind to heregulin (HRG) with high affinity and effectively block HRG binding to cell surface receptors.
  • HRG heregulin
  • p85-sErbB3 binding to HRG with high affininity substantially blocks HRG binding to cell surface receptors.
  • Other embodiments of the present invention pertain to the use of an sErbB3 agent to regulate ErbB receptor signal transduction pathways and biological effects on cellular proliferation, differentiation, and metastasis.
  • Embodiments of the present invention also pertain to the risk assessment and prevention, screening, diagnosis, prognosis, theragnosis, treatment, and evaluation of responsiveness to treatment, and monitoring of disease progression, recurrence, or metastasis of cancer cells using sErbB3 isoforms, such as p45-sErbB3 or p85-sErbB3.
  • Another embodiment of the present invention pertains to an expression vector, such as a plasmid or virus, containing an isolated cDNA encoding an sErbB3 isoform, such as p45-sErbB3 or p85-sErbB3, as well as a eukaryotic or prokaryotic cell containing the expression vector.
  • an expression vector such as a plasmid or virus, containing an isolated cDNA encoding an sErbB3 isoform, such as p45-sErbB3 or p85-sErbB3, as well as a eukaryotic or prokaryotic cell containing the expression vector.
  • Embodiments of the present invention also pertain to a process for producing the p85-sErbB3 isoform and other sErbB3 isoforms, which comprises the steps of ligating the isolated cDNA into an expression vector capable of expressing the isolated cDNA in a suitable host; transforming the host with the expression vector; culturing the host under conditions suitable for expression of the isolated cDNA and production of the p85-sErbB3 protein or other sErbB3 isoforms, and isolating the protein from the host.
  • the host cell may be a prokaryote or a eukaryote.
  • Additional embodiments of the present invention relate to molecular probes and antibody reagents specific for the unique region of p85-sErbB3 or other sErbB3 isoform epitopes.
  • polyclonal and monoclonal antibodies specific to p85-sErbB3 are generated using a unique C-terminal sequence of the p85- sErbB3 as an antigen.
  • the affinity-purified antibody can be used to detect p85- sErbB3 using immunoblot, immunohistochernistry, immunoassay and other detection methods.
  • One embodiment of the present invention pertains to irnmunoprecipitation followed by immunoblot analysis to detect p85-sErbB3 using anti-sErbB3 antibodies.
  • Another embodiment of the invention relates to a system and method of detecting p85-sErbB3 and other sErbB3 isoforms in a mammalian biological specimen selected from the group consisting of fluids, such as saliva, blood, serum, plasma, urine and ascites, solid tissues, and their derivatives.
  • Yet another embodiment of the invention relates to a vector for gene therapy, comprising a nucleic acid molecule having i) a transcription regulatory sequence; and ii) a second sequence coding for p85-sErbB3 or another sErbB3 isoform under transcriptional control of the transcription regulatory sequence; and iii) a delivery vehicle for delivering the nucleic acid molecule.
  • Fig, 1. is a diagram of soluble ErbB3 (sErbB3) proteins.
  • ErbB3 is composed of a 19 amino acid (aa) signal peptide sequence that is cleaved (gray box), an extracellular ligand-binding domain (aa 1-620), a transmembrane domain (aa 621- 646; indicated as TM), and an intracellular domain (aa 647-1323).
  • the extracellular domain of the receptor can be further divided into four subdomains (I-TV), as noted in the text.
  • the alternate c-erbB3 transcripts arise from read-through of an intron and the use of alternative polyadenylation signals.
  • p45-sErbB3 contains the amino-terminal 310 amino acids of ErbB3 and two unique carboxy-terminal amino acid residues.
  • p50-sErbB3 contains the amino-terminal 351 amino acids of ErbB3 and 30 unique carboxy-terminal amino acid residues.
  • p75-sErbB3 contains the amino-terminal 474 amino acids of ErbB3 and 41 unique carboxy-terminal amino acid residues.
  • p85- sErbB3 contains the amino-terminal 519 amino acids of ErbB3 and 24 unique carboxy-terminal amino acid residues.
  • the carboxy-terminal unique sequences are denoted as black boxes.
  • Fig. 2. demonstrates that p45-sErbB3 and p85-sErbB3 in conditioned media can block HRG-induced activation of ErbB3.
  • p45-sErbB3 and p85-sErbB3 in the concentrated conditioned media were detected by Western blotting using an anti-ErbB3 antibody recognizing the extracellular region of ErbB3.
  • Increasing volumes (5, 10, 20 ⁇ l; left to right) of the concentrated conditioned media (xl5) were loaded on an SDS-PAGE gel.
  • Binding assays were performed in a 96-well plate format as described below in more detail in the Examples. Binding results were analyzed by using Scatchard method and by plotting the displacement of 125 I-HRG ⁇ 177-244 binding by unlabeled HRG ⁇ I w -244 (Inset).
  • Fig. 4. is a graph showing inhibition of HRG ⁇ binding by p85-sErbB3 and by 2C4, a monoclonal antibody specific for ErbB2.
  • T47D cells were incubated with the indicated concentrations of p85-sErbB3 and 2C4 at room temperature for 30 min.
  • I25 I-HRG ⁇ i 77-244 (0.1 nM) was then added and binding reactions were performed as described below in more detail in the Examples.
  • 125 I-HRG ⁇ I77-244 bound to the cell surface was measured using a gamma counter.
  • Fig. 5. demonstrates p85-sErbB3 blocks HRG-induced activation of
  • ErbB2 and ErbB3 in the Ba/F3 (ErbB2+ErbB3) cells were untreated or stimulated with HRG ⁇ i -24 i alone or HRG ⁇ i- 24 i plus purified p85-sErbB3 for 10 min at room temperature.
  • Receptor phosphorylation levels and ErbB2 and ErbB3 receptor levels were determined by anti-ErbB2 (A) and anti-ErbB3 (B) immunoprecipitation followed by Western blotting as described in Fig. 2.
  • Fig. 6. demonstrates p85-sErbB3 blocks HRG-induced activation of
  • p85- sErbB3 blocks HRG-induced activation of ErbB2, ErbB3, and ErbB4 in T47D and MCF7 breast carcinoma cells. Serum-starved cells were stimulated with no HRG ⁇ , HRG ⁇ alone, or 6 iiM HRG ⁇ plus 36 nM p85-sErbB3 for 10 min at room temperature. Receptor phosphorylation levels and ErbB2, ErbB3, and ErbB4 receptor levels were determined by immunoprecipitation followed by Western blotting.
  • p85-sErbB3 inhibits HRG-induced association of PI3K (p85) with ErbB3 and activation of MAPK and Mt in T47D cells.
  • Cells were treated with 1 nM HRG ⁇ and 10 nM p85-sErbB3 for 10 min or 30 min and analyzed for activation of ErbB3.
  • Association of PI3K (p85) with ErbB3 was analyzed by immunoprecipitation of cell lysates using an anti-ErbB3 antibody followed by Western blotting of anti-PI3K (p85) antibody.
  • Activation of MAPK and Akt was examined by Western blotting of cell lysates using antibodies specific to phospho-MAPK and phospho-Akt.
  • Fig. 7. demonstrates p85-sErbB3 inhibits cell growth stimulation by
  • MCF7 cells were trypsinized, washed, and plated at a density of 5,000 (squares) or 8,000 cells/well (triangles) in 96-well plates with increasing concentrations of HRG ⁇ in serum-free medium and growth was measured after 3 days (inset). MCF7 cells were trypsinized, washed, incubated with p85-sErbB3 for 30 min, and plated with or without 0.4 nM HRG ⁇ in serum-free medium.
  • Fig. 8 is an immunoblot demonstrating specificity of the p85-sErbB3 antibody.
  • soluble ErbB3 means that the polypeptide is found in a form that is not anchored to the membrane of a cell via a typical transmembrane domain, i.e., a portion of the sErbB3 is not found physically embedded in the lipid bilayer which comprises the cell membrane.
  • biological activity is defined to mean a polypeptide or a variant or fragment thereof, which has at least about 10%, preferably at least about 50%, and more preferably at least about 90% of the biological activity of the related polypeptide, such as for example the proteins having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 4.
  • polypeptide of the invention can be measured by methods well known in the art including, but not limited to, the ability to bind heregulins or inhibit ErbB receptor mediated signaling.
  • protein is a polypeptide, and the term
  • polypeptide comprises at least two amino acids with no predefined limitation in length.
  • sErbB3 polypeptides may be the complete sequence, for example SEQ ID NO:2 or SEQ ID NO:4, or may be corresponding fragments or variants thereof, such as the unique carboxy terminal region.
  • recombinant DNA sequence or segment or “preselected DNA sequence or segment” refer to a nucleic acid that has been derived or isolated from any appropriate tissue source and that may be subsequently chemically altered, typically in vitro, so that its sequence is not naturally occurring, or corresponds to naturally occurring sequences that are not positioned as they would be positioned in a genome which has not been transformed with exogenous DNA.
  • preselected DNA "derived” from a _ source would be a DNA sequence that is identified as a useful fragment within a given organism and which is then chemically synthesized in essentially pure form.
  • RNA or DNA sequences necessary for the expression, post-transcriptional modification, translation, and post- translational modification of an operably linked coding sequence in a particular host organism include a promoter, and optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, stop sequences, enhancers, splicing, and polyadenylation signal sequences, as well as glycosylation and secretory signal sequences.
  • the term "cell line” or "host cell” is intended to refer to well-characterized homogenous, biologically pure populations of cells. These cells may be eukaryotic cells that are neoplastic or which have been “immortalized” in vitro by methods known in the art, as well as primary cells, or prokaryotic cells.
  • the cell line or host cell is preferably of mammalian origin, but cell lines or host cells of non- mammalian origin may be employed, including avian, plant, insect, yeast, fungal or bacterial sources.
  • the preselected DNA sequence is related to a DNA sequence that is resident in the genome of the host cell but is not expressed, or not highly expressed, or, alternatively, over-expressed.
  • transfected or transformed are used herein to include any host cell or cell line, the genome of which has been altered or augmented by the presence of at least one preselected DNA sequence, which DNA is also referred to in the art of genetic engineering as “heterologous DNA,” “recombinant DNA,” “exogenous DNA,” “genetically engineered DNA,” “non-native DNA,” or “foreign DNA,” wherein the DNA was isolated and introduced into the genome of the host cell or cell line by the process of genetic engineering.
  • the host cells of the present invention are typically produced by transfection with a DNA sequence in a plasmid expression vector, a viral expression vector, or as an isolated linear DNA sequence.
  • the transfected DNA is a chromosomally integrated recombinant DNA sequence which comprises a DNA encoding an sErbB3 isoform, for which the host cell may or may not express significant levels of autologous or "native" sErbB3.
  • a recombinant, functionally dominant-negative ErbB3 mutant with a deleted cytoplasmic domain, but which retains its transmembrane domain, can inhibit full-length ErbB2 and ErbB3 receptor activation(Ram, T.G., et al., 2000).
  • expression of a naturally occurring sEGFR/sErbBl isoform inhibits TGF ⁇ dependent transformation.
  • soluble EGFR secreted by the A431 human carcinoma cell line also has been shown to inhibit the kinase activity of full-length EGFR.
  • These soluble EGFR/ErbBl receptors do not function as antagonists through high affinity ligand-binding.
