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WO1999043811A1 - Adn complementaires codant un gene bog (b5t-over-expressed gene) et son produit proteinique - Google Patents

Adn complementaires codant un gene bog (b5t-over-expressed gene) et son produit proteinique Download PDF

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Publication number
WO1999043811A1
WO1999043811A1 PCT/US1999/004142 US9904142W WO9943811A1 WO 1999043811 A1 WO1999043811 A1 WO 1999043811A1 US 9904142 W US9904142 W US 9904142W WO 9943811 A1 WO9943811 A1 WO 9943811A1
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bog
cells
polypeptide
prb
polypeptide fragment
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PCT/US1999/004142
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English (en)
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Snorri S. Thorgeirsson
Joseph T. Woitach
Minghuang Zhang
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The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services
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Priority to AU27905/99A priority Critical patent/AU2790599A/en
Publication of WO1999043811A1 publication Critical patent/WO1999043811A1/fr
Priority to US09/637,746 priority patent/US6727079B1/en
Priority to US10/772,988 priority patent/US7342099B2/en
Priority to US11/956,680 priority patent/US20100129791A1/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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates generally to the isolation and characterization of novel DNA and polypeptides, designated herein as "BOG".
  • RB retinoblastoma susceptibility gene
  • pRb gene product
  • RB retinoblastoma susceptibility gene
  • pRb is a nuclear protein that acts as a cell cycle control checkpoint by inhibiting Gl/S progression.
  • pRb contributes to maintaining the quiescent state of the cell by repressing transcription of genes required for the cell cycle through interaction with transcription factors, such as E2F (see e.g. Hiebert et al., Genes Develop., 6, 177-185 (1992)).
  • pRb Upon entrance into the cell cycle, pRb is phosphorylated by cell cycle-dependent kinases (see e.g. Matsushime et al., Nature, 35, 295- 300 (1992)) which is thought to permit its dissociation from transcription factors and, hence, the expression of genes required for progression through the cell cycle.
  • cell cycle-dependent kinases see e.g. Matsushime et al., Nature, 35, 295- 300 (1992)
  • the association of pRb with cell cycle regulators like cyclins and cell cycle-dependent kinases suggests a 2 universal character to its function.
  • the loss of this activity can induce cell transformation as evidenced by the reversion of the transformed phenotype in pRb cells after replacement of a functional pRb.
  • injection of the RB gene product, pRb, into GI cells can block cell cycle progression.
  • the identification of factors that interfere with and/or control pRb function is critical for understanding both cell cycle control and oncogenesis.
  • pRb is a target for oncogenic products of DNA tumor viruses.
  • Certain transforming proteins such as SV40T and E7, which are derived from the transforming or tumor-associated subtypes of adenoviruses and human papilloma viruses (HPV) are also capable of binding to pRb, thereby blocking its normal function. All these viral oncoproteins interact with pRb through an LXCXE motif (where X is any amino acid).
  • TGF- ⁇ l transforming growth factor- ⁇ l
  • the mechanism(s) by which TGF- ⁇ l inhibits cellular proliferation involves an attenuation of phosphorylation of pRb at the Gl/S transition of the cell cycle. See e.g. I. Reynisdottir, et al.
  • Adenovirus EIA, SV40 T antigen, and papillomarivus E7 are three exemplary viral proteins which have been found to bind to pRb. This binding is responsible for the release of transcription factors required for the expression of cell cycle genes (see e.g. Nevins, Science,
  • the viral oncoprotein-binding domain in pRb, pi 07 and pi 30 is a conserved region termed the "pocket region" (see e.g. Ewin et al., Cell, 66, 1155-1164 (1991)), and it is thought to play a primary role in the function of these proteins.
  • the pocket is structurally formed by two regions A and B, which are conserved in pRb, pi 07 and pi 30 and separated by nonconserved spacers of different sizes in pRb, pi 07 and pi 30.
  • pRb In addition to its interaction with viral oncoproteins, pRb is known to bind at least two dozen cellular proteins. J. Wang, Curr Opin Gen. Dev, 7:39 (1997); Y. Taya, et al., Trend Biochem. Sci., 22: 14 (1997). Included in these is the transcriptional factor E2F-1 which is required for the transcription of certain cellular genes that participate in growth control and DNA synthesis (see e.g. W.B. La Thangue, et al., Biochem. Soc Trans., 24:54 (1996).
  • E2F is composed of a family of closely related proteins, E2F-1 to -5, and a set of partner proteins belonging to the DP family of transcriptional factors (see e.g. J.E. Slansky, et al., Curr. Top. Microbiol. Immunol, 208:1 (1996).
  • E2F binds preferentially to the hypophosphorylated form of pRb and to several Rb related proteins, including pi 07 and pi 30.
  • E2F can be dissociated from pRb and related protein complexes by two mechanisms: hyperphosphoylation of pRb or by the competitive binding with viral proteins. Treatment of 4 cells with TGF- ⁇ l results in the accumulation of pRb in the hypophosphorylated form.
  • hypophosphorylated pRb binds E2F-1 thereby blocking E2F-1 growth promoting activity whereas phosphorylation of pRb or complexing with viral oncoproteins releases E2F from pRb and leads to transcriptional activation of growth promoting genes.
  • B5T Over-expressed Gene B5T Over-expressed Gene
  • BOG is a previously undescribed gene whose product binds the pRb tumor suppressor and is involved in regulating cellular growth. As BOG is shown to bind pRb, a factor known to regulate cellular growth and development, it is a prototype for molecules that function as endogenous regulators of cellular growth. As such, this novel protein has a variety of applications in the identification, characterization and regulation of activities associated with cellular regulation as well as processes associated with oncogenesis. 5 BOG is over-expressed in a number transformed rat liver epithelial (RLE) cell lines resistant to the growth inhibitory effect of TGF- ⁇ l as well as in primary liver tumors.
  • RLE rat liver epithelial
  • BOG Over-expression of BOG in non-transformed TGF- ⁇ l sensitive RLE cell lines can confer resistance to the growth inhibitory effect of TGF- ⁇ l .
  • continuous over-expression of BOG in normal RLE cells typically leads to rapid transformation and the transformed cells can form hepatoblastoma-like tumors when transplanted into nude mice.
  • Incubation of BOG over-expressing cells with BOG antisense oligonucleotides can restore sensitivity to TGF- ⁇ .
  • BOG typically binds to pRb to form complex that does not contain E2F-1 and, in vitro, BOG can displace E2F-1 from E2F-l/pRb complexes. It is believed that BOG may be important in the transformation process, due in part, to the capacity of BOG to confer resistance to the growth inhibitory effects of TGF- ⁇ l through interaction with pRb and the subsequent displacement of E2F-1.
  • the invention provides an isolated nucleic acid molecule includes DNA which includes nucleotides which encode a BOG polypeptide.
  • the isolated nucleic acid can include DNA encoding BOG polypeptide having amino acid residues 1 to 173 of Tables 1, 5 or 7 or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides a vector comprising a gene encoding a BOG polypeptide.
  • a host cell comprising such a vector is also provided.
  • the host cells may be E. coli, yeast or mammalian cells.
  • a process for producing BOG polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of BOG. If desired, the BOG may be recovered.
  • the invention provides methods for using BOG-RELATED polypeptides and nucleic acids for studying and modulating mechanisms involved in cellular proliferation.
  • the invention provides a method of modulating cellular phenotype by controlling the level of BOG expression within the cell.
  • the invention provides a method of generating a transformed cellular phenotype by overexpressing BOG.
  • the invention provides a method of reducing BOG expression via antisense oligonucleotides in order to effect a cellular phenotype such as TGF- ⁇ sensitivity.
  • the invention provides methods for effecting the interaction between pRb and pRb binding proteins.
