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WO2008012237A1 - Construction de multiples antigènes et leur utilisation - Google Patents

Construction de multiples antigènes et leur utilisation Download PDF

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WO2008012237A1
WO2008012237A1 PCT/EP2007/057358 EP2007057358W WO2008012237A1 WO 2008012237 A1 WO2008012237 A1 WO 2008012237A1 EP 2007057358 W EP2007057358 W EP 2007057358W WO 2008012237 A1 WO2008012237 A1 WO 2008012237A1
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cea
sequence
her2
vector
nucleotides
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Luigi Aurisicchio
Gennaro Ciliberto
Nicola La Monica
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Istituto di Ricerche di Biologia Molecolare P Angeletti SpA
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Istituto di Ricerche di Biologia Molecolare P Angeletti SpA
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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
    • 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
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • 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
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • 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
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates generally to the therapy of cancer. More specifically, the present invention relates to dicistronic adenovirus and plasmid vectors expressing both the carcinoembryonic antigen (CEA) tumor-associated antigen, or portion thereof, and the human epidermal growth factor 2/neu antigen (HER2/neu), or portion thereof.
  • CEA carcinoembryonic antigen
  • HER2/neu human epidermal growth factor 2/neu antigen
  • the present invention also provides methods for inducing an immune response against CEA and/or HER2-associated tumors using the compositions and molecules disclosed herein.
  • Vaccination has become a standard procedure for the prevention of numerous infectious diseases.
  • the use of vaccines for the prevention and/or treatment of other types of diseases, such as cancer, is now an attractive possibility due to recent advances in molecular engineering and an increased understanding of tumor immunology.
  • Cancer is one of the leading causes of mortality worldwide. Despite an abundance of cancer-related research, conventional therapies that combine surgery, radiation, and chemotherapy, often fail to effectively treat established cancers. Reliable methods of prevention also remain unavailable. Cancer typically involves the malfunction of genes that encode products that contribute to the regulation of the cell cycle or cell proliferation, such as growth factors and their receptors, oncogenes, and tumor suppressor genes. Many of these gene products are expressed on the surface of a variety of tumor cells; and, hence, are designated tumor-associated antigens (TAAs).
  • TAAs tumor-associated antigens
  • the introduction of genes encoding TAAs directly into a subject has been shown to generate a protective immune response against the TAA in many experimental models, making these molecules a target for vaccine therapy. However, because many of these gene products are also expressed in normal cells, albeit at lower levels, many immunological therapies targeting TAAs have proven ineffective due to self-tolerance.
  • epidermal growth factor 2 is a transmembrane tumor associated antigen encoded by the HER2/neu proto-oncogene (also called c-erbB-2), and carcinoembryonic antigen (CEA).
  • EAA carcinoembryonic antigen
  • Low levels of expression of the HER2/neu transcript and the encoded 185 kD protein are normally detected in adult epithelial cells of various tissues, including the skin and breast, and tissues of the gastrointestinal, reproductive, and urinary tracts (Press et al., Oncogene 5: 953-962 (1990)). Higher levels of HER2/neu expression are also detected in the corresponding fetal tissues during embryonic development (Press et al., supra).
  • the HER2 antigen is an attractive target for active specific immunotherapy.
  • the HER2/neu gene is commonly overexpressed or amplified in various malignancies, such as carcinomas of the breast, ovary, uterus, colon, and prostate, and adenocarcinomas of the lung (reviewed in Disis and Cheever, Adv. Cancer Research 71 : 343-371 (1997)).
  • Overexpression of HER2/neu correlates with a poor prognosis and a higher relapse rate for cancer patients (Slamon et al., Science 244: 707-712 (1989)).
  • HER2 HER2-like kinase activity and cell proliferation, and contributes to the aggressive behavior of tumor cells (Ben-Levy et al. Embo J 13(14): 3302-11 (1994)).
  • the high expression level of HER2 observed in tumors is in direct contrast with the low levels associated with normal adult tissues.
  • Anti-hHER2 cytotoxic T lymphocytes have been isolated from breast and ovarian cancer patients (Ioannides et al. Cell Immunol 151(1): 225-34 (1993); Peoples et al. Proc Natl Acad Sci USA 92 (14): 6547-51 (1995)).
  • CTL cytotoxic T lymphocytes
  • HLA-A2.1 -associated hHER2 peptides have been defined and peptide-specific T cells can be generated in vitro (Fisk et al. Cancer Res 57(1): 8-93 (1997); Yoshino et al. Cancer Res 54(13): 3387-90 (1994); Lustgarten et al. Hum Immunol 52(2): 109- 18 (1997)).
  • HER2/neu has been pursued as a target for the development of immunological treatments of malignancies.
  • Anti-HER2 monoclonal antibodies have been investigated as therapies for breast cancer, with each antibody approach demonstrating various levels of success (for discussion, see Yarden, Oncology 61(suppl 2): 1-13 (2001)).
  • HER2/neu peptide-based vaccines have been studied in rodent models (for review, see Disis and Cheever, Adv. Cancer Res. 71 :343-71 (1997)).
  • Vectors encoding human HER2 and/or the extracellular and transmembrane domains of HER, and their use as vaccines have been disclosed (WO 05/012527), as have vectors encoding rhesus monkey HER2 (WO 04/061105) for use as vaccines.
  • the CEA gene is a TAA commonly associated with cancer.
  • the CEA gene encodes a protein that acts as an intercellular adhesion molecule (Benchimol et al, Cell 57: 327-334 (1989)).
  • CEA can also inhibit cell death resulting from detachment of cells from the extracellular matrix and can contribute to cellular transformation associated with certain proto -oncogenes such as Bcl2 and C-Myc ⁇ see Berinstein, J. Clin Oncol. 20(8): 2197-2207 (2002)).
  • CEA is normally expressed during fetal development and in adult colonic mucosa. Aberrant CEA expression has long been correlated with many types of cancers, with the first report describing CEA overexpression in human colon tumors over thirty years ago (Gold and Freedman, J. Exp. Med. lll A ⁇ -A ⁇ l (1965)). Overexpression of CEA has since been detected in nearly all colorectal tumors, as well as in a high percentage of adenocarcinomas of the pancreas, liver, breast, ovary, cervix, and lung.
  • Therapeutic approaches targeting CEA include the use of anti-CEA antibodies (see Chester et al., Cancer Chemother. Pharmacol. 46 (Suppl): S8-S12 (2000)), as well as CEA-based vaccines (for review, see Berinstein, supra).
  • the carcinoembryonic antigen (CEA) and the HER2/neu genes are commonly associated with the development or presence of adenocarcinomas, including colorectal carcinomas.
