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WO2015126128A1 - Agent pour la thérapie du cancer - Google Patents

Agent pour la thérapie du cancer Download PDF

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Publication number
WO2015126128A1
WO2015126128A1 PCT/KR2015/001566 KR2015001566W WO2015126128A1 WO 2015126128 A1 WO2015126128 A1 WO 2015126128A1 KR 2015001566 W KR2015001566 W KR 2015001566W WO 2015126128 A1 WO2015126128 A1 WO 2015126128A1
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Prior art keywords
mucl
seq
immunogenic
polynucleotide according
amino acid
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English (en)
Inventor
Jaesung Lee
Je-heon MAENG
Soondong LEE
Kil Jung Kim
Ji Won Chung
Hunsik CHOI
Sujeong Kim
Bumsup Lee
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Kolon Life Science Inc
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Kolon Life Science Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001169—Tumor associated carbohydrates
    • A61K39/00117—Mucins, e.g. MUC-1
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/10041—Use of virus, viral particle or viral elements as a vector

Definitions

  • the present invention relates to an anti-tumor agent comprising modified human MUCl polynucleotide or a functional fragment thereof, or a modified human MUCl polypeptide encoded by the said polynucleotide, and a use of the anti-tumor agent for preventing or treating MUCl over-expressing cancer.
  • MUCl The Mucin- 1 (MUCl) gene was cloned in the early 1990s. It belongs to the mucin family, comprising 21 members. MUC l encodes a highly glycosylated, type I transmembrane glycoprotein, with a variable number of 20-amino-acid repeat sequences referred to as "variable number tandem repeat" (VNTR). The number of VNTR is variable from one allele to another, varying from 25 to 120 VNTR per MUCl molecule, with the alleles containing 40 and 66 VNTR being the most frequent .
  • VNTR variable number tandem repeat
  • MUCl glycoprotein is highly over-expressed and hypo-glycosylated in tumor cells from numerous cancer types. MUCl belongs to the "overexpressed tumor- associated antigen (TAA)" category, even if this over-expression is not the only hallmark of MUCl in tumor cells, since it is often accompanied by modification of the MUCl glycosylation status.
  • TAA tumor-associated antigen
  • MUCl is a glycoprotein expressed at the apical surface of epithelial cells and characterized by a high glycosylation level (Roulois et al., BioMed Research International, 1-10, 2013).
  • TAA tumor-associated antigen
  • MUCl -based immunogens have been evaluated as potential cancer vaccines. These include whole cells expressing MUCl, MUCl purified from tumor cells, and peptide or glycopeptide fragments derived from the tandem repeat region of MUCl (U.S. Pat. Nos. 5,744,144, 5,827,666 WO 88/05054, U.S. Pat. Nos. 4,963,484 and 6,344,203) and plasmid DNA vaccine (Zhang et al., Human Immunol., 69: 250-258, 2008).
  • Vaccines using the MUC 1 VNTR region have been employed for MUC 1 -based immunotherapy in clinical trials for different types of cancers. More than sixty clinical trials interested in MUCl protein are currently in progress (http://www.clinicaltrials.gov/). The majority of these are developing vaccination strategies against MUCl to treat cancer. Only three clinical studies have reached the phase IIB or III stage. Some of these studies have shown a possible clinical efficacy exerted by this vaccine in inducing an MUCl- specific T-cell response although tumor response rate using the vaccine only was not evident.
  • the BLP25 liposome vaccine (stimuvax or L-BLP25) is a liposomal vaccine containing a 25-amino-acid MUCl peptide corresponding to the core peptide of MUCl (STAPPAHGVTSAPDTRPAPGSTAPP).
  • STAPPAHGVTSAPDTRPAPGSTAPP The phase III clinical trial (START) did not meet its primary endpoint (Overall survival) in non-small-cell lung carcinoma (NSCLC).
  • NSCLC non-small-cell lung carcinoma
  • the company MERCK Sereno is in progress to perform a separate clinical trial (INSPIRE).
  • the vaccines using the VNTR region like L-BLP25 has not been successful to extend overall survival in clinical trials.
  • TG4010 is a recombinant virus of the Modified Vaccinia Ankara (MVA), encoding MUCl and IL-2 (MVA MUC1 -IL2).
  • MUCl Modified Vaccinia Ankara
  • IL-2 IL-2
  • a best clinical responses using TG4010 were observed for NSCLC.
  • TG4010 enhanced the effect of chemotherapy, with an increase in six-month progression-free survival for patients who received chemotherapy in combination with the vaccine as compared with patients given chemotherapy.
  • a phase IIB/III trial is currently being performed to evaluate the clinical efficacy by combining chemotherapy plus TG4010 in NSCLC.
  • TG-4010 alone does not seem to extend overall survival.
  • an immunogenic peptide including a modified MUCl polypeptide or a functional fragment thereof, or a modified MUCl polynucleotide encoding the modified MUCl polypeptide.
  • the modified MUCl polynucleotides or polypeptides have increased immunogenicity.
  • an anti-tumor agent which comprises modified MUCl polypeptide or a functional fragment thereof, or a modified MUCl polynucleotide encoding the modified MUCl polypeptide, and a use of the anti-tumor agent for preventing or treating MUCl -expressing cancer or MUC 1 -positive cancer.
  • composition comprising the anti-tumor agent, in which the composition is used for preventing or treating MUCl -expressing cancer or MUCl -positive cancer.
  • a genetic vaccine comprising the immunogenic polynucleotide, wherein said composition elicits a cellular immune response against MUCl antigen in a MUCl -positive cancer cell.
  • kits for preventing or treating MUC1- expressing cancer or MUCl -positive cancer comprising the said agent.
  • a vector including the said agent, or a cell or an organism that is transfected or transduced with the polynucleotide, polypeptide or a pharmaceutical composition.