  • Herstatin, a naturally occurring soluble ErbB2 isoform inhibits ErbB2 receptor activation by blocking ErbB2 dimerization.
  • sErbB3 for example p85-sErbB3 and p45-sErbB3, inhibits HRG-induced stimulation of ErbB2, ErbB3, and ErbB4, at least in part, by neutralizing ligand activity through competitive binding.
  • the present invention provides several novel isolated and purified nucleic acid sequences which encode isoforms of sErbB3 and nucleic acid sequences encoding engineered variants of these proteins.
  • nucleic acids which specifically encode isoforms of sErbB3 whose amino acid sequences comprise the sequences of SEQ ID NO: 2 and SEQ ID NO: 4.
  • Embodiments of the present invention relate to the use of sErbB3 as a unique HRG inhibitor because it can block HRG binding to cell surface ErbB receptors via binding to HRG with high affinity, thereby inhibiting HRG-induced stimulation of ErbB2, ErbB3, and ErbB4. This inhibition is sufficient to effectively block HRG-stimulated cell growth.
  • These novel sErbB3 isoforms therefore, are potent modulators of HRG regulated cell functions, such as proliferation, migration, and differentiation in normal mammalian and human tissues.
  • Embodiments of the present invention also relate to regulation, either upregulation or downregulation, of heregulin activity using an sErbB3 agent.
  • sErbB3 agent for expression of sErbB3 or regulation, either upregulation or downregulation, of sErbB3 activity or expression (either transcription or translation).
  • sErbB3 agent include without limitation a polypeptide, a nucleic acid sequence (RNA or DNA, sense or antisense), an sErbB3 antibody, an expression vector, a small molecule, or a polypeptide agonist or antagonist.
  • Soluble ErbB3 also binds to ErbB receptors and regulates ErbB receptor signaling activities through downstream effectors such as MAPK, Akt, and PI3K. Additional embodiments of the present invention relate to regulation, either upregulation or downregulation, of ErbB receptor activities using an sErbB3 agent.
  • sErbB3 is expressed in most epithelial tissues of many organs, including esophagus, liver, colon, stomach, thyroid, head and neck, kidney, bladder, pancreas, lung, skin, breast, ovary, uterus (cervix and endoflowerim), , prostate gland, brain, intestine, or testis.
  • Various embodiments of the present invention also relate to methods for treating cancer, such as carcinomas and gliomas, and cancer therapeutics comprising sErbB3 agents which regulate sErbB3 expression and/or function. Nucleic acid and amino acid sequences.
  • polypeptides in embodiments of the present invention may be in substantially pure form, isolated as a recombinant form, and may be fused to other moieties. Additional N-terminal or C-terminal sequences may be added to the polypeptides for various reasons, for example to improve expression or regulation of expression in particular expression systems, to provide protection against proteolytic cleavage, or to aid in identification as fusion proteins or purification such as affinity chromatography using fusion proteins. Techniques for providing such additional sequences are well known in the art. Furthermore polypeptides with additional N- terminal or C-terminal sequences may simply result from the technique used to obtain the polypeptide without providing any advantageous characteristics and are also within the scope of the present invention. Whatever sequence is added, the resultant polypeptide preferably exhibits the biological activity of the cognate sErbB3, for example a polypeptide having the amino acid sequence SEQ ED NO: 2 or SEQ ID NO: 4.
  • polypeptides of the present invention include post-translational modifications, for example and without limitation, phosphorylation, glycosylation and farnesylation.
  • alterations can occur in the amino acid sequence of a polypeptide, which likely do not affect the function.
  • Such alterations include amino acid deletions, insertions, and substitutions.
  • Such alterations can result from alternative splicing and/or the presence of multiple translation start sites and/or stop sites. Polymorphisms may also arise as a result of the infidelity which is inherent in the translation process. Conservative substitutions are preferred.
  • the amino acids glycine, alanine, valine, leucine and isoleucine are often substituted for one another.
  • glycine and alanine are used to substitute for one another because they have relatively short aliphatic side chains and that valine, leucine and isoleucine are used to substitute for one another because they have larger hydrophobic aliphatic side chains.
  • amino acids that are often substituted for one another include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and cysteine and methionine (amino acids having sulphur-containing side chains). Aspartic acid and glutamic acid can substitute for phosphoserine and phosphothreonine, respectively (amino acids with acidic side chains). Amino acid substitutions or insertions can be made using naturally occurring or non-naturally occurring amino acids; however, L- amino acids are preferred in one embodiment.
  • polypeptides embodied within embodiments of the present invention have at least 50% sequence identity with the related sErbB3 isoform, for example isoforms comprising SEQ ID NO: 2 or SEQ ID NO: 4, and in various embodiments the degree of sequence identity is at least 75%. In various embodiments, sequence identities of at least 80%, 85%, 90%, 95%, 98% or 99% are used.
  • Amino acid sequence variants of an sErbB3 or fragments thereof can be prepared by a number of techniques, such as random mutagenesis of DNA which encodes an sErbB3 or a region thereof. Useful methods also include PCR mutagenesis, cassette mutagenesis, and saturation mutagenesis. A library of random amino acid sequence variants can also be generated by the synthesis of a set of degenerate oligonucleotide sequences. Non-random or directed mutagenesis techniques can be used to provide specific sequences or mutations in specific regions.
  • Alanine scanning mutagenesis is a useful method for identification of certain residues or regions of the desired protein that are preferred locations or domains for mutagenesis, see for example Cunningham and Wells (Science 244:1081-1085, 1989).
  • Oligonucleotide-mediated mutagenesis is a useful method for preparing substitution, deletion, and insertion variants of DNA, see, e.g., Adelman et al, (DNA 2:183, 1983). Additionally, in another example, combinatorial mutagenesis is used to generate variants.
  • Embodiments of the present invention further relate to variants of the nucleic acid sequences, for example nucleic acid sequences SEQ ID NO: 1 and SEQ ID NO: 3, which encode proteins, analogs or derivatives of an sErbB3, for example p85-sErbB3 and p45-sErbB3.
  • nucleic acid valiants are produced by nucleotide substitutions, deletions, or additions and may involve one or more nucleotides.
  • the nucleic acid sequence is a genomic sequence or a cDNA sequence.
  • the nucleotide sequence includes, for example: an sErbB3 coding region; a promoter sequence, such as a promoter sequence from an sErbB3 gene or from another gene; an enhancer sequence; untranslated regulatory sequences either 5' or 3' from an sErbB3 gene or from another gene; a polyadenylation site; and an insulator sequence.
  • the nucleotides of embodiments of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve the stability, hybridization, or solubility of the molecules.
  • the deoxyribose phosphate backbone of the polynucleotide molecules is modified to generate peptide polynucleotides (see, for example Hyrup et al, Bioorganic & Medicinal Chemistry, 4:523, 1996).
  • peptide polynucleotides or "PNAs” refer to polynucleotide mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • PNA oligomers are synthesized using standard solid phase peptide synthesis protocols. PNAs are used in therapeutic and diagnostic applications. For example, PNAs are used as antisense agents for sequence-specific modulation of gene expression.
  • the unique sErbB3 sequences are also used as a target for selective inhibition of expression (stability, transcription, or translation) using siRNA, RNAi, short hairpin RNA, microRNAs, ribozyme, and triple helix methodologies, as well as antisense sequences, including antisense oligonucleotides.
  • Useful fragments of the sErbB3 nucleic acid sequences include antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target sErbB3 mRNA or sErbB3 DNA sequences.
  • Antisense or sense oligonucleotides comprise a fragment of the coding region of an sErbB3, for example to a unique region such as the C-terminus. Such a fragment generally comprises at least about 5 nucleotides, and typically 14 to 30 nucleotides.
  • the ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is well known in the art and is described in, for example, Stein and Cohen (Cancer Res. 48:2659, 1988), van der Kiol et al. (Bio Techniques 6:958, 1988), Izant J. G. and Weintraub H., (Cell, 36: 100.7-1015, 1984) and Rosenberg et al. (Nature, 313:703-706, 1985).
  • binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block transcription or translation of the target sequence by one of several means, including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means.
  • the antisense oligonucleotides are used for example to block expression of sErbB3 proteins.
  • Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiester backbones or other sugar linkages, and wherein such sugar linkages are resistant to endogenous nucleases.
  • Such oligonucleotides with resistant sugar linkages are stable in vivo, i.e., capable of resisting enzymatic degradation, but retain sequence specificity to be able to bind to target nucleotide sequences.
  • sense or antisense oligonucleotides include oligonucleotides which are covalently linked to other organic moieties, such as for example organic moieties that increase affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lysine).
  • intercalating agents such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence.
  • sense or antisense oligonucleotides are introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, for example cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors.
  • a ligand binding molecule for example cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors.
  • conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.
  • sequences embodied herein relate specifically to sErbB3 isoforms, for example SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.
  • Various embodiments relate to the unique C-terminal region of the sErbB3 isoforms, for example valiants and fragments specific to the unique region.
  • inventions comprise an expression vector containing a nucleic acid sequence that expresses an sErbB3 polypeptide, for example a polypeptide having an amino acid sequence comprising SEQ ID NO:2 or SEQ ID NO:4, in a suitable host.
  • the nucleic acid sequence has a promoter operably linked to the polypeptide coding region, the promoter being inducible or constitutive and, optionally, cell-type or tissue-specific.
  • the promoter may also be a heterologous promoter.
  • the vector may be, for example, a plasmid, a single or double-stranded phage vector, or a single or double-stranded viral RNA or DNA molecule.
  • an inducible vector is a vector induced for expression by environmental factors that are easy to manipulate, such as temperature and nutrient additives.
  • viral vectors include viruses such as baculoviruses, papova viruses such as SV40, vaccinia viruses, adenoviruses including adeno-associated viruses, fowl pox viruses, lentiviruses, parvoviruses, herpes simplex viruses, pseudorabies viruses, and retroviruses, as well as vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • Embodiments of the present invention also include therapeutic expression of genetic material, including gene therapy expression of an sErbB3 isoform, variant, or fragment.
