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WO2004041065A2 - Procedes de prevention et de traitement de tumeurs de her-2/neu - Google Patents

Procedes de prevention et de traitement de tumeurs de her-2/neu Download PDF

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Publication number
WO2004041065A2
WO2004041065A2 PCT/US2003/034362 US0334362W WO2004041065A2 WO 2004041065 A2 WO2004041065 A2 WO 2004041065A2 US 0334362 W US0334362 W US 0334362W WO 2004041065 A2 WO2004041065 A2 WO 2004041065A2
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neu
cells
vector
ofthe
composition
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WO2004041065A3 (fr
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John J. Morris
Jay A. Berzofsky
Yoshio Sakai
Jong-Myun Park
Masake Terabe
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US Department of Health and Human Services
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US Department of Health and Human Services
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Priority to AU2003286770A priority Critical patent/AU2003286770A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

  • the invention provides a method for treating a subject suffering from cancer or is identified as being susceptible to developing cancer.
  • a recombinant E1-, E3-deleted non-replicating adenoviral gene transfer vector expressing a truncated ⁇ ER.-2/neu oncogene is effective as a vaccine against ⁇ ER.-2/neu expressing breast cancer cells and other tumors.
  • NCI National Cancer Institute
  • NCHS National Center for Health Statistics
  • proto-oncogenes a number of human malignancies have been identified as correlated with the presence and expression of "oncogenes" in the human genome. More than twenty different oncogenes have now been implicated in tumorigenesis, and are thought to play a direct role in human cancer. Many of these oncogenes apparently evolve through mutagenesis of a normal cellular counterpart, termed a "proto-oncogene", which leads to either an altered expression or activity ofthe expression product. There is considerable data linking proto-oncogenes to cell growth, including their expression in response to certain proliferation signals and expression during embryonic development. Moreover, a number ofthe proto-oncogenes are related to either a growth factor or a growth factor receptor.
  • neu oncogene referred to variously as c-erbB-2, HER-2 or neu oncogene (referred to herein simply as the neu oncogene), is now known to be intimately involved in the pathogenesis of cancers ofthe human female breast and genital tract.
  • the neu oncogene which encodes a pi 85 tumor antigen, was first identified in transfection studies in which NTH 3T3 cells were transfected with DNA from chemically induced rat neuroglioblastomas.
  • the pi 85 protein has an extracellular, transmembrane, and intracellular domain, and therefore has a structure consistent with that of a growth factor receptor.
  • the human neu gene was first isolated due to its homology with v-erbB and EGF-r probes (Semba K et al., PNAS, USA 82(19):6497-501 (Oct.1985); King, CR et al, Science 229(4717):974-6 (Sept. 6,1985)).
  • the neu oncogene is of particular importance to medical science because its presence is correlated with the incidence of cancers ofthe human breast and female genital tract. Moreover, amplification/overexpression of this gene has been directly correlated with relapse and survival in human breast cancer (Slamon DJ, et al. Science 235(4785): 177-82 (Jan. 9, 1987). Therefore, it is an extremely important goal of medical science to produce a vaccine that is prophylactic for individuals identified as being in a high risk group, such as for example, a familial history of breast cancer, and/or treating an individual diagnosed with breast cancer.
  • the present invention provides compositions and methods for treating an individual suffering from or susceptible to developing ⁇ ER.-2/neu positive cancer cells such as breast cancer cells.
  • Non-replicating vectors comprising an oncogene lacking kinase signaling or intracellular domains were used to transduce antigen presenting cells which present antigenic fragments to the immune effector cells resulting in a dramatic decrease of breast cancer cells.
  • the invention provides for methods and compositions for treating an individual suffering from or susceptible to, tumor growth development.
  • the tumor to be treated by the methods and compositions ofthe invention are directed to HER-2 expressing tumors, such as for example breast cancer.
  • a ⁇ ER.-2lneu molecule which lacks the intracellular domain, responsible for kinase signaling is cloned into a non-replicating vector such as for example adenoviral vector.
  • the tumor antigen and vector is not limited to HER- 2 or adenoviral vectors but, can include vectors such as plasmids, viral vectors, naked DNA and the like. Any antigen that is oncogenic may be used such as for example, CEA, K-ras, p53 and the like.
  • compositions as referred to herein, are intended to include any nucleic acid molecules, including naked DNA, and express a desired tumor antigen.
  • the invention provides a composition comprising an effective amount of a truncated ⁇ ER.-2/neu molecule in a pharmaceutically accepted carrier.