  • One aspect of the present invention relates to a modified human MUCl polynucleotide or a functional fragment thereof, or a modified human MUCl polypeptide encoded by the said polynucleotide or a functional fragment thereof, and a method of producing the polypeptide or a functional fragment, a polypeptide encoding the protein or a functional fragment.
  • An embodiment of the present invention is to provide relates to an anti-tumor agent comprising a modified human MUCl polynucleotide or a functional fragment thereof, or a modified human MUCl polypeptide encoded by the said polynucleotide.
  • the modified human MUCl polynucleotide or a functional fragment thereof may be isolated or prepared by using a living cell or organism, or may be non-naturally occurring. Also, the modified human MUCl polynucleotide or a functional fragment thereof may be recombinant or synthetic.
  • the present invention relates to a polynucleotide encoding immunogenic MUCl peptide or an immunogenic fragment thereof which includes a substitution of at least a tyrosine in C-terminal domain of MUCl peptide.
  • the modified polynucleotide can be a single stranded or a double stranded with a complementary polynucleotide thereto, or preferably double stranded.
  • the immunogenic polynucleotide can be the immunogenic fragment which encodes the peptide fragment with a size length of less than 300 amino acids in length., for example 250 amino acids, or 74 to 222 amino acids.
  • the modified polypeptide comprises 74 amino acids modified polypeptide which at least a tyrosine in the cytoplasmic tail domain shown in SEQ ID NO: 3 is substituted, and 1 to 148 amino acids consisting of at least a consecutive amino acid selected from 1034 to 1 181 amino acids in the amino acid sequence of SEQ ID NO: 1.
  • the immunogenic polynucleotide, immunogenic polypeptide, or DNA vaccine has reduce tumor size and/or to extend overall survival, thereby causing the prevention and/or the treatment the MUC1- expressing cancer.
  • Most of animal studies shows an efficacy in a therapeutic models where tumor cells were injected prior to cancer vaccine administration have been failed. Therefore, recent studies in therapeutic cancer vaccine field showed cytotoxic T- lymphocytes (CTL) reaction assay and prophylactic efficacy experiments to show its efficacy. It has been recently known that the essential role of cancer vaccines is not to reduce tumor size but to extend overall survival and improve quality of life.
  • CTL cytotoxic T- lymphocytes
  • FIG. 1 shows a schematic structure of a human full-length MUC1 and a detailed sequence modification in a cytoplasmic tail domain of MUC1.
  • FIG. 2 shows the map of plasmid encoding modified MUC1 peptide (pVAXl- MUC 1) obtained in Example 1.
  • FIG. 3 shows the map of adenoviral vector construct expressing MUC1 named as Ad-MUCl obtained in Example 2.
  • FIG. 4 shows the map of retroviral vector construct expressing MUC1 named as pMSCV-puro-MUCl obtained in Example 4.
  • FIG. 5 shows protein expression and secretion of MUCl-wt and mutants by Western blot analysis.
  • FIG. 6 demonstrates the anti-tumor effect of recombinant plasmids containing MUCl-wt and mutants of MUC l-2mA, MUCl-2mB, MUCl-2mA, MUCl-3mB and MUCl-4m.
  • FIG. 7 demonstrates the anti-tumor effect of recombinant plasmids containing
  • MUC 1 mutants of MUC 1-S 1 , MUC 1 -S2, MUC 1-S3, MUC1-S4 and MUCl-2mA are shown in FIG. 1 .
  • FIG. 8 demonstrates the anti-tumor effect of adenoviral vector containing MUCl mutant MUC1-S2, compared with Ad-GFP.
  • Fig. 9 shows the number of the counted spots evaluated as IFN- ⁇ secreting cell number where the splenocytes were treated with MUCl epitope no. 1 - 5 respectively.
  • MUCl is a Type I membrane glycoprotein of the mucin family having an extensive extracellular domain consisting of hundreds of tandem repeat units, a single transmembrane domain and a C-terminal cytoplasmic tail.
  • the human MUCl has various isoforms.
  • the isoform 1 of human MUCl can include an amino acid sequence of Uniprot accession number PI 5941.
  • the human full amino acid sequence of wild type MUCl is represented in SEQ ID NO: l and the corresponding nucleotide sequence is represented in SEQ ID NO:2.
  • the position number of modified Tyrosine residue is represented in the SEQ ID NO: l which is the human wild type isoform 1 of MUCl .
  • at least one Tyrosine residue in C-terminal domain of MUCl can be modified according to the present invention, but the position of Tyrosine can be changed depending on the isoform of MUC 1.
  • the gene MUC l is composed of seven exons that together comprise 4,2 to 7,0 kb of genomic DNA.
  • the alternative splicing of mRNA gives rise to transcripts of 3.7 to 6.4 kb coding for the different isoforms of the MUCl protein, transmembrane, secretory, and MUCl X/Y/Z isoforms.
  • MUCl includes any kind of isoform, but for example, the position number of amino acid or nucleic acid disclosed herein is based on the MUCl /REP or MUCl/TM.
  • the schematic structure of a human full-length MUCl is shown in Fig. 1 for example.
  • the full length MUCl protein consists of 1255 amino acids of SEQ ID NO: l which includes five major domains of signal peptide (a.a. 1-23), extracellular domain (a.a 23-1 158), SEA domain (a.a. 1034-1 151), transmembrane domain (1 159-1 181), and cytoplasmic tail domain (a.a. 1 182-1255) (www.uniprot.or/uniprot/P 15941).
  • the immunogenic fragment of MUCl comprises the C-terminal domain, and for example, corresponds to the fragment comprising a consecutive sequence of position 1 182 to position 1255 represented by an amino acid sequence of SEQ ID NO: 1.
  • the full nucleotide sequence of wild type MUC 1 is represented in SEQ ID NO:2.
  • the immunogenic polynucleotide can be a nucleotide sequence encoding the fragment of MUC 1 including the C-terminal domain and may further includes at least an additional domain selected from the group consisting of a transmembrane domain (TM), and SEA domain of MUCl .