  • Figure 2(A) shows the yeast two-hybrid system as used to demonstrate the interaction of Rb and BOG in vivo (Matchmaker Gal4 Two-hybrid System, Clontech). Vectors containing only BOG, Rb, or vector not containing insert were used as negative controls to demonstrate lack of growth on selective media and lack of ⁇ -galactosidase activity. SV40 large T was used as a positive control to demonstrate interaction with Rb. Growth rates and ⁇ - galactosidase activity for BOG and Rb were similar to those exhibited by SV40T and Rb.
  • Figure 3(A) shows colony formation of a heterogeneous population of transfected RLE and B7 cells expressing BOG.
  • Two RLE cell lines, phi 13 and B7, which are untransformed and known to be sensitive to TGF- ⁇ were transfected with BOG or empty vector. After the cells were allowed to recover, they were cultured in the presence of TGF- ⁇ l for 14 days. Most cells that contained the empty vector control did not grow or died during the two weeks in culture. Colonies grow in cells overexpressing BOG were stained with crystal violet.
  • Figure 3(B) shows growth curves in the presence of TGF- ⁇ l .
  • RLE phi 13 cells were transfected with BOG and cell lines overexpressing BOG were selected.
  • Two clones, BOG- A and BOG-B, representing the two extremes of BOG expression in these lines were chosen and expanded for further experiments.
  • the effect of overexpression of BOG on the cells ability to proliferate in the presence of TGF- ⁇ l was examined.
  • the effect of TGF- ⁇ l on growth is expressed as a percent inhibition of growth of cells grown in the presence of TGF- ⁇ l versus cells grown in normal media for the same period of time. Values and standard error presented represent two separate experiments done in quadruplicate.
  • Figure 3(C) shows the effect of BOG expression on the TGF- ⁇ l receptor.
  • the same cell lines used to examine growth were expanded and their ability to bind TGF- ⁇ l was examined. Binding curves were generated using samples analyzed in triplicate for each concentration of TGF- ⁇ l . Tenfold excess of unlabeled TGF- ⁇ l was used to determine nonspecific binding and subtracted to obtain specific cpm bound. Saturation curves were generated for phi 13, 8 BOG-A and BOG-B with standard error calculated from two separate experiments. To further evaluate the effect of BOG on the TGF- ⁇ receptor expression, Northern blot analysis of TGF- ⁇ receptor II was performed (inset). BOG expression also was tested to confirm previous characterizations of the transgene expression (inset).
  • Figure 3(D) shows the restoration of TGF- ⁇ l sensitivity by antisense oligonucleotides to BOG.
  • BOG protein In order to decrease the amount of BOG protein, cell lines were incubated with antisense oligonucleotides to BOG. Oligonucleotide alone was not toxic to any of the cell lines as demonstrated by 100% growth as compared to the cells grown in control media.
  • Figure 4(B) shows BOG expression in rat liver tumors. Tumors were derived utilizing the Solt-Farber carcinogenesis protocol (17) and were analyzed by Northern blot analysis using the 1.9 kb BOG cDNA as a probe.
  • Figure 5 shows the results of a BLAST search and sequence analysis showing that a Blast search revealed only matches with sequences in EST database. Search results were obtained by using the open reading frame of rat BOG cDNA. The non-coding region of rat and mouse BOG both contain a B-l like repeat and homology searches with this region produce many matches. Only the open reading frame of the human BOG sequence is available. P(N) value calculated by BLASTN search program with default values (reference Altschul, S.F., et al.). 9
  • Figure 6(A) shows in vitro translation data and Western Blot data characterizing BOG.
  • In vitro translation of rBOG produces a 20 kDA protein and Western blot analysis identifies a protein of the same molecular weight.
  • Figure 6(B) is a multi-tissue blot analysis showing that rBOG is expressed ubiquitously in all tissues analyzed.
  • Figure 7 shows a genomic southern blot examining BOG in different species and demonstrating that the BOG gene is present in all mammals tested.
  • Figure 8(C) is a representational map of the SB6 murine genomic clone.
  • Figure 9(A) shows that rat BOG is localized to the nucleus as shown by staining with polyclonal antibodies.
  • Figure 9(B) shows that preincubating the anti-BOG polyclonal antibodies with BOG antigen inhibits nuclear staining.
  • Figures 10(A) and 10(B) show that the expression of BOG is cell cycle regulated.
  • RLE and B5T cells were synchronized at various points in the cell cycle, both cell lines show a similar pattern of expression of BOG during the cell cycle, with peak expression being seen during late GI .
  • Figure 11 shows the expression of rat BOG after partial hepotectomy.
  • a 2/3 partial hepotectomy was performed on rats to obtain a synchronized population of primary hepatocytes.
  • the peak expression of BOG corresponds to late Gl-S.
  • Figure 12 shows the derivation of RLE cell lines.
  • the RLE cell lines were derived from parental cells isolated from a 10 day old Fisher 344 rat and untransformed and transformed cell lines were derived from this original population. 10
  • Figure 13(A)-13(F) shows the sequence and a TESS-String based search partial characterization of the murine 5' genomic BOG DNA sequence (SEQ ID NO: 11) located approximately 10 nucleotides upstream of the ATG initiation codon.
  • BOG polypeptide and “BOG” when used herein encompass native sequence BOG and BOG variants (which are further defined herein).
  • the BOG may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods.
  • the BOG polypeptide which may be a fragment of a native sequence, contains a retinoblastoma gene product (pRb) binding domain having the motif LXCXE.
  • the BOG polypeptide also includes at least one casein kinase II phosphorylation motif that is typically located downstream (i.e. closer to the C-terminus of the polypeptide) from the pRb binding domain.
  • the BOG polypeptide may contain a second casein kinase II phosphorylation motif that is upstream from the pRb binding domain.
  • a “native sequence BOG” is a polypeptide having the same amino acid sequence as an BOG derived from nature. Such native sequence BOG can be isolated from nature or can be produced by recombinant or synthetic means.
  • the term "native sequence BOG” specifically encompasses naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants of the BOG.
  • the native sequence BOG is a mature or full-length native sequence BOG polypeptide comprising amino acids 1 to 173 of Table 1.
  • the BOG polypeptide comprises amino acid residues 1 to 173 of Tables 5 or 7.
  • Percent (%) amino acid sequence identity with respect to the BOG sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the BOG sequence, after aligning the sequences in the same reading frame and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • Percent (%) nucleic acid sequence identity with respect to the BOG sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the BOG sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • epitope tagged when used herein refers to a chimeric polypeptide comprising BOG, or a functional fragment thereof, fused to a "tag polypeptide".
  • the tag polypeptide has enough residues to provide an epitope against which an antibody can be made, or which can be identified by some other agent, yet is short enough such that it does not interfere with activity of the BOG.
  • the tag polypeptide preferably also is sufficiently unique so that the antibody does not substantially cross-react with other epitopes.
  • Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 to about 50 amino 12 acid residues (preferably, between about 10 to about 20 residues).
  • Isolated when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the polypeptide will be purified to a degree sufficient to obtain N- terminal or internal amino acid sequence by use of a spinning cup sequenator, or to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or silver stain.
  • Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the BOG natural environment will not be present.
  • isolated polypeptide will be prepared by at least one purification step (referred to herein as an "isolated and purified polypeptide").
  • An "isolated" BOG nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the BOG nucleic acid.
  • An isolated BOG nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated BOG nucleic acid molecules therefore are distinguished from the BOG nucleic acid molecule as it exists in natural cells.
  • an isolated BOG nucleic acid molecule includes BOG nucleic acid molecules contained in cells that ordinarily express BOG where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence 13 if it affects the transcription of the sequence: or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking may be accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers may be used in accordance with conventional practice.