  • the HBK-2/neu molecule is cloned into a vector which expresses HER-2/ neu gene products, fragments or complementary sequences thereof.
  • the composition comprises the BER-2/neu molecule, fragments or complementary sequences thereof, cloned into a vector which expresses HER-2/ neu gene products.
  • the vector is an adenovirus shuttle vector and comprises deletions in El and E3 early region genes.
  • the composition comprises truncated HER- 2lneu transmembrane domains and or extracellular domains or fragments and complements thereof.
  • the truncated rat ⁇ ER2/neu oncogene comprising the extracellular and transmembrane domains of ⁇ ER2/neu [rHER2/ «ew(ECDtm)], complements or fragments thereof, are cloned into a shuttle plasmid.
  • the composition comprises an adenovirus vector wherein the vector is homologously recombined with the plasmid comprising the extracellular and transmembrane domains of ⁇ ER2/neu by co-transfection into 293 HEK cells.
  • the homologously recombined adenovirus vector and the plasmid produce a recombinant adenovirus vector expressing the extracellular and transmembrane domains of ⁇ ER2/neu (Ad.rHER2/new(ECDtm)), or fragments thereof.
  • the recombinant adenovirus vector expresses at least about 50% to about 99% ofthe extracellular domain and at least about 50% to about 99% ofthe transmembrane domain of BERl/neu, or any fragment or combination thereof.
  • the composition comprising the recombinant adenovirus vector expresses at least about 50% to about 99% ofthe extracellular domain of ⁇ ER2/neu, complements or fragments thereof.
  • the composition comprising the truncated HER- 2/neu lacks a ⁇ ER.-2/neu intracellular domain and/or the HER-2/neu vector does not encode for intracellular domain gene products with kinase activity.
  • composition comprising ti ⁇ K-2/neu is encoded by a sequence having at least about 80% up to about 99.9% sequence identity to a molecule identified by SEQ ID NO: 1, complements, species variants or fragments thereof.
  • composition comprising ⁇ ER.-2/neu is encoded by a sequence having at least about 90% sequence identity to a molecule identified by SEQ ID NO: 1, complements, species variants, such as human HER-2/neu or fragments thereof.
  • compositions ofthe invention that are administered to an individual suffering from or susceptible to tumor growth are taken up by antigen presenting cells such as dendritic cells which present the antigen on their cell surface.
  • Dendritic cells are powerful antigen presenting cells can present micro or nanomolar amounts of antigen on their surface. Amounts of peptides required for antigen presentation are well known in the art.
  • the immune effector cells are T and B lymphocytes, and in particular CD4+ and CD8+ T cells are activated.
  • Activated T cells recognize, for example, HER-2 tumor cells, resulting in the lysis of such tumor cells.
  • Antigen presentation to the cells ofthe immune system are also primed to recognize the antigen administered to an individual, thereby, becoming activated when any of these immune effector cells recognize such tumors.
  • compositions ofthe invention are adrninistered to individuals diagnosed with cancer, in post-operative procedures, as a prophylactic to any individual, especially for example, individuals who are at high risk of developing cancer identified by any means, such as for example a family history.
  • compositions ofthe invention can be co- administered with adjuvants, such as cytokines, and/or chemotherapy.
  • the antigen presenting cells are dendritic cells.
  • compositions ofthe invention comprise non-replicating adenoviral vectors with deletions in the E1-, and E3-regions and express a ⁇ ER.-2/neu molecule that lacks the intracellular domain and/or kinase activity.
  • the recombinant adenoviral vector expressing HER-2 comprises HER-2 transmembrane domains and/or extracellular domains.
  • the vector can express the entire ⁇ ER-2/neu molecule, or fragments thereof.
  • the activated immune effector cells recognize and kill HER-2/neu positive tumor cells as measured by cytotoxic T cell assays.
  • the HER-2/neu is encoded by a sequence having at least about 80% sequence identity to a molecule identified by SEQ ID NO: 1, more preferably, the HER-2/neu is encoded by a sequence having at least about 90% sequence identity to a molecule identified by SEQ ID NO: 1, most preferably, the HER- 2/neu is encoded by a sequence having at least about 95% sequence identity to a molecule identified by SEQ TD NO: 1.
  • any variants, fragments, substantial fragments mutants and the like, of HER-2 or any other tumor antigen can be expressed by a desired vector such as for example, the recombinant adenovirus vector lacking the E1-, E3-early region genes.
  • peptides are administered to an individual suffering from or susceptible to cancers.