  • the additional domain can includes 1 to 148 amino acids consisting of at least a consecutive amino acid selected from 1034 to 1181 amino acids in the amino acid sequence of SEQ ID NO: 1.
  • the C-terminal domain or cytoplasmic tail domain is represented in an amino acid sequence of SEQ ID NO: 3 and the corresponding nucleotide sequence is represented in the SEQ ID NO:4.
  • the MUCl fragment including the C-terminal domain and a transmembrane domain is represented by an amino acid sequence of SEQ ID NO: 5 and the corresponding nucleotide sequence is represented by SEQ ID NO: 6.
  • the amino acid sequence of SEQ ID NO: 7 are the amino acid sequence of SEQ ID NO: 6 and the signal peptides of 26 amino acids in the N-terminus, and the corresponding nucleotide sequence is represented in the SEQ ID NO: 8.
  • the immunogenic polynucleotide can encode the peptide fragment including C- terminal domain and a transmembrane domain which comprises a consecutive sequence of position 1 159 to position 1255 represented by an amino acid sequence of SEQ ID NO: l .
  • the nucleotide sequence of said polynucleotide is represented in SEQ ID NO: 2.
  • the immunogenic polynucleotide of the present invention includes at least a substituted amino acids of which is located at least one tyrosine selected from the group consisting of positions 10, 22, 28, 31 , 37, 48 and 62 of SEQ ID NO: 3, or preferably 31 , 37, 48 and 62 positions of SEQ ID NO: 3.
  • the positions of 10, 22, 28,31 , 37, 48 and 62 of SEQ ID NO: 3 correspond to the positions of 1 191, 1203, 1212, 1209, 1218, 1229, and 1243 of SEQ ID NO: 1 respectively.
  • the immunogenic polynucleotide of the present invention includes at least a substituted amino acids of which is located at least one position selected from the group consisting of positions 1 191 , 1203, 1212, 1209, 1218, 1229, and 1243 of SEQ ID NO: 1 , preferably tyrosines at a least one position selected from the group consisting of positions 1203, 1218, 1229, and 1243 of SEQ ID NO: 1.
  • At least tyrosine reside in the C-terminal region of MUC 1 can be substituted independently by at least one amino acid which can be selected from the group consisting of phenylalanine, alanine, arginine, and aspartic acid.
  • the modified polynucleotide derived from MUCl can be prepared by site-directed mutagenesis using specific PCR primers shown in Table 1 where the primer sequences are described. A list of the primers used in each construct is shown in Table 2.
  • nucleotide sequences encoding the polypeptides according to the present invention.
  • Nucleotide sequences may be in the form of DNA, RNA or mixtures thereof.
  • Nucleotide sequences or isolated nucleic acids may be inserted into DNA, RNA or DNA/RNA vectors as are well known in the art, such as plasmids, viral vectors, and the like.
  • the nucleotide sequences of modified MUCl fragment further include Kozak sequence (GCCGCCACC) as shown in SEQ ID NO: 33 and further modified nucleotides for the codon optimization.
  • the modified polypeptide can include signal peptide such as TPA sequence.
  • the TPA sequence can be a signal peptide shown in SEQ ID NO: 7. in another aspect, in the amino acid sequence of SEQ ID NO: 7, first methionine and 27th methionine are introduced to improve the efficient expression of the MUCl fragment.
  • the amino acid sequence corresponding to the codon-optimized nucleotide sequence of SEQ ID NO: 8 is represented in SEQ ID NO: 7.
  • an isolated nucleic acid molecule that encodes a polypeptide and can be expressed in a eukaryotic cell, and said molecule comprises:
  • a second nucleic acid sequence that is linked in frame to the first nucleic acid and is operably linked thereto, a promoter active in said eukaryotic cell and, optionally, one or more regulatory elements that enhance expression of said nucleic acid in said cell, and wherein the first polypeptide can be immunogenic polynucleotide.
  • nucleic acid molecules can be included in a part of a vector.
  • a vector is a replicating polynucleotide, such as a plasmid, phage, or cosmid, to which another polynucleotide may be attached so as to bring about the replication of the attached polynucleotide.
  • Construction of vectors containing a polynucleotide of the invention employs standard ligation techniques known in the art.
  • a vector can provide for further cloning (amplification of the polynucleotide), i.e., a cloning vector, or for expression of the polynucleotide, i.e., an expression vector.
  • vector includes, but is not limited to, plasmid vectors, viral vectors, cosmid vectors, transposon vectors, and artificial chromosome vectors.
  • viral vectors include, for instance, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, herpes virus vectors, polio virus, alphavirus, baculovirus, and Sindbis virus.
  • the vector can be a plasmid such as pVAXl , and viral vectors such as adenovirus, vaccinia virus, retrovirus, lentivirus, adeno-associated virus (AAV), herpes simplex virus and the like.
  • AAV adeno-associated virus
  • Nucleotide sequences encoding a polypeptide of the present invention may include promoters, enhancers and other regulatory sequences necessary for expression, transcription and translation. Vectors encoding such sequences may include restriction enzyme sites for the insertion of additional genes and/or selection markers, as well as elements necessary for propagation and maintenance of vectors within cells.
  • vector as used herein is defined as a nucleic acid vehicle for the delivery of a nucleic acid of interest into a cell.
  • the vector may be a linear molecule or a circular molecule.
  • a cell or organism that is transfected or transduced with the polynucleotide, polypeptide or a pharmaceutical composition.
  • the polynucleotide and the polypeptide can be an immunogenic polypeptide or peptide according to the present invention.
  • modified MUCl protein or a functional fragment thereof for immunotherapy of MUCl over-expressing cancer and a method of producing the protein or a functional fragment.
  • the modified MUCl protein has the increased immunogenicity and reduced carcinogenecity.