  • Polynucleotide and “nucleic acid” refer to single- or double-stranded molecules which may be DNA, comprised of the nucleotide bases A, T, C and G, or RNA, comprised of the bases A, U (substitutes for T) , C, and G .
  • the polynucleotide may represent a coding strand or its complement.
  • Polynucleotide molecules may be identical in sequence to the sequence which is naturally occurring or may include alternative codons which encode the same amino acid as that which is found in the naturally occurring sequence (See, Lewin “Genes V" Oxford University Press Chapter 7, pp. 171-174 (1994)). Furthermore, polynucleotide molecules may include codons which represent conservative substitutions of amino acids as described.
  • the polynucleotide may represent genomic DNA or cDNA.
  • Polypeptide refers to a molecule comprised of amino acids which correspond to those encoded by a polynucleotide sequence which is naturally occurring.
  • the polypeptide may include conservative substitutions where the naturally occurring amino acid is replaced by one having similar properties, where such conservative substitutions do not alter the function of the polypeptide (See, Lewin “Genes V” Oxford University Press Chapter 1, pp.: 9-13 (1994)).
  • antibody is used in the broadest sense and specifically covers single anti-BOG monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies) and anti- BOG antibody compositions with polyepitopic specificity.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts.
  • mammal refers to any mammal classified as a mammal, including humans, cows, horses, dogs and cats. In a preferred embodiment of the invention, the 14 mammal is a human.
  • BOG Nucleic Acids and Polypeptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as BOG.
  • BOG nucleotide sequences encoding polypeptides referred to in the present application as BOG.
  • Applicants have identified and isolated genes and cDNA encoding BOG polypeptides, as disclosed in further detail in the Examples below.
  • sequence homology searches Applicants found that BOG (as shown in Tables 1 , 5 and 7) contains the pRb binding motif LXCXE and shares certain amino acid sequence identity with pRb binding proteins including a number of viral oncoproteins (see Table 2).
  • BOG polypeptide was found to bind pRb and to be able to displace other proteins complexed with pRb.
  • BOG variants can be prepared.
  • BOG variants can be prepared by introducing appropriate nucleotide changes into the BOG nucleotide sequence, or by synthesis of the desired BOG polypeptide.
  • amino acid changes may alter post-translational processes of the BOG, such as changing the number or position of glycosylation sites or altering the protein binding characteristics.
  • Variations in the native full-length sequence BOG or in various domains of the BOG described herein can be made, for example, using any of the techniques and guidelines for conservative and non- conservative mutations set forth, for instance, in U.S. Patent No. 5,364,934.
  • amino acid substitutions at the H and/or D residues of the LHCDE motif are contemplated, such as conservative substitutions at one or both of these residues.
  • Insertions or deletions may optionally be in the range of 1 to 5 amino acids.
  • the variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity in any of the in vitro assays described in the Examples below.
  • BOG molecules of the invention can have amino acid substitutions in the amino acid sequence shown in Tables 1, 5 and 7 (Current Protocols In Molecular Biology, Volume I, Unit 8, Frederick M. Ausubul et al. eds., 1995). Such substitutions result in BOG that retains the ability to bind pRb.
  • amino acid substitutions include, but are not necessarily limited to, amino acid substitutions known in the art as "conservative”.
  • Methionine (M) which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered "conservative" in particular environments.
  • Variations can be made using methods known in the art such as site-directed mutagenesis, alanine scanning, and PCR mutagenesis.
  • Site-directed mutagenesis [Carter et al., Nucl. Acids
  • Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence.
  • preferred scanning amino acids are relatively small, neutral amino acids.
  • Such amino acids include alanine, glycine, serine, and cysteine.
  • Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.
  • codon optimized sequences typically have rare codons (i.e., codons having a useage frequency of less than about 20% in known sequences of the desired host) replaced with higher frequency codons.
  • Codon preferences for a specific organism may be calculated, for example, by utilizing codon usage tables available on the INTERNET at the following address: http://www.dna.affrc. go.jp/ ⁇ nakamura/codon.html.
  • Nucleotide sequences which have been optimized for a particular host species by replacing any codons having a useage frequency of less than about 20% are referred to herein as "codon optimized sequences.”
  • Additional sequence modifications are known to enhance protein expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon/intron splice site signals, transposon-like repeats, and/or other such well-characterized sequences which may be deleterious to gene expression.
  • the GC content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Where possible, the sequence may also be modified to avoid predicted hairpin secondary mRNA structures.
  • Other useful modifications include the addition of a translational initiation consensus sequence at the start of the open reading frame, as described in Kozak, Mol. Cell Biol., 9:5073-5080 (1989).
  • Nucleotide sequences which have been optimized for expression in a given host species by elimination of spurious polyadenylation sequences, elimination of exon/intron splicing signals, elimination of 17 transposon-like repeats and/or optimization of GC content in addition to codon optimization are referred to herein as an "expression enhanced sequence.”
  • B. Modifications of BOG Covalent modifications of BOG are included within the scope of this invention.
  • One type of covalent modification includes reacting targeted amino acid residues of the BOG with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the BOG.
  • Derivatization with bifunctional agents is useful, for instance, for crosslinking BOG to a water-insoluble support matrix or surface for use in the method for purifying anti-BOG antibodies, and vice-versa.
  • crosslinking agents include, e.g., l,l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxy- succinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3.3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-l,8-octane and agents such as methyl-3- [(p-azidophenyl)dithio]propioimidate.
  • Another type of covalent modification of the BOG polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide.
  • "Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence BOG, and/or adding one or more glycosylation sites that are not present in the native sequence BOG. Addition of glycosylation sites to the BOG polypeptide may be accomplished by altering the amino acid sequence. The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence BOG (for O-linked glycosylation sites).
  • the BOG amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the BOG polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
  • Another means of increasing the number of carbohydrate moieties on the BOG polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 11 September 1987, and in Aplin and Wriston, CRC Crit. 18
  • the chimeric molecule may comprise a fusion of the BOG with an immunoglobulin or a particular region of an immunoglobulin.
  • the BOG may be fused any one of a variety of known fusion protein partners that are well known in the art such as maltose binding protein, LacZ, thioredoxin or an immunoglobulin constant region (Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley, P.S., Brady, W., Urnes, M., Grosmaire, L., Damle, N., and Ledbetter, L.(1991) J.Exp. Med. 174, 561-566).
  • this fusion partner is a non-BOG binding molecule so as to prevent difficulties associated with intramolecular interactions.
  • a bivalent form of the chimeric molecule such a fusion could be to the Fc region of an IgG molecule.
  • Other fusion proteins and tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell.
  • tag polypeptides include the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; an "-tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].
  • the description below relates primarily to production of BOG by culturing cells transformed
  • BOG sequence or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques [see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J. Am. Chem. Soc, 85:2149-2154 (1963)].
  • In vitro protein synthesis may be performed using manual techniques or by automation.
  • Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using manufacturer's instructions.
  • Various portions of the BOG may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length BOG.
  • DNA encoding BOG may be obtained from a cDNA library prepared from tissue expressing a BOG mRNA. Accordingly, human BOG DNA can be conveniently obtained from a cDNA library prepared from human tissue.
  • the BOG-encoding gene may also be obtained from a genomic library or by oligonucleotide synthesis. Libraries can be screened with probes (such as antibodies to the BOG or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it.
  • Illustrative libraries include mouse kidney cDNA library (mouse kidney 5'-stretch cDNA, Clonetech laboratories, Inc.) and human liver cDNA library (human liver 5' stretch plus cDNA, Clonetech Laboratories, Inc.). Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). AJ alternative means to isolate the gene encoding BOG is to use PCR methodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].
  • the oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized.