  • the peptides of BER-2/neu are encoded by a sequence having at least about 80% sequence identity to a molecule identified by SEQ ID NO: 1 or fragments thereof, more preferably the peptides of HER- 2/neu are encoded by a sequence having at least about 90% sequence identity to a molecule identified by SEQ ID NO: 1 or fragments thereof, even more preferably the peptides of BE -2/neu are encoded by a sequence having at least about 95% sequence identity to a molecule identified by SEQ ID NO: 1, or fragments thereof.
  • any tumor antigen polypeptide can be administered to an individual diagnosed as suffering from or susceptible to cancers.
  • the polypeptides. corresponding to identified tumor antigens can be used to stimulate the cells ofthe immune system to recognize and lyse tumor cells expressing tumor antigens.
  • FIG. 1 is a schematic illustration of he recombinant Adenovirus vectors comprising truncated rat EERl/neu oncogenes with extracellular and transmembrane domains [rHER2/ «e «(ECDtm)] or the extracellular domain only [rHER2/ « ⁇ u(ECD)].
  • Ad.EYFP is a vector expressing enhanced yellow fluorescent protein.
  • Figure 2 is a schematic illustration showing that mice progressively develop cancers in mammary glands between 15 and 25 weeks of age.
  • Figure 3 is a cell sorter fluorescence graph showing murine bone marrow cells cultured for 8-10 days under conditions described infra, generated a CDI lc(+), CDI lb(+), CD8a(-) myeloid dendritic cells (DC) phenotype.
  • Figure 4 is a cell sorter fluorescence graph showing on day 8, DCs were infected with Ad.EYFP at varying MOFs. On day 10, 70% and 56% of DCs expressed EYFP at MOFs 30 and 3, respectively.
  • Figure 6 is a photograph ofthe results of a Northern Blot analysis of RNA isolated from DCs infected with Ad.rHER2/new(ECD-tm) or Ad.rHER2/we ⁇ (ECD), and probed with 32 P-labeled fragment of rat Her2/rae « cDNA showed the expected ⁇ ERl/neu RNA's.
  • Figure 7 is a graph showing co-culturing of irradiated DCs infected with either of the two Ad. ⁇ ER2/neu vectors with splenic lymphocytes from na ⁇ ve mice resulted in a greater level of stimulation than culture with uninfected DCs or DCs infected with the Ad.null vector.
  • Figure 8 is a graph showing the results of three weekly vaccinations of BALB/c- neu ⁇ transgenic mice with allogeneic DCs infected with Ad.rHER2/ «ew(ECDtm) or Ad.rHER2/neM(ECD) beginning at 5-6 weeks of age.
  • Figure 9 is a graph showing the results of protection against tumors of mice vaccinated with various rAd vectors.
  • Ad.rHER2/ «eM(ECDtm) conferred significant protection over all other treatment groups.
  • Figure 10 is a schematic representation showing the restriction map of Ad.HER- 2lneu, an E1-, E3-deleted non-replicating type 5 adenoviral gene transfer vector expressing a truncated ⁇ ER.-2/neu.
  • Figure 11 A and 1 IB are graphs showing the average number of tumors in Rer2/neu transgenic mice with Ad-Null, Ad-ECD(extracellular domain of Her2/neu), Ad-TM (ECD and transmembrane domain of Her2/neu) or none, starting at the age of 4-6 weeks (Fig. 11A;5 mice per group) or 6-8 weeks (Fig. 1 lB;3-4 mice per group). Mice were immunized totally five times with an interval of 3-4 weeks.
  • Figure 12 are graphs showing the titrating dose of Ad- ⁇ er2/neu vaccine.
  • Figure 13A-13F are graphs showing that CD4 + T cells are important for eliciting protective immune responses induced by Ad-Her2/ «ew vaccine. Lymphocytes were depleted with anti-CD4 (Figure 13D), anti-CD8 ( Figure 13E) or anti-CD4/anti-CD8 ( Figure 13F) treatment (0.5mg/injection, i.p.) at day 0, 1, 2, 5 of immunization with Ad- Her2/neu. Each group was immunized with 10 pfu adenovirus twice from the age of 6-8 weeks at an interval of 3 weeks, except for the group in figure 13A (control group; no immunization). Rat Ig ( Figure 13C) was used as antibody control.
  • Figures 14A-14D are graphs showing protection of tumor-injected mice by administration of Ad- ⁇ er2/neu vaccine. Protection was observed in mice immumzed at day-11 ( Figure 14C) and day 5 ( Figure 14D) of TUBO cell injection but not from na ⁇ ve (A) or Ad-null group ( Figure 14B).
