WO2001016158A2 - Molecules d'acide nucleique et proteines d'integrateur 2 de pontage et utilisations associees - Google Patents

Molecules d'acide nucleique et proteines d'integrateur 2 de pontage et utilisations associees Download PDF

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WO2001016158A2
WO2001016158A2 PCT/US2000/023723 US0023723W WO0116158A2 WO 2001016158 A2 WO2001016158 A2 WO 2001016158A2 US 0023723 W US0023723 W US 0023723W WO 0116158 A2 WO0116158 A2 WO 0116158A2
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bin2
protein
binl
peptide
nucleic acid
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PCT/US2000/023723
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WO2001016158A3 (fr
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George C. Prendergast
Kai Ge
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The Wistar Institute Of Anatomy And Biology
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Priority to US10/069,540 priority Critical patent/US6831063B1/en
Priority to AU73367/00A priority patent/AU7336700A/en
Publication of WO2001016158A2 publication Critical patent/WO2001016158A2/fr
Publication of WO2001016158A3 publication Critical patent/WO2001016158A3/fr
Priority to US10/976,492 priority patent/US7150968B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates generally to cancer diagnosis and therapy, and more specifically, to cancers associated with over- or underexpression of Binl or other members of the BAR family of adaptor proteins.
  • Binl/Amphiphysin/RVS (BAR) proteins are a family of adaptor proteins implicated in a diverse set of cellular processes, including tumorigenesis, cell survival, differentiation, and nerve synaptic activity. BAR proteins share a common N-terminal BAR domain also termed the RVS domain. While BAR proteins share a common domain (BAR), they appear to have divergent physiological functions. As one example, amphiphysin is a neuronal protein of this family which is implicated in synaptic vesicle endocytosis [Wigge and McMahon, Trends Neurosci. 21: 339-344 (1998)].
  • Amphiphysin is also a paraneoplastic autoimmune antigen in cancers of the breast, lung, and other tissues [Antoine et al, Arch. Neurol. 56: 172-177 (1999); Dropcho, Ann. Neurol 39: 659-667 (1996); Folli et al, N. Engl. J. Med. 328: 546-51 (1993)].
  • Binl (Bridging LNtegrator-1) is a second, ubiquitous BAR protein that was initially identified in mammalian cells through its ability to interact with and inhibit the oncogenic properties of c-Myc [Sakamuro et al, Nature Genet. 14: 69-77 (1996)].
  • Ubiquitous Binl isoforms that localize to the nucleus have tumor suppressor properties and have been implicated in growth control, differentiation, and programmed cell death [Mao et al, Genomics 56: 51-58 (1999); Prendergast, Oncogene 18: 2966-2986 (1999); Sakamuro et al. 1996, cited above; Wechsler-Reya et al, Mol. Cell. Biol 18: 566-575 (1998)].
  • Bin2 proteins, nucleic acids, and other Bin2 compositions of the invention have a variety of uses relating to regulation of cell growth control, cell survival, differentiation, endocytosis and actin organization, as well for diagnosis and treatment of conditions associated with aberrant cell behavior.
  • the present invention provides a Bin2 protein.
  • the protein has the 564 amino acid sequence of SEQ ID NO:2.
  • the present invention provides a Bin2 peptide or protein selected from the group consisting of a fragment of Bin2 comprising at least 8 amino acids in length.
  • a fragment of this invention is at least 8 contiguous amino acids in length and is selected from amino acids 1 to 13 of SEQ ID NO:2. In another embodiment, a fragment of this invention is at least 14 amino acids in length and includes amino acids 23 to 35 of SEQ ID NO:2, and preferably amino acids 23-45 of SEQ ID NO: 2. In still another embodiment, a fragment includes amino acids 138 - 155 of SEQ ID NO:2 and comprises at least 19 amino acids in length. In still another embodiment, a fragment includes amino acids 179-336, or a smaller fragment of at least 8 amino acids contained therein. Still other fragments may be selected from the sequence. In yet another embodiment, the invention provides analogs or homologs of SEQ ID NO:2.
  • the invention provides a fusion protein comprising the amino acid sequence of SEQ ID NO: 2, a fragment, analog or homolog thereof, and a fusion partner.
  • the invention provides a deletion protein comprising the amino acid sequence of SEQ ID NO:2 with one to twenty amino acids deleted therefrom.
  • the present invention provides a Bin2 nucleic acid sequence.
  • the Bin2 nucleic acid sequence encodes a protein or fragment of the invention (such as those mentioned above) and contains SEQ ID NO:l or a fragment thereof.
  • the Bin2 nucleic acid sequence hybridizes to the sequence of SEQ ID NO:l under stringent conditions.
  • the invention provides a nucleic acid sequence complementary to the nucleic acid sequence of SEQ ID NO: 1.
  • the invention provides a nucleic acid sequence encoding a fusion protein of the invention.
  • the invention provides an allelic variant of any of the Bin2 nucleic acid sequences of the invention.
  • the nucleic acid sequence is an antisense sequence to the sequences described above.
  • the invention provides a vector comprising a Bin2 nucleic acid sequence of the invention under the control of regulatory sequences which direct expression of the Bin2 protein.
  • the invention provides a host cell transformed with the vector of the invention.
  • the invention provides a diagnostic reagent comprising a Bin2 nucleic acid sequence of the invention and a detectable label which is associated with said sequence. Methods of diagnosing conditions associated with inappropriate functional levels, the loss of expression of Bin2 or altered expression of Bin2, e.g., cancers, which use this reagent are also provided.
  • the invention provides a diagnostic reagent comprising a Bin2 protein or peptide of the invention and a detectable label which is associated with that protein.
  • Bin2 protein for diagnosing cancers associated with inappropriate expression (e.g., overexpression or underexpression or altered expression) of Binl, to which Bin2 binds.
  • This method involves the steps of contacting a sample from a human or animal to be diagnosed with the Bin2 protein of the invention, or the diagnostic reagent containing this protein, whereby in the presence of Binl in the sample, a complex is formed between Binl and the Bin2 protein or reagent, and analyzing for the presence of said complex.
  • the invention provides an isolated anti-Bin2 antibody which is specific for the Bin2 protein of the invention.
  • the invention provides a diagnostic reagent comprising the anti-Bin2 antibody of the invention and a detectable label.
  • a method of diagnosing cancer or hyperplastic disease characterized by inappropriate levels or altered expression of functional Bin2 in a human or an animal using the anti-Bin2 antibody or diagnostic reagent of the invention involves contacting an anti-Bin2 antibody or a diagnostic reagent containing same with a sample from a human or animal to be diagnosed, whereby in the presence of Bin2, a detectable complex is formed with the Bin2 protein or diagnostic reagent, analyzing for the presence or absence of said complex; and comparing the level of complex to a standard, wherein the absence of said detectable label indicates the absence of functional Bin2.
  • the invention provides a kit for diagnosing a condition associated with Bin2 comprising a diagnostic reagent of the invention.
  • the invention provides an anti-idiotype antibody specific for the anti-Bin2 antibody of the invention.
  • the invention provides a composition comprising an effective amount of a Bin2 protein or anti-idiotype of the invention and a pharmaceutically acceptable carrier.
  • the invention provides a method of detecting inappropriate expression of Box-dependent myc-interacting protein-2 (Bin2) in a patient comprising providing a sample from a patient suspected of having said inappropriate (over- or under-expression) or altered expression; incubating said sample in the presence of an anti-Bin2 antibody or a diagnostic reagent containing same; and comparing levels of expression to a normal Bin2 control.