  • the oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are 20 well known in the art, and include the use of radiolabels like 32 P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., supra.
  • Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined through sequence alignment using computer software programs which employ various algorithms to measure homology.
  • Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA.
  • Host cells are transfected or transformed with expression or cloning vectors described herein for BOG production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the culture conditions such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.
  • transfection is known to the ordinarily skilled artisan, for example by using lipofectin, CaPO 4 or electroporation.
  • transformation is performed using standard techniques appropriate to such cells.
  • the calcium treatment employing calcium chloride, as described in Sambrook et al., supra, or electroporation is generally used for prokaryotes or other cells that contain substantial cell-wall barriers.
  • Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al., Gene. 23:315 (1983) and WO 89/05859 published 29 June 1989.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells.
  • Suitable prokaryotes include but are not limited to eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli.
  • Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W31 10 (ATCC 27,325) and K5 772 (ATCC 53,635).
  • Suitable host cells for the expression of glycosylated BOG are derived from multicellular organisms.
  • invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9 cells. See e.g. Current Protocols In Molecular Biology, Volume I, Unit 16, Frederick M. Ausubul et al. eds., 1995.
  • useful mammalian host cell lines include rat liver epithelial cells. Hugget, A. C. et. al. Supra, Chinese hamster ovary (CHO) and COS cells.
  • monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol.. 36:59 (1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA. 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.. 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCC CCL51).
  • the selection of the appropriate host cell is deemed to be within the skill in the art. 22 3. Selection and Use of a Replicable Vector
  • the nucleic acid (e.g., cDNA or genomic DNA) encoding BOG may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression.
  • a replicable vector for cloning (amplification of the DNA) or for expression.
  • the vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage.
  • the appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuciease site(s) using techniques known in the art.
  • Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.
  • the BOG may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • a heterologous polypeptide which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • the signal sequence may be a component of the vector, or it may be a part of the BOG DNA that is inserted into the vector.
  • the signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat- stable enterotoxin II leaders.
  • yeast secretion the signal sequence may be, e.g.
  • yeast invertase leader the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces "-factor leaders, the latter described in U.S. Patent No. 5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990.
  • mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders.
  • Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses.
  • the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2: plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.
  • Selection genes will typically contain a selection gene, also termed a selectable marker.
  • Typical selection genes encode proteins that (a) confer resistance to 23 antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
  • suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the BOG nucleic acid, such as Neomycin, DHFR or thymidine kinase.
  • An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980).
  • a suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene.
  • the trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].
  • Expression and cloning vectors usually contain a promoter operably linked to the BOG nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known.
  • Promoters suitable for use with prokaryotic hosts include the ⁇ -lactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:2X25 (1983)]. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding BOG.
  • S.D. Shine-Dalgarno
  • Suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv.
  • yeast promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with 24 nitrogen metabolism, metallothionein, glyceraldehyde-3 -phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
  • BOG transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus
  • viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus
  • SV40 from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription.
  • Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, "- fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus.
  • Examples include the SV40 enhancer on the late side of the replication origin (bp 100- 270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the enhancer may be spliced into the vector at a position 5' or 3' to the BOG coding sequence, but is preferably located at a site 5' from the promoter.
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding BOG.
  • Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA
  • antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.
  • the antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
  • Gene expression may be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product.
  • Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native sequence BOG polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to BOG DNA and encoding a specific antibody epitope.
  • BOG BOG from recombinant cell proteins or polypeptides.
  • the following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms of the BOG.
  • Hybridization probes may be labeled by a variety of labels, including radionucleotides such as 3 P or 5 S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems. Labeled probes having a sequence complementary to that of the BOG gene of the present invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine which members of such libraries the probe hybridizes to. Hybridization techniques are described in further detail in the Examples below.
  • Nucleotide sequences encoding a BOG can also be used to construct hybridization probes for mapping the gene which encodes that BOG and for the genetic analysis of individuals with genetic disorders.
  • the nucleotide sequences provided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization 27 screening with libraries.
  • Screening assays can be designed to find lead compounds that mimic the biological activity of a native BOG or a ligand or receptor for BOG. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates. Small molecules contemplated include synthetic organic or inorganic compounds.
  • the assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art.
  • Antisense technology entails the administration of exogenous oligonucleotides which bind to a target polynucleotide located within the cells.
  • the term "antisense” refers to the fact that such oligonucleotides are complementary to their intracellular targets, e.g., BOG . See for example, Jack Cohen, OLIGODEOXYNUCLEOTIDES, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1 :1-5 (1988).
  • the BOG antisense oligonucleotides of the present invention include derivatives such as S-oligonucleotides (phosphorothioate derivatives or S-oligos, see, Jack Cohen, supra) which exhibit enhanced cancer cell growth inhibitory action.
  • the BOG antisense oligonucleotides of the present invention may be RNA or DNA which is complementary to and stably hybridizes with the first 100 N-terminal codons or last 100 C- terminal codons of the BOG genome or the corresponding mRNA. While absolute complementarity is not required, high degrees of complementarity are preferred. Use of an oligonucleotide complementary to this region allows for the selective hybridization to BOG 28 mRNA and not to mRNA specifying other regulatory subunits of protein kinase.
  • the BOG antisense oligonucleotides of the present invention are a 15 to 30-mer fragment of the antisense DNA molecule having a sequence which hybridizes to BOG mRNA.
  • BOG antisense oligonucleotide is a 30-mer oligonucleotide which is complementary to a region in the first 10 N-terminal codons and last 10 C-terminal codons of
  • the antisense molecules are modified to employ ribozymes in the inhibition of BOG expression.
  • the BOG antisense oligonucleotide is coadministered with an agent which enhances the uptake of the antisense molecule by the cells.
  • the BOG antisense oligonucleotide may be combined with a lipophilic cationic compound which may be in the form of liposomes.
  • liposomes to introduce nucleotides into cells is taught, for example, in U.S. Pat. Nos. 4,897,355 and 4,394,448, the disclosures of which are incorporated by reference in their entirety. See also U.S. Pat. Nos.
  • the BOG antisense oligonucleotide may be combined with a lipophilic carrier such as any one of a number of sterols including cholesterol, cholate and deoxycholic acid.
  • the BOG antisense oligonucleotide may be coadministered with a second agent that is effected by BOG expression.
  • this second agent is one or more isoforms of TGF- ⁇ .
  • a combination of BOG antisense oligonucleotides and TGF- ⁇ l are administered to cells which have reduced sensitivity to TGF- ⁇ due to BOG overexpression.
  • a combination of these two molecules may be used to synergistically induce TGF- ⁇ l mediated apoptosis. Methods pertaining to these embodiments are well known in the art.
  • the antisense oligonucleotides of the present invention may be prepared according to any of the methods that are well known to those of ordinary skill in the art.
  • the antisense oligonucleotides are prepared by solid phase synthesis. See, Goodchild, J., Bioconjugate
  • the antisense oligonucleotides can be obtained from a number of companies which specialize in the custom synthesis of oligonucleotides.
  • Nucleic acids which encode BOG or its modified forms can also be used to generate either transgenic animals or "knock out" animals which, in turn, are useful in the development and screening of therapeutically useful reagents.
  • a transgenic animal e.g., a mouse or rat
  • a transgenic animal is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage.
  • a transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops.
  • cDNA encoding BOG can be used to clone genomic DNA encoding BOG in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which express DNA encoding BOG.
  • Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009.
  • particular cells would be targeted for BOG transgene incorporation with tissue-specific enhancers.
  • Transgenic animals that include a copy of a transgene encoding BOG introduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased expression of DNA encoding BOG.
  • Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression.