  • an immunogenic peptide comprising a MUCl peptide or an immunogenic fragment thereof which comprises a substitution of at least a tyrosine in C-terminal domain of MUCl peptide or a fragment thereof is substituted.
  • the immunogenic peptide can be the peptide fragment with a size length of less than 300 amino acids in length, or less than 70 amino acids.
  • the immunogenic fragment of MUCl comprises the C-terminal domain, and for example, corresponds to the fragment comprising a consecutive sequence of position 1 183 to position 1255 represented by an amino acid sequence of SEQ ID NO: 1.
  • the immunogenic peptide having the C-terminal domain as represented by an amino acid sequence of SEQ ID NO:3 and the corresponding nucleotide sequence is represented in SEQ ID NO: 4.
  • the immunogenic peptide can be the fragment of MUCl including the C- terminal domain and a transmembrane domain, or the fragment of MUC 1 including the C-terminal domain, a transmembrane domain and a SEA domain.
  • the immunogenic peptide includes C-terminal domain and a transmembrane domain which comprises a consecutive amino acid sequence of position 1 159 to position 1255 represented by an amino acid sequence of SEQ ID NO: 1.
  • the immunogenic polynucleotide having a nucleotide sequence encoding the fragment of MUCl comprises the C-terminal domain and a transmembrane domain as set forth in SEQ ID NO: 5 and the corresponding nucleotide sequence is represented in SEQ ID NO: 6.
  • the immunogenic peptide of the present invention includes at least a substituted tyrosine which locates at a least one position selected from the group consisting of position 1 191 , 1203, 1209, 1212, 1218, 1229, and 1243 of SEQ ID NO: 1, or preferably at a least one position selected from the group consisting of position 1203, 1218, 1229, and 1243 of SEQ ID NO: 1.
  • the examples of the immunogenic peptide can be shown in Table 1.
  • An embodiment of the present invention relates to an immunogenic agent comprising a modified MUCl protein or a functional fragment thereof, or a polynucleotide encoding the same, and a use of the immunogenic agent for immunotherapy of cancer.
  • Another aspect of the present invention is a method for preventing or treating cancer in a subject.
  • the method includes administering to a subject an effective amount of a MUCl cytoplasmic tail peptide of SEQ ID NO: 3 or portion thereof so that cancer is prevented or treated in the subject.
  • the immunogenic agent comprises a modified MUCl protein or a functional fragment thereof, or a polynucleotide encoding the same.
  • Still aspect of the present invention is a method of eliciting an anti-tumor activity, and immune response in the subject in need by administering the immunogenic agent of the present invention.
  • An embodiment of the present invention provides a method of prevention and/or treating a cancer in a subject, the method including immunizing the subject in need with an immunogenic agent including at least one substitution of amino acid in MUCl protein or an immunogenic fragment thereof.
  • the present invention includes a method of reducing the tumor burden in a subject, the method including immunizing the subject with an immunogenic peptide including at least one substitution of amino acid in MUCl protein or an immunogenic fragment thereof.
  • the present invention includes a method of preventing tumor recurrence in a subject, the method including immunizing the subject with an immunogenic peptide including at least one substitution of amino acid in MUCl protein or an immunogenic fragment thereof.
  • the present invention includes a method of preventing cancer in a subject, the method including immunizing the subject with an immunogenic peptide including at least one substitution of amino acid in MUCl protein or an immunogenic fragment thereof.
  • An embodiment of the present invention is to provide a method of inhibiting the proliferation of a MUC 1 -expressing cancer cell comprising contacting said MUC1- expressing cancer cell with an effective amount of a MUC 1.
  • a further embodiment of the present invention is to provide a method of killing a MUCl -expressing cancer cell comprising contacting said MUCl -expressing cancer cell with an effective amount of a MUCl .
  • a still embodiment is to provide a method of treating a cancer in a patient comprising administering an effective amount of MUCl .
  • the cancer may relate to overexpression and/or abnormal activation of MUCl .
  • the cancer cell expresses an elevated amount of MUCl or the modified MUCl .
  • MUCl is normally expressed at the apical border of healthy epithelia in the respiratory, reproductive and gastrointestinal tracts. In sharp contrast to the healthy pattern of expression that restricts MUCl to luminal surface, the cancerous tissues display an aberrant expression pattern where MUCl is uniformly distributed over the entire tissue surface.
  • the cancer may be a solid cancer or a blood cancer.
  • the cancer may be, but not limited to, one or more selected from the group consisting of a lung cancer cell, a brain cancer cell, a head & neck cancer cell, a breast cancer cell, a skin cancer cell, a liver cancer cell, a pancreatic cancer cell, a stomach cancer cell, a colon cancer cell, a rectal cancer cell, a uterine cancer cell, a cervical cancer cell, an ovarian cancer cell, a testicular cancer cell, a skin cancer cell or a esophageal cancer cell.
  • the cancer may be a primary cancer or a metastatic cancer.
  • prevention and/or treatment of cancer may be used to refer not only to inhibition of cancer cell proliferation and/or cancer cell death, but also to inhibition of metastasis and/or invasion of cancer.
  • the cancer or tumor is an ovary cancer, colon cancer, rectal cancer, non-small lung carcinoma (NSCLC), renal cell carcinoma (RCC), or pancreatic cancer.
  • the cancer or tumor expresses the MUCl protein sequence including a C-terminal domain of the MUCl protein and including tyrosine residues substituted with another amino acid.
  • a "subject” or a “patient,”as used herein, refers to any mammal or preferably human that may be susceptible to tumorigenesis or carcinogenesis associated with the aberrant expression of MUCl .
  • subject or patient include a human, a non- human primate, a cow, a horse, a pig, a sheep, a goat, a dog, a cat or a rodent such as a mouse, a rat, a hamster, or a guinea pig.
  • the invention is directed toward use with humans.