  • Bin2 Box-dependent myc-interacting protein-2
  • the invention provides a method of detecting inappropriate expression of Box-dependent myc-interacting peptide-2 (Bin 2) in a patient comprising providing a sample from a patient suspected of having said inappropriate expression and performing nucleic acid amplification using a Bin2 nucleic acid sequence of the invention.
  • the invention provides a method of identifying compounds which specifically bind to Bin2 or which specifically inhibit or block the binding of Bin2 to its ligand
  • the method involves comprising the steps of contacting said Bin2 or a fragment thereof with a test compound to permit binding of the test compound to Bin2; and determining the amount of test compound which is bound to Bin2
  • the invention provides a method of contacting an amount of immobilized first Bin peptide or a fragment thereof with a test compound and an amount of labeled second Bin peptide or fragment, wherein said first Bin peptide is either a Bin l or a Bin2 peptide, and the second Bin peptide is the Bin peptide that binds said first Bin peptide Unbound material is separated from immobilized material and the amount of label on said immobilized material is measured A decrease in the amount of label immobilized in the presence of test compound compared to the amount of label immobilized in the presence of a control peptide or protein, indicates that said test compound
  • Figs 1 A- 1 C illustrate the continuous sequence of full-length Bin2 cDNA
  • FIG. 2 provides an alignment of the BAR domains of Bin2 [SEQ ID NO: 2] and Binl [SEQ ID NO: 3].
  • Fig. 3 is an illustration comparing Bin2 structure with other BAR family members.
  • Fig. 4A provides the amino acid sequence alignment of Binl [SEQ ID NO:
  • Fig. 4B is an illustration of the domains in Binl, Bin2, and Daxx and the relative region of similarity between Bin2 and Daxx (dark shaded).
  • Fig. 5 A is a bar graph illustrating the results of a colony formation assay.
  • HepG2 and A549 cells were transfected with expression vectors and stable transformants were selected by culturing cells in G418. Colonies were scored by methanol fixation and crystal violet staining 2-3 weeks later. The data represent the mean and standard error from three trials.
  • Fig. 5B is a bar graph that illustrates that Bin2 does not affect the tumor suppressor activity of Binl .
  • the colony formation assay was performed as above using 10 ⁇ g vector, 5 ⁇ g vector + 5 ⁇ g Binl plasmid, or 5 ⁇ g Binl + 5 ⁇ g Bin2 plasmids.
  • the present invention provides novel, isolated mammalian Bin2 nucleic acid sequences, fragments thereof and proteins and peptides encoded thereby.
  • the invention further provides methods of using these sequences, proteins, and compositions containing them for diagnosis and treatment of disorders associated with deregulation, deficiency or amplification of the c-myc oncogenes.
  • the present invention further provides methods of using these sequences to generate antibodies and new compositions useful for such diagnosis and treatments.
  • Bin2 (Bridging LNtegator-2), which is a novel member of the BAR family. Bin2 is expressed predominantly in hematopoietic cells and can form a stable complex with Binl . Bin2 is upregulated during differentiation of granulocytes, thereby functioning in that cell lineage. Bin2 lacks sequences found in Binl that mediate c-Myc interaction. Bin2 also lacks sequences found in amphiphysin that mediate interaction with endocytotic machinery in the brain. In addition, Bin2 lacks a C-terminal region SH3 domain, instead including a C-terminal extension that is unrelated to other members of the BAR family.
  • Bin2 Database comparisons with Bin2 revealed a previously unrecognized region of similarity between BAR family proteins and Daxx, a nucleocytoplasmic adaptor implicated in programmed cell death, JNK signaling, and chromosomal regulation [Chang et al, Science 281: 1860-1863 (1998); Pluta et ⁇ /., J. Cell Sci. Ill: 2029-2041 (1998); Yang et al, Cell 89: 1067-1076 (1997)], within the central part of these proteins.
  • the human Bin2 gene was mapped to chromosome 4q22.1 , within a region that is frequently deleted in breast and liver cancers.
  • Bin2 is a novel BAR protein which has nonredundant functions relative to other members of the BAR family.
  • compositions containing Bin2 proteins and nucleic acid sequences are useful for a variety of purposes. These aspects of the invention are discussed in more detail below.
  • the present invention provides mammalian nucleic acid sequences encoding a
  • Bin2 protein or peptide The nucleic acid sequences of this invention may be isolated from cellular materials with which they are naturally associated or produced using techniques known in the art. In one embodiment, the present invention provides Bin2 nucleic acid sequence SEQ ID NO: l and fragments of at least eight contiguous amino acids thereof. However, the present invention is not limited to these nucleic acid sequences.
  • Bin2 DNA [SEQ ID NO: 1]
  • sequences of the Bin2 DNA [SEQ ID NO: 1]
  • sequences of the Bin2 DNA [SEQ ID NO: 1]
  • sequences of the Bin2 DNA [SEQ ID NO: 1]
  • sequences of the Bin2 DNA [SEQ ID NO: 1]
  • sequences of the Bin2 DNA [SEQ ID NO: 1]
  • sequences of the Bin2 DNA [SEQ ID NO: 1]
  • allelic variants of these sequences within a species may also be readily obtained given the knowledge of the nucleic acid sequence provided by this invention.
  • the present invention further encompasses nucleic acid sequences capable of hybridizing under stringent conditions [see, J. Sambrook et al, Molecular Cloning: A Laboratory Manual. 2d ed., Cold Spring Harbor Laboratory (1989)] to the sequences of the invention, SEQ ID NO: 1, their anti-sense strands, or biologically active fragments thereof.
  • An example of a highly stringent hybridization condition is hybridization at 2XSSC at 65°C, followed by a washing in 0.1XSSC at 65°C for an hour.
  • an exemplary highly stringent hybridization condition is in 50% formamide, 4XSSC at 42 °C.
  • Moderately high stringency conditions may also prove useful, e.g. hybridization in 4XSSC at 55 °C, followed by washing in 0.1XSSC at 37 °C for an hour.
  • An alternative exemplary moderately high stringency hybridization condition is in 50% formamide, 4XSSC at 30°C.
  • the nucleic acid sequences may be modified. Utilizing the sequence data provided herein, it is within the skill of the art to obtain or prepare synthetically or recombinantly other polynucleotide sequences, or modified polynucleotide sequences, encoding the full-length proteins or useful fragments of the invention. Such modifications at the nucleic acid level include, for example, modifications to the nucleotide sequences which are silent or which change the amino acids, e.g. to improve expression or secretion. Also included are allelic variations, caused by the natural degeneracy of the genetic code.
  • nucleotide sequences encoding mutants of the Bin2 peptides and proteins provided herein.
  • Such mutants include amino terminal, carboxy terminal or internal deletions, which substantially retain the biological activity (e.g., the antigenicity and/or the ability to bind Binl) of the full-length Bin2 or other proteins or fragments.
  • Such a truncated, or deletion mutant may be expressed for the purpose of affecting the activity of the full-length or wild-type gene or gene fragments.
  • the invention provides nucleic acid sequence fragments that encode a desirable fragment of Bin2, e.g., a Binl binding region.
  • these oligonucleotide fragments are at least 15 nucleotides in length.
  • oligonucleotide fragments of varying sizes may be selected as desired.
  • Such fragments may be used for such purposes as performing polymerase chain reaction (PCR), e.g., on a biopsied tissue sample.
  • suitable nucleic acid fragments include those encoding the complete BAR motif or domain (amino acid 1 to 221 or amino acid 1 to 249 of SEQ ID NO:2) and the putative BAR effector region (amino acid 138 - 155 of SEQ ID NO:2).
  • a fragment of this invention encodes at least 8 contiguous amino acids in length, e.g., a fragment selected from amino acids 1 to 13 of SEQ ID NO:2.