  • Figurel5 is a series of graphs showing the therapeutic effects of Ad- ⁇ .er2/neu vaccine.
  • BALB/c mice were immumzed with Ad-Her2/neu (10 8 pfu, i.p., once) on days 2, 4, 7, 10 or 15, respectively, after injection with Tubo cells (10 6 cells, s.c).
  • Figures 16A- 16F are graphs showing results obtained by Ad-Her2/neu vaccination.
  • BALB/c mice were subcutaneously injected with TUBO cells (10 6 cell/injection) and were grouped by their tumor sizes after day 17 of TUBO cell injection. Mice having the smallest tumor were used for non-treated-control ( Figure 16A) or Ad- null treated group ( Figure 16B). Others were immunized once with Ad-Ker2/neu, 10 7 pfu ( Figure 16C) or 10 8 pfu ( Figures 16D, 16E, and 16F).
  • FIGS 17A-17E are graphs showing CD4 T cells responses protected against tumor growth in mammary tumor -injected mice model.
  • BALB/c mice were immunized with Ad-TM (i.p.,10 8 pfu/injection, one injection) 7 days before they were injected with TUBO cells (s.c.,10 6 cells/inj.).
  • T cells were depleted with ⁇ -CD4 or/and ⁇ -CD8 (i.p., 0.5 mg/inj) from the day of immunization.
  • CD4-depleted group was not protected from tumor growth (Figure 17C) but CD8-depleted group showed tumor prevention (Figure 17D).
  • Rat IgG was used as a control antibody (Figure 17A). Depletion of T lymphocytes were confirmed as less than 1% on day 10 of tumor injection by FACS analysis.
  • Figures 18A-18C are graphs showing Ad-Her2/new vaccination induced serum antibodies against Her2/neu and prevented tumor development.
  • BALB-neu T mice were immumzed with 10 8 pfu of vaccine at the age of 8 weeks and 11 weeks.
  • CD4 T cells ( Figure 18B) or CD8 T cells ( Figure 18C) were depleted when immunized.
  • Serum antibodies against Her2/neu were detected from tumor-protected groups ( Figures 18A and 18C) even after 23 weeks ofthe final immunization but not from non-protected group ( Figure 18B).
  • Figure s 19A and 19B are flow cytometer plots showing the DC phenotype.
  • Figure 19A shows the expression of various surface markers of bone marrow cells from the femures of BALB/c female mice cultured under GM-CSF for 10 days and examined by flow cytometry analysis. Open curves: isotype controls.
  • Figure 19B shows the results from DC on day 8, infected with Ad.HER2 or Ad.null at MOI 30, harvested on day 10 and examined for the expression level of surface markers.
  • Figures 20A is a gel showing Northern blotting analysis of ⁇ ER-2/neu expression by Ad.HER2.
  • Figure 20B is a scattergram showing the flow cytomery analysis of HER- 2/neu expression by Ad.HER2. Open curves: isotype controls.
  • Figure 21 is a graph showing stimulation of splenocytes proliferation by DC A ⁇ I . HER2 . Vertical bar: standard error.
  • Figures 22A and 22B are graphs showing the antitumor vaccine efficacy of DC Ad .
  • Figure 23 A is a graph showing anti-HER2/neu antibody in sera.
  • Figure 23B shows a scatter gram of IFN- ⁇ producing cells in spleen of vaccinated BALB-newT mice.
  • Figure 24 are photographs of immunohistochemical stains showing infiltration of CD4+ and CD8+ cells in mammary glands of mice vaccinated with DCACI.HE R 2.
  • Figure 25 is a graph showing protection against tumors by DCA ⁇ I.HER 2 vaccination requires CD4+ cells.
  • Figure 26A and 26B are graphs showing that DCA ⁇ I.H E R 2 vaccination is not affected by pre-existing anti-adenovirus immunity.
  • the present invention provides compositions and methods for the treatment of tumors.
  • the methods include both, prophylaxis and treatment of individuals suffering from or susceptible to, tumors expressing HER-2 oncogene.
  • the compositions preferably comprise the use of non-replicating vectors encoding a non-signaling modified tumor antigen.
  • the methods involve the transfer of a gene for the target antigen into dendritic cells.
  • the advantages include: expression ofthe tumor antigen by the gene transfer vector without requiring detailed knowledge ofthe peptide epitopes or binding affinities for a particular HLA molecule; the dendritic cells can process the antigen naturally leading to a more effective antigen presentation and an improved immune response ofthe desired type.