  • the present invention generally involves a composition related to cancers and methods of treatment of cancers characterized by the aberrant expression of a class of cell surface receptors characterized by interchain binding regions.
  • One such set of cancers are those cancers characterized by the aberrant expression of MUCl .
  • Much of the description of the invention herein involves cells that aberrantly express MUCl . It is to be understood that in these instances the description is to be considered example, and that the principles of the invention apply to other cell surface receptors that function by a similar mechanism. With the disclosure herein, . those of ordinary skill in the art will readily be able to identify other cell surface receptors that function by this or a similar mechanism, and to apply the invention to those cancers characterized by aberrant expression of those receptors.
  • compositions or agents as described herein. That is, one aspect of the invention involves a series of compositions or agents useful for treatment of cancer or tumor characterized by the aberrant expression of MUC1. These compositions may also be packaged in kits, optionally including instructions for use of the composition for the treatment of such conditions. These and other embodiments of the invention may also involve promotion of the treatment of cancer or tumor according to any of the techniques and compositions and combinations of compositions described herein.
  • An effective amount is generally an amount sufficient to inhibit MUC1-associated cancer within the subject.
  • a pharmaceutical composition according to the invention may be administered by any conventional route, especially the intramuscular route, for example in the form of an injectable solution or suspension.
  • Intramuscular administration elicited a more Thl -biased immune response (Feltquate et al., J. Immunol., 158: 2278-2284, 1997).
  • a composition according to the invention may be administered according to the modes conventionally implemented for already known vaccines, for example in a single dose or dose repeated one or several times after a certain lapse of time.
  • a composition according to the invention may be administered frequently for a sufficient period for the treatment to be effective.
  • Such a composition may advantageously be injected intratumorally.
  • a pharmaceutical composition according to the invention may be prepared according to conventional techniques.
  • the present invention relates to methods of cancer therapy which involve the peptides or polynucleotides according to the present invention.
  • vaccines which include peptides according to the invention can be administered to cancer patients to induce immunity
  • retrovirus as used herein is defined as an RNA virus of the retroviridae family, which includes the subfamilies Oncovirinae, Lentivirinae and Spumavirinae.
  • DNA sequences encoding therapeutic agents which may be contained in the vector include, but are not limited to, DNA sequences encoding the immunogenic.
  • the DNA sequence may further include a leader sequence or portion thereof, a secretory signal or portion thereof and/or may further include a trailer sequence or portion thereof.
  • Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the Rous Sarcoma Virus (RSV) promoter; inducible promoters, such as the MMR promoter, the metallothionein promoter; heat shock promoters; the albumin promoter, and the ApoAI promoter.
  • CMV cytomegalovirus
  • RSV Rous Sarcoma Virus
  • inducible promoters such as the MMR promoter, the metallothionein promoter
  • heat shock promoters such as the albumin promoter, and the ApoAI promoter.
  • expression vector or "expression cassette” as used herein refers to a nucleotide sequence which is capable of affecting expression of a protein coding sequence in a host compatible with such sequences.
  • Expression cassettes include at least a promoter operably linked with the polypeptide coding sequence; and, optionally, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression may also be included, e.g., enhancers.
  • operably linked means that the coding sequence is linked to a regulatory sequence in a manner that allows expression of the coding sequence. Known regulatory sequences are selected to direct expression of the desired protein in an appropriate host cell.
  • regulatory sequence includes promoters, enhancers and other expression control elements. Such regulatory sequences are described in, for example, Goeddel, Gene Expression Technology. Methods in Enzymology, vol. 185, Academic Press, San Diego, Calif. ( 1990)).
  • DNA vaccines have emerged as an attractive approach for antigen-specific cancer immunotherapy.
  • DNA vaccines offer many advantages over more conventional vaccines, such as peptide or attenuated live pathogens.
  • One advantage is that DNA vaccines are reasonably stable and can be easily prepared and harvested in large quantities.
  • Intramuscular immunization with a DNA vaccine encoding an immunogenic agent comprising a modified MUC1 protein or a functional fragment thereof, or a polynucleotide encoding the same and a use of the immunogenic agent for immunotherapy of MUC1 over-expressed cancer.
  • the isolated or recombinant nucleic acid molecule is operatively linked to a promoter, such as, e.g., a constitutive, an inducible or a tissue- specific promoter.
  • a promoter such as, e.g., a constitutive, an inducible or a tissue- specific promoter.
  • the promoter can be expressed in any cell, including cells of the immune system, including, e.g., antigen presenting cells (APCs), e.g., in a constitutive, an inducible or a tissue-specific manner.
  • APCs antigen presenting cells
  • antigen or "immunogen” as used herein refers to a compound or composition comprising a peptide, polypeptide or protein which is “antigenic” or “immunogenic” when administered (or expressed in vivo by an administered nucleic acid, e.g., a DNA vaccine) in an appropriate amount (an “immunogenically effective amount"), i.e., capable of inducing, eliciting, augmenting or boosting a cellular and/or humoral immune response either alone or in combination or linked or fused to another substance (which can be administered at once or over several intervals).
  • An immunogenic composition can comprise an antigenic peptide of at least about 5 amino acids, a peptide of 10 amino acids in length, a polypeptide fragment of 15 amino acids in length, 20 amino acids in length or longer. Smaller immunogens may require presence of a "carrier" polypeptide e.g., as a fusion protein, aggregate, conjugate or mixture, preferably linked (chemically or otherwise) to the immunogen.
  • the immunogen can be recombinantly expressed from a vaccine vector, which can be naked DNA comprising the immunogen's coding sequence operably linked to a promoter, e.g., an expression cassette as described herein.
  • the immunogen includes one or more antigenic determinants or epitopes which may vary in size from about 3 to about 15 amino acids.
  • epitope refers to. an antigenic determinant or antigenic site that interacts with an antibody or a T cell receptor (TCR), e.g., the MHC class I-binding peptide compositions (or expressed products of the nucleic acid compositions of the invention) used in the methods of the invention.