  • a nucleic acid sequence of this invention encodes at least 14 amino acids in length. Exemplary fragments include those encoding amino acids 23 to 35 or a peptide within amino acids 23 to 45 of SEQ ID NO:2.
  • a nucleic acid sequence encodes a BIN2 fragment within amino acids 138 - 155 of SEQ ID NO:2 and comprises at least 19 amino acids in length.
  • Still another nucleic acid sequence of the invention encodes a peptide of at least 8 amino acids in length within amino acids 179 to 336 of SEQ ID NO: 2.
  • nucleic acid sequence of the invention encodes a peptide of at least 8 amino acids in length within amino acids 250 to 564 of SEQ ID NO: 2, which is the region unrelated to the other BAR family proteins.
  • nucleotide sequences of the invention encode analogs or homologs of SEQ ID NO:2.
  • nucleotide sequences of this invention encode fusion proteins comprising the amino acid sequence of SEQ ID NO: 2, a fragment, analog or homolog thereof, and a fusion partner.
  • a nucleotide sequence of this invention encodes a deletion protein comprising the amino acid sequence of SEQ ID NO:2 with one to twenty amino acids deleted therefrom. Other useful fragments may be readily identified by one of skill in the art by resort to conventional techniques.
  • nucleotide sequences of the invention may be isolated by conventional uses of polymerase chain reaction or cloning techniques such as those described in obtaining the murine and human sequences, described below. Alternatively, these sequences may be constructed using conventional genetic engineering or chemical synthesis techniques.
  • nucleic acid sequences are useful for a variety of diagnostic, prophylactic and therapeutic uses.
  • the nucleic acid sequences are useful in the development of diagnostic probes and antisense probes for use in the detection and diagnosis of cancers and other conditions associated with inappropriate levels of functional Bin2 (and/or its binding partner, Binl) or altered expression of Bin2 by utilizing a variety of known nucleic acid assays, e.g., Northern and Southern blots, polymerase chain reaction (PCR), and other assay techniques known to one of skill in the art.
  • the nucleic acid sequences of the invention may optionally be associated with a detectable label, such as are described in detail below.
  • the nucleic acid sequences of this invention are also useful in the production of the peptides and proteins of the invention in vitro, in vivo, and ex vivo.
  • Bin2 polypeptides or proteins For convenience throughout this specification, reference will be made to "Bin2 peptides and proteins", but it will be understood that this term encompasses the fragments, analogs, modified peptides and proteins, fusion proteins, and other amino acid constructs of the invention, except where otherwise specified.
  • Bin2 peptides and proteins may be isolated in a form substantially free from other proteinaceous and non-proteinaceous cellular materials (e.g., hepatocytes) or from cell extracts.
  • these peptides and proteins may be isolated from the cellular materials and optionally, further purified using any of a variety of conventional methods including, liquid chromatography such as normal or reversed phase, using HPLC, FPLC and the like; affinity chromatography (such as with inorganic ligands or monoclonal antibodies); size exclusion chromatography; immobilized metal chelate chromatography; gel electrophoresis; and the like.
  • liquid chromatography such as normal or reversed phase, using HPLC, FPLC and the like
  • affinity chromatography such as with inorganic ligands or monoclonal antibodies
  • size exclusion chromatography such as with inorganic ligands or monoclonal antibodies
  • size exclusion chromatography such as with inorganic ligands or monoclonal antibodies
  • Bin2 peptides and proteins of the invention may be produced recombinantly following conventional genetic engineering techniques [see e g , Sambrook et al. Molecular Cloning' A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY and the detailed description of making the proteins below]
  • the peptides and proteins of the invention may be produced using conventional chemical synthesis techniques, such as those described in G Barony and R.B. Merrifield, The Peptides: Analysis, Synthesis & Biology, Academic Press, pp. 3-285 (1980), among others.
  • the term “artificial” is used herein to denote the preparation of the construct (e.g., a peptide, protein, nucleic acid, or antibody of the invention) by chemical synthesis, recombinant technology, or other similar means
  • the present invention further provides analogs, fragments, and mutant peptides, as well as proteins containing Bin2, or such analogs, fragments or mutants, as described below
  • Analogs or modified versions of the Bin2 proteins and peptides are provided Typically, analogs differ from the specifically identified proteins by only one to four codon changes Examples include polypeptides with minor amino acid variations from the illustrated amino acid sequence of Bin2 having conservative amino acid replacements Conservative replacements are those that take place within a family of amino acids that are related in their side chains and chemical properties. Also provided are homologs of the proteins of the invention which are characterized by ha ⁇ 'ing at least 90% identity, and more preferably 95-99% identity with Bin2 sequences Based on the sequence information provided herein, one of skill in the art can readily obtain full-length homologs and analogs
  • identity means the degree of sequence relatedness between two peptide or two nucleotide sequences as determined by the identity of the match between two lengths of such sequences
  • identity can be readily calculated by methods extant in the prior art [See, e.g., COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A M , ed , Oxford University Press, New York, (1988), BIOCOMPUTING INFORMATICS AND GENOME PROJECTS, Smith, D W , ed , Academic Press, New York, ( 1993), COMPUTER ANALYSIS OF SEQUENCE DATA, PART 1, Griffin, A M , and Griffin, H.G., eds., Humana Press, New Jersey, ( 1994), SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G , Academic Press, (1987), and SEQUENCE ANALYSIS PRIMER, Gribskov, M and Devereux, J , e
  • Methods commonly employed to determine identity or homology between two sequences include, but are not limited to, those disclosed in GUIDE TO HUGE COMPUTERS, Martin J Bishop, ed , Academic Press, San Diego, 1994 Preferred methods to determine identity or homology are designed to give the largest match between the two sequences tested Methods to determine identity and similarity are codified in computer programs Preferred computer program methods to determine identity and homology between two sequences include, but are not limited to, the algorithm BESTFIT from the GCG program package [J Devereux et al, Niic Acids Res , 12( 1 ) 387 ( 1984)], the related MACVECTOR program
  • Bin2 peptide or protein of the piesent invention may also be modified to increase its ability to bind and thus, complex with, Binl
  • the Bin2 peptide or protein may be coupled to a chemical compounds or non- proteinaceous carriers.
  • the coupling is designed not to interfere with the desired biological activity of either the Bin2 peptide or protein or the carrier.
  • the carrier may be selected which facilitates cell penetration, e.g. a lipid or a carbohydrate.
  • the carrier may be selected to deliver a toxin to Binl , to which the Bin2 peptide binds.
  • toxins are known to those of skill in the art and may include, e.g., chemical compounds including, without limitation, dinitrophenol groups and arsonilic acid.
  • Yet other carriers may be selected simply to facilitate production or delivery of the Bin2 peptide or protein.
  • useful carriers known in the art include, without limitation, keyhole limpet hemocyanin (KLH); bovine serum albumin (BSA), ovalbumin, agarose beads; activated carbon; or bentonite.
  • Bin2 peptides and proteins of the invention may also be modified by other techniques, such as by denaturation with heat and/or SDS.
  • the peptides and proteins of the invention may be modified to provide an additional N - or C-terminal amino acid sequence suitable for biotinylation, e.g., cysteine or lysine.
  • fragments of the Bin2 peptide or of the other proteins identified herein are desirably characterized by having a biological activity similar to that displayed by the complete protein, including, e.g., the ability to bind and complex with Binl .
  • These fragments may be designed or obtained in any desired length, including as small as about 5 to about 8 amino acids in length, about 14 or 15 amino acids in length, about 19 to 20 amino acids in length, or longer. Such a fragment represents less than the full-length
  • Bin2 protein and may represent as little as a single epitope of the protein.
  • one particularly desirable fragment of the invention is the BAR domain (amino acid 1 to 221 or amino acid 1 to 249 of SEQ ID NO:2), which contains dimerization signals.