  • Gene therapy is either by in vivo gene therapy, which is direct delivery of the nucleic acid or nucleic acid-carrying vector into a patient, or ex vivo gene therapy, which is indirect delivery to the patient via transplanted cells that were first transformed with the nucleic acid sequences or nucleic acid-carrying vector in vitro.
  • viral vectors such as the examples listed herein can be used for in vivo and ex vivo gene therapy.
  • Plasmid DNA can be delivered with the help of, for example and without limitation: cationic liposomes such as lipofectin, or derivatized (e.g. antibody conjugated) polylysine conjugates, nanoparticles, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO.sub.4 precipitation earned out in vitro.
  • cationic liposomes such as lipofectin, or derivatized (e.g. antibody conjugated) polylysine conjugates, nanoparticles, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO.sub.4 precipitation earned out in vitro.
  • Non-viral delivery vehicle also referred to herein as “non-viral vector” as used herein is meant to include chemical formulations containing naked or condensed polynucleotides (e.g., a formulation of polynucleotides and cationic compounds, for example dextran sulfate), and naked or condensed polynucleotides mixed with an adjuvant such as a viral particle (i.e., the polynucleotide of interest is not contained within the viral particle, but the transforming formulation is composed of both naked polynucleotides and viral particles, see, for example Curiel et al.
  • non- viral delivery vehicle includes vectors composed of polynucleotides plus viral particles where the viral particles do not contain the polynucleotide of interest.
  • Non-viral delivery vehicles include bacterial plasmids, viral genomes or portions thereof, wherein the polynucleotide to be delivered is not encapsidated or contained within a viral particle, and constructs comprising portions of viral genomes and portions of bacterial plasmids and/or bacteriophages.
  • the term also encompasses natural and synthetic polymers and co-polymers.
  • the term further encompasses lipid- based vehicles.
  • Lipid-based vehicles include cationic liposomes such as disclosed for example by Feigner et al (U.S. Pat. Nos. 5,264,618 and 5,459,127; PNAS 84:7413- 7417, 1987; Annals N. Y. Acad. Sci. 772:126-139, 1995), DDAB, DOPC, and phospholipids such as phophatidylcholine.
  • lipid based vehicles consist of neutral or negatively charged phospholipids or mixtures thereof including artificial viral envelopes as disclosed for example by Schreier et al. (U.S. Pat. Nos. 5,252,348 and 5,766,625).
  • Non-viral delivery vehicles include polymer-based carriers, including natural and synthetic polymers and co-polymers.
  • the polymers are biodegradable or are readily eliminated from the subject.
  • Naturally occurring polymers include polypeptides and polysaccharides.
  • Synthetic polymers include, but are not limited to, polylysines and polyethyleneimines (PEI; see for example Boussif et al., PNAS 92:7297-7301, 1995) which molecules can also serve as condensing agents. These carriers are dissolved, dispersed or suspended in a dispersion liquid such as water, ethanol, saline solutions and mixtures thereof.
  • a dispersion liquid such as water, ethanol, saline solutions and mixtures thereof.
  • a wide variety of synthetic polymers are known in the art.
  • naked DNA or RNA molecules are used where they are in a form which is resistant to degradation, such as by modification of the ends, by the formation of circular molecules, or by the use of alternate bonds including phosphothionate and thiophosphoryl modified bonds.
  • the delivery of nucleic acids is facilitated by transport where the nucleic acid molecules are conjugated to polylysine or transferrin.
  • Antisense or sense oligonucleotides are introduced into a cell containing the target nucleic acid sequence by any genetic material transfer method, including, for example, CaPO.sub.4-mediated DNA transfection, lipid mediated transfection, electroporation, or by using gene transfer vectors or methods described above.
  • an antisense or sense oligonucleotide is inserted into a suitable viral vector, such as those described previously.
  • a cell containing the target nucleic acid sequence is contacted with the recombinant viral vector, either in vivo or ex vivo.
  • Other embodiments of the present invention relate to methods for detecting an sErbB3 and assessing the risk of developing a preneoplastic lesion or cancer, screening for a cancer, or diagnosing a cancer. Additional embodiments of the present invention relate to assaying a biological sample for an sErbB3 to evaluate prognosis, theragnosis, responsiveness to a treatment of cancer, prophylactic selection of a cancer prevention regimen, early detection of a cancer,or cancer progression, recurrence, or metastasis.
  • One embodiment relates to a method of assaying for an sErbB3 and/or diagnosing a cancer in a subject, for example a human or other mammal, which comprises the step of detecting and/or quantifying a concentration of a polypeptide or nucleic acid sequence of the invention in a biological sample obtained from said subject.
  • biological samples include fluids, such as saliva, blood, serum, plasma, urine and ascites, solid tissues, and their derivatives.
  • antibodies which recognize a polypeptide of the invention are used to detect the amount of the polypeptide in a biological sample such as serum.
  • binding of an sErbB3 antibody in tissue sections can be used to detect aberrant polypeptide localization or an aberrant concentration of polypeptide.
  • an antibody to a polypeptide of the invention can be used to assay a subject sample, for example tissue or serum, for the concentration of the polypeptide where an aberrant amount of polypeptide is indicative of a risk of developing a preneoplastic lesion or cancer, prognosis, tlieragnosis, responsiveness to treatment, prophylactic selection of a cancer prevention regimen, early detection of a cancer, or cancer progression, recurrence, or metastasis.
  • an "aberrant amount” means an amount that is increased or decreased compared with the amount in a subject free from cancer or an established reference level.
  • suitable immunoassays for detecting or assaying sErbB3 include, without limitation, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), acridinium-linked immunosorbent assays (ALISA), "sandwich” immunoassays, immunohistochemical assays, immunofluorescent detection assays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement- fixation assays, immunoradiometric assays, fluorescent immunoassays and protein A immunoassays.
  • competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), acridinium-linked immunosorbent assays (ALISA), "sandwich” immunoas
  • Immunoassays to detect and quantitate an sErbB3, for example p85- sErbB3, have been developed for use on formalin-fixed, paraffin-embedded tissue and tumor samples and for use on frozen-section tissue and tumor samples.
  • antibodies can be used to selectively quantitate an sErbB3 polypeptide in other tissues, including for example saliva, blood, serum, plasma, and urine, using enzyme-linked immunosorbent assays, acridinium-linked immunosorbent assays, and radioimmunoassay.
  • Such assays may be combined with the quantitive assessment of other biomarkers on a similar platform (e.g., multiplex assays) to increase the biological or clinical information obtained.
  • RNA in situ hybridization RNA ISH
  • RNAse protection assays RNAse protection assays.
  • Genetic aberrations in the relative copy number of the sequence can be performed by any genomic DNA detection method, for example FISH.
  • Soluble ErbB3 polypeptides embodied within the present invention can be "antigenic” and/or “immunogenic”.
  • antigenic means that the polypeptide is capable of being used to generate antibodies or indeed is capable of inducing an antibody response in a subject.
  • immunogenic means that the polypeptide is capable of eliciting an immune response in a subject.
  • the polypeptide could not only generate an antibody or anti-idiotypic antibody response but, in addition, non-antibody based immune responses, and also could be used to produce a therapeutic vaccine.
  • the unique C-te ⁇ ninal region of an sErbB3 is a target of the vaccine.
  • Further embodiments relate to antibodies, which specifically bind an sErbB3 isoform, generated using an immunogen derived from the sErbB3 isoform, such as from an isoform having SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.
  • an antibody specific to the sErbB3 is generated using the unique C-terminal region (described herein) of the specific sErbB3, such as a 23 aa sequence (Ser Lys GIy Ser GIn Ser Aig Met GIy GIy GIy GIy Ala Leu GIn Trp Asn Cys Ser GIy GIy He GIn) from the unique C-terminal sequence of p85-sErbB3.
  • Such antibodies include, but are not limited to polyclonal, monoclonal, bispecific, humanized or chimeric antibodies, single chain antibodies, Fab fragments and F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, affibodies, and epitope-binding fragments of any of the above.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen.
  • the immunoglobulin molecules can be of any class, e.g., IgG, IgE, IgM, IgD and IgA, or subclass of immunoglobulin molecule. As described above, such antibodies are used for detection and quantification as well as in methods of treating cancer, methods of regulating sErbB3 isoforms, and cancer therapeutics.
  • ELISA enzyme-linked immunosorbent assay
  • Polyclonal antibodies directed towards a sErb3 polypeptide are generated by stimulating their production in a suitable animal host (e.g. a chicken, mouse, rat, guinea pig, rabbit, sheep, goat or monkey) when a polypeptide of embodiments of the present invention is injected into the animal. If necessary, an adjuvant may be administered together with the polypeptide of the invention. The antibodies are then purified by virtue of high affinity binding to the associated polypeptide of the invention.
  • a suitable animal host e.g. a chicken, mouse, rat, guinea pig, rabbit, sheep, goat or monkey
  • Monoclonal antibodies (mAbs) directed toward an sErbB3 polypeptide may be generated by any technique known to those skilled in the art to provide for the production of antibody molecules by continuous cell lines in culture.
  • Some examples for producing mAbs include the hybridoma technique (Kohler and Milstein , 1975, Nature 256:495497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.
  • the hybridoma producing the mAbs of the invention may be cultivated in vitro or in vivo.
  • the mAbs to sErbB3 include but are not limited to human mAbs and chimeric mAbs (e.g., human-mouse chimeras).
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a human immunoglobulin constant region and a variable region derived from a murine mAb. See, for example, U.S. Pat. No. 4,816,567; and U.S. Pat. No. 4,816,397.
  • Humanized antibodies are antibody molecules from non- human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. See, for example, U.S. Pat. No. 5,585,089.
  • CDRs complementarity determining regions
  • Chimeric and humanized mAbs can be produced by recombinant DNA techniques known in the art, for example and without limitation using methods described in WO 87/02671; EP 184,187; EP 171,496; EP 173,494; WO 86/01533; U.S. Pat. No. 4,816,567; EP 125,023; Better et al., 1988, Science 240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci.