  • the present invention relates to compositions and methods to elicit or enhance immunity to the protein products expressed by the CEA and/or HER2/neu genes.
  • the present invention provides expression vectors comprising a first sequence of nucleotides and a second sequence of nucleotides; wherein the first sequence of nucleotides encodes a human CEA protein, or variant thereof, and the second sequence of nucleotides encodes a human HER2/neu protein, or variant thereof.
  • the expression vectors described herein are useful as therapeutic vaccines in individuals suffering from cancer, particularly those individuals in which CEA and/or HER2 are overexpressed at the tumor site.
  • Said vaccines are useful as a monotherapy or as part of a therapeutic regime, said regime comprising administration of a second genetic vaccine or other vaccine such as a cell-based, protein, or peptide-based vaccine, or comprising radiotherapy or chemotherapy.
  • the first and/or second sequence of nucleotides comprises codons that have been optimized for high levels of expression in a human host cell.
  • the present invention further provides an adenoviral vector comprising a first sequence of nucleotides and a second sequence of nucleotides; wherein the first sequence of nucleotides encodes a human CEA protein, or variant thereof, and the second sequence of nucleotides encodes a human HER2/neu protein, or variant thereof.
  • the use of the adenovirus vector in immunogenic compositions and vaccines for the prevention and/or treatment of CEA and or HER2-associated cancer is provided.
  • the adenovirus vector is an Ad6 vector.
  • the first sequence of nucleotides encodes a variant of a human CEA protein fused to the B subunit of the heat labile enterotoxin o ⁇ E.coli (LTB), or substantial portion thereof, wherein the CEA portion of the encoded CEA fusion protein is deleted of its C-terminal anchoring domain.
  • LTB heat labile enterotoxin o ⁇ E.coli
  • the second sequence of nucleotides encodes a variant of a human HER2/neu protein, wherein the variant HER2 protein is a truncated form of human HER2 protein which comprises the extracellular and transmembrane domains of the HER2 protein, but not the intracellular domain.
  • an exemplary expression vector of the present invention comprising an Ad6 vector comprising nucleotide sequences encoding a CEA-LTB fusion as well as the extracellular and transmembrane domains of HER2 (HER2ECDTM) was confirmed in accordance with the present invention.
  • the elicited immune response was measured in two different strains of mice against both the encoded antigens and in mice double-transgenic for CEA and HER2/neu, thus resulting in breakage of immune tolerance.
  • promoter refers to a recognition site on a DNA strand to which the RNA polymerase binds.
  • the promoter forms an initiation complex with RNA polymerase to initiate and drive transcriptional activity.
  • the complex can be modified by activating sequences termed “enhancers” or inhibiting sequences termed “silencers”.
  • cassette refers to a nucleotide or gene sequence that is to be expressed from a vector, for example, the nucleotide or gene sequence encoding the hCEA-LTB fusion or the nucleotide sequence encoding the HER2 extracellular and transmembrane domains (hereinafter HER2ECDTM).
  • a cassette comprises a gene sequence that can be inserted into a vector, which in some embodiments, provides regulatory sequences for expressing the nucleotide or gene sequence.
  • the nucleotide or gene sequence provides the regulatory sequences for its expression.
  • the vector provides some regulatory sequences and the nucleotide or gene sequence provides other regulatory sequences.
  • the vector can provide a promoter for transcribing the nucleotide or gene sequence and the nucleotide or gene sequence provides a transcription termination sequence.
  • the regulatory sequences that can be provided by the vector include, but are not limited to, enhancers, transcription termination sequences, splice acceptor and donor sequences, introns, ribosome binding sequences, and poly(A) addition sequences.
  • vector refers to some means by which DNA fragments can be introduced into a host organism or host tissue.
  • vectors including plasmid, virus (including adenovirus), bacteriophages and cosmids.
  • first generation describes adenoviral vectors that are replication-defective.
  • First generation adenovirus vectors typically have a deleted or inactivated El gene region, and preferably have a deleted or inactivated E3 gene region.
  • the abbreviation “LT” refers generally to the heat labile enterotoxin of E. coli.
  • LT may refer to the complete enterotoxin, comprising subunits A and B or a substantial portion of subunit A, or a substantial portion of subunit B.
  • the abbreviation “LTB” refers to the B subunit of the heat labile enterotoxin of E. coli, or substantial portion thereof, including subunits which are truncated on the C-terminal or N-terminal end but maintain biological activity, as well as subunits that contain internal amino acid insertions, deletions, or substitutions but maintain biological activity.
  • Ad6 CEA-LTBopt.HER2ECDTM refers to a construct, disclosed herein, which comprises an Ad6 adenoviral genome deleted of the El and E3 regions.
  • the El region is replaced by a codon- optimized human CEA-LTB gene in an El parallel orientation under the control of a human CMV promoter without intron A, followed by a bovine growth hormone polyadenylation (bGH) signal.
  • bGH bovine growth hormone polyadenylation
  • the codon-optimized human CEA sequence is devoid of the GPI anchor coding sequence and is fused at its C-terminus to the B subunit of E. coli heat labile enterotoxin.
  • fusion protein refers to a protein having at least two polypeptides covalently linked in which one polypeptide comes from one protein sequence or domain and the other polypeptide comes from a second protein sequence or domain.
  • the fusion proteins of the present invention comprise a human CEA polypeptide or fragment or variant thereof, and a second polypeptide, which comprises the B subunit of E. coli heat labile enterotoxin (LT).
  • LT heat labile enterotoxin
  • CEA-LTB fusion proteins of the present invention are preferably linked N- terminus to C-terminus.
  • the CEA polypeptide and the LTB polypeptide can be fused in any order.
  • the C-terminus of the CEA polypeptide is fused to the N-terminus of LTB, as exemplified in FIGURE 9.
  • CEA-LTB fusion proteins in which the LTB polypeptide is fused to the N-terminus of the CEA polypeptide are also contemplated.
  • CEA-LTB fusion refers to a nucleic acid sequence in which at least a portion of the CEA gene is fused to a substantial portion of the LTB subunit of E. coli heat labile enterotoxin.
  • CEA-LTB fusion protein refers to a polypeptide encoded by a CEA-LT fusion as described.
  • CEA-LTB fusion and CEA-LTB fusion protein are also understood to refer to fragments thereof, homologs thereof, and functional equivalents thereof (collectively referred to as "variants"), such as those in which one or more amino acids is inserted, deleted or replaced by other amino acid(s).
  • the CEA-LT fusions of the present invention upon administration to a mammal such as a human being, can stimulate an immune response by helper T cells or cytotoxic T cells, or stimulate the production of antibodies at least as well as a "wild- type" CEA sequence.