  • BAR domain amino acid 1 to 221 or amino acid 1 to 249 of SEQ ID NO:2
  • fragments of the BAR domain are composed of at least 14 amino acids in length with respect to any fragment which encompasses all of amino acids 1 to 13 or which encompasses all of amino acids 23 to 35 or amino acids 23 to 45 of SEQ ID NO 2
  • Another desirable fragment encompasses the putative BAR effector region (amino acid 138 to 155 of SEQ ID NO 2), which is implicated in Binl in tumor suppressor and programmed cell death signaling
  • Still other desirable fragments include fragments of about 8 or more amino acids from amino acids 250 to 564 of SEQ ID NO 2, the C terminal region unrelated to other BAR family proteins.
  • any fragments of Bin2 containing these latter regions are at least 19 amino acids in length
  • a Bin2 fragment may be a T cell epitope.
  • T cell epitope may be readily identified may be readily identified using available computer modelling programs
  • the peptides of the invention may be modified to create deletion mutants, for example, by truncation at the amino or carboxy termini, or by elimination of one or more amino acids
  • Still other modified fragments of Bin2 may be prepared by any number of now conventional techniques to improve production thereof, to enhance protein stability or other characteristics, e g binding activity or bioavailability, or to confer some other desired property upon the protein.
  • Other useful fragments of these polypeptides may be readily prepared by one of skill in the art using known techniques, such as deletion mutagenesis and expression.
  • Bin2 peptides and protein of the present invention may also be constructed, using conventional genetic engineering techniques as part of a larger and/or multimeric protein or protein compositions
  • a fusion protein may be desirable in order to improve yield on expression and/or purification
  • Suitable fusion partners for such a purpose are well known to those of skill in the art and include, e g , glutatione-S- transferase and maltose binding protein
  • a fusion protein of the invention may be composed of a Bin2 fragment, such as a fragment corresponding to a T cell epitope or to the Binl binding region, which is fused to an active agent.
  • the active agent may be composed of other Bin2 peptides and proteins of this invention, or may be other proteinaceous materials or nucleic acid molecule.
  • a Bin2 peptide or protein of the invention may be desirable to fuse a Bin2 peptide or protein of the invention with a proteinaceous molecule which facilitates its cell penetration, e.g., Drosophila antemiapedia, HIV Rev peptides, which are known to those of skill in the art.
  • a Bin2 peptide or protein of the invention to a molecule which is to be targeted to a particular cell type and/or to Binl .
  • Particularly suitable are toxins and anti-cancer agents, any of which are known to those of skill in the art.
  • suitable examples include, without limitation, tetanus toxoid, cholera toxoid, PPD (purified protein derivative of tuberculin), and molecular toxins such as imidazole protein cross-linkers or other conjugates (which would kill a bound Binl molecule). These proteins are effective in the prevention, treatment and diagnosis of cancers associated with inappropriate levels of functional Bin2 and/or inappropriate Bin l function.
  • the fusion proteins of the invention are constructed for use in the methods and compositions of this invention. These fusion proteins may be produced recombinantly, or may be synthesized chemically.
  • a protein composition which may be a preferred alternative to the fusion proteins described above is a cocktail (i.e., a simple mixture) containing a Bin2 peptide or protein, or different mixtures of the Bin2 peptides and proteins of this invention.
  • the peptide and proteins of the invention may be provided with a detectable label, such as are described in detail below.
  • a peptide or protein antigen of the present invention may also be used in the form of a pharmaceutically acceptable salt.
  • Suitable acids and bases which are capable of forming salts with the polypeptides of the present invention are well known to those of skill in the art, and include inorganic and organic acids and bases.
  • the proteins and nucleic acid molecules of the invention may be isolated from natural sources as described above, or may be produced using chemical synthesis techniques, such as are well known to those of skill in the art.
  • the peptides and proteins of the invention may be produced using conventional chemical synthesis techniques, such as those described in G. Barony and R.B. Merrifield, THE PEPTIDES: ANALYSIS, SYNTHESIS & BIOLOGY, Academic Press, pp. 3-285 (1980), among others.
  • a DNA sequence of the invention is inserted into a suitable expression system.
  • a recombinant molecule or vector is constructed in which the polynucleotide sequence encoding Bin2 is operably linked to a heterologous expression control sequence permitting expression of the Bin2 protein.
  • appropriate expression vectors are known in the art for protein expression, by standard molecular biology techniques. Such vectors may be selected from among conventional vector types including insects, e.g., plasmids, yeast, fungal, bacterial, insect (e.g., baculovirus expression) or viral expression systems. Other appropriate expression vectors, of which numerous types are known in the art, can also be used for this purpose.
  • Suitable host cells or cell lines for transfection by this method include mammalian cells, such as human 293 cells, Chinese hamster ovary cells (CHO), the monkey COS-1 cell line or murine 3T3 cells derived from Swiss, Balb-c or NIH mice may be used. Another suitable mammalian cell line is the CV-1 cell line. Still other suitable mammalian host cells, as well as methods for transfection, culture, amplification, screening, production, and purification are known in the art. [See, e.g., Gething and Sambrook, Nature, 293:620-625 (1981 ), or alternatively, Kaufman et al, Mol. Cel Bio , 5(7): 1750-1759 ( 1985) or Howley et al, U. S.
  • bacterial cells are useful as host cells for the present invention.
  • E. coli e.g., HB 101 , MCI 061, and strains used in the following examples
  • Various strains of B. sublilis, Psevdomonas, other bacilli and the like may also be employed in this method.
  • Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of the polypeptides of the present invention.
  • Other fungal cells may also be employed as expression systems.
  • insect cells such as Spodoptera frugipedera (Sf9) cells may be used, e.g., in the baculovirus expression system.
  • Sf9 Spodoptera frugipedera
  • the present invention provides a method for producing a recombinant Bin2 protein which involves transfecting a host cell with at least one expression vector containing a recombinant polynucleotide encoding a Bin2 protein under the control of a transcriptional regulatory sequence, e.g., by conventional means such as electroporation.
  • the transfected host cell is then cultured under conditions that allow expression of the Bin2 protein.
  • the expressed protein is then recovered, isolated, and optionally purified from the culture medium (or from the cell, if expressed intracellularly) by appropriate means known to one of skill in the art.
  • the proteins may be isolated in soluble form following cell lysis, or may be extracted using known techniques, e.g., in guanidine chloride.
  • the Bin2 proteins of the invention may be produced as a fusion protein.
  • Suitable fusion partners for the Bin2 proteins of the invention are well known to those of skill in the art and include, among others, ⁇ -galactosidase, glutathione-S-transferase, and poly- histidine.
  • an appropriate vector for delivery of Bin2, or fragment thereof may be readily selected by one of skill in the art.
  • Exemplary vectors are readily available from a variety of academic and commercial sources, and include, e.g., DNA vectors (including "naked” DNA and plasmid systems), adeno-associated virus, adenovirus vectors, or other viral vectors, e.g., various poxviruses, vaccinia, etc.
  • Methods for insertion of a desired gene, e.g. Bin2, and obtaining in vivo expression of the encoded protein are well known to those of skill in the art.
  • Bin2 proteins of this invention are also useful as antigens for the development of anti-Bin2 antisera and antibodies to Bin2 or to a desired fragment of a Bin2 protein.
  • Specific antisera may be generated using known techniques. See, Sambrook, cited above, Chapter 18, generally, incorporated by reference.
  • antibodies of the invention, both polyclonal and monoclonal may be produced by conventional methods, including the Kohler and Miistein hybridoma technique and the many known modifications thereof.