  • sErbB3 antibodies are desirable. Such antibodies can be generated, for example, using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but which can express human heavy and light chain genes.
  • transgenic mice are immunized by methods known to those skilled in the art with a selected antigen, e.g., all or a portion of an sErbB3 specific polypeptide, for example SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or a unique C-terminal sequence, such as the unique 24 aa C-terminal sequence of p85-sErbB3 (for example, Ser Lys GIy Ser GIn Ser Arg Met GIy GIy GIy GIy Ala Leu GIn Trp Asn Cys Ser GIy GIy lie GIn).
  • a selected antigen e.g., all or a portion of an sErbB3 specific polypeptide, for example SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or a unique C-terminal sequence, such as the unique 24 aa C-terminal sequence of p85-sErbB3 (for example, Ser
  • mAbs directed against the antigen can be obtained using conventional hybridoma technology where the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic selection.
  • therapeutically useful IgG, IgA, IgM, IgD and IgE antibodies can be produced.
  • references and protocols for producing human antibodies see, for examples Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93); U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No. 5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806.
  • completely human antibodies which recognize a selected epitope can be produced, for example, by the "guided selection” technique in which a selected non-human mAb, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope such as disclosed inJespers et al. (1994) Biotechnology 12:899-903.
  • a selected non-human mAb e.g., a mouse antibody
  • the sErbB3 antibodies embodied herein also can be generated using various phage display methods known in the art whereby functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding the functional antibody domains.
  • phage selection which is performed for example by using labeled antigen or antigen bound or captured to a solid surface or bead
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria.
  • Single-chain Fvs and antibodies which bind an sErbB3 also can be produced by methods known in the art, such as for example those disclosed in U.S. Pat. No. 4,946,778 and U.S. Pat. No. 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al, Science 240:1038-1040 (1988).
  • bispecific antibodies which bind to an sErbB3 can be made by methods known in the art and are embodied herein.
  • bispecific antibodies comprise a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair with a second binding specificity in the other arm. See, for example WO 94/04690 and Suresh et al., Methods in Enzymology, 1986, 121:210.
  • Embodiments of the present invention include functionally active fragments, derivatives or analogs of the anti-polypeptide immunoglobulin molecules.
  • “Functionally active” means that the fragment, derivative or analogue is able to elicit anti-anti-idiotype antibodies (i.e., tertiary antibodies) that recognize the same antigen that is recognized by the antibody from which the fragment, derivative or analogue is derived.
  • the antigenicity of the idiotype of the immunoglobulin molecule is enhanced by deletion of framework and CDR sequences that are C- terminal to the CDR sequence that specifically recognizes the antigen.
  • synthetic peptides containing the CDR sequences are used in binding assays with the antigen by any binding assay method known in the art.
  • Embodiments of the present invention include antibody fragments such as, but not limited to, F(ab')2 fragments and Fab fragments.
  • antibody fragments which recognize specific epitopes are generated by known techniques to those skilled in the art.
  • F(ab')2 fragments consist of the variable region, the light chain constant region and the CHl domain of the heavy chain and are generated by pepsin digestion of the antibody molecule.
  • Fab fragments are generated by reducing the disulphide bridges of the F(ab').sub.2 fragments.
  • any other molecule with the same specificity as the antibodies and antibody fragments of embodiments of the present invention are embodied herein.
  • Embodiments of the present invention also relate to heavy chain and light chain dimers of the antibodies of the invention, or any minimal fragment thereof such as Fvs or single chain antibodies (SCAs).
  • Fvs or single chain antibodies See for example U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:42342; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54.
  • Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may be used such as the method disclosed in Skerra et al, (1988, Science 242: 1038-1041).
  • Additional embodiments of the present invention provide for fusion polypeptides of the immunoglobulins of embodiments of the invention, or functionally active fragments thereof, for example in which the immunoglobulin is fused via a covalent bond (e.g., a peptide bond), at either the N-terminus or the C-terminus to an amino acid sequence of another polypeptide (or portion thereof, which is at least 10, 20 or 50 amino acids in length) that is not the immunoglobulin.
  • the immunoglobulin, or fragment thereof may be covalently linked to the other polypeptide at the N- terminus of the constant domain.
  • Such fusion polypeptides may facilitate purification, increase half-life in vivo, and enhance the delivery of an antigen across an epithelial barrier to the immune system.
  • the immunoglobulins of embodiments of the invention include analogues and derivatives that are modified, such as by the covalent attachment of any type of molecule as long as such covalent attachment does not impair specific binding.
  • the derivatives and analogues of the immunoglobulins include those that have been further modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, hi examples, chemical modifications of the analogues and derivatives are carried out by known techniques including, but not limited to, specific chemical cleavage, acetylation, and formylation.
  • the analogues or derivatives contain one or more non-classical amino acids.
  • the sErbB3 antibodies described herein are used in methods known in the art relating to the localization and activity of the polypeptides of embodiments of the invention. Examples of use include without limitation, imaging or radioimaging these polypeptides, measuring amounts thereof in appropriate biological samples, in diagnostic, prognostic, and theragnostic methods, and for radiotherapy.
  • the antibodies of embodiments of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression, and may be produced by recombinant expression techniques.
  • Recombinant expression of antibodies, or fragments, derivatives or analogs thereof requires construction of a nucleic acid that encodes the antibody. If the nucleotide sequence of the antibody is known, a nucleic acid sequence encoding the antibody may be assembled from chemically synthesized oligonucleotides, as described for example in Kutmeier et al.
  • the nucleic acid encoding the antibody is obtained by cloning the antibody.
  • a nucleic acid encoding the antibody may be obtained from a suitable source (e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the antibody) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence.
  • a suitable source e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the antibody
  • nucleic acid encoding at least the variable domain of the antibody molecule may be introduced into a vector containing the nucleotide sequence encoding the constant region of the antibody molecule.
  • Vectors containing the complete light or heavy chain for co-expression with the nucleic acid to allow the expression of a complete antibody molecule are also available.
  • the nucleic acid encoding the antibody can be used to introduce the nucleotide substitution(s) or deletion(s) necessary to substitute or delete the one or more variable region cysteine residues participating in an intrachain disulphide bond with an amino acid residue that does not contain a sulfhydryl group.
  • the vector for the production of the antibody may be produced by recombinant DNA technology using techniques well known in the art. Such methods can be used to construct expression vectors containing an antibody molecule coding sequence and appropriate transcriptional and translational control signals. These methods include, for example without limitation, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
  • host-expression vector systems can be utilized to express an antibody molecule of embodiments of the invention.
  • Such host-expression systems represent vehicles by which the coding sequences of interest may be produced in large quantity and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the antibody molecule of the invention in situ.
  • These include but are not limited to microorganisms such as bacteria (e.g., E. coli or B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces or Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, HEK 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter)
  • a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a polypeptide is to be produced for the generation of pharmaceutical compositions comprising an antibody molecule, vectors which direct the expression of high levels of fusion polypeptide products that are readily purified may be desirable.
  • vectors include but are not limited to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J.
  • pGEX vectors may also be used to express foreign polypeptides as fusion polypeptides with glutathione S- transferase (GST).
  • fusion polypeptides are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix of glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • AcNPV is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the antibody coding sequence may be cloned individually into nonessential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • an AcNPV promoter for example the polyhedrin promoter.
  • a number of viral-based expression systems e.g., an adenovirus expression system or those described above may be utilized.
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in a specific way. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of polypeptide products may be important for the function of the polypeptide.
  • cell lines that stably express an antibody of interest can be produced by transfecting the cells with an expression vector comprising the nucleotide sequence of the antibody and the nucleotide sequence of a selectable marker (e.g., neomycin or hygromycin), and selecting for expression of the selectable marker.
  • a selectable marker e.g., neomycin or hygromycin
  • Such engineered cell lines may be particularly useful for screening and evaluation of agents that interact directly or indirectly with the antibody molecule.
  • the expression levels of the antibody molecule can be increased by vector amplification.
  • the host cell may be co-transfected with two expression vectors, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
  • the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • a single vector may be used which encodes both heavy and light chain polypeptides.
  • the light chain is preferably placed before the heavy chain to avoid an excess of toxic free heavy chain (see, e.g. Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197).
  • the coding sequences for the heavy and light chains comprise either cDNA or genomic DNA.
  • the antibody molecule may be purified by any method known in the ait for purification of an antibody molecule, for example without limitation, by chromatography (e.g., ion exchange chromatography, affinity chromatography such as with protein A or specific antigen, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of polypeptides.