  • the CEA-LTB fusions can enhance the immune response as compared to a wild-type CEA.
  • AD refers to the anchoring domain of a CEA gene or protein.
  • the anchoring domain of the wild-type human CEA is located from about amino acid 679 to about amino acid 702 of SEQ ID NO:6 (FIGURE 10).
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in which the disorder is to be prevented.
  • a “disorder” is any condition that would benefit from treatment with the molecules of the present invention, including the nucleic acid molecules described herein and the fusion proteins that are encoded by said nucleic acid molecules. Encompassed by the term “disorder” are chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.
  • the molecules of the present invention are intended for use as treatments for disorders or conditions characterized by aberrant cell proliferation, including, but not limited to, breast cancer, colorectal cancer, and lung cancer.
  • the term "effective amount” means sufficient vaccine composition is introduced to produce the adequate levels of the polypeptide, so that a protective or therapeutic immune response results. One skilled in the art recognizes that this level may vary.
  • a “conservative amino acid substitution” refers to the replacement of one amino acid residue by another, chemically similar, amino acid residue. Examples of such conservative substitutions are: substitution of one hydrophobic residue (iso leucine, leucine, valine, or methionine) for another; substitution of one polar residue for another polar residue of the same charge (e.g., arginine for lysine; glutamic acid for aspartic acid).
  • hCEA and hCEAopt refer to a human carcinoembryonic antigen and a human codon-optimized carcinoembryonic antigen, respectively.
  • hHER2.wt and hHER2.opt refer to a human epidermal growth factor 2 antigen and a human codon-optimized epidermal growth factor 2 antigen, respectively.
  • hHER2ECDTM.wt and hHER2ECDTM.opt refer to a truncated human epidermal growth factor 2 antigen and a truncated human codon-optimized epidermal growth factor 2 antigen, respectively.
  • the truncated forms of HER2, "hHER2ECDTM.wt” and “hHER2ECDTM.opt,” comprise the extracellular and transmembrane domains of the human HER2 protein.
  • Substantially similar means that a given nucleic acid or amino acid sequence shares at least 75%, preferably 85%, more preferably 90%, and even more preferably 95% identity with a reference sequence.
  • the reference sequence can be relevant portions of the wild-type human CEA nucleotide or amino acid sequence, the wild-type HER2 or HER2ECDTM nucleotide or amino acid sequence, or the wild-type nucleotide or amino acid sequence of the LTB subunit of the E.coli heat labile enterotoxin, as dictated by the context of the text.
  • a CEA protein sequence that is "substantially similar" to the wild-type human CEA protein or fragment thereof will share at least 75% identity with the relevant fragment of the wild-type human CEA, along the length of the fragment, preferably 85% identity, more preferably 90% identity and even more preferably 95% identity.
  • Whether a given CEA, HER2, or LTB protein or nucleotide sequence is "substantially similar" to a reference sequence can be determined for example, by comparing sequence information using sequence analysis software such as the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG).
  • the GAP program utilizes the alignment method of Needleman and Wunsch (J. MoI. Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math. 2:482, 1981).
  • a "substantial portion" of a gene, variant, fragment, or subunit thereof means a portion of at least 50%, preferably 75%, more preferably 90%, and even more preferably 95% of a reference sequence.
  • a “gene” refers to a nucleic acid molecule whose nucleotide sequence codes for a polypeptide molecule. Genes may be uninterrupted sequences of nucleotides or they may include such intervening segments as introns, promoter regions, splicing sites and repetitive sequences. A gene can be either RNA or DNA. A preferred gene is one that encodes the invention peptide.
  • the term "nucleic acid” or “nucleic acid molecule” is intended for ribonucleic acid
  • RNA deoxyribonucleic acid
  • DNA probes, oligonucleotides, fragment or portions thereof, and primers.
  • DNA can be either complementary DNA (cDNA) or genomic DNA, e.g. a gene encoding a CEA fusion protein.
  • Wild-type protein or "wt protein” refers to a protein comprising the major sequence of amino acids that occurs in nature, often designated as the reference sequence or "normal” allele.
  • the amino acid sequence of wild-type human CEA is shown in FIGURE 10 (SEQ ID NO:6).
  • Wild-type CEA gene refers to any gene comprising a sequence of nucleotides that encodes a naturally occurring CEA protein, including proteins of human origin or proteins obtained from another organism, including, but not limited to, other mammals such as rat, mouse and rhesus monkey.
  • FIGURE 11 The amino acid sequence of wild-type human HER2 protein is shown in FIGURE 11 (SEQ ID NO:7) and has been reported (Coussens et al, Science 230: 1132-39 (1985); King et al., Science 229: 974-76 (1985)).
  • mamalian refers to any mammal, including a human being.
  • Ag refers to an antigen.
  • TAA refers to a tumor-associated antigen.
  • ORF refers to the open reading frame of a gene.
  • FIGURE 1 shows a schematic representation of an exemplary Ad6-CEA-HER2 dicistronic cassette of the present invention, as described in EXAMPLE 1.
  • the two expression cassettes are oriented in tandem and hCMV and mCMV drive the expression of CEA-LTB and
  • FIGURE 2 shows the results of an analysis of the genomic stability of the
  • MRKAd ⁇ CEA-LTB/HER2-ECDTM construct Three plaque isolates were amplified and repeatedly passaged in PERC6 cells prior to analysis of the genomic structure, as described in
  • FIGURE 3 demonstrates the immunogenic potency of MRKAd ⁇ CEA- LTB/HER2-ECDTM.
  • Groups of BALB/c and C57BL/6 mice were immunized with two injections of MRKAd ⁇ vectors encoding the human CEA-LTB and HER2-ECDTM (109 and I ⁇ 8 vp, see EXAMPLE 2).
  • Immune response was measured by IFN ⁇ ELIspot assay two weeks after the last injection.
  • FIGURE 4 shows a comparison of an exemplary Ad6 dicistronic (MRKAd ⁇ CEA- LTB/HER2-ECDTM) vector to monocistronic vectors encoding HER2ECDTM and CEA.
  • C57BL/6 mice (5-10/group) received two injections of 108 vp, two weeks apart. Two weeks later, mice were sacrificed and splenocytes were analyzed by intracellular staining upon stimulation with pools of peptides covering CEA, LTB and the ECD. TM portion of Her2/Neu.
  • Panel A shows the CD8+ specific immune response; and panel B shows the CD4+ specific immune response. Black dots represent the immune response per each single mouse; white circles represent the geometric mean of the group.
  • FIGURE 5 demonstrates that MRKAd ⁇ CEA-LTB/HER2-ECDTM can break tolerance to both target antigens.