  • desirable antibodies are generated by applying known recombinant techniques to the monoclonal or polyclonal antibodies developed to these antigens [see, e.g., PCT Patent Application No.
  • PCT/GB85/00392 British Patent Application Publication No. GB2188638A; Amit et al, Science, 233:747-753 ( 1986); Queen et al., Proc. Nal'l. Acad. Sci. USA, 86: 10029-10033 (1989); PCT Patent Application No. PCT ⁇ VO900786I ; and Riechmann el al. Nature, 332:323-327 ( 1988); Huse el al. Science, 246: 1275-1281 ( 1988)], or any other techniques known to the art.
  • one of skill in the art may generate chimeric, humanized or fully human antibodies directed against a Bin2 peptide or protein of the invention by resort to known techniques by manipulating the complementarity determining regions of animals or human antibodies to the Bin2 protein of the invention. See, e.g., E. Mark and Padlin, "Humanization of Monoclonal Antibodies", Chapter 4, THE HANDBOOK OF EXPERIMENTAL PHARMACOLOGY, Vol. 113, The Pharmacology of Monoclonal Antibodies, Springer-Verlag (June, 1994).
  • the antigens may be assembled as multi-antigenic complexes
  • Anti-idiotype antibodies (Ab2) and anti-anti-idiotype antibodies (Ab3).
  • Ab2 are specific for the target to which anti- Bin2 antibodies of the invention bind and Ab3 are similar to Bin2 antibodies (Abl) in their binding specificities and biological activities [see, e.g., M.
  • anti-idiotype and anti-anti- idiotype antibodies may be produced using techniques well known to those of skill in the art.
  • Such anti-idiotype antibodies (Ab2) can bear the internal image of the Binl and bind to it in much the same manner as Bin2 and are thus useful for the same purposes as Bin2.
  • polyclonal antisera, monoclonal antibodies and other antibodies which bind to Bin2 as the antigen are useful to identify epitopes of Bin2, to separate Bin2 from contaminants in living tissue (e.g., in chromatographic columns and the like), and in general as research tools and as starting material essential for the development of other types of antibodies described above.
  • Anti-idiotype antibodies are useful for binding Bin2 and thus may be used in the treatment of cancers in which Bin2 is part of a biochemical cascade of events leading to carcinoma.
  • the Ab3 antibodies may be useful for the same reason the Abl are useful.
  • Other uses as research tools and as components for separation of Bin2 from other contaminant of living tissue, for example, are also contemplated for these antibodies.
  • the antibodies are associated with conventional labels which are capable, alone or in concert with other compositions or compounds, of providing a detectable signal.
  • the labels are desirably interactive to produce a detectable signal.
  • the label is detectable visually, e.g. colorimetrically.
  • enzyme systems have been described in the art which will operate to reveal a colorimetric signal in an assay.
  • peroxidase which reacts with peroxide and a hydrogen donor such as tetramethyl benzidine (TMB), produces an oxidized TMB that is seen as a blue color.
  • TMB tetramethyl benzidine
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • hexokinase in conjunction with glucose-6-phosphate dehydrogenase which reacts with ATP, glucose, and NAD+ to yield, among other products, NADH that is detected as increased absorbance at 340 nm wavelength.
  • glucose oxidase which uses glucose as a substrate
  • peroxide as a product.
  • label systems that may be utilized in the methods of this invention are detectable by other means, e.g., colored latex microparticles [Bangs Laboratories, Indiana] in which a dye is embedded may be used in place of enzymes to form conjugates with the antibodies and provide a visual signal indicative of the presence of the resulting complex in applicable assays.
  • Still other labels include fluorescent compounds, radioactive compounds or elements.
  • Detectable labels for attachment to antibodies useful in diagnostic assays of this invention may be easily selected from among numerous compositions known and readily available to one skilled in the art of diagnostic assays.
  • the methods and antibodies of this invention are not limited by the particular detectable label or label system employed. Suitably, these detectable systems may also be utilized in connection with diagnostic reagents composed of the peptides, proteins, and nucleic acid sequences of the invention.
  • the present invention provides reagents and methods useful in detecting and diagnosing a deficiency in normal Bin2 levels and/or abnormal levels of non-functional Bin2, and particularly deficiencies or excess production thereof, in a patient. Further, the present invention provides reagents which bind Binl , and therefore as defined herein, a deficiency of Bin2 is an inadequate level of functional Bin2 to compensate for the levels of Binl in a patient. A deficiency of Binl is an inadequate level of functional Binl to compensate for the levels of c-Myc in a patient.
  • Conditions associated with deficiencies of Bin2 include hepatocarcinoma; conditions associated with Binl include a variety of cancers, e.g., epithelial cell cancer, breast cancer, melanoma, prostate cancer, liver cancer and colon cancer, and hyperplastic disease states, e.g., benign prostate hyperplasia. Conditions associated with altered expression or loss of expression of normal Bin2 include myeloid and lymphoid leukemias.
  • Bin2 proteins For convenience, reference will be made to Bin2 proteins throughout this and the following section. However, it will be understood that Bin2 nucleic acids (including anti-sense sequences and oligonucleotide fragments, among others), peptides, analogs and diagnostic compositions containing these molecules may be useful in these methods.
  • this method involves detecting the presence of Binl (or other ligand for Bin2) which is produced by the affected human or animal patient's system and which are capable of binding to the Bin2 peptides and proteins (or Ab2) of this invention or combinations thereof.
  • This method comprises the steps of incubating a Bin2 peptide or protein of this invention with a sample of biological fluids from the patient. Binl present in the samples will form a complex with the Bin2 peptide or protein. Subsequently the reaction mixture is analyzed to determine the presence or absence of these complexes. The step of analyzing the reaction mixture comprises contacting the reaction mixture with a labeled specific binding partner for the Bin2 ligand.
  • the Bin2 peptide or protein, or a mixture of the peptides and proteins of the invention is electro- or dot-blotted onto nitrocellulose paper.
  • the biological fluid e.g. serum or plasma
  • ligand e.g., Binl
  • latex beads are conjugated to the Bin2 peptide or protein of this invention.
  • the biological fluid is incubated with the bead/protein conjugate, thereby forming a reaction mixture.
  • the reaction mixture is then analyzed to determine the presence of Binl or other Bin2 ligand.
  • the diagnostic method of the invention involves detecting the presence of the naturally occurring Bin2 peptide or protein itself in its association with hepatocytes in the biological fluids of an animal or human infected by the pathogen.
  • This method includes the steps of incubating a ligand specific for Bin2 (e.g., Binl or an antibody of this invention, e.g. produced by administering to a suitable human and/or animal an antigen of this invention), preferably conventionally labelled for detection, with a sample of biological fluids from a human or an animal to be diagnosed.
  • a complex is formed (specific binding occurs).
  • excess labeled antibody or other ligand
  • the reaction mixture is analyzed to determine the presence or absence of the antigen-antibody complex and the amount of label associated therewith.
  • Assays employing a peptide or protein of the invention can be heterogenous (i.e., requiring a separation step) or homogenous. If the assay is heterogenous, a variety of separation means can be employed, including centrifugation, filtration, chromatography, or magnetism.
  • Bin2 peptide or protein of the invention is adsorbed to the surface of a microtiter well directly or through a capture matrix (i.e., antibody). Residual protein-binding sites on the surface are then blocked with an appropriate agent, such as bovine serum albumin (BSA), heat-inactivated normal goat serum (NGS), or BLOTTO (a buffered solution of nonfat dry milk which also contains a preservative, salts, and an antifoaming agent).
  • BSA bovine serum albumin
  • NGS heat-inactivated normal goat serum
  • BLOTTO a buffered solution of nonfat dry milk which also contains a preservative, salts, and an antifoaming agent
  • the well is then incubated with a biological sample suspected of containing Binl or another ligand specific for Bin2.