  • an animal is treated with the reagent and a reduced incidence of the pathological condition, compared to untreated animals bearing the transgene, would indicate a potential therapeutic intervention for the pathological condition.
  • non-human homologues of BOG can be used to construct a BOG "knock out" animal which has a defective or altered gene encoding BOG as a result of homologous recombination between the endogenous gene encoding BOG and altered genomic DNA encoding BOG introduced into an embryonic cell of the animal.
  • cDNA encoding BOG can be used to clone genomic DNA encoding BOG in accordance with 30 established techniques.
  • a portion of the genomic DNA encoding BOG can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration.
  • flanking DNA typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector [see e.g., Thomas and Capecchi, Cell. 51:503 (1987) for a description of homologous recombination vectors].
  • the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected [see e.g., Li et al., Cell. 69:915 (1992)].
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152].
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal.
  • Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA.
  • Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of the BOG polypeptide.
  • genomic BOG control sequences of the present invention may be employed in numerous various combinations and organizations to assess the regulation of BOG.
  • each unit comprise the same positive or negative element. All that is required is that such sequences be located upstream of and sufficiently proximal to a transcription initiation site.
  • TATA sequences normally associated with the heterologous gene are most desirably located between about 20 and 30 nucleotides upstream of transcription initiation.
  • the heterologous gene is a reporter gene which encodes an enzyme which produces colorimetric or fluorometric change in the host cell which is detectable by in situ analysis and which is a quantitative or semi-quantitative function of transcriptional activation.
  • exemplary enzymes include esterases, phosphatases, proteases (tissue plasminogen activator or urokinase) and other enzymes capable of being detected by activity which generates a chromophore or fluorophore as will be known to those skilled in the art.
  • a preferred example is E. coli beta-galactosidase.
  • This enzyme produces a color change upon cleavage of the indigogenic substrate indolyl-B-D-galactoside by cells bearing beta-galactosidase (see, e.g., Goring et al., Science, 235:456-458 (1987) and Price et al., Proc. Natl. Acad. Sci. U.S.A., 84:156-160 (1987)).
  • this enzyme facilitates automatic plate reader analysis of BOG control sequence mediated expression directly in microtiter wells containing transformants treated with candidate activators.
  • the endogenous beta-galactosidase activity in mammalian cells ordinarily is quite low, the analytic screening system using ⁇ -galactosidase is not hampered by host cell background.
  • CAT chloramphenicol acetyltransferase
  • the illustrative co-immunoprecipitation and Gal4 protein-protein interaction assays may useful in screening for compounds modulating BOG activity, or in screening for compounds altering BOG activity in a cell.
  • BOG participates with pRb in controlling the proliferative response of a cell to its environment.
  • binding of a ligand at a molecular binding site can be modulated in a direct manner (e.g., by blocking the site), as well as altered in an indirect manner (e.g., by conformational changes induced following binding of a second (different) ligand at a distant site).
  • binding site specificity of BOG for a particular pRb family member can be completely altered (i.e., to bind a different ligand) by agents that bind at distant sites in the pRb polypeptide.
  • a number of exemplary protocols which may be used in these studies are known in the art, see e.g. U.S. Patent No. 5,625,031.
  • Such regulatory factors can include, at least: a) cofactors that bind to the complex and exert regulatory action by destabilizing or stabilizing the complex; b) agents that modulate or alter the activity of the complex by inducing conformational changes in the BOG polypeptides as they are bound together in the complex; c)enzymes that inactivate one or both members of the complex; and, d) cellular control factors (e.g., signal transduction second messengers, transcription regulatory factors, and the like) that bind pRb or pRb complexes and modulate or alter functional activity.
  • polypeptides such as truncated BOG polypeptides can be constructed that control the activity of the BOG/pRb complexes in the cell by inhibiting or promoting the activities of such regulatory factors.
  • BOG genes can be incorporated into any standard cloning vector.
  • vector is well understood in the art and is synonymous with the often-used phrase
  • a suitable vector is a non-chromosomal double-stranded DNA comprising an intact replicon such that the vector is replicated when placed within a unicellular organism.
  • Viral vectors include retroviruses, adenoviruses, herpes virus, papovirus. etc.
  • Other suitable vectors include plasmids. Plasmids and retroviruses are generally preferred as vectors.
  • pBKCMV-BOG pBKCMV purchased from Stratagene'" 1
  • CMV promoter This promoter is suitable for expression of the BOG genes in a wide variety of cells.
  • the CMV promoter is not specifically required for transcription and expression of the BOG genes.
  • the promoter DNA can be amplified using PCR technology while concurrently providing restriction sites at the 5' and 3' ends of the promoter DNA.
  • the amplified promoter DNA can then be inserted into a cloning vehicle (for example pBKCMV) using conventional endonucleases and known recombinant DNA technology.
  • a cloning vehicle for example pBKCMV
  • Cloning vectors containing the desired promoter upstream of the 5' end of BOG genes are constructed in this manner.
  • cloning vectors containing an appropriate promoter and the BOG gene may be constructed using PCR technology in a manner analogous to the preparation of vectors containing exogenous genes as is well known in the art.
  • Cloning vectors containing BOG genes are transfected into host cells using known transfection processes. Suitable transfection processes include lipofection, electroporation and retrovirus infection. When transfecting cells with BOG, the desired cells are isolated and cultured in suitable media.
  • Transfection of cells using lipofection may conducted according to standard lipofection procedures. See Feigner et al. 1987. Proc. Natl. Acad. Sci. (USA). 84:7413-7417.
  • 34 liposome-mediated DNA transfection is accomplished by exposing 1-20 micrograms of plasmid DNA and commercially available liposomes (Bethesda Research Laboratories) in culture medium. The transfected cells are then repeatedly passaged in culture medium and the desired clones are isolated.
  • Retrovirus infection may also be accomplished using previously described procedures. See for example Miller et al. J. Virol. 62:4337-4345 and Halbert et al. J. Virol. 65:473-378. 1991.
  • plasmid DNA is transfected into a desired packaging cell line such as Psi-2 or other cell lines, using standard calcium phosphate precipitation.
  • Viruses produced from the Psi-2 cells or equivalent cells are then used to infect an amphotropic packaging cell line, for example PA317.
  • Viruses produced by the amphotropic packaging cell line are used to infect the desired host parent cells of the present invention.
  • Selection of clones with a modulated phenotype may be undertaken by a variety of protocols that are well known in the art (see e.g. U.S. Patent No. 5,376,542).
  • the cells may be selected for their ability to respond to factors such as TGF- ⁇ .
  • cells can be selected by their ability to form colonies and grow in soft agar.
  • the cells can be selected by their ability to form tumors in animal models such as in nude mice.
  • the desired clones are selected by culturing in optimal media and repeated passaging. Generally, 10-20 passages are required to eliminate spurious cells and obtain pure clonal cells.
  • Optimal media are selected according to the type of parent cell which is utilized.
  • RPMI media is preferred; for fibroblasts, DMEM media is preferred; and for epithelial cells, a serum-free medium such as keratinocyte growth medium (KGM) or SFM (Gibco Company) is preferred.
  • KGM keratinocyte growth medium
  • SFM Gabco Company
  • Example 8 chromosomal localization of the human and murine BOG genes is described. Chromosomal localization was done by FISH analysis (Stokke, T. et al., Genomics 26: 134- 35 137 (1995)). Murine BOG maps to chromosome 2 and in human to the syntenic region of chromosome 20.
  • the invention provides diagnostic assays for determining chromosomal rearrangement of BOG genes in a cell.
  • the chromosomal location of BOG genes is conveniently determined in chromosomal smears by in situ hybridization with oligonucleotide probes or cDNA and the like.