  • a “genetic modification” refers to any addition, deletion or disruption to a cell's normal nucleotides. Any method which can achieve the genetic modification of APCs are within the spirit and scope of this invention. Art recognized methods include viral mediated gene transfer, liposome mediated transfer, transformation, transfection and transduction.
  • nucleic acid molecule refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules”), or any phosphoester analogues thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix.
  • Double stranded DNA--DNA, DNA-RNA and RNA-RNA helices are possible.
  • nucleic acid molecule and in particular DNA or RNA molecule, refers only to the primary and secondary structure ofthe molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • a "recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.
  • fragment or segment as applied to a nucleic acid sequence, gene or polypeptide, will ordinarily be at least about 5 contiguous nucleic acid bases (for nucleic acid sequence or gene) or amino acids (for polypeptides), typically at least about 10 contiguous nucleic acid bases or amino acids, more typically at least about 20 contiguous nucleic acid bases or amino acids, usually at least about 30 contiguous nucleic acid bases or amino acids, preferably at least about 40 contiguous nucleic acid bases or amino acids, more preferably at least about 50 contiguous nucleic acid bases or amino acids, and even more preferably at least about 60 to 80 or more contiguous nucleic acid bases or amino acids in length.
  • “Overlapping fragments” as used herein, refer to contiguous nucleic acid or peptide fragments which begin at the amino terminal end of a nucleic acid or protein and end at the carboxy terminal end ofthe nucleic acid or protein. Each nucleic acid or peptide fragment has at least about one contiguous nucleic acid or amino acid position in common with the next nucleic acid or peptide fragment, more preferably at least about three contiguous nucleic acid bases or amino acid positions in common, most preferably at least about ten contiguous nucleic acid bases amino acid positions in common.
  • a significant "fragment" in a nucleic acid context is a contiguous segment of at least about 17 nucleotides, generally at least 20 nucleotides, more generally at least 23 nucleotides, ordinarily at least 26 nucleotides, more ordinarily at least 29 nucleotides, often at least 32 nucleotides, more often at least 35 nucleotides, typically at least 38 nucleotides, more typically at least 41 nucleotides, usually at least 44 nucleotides, more usually at least 47 nucleotides, preferably at least 50 nucleotides, more preferably at least 53 nucleotides, and in particularly preferred embodiments will be at least 56 or more nucleotides.
  • a “vector” is a composition which can transduce, transfect, transform or infect a cell, thereby causing the cell to express nucleic acids and/or proteins other than those native to the cell, or in a manner not native to the cell.
  • a cell is "transduced” by a nucleic acid when the nucleic acid is translocated into the cell from the extracellular environment. Any method of transferring a nucleic acid into the cell may be used; the term, unless otherwise indicated, does not imply any particular method of delivering a nucleic acid into a cell.
  • a cell is "transformed” by a nucleic acid when the nucleic acid is transduced into the cell and stably replicated.
  • a vector includes a nucleic acid (ordinarily RNA or DNA) to be expressed by the cell.
  • a vector optionally includes materials to aid in achieving entry ofthe nucleic acid into the cell, such as a viral particle, liposome, protein coating or the like.
  • a "cell transduction vector” is a vector which encodes a nucleic acid capable of stable replication and expression in a cell once the nucleic acid is transduced into the cell.
  • downstream when used in reference to a direction along a nucleotide sequence means in the direction from the 5' to the 3' end.
  • upstream means in the direction from the 3' to the 5' end.
  • the term "gene” means the gene and all currently known variants thereof and any further variants which may be elucidated.
  • variants when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to a wild type gene. This definition may also include, for example, "allelic”, “splice,” “species,” or “polymorphic” variants.
  • a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
  • the corresponding polypeptide may possess additional functional domains or an absence of domains.
  • Species variants are polynucleotide sequences that vary from one species to another. Of particular utility in the invention are variants of wild type target genes.
  • Variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes that give rise to variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • variant of polypeptides refers to an amino acid sequence that is altered by one or more amino acid residues.
  • the variant may have "conservative” changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have "nonconservative” changes (e.g., replacement of glycine with tryptophan).
  • Analogous minor variations may also include amino acid deletions or insertions, or both.
  • Guidance in determimng which amino acid residues may be substituted, inserted, or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR).
  • polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs,) or single base mutations in which the polynucleotide sequence varies by one base.
  • SNPs single nucleotide polymorphisms
  • complementary or “complements” are used interchangeably throughout and mean that two sequences are complementary when the sequence of one can bind to the sequence ofthe other in an anti-parallel sense wherein the 3'-end of each sequence binds to the 5'-end ofthe other sequence and each A, T(U), G, and C of one sequence is then aligned with a T(U), A, C, and G, respectively, ofthe other sequence.