  • chromatography e.g., ion exchange chromatography, affinity chromatography such as with protein A or specific antigen, and sizing column chromatography
  • centrifugation e.g., centrifugation, differential solubility
  • differential solubility e.g., differential solubility
  • antibodies of the invention or fragments thereof are conjugated to a diagnostic or therapeutic moiety. The antibodies are used for diagnosis or to determine the efficacy of a given treatment regimen. Detection is facilitated by coupling the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive nuclides, positron emitting metals (for use in positron emission tomography), and nonradioactive paramagnetic metal ions. See generally U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics.
  • Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin and biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and aequorin; and suitable radioactive nuclides include .sup.1251, .sup.1311, .sup.l l 1In and .sup.99Tc.
  • the therapeutic moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a polypeptide such as tumour necrosis factor, .alpha.-interferon, .beta.-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, a biological response modifier such as a lymphokine, interleukin-1 (IL-I), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM- CSF) 5 granulocyte colony stimulating factor (G-CSF), nerve growth factor (IL-I),
  • an antibody is conjugated to a second antibody to form an antibody heteroconjugate as disclosed for example in U.S. Pat. No. 4,676,980.
  • An antibody with or without a therapeutic moiety conjugated to it can be used as a therapeutic that is administered alone or in combination with cytotoxic factor(s) and/or cytokine(s). Screening
  • Embodiments of the invention include methods for identifying active agents (e.g., chemical compounds, proteins, or peptides) that bind to an sErbB3 polypeptide of embodiments of the invention and/or have a stimulatory or inhibitory effect on the expression or activity of an sErbB3 polypeptide of the invention.
  • active agents include, but are not limited to, nucleic acids (e.g., DNA and RNA), carbohydrates, lipids, proteins, peptides, peptidomimetics, agonists, antagonists, small molecules and other drugs.
  • Active agents can be obtained using any of the numerous suitable approaches in combinatorial library methods known in the art, including without limitation: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. See, for examples, methods disclosed in Lam, 1997, Anticancer Drug Des. 12:145; U.S. Pat. No. 5,738,996; and U.S. Pat. No. 5,807,683.
  • polypeptides that interact with a protein such as sErbB3.
  • polypeptides include synthetic peptides, small molecular weight peptides (e.g., linear or cyclic peptides) or generated mutant gene products.
  • Techniques for screening large gene libraries often include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the genes under conditions in which detection of a desired activity, assembly into a trimeric molecule, binding to natural ligands, e.g., a receptor or substrate, facilitates relatively easy isolation of the vector encoding the gene whose product was detected.
  • Examples include without limitation: two hybrid (interaction trap) assays, display libraries in which the candidate peptides are displayed on the surface of a cell, plasmid, or viral particle, and the ability of particular cells or viral particles to bind an appropriate receptor protein via the displayed product is detected in a "panning assay".
  • high through-put assays are followed or substituted by secondary screens, such as binding assays, to determine biological activities and differentiate agonists from antagonists.
  • cancers such as carcinomas and gliomas, with aberrant sErbB3 expression or activity or which are regulated by HRG signaling or ErbB activity are embodied herein.
  • cancers include without limitation esophageal, liver, colon, gastric, thyroid, head and neck, kidney, bladder, pancreatic, lung, skin, breast, ovarian, cervical, endometrial, prostate, brain, intestinal, or testicular.
  • Various embodiments of the present invention include methods and therapeutics to treat cancer, including carcinomas and gliomas, using an sErbB3 agent to regulate an sErbB3 by any available means including without limitation nucleic acids (e.g., DNA and RNA), carbohydrates, lipids, proteins, peptides, peptidomimetics, agonists, antagonists, small molecules and other drugs.
  • nucleic acids e.g., DNA and RNA
  • carbohydrates lipids, proteins, peptides, peptidomimetics
  • agonists, antagonists small molecules and other drugs.
  • various embodiments of the present invention pertain to regulation of p85-sErbB3 and/or p45-sErbB3 or other sErbB3 isoforms.
  • the methods and therapeutics of the present invention comprise administering the nucleic acid sequences SEQ ID NO:1 or SEQ ID NO: 3, or a complementary sequence thereof, or the amino acid sequences SEQ ID NO: 2 or SEQ ID NO: 4.
  • the sequences include for example, without limitation and as more fully described supra, the complete sequence, fragments and variants, as well as antisense oligonucleotides.
  • Additional embodiments of the present invention include methods to regulate heregulin or ErbB signaling activity through regulation of an sErbB3 agent.
  • Effective amounts of an sErbB3 or an agonist or antagonist of an sErbB3 can be determined, for example, by decreased or increased heregulin activity, respectively as well as by effects on other biological endpoints such as receptors, downstream mediators, or biochemical targets such as cell survival, cell growth, or metastasis.
  • One embodiment of the present invention relates to a method of treating cancer in a subject, such as a human.
  • the method includes administering to the subject an agent that regulates an sErbB3 (function or expression), such as p85- sErbB3 or p45-sErbB3, in an amount sufficient to reduce or prevent carcinoma cell growth and/or metastasis.
  • the sErbB3 agent may be administered in an amount effective to regulate heregulin activation of ErbB receptor signaling activity, as well as by effects on other biological endpoints such as receptors, downstream mediators, or biochemical targets such as cell survival, cell growth, or metastasis.
  • the agent comprises any of the previously described polypeptides, nucleic acid sequences, or variants thereof or previously described instruments.
  • Examples include without limitation an sErbB3 polypeptide, or a functional fragment, variant or analog thereof having an sErbB3 activity; a polypeptide agonist or antagonist of sErbB3 that increases or decreases respectively the activity of an sErbB3 or the binding of an sErbB3 to a binding partner; a small molecule that increases or decreases expression of an sErbB3, for example by binding to the promoter region of the ErbB3 gene; an antibody, for example an antibody that binds to and stabilizes or assists the binding of sErbB3 to an sErbB3 binding partner or an antibody that inhibits binding to and destabilizes the binding of s ⁇ rbB3 to an sErbB3 binding partner; or a nucleotide sequence encoding an sErbB3 polypeptide or functional fragment or analog thereof.
  • the agent and instruments described herein also may comprise any compositions or methods in molecular medicine or therapeutic transfer of genetic material known to those skilled in the art such as those previously described above to achieve a therapeutic effect.
  • the amount of sErbB3 protein is increased by elevated transcriptional expression of the endogenous sErbB3 gene and translation of the sErbB3 isoform from its alternate mRNA or by increasing sErbB3 rnRNA stability.
  • transcription of the sErbB3 gene is increased for example by altering its regulatory sequence such as by the addition of a positive regulatory element (such as an enhancer or a DNA-binding site for a transcriptional activator); the deletion of a negative regulatory element (such as a DNA-binding site for a transcriptional repressor); and/or replacement of the endogenous regulatory sequence, or elements therein, with that of another gene, thereby allowing the coding region of the ErbB3 gene to be transcribed more efficiently.
  • a positive regulatory element such as an enhancer or a DNA-binding site for a transcriptional activator
  • a negative regulatory element such as a DNA-binding site for a transcriptional repressor
  • replacement of the endogenous regulatory sequence, or elements therein with that of another gene, thereby allowing the coding region of the ErbB3 gene to be transcribed more efficiently.
  • the amount of sErbB3 protein is regulated post-transcriptionally by microRNAS or post- transcriptional regulatory elements
  • the agent is a vector that includes a nucleic acid sequence encoding an sErbB3, preferably a human sErbB3, for example SEQ ID NO: 2 or SEQ ED NO:4.
  • the vector can be any vector suitable for transfer of genetic material such as those listed previously or that are known in the art.
  • a therapeutic treatment or composition comprises essentially the agent.
  • the agent can be administered by any method known in the art, for example by direct administration, e.g., injection, intravenous or intramuscular.
  • the agent is delivered directly to an affected tissue.
  • the agent can be coupled to a second agent, for example a delivery agent (e.g., an agent that protects the agent from degradation) or a targeting agent (e.g., for targeting to the cancer or affected tissue or targeting to the inside of a cell).
  • a delivery agent e.g., an agent that protects the agent from degradation
  • a targeting agent e.g., for targeting to the cancer or affected tissue or targeting to the inside of a cell.
  • Targeting may occur by means of, for example, the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof.
  • compositions suitable for administration to a subject for example a human.
  • Such compositions may include the agent and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances are known. Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the invention. Supplementary active compounds can also be incorporated into the compositions.
  • Suitable carriers, excipients, and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoates, propylhydroxybenzoates, talc, magnesium stearate and mineral oil.
  • compositions may additionally include solubilizing agents, lubricating agents, wetting agents, sweeteners, colorants, odorants, salts (polypeptides of embodiments of the present invention may themselves be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents, antioxidants, flavoring agents, emulsifiers, preservatives and the like.
  • a pharmaceutical carrier for hydrophobic compounds of embodiments of the invention is a co-solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.
  • the pharmaceutical composition may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route.
  • Such compositions may be prepared by any method known in the art of pharmacy, for example by admixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.
  • Solutions or suspensions used for parenteral application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents, suspending agents, or thickening agents; antibacterial agents such as benzyl alcohol or methyl parabens; antifungal agents such as parabens or tbimerosal; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents, suspending agents, or thickening agents
  • antibacterial agents such as benzyl alcohol or methyl
  • pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral composition can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • the composition may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets. Prolonged adsorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., an agent described herein) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g., an agent described herein