  • the sample can be applied neat, or more often, it can be diluted, usually in a buffered solution which contains a small amount (0.1-5.0% by weight) of protein, such as BSA, NGS, or BLOTTO.
  • a buffered solution which contains a small amount (0.1-5.0% by weight) of protein, such as BSA, NGS, or BLOTTO.
  • Hulg labeled anti-human immunoglobulin
  • labeled antibodies to other species, e.g., dogs.
  • the label can be chosen from a variety of enzymes, including horseradish peroxidase (HRP), ⁇ -galactosidase, alkaline phosphatase, and glucose oxidase, as described above. Sufficient time is allowed for specific binding to occur again, then the well is washed again to remove unbound conjugate, and the substrate for the enzyme is added. Color is allowed to develop and the optical density of the contents of the well is determined visually or instrumentally.
  • HRP horseradish peroxidase
  • ⁇ -galactosidase alkaline phosphatase
  • glucose oxidase glucose oxidase
  • MAbs or other antibodies of this invention which are capable of binding to Bin2 peptides and proteins can be bound to ELISA plates.
  • the biological fluid is incubated on the antibody-bound plate and washed. Detection of any antigen-antibody complex, and qualitative measurement of the labeled MAb is performed conventionally, as described above.
  • Bin2 peptide or protein The dipstick assay involves fixing an antigen or antibody to a filter, which is then dipped in the biological fluid, dried and screened with a detector molecule.
  • diagnostic assays can employ the antigen(s) or fragments of this invention as nucleic acid probes or as anti-sense sequences, which can identify the presence of infection in the biological fluid by hybridizing to complementary sequences produced by the pathogen in the biological fluids.
  • Such techniques such as PCR, Northern or Southern hybridizations etc. are well known in the art.
  • any number of conventional protein assay formats, particularly immunoassay formats, or nucleic acid assay formats may be designed to utilize the isolated antigens and antibodies or their nucleic acid sequences or anti-sense sequences of this invention for the detection of disorders associated with inappropriate/altered levels of functional Bin2 and/or for monitoring inappropriate levels of Binl in animals and humans.
  • This invention is thus not limited by the selection of the particular assay format, and is believed to encompass assay formats which are known to those of skill in the art.
  • kits are useful for diagnosing conditions associated with dysfunctional Bin2 levels and/or Binl levels, including cancers in a human or an animal sample.
  • a diagnostic kit contains an antigen of this invention and/or at least one antibody capable of binding an antigen of this invention, or the nucleic acid sequences encoding them, or their anti-sense sequences.
  • such kits may contain a simple mixture of such antigens or sequences, or means for preparing a simple mixture.
  • kits can include microtiter plates to which the Bin2 peptides, proteins, antibodies, or nucleic acid sequences of the invention have been pre-adsorbed, various diluents and buffers, labeled conjugates for the detection of specifically bound antigens or antibodies, or nucleic acids and other signal-generating reagents, such as enzyme substrates, cofactors and chromogens.
  • Other components of these kits can easily be determined by one of skill in the art.
  • Such components may include polyclonal or monoclonal capture antibodies, antigen of this invention, or a cocktail of two or more of the antibodies, purified or semi -purified extracts of these antigens as standards, MAb detector antibodies, an anti-mouse or anti-human antibody with indicator molecule conjugated thereto, an ELISA plate prepared for absorption, indicator charts for colorimetric comparisons, disposable gloves, decontamination instructions, applicator sticks or containers, and a sample preparator cup.
  • Such kits provide a convenient, efficient way for a clinical laboratory to diagnose Bin2- associated conditions.
  • Bin2 is expressed predominantly in blood, e.g., hematopoietic, cells and is upregulated during monocytic differentiation.
  • Bin2 levels have been detected in spleen and peripheral blood leukocytes, and in thymus, colon and placenta, and Bin2 RNA has been strongly expressed in several human lymphoid and lymphoid cell lines, including GM1500, ALL200, BV173 and HL60.
  • Bin2 was induced during granulocytic differentiation of HL60 cells, a promyelocytic leukemia cell line.
  • Bin2 includes disorders associated with blood cells and hepatocytes, including hepatocarcinomas and certain myeloid or lymphoid leukemias. Further, Bin2 may be useful in directing treatment to disorders associated with inappropriate Binl functions, and particularly, for inhibiting excessive Binl levels. These Binl- associated disorders include liver, colorectal, prostate, and breast cancers, epithelial cell cancers, melanoma, and hyperplastic disease states.
  • the therapeutic compositions of the invention may be formulated to contain an anti-idiotype antibody of the invention, the Bin2 protein itself or a fragment thereof, or nucleic acid sequences which direct expression of these antibodies, proteins or fragments thereof, including anti-sense sequences.
  • the therapeutic composition desirably contains 0.01 ⁇ g to 10 mg protein.
  • These compositions may contain a pharmaceutically acceptable carrier, which facilitate administration of the compositions but are physiologically inert and/or nonharmful. Suitable carriers are well known to those of skill in the art and include, for example, saline.
  • such compositions may include conventional delivery systems into which protein of the invention is incorporated.
  • these compositions may contain other active ingredients, e.g., chemotherapeutics.
  • Carriers may be selected by one of skill in the art.
  • Exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, olive oil, sesame oil, and water.
  • the carrier or diluent may include a time delay material, such as glycerol monostearate or glycerol distearate alone or with a wax.
  • slow release polymer formulations can be used.
  • this composition may also contain conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers.
  • suitable ingredients which may be used in a therapeutic composition in conjunction with the antibodies include, for example, casamino acids, sucrose, gelatin, phenol red, N-Z amine, monopotassium diphosphate, lactose, lactalbumin hydrolysate, and dried milk.
  • agents useful in treating hepatocarcinoma or other conditions associated with dysfunctional Bin2 levels are expected to be useful in reducing or eliminating disease symptoms.
  • Such agents may operate in concert with the therapeutic compositions of this invention. The development of therapeutic compositions containing these agents is within the skill of one in the art in view of the teachings of this invention.
  • a human or an animal may be treated for hepatocarcinoma by administering an effective amount of such a therapeutic composition.
  • An "effective amount" may be between about 0.05 to about 1000 ⁇ g/mL of a Bin2 peptide, protein or antibody of the invention.
  • a suitable dosage may be about 1.0 mL of such an effective amount.
  • Such a composition may be administered 1 - 3 times per day over a 1 day to 12 week period. However, suitable dosage adjustments may be made by the attending physician or veterinarian depending upon the age, sex, weight and general health of the human or animal patient.
  • such a composition is administered parenterally, preferably intramuscularly or subcutaneously.
  • compositions may also be formulated to be administered by any other suitable route, including orally or topically.
  • Still another composition contains Bin2 polynucleotide sequences which contain regulatory sequences which regulate expression of the proteins of the invention in vivo or in vitro.
  • a DNA-based composition contains 0.05 ⁇ g to 1 mg DNA per mL dose.
  • a dose may be in the range of lxlO "3 pfu to lxlO 13 pfu per dose.
  • the dose, timing and mode of administration of these compositions may be determined by one of skill in the art.
  • a therapeutic composition of the invention is preferably administered in a site-directed manner and is repeated as needed.
  • therapy may be administered in conjunction with conventional therapies, including radiation and/or chemotherapeutic treatments.
  • Bin2 peptides, antibodies and polynucleotide sequences of the present invention are also useful in the screening and development of chemical compounds or proteins which have utility as therapeutic drugs for the treatment of cancers associated with inappropriate Bin2 levels.