  • the complementary sequence ofthe oligonucleotide has at least 80% or 90%, preferably 95%, most preferably 100%, complementarity to a defined sequence.
  • alleles or variants thereof can be identified.
  • a BLAST program also can be employed to assess such sequence identity.
  • complementary sequence as it refers to a polynucleotide sequence, relates to the base sequence in another nucleic acid molecule by the base-pairing rules. More particularly, the term or like term refers to the hybridization or base pairing between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid to be sequenced or amplified.
  • Complementary nucleotides are, generally, A and T (or A and U), or C and G.
  • Two single stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 95% ofthe nucleotides ofthe other strand, usually at least about 98%, and more preferably from about 99 % to about 100%.
  • Complementary polynucleotide sequences can be identified by a variety of approaches including use of well-known computer algorithms and software; " for example the BLAST program.
  • heterologous component refers to a component that is introduced into or produced within a different entity from that in which it is naturally located.
  • a polynucleotide derived from one organism and introduced by genetic engineering techniques into a different organism is a heterologous polynucleotide which, if expressed, can encode a heterologous polypeptide.
  • a promoter or enhancer that is removed from its native coding sequence and operably linked to a different coding sequence is a heterologous promoter or enhancer. Possible alternative terminology includes “foreign” or "exogenous”.
  • a heterologous nucleotide sequence may encode a sequence of amino acids, i.e. a peptide or a polypeptide.
  • promoter refers to a polynucleotide sequence that controls transcription of a gene or coding sequence to which it is operably linked.
  • a large number of promoters, including constitutive, inducible and repressible promoters, from a variety of different sources, are well known in the art and are available as or within cloned polynucleotide sequences (from, e.g., depositories such as the ATCC as well as other commercial or individual sources).
  • an “enhancer,” as used herein, refers to a polynucleotide sequence that enhances transcription of a gene or coding sequence to which it is operably linked.
  • enhancers from a variety of different sources are well known in the art and available as or within cloned polynucleotide sequences (from, e.g., depositories such as the ATCC as well as other commercial or individual sources).
  • a number of polynucleotides comprising promoter sequences (such as the commonly-used CMV promoter) also comprise enhancer sequences.
  • "Operably linked” refers to a juxtaposition, wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a promoter is operably linked to a coding sequence if the promoter controls transcription ofthe coding sequence. Although an operably linked promoter is generally located upstream ofthe coding sequence, it is not necessarily contiguous with it.
  • An enhancer is operably linked to a coding sequence if the enhancer increases transcription ofthe coding sequence. Operably linked enhancers can be located upstream, within or downstream of coding sequences.
  • a polyadenylation sequence is operably linked to a coding sequence if it is located at the downstream end ofthe coding sequence such that transcription proceeds through the coding sequence into the polyadenylation sequence.
  • Gene delivery are terms referring to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgenes") into a host cell, irrespective ofthe method used for the introduction.
  • exogenous polynucleotide sometimes referred to as a "transgenes”
  • transgenes exogenous polynucleotide
  • Such methods include a variety of well-known techniques such as vector-mediated gene transfer (by, e.g., viral infection/transfection, or various other protein-based or lipid- based gene delivery complexes) as well as techniques facilitating the delivery of "naked" polynucleotides (such as electroporation, "gene gun” delivery and various other techniques used for the introduction of polynucleotides).
  • the introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon ofthe host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
  • a replicon ofthe host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
  • a number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art and described herein.
  • a target cell or “recipient cell” refers to an individual cell or cell which is desired to be, or has been, a recipient of exogenous nucleic acid molecules, polynucleotides and/or proteins. The term is also intended to include progeny of a single cell.
  • "In vivo" gene delivery, gene transfer, gene therapy and the like as used herein, are terms referring to the introduction of a vector comprising an exogenous polynucleotide directly into the body of an organism, such as a human or non-human mammal, whereby the exogenous polynucleotide is introduced to a cell of such organism in vivo.
  • a cell is "transduced" by a nucleic acid when the nucleic acid is translocated into the cell from the extracellular environment. Any method of transferring a nucleic acid into the cell may be used; the term, unless otherwise indicated, does not imply any particular method of delivering a nucleic acid into a cell.
  • a cell is "transformed” by a nucleic acid when the nucleic acid is transduced into the cell and stably replicated.
  • a vector includes a nucleic acid (ordinarily RNA or DNA) to be expressed by the cell.