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation may include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • compositions comprised of an sErbB3 agent adapted for oral administration may be presented as discrete units such as capsules or tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous or nonaqueous liquids; or as edible foams or whips; or as emulsions).
  • Suitable excipients for tablets or hard gelatine capsules include lactose, maize starch or derivatives thereof, stearic acid or salts thereof.
  • Suitable excipients for use with soft gelatine capsules include, for example, vegetable oils, waxes, fats, semi-solid, or liquid polyols etc.
  • excipients which may be used include, for example, water, polyols and sugars.
  • oils e.g. vegetable oils
  • Pharmaceutical compositions comprised of the sErbB3 agent adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.
  • the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3(6):318 (1986).
  • compositions comprised of an sErbB3 agent adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.
  • the active ingredient When formulated in an ointment, the active ingredient may be employed with either a paraffmic or a water- miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.
  • Pharmaceutical compositions adapted for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.
  • Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.
  • compositions comprised of an sErbB3 agent adapted for rectal administration may be presented as suppositories or enemas.
  • Pharmaceutical compositions comprised of an sErbB3 agent adapted for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range of 20 to 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • Suitable compositions wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops include aqueous or oil solutions of the active ingredient.
  • compositions comprised of an sErbB3 agent adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurised aerosols, nebulizers or insufflators.
  • compositions comprised of an sErbB3 agent adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray compositions.
  • compositions of dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • compositions described herein can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to a patient by employing any of the procedures well known in the art.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • Liposomal suspensions for example liposomes targeted to infected cells with monoclonal antibodies to viral antigens
  • compositions of embodiments of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising at least one agent of embodiments of the invention, optionally together with one or more pharmaceutically acceptable excipients, carriers or diluents.
  • the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • the pharmaceutical composition may be provided in unit dosage form, and may generally be provided in a sealed container, and may be provided as part of a kit. Such a kit may include instructions for use and a plurality of the unit dosage forms.
  • Dosages of the active agent of embodiments of the present invention can vary between wide limits, depending upon the disease or disorder to be treated, and the age, condition, and response of the individual to be treated. A physician will ultimately determine appropriate dosages to be used. This dosage may be repeated as often as appropriate. If side effects develop the amount and/or frequency of the dosage can be reduced in accordance with normal clinical practice.
  • Embodiments of the invention further provide quantitative diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of embodiments of the invention.
  • kit may contain separate containers, dividers or compartments for the composition and informational material.
  • the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet, hi other embodiments, the separate elements of the kit are contained within a single, undivided container.
  • the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label.
  • the kit includes a plurality of individual containers, each containing one or more unit dosage forms of the agent.
  • the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of the agent.
  • the containers of the kits can be air tight and/or waterproof.
  • the kit optionally includes a device suitable for administration of the composition.
  • the present invention provides a method for the treatment of cancer in a subject, for example a human, which comprises administering to the subject a therapeutically effective amount of at least one active agent of the invention.
  • agent may be in the form of a pharmaceutical composition as described herein.
  • the method may include more than one sErbB3 agent, more than one sErbB3 isoform, as well as additional therapeutics or combinations thereof.
  • Conditioned media containing sErbB3 inhibited HRG ⁇ - stimulated ErbB3 activation by 40% (p45-sErbB3) and 80% (p85-sErbB3) at 1 ⁇ g/ml HRG ⁇ , as determined by densitometric analysis. However, at a higher concentration (2 ⁇ g/ml), conditioned media containing p85-sErbB3 decreased activation by 30%, although inhibition by conditioned media containing p45-sErbB3 was negligible (See Fig. 2A).
  • p85-sErbB3 was isolated from a concentrated conditioned medium of cells stably transfected with a cDNA clone R31F encoding p85-sErbB3 and was purified in two steps. The first step was lectin affinity chromatography with a Con A column (Sigma). The bound p85-sErbB3 was washed with column buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM MnCl 2 , and 1 mM CaCl 2 ) and eluted using column buffer containing 1 M ⁇ -methyl D-mannoside, then dialyzed against 20 mM Tris-HCl, pH 7.5 overnight.
  • column buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM MnCl 2 , and 1 mM CaCl 2
  • the second step of purification was accomplished using a Mono Q® anion exchange chromatography column for resolution of proteins and peptides on an FPLC® system, i.e., a microprocessor controlled, solvent delivery apparatus used in purification of biologically active compounds column (Pharmacia).
  • the bound p85-sErbB3 was eluted from the column with 0-500 mM NaCl gradient containing 20 mM Tris-HCl, pH 7.5.
  • Samples taken from each step were subjected to SDS-PAGE in duplicate and analyzed by Coomassie staining and by Western blot using anti-ErbB3 236 antibody recognizing the extracellular domain of the ErbB3 (Lee and Maihle 1998).
  • the final p85-sErbB3 pool was homogeneous on SDS-PAGE, and the identity of the purified protein was confirmed by Western blot analysis. Purified preparations of p85-sErbB3 were used in all subsequent experiments.
  • p85-sErbB3 Binds to HRG with High Affinity.
  • Previous reports based the assignation of the subdomain boundaries for the ErbB3 extracellular domain on the subdomain boundaries of EGFR (Lee and Maihle 1998) as defined by the genomic structure of avian ErbBl(Callaghan, Antczak et al. 1993).
  • p85- sErbB3 is composed of subdomains I through III and includes the first 45 amino acids of subdomain IV (aa 1-519), and a unique twenty-four amino acid sequence at the carboxy-terminus. Binding studies using heregulins indicate that subdomains I and II are required for heregulin binding (Singer, Landgraf et al. 2001).
  • subdomains I and III are low and high affinity binding sites, respectively (Lax, Bellot et al. 1989).
  • p85-sErbB3 effectively limits binding of heregulin to cell surface receptors in the breast carcinoma cell line T47D. This cell line expresses all four ErbB receptors at moderate levels. Cells were incubated with varying concentrations of p85-sErbB3 in the presence of 125 I-labeled HRG ⁇ 177 . 244 . Simultaneously, a separate group of cells was incubated with 125 I-HRGp H7-244 in the presence of varying concentrations of 2C4, a monoclonal antibody specific for the ErbB2 extracellular domain (Lewis, Lofgren et al. 1996). As shown by the inhibition curves (See Fig.
  • p85-sErbB3 and 2C4 inhibit HRGp 177-244 binding to cell surface receptors with similar IC 50 values (0.45 + 0.03 nM and 0.55 + 0.03 nM, respectively) although the mechanism of inhibition by these two molecules is distinct.
  • 2C4 inhibits heregulin binding to cell surface receptors by blocking ErbB2-ErbB3 heterodimerization via binding to the ErbB2 extracellular domain (Fitzpatrick, Pisacane et al. 1998)
  • p85-sErbB3 inhibits receptor activation and heterodimerization, at least in part, by competing for heregulin binding to the cell surface.
  • p85-sErbB3 Blocks HRG-Induced Activation of ErbB2, ErbB3, and ErbB4.
  • the ability of p85-sErbB3 to modulate HRG-stimulated receptor activation in the Ba/F3 (ErbB2+ErbB3) cell line was examined using purified p85- sErbB3. This allowed an analysis of the mechanism of p85-sErbB3 mediated inhibition in a quantitative manner. As shown in Fig. 5, when Ba/F3 (ErbB2+ErbB3) cells were treated with p85-sErbB3 at a 10-fold molar excess over HRGp 1-241 , ErbB3 phosphorylation levels were reduced to basal levels.
  • the anti-ErbB antibodies used in these experiments recognize the carboxy-terminal 17 aa (ErbB3) and 18 aa (ErbB2 and ErbB4) sequences of these receptors. Each of these sequences contains one tyrosine residue, hnmunoblot detection by the anti-ErbB2 and ErbB4 antibodies used in this study, therefore, may reflect either the level of receptor expression or the unphosphorylated fraction of these receptors.
  • HRG-stimulated activation of ErbB2, ErbB3, and ErbB4 leads to activation of two major signal transduction pathways: the PI3K pathway and the MAPK pathway (Wallasch, Weiss et al. 1995).
  • MAPK p85 phosphatidylinositide 3-kinase
  • HRG-induced phosphorylation of ErbB receptors by p85-sErbB3 correlates with the modulation of HRG's biological effects. Specifically, a cell growth assay using MCF7 cells stimulated with HRG ⁇ was performed and showed that, within the concentration range tested, growth of this cell line was dose-dependent (See Fig. 7). It was observed that at a concentration of 0.4 nM HRG ⁇ the cell growth rate was half of the rate observed at saturating levels of HRG ⁇ .
  • p85-sErbB3 inhibited cell growth by 75% and 90%, at densities of 5,000 and 8,000 cells/well, respectively, whereas the same concentration of p85-sErbB3 did not affect cell growth in the absence of HRG ⁇ (See Fig. 7).
  • the present invention discloses the use of p85-sErbB3 as a potent inhibitor of HRG-dependent breast carcinoma cell growth in vitro.
  • p85-sErbB3 antibodies Soluble ErBb3 antibodies are directed to the unique C-terminal regions. Rabbit polyclonal antibodies specific for p85-sErbB3 were generated using a 23 aa polypeptide from the unique carboxy-terminal region (Ser Lys GIy Ser GIn Ser Arg Met GIy GIy GIy GIy Ala Leu GIn Trp Asn Cys Ser GIy GIy He GIn) and methods known in the art.
  • p85-sErbB3 expression in tissues Immunohistochemistry analysis using the p85-sErbB3 specific antibody, and using both pre-immune and peptide competition controls, showed p85-sErbB3 expression in most tissues or organs, including the esophagus, stomach, liver, gall bladder, small bowel, ureter, colon, thyroid gland, tonsils, lymph nodes, spleen, thymus, skeletal muscle, bronchioles, heart (epicardium and myocardium), hippocampus, head and neck, kidney, bladder, pancreas, adrenal gland, lung, skin, breast, ovary, uterus (cervix and endometrium), fallopian tube, placenta, prostate gland, brain, intestine, or testis, (data not shown).
  • tissues or organs including the esophagus, stomach, liver, gall bladder, small bowel, ureter, colon, thyroid gland, tonsils, lymph nodes, spleen, th