  • TCR T cell receptor
  • An "antigen” is a molecule or chemical structure that either induces an immune response or is specifically recognized or bound by the product or mediator of an immune response, such as an antibody or a TCR.
  • TCRs bind to peptide epitopes which are physically associated with a third molecule, a major histocompatibility complex (MHC) class I or class II protein.
  • MHC major histocompatibility complex
  • Example 1 Preparation of mutated polynucleotide of MUClCloning MUC1 into pVAXl
  • Codon-optimized DNA encoding MUC1 transmembrane (TM) and C-terminal (CT) domain which corresponds to the fragment of an amino acids 1 159 to 1255 in SEQ ID NO: l was synthesized at Bioneer in South Korea and cloned into the Hindlll/EcoRI site of pVAXl vector.
  • the codon-optimized nucleotide sequence of MUC1 fragment including the signal peptide of tissue plasminogen activator (TP A, 23 amino acids), TM (23 amino acids) and CT domain (74 amino acids), and one methionine is represented in SEQ ID NO: 8
  • the amino acid sequence corresponding to the nucleotide sequence (SEQ ID NO: 8) is represented in SEQ ID NO:7.
  • 24th methionine are introduced to improve the efficient expression of the MUC1 fragment
  • the fragment of 1 st to 23th amino acids is a TPA signal sequences
  • the fragment of 25th amino acid to 48th amino acid is TM domain
  • the fragment 49th amino acid to 121th amino acid is CT domain.
  • Said construct pVAXl-MUCl (WT) was served as a template vector for site-directed mutagenesis.
  • the C-terminal region of the MUC1 fragment was modified by substitution of at least one amino acid sequence, producing thirteen kinds of mutated polynucleotides derived from MUC1.
  • the list of mutated MUC1 fragments is summarized in Table 1.
  • the primers used in the site-directed mutagenesis are listed in Table 2, and the combinations of primer pairs are described in Table 3.
  • the mutated MUCl fragment including two substitutions named as 2m- A and 2m-B, two kinds of three amino acids-substituted DNA constructs named as 3m-A and 3m-B, and a four amino acid substituted gene construct were obtained and shown in Table 1.
  • the modified MUCl SI to S4 were obtained by using oligonucleotide primers for 1 st mutagenesis (SEQ ID NOS: 19-20) as listed in Table 3.
  • the modified MUCl 2m- A was obtained by using oligonucleotide primers for 1st mutagenesis(SEQ ID NOS: 19-20) and oligonucleotide primers for 2nd mutagenesis(SEQ ID NOS: 21-22).
  • the modified MUCl 2m-B was obtained by using oligonucleotide primers for 1 st mutagenesis(SEQ ID NOS: 25-26) and oligonucleotide primers for 2nd mutagenesis(SEQ ID NOS: 21-22).
  • the modified MUCl 3m- A was obtained by using oligonucleotide primers for 1st mutagenesis(SEQ ID NOS: 19-20), oligonucleotide primers for 2nd mutagenesis (SEQ ID NOS: 21-22), and oligonucleotide primers for 3rd mutagenesis(SEQ ID NOS: 23-24).
  • the modified MUCl 3m-B was obtained by using oligonucleotide primers for 1 st mutagenesis(SEQ ID NOS: 19-20), oligonucleotide primers for 2nd mutagenesis (SEQ ID NOS: 21-22), and oligonucleotide primers for 3rd mutagenesis(SEQ ID NOS:
  • the modified MUCl 4m was obtained by using four sets of primers which include oligonucleotide primers for 1st mutagenesis(SEQ ID NOS: 19-20), oligonucleotide primers for 2nd mutagenesis (SEQ ID NOS: 21-22), oligonucleotide primers for 3rd mutagenesis(SEQ ID NOS: 23-24), and oligonucleotide primers for 4th mutagenesis(SEQ ID NOS: 25-26).
  • modified polynucleotides derived from MUCl were obtained and sequences were confirmed.
  • the amino acid sequences of S 1 to S4 and 2m modified MUCl fragments are shown in SEQ ID NOs: 9, 1 1 , 13, 15 and 17, and the nucleotide sequences are shown in SEQ ID NOS: 10, 12, 13, 14, 16 and 18.
  • the amino acid sequences and the nucleotide sequences of 2m-B, 3m-A, 3m-B, and 4m are not given, but these sequences can be easily reasoned from Tables 1 and 3.
  • the nucleotide sequences of SEQ ID NOS: 10, 12, 13, 14, 16 and 18 can include further the Kozak sequence (GCCGCCACC).
  • the construct pVAXl-MUCl including mutants of MUCl was shown in Fig. 2.
  • Recombinant adenoviruses were constructed by using AdEasy XL adenoviral system (Agilent) according to the manufacturer's instructions.
  • Mutated MUCl polynucleotides (S2, 2m- A, or 2m-B) were obtained by digesting with using restriction enzymes of HindEI and EcoRV and cloned into the
  • BJ5183-AD-1 competent cells were thawed on ice.
  • 1 ⁇ g of the linearized shuttle vector obtained in 2.1 was added and mixed by tapping the tube gently, and the tube was kept on ice for 20 min.
  • the resulting product was heat-shocked by incubating at 42°C for 90 sec, and then 1 ml of LB broth was added immediately.
  • the tube was incubated at 37°C for 1 hr by shaking at 150 rpm.
  • the entire volume of recovered cells was plated on LB-kanamycin agar plate, and incubated at 37 °C overnight.
  • Mercaptoethanol was added to 100 ⁇ of cells. Then, 50 ng of the Ad-MUCl plasmid was added to the cells and mixed by tapping the tube gently. The cells were kept on ice for 20 min, and heat-shocked by incubating at 42°C for 90 sec. One mililiter of sterile LB broth was added to the cells, and the tube was incubated at 37°C for 1 hr by shaking at 150 rpm. The entire volume of recovered cells was plated on LB-kanamycin agar plate, and incubated at 37°C overnight.