  • a vector optionally includes materials to aid in achieving entry ofthe nucleic acid into the cell, such as a viral particle, liposome, protein coating or the like.
  • a "cell transduction vector” is a vector which encodes a nucleic acid capable of stable replication and expression in a cell once the nucleic acid is transduced into the cell.
  • homologous recombination means a nucleotide sequence on one vector is homologous to a nucleotide sequence on another vector. Using restriction enzymes to cut the two sequences and ligating the two sequences results in the two vectors combining.
  • the plasmids used herein such as pDC316, encodes the adenoviral 5 '-inverted terminal repeat sequences and can homologously recombine of with the adenoviral backbone plasmid pBGH.lox.deltaElE3Cre (MicrobixTM, Toronto, Ontario).
  • flanking DNA both at the 5' and 3' ends
  • flanking DNA both at the 5' and 3' ends
  • homologous nucleic acid sequences when compared, exhibit significant sequence identity or similarity.
  • the standards for homology in nucleic acids are either measures for homology generally used in the art by sequence comparison or based upon hybridization conditions. The hybridization conditions are described in greater detail below.
  • Stringency is meant the combination of conditions to which nucleic acids are subject that cause the duplex to dissociate, such as temperature, ionic strength, and concentration of additives such as formamide. Conditions that are more likely to cause the duplex to dissociate are called “higher stringency”, e.g. higher temperature, lower ionic strength and higher concentration of formamide.
  • relatively stringent conditions For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g., one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50° C. to about 70° C.
  • hybridization may occur even though the sequences of probe and target strand are not perfectly complementary, but are mismatched at one or more positions.
  • Conditions may be rendered less stringent by increasing salt concentration and decreasing temperature.
  • a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37° C. to about 55° C, while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20° C. to about 55° C.
  • a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37° C. to about 55° C
  • a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20° C. to about 55° C.
  • hybridizing conditions when used with a maintenance time period, indicates subjecting the hybridization reaction admixture, in context ofthe concentration ofthe reactants and accompanying reagents in the admixture, to time, temperature, pH conditions sufficient to allow the polynucleotide probe to anneal with the target sequence, typically to form the nucleic acid duplex.
  • Such time, temperature and pH conditions required to accomplish the hybridization depend, as is well known in the art on the length ofthe polynucleotide probe to be hybridized, the degree of complementarity between the polynucleotide probe and the target, the guanidine and cytosine content ofthe polynucleotide, the stringency ofthe hybridization desired, and the presence of salts or additional reagents in the hybridization reaction admixture as may affect the kinetics of hybridization.
  • Methods for optimizing hybridization conditions for a given hybridization reaction admixture are well known in the art.
  • nucleic acid sequence comparison context means either that the segments, or their complementary strands, when compared, are identical when optimally aligned, with appropriate nucleotide insertions or deletions, in at least about 50% ofthe nucleotides, generally at least 56%, more generally at least 59%, ordinarily at least 62%, more ordinarily at least 65%, often at least 68%, more often at least 71%, typically at least 74%, more typically at least 77%, usually at least 80%, more usually at least about 85%, preferably at least about 90%, more preferably at least about 95 to 98% or more, and in particular embodiments, as high at about 99% or more ofthe nucleotides.
  • substantial homology exists when the segments will hybridize under selective hybridization conditions, to a strand, or its complement, typically using a fragment derived from SEQ ID NO: 1.
  • selective hybridization will occur when there is at least about 55% homology over a stretch of at least about 14 nucleotides, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90%. See Kanehisa (1984) Nuc. Acids Res. 12:203-213.
  • the length of homology comparison may be over longer stretches, and in certain embodiments will be over a stretch of at least about 17 nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 40 nucleotides, preferably at least about 50 nucleotides, and more preferably at least about 75 to 100 or more nucleotides.
  • the endpoints ofthe segments may be at many different pair combinations.
  • an “antigen” is any substance that reacts specifically with antibodies or T lymphocytes (T cells).
  • An “antigen-binding site” is the part of an immunoglobulin molecule that specifically binds an antigen. Additionally, an antigen-binding site includes any such site on any antigen-binding molecule, including, but not limited to, an MHC molecule or T cell receptor.
  • Antigen processing refers to the degradation of an antigen into fragments (e.g., the degradation of a protein into peptides) and the association of one or more of these fragments (e.g., via binding) with MHC molecules for presentation by "antigen-presenting cells" to specific T cells.