  • Translocation of a BOG gene i.e., from a chromosomal location found in a normal cell to a location found in a transformed cell, may contribute to a phenotype of uncontrolled cell growth by removing normal transcription regulatory control of either gene expression of a BOG or RB polypeptide.
  • the cell may acquire a malignant (i.e., uncontrolled) growth phenotype, and in the case where the rearrangement induces under- expression the cell may undergo premature senescence. Screening cellular samples from individuals for the potential of BOG chromosomal rearrangement may indicate a relative risk factor for the possibility of developing cancer.
  • the present invention further provides anti-BOG antibodies.
  • Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies.
  • the BOG antibodies may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections.
  • the immunizing agent may include the BOG polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized.
  • immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
  • polyclonal antibodies may be generated commercially, for example by Genemed Synthesis, Inc. using art accepted methods. 36
  • the BOG antibodies may, alternatively, be monoclonal antibodies.
  • Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature. 256:495 (1975).
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro.
  • rat or mouse myeloma cell lines are employed.
  • the hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center,
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against BOG.
  • the binding specificity 37 of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells of the invention serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Patent No. 4,816,567) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide.
  • non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
  • the antibodies may be monovalent antibodies.
  • Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. 38
  • cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.
  • in vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.
  • the BOG antibodies of the invention may further comprise humanized antibodies or human antibodies.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol, 2:593- 596 (1992)].
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import” variable domain.
  • Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al., J. Mol. Biol.. 222:581 (1991)].
  • the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J Immunol, 7 ⁇ 7(7):86-95 (1991)].
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.
  • one of the binding specificities is for the BOG
  • the other one is for any other antigen, and preferably for a cell- surface protein or receptor or receptor subunit.
  • Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature, 305:537-539 (1983)].
  • Heteroconjugate antibodies are also within the scope of the present invention.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Patent No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089].
  • the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
  • BOG antibodies of the invention have various utilities.
  • BOG antibodies may be used in diagnostic assays for BOG, e.g., detecting its expression in specific cells, tissues, or serum.
  • diagnostic assay techniques known in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987) pp. 147-158].
  • the antibodies used in the diagnostic assays can be labeled with a detectable moiety.
  • the detectable moiety should be capable of producing, either directly or indirectly, a detectable signal.
  • the detectable moiety may be a radioisotope, such as 3 H, 14 C, 32 P, 35 S, or *25 I, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.
  • a radioisotope such as 3 H, 14 C, 32 P, 35 S, or *25 I
  • a fluorescent or chemiluminescent compound such as fluorescein isothiocyanate, rhodamine, or luciferin
  • an enzyme such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.
  • Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al., Nature, 144:945 (1962); David et
  • BOG antibodies also are useful for the affinity purification of BOG from recombinant cell culture or natural sources.
  • the antibodies against BOG are immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well known in the art.
  • the immobilized antibody then is contacted with a sample containing the BOG to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the BOG, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the BOG from the antibody.
  • TGF- ⁇ l induced growth inhibition is an early event during spontaneous transformation of RLE cells.
  • This resistance to the growth inhibitory effects of TGF- ⁇ l can clearly be caused by multiple factors.
  • a 1897 bp cDNA clone was obtained which encoded a protein of 173 amino acids with a predicted Mr ⁇ 19,000 Daltons (Tables 1 , 5 and 7).
  • DNA homology searches of Genba k revealed BOG to be a unique transcript.
  • BOG mRNA was translated in vitro and the product analyzed by polyacrylamide gel electrophoresis, which confirmed the protein size as 19.3 KDa protein.
  • the deduced protein sequence contained a pRb-binding motif LXCXE (Table 1 , double underlined) and two casein kinase II phosphorylation sites (Table 1 , underline). Alignment of the pRb-binding motif in BOG protein with that in HPV16 E7, SV40 large T antigen, adenovirus EIA and Rb-binding protein is shown in Table 2. In addition to the pRb-binding region, BOG and HPV16 E7 exhibited 29.6% identity and 7.1% similarity over the whole sequence BOG (Table 3). Expression of BOG in the rat was analyzed using a multi-tissue northern blot ( Figure 1). Transcript for BOG could be detected in all tissues but varied in expression levels, demonstrating the highest expression in the spleen, testis and kidney. The ubiquitous expression pattern suggests a general rather than cell specific function for BOG.
  • Protocols for the isolation of rat BOG cDNA Poly(A)+ was isolated from RLE- 13 and B5T cells by using oligo (dt) cellulose, as previously described (Bradley JE, Bishop GA, St John T, Frelinger JA (1988): Biotechniques 6:114-116.
  • the cDNAs were synthesized using Superscript reverse transcriptase (BRL) as recommended by the vendor; added with BstXI adaptor (InVitrogen) and used to produce RLE- 13 and B5T cDNA libraries in pcDNAIneo.
  • B5T cDNA enriched subtractive cDNA library construction B5T and RLE- 13 cDNAs were digested with Hindlll+Xbal and BamHI+XhoI, respectively and subjected to agarose gel purification. Digested RLE- 13 cDNA fragments (20 ⁇ g) were digested further with 43 Alul+Rsal and dephosphorylated, then hybridized with Hindlll+Xbal digested B5T cDNA fragments (0.4 ⁇ g). The hybridization mixture was used to construct the B5T cDNA enriched subtractive cDNA library in BluescriptM13 with Hindlll+Xbal protruding termini. The enriched library was plated and single colonies were isolated. The plasmid from each clone/colony was sequenced, and novel sequences were used to screen a panel of rat tumor samples and synchronized cells.
  • the cDNA sequence of rat BOG was determined by double strand sequence analysis of a 1.9kb clone. Sequencing of plasmids was performed using one of two kits; Dye Terminator Ready Reaction Kits-FS (cat# 402080) or BigDye Terminator Ready Reaction Kits (cat# 4303149), Perkin-Elmer Applied Biosystems. The reactions were purified using Centrisep Spin Columns (cat# CS-901), Princeton Separations. The samples were analyzed on a 377 ABI Prism DNA sequencer, Perkin-Elmer Applied Biosystems. The predicted amino acid sequence was derived using PCGENE and indicated below with single letter code.
  • BOG contains the LXCXE pRb-binding motif found in many of the Rb binding proteins.
  • yeast two- hybrid system To test the interaction between BOG and pRb, we employed the yeast two- hybrid system. The appropriate plasmid constructs were sequentially transformed into yeast reporter strains GAL1 promoter activity was analyzed by growth on plates lacking histidine. CYC1 promoter activity also was analyzed by induction of lacZ by staining clones on X-gal containing media ( Figure 2A). As a positive control for both promoters SV40 large T antigen (pTDl) interaction with pRb (pGBT9/Rb) was used. The intensity of X-gal staining for Rb and BOG as compared to pRb and SV40 large T suggests that their degree of interaction is comparable.
  • yeast clones with plasmids containing only Rb, BOG, or both expression vectors without insert served as negative controls, while the interaction of SV40T and Rb, and wild type GAL4 served as positive controls.
  • the yeast Matchmaker Gal4 Two-hybrid System (cat# Kl 605-1), Clontech Laboratories, Inc., contains the vectors pGAD424, pGBT9, pTDl and pCLl.
  • Immunoprecipitates were separated on a 10% SDS-PAGE gel and analyzed by Western blot analysis using anti-Rb, anti-BOG and anti-E2F-l antibodies (Santa Cruz Biotechnology).
  • a chimeric protein was constructed using the Protein and Purification System (New England Biolabs, Inc.).
  • the pMAL-P2 vector used produced a protein in which BOG is fused to the carboxy-terminal of the maltose binding protein (MBP).
  • MBP maltose binding protein
  • the chimeric protein was purified using an amylose resin and tested for its ability to precipitate pRb from whole cell lysates.