  • TransIT-293 reagent was prepared according to the manufacturer's instruction. Briefly, it was heated to room temperature and was vortexed gently before using. Two hundred and fifty microliter of serum free DMEM was placed in a sterile tube and 2.5 ⁇ g of Ad- MUCl plasmid treated with Pad was added gently to serum free DMEM. Seven point five microliter of TransIT-293 reagent was added to the diluted DNA mixture in tube, mixed and incubated at room temperature for 15-30 min. The TransIT-293 reagen DNA complexes were added to 293 cells, and the culture was incubated until cytopathic effect (CPE) was shown. 2.6. Harvesting recombinant adenovirus containing mutated MUCl
  • the cells and culture medium were harvested and lysed by 3 rounds of freeze-and-thaw method. Then, the tube was centrifuged, and the supernatant was harvested and stored. This supernatant contained the Ad-MUCl .
  • Ad-MUCl was amplified and purified. Thus, the adenoviral vectors containing the mutated MUCl of Ad-MUCl -S2, Ad-MUCl - 2m- A, and Ad-MUCl - 2m-B were obtained.
  • MUC1-S2, pVAXl-MUCl-S3, pVAXl-MUCl-S4, pVAXl-MUCl-2m-A, pVAXl - MUCl -2m-B, pVAXl-MUCl-3m-A, pVAXl-MUCl-3-B and pVAXl-MUCl-4m were transfected into 293T cells.
  • 293T cells were grown to approximately 70% confluence in DMEM supplemented with 10% FBS in 60 mm culture dishes.
  • Reverse transcription reaction mixtures consisted of 50 ⁇ oligo dT, 10 mM dNTP, 5 ⁇ g RNA. Mixtures were incubated at 65 ° C for 5 min and chilled in ice at least 1 min.
  • Second reaction mixtures consisted of the first reaction mixtures, 2 ⁇ of 10X RT buffer, 4 ⁇ of 25 mM MgC12, 2 ⁇ of 0.1 M DTT, 1 ⁇ of RNaseOUT and 1 ⁇ of SuperScriptrH (200U/ml).
  • the reaction mixtures were incubated at 50 ° C for 50 min and heated to 85 ° C for 5 min, and then chilled in ice. After cooling, 1 ⁇ of RNaseH was added to mixtures and incubated at 37 ° C and then stored at 4 ° C until analysis.
  • PCR reaction mixture contained 4 ⁇ of 5X Phusion HF buffer, 0.4 ⁇ of 10 mM dNTP, 0.5 ⁇ sense and antisense primers for target genes, and 0.02 ⁇ Phusion DNA polymerase in a total volume of 20 ⁇ .
  • PCR cycling was as follows: 98°C 30 sec, 25 cycles of 98°C 30 sec, 54°C 30 sec, 72°C 15 sec.
  • PCR products were analyzed in 1.5% agarose gel and visualized with LAS4000 (Fuji Film). The result was shown in Fig. 5C.
  • the sequences of primers and sizes of PCR products are listed in Table 4.
  • PCR reaction mixture contained 2 ⁇ of 10X Ex buffer, 1.6 ⁇ of 2.5 mM dNTP, 0.5 ⁇ sense and antisense primers for target genes, and 0.2 ⁇ of Ex taq polymerase in a total volume of 20 ⁇ .
  • PCR cycling was as follows: 98°C 5 min., 35 cycles of 98°C 10 sec, 55°C 30 sec, 72°C 1 min.
  • the sequences of primers are listed in Table 5 and are presented As SEQ Id NO: 31 and 321 in the attached Sequence Listing.
  • PCR product was cloned into the Xhol and EcoRI restriction enzymes sites of pMSCVpuro vector (Clontech) to generate pMSCV-puro-MUCl (see Fig.4).
  • pMSCV-puro-MUCl see Fig.4
  • MUC1 retroviruses were produced by using three (3) plasmid transfection method.
  • 2x105 cells of 293T were plated onto a 6-well plate.
  • 250 ⁇ of serum-free media, 1.5 ⁇ g of pGP vector, 1.5 g of pE-ampho vector, 2.5 ⁇ g of pMSCV-puro-MUCl were mixed with 7.5 ⁇ of TransIT-293 reagent and incubated at RT for 30 min. This mixture was added to the cells and the cells were incubated at 37°C for 2 days in 5% C02. After 2 days, culture supernatant containing retroviral vector was harvested and filtered through 0.45 ⁇ syringe filter.
  • LLC Lewis lung carcinoma
  • CT26.WT cells were transduced with the MUCl retroviral vector. Transduction conditions were as follows:
  • 1x106 cells were seeded into 10 cm plate. Two mililiter of MUCl retroviral vector was added to the cells in the presence of polybrene (8 ⁇ g/mL) and cultured for 2 days. Then, stably transduced cells were selected by adding puromycin (2 ⁇ g/mL for LLCl-MUCl, 10 ⁇ for CT26.WT-MUC1) to the culture medium for 2 weeks.
  • mutant MUCls The anti-tumor effect of mutant MUCls was compared with wild-type MUCl in C57BL/6 mice.
  • Five microgram of pVAXl plasmids containing MUC l wt or mutant genes (prepared in Example 1) were injected intramuscularly into C57BL/6 mice (6 week old) using the BTX electroporator four times in one week-interval.
  • the blank pVAXl plasmid was used as a control.
  • 1x105 cells of Lewis lung carcinoma over- expressing MUCl (LLCl -MUCl) was injected subcutaneously after 3rd injection of plasmid. Tumor size was measured using Vernier calipers after LLCl-MUCl administration. Tumor volume was calculated using below formula.
  • Tumor volume length x width x width/2
  • the MUC1 -S2 has more effective anti-tumor activity among single mutants (SI , S2, S3, S4), and 2m-A has more effective anti-tumor activity than other multiple mutants (3m-A, 3m-B, 4m).