  • Bin2 may be immobilized directly or indirectly (e.g., via an anti-Bin2 antibody) on a suitable surface, e.g., in an ELISA format. Such immobilization surfaces are well known. For example, a wettable inert bead may be used. Alternatively, Bin2 may be used in screening assays which do not require immobilization, e.g., in the screening of combinatorial libraries. Assays and techniques exist for the screening and development of drugs capable of binding to selected regions of Bin2. These include the use of a phage display system for expressing the Bin2 proteins, and using a culture of transfected E.
  • a method for identifying compounds which specifically bind to a Bin2 protein can include simply the steps of contacting a selected Bin2 protein with a test compound to permit binding of the test compound to the Bin2 protein; and determining the amount of test compound, if any, which is bound to the Bin2 protein. Such a method may involve the incubation of the test compound and the Bin2 protein immobilized on a solid support.
  • a surface containing the immobilized ligand is permitted to come into contact with a solution containing the Bin2 protein and binding is measured using an appropriate detection system.
  • Suitable detection systems include, without limitation, the streptavidin horseradish peroxidase conjugate and direct conjugation by a tag, e.g, fluorescein. Other systems are well known to those of skill in the art. This invention is not limited by the detection system used.
  • Another method of identifying compounds which specifically bind to a Bin2 protein can include the steps of contacting a Bin2 protein immobilized on a solid support with both a test compound and the protein sequence which is a receptor for Bin2 to permit binding of the receptor to the Bin2 protein; and determining the amount of the receptor which is bound to the Bin2 protein. The inhibition of binding of the normal protein by the test compound thereby indicates binding of the test compound to the Bin2 protein.
  • interaction blockers may be identified.
  • a simple plate assay could be used to screen for such blockers.
  • one peptide/protein is bound to the dish and the other is added in an aqueous buffer (physiological KC1, e.g. 150 mM, and pH, eg. 7.5). Binding is monitored using an antibody to the peptide which is conjugated to a fluorescent or enzymological marker (e.g., fluorescein or HRP).
  • a fluorescent or enzymological marker e.g., fluorescein or HRP
  • the primary antibody could also be detected by using an anti-primary antibody that is so tagged.
  • the binding conditions are empirically optimized for salt, pH, metal, and detergent conditions. Under optimized binding conditions, the assay is run in such a fashion that peptides or peptidomimetic drugs are added to the binding buffer.
  • the positive control for blocking binding is provided by addition of excess Bin protein, whereas the negative control is an unrelated protein or scrambled peptide.
  • a GST-Bin 1 protein is used to coat a plate and a Bin2 peptide used for binding.
  • the peptide is directly identified by an antibody; alternatively, it is epitope tagged.
  • the peptide itself is conjugated to the marker desired. This type of assay is amenable to high throughput screening since it can be configured in a 96-well format.
  • the present invention provides compounds capable of interacting with Bin2, or selected portions thereof, and either enhancing or inhibiting its biological activity, as desired.
  • the assay methods described herein are also useful in screening for inhibition of Bin2.
  • the solution containing the inhibitors may be obtained from any appropriate source, including, for example, extracts of supernatants from cultures of bioorganisms, extracts from organisms collected from natural sources, chemical compounds, and mixtures thereof.
  • a TBLASTN search of the expressed sequence tag (EST) database with the amino acid sequence containing this epitope identified a germinal B cell cDNA (Genbank accession number AA452680) which encoded a polypeptide related to but nonidentical to amphiphysin or Binl .
  • Bin2 differs in that it includes a large C- terminus that extends beyond the BAR domain
  • Bin2 lacked sequences implicated in nonneuronal isoforms of Bin l in interaction with c-Myc or TATA- binding protein in the nucleus [Sakamuro el al, cited above] or in alternately spliced neuronal isoforms in interaction with clathrin [Ramjaun and McPherson, J. Neiirochem. 70: 2369-2376 ( 1998)] or AP-2 (P de Camilli, pers comm )
  • the structure of Bin2 suggested strongly that it represented a nonredundant function.
  • Bin2 was a novel mammalian member of the BAR family of adaptor proteins
  • FISH fluorescence in situ hybridization
  • genomic DNA isolated from human foreskin, HepG2 or HLF hepatoma cells, or DU145 prostate carcinoma cells was analyzed by standard Southern analysis using the Bin2 cDNA as a hybridization probe. Both HepG2 and HLF exhibited normal and aberrant banding patterns relative to normal foreskin and DU145 cells, which showed the same pattern as foreskin, consistent with alteration of at least one allele of the Bin2 gene in certain liver tumors. Similar to DU145, genomic DNAs isolated from a panel of nonmalignant and malignant B lymphoid cell lines (Daudi, Raji, 380, GM1500, G97, BV173) also did not exhibit aberrent organization of the Bin2 gene. It was concluded that the Bin2 gene localized to chromosome 4q22.1 within a region that was altered in hepatocellular tumors.
  • EXAMPLE 3 - EVIDENCE OF SIMILARITY BETWEEN BLN2. BTN1. AND DAXX In an effort to gain insight into the function of Bin2, the sequences in its central and C-terminal regions were compared to the DNA database using the BLAST algorithm.
  • Table I illustrates the similarity between central regions of Bin2 and Daxx. Amino acid sequences from the central region of Bin2 were used to query the DNA database for related sequences using the TBLASTN algorithm. This figure provides the selected hits and significance scores. The query sequence was amino acid 179-336 in Bin2 [SEQ ID NO: 2 ]. The score for the Daxx hit was more significant than the hit to the related yeast BAR protein RVS161 (underlined). See, also, Figs. 4A and 4B.
  • Daxx through its ability to interact with CENP-C, a centromere-binding protein While the meaning of this interaction is not yet known, consistent with some nuclear role Daxx has also been found to interact with the cancer-related protein Pml and to localize to the subnuclear domain ND10 where Pml can be found (G Maul, pers co m ) Interestingly, Pml and Binl have each been demonstrated to have a role in certain types of programmed cell death [Quignon et al. Nat. Genet. 20: 259-265 (] 998), Wang el al, Nat. Genet. 20: 266-272 ( 1998)] Therefore, it is believed that the relationship between Daxx and Bin2 may reflect some related connection to cell survival controls This possibility would be consistent with the apparent role of RVS proteins in yeast survival, following stresses which result from nutrient starvation
  • Bin2 Northern hybridization of human tissue blots obtained from Clontech initially suggested a broad range of expression, similar to Binl and in contrast to amphiphysin (results not shown).
  • Two Bin2 messages were noted in most tissues, suggesting alternate splicing as occurs in Binl [Wechsler-Reya et al, J. Biol Chem. 272: 31453- 31458 (1997)].
  • the highest levels of Bin2 message were seen in spleen, peripheral blood leukocytes, thymus, placenta, testis, colon, liver, and lung.
  • the pattern of expression more resembled Binl, which is ubiquitous but expressed at highest levels in skeletal muscle, than amphiphysin, which is essentially specific to the central nervous system.
  • Bin2 might be preferentially expressed in hemapoietic cells and that the apparent pattern of ubiquitous expression reflected contamination of various tissues with hematopoietic cells, where Bin2 was expressed at high levels.
  • Bin2 message was barely detectable or undetectable by Northern analysis in 21 human cell lines derived from a variety of tissues, including breast, lung, prostate, brain, connective tissue (fibroblast), liver, and colon, despite the detection of Bin2 in these tissues.
  • Bin2 message was strongly expressed in several lymphoid cell lines (GM1500, ALL200, BV173, Jurkat) and in myeloid cells (HL60) that are derived from the hematopoietic lineage.
  • Bin2 may be altered in the cell lines examined, which were immortalized or malignant, it was possible that the inability to detect expression was due to functional loss.