  • Protein preparations capable of precipitating pRb were used for displacement experiments.
  • the purified MBP without BOG did not precipitate any proteins from whole cell lysates under similar conditions.
  • Displacement of E2F-1 from pRb/E2F-l complexes by BOG were performed with RLE phi 13 cells treated with TGF- ⁇ l (500 pmol) for 24 hours to maximize pRb/E2F-l complex formation.
  • Cells were harvested and whole cell extracts were immunoprecipitated with anti-Rb and anti-E2F-l antibodies (Santa Cruz Biotechnology). Immunocomplexes were aliquoted into two factions, and to one fraction the chimeric BOG-MBP was added, the second fraction served as a control and received only buffer.
  • the cells transfected with either pcDNA-BOG or control plasmid pcDNA were subjected to neomycin selection, and pools of neomycin-resistant RLE, RLE/BOG, B7 and B7/BOG cells were generated. The four pools were then cultured in the presence of 0.5 ng/ml TGF- ⁇ 1.
  • the presence of pcDNA- BOG plasmid in RLE/BOG and B7/BOG cells significantly decreased the sensitivity of these cells to growth inhibition by TGF- ⁇ l growth ( Figure 3 A).
  • BOG in the tumors may be due in part to the increased mitotic activity in the tumor
  • an increased expression of BOG in all the tumors may reflect the importance of BOG in the selection of the clonal phenotype that maintained a growth advantage during neoplastic development in the liver.
  • Protocols for evaluating constitutive over-expression of BOG in RLE cell lines Colony formation of heterogeneous population of transfected RLE and B7 cells expressing BOG.
  • the cDNA Hindlll-Xbal cDNA fragment from subtractive clone in pBluescriptM13 was subcloned into the mammalian expression vector pCDNAIneo (Invitrogen), which contains the CMV promoter and a neomycin selectable marker.
  • the expression plasmid pCDNAI- BOG and the control vector pCDNACMV were transfected into phi 13 and B7 cells by lipofectin (Gibco BRL). After a 3 weeks selection in Ham's F-12 media containing G418 400 ⁇ g activity/ml (726 ⁇ g/ml) (Sigma), colonies were harvested as mixed colonies and maintained in selective media.
  • 10 5 cells for phi 13CMV phi 13 transfected with the empty vector pCDNAIneo as a control
  • phi 13 BOG phi 13 transfected with expression plasmid pCDNAI-BOG
  • 5x10 4 cells for B7CMV B7 cells with pCDNAIneo
  • B7BOG B7 cells with pCDNAI-BOG
  • the cells were allowed to attach and media containing 0.5ng/ml TGF- ⁇ l (R&D Systems) was added into the dishes 18 hours after plating.
  • the medium containing 0.5 ng/ml TGF- ⁇ l was changed every 3 days for 15 days on all plates.
  • BOG-A and BOG-B cells were plated at 5x10 3 into 96 well plates. 50 After 6 hours when the cells were attached, the media was changed to Ham's F-12 media containing either 0, 100 pg/ml or 1000 pg/ml of TGF- ⁇ l . The cultures were maintained for
  • TGF- ⁇ l on growth is expressed as a percent inhibition of growth of cells grown in the presence of TGF- ⁇ l versus cells grown in normal media for the same period of time.
  • BOG-A and BOG-B cells were plated in 24-well plates and grown to 90% confluency.
  • TGF- ⁇ and anti-E2F-l were both iodinated by the method of Sambrook J, Fritsch EF, Maniatis T (1989): "Molecular
  • Protocols for assessing colony formation in agar and tumor formation in nude mice were assessed for assessing colony formation in agar and tumor formation in nude mice.
  • sensitivity to TGF- ⁇ l could also be restored in the B5T cell line, a transformed cell line that was less sensitive to TGF- ⁇ l , but is known to contain the full compliment of TGF- ⁇ receptors.
  • BOG the overexpression of BOG.
  • sensitivity to TGF- ⁇ may be restored in transformed cells which still maintain signaling through the TGF- ⁇ pathway, through methods that decrease the expression of BOG.
  • overexpression of BOG decreases sensitivity to TGF- ⁇ even in the presence of an intact TGF- ⁇ signaling pathway, BOG may be an early indicator or selection for cells destined to become transformed in vivo.
  • Protocols for the inhibition of BOG by anti-sense oligonucleotides Phosphothionate- modified antisense oligonucleotides corresponding to the 5' end (AATCACTGCCTTGGTAGGGGACACCATTAA) and the 3' end of the one reading frame (TCAGTTCATGGAACTCTGTGTTCTGAAAGTGAC) of BOG were synthesized and purified by OPC column (Bioserve Biotechnology, Inc.). Cells were plated at 50% confluency and allowed to attach in Ham-F12 media containing 10% Fetal Bobine Serum (FBS).
  • FBS Fetal Bobine Serum
  • the cells were washed once with DPBS and media containing antisense oligonucleotides (340 ⁇ g/ml each) and/or TGF- ⁇ l (250 pg/ml) was added to the cells, and the cultures were allowed to grow for 48 hours. Cell growth was assayed using the Cell Titer 96 Non-radioactive Cell Proliferation Assay (CellTiter 96 Non- Radioactive Cell Proliferation Assay (cat# G4000), Promega Corporation).
  • TGF- ⁇ concentration of TGF- ⁇ was determined by art accepted methods. In particular, the dose response of the cells to TGF- ⁇ was previously determined (Hugget, A. C. et. al.). 52 Development of resistance to the growth inhibitory effects of transforming growth factor beta
  • TGF- ⁇ a dose of 0.5 ng/ml TGF- ⁇ was chosen because it was an effective dose for the cytostatic effect in all cell lines analyzed.
  • a chimeric protein (BOG-MBP) was constructed using the Protein Fusion and Purification System (New England Biolabs, Inc.). PCR protocols were utilized to generate a BOG polynucleotide having restriction endonuciease sites that allowed the complete open reading frame to be linked to the maltose binding protein (MBP). See e.g. Current Protocols In Molecular Biology, Volume I, Unit 17, Frederick M. Ausubul et al. eds.. 1995.
  • the pMAL- P2 vector used produced a protein in which the complete rBOG protein is fused to the carboxy-terminal of MBP (pMAL-P2-BOG).
  • the chimeric protein was purified using an amylose resin and tested for its ability to precipitate pRb from whole cell lysates. The DNA sequence encoding an extracellular region of the BOG polypeptide.
  • Polyclonal antibodies to BOG was generated commercially by Genemed Synthesis, Inc. using art accepted methods. Initially, the rabbits were immunized with the fusion protein (BOG- MBP) and subsequent booster shots were done with purified 20kDa BOG. The final serum generated recognized both BOG and MBP. The following example illustrates preparation of monoclonal antibodies which can specifically bind BOG.
  • Immunogens that may be employed include purified BOG, fusion proteins containing BOG, and cells expressing recombinant BOG on the cell surface. Selection of the immunogen can be made by the skilled artisan without undue experimentation. 53 Mice, such as Balb/c, are immunized with the BOG immunogen emulsified in complete
  • the immunogen is emulsified in MPL-TDM adjuvant (Ribi
  • mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice may also be boosted with additional immunization injections. Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect BOG antibodies.
  • the animals "positive" for antibodies can be injected with a final intravenous injection of BOG.
  • the mice Three to four days later, the mice are sacrificed and the spleen cells are harvested.
  • the spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU.l, available from ATCC, No. CRL 1597.
  • the fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
  • HAT hyperxanthine, aminopterin, and thymidine
  • the hybridoma cells will be screened in an ELISA for reactivity against BOG. Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against B
  • the positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the anti-BOG monoclonal antibodies.