  • Groups of 5 CEA/HER2 double transgenic mice were immunized with 1010 vp of the MRKAd ⁇ vector encoding both target antigens. Immune response was measured by IFN ⁇ intracellular staining on mouse splenocytes 14 days after the last injection. Average values are also shown (black line).
  • FIGURE 6 shows the immune response elicited by MRKAd ⁇ CEA-LTB/HER2- ECDM in engrafted NOD/scid-DRl mice.
  • CEA and Neu responses were measured with 15mer peptides covering the antigen sequence. PMA at 5 ng/ml was added as costimulus.
  • FIGURE 7 shows the immune response elicited by MRKAd ⁇ CEA-LTB/HER2-
  • mice ECDM in engrafted NOD/scid-DRl mice.
  • Mice engrafted under the kidney capsule with CBMNC received two injections of lOlO vp dicistronic Ad ⁇ two weeks apart. Intracellular staining for IFN ⁇ was performed two weeks after last boost.
  • CEA and Neu responses were measured with 15mer peptides covering the antigen sequence.
  • PMA at 5 ng/ml was added as costimulus.
  • FIGURE 8 panel A, shows the nucleotide sequence of a codon-optimized polynucleotide (hHER2ECDTM.opt, SEQ ID NO:1) that encodes a truncated human HER2 protein, said protein comprising the extracellular and transmembrane domains of the HER2 protein.
  • Panel B shows a second polynucleotide that encodes the extracellular and transmembrane domains of the HER2 protein, the second polynucleotide comprising "wild-type" nucleotide sequences, which have not been codon optimized (hHER2ECDTM.wt, SEQ ID NO:2).
  • FIGURE 9 shows the nucleotide sequence (SEQ ID NO:3) of an exemplary hCEA-LTB fusion (Panel A). Also shown is the nucleotide sequence of an exemplary hCEAopt- LTB fusion (SEQ ID NO:4, Panel B). Panel C shows the nucleotide sequence (SEQ ID NO:5) of an exemplary fully optimized hCEA-LTB fusion, designated herein hCEAopt-LTBopt. Nucleotide sequences encoding LTB are shown in bold. Junction sequences, created by the cloning strategy employed to fuse the CEA and LTB sequences are underlined.
  • FIGURE 10 shows the amino acid sequence of wild-type human CEA (SEQ ID NO:6), which was previously described (see, e.g., U.S. Patent No. 5,274,087).
  • FIGURE 11 shows the amino acid sequence of the wild-type human HER2 protein (SEQ ID NO:7; see, e.g. Coussens et al, Science 230: 1132-39 (1985); King et al, Science 229: 974.76 (1985)).
  • FIGURE 12 shows the nucleotide sequence of a portion of the wild-type human CEA cDNA from nt 1 to nt 2037 (SEQ ID NO:8, Panel A), encoding a portion of the hCEA protein from aa 1 to aa 679 (SEQ ID NO:9, Panel B).
  • Carcinoembryonic antigen (CEA) and HER2/neu are commonly associated with the development of adenocarcinomas.
  • the present invention relates to compositions and methods to elicit or enhance immunity to the protein product expressed by the CEA and/or HER2/neu tumor-associated antigens, wherein aberrant CEA and/or HER2 expression is associated with the carcinoma or its development. Association of aberrant CEA and/or HER2 expression with a carcinoma does not require that the CEA and/or HER2 protein be expressed in tumor tissue at all timepoints of its development, as abnormal CEA and/or HER2 expression may be present at tumor initiation and not be detectable late into tumor progression or vice-versa.
  • the present invention provides compositions and methods to elicit or enhance immunity to the protein products expressed by the CEA and/or HER2/neu genes.
  • the present invention provides expression vectors comprising a first sequence of nucleotides and a second sequence of nucleotides; wherein the first sequence of nucleotides encodes a human CEA protein, or variant thereof, and the second sequence of nucleotides encodes a human HER2/neu protein, or variant thereof. It is believed that the dicistronic vectors of the present invention, which comprise nucleotide sequences encoding two tumor-associated antigens (specifically CEA and HER2/neu) will elicit a more efficacious immune response than a vaccine that targets only a single TAA.
  • a tumor that initially expresses one TAA at tumor initiation or during early tumor development may not express that same TAA as tumor development progresses.
  • a tumor that expresses numerous tumor markers at one time point may lose expression of one or more tumor antigens later in time.
  • a vaccine that elicits an immune response against more than one TAA would be more likely to induce tumor regression and/or halt tumor growth than a vaccine that targets only a single TAA because a tumor that expresses any one of the TAA's targeted by the vaccine would be susceptible to lysis by CTLs and/or antibodies.
  • the dicistronic vectors of the present invention are more cost- effective to develop as vaccines, requiring less production materials (e.g. controls) than the use of multiple monocistronic vectors.
  • the expression vectors described herein are useful as therapeutic vaccines in individuals suffering from cancer, particularly those individuals in which CEA and/or HER2 are overexpressed at the tumor site.
  • Said vaccines are useful as a monotherapy or as part of a therapeutic regime, said regime comprising administration of a second genetic vaccine or other vaccine such as a cell-based, protein, or peptide-based vaccine, or comprising radiotherapy or chemotherapy.
  • the CEA and HER2 nucleotide sequences of the present invention encode the full- length human CEA and human HER2 proteins; or variants thereof, as stated above.
  • Contemplated variants include, but are not necessarily limited to: sequences that are C- or N- terminally truncated, sequences with conservative substitutions, and sequences with internal deletions or insertions; relative to the wild-type CEA and HER2 protein sequences.
  • the encoded CEA and HER2 protein variants of the present invention must be sufficient to elicit a CEA or HER2-specific immune response in a patient in need thereof. Reference sequences for the wt human CEA and wt human HER2 have been reported (see, e.g., U.S. Patent No.
  • the full length CEA gene, or variant thereof is fused in- frame to nucleotide sequences encoding the B subunit of E. coli heat labile enterotoxin (LTB).
  • Exemplary CEA-LTB sequences are shown in FIGURES 9A - 9C and set forth as SEQ ID NOs: 3-5.
  • the LTB subunit, or substantial portion thereof, of the CEA-LTB fusion of may be fused to the amino terminus or the carboxy terminus of the CEA sequence.
  • the LTB sequence and the CEA sequence can be fused N-terminus to N-terminus, C- terminus to C-terminus, C-terminus to N-terminus or N-terminus to N-terminus.
  • the C-terminus of the CEA polypeptide is fused to the N- terminus of LTB.
  • contemplated for use in the dicistronic vectors of the present invention are nucleotide sequences encoding variants or mutants of the CEA, HER2 and/or LTB sequences described herein.