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Oncology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Urology & Nephrology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Veterinary Medicine (AREA)
  • Hospice & Palliative Care (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne la régulation d’isoformes de sErbB3 dans des procédés de régulation de l’activité héréguline ou les activités de récepteur de ErbB. La présente invention concerne en outre des thérapies du cancer et des procédés de traitement thérapeutique du cancer comprenant sErbB3. La présente invention concerne en outre la détection de sErbB3 dans des échantillons biologiques pour l’évaluation du risque et la prévention, le dépistage, le diagnostic, le pronostic, la theragnostique, l’évaluation de la réponse au traitement, et/ou la surveillance de la progression ou la récurrence d’une maladie, ou la métastase d’un cancer. Dans des exemples, des séquences d’acide nucléique, des polypeptides, des sondes moléculaires, et des anticorps de sErbB3 sont des agents utiles pour la régulation, l’expression, la détection, et la thérapie du cancer associés à sErbB3.
PCT/US2008/067910 2008-06-23 2008-06-23 Détection de erbb3 soluble, régulation et traitement du cancer WO2009157919A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/US2008/067910 WO2009157919A1 (fr) 2008-06-23 2008-06-23 Détection de erbb3 soluble, régulation et traitement du cancer

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2008/067910 WO2009157919A1 (fr) 2008-06-23 2008-06-23 Détection de erbb3 soluble, régulation et traitement du cancer