  • the hybridoma cells can be grown in tissue culture flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed. 54 EXAMPLE 8
  • DNA comprising the coding sequence of BOG was employed for use as both primers and probes to screen for homologous DNAs (such as those encoding naturally-occurring variants of BOG) in human tissue cDNA libraries, human tissue genomic libraries. Analysis of cDNA libraries from human and mouse indicate that BOG is a highly conserved gene in both these species.
  • the coding region of the human cDNA was isolated from a human liver cDNA library (human liver 5' stretch plus cDNA, Clonetech Laboratories, Inc.) using PCR. See Table 4. Primers were designed to the 5' (ATGGTCTCTCCTAGC) and 3' (GAGTTCCATGAAC) portions of the open reading frame of mouse and rat BOG, and PCR was done using PCR SuperMix (Gibco, BRL Life Technologies) using company recommended conditions. The predicted 500 bp PCR fragment was cloned into the TA vector pCR2.1 (In VitrogenTM), for further analysis (pCR2.1huBOG). Five clones were isolated and double strand sequenced obtained to confirm hBOG sequence.
  • mice cDNA clone was isolated from a mouse kidney cDNA library (mouse kidney 5'- stretch cDNA, Clonetech laboratories, Inc.). See Table 6. Phage were plated at 5x10 5 pfu/150mm plate and duplicate filters were screened using a 500 bp fragment corresponding to the coding region of rBOG. Seven plaques were identified and plaque purified. The insert from these lambda clones were subcloned into the pBluescript vector (StratageneTM) for further analysis (pBS ⁇ mBOG). All clones were double strand sequenced and used to confirm overall sequence of mBOG.
  • pBluescript vector StratageneTM
  • Chromosomal localization was done by FISH analysis (Stokke, T., Collins, C, Kuo, W., Kowbel, D., Shadrvan, F., Tanner, M., Kallionienmi, A., Kallioniemi, O., Pinkel, D., Deaven,
  • TGF- ⁇ l(250pg/ml) was added in the fresh medium.
  • Cells were harvested at the indicated times using 0.1% trypsin solution in versene for flow cytometer analysis
  • cells were incubated with Ham's F12 medium containing 0.2%) defined fetal bovine serum for 72 hours. At time 0 hours, the low serum medium was removed and the cells were stimulated by addition of complete medium containing 10% serum.
  • RNA isolation cells were lysed in 4M guanidine thiocyanate solution.
  • flow cytometer analysis cells were fixed by dehydration in 70% ethanol and stored at 4°C prior to analysis. Cells were then washed once with ice-cold PBS, treated with 500 units of RNase A (Boehringer Mannheim) per ml for 15' at 37°C.
  • Table 1 shows the cDNA (SEQ ID NO: 1) and amino acid (SEQ ID NO: 2) sequences of rat BOG as determined by double strand sequence analysis of a 1.9 kb clone.
  • the potential Rb binding domain is indicated by the underline and the two putative casein kinase II phosphorylation sites by boldface type.
  • AAAGCAGGGT CAGGAGGGAA GGCTATCTTG ACCCTGTCTC AGAAAGAGTG AGCAAGAAGA 946
  • Table 2 shows amino acid sequence homology of the BOG pRb binding domain (SEQ ID NO: 2) and casein kinase II consensus sequence to the Rb binding proteins Human Papilloma Virus E7 (SEQ ID NO: 3), Simian Virus 40 large T antigen (SEQ ID NO: 4), Adenovirus EIA (SEQ ID NO: 5) and RBP-1 (SEQ ID NO: 6) is illustrated by alignment of the Rb binding domain (LXCXE) (bold) and casein kinase II phosphorylation sites (underlined).
  • Table 3 shows the total amino acid sequence homology of rBOG (SEQ ID NO: 2) and E7
  • Table 4 shows the cDNA coding sequence of human BOG (SEQ ID NO: 7).
  • Table 5 shows the amino acid sequence of human BOG protein (SEQ ID NO: 8).
  • Table 6 shows the cDNA coding sequence of murine BOG (SEQ ID NO: 9). ATGGCGTCCC CCAACAAGGC AGTGATTGTT CCTGGGAACG GAGGCGGGGA TGTGGCCACC 060
  • Table 7 shows the amino acid sequence of murine BOG protein (SEQ ID NO: 10).
  • Table 8 shows a comparison of the amino acid sequence of the murine (SEQ ID NO: 10), human (SEQ ID NO: 8) and rat BOG (SEQ ID NO: 2) proteins.

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Abstract

La présente invention concerne des acides nucléiques codant de nouveaux polypeptides, appelés dans cette invention 'BOG' (B5T Over-expressed Gene). Le BOG se lie au pRb et est surexprimé en grand nombre dans des lignées cellulaires épithéliales hépatiques de rat transformées (RLE) résistant à l'effet inhibiteur de croissance du TGF-β1 ainsi qu'aux tumeurs de foie primitives. Cette invention concerne par ailleurs des compositions renfermant des chimères de BOG, des acides nucléiques codant le BOG et des anticorps du BOG. Enfin, cette invention concerne des procédés d'utilisation du BOG pour moduler les interactions pRb/protéine et pour modifier le phénotype cellulaire.
PCT/US1999/004142 1998-02-25 1999-02-25 Adn complementaires codant un gene bog (b5t-over-expressed gene) et son produit proteinique WO1999043811A1 (fr)

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US10/772,988 US7342099B2 (en) 1998-02-25 2004-02-05 cDNA encoding a gene BOG (B5T over-expressed gene) and its protein product
US11/956,680 US20100129791A1 (en) 1998-02-25 2007-12-14 cDNA Encoding a Gene Bog (B5T Over-Expressed Gene) and its Protein Product

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001029228A1 (fr) * 1999-10-18 2001-04-26 Shanghai Bio Road Gene Development Ltd. Nouveau polypeptide, caseine kinase humaine 48, et polynucleotide codant pour ce polypeptide
WO2001066739A1 (fr) * 2000-03-07 2001-09-13 The Government Of The United States Of America As Represented By The Secretary, Department Of Health And Human Services ADNC CODANT UN GENE TRAG (GENE ASSOCIE A LA RESISTANCE CONTRE TGF-β) ET SON PRODUIT PROTEIQUE
WO2003000929A3 (fr) * 2001-06-26 2003-11-27 Sankyo Co Polynucleotides, polypeptides et cancer

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL- EMEST1 11 September 1997 (1997-09-11), MARRA, M. ET AL.: "vf65e09.r1 Barstead MPLRB1 Mus musculus cDNA clone 848680 5'.", XP002106856 *
TAYA Y ET AL: "RB kinases and RB-binding proteins: new points of view", TIBS TRENDS IN BIOCHEMICAL SCIENCES, vol. 22, no. 1, 1 January 1997 (1997-01-01), pages 14-17, XP004016856 *
WOITACH, J.T. ET AL.: "A retinoblastoma-binding protein that affects cell-cycle control and confers transforming ability.", NATURE GENETICS, vol. 19, August 1998 (1998-08-01), pages 371 - 4, XP002106855 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001029228A1 (fr) * 1999-10-18 2001-04-26 Shanghai Bio Road Gene Development Ltd. Nouveau polypeptide, caseine kinase humaine 48, et polynucleotide codant pour ce polypeptide
WO2001066739A1 (fr) * 2000-03-07 2001-09-13 The Government Of The United States Of America As Represented By The Secretary, Department Of Health And Human Services ADNC CODANT UN GENE TRAG (GENE ASSOCIE A LA RESISTANCE CONTRE TGF-β) ET SON PRODUIT PROTEIQUE
WO2003000929A3 (fr) * 2001-06-26 2003-11-27 Sankyo Co Polynucleotides, polypeptides et cancer

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