  • any such biologically active fragment and/or mutant will encode either a protein or protein fragment which at least substantially mimics the pharmacological properties of the hCEA protein and the hHER2 protein, including but not limited to the hCEA ⁇ AD protein as set forth in SEQ ID NO: 9 and the HER2ECDTM protein encoded by the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.
  • the CEA and/or HER2 variants encoded by the nucleotide sequences in the dicistronic vectors of the present invention are sufficient to elicit an immune response against CEA and HER2.
  • the dicistronic vectors of this embodiment of the present invention are, therefore, useful as prophylactic and/or therapeutic vaccines against cancers that overexpress CEA and/or HER2.
  • an LTB variant is fused to the CEA sequence of the CEA fusion, wherein the LTB variant is truncated of its signal sequence (see, e.g. SEQ ID NO's: 3-5). While not being bound by theory, deletion of the toxin signal sequence, e.g. the LTB signal sequence, ensures that posttranslational processing of the CEA fusion is driven by the CEA signal sequence.
  • the CEA protein of the expression vector is human CEA (SEQ ID NO: 6) or a functional equivalent thereof, for example, a human CEA deleted of its C-terminal anchoring domain (CEA ⁇ AD) (SEQ ID NO: 9), which is located from about amino acid 679 to about amino acid 702 of the full-length human CEA. While not being bound by theory, deletion of the anchoring domain increases secretion of the CEA fusion protein, thereby enhancing cross priming of the CEA-LTB immune response.
  • the dicistronic vectors of the present invention comprise nucleotide sequences expressing CEA, HER2 and/or variants thereof that are assembled into an expression cassette.
  • the cassette preferably contains CEA and HER2 protein-encoding gene sequences, with related transcriptional and translational control sequences operatively linked to it, such as a promoter, and termination sequences.
  • the expression of the CEA nucleotide sequence, or CEA variant thereof is driven by a first promoter while the expression of the HER2 nucleotide sequence, or HER2 variant thereof, is driven by a second promoter.
  • the first promoter and the second promoter are not the same. While not wishing to be bound by theory, the use of two different promoters reduces the possibility of internal recombination events and avoids the reduction of gene expression due to squelching of transcription factors.
  • CEA-LTB nucleotide sequence for example CEA-LTBopt (SEQ ID NO:5)
  • CMV human cytomegalovirus
  • HER2ECDTM nucleotide sequence for example HER2ECDTMopt (SEQ ID NO: 1)
  • IE CMV immediate-early
  • HER2ECDTM is driven by the murine CMV promoter.
  • promoters include naturally occurring promoters such as the EFl alpha promoter, Rous sarcoma virus promoter, and SV40 early/late promoters and the p- actin promoter; and artificial promoters such as a synthetic muscle specific promoter and a chimeric muscle- specific/CMV promoter (Li et al., Nat. Biotechnol. 17:241-245 (1999); Hagstrom et al., Blood 95:2536-2542 (2000)).
  • naturally occurring promoters such as the EFl alpha promoter, Rous sarcoma virus promoter, and SV40 early/late promoters and the p- actin promoter
  • artificial promoters such as a synthetic muscle specific promoter and a chimeric muscle- specific/CMV promoter (Li et al., Nat. Biotechnol. 17:241-245 (1999); Hagstrom et al., Blood 95:2536-2542 (2000)).
  • the expression cassette of the dicistronic vector of the present invention comprises a termination sequence(s)/polyadenylation signal following the gene-encoding nucleotide sequences.
  • the polyadenylation signal is responsible for cleaving the transcribed RNA and the addition of a poly (A) tail to the RNA.
  • the poly (A) tail is important for the mRNA processing.
  • Polyadenylation signals that can be used as part of a gene expression cassette include the minimal rabbit -globin polyadenylation signal and the bovine growth hormone polyadenylation (BGH). (Xu et al, Gene 272: 149-156 (2001), Post et al, U.S. Patent U. S. 5,122,458.) Additional examples include the Synthetic Polyadenylation Signal (SPA) and SV40 polyadenylation signal. In preferred embodiments of the present invention, nucleotide sequences coding for the CEA and HER2 variants are followed by the BGH and SV40 polyadenylation signals.
  • BGH bovine growth hormone polyadenylation
  • dicistronic vectors comprising variant nucleotide sequences that encode CEA and HER2, or variants thereof, the variant nucleotide sequences including but not necessarily limited to: nucleotide substitutions, deletions, additions, amino -terminal truncations and carboxy-terminal truncations.
  • mutations in the DNA sequence that do not substantially alter the ultimate physical properties of the expressed protein.
  • the mutations of the present invention encode mRNA molecules that express a CEA, CEA fusion protein, HER2, or variants thereof that are sufficient to elicit an immune response against CEA and/or HER2.
  • the dicistronic vectors comprise nucleotide sequences that are codon-optimized for high level expression in a human host cell.
  • the codons of the CEA and/or HER2 nucleotide sequence within the dicistronic vector are designed so as to use the codons preferred by the projected host cell, which in preferred embodiments is a human cell.
  • the dicistronic vectors comprising codon-optimized sequences may be used for the development of recombinant adenovirus or plasmid-based DNA vaccines and immunogenic compositions, which provide effective immunoprophylaxis against CEA and/or HER2-associated cancer through neutralizing antibody and cell-mediated immunity.
  • the dicistronic vectors comprise nucleic acid sequences that encode a desired CEA, HER2, or variant thereof, and that are converted to a polynucleotide sequence having an identical translated sequence but with alternative codon usage as described by Lathe, "Synthetic Oligonucleotide Probes Deduced from Amino Acid Sequence Data: Theoretical and Practical Considerations" J. Molec. Biol. 183:1-12 (1985), which is hereby incorporated by reference.
  • the methodology generally consists of identifying codons in the wild-type sequence that are not commonly associated with highly expressed human genes and replacing them with optimal codons for high expression in human cells.
  • the new gene sequence is then inspected for undesired sequences generated by these codon replacements (e.g., "ATTTA" sequences, inadvertent creation of intron splice recognition sites, unwanted restriction enzyme sites, etc.). Undesirable sequences are eliminated by substitution of the existing codons with different codons coding for the same amino acid.
  • the synthetic gene segments are then tested for improved expression.
  • the sequence of nucleotides that encodes the CEA protein, or variant or fusion thereof is codon-optimized for high-level expression in human cell ⁇ see, e.g. FIGURE 9).
  • the nucleotide sequence encoding the HER2 protein or HER2 variant, such as HER2ECDTM is codon- optimized for high-level expression in human cells ⁇ see, e.g. FIGURE 8A).
  • both the first sequence of nucleotides and the second sequence of nucleotides, encoding the CEA and HER2 proteins or variants are codon-optimized for high-level expression in human cells.