Publications (1)

Publication Number Publication Date
WO2009157919A1 true WO2009157919A1 (fr) 2009-12-30

Family

ID=39938128

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2008/067910 WO2009157919A1 (fr) 2008-06-23 2008-06-23 Détection de erbb3 soluble, régulation et traitement du cancer

Country Status (1)

Country Link
WO (1) WO2009157919A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9220775B2 (en) 2011-11-23 2015-12-29 Medimmune Llc Binding molecules specific for HER3 and uses thereof
WO2016011432A3 (fr) * 2014-07-17 2016-04-28 Czerniecki Brian J Identification de peptides immunogènes de classe ii du complexe majeur d'histocompatibilité pour une immunothérapie
US10745490B2 (en) 2014-04-11 2020-08-18 Celldex Therapeutics, Inc. Anti-ErbB antibodies and methods of use thereof
US11305012B2 (en) 2013-09-24 2022-04-19 Medimmune, Llc Binding molecules specific for HER3 and uses thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003080835A1 (fr) * 2002-03-26 2003-10-02 Zensun (Shanghai) Sci-Tech. Ltd. Procedes fondes sur l'utilisation de erbb3 et compositions associees de traitement des neoplasmes
US20030190702A1 (en) * 1999-09-30 2003-10-09 Maihle Nita J. System and method to inhibit heregulin activated processes and other methods using soluble ErbB3 and method to produce soluble ErbB3
US6639060B1 (en) * 1989-12-01 2003-10-28 The United States Of America As Represented By The Department Of Health And Human Services erbB-3 nucleic acids
US20070122407A1 (en) * 1996-03-27 2007-05-31 Robert Akita ISOLATED NUCLEIC ACIDS, VECTORS AND HOST CELLS ENCODING ErbB3 ANTIBODIES

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6639060B1 (en) * 1989-12-01 2003-10-28 The United States Of America As Represented By The Department Of Health And Human Services erbB-3 nucleic acids
US20070122407A1 (en) * 1996-03-27 2007-05-31 Robert Akita ISOLATED NUCLEIC ACIDS, VECTORS AND HOST CELLS ENCODING ErbB3 ANTIBODIES
US20030190702A1 (en) * 1999-09-30 2003-10-09 Maihle Nita J. System and method to inhibit heregulin activated processes and other methods using soluble ErbB3 and method to produce soluble ErbB3
WO2003080835A1 (fr) * 2002-03-26 2003-10-02 Zensun (Shanghai) Sci-Tech. Ltd. Procedes fondes sur l'utilisation de erbb3 et compositions associees de traitement des neoplasmes

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9220775B2 (en) 2011-11-23 2015-12-29 Medimmune Llc Binding molecules specific for HER3 and uses thereof
US10040857B2 (en) 2011-11-23 2018-08-07 Medimmune, Llc Binding molecules specific for HER3 and uses thereof
US11091554B2 (en) 2011-11-23 2021-08-17 Medlmmune, Llc Binding molecules specific for HER3 and uses thereof
US11305012B2 (en) 2013-09-24 2022-04-19 Medimmune, Llc Binding molecules specific for HER3 and uses thereof
US10745490B2 (en) 2014-04-11 2020-08-18 Celldex Therapeutics, Inc. Anti-ErbB antibodies and methods of use thereof
WO2016011432A3 (fr) * 2014-07-17 2016-04-28 Czerniecki Brian J Identification de peptides immunogènes de classe ii du complexe majeur d'histocompatibilité pour une immunothérapie
US10829538B2 (en) 2014-07-17 2020-11-10 The Trustees Of The University Of Pennsylvania Identification of immunogenic MHC class II peptides for immune-based therapy

Similar Documents

Publication Publication Date Title
US7744882B2 (en) Soluble ErbB3 methods of detection and antibodies
Wong et al. The TGF-α precursor expressed on the cell surface binds to the EGF receptor on adjacent cells, leading to signal transduction
Peles et al. Cell‐type specific interaction of Neu differentiation factor (NDF/heregulin) with Neu/HER‐2 suggests complex ligand‐receptor relationships.
Kleeff et al. The cell-surface heparan sulfate proteoglycan glypican-1 regulates growth factor action in pancreatic carcinoma cells and is overexpressed in human pancreatic cancer.
US5820859A (en) Method of targeting a therapeutic agent to cells expressing the erb B-3 receptor
US20030028905A1 (en) Mutant forms of the TGF-beta type II receptor which bind all TGF-beta isoforms
KR101009501B1 (ko) 항-라이실 티알엔에이 합성효소 항체 및 항-라미닌 수용체 항체를 유효성분으로 포함하는 암 진단용 조성물
US8962808B2 (en) EGFR-related polypeptides and methods of use
WO2009157919A1 (fr) Détection de erbb3 soluble, régulation et traitement du cancer
US7705117B2 (en) EGFH2 genes and gene products
US8703917B2 (en) Epidermal growth factor receptor variants and pharmaceutical compositions thereof
EP0828843A1 (fr) Heregulines de recombinaison et leurs fonctions biologiques lors de l'activation d'un recepteur
US7745398B2 (en) Soluble ErbB3 and treatment of cancer
CA2495849A1 (fr) Proteine impliquee dans le carcinome
US8389684B2 (en) Tumor biomarker
WO1995025166A1 (fr) Methodes de traitement ou de diagnostic de maladies ou d'etats associes a une transduction anormale des signaux
QIAN et al. Identification of pl85neu sequences required for monoclonal antibody-or ligand-mediated receptor signal attenuation
WO2001014415A2 (fr) Gènes egfh2 et produits géniques
KR102268041B1 (ko) 구강암의 예후 예측용 조성물
US20050032689A1 (en) Methods for treatment or diagnosis of disease or disorders associated with an APB domain
JPH10507362A (ja) Ｈｅｒ４ヒトレセプタチロシンキナーゼ
WO2004016646A2 (fr) Modulateurs peptidiques d'un sous-type de pyruvate kinase m2 (m2-pk) specifique de tumeurs
JP5208105B2 (ja) ＥｒｂＢ２を介するシグナル伝達経路のダウンレギュレーションの有無を判定する方法、抗癌剤による治療の要否の判断を補助する方法、およびシグナル伝達阻害剤
US7297760B2 (en) Cancer associated protein
KR20200067419A (ko) 구강암의 예후 예측용 조성물

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08874836

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08874836

Country of ref document: EP

Kind code of ref document: A1