  • the likelihood that Bin2 was predominantly expressed in hematopoietic cells was supported by "virtual" Northern analyses, performed by comparing Bin2 sequences to the EST database, which provides information about the source of the cDNA.
  • the majority of identical ESTs identified in this manner had been cloned from cDNA libraries derived from germinal B lymphoid cells, fetal liver (which is rich in B lymphoid cells), and placenta (which is rich in myeloid cells), although kidney libraries were also represented.
  • Bin2 One cell line identified to be positive for Bin2 expression was HL60 promyelocytic leukemia cells, which can be induced to differentiate to monocytes by treatment with dimethyl sulfoxide (DMSO) or to granulocytes by treatment with retinoic acid (RA).
  • DMSO dimethyl sulfoxide
  • RA retinoic acid
  • Bin2 The elevation of Bin2 during differentiation was reminiscent of a similar elevation of Binl which occurs during differentiation of skeletal myoblasts and certain other cells including smooth muscle and keratinocytes [Mao et al,Genomics 56: 51-58 (1999); Wechsler- Reya et al. Mol. Cell. Biol 18: 566-575 (1998)]. It was concluded that Bin2 was expressed predominantly in hematopoietic cells and was likely to function in the myeloid lineage.
  • Bin2 is expressed predominantly in hemapoietic cells. This pattern of expression contrasts with amphiphysin, which is largely restricted to the central nervous system, as well as with Binl, which is highly expressed in muscle but otherwise ubiquitous. Induction of Bin2 message was documented in a model system for monocytic differentiation, in support of the notion that Bin2 is likely to have an important function(s) in the hematopoietic lineage. Whether the Bin2 gene has important functions in other tissues is not yet clear. While expression was documented by Northern analysis in many normal tissues, these results could be ascribed to contamination of tissues with hemapoietic cells: while Bin2 message was abundant in lymphoid and myeloid cells, it was undetectable in most benign and malignant non-hemapoietic cell lines examined.
  • Bin2 the Bin2 gene is located at chromosome 4q22.1, a region frequently disrupted in hepatocarcinoma, and aberrant organization of the gene was observed in two liver tumor lines. Bin2 was not expressed in these lines but little effect of ectopic Bin2 on the proliferation of these cell lines in vitro was noted, so the significance of the aberrant gene organization and possible loss of expression remains unclear.
  • Binl is expressed in hematopoietic cells, whether Bin2 and Binl could also form a stable biochemical complex was investigated. For coimmunoprecipitation experiments, Binl was in vitro translated in the presence or absence of Bin2 and complex formation was assessed by SDS-PAGE and fluorography after immunoprecipitation with the Binl monoclonal antibody 99D [Wechsler-Reya et al, Cancer Res. 57: 3258-3263 (1997)].
  • Bin2 and Binl were in vitro translated with empty vector or cotranslated with each other in the presence of 35S-methionine. Products were fractionated on SDS- PAGE gels and fluorographed or subjected to immunoprecipitation with the anti-Binl antibody 99D [Wechsler-Reya et al, cited above] before fractionation. Bin2 migrated with an apparent MW ⁇ 82 kD, which is greater than the predicted MW ⁇ 61 kD, reminiscent of a similar aberrent mobility displayed by Binl, which has an apparent MW -68 kD and a predicted MW -50 kD. Bin2 was not immunoprecipitated by 99D unless cotranslated with Binl .
  • Binl and Bin2 formed a BAR domain-dependent complex in cells.
  • Binl and Bin2 were shown to form a stable biochemical complex, in the manner of RVS 161 and RVS 167 in yeast or amphiphysin and neuronal splice isoforms of Binl in mammalian cells [Navarro et al, cited above (1997); Wigge et al, cited above (1997)], and the association depended upon the integrity of the BAR domain. Bin2 did not affect the tumor suppressor properties of Binl that are manifested in HepG2 cells [Sakamuro et al, Nature Genet. 14: 69-77 (1996)].
  • Bin2 association rested on an N-terminal BAR determinant whereas the tumor suppressor activity of Binl rests upon a C-terminal BAR determinant.
  • Evidence that BAR domains encode unique activities an are not functionally equivalent is provided by domain swapping studies performed in yeast [Sivadon et al, FEBS Lett. 417: 21-27 (1997)].
  • the BAR domain of Bin2 may have unique features, perhaps related to Binl regulation rather than effector signaling. In future work, it will be important to determine the physiological functions of Bin2 and how they are manifested independently or in an integrated manner with the functions of Binl .
  • Binl has tumor suppressor properties in certain malignant cells [Sakamuro et al. 1996, cited above], so whether Bin2 had any similar effects and/or whether it could influence the growth inhibitory activity of Binl was investigated.
  • HepG2 hepatoma cells lack endogenous Binl and Bin2 expression so they provided a useful background to perform these experiments.
  • Bin2 could relieve or augment growth suppression by Binl
  • a similar set of colony formation experiments was performed in HepG2 except that untagged empty vector or Bin2 vector was cotransfected with a neomycin resistance gene-tagged Binl vector [Sakamuro et al. 1996, cited above].
  • the number of colonies which emerged using Bin2 vectors was similar to those produced with control vector, indicating that Bin2 did not affect the ability of Binl to suppress HepG2 cell growth (see Fig. 5B). It was concluded that Bin2 lacked the strong growth inhibitory properties in Hep G2 cells that are inherent to Binl .

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Abstract

La présente invention concerne des séquences Bin2 et des protéines codées par ces dernières; des compositions et des procédés dans lesquels on utilise ces séquences et ces protéines dans le diagnostic et le traitement des troubles sanguins, y compris l'hépatocarcinome; des oligonucléotides dérivés de séquences codant bin2, ainsi que des compositions et des procédés dans lesquels on les utilise à des fins de diagnostic et de traitement thérapeutique.
PCT/US2000/023723 1999-08-31 2000-08-30 Molecules d'acide nucleique et proteines d'integrateur 2 de pontage et utilisations associees WO2001016158A2 (fr)

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Publication number Priority date Publication date Assignee Title
EP2518166A2 (fr) 2005-05-20 2012-10-31 Veridex, LLC Dosage moléculaire par aspiration de la thyroïde au moyen d'une aiguille fine
CN112592403A (zh) * 2020-12-31 2021-04-02 中南大学湘雅二医院 cBIN1抗体及其应用
CN113957086A (zh) * 2021-12-11 2022-01-21 浙江大学 SlBIN2基因在调控番茄果实成熟和类胡萝卜素合成中的应用

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US5958753A (en) * 1996-08-29 1999-09-28 The Wistar Institute Of Anatomy And Biology Nucleic acid sequences encoding Bau, a Bin1 interacting protein, and vectors and host cells containing same

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US5958753A (en) * 1996-08-29 1999-09-28 The Wistar Institute Of Anatomy And Biology Nucleic acid sequences encoding Bau, a Bin1 interacting protein, and vectors and host cells containing same

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2518166A2 (fr) 2005-05-20 2012-10-31 Veridex, LLC Dosage moléculaire par aspiration de la thyroïde au moyen d'une aiguille fine
CN112592403A (zh) * 2020-12-31 2021-04-02 中南大学湘雅二医院 cBIN1抗体及其应用
CN112592403B (zh) * 2020-12-31 2022-04-15 中南大学湘雅二医院 cBIN1抗体及其应用
CN113957086A (zh) * 2021-12-11 2022-01-21 浙江大学 SlBIN2基因在调控番茄果实成熟和类胡萝卜素合成中的应用
WO2023103580A1 (fr) * 2021-12-11 2023-06-15 浙江大学 Utilisation du gène slbin2 dans la régulation et le contrôle de la maturation des fruits de solanum lycopersicum et de la synthèse des caroténoïdes

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