  • the present invention also relates to host cells transformed or transfected with the dicistronic vectors of the present invention.
  • Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to, bacteria such as E. coli, fungal cells such as yeast, mammalian cells including, but not limited to, cell lines of bovine, porcine, monkey and rodent origin; and insect cells including but not limited to Drosophila and silkworm derived cell lines.
  • Such recombinant host cells can be cultured under suitable conditions to produce a CEA and HER2 protein, or biologically equivalent variants.
  • the host cell is human.
  • the term "host cell" is not intended to include a host cell in the body of a transgenic human being, human fetus, or human embryo.
  • the dicistronic vectors of the present invention are preferably adenoviral or plasmid vectors, although linear DNA linked to a promoter, or other vectors, such as adeno- associated virus or a modified vaccinia virus, retroviral or lentiviral vector may also be used.
  • the vector is an adenovirus vector (used interchangeably herein with "adeno vector").
  • Adenovectors can be based on different adenovirus serotypes such as those found in humans or animals. Examples of animal adenoviruses include bovine, porcine, chimp, murine, canine, and avian (CELO).
  • Preferred adeno vectors are based on human serotypes, more preferably Group B. C, or D serotypes. Examples of human adenovirus Group B.
  • C, D, or E serotypes include types 2 ("Ad2"), 4 ("Ad4"), 5 (“Ad5"), 6 (“Ad6"), 24 ("Ad24”), 26 ("Ad26”), 34 ("Ad34") and 35 ("Ad35”).
  • the expression vector is an adenovirus type 6 (Ad6) vector.
  • the vector chosen is an adenovirus
  • These adenoviral vectors are characterized by having a nonfunctional El gene region, and preferably a deleted adenoviral El gene region. Adeno vectors do not need to have their El and E3 regions completely removed. Rather, a sufficient amount the El region is removed to render the vector replication incompetent in the absence of the El proteins being supplied in bans; and the El deletion or the combination of the El and E3 deletions are sufficiently large enough to accommodate a gene expression cassette.
  • the expression cassette is inserted in the position where the adenoviral El gene is normally located.
  • these vectors optionally have a nonfunctional or deleted E3 region. It is preferred that the adenovirus genome used be deleted of both the El and E3 regions ( ⁇ E1 ⁇ E3).
  • the adenoviruses can be multiplied in known cell lines which express the viral El gene, such as 293 cells, or PERC.6 cells, or in cell lines derived from 293 or PERC.6 cell which are transiently or stably transformed to express an extra protein.
  • the cell line may express components involved in the regulatory system.
  • T-Rex-293 is T-Rex-293; others are known in the art.
  • the adenovirus may be in a shuttle plasmid form.
  • This invention is also directed to a shuttle plasmid vector which comprises a plasmid portion and an adenovirus portion, the adenovirus portion comprising an adenoviral genome which has a deleted El and optional E3 deletion, and has an inserted expression cassette comprising a first sequence of nucleotides that encodes a CEA protein, or variant thereof, and a second sequence of nucleotides that encodes a HER2 protein, or variant thereof.
  • there is a restriction site flanking the adenoviral portion of the plasmid so that the adenoviral vector can easily be removed.
  • the shuttle plasmid may be replicated in prokaryotic cells or eukaryotic cells.
  • the expression cassette is inserted into the pMRKAd6-HV0 adenovirus plasmid (See Emini et al, WO2003031588A2), which is hereby incorporated by reference).
  • This plasmid comprises an Ad6 adenoviral genome deleted of the El and E3 regions.
  • this enhanced adenoviral vector is capable of maintaining genetic stability following high passage propagation. Standard techniques of molecular biology for preparing and purifying DNA constructs enable the preparation of the adenoviruses, shuttle plasmids, and DNA immunogens of this invention.
  • the dicistronic CEA-HER2 vectors of the present invention allow for the development of a therapeutic or prophylactic cancer vaccine by providing a dicistronic expression cassette that encodes both a CEA and a HER2 protein, or variants thereof, which can elicit an immune response against both the CEA and HER2 TAAs when administered to a mammal such as a human being.
  • one aspect of the instant invention is a method of preventing or treating CEA and/or HER2-associated cancer comprising administering to a mammal a vaccine vector comprising a first sequence of nucleotides and a second sequence of nucleotides; wherein the first sequence of nucleotides encodes a human CEA protein, or variant thereof, and the second sequence of nucleotides encodes a human HER2/neu protein, or variant thereof.
  • the method comprises administering an expression vector comprising a first sequence of nucleotides that encodes a CEA fusion protein, wherein the CEA fusion protein comprises a CEA protein or variant thereof, fused to a B subunit of E. coli heat labile enterotoxin (LTB); and a second sequence of nucleotides that encodes a truncated human HER2/neu protein, wherein the truncated HER2 protein comprises the extracellular and transmembrane domains of HER2/neu.
  • LTB E. coli heat labile enterotoxin
  • the dicistronic vaccine vector may be administered for the treatment or prevention of a cancer in any mammal, including but not limited to: lung cancer, breast cancer, and colorectal cancer.
  • the mammal is a human.
  • Suitable vectors can deliver nucleic acid into a target cell without causing an unacceptable side effect.
  • the vector is an adenovirus vector or a plasmid vector.
  • the vector is an adenoviral vector comprising an adenoviral genome with a deletion in the adenovirus El region, and an insert in the adenovirus El region, wherein the insert comprises an expression cassette comprising: a first sequence of nucleotides and a second sequence of nucleotides; wherein the first sequence of nucleotides encodes a human CEA protein, or variant thereof, and the second sequence of nucleotides encodes a human HER2/neu protein, or variant thereof.
  • the instant invention further relates to an adenovirus vaccine vector comprising an adenoviral genome with a deletion in the El region, and an insert in the El region, wherein the insert comprises an expression cassette comprising: (a) a first sequence of nucleotides that encodes a human CEA protein, or variant thereof; (b) a second sequence of nucleotides that encodes a human HER2/neu protein, or variant thereof; (c) a first promoter operably linked to the first sequence of nucleotides and (d) a second promoter operably linked to the second sequence of nucleotides.
  • the first promoter and the second promoter are not the same.
  • the adenovirus vector is an Ad 6 vector.
  • the recombinant adenovirus and plasmid- based polynucleotide vaccines disclosed herein are used in various prime/boost combinations in order to induce an enhanced immune response.
  • the two vectors are administered in a "prime and boost" regimen.
  • the first type of vector is administered one or more times, then after a predetermined amount of time, for example, 2 weeks, 1 month, 2 months, six months, or other appropriate interval, a second type of vector is administered one or more times.