  • Ad-MUCl -S2 1x1010 viral particles of Ad-MUCl -S2 were subcutaneously injected to the right flank of mice twice in one-week interval.
  • the recombinant adenovirus containing GFP gene Ad-GFP
  • CT26.WT-MUC1 carcinoma 1x105 cells of CT26.WT-MUC1 carcinoma was injected subcutaneously to the left flank.
  • Mouse colonic carcinomas designated CT 26 is one of the most extensively used syngeneic mouse tumor models. Tumor size was measured by Vernier calipers. Tumor volume was calculated as described above. The test groups and the test results were shown in Table 8 and Fig 8, respectively.
  • Ad-MUCl -S2 has anti-tumor effects of inhibiting CT26.WT-MUC1 cell's growth compared with Ad-GFP, as shown in Fig. 8.
  • FIG. 8 demonstrates the anti -tumor effect of adenoviral vector containing MUCl mutants of Ad-MUCl-S2, compared with Ad-GFP.
  • Ad MUC l S2 contributed to a 3-fold decrease in the magnitude of tumor growth compared with Ad-GFP. This finding proves that MUCl S2 is potential antigen to gene therapy because the application of the Ad MUCl S2 tumor-specific gene therapy enhances antitumor effects.
  • mice Six week-old C57BL/6 mice were immunized intramuscularly with plasmid DNAs of pVAXl, pVAXl-MUCl-wt, and pVAXl-MUCl-2mA obtained in Example 1 , by using the BTX electroporator four times in one-week interval. After two months from last immunization, ELISPOT assay was performed to verify MUC1 -specific T cell immune response. ELISPOT measures the frequency of T cells releasing IFN-gamma in response to stimulation with the plasmid DNA.
  • 96-well ELISPOT plate was coated by anti-mouse IFN- ⁇ capture antibody.
  • ELISPOT plate coated with anti- mouse IFN- ⁇ capture antibody was blocking with RPMI-1640 containing 10% FBS for 2 hrs.
  • spleen was extracted from immunized mice and was dissociated into single cell suspension in RPMI-1640 containing 10% FBS.
  • Splenocytes were put in each well of ELISPOT plate with the cell number of 5x105.
  • the wells of ELIPSOT plate containing splenocytes were treated with MUC 1 epitope no. 1 - 5 and incubated for 18 hrs. Concanavalin A was used as a positive control.
  • the sequence of MUC1 epitope no. 1 - 5 was shown in Table 9.
  • splenocytes and eptiopes in wells of ELISPOT plate were aspirated and washed with distilled water twice and PBS containing 0.05% tween-20 three times.
  • Biotinylated anti-mouse IFN- ⁇ was added to each of wells for detection and incubated for 2 hrs at room temperature.
  • contents in the wells of ELISPOT plate was removed and the wells of ELISPOT plate was washed with PBS containing 0.05% tween-20 three times.
  • the wells was treated with streptavidin-horseradish peroxidase (HRP) and incubated for 1 hr.
  • HRP streptavidin-horseradish peroxidase
  • ELISPOT plate was aspirated and washed with PBS containing 0.05% tween-20 four times and PBS twice. The spot was visualized by adding AEC substrate into wells. The substrate reaction was stopped by washing the wells with distilled water. The spots in the well of ELISPOT plate was counted by ELISPOT reader. The counted spots were evaluated as IFN- ⁇ secreting cell number. The result was shown in Fig 9. Fig. 9 shows the number of the counted spots evaluated as IFN- ⁇ secreting cell number where the splenocytes were treated with MUC1 epitope no. 1 - 5 respectively.
  • the number of cells secreting IFN- ⁇ showed similar trend between pVAXl group and pVAXl-MUC l-wt in ELISPOT assay using mice immunized after a lapse of two months from the last injection.
  • the increase of IFN- ⁇ secreting cells was observed in pVAXl-MUCl-2mA group passed through two months from the last injection.
  • splenocytes stimulated with MUC1 epitopes No. 3 and No. 5 showed 5- and 4-fold higher cell population secreting IFN- ⁇ compared with pVAXl group, respectively.

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Abstract

La présente invention concerne un agent immunogène comprenant une protéine MUC1 modifiée ou un fragment fonctionnel de celui-ci, ou un polynucléotide codant correspondant, et une utilisation de l'agent immunogène pour l'immunothérapie du cancer exprimant MUC1.
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Citations (2)

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US20120270798A1 (en) * 2003-07-11 2012-10-25 Michael Anthony Hollingsworth Compositions and Methods for Preventing or Treating Cancer
US8329639B2 (en) * 2011-02-24 2012-12-11 Oncothyreon Inc. MUC1 based glycolipopeptide vaccine with adjuvant

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120270798A1 (en) * 2003-07-11 2012-10-25 Michael Anthony Hollingsworth Compositions and Methods for Preventing or Treating Cancer
US8329639B2 (en) * 2011-02-24 2012-12-11 Oncothyreon Inc. MUC1 based glycolipopeptide vaccine with adjuvant

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Title
BEHRENS ET AL.: "The reactive tumor microenvironment: MUC1 signaling directly reprograms transcription of CTGF", ONCOGENE, vol. 29, no. 42, 2010, pages 5667 - 5677 *
DATABASE NCBI, GenBank [O] 26 April 2013 (2013-04-26), XP055221485, Database accession no. ABS01309.1 *
WANG ET AL.: "Identification of four sites of stimulated tyrosine phosphorylation in the MUC1 cytoplasmic tail", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 310, no. Issue 2, 2003, pages 341 - 346, XP004458948 *
WRIGHT ET AL.: "Retention of immunogenicity produced by mucin1 peptides with glycosylation site substitutions", IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY, vol. 32, no. 4, 2010, pages 647 - 655 *

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