  • the vectors carry expression cassettes encoding the same TAAs or combination of
  • the vector contain one or more promoters recognized by mammalian or insect cells.
  • the plasmid would contain a strong promoter such as, but not limited to, the CMV promoter.
  • the synthetic CEA fusion gene or other gene to be expressed would be linked to such a promoter.
  • An example of such a plasmid would be the mammalian expression plasmid VlJns as described (J. Shiver et. al. in DNA Vaccines, M. Liu et al. eds., N.Y. Acad. Sci., N.Y., 772:198-208 (1996), which is herein incorporated by reference).
  • dicistronic vectors described herein as part of a therapeutic regime, said therapeutic regime including administration of a second dicistronic vector as described herein or, alternatively, administration of a second genetic vaccine or other vaccine such as a cell-based, protein, or peptide-based vaccine, or comprising radiotherapy or chemotherapy.
  • an adenoviral vector vaccine and a plasmid vaccine may be administered to a vertebrate as part of a therapeutic regime to induce an immune response.
  • the present invention relates to a method of treating a mammal with a CEA and/or
  • HER2-associated cancer comprising: (a) introducing into the mammal a first vector comprising: i) a first sequence of nucleotides and a second sequence of nucleotides; wherein the first sequence of nucleotides encodes a human carcinoembryonic antigen (CEA) protein, or variant thereof, and the second sequence of nucleotides encodes a human HER2/neu protein, or variant thereof; and ii) a promoter operably linked to the polynucleotide; (b) allowing a predetermined amount of time to pass; and (c) introducing into the mammal a second vector comprising: i) a first sequence of nucleotides and a second sequence of nucleotides; wherein the first sequence of nucleotides encodes a human carcinoembryonic antigen (CEA) protein, or variant thereof, and the second sequence of nucleotides encodes a human HER2/neu protein, or variant thereof and ii) a
  • the first vector is a plasmid and the second vector is an adenovirus vector.
  • the first vector is an adenovirus vector and the second vector is a plasmid.
  • the first type of vector may be administered more than once, with each administration of the vector separated by a predetermined amount of time. Such a series of administration of the first type of vector may be followed by administration of a second type of vector one or more times, after a predetermined amount of time has passed. Similar to treatment with the first type of vector, the second type of vector may also be given one time or more than once, following predetermined intervals of time.
  • an immunologically or prophylactically effective dose of about 1 ng to 100 mg, and preferably about 10 ⁇ g to 300 ⁇ g of a plasmid vaccine vector is administered directly into muscle tissue.
  • An effective dose for recombinant adenovirus is approximately 106 - 1012 particles and preferably about 107 — 10l 1 particles.
  • Subcutaneous injection, intradermal introduction, impression though the skin, and other modes of administration such as intraperitoneal, intravenous, intramuscular or inhalation delivery are also contemplated.
  • the vaccine vectors are introduced to the recipient through intramuscular injection.
  • the vaccine vectors of this invention may be naked, i.e., unassociated with any proteins, or other agents which impact on the recipient's immune system.
  • it is desirable for the vaccine vectors to be in a physiologically acceptable solution such as, but not limited to, sterile saline or sterile buffered saline.
  • a physiologically acceptable solution such as, but not limited to, sterile saline or sterile buffered saline.
  • an agent which assists in the cellular uptake of DNA such as, but not limited to calcium ion.
  • transfection facilitating reagents and pharmaceutically acceptable carriers are generally referred to as transfection facilitating reagents and pharmaceutically acceptable carriers.
  • the vaccines and immunogenic compositions described herein may be formulated with an adjuvant in order to primarily increase the immune response elicited by administration of the expression vectors described herein.
  • Adjuvants which may be used in conjunction with the expression vectors of the present invention, include, but are not limited to, adjuvants containing CpG oligonucleotides, or other molecules acting on toll-like receptors such as TLR9 (for review, see, Daubenberger, C.A., Curr. Opin. MoI. Ther. 9(l):45-52 (2007)), T- helper epitopes, lipid-A and derivatives or variants thereof, liposomes, cytokines, (e.g.
  • GMCSF granulocyte macrophage-colony stimulating factor
  • CD40 CD28
  • CD70 CD70
  • IL-2 heat- shock protein
  • HSP heat- shock protein
  • OF40 CD 134
  • non ionic block copolymers incomplete
  • adjuvants for use with the compositions described herein are adjuvants containing saponins ⁇ e.g. QS21), either alone or combined with cholesterol and phospholipid in the characteristic form of an ISCOM ("immune stimulating complex,"yor review, see Barr and Mitchell, Immunology and Cell Biology 74: 8-25 (1996); and Skene and Sutton, Methods 40: 53-59 (2006)).
  • aluminum-based compounds such as aluminum hydroxide (Al(OH) 3 ), aluminum hydroxyphosphate (AlPO 4 ), amorphous aluminum hydroxyphosphate sulfate (AAHS) or so-called “alum” (KAl(SO 4 )' 12H 2 O), many of which have been approved for administration into humans by regulatory agencies worldwide, may be combined with the compositions provided herein.
  • EXAMPLE 1 Construction, rescue and genomic stability of Ad6- CEA-LTB/HER2-ECDM dicistronic vector.
  • a CEA-LTB fusion was engineered by joining the cDNA of the CEA protein deleted of the anchoring sequence (nucleotide 1 to 2037) to the LTB subunit of the E. coli heat labile enterotoxin to which the signal peptide coding sequence had been removed (nucleotide 64 to 375), as previously described (WO 2005/077977).
  • a C-terminal deletion mutant of p 185 HEPv2/neu protein
  • HER2ECDTM was constructed as previously described (WO 2005/012527). Nucleotide sequences encoding both the CEA-LTB fusion protein and the HER2ECDTM were codon- optimized for optimal expression in human cells.
  • a dicistronic cassette was assembled in tandem (FIGURE 1).
  • the specific design of the cassette included both a human cytomegalovirus (CMV) promoter and a mouse CMV promoter, which enable the expression of the CEA-LTB fusion protein and the HER2ECD.TM protein, respectively.
  • CMV human cytomegalovirus
  • Two different promoters were chosen to reduce the possibility of internal recombination events and to avoid the reduction of gene expression due to squelching of transcription factors.
  • the nucleotide sequences coding for the CEA and HER2 variants were followed by the BGH and SV40 polyadenylation signals, respectively.
  • Shuttle plasmid pNEBAd6-CEA-LTB/HER2ECDTM was constructed by removing the dicistronic expression cassette from polyMRK-CEA-LTB/HER2ECDTM-SV40 by Spel and AflII and inserting it in the same restriction sites of pNEBAd ⁇ - 2HCMVnefMCMVgagpol.
  • the genetic structure of pNEBAd6-CEA-LTB/HER2ECDTM was verified by restriction enzyme analysis.
  • transgene containing fragment was liberated from shuttle plasmid pNEBAd ⁇ - CEA-LTB/HER2ECDTM by digestion with restriction enzymes Pad and Pmel and gel purified.
  • the purified transgene fragment was then co -transformed into E. coli strain BJ5183 with linearized (C/ ⁇ l-digested) adenoviral backbone plasmid, pAd6MRKDElDE3. Plasmid DNA isolated from BJ5183 transformants was then transformed into competent E. coli DH5 ⁇ for screening by restriction analysis.
  • the desired plasmid pMRKAd6CEA-LTB-HER2ECDTM was verified by restriction enzyme digestion and DNA sequence analysis. This plasmid was cut with Pad to release the Ad ITRs and subsequently transfected in PerC-6 cells, which were serially passaged until a cytopathic effect was observed.
  • Red blood cells were removed by osmotic lysis by adding 1 ml of 0.1X PBS to the cell pellet and vortexing for approximately 15s. One ml of 2x PBS was then added and the volume was brought to 4ml with Ix PBS. Cells were pelleted by centrifugation at 1200 rpm for 10 min at RT, and the pellet was resuspended in 1 ml RlO medium. Viable cells were counted using Turks staining.
  • Splenocytes were plated at 5x105 and 2.5x105 ells/well in duplicate and incubated for 2Oh at 37°C with l ⁇ g/ml suspension of each peptide.
  • Concanavalin A (ConA) was used as positive internal control for each mouse at 5 ⁇ g/ml. After washing with PBS, 0.05% Tween 20, plates were incubated O/N at 4°C with 50 ⁇ l/well of biotin-conjugated rat anti-mouse IFN ⁇ (RatlgGl, clone XMG 1.2, PharMingen) diluted to 1 :2500 in assay buffer.
  • Ad ⁇ monocistronic vectors were constructed: one encoding CEA-LTB under the transcriptional control of the human CMV promoter, and one encoding the ECD. TM portion of HER2/Neu controlled by the mouse CMV promoter. The vectors were rescued and amplified. Subsequently, the genomic structures of the vectors were compared by restriction analysis, revealing no major rearrangements. Expression of each of the antigens was verified by ELISA and by FACS staining upon infection of HeLa cells (not shown). The expression levels of each of the monocistronic Ad6 vectors encoding the single TAAs was comparable to that of the dicistronic Ad6 vector encoding both the CEA and HER2 variants.
  • mice Groups of C57BL/6 mice were immunized with: 1) Ad6-CEA-LTB; 2) Ad6- ECD.TM; 3) Ad6-CEA-LTB+Ad6-ECD.TM; 4) Ad6-dicistronic. Each mouse received IM injections of 108 viral particles, two weeks apart and immune response was measured by intracellular staining for IFN ⁇ 14 days later. Both a CD8+ (FIGURE 4A) and a CD4+ (FIGURE 4B) immune response was elicited in immunized mice.
  • Ad6 monocistronic vectors resulted in comparable immunogenicity for each relative antigen, thus showing that efficacy of Ad6 dicistronic is not affected by mechanisms such as transcription factors squelching between human and mouse CMV promoter within the same antigen presenting cell.
  • CEA-HER2.Tg human HER2 and human CEA double transgenic mice were generated and used as animal model for immunization studies.
  • CEA-HER2.Tg mice were derived by crossing human CEA transgenic mice with a line of human HER2 transgenic mice developed by Wei-Zen Wei (Wayne State University, Detroit).
  • CEA.tg mice (H-2b) were provided by J. Primus (Vanderbilt University) and kept in standard conditions (Clarke et al. Cancer Res. 58:1469-77 (1998)).
  • the HER2 transgenic mice carry the full length wild type cDNA of human HER2 under the control of the whey acidic protein promoter (WAP) (Piechocki et al. Human ErbB-2 (Her-2) transgenic mice: a model system for testing Her-2 based vaccines. J Immunol. 171(11):5787-94 (2003)). These mice express HER2 protein in the secretory mammary epithelia during pregnancy and lactation, and constitutively express HER2 in the Bergman glial cells within the molecular layer of the cerebellum (Piechocki et al., supra). No neoplastic transformation was detected in any tissue of the HER2 transgenic mice (Piechocki et al.).
  • WAP whey acidic protein promoter
  • CEA-HER2.Tg mice were immunized with two intramuscular (IM) injections of lxlOlO vp of the MRKAd ⁇ vector two weeks apart. Fifteen days later, mice were euthanized and splenocytes were analyzed by intracellular staining for HER2 and CEA specific IFN ⁇ production. A significant immune response was measured against CEA and HER2 peptide pools (see FIGURE 5), particularly against the C-terminal region of CEA (CEA-2) and of HER2- ECDTM (Neu-2). The immune response against LTB was similarly high.
  • Ad6 dicistronic vector induces immune response in mice engrafted with human immune system.
  • NOD/scid-DRl chimeric mice were engrafted with HLA- A2 human cord blood MNC (CBMNC), either at 5 days old or 8-12 weeks old. 2 (adult) or 6-8 (neonates) weeks after engraftment, mice were immunized twice with MRKAd ⁇ CEA-LTB/HER2-ECDM IM (10l0 viral particles), 2 weeks apart.
  • CBMNC human cord blood MNC

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Abstract

L'invention concerne les vecteurs de l'adénovirus discistronique et plasmidiques exprimant tous deux l'antigène spécifique des tumeurs CEA à antigène carcinoembryonnaire ou une partie de celui-ci et l'antigène 2/neu du facteur de croissance de l'épiderme humain (HER2/neu) ou une partie de celui-ci. La présente invention concerne également des procédés d'induction d'une réponse immunitaire contre les tumeurs spécifiques CEA et/ou HER2 en utilisant les compositions et les molécules décrites ici. Les gènes CEA et HER2 codent les antigènes généralement associés au développement des carcinomes humains. La présente invention concerne des compositions et des procédés de déclenchement ou de stimulation de l'immunité aux produits protéiques exprimés par les antigènes spécifiques des tumeurs CEA et/ou HER2/neu, une expression de CEA et/ou d'HER2 aberrante étant associée à un carcinome ou à son développement. Les vecteurs dicistroniques de la présente invention sont utilisés dans des vaccins et des compositions pharmaceutiques dans la prévention et/ou le traitement du cancer.
PCT/EP2007/057358 2006-07-24 2007-07-17 Construction de multiples antigènes et leur utilisation Ceased WO2008012237A1 (fr)

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