  • DC Densenchymal cells
  • TCR/CD3 T-cell receptor/CD3
  • MHC major histocompatibility complex
  • the second type of signal is neither antigen-specific nor MHC- restricted, and can lead to a full proliferation response of T cells and induction of T cell effector functions in the presence ofthe first type of signals.
  • This two-fold signaling can, therefore, result in a vigorous immune response.
  • DC arise from bone marrow-derived precursors. Immature DC are found in the peripheral blood and cord blood and in the thymus. Additional immature populations may be present elsewhere. DC of various stages of maturity are also found in the spleen, lymph nodes, tonsils, and human intestine. Avian DC may also be found in the bursa of Fabricius, a primary immune organ unique to avians.
  • the dendritic cells ofthe present invention are mammalian, preferably human, mouse, or rat.
  • a "co-stimulatory molecule” encompasses any single molecule or combination of molecules which, when acting together with a peptide MHC complex bound by a T cell receptor on the surface of a T cell, provides a co-stimulatory effect which achieves activation ofthe T cell that binds the peptide.
  • a "transgenic animal” is created when gene manipulation is used to modify the germ cells of animals permanently. Typically, foreign genes are placed in the gei ⁇ ome by "transgenesis” generating a transgenic organism.
  • An example of a transgenic mouse model of breast cancer is the neuT transgenic mouse [Sacco et al, Breast Cancer Res. Treat., 47:171-180 (1998)] wherein the rat ⁇ ER-2/neu oncogene is under the control of an MMTV promoter.
  • Diagnostic or “diagnosed” means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity.
  • the "sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives”). Diseased individuals not detected by the assay are “false negatives.” Subjects who are not diseased and who test negative in the assay, are termed “true negatives.”
  • the "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive” rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
  • patient or “individual” are used interchangeably herein, and is meant a mammalian subject to be treated, with human patients being preferred.
  • the methods ofthe invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates.
  • fresh tumors refer to tumors removed from a host by surgical or other means.
  • proliferative growth disorder refers to a condition characterized by uncontrolled, abnormal growth of cells.
  • the cancer to be treated is breast cancer and the abnormal proliferation of cells in the breast can be any cell in the organ.
  • cancer include but are not limited to, carcinoma, blastoma, and sarcoma.
  • carcinoma refers to a new growth that arises from epithelium, found in skin or, more commonly, the lining of body organs.
  • Treatment is an intervention performed with the intention of preventing the development or altering the pathology or symptoms of a disorder. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. “Treatment” may also be specified as palliative care. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. In tumor (e.g., cancer) treatment, a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e.g., radiation and/or chemotherapy.
  • other therapeutic agents e.g., radiation and/or chemotherapy.
  • in need of such treatment refers to a judgment made by a care giver such as a physician, nurse, or nurse practitioner in the case of humans that a patient requires or would benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a care giver's expertise, but that include the knowledge that the patient is ill, or will be ill, as the result of a condition that is treatable by the compositions ofthe invention.
  • Cells ofthe immune system or “immune cells” as used herein, is meant to include any cells ofthe immune system that may be assayed, including, but not limited to, B lymphocytes, also called B cells, T lymphocytes, also called T cells, natural killer (NK) cells, lymphokine-activated killer (LAK) cells, monocytes, macrophages, neutrophils, granulocytes, mast cells, platelets, Langerhans cells, stem cells, dendritic cells, peripheral blood mononuclear cells, tumor-infiltrating (TJX) cells, gene modified immune cells including hybridomas, drug modified immune cells, and derivatives, precursors or progenitors ofthe above cell types.
  • B lymphocytes also called B cells
  • T lymphocytes also called T cells
  • NK natural killer
  • LAK lymphokine-activated killer
  • monocytes monocytes
  • macrophages macrophages
  • neutrophils granulocytes
  • mast cells platelets
  • Langerhans cells
  • Immuno effector cells refers to cells capable of binding an antigen and which mediate an immune response. These cells include, but are not limited to, T cells (T lymphocytes), B cells (B lymphocytes), monocytes, macrophages, natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), for example CTL lines, CTL clones, and CTLs from tumor, inflammatory, or other infiltrates.
  • T cells or "T lymphocytes” are a subset of lymphocytes originating in the thymus and having heterodimeric receptors associated with proteins of the CD3 complex (e.g., a rearranged T cell receptor, the heterodimeric protein on the T cell surfaces responsible for antigen/MHC specificity ofthe cells).
  • T cell responses may be detected by assays for their effects on other cells (e.g., target cell killing, macrophage, activation, B-cell activation) or for the cytokines they produce.
  • CD4 is a cell surface protein important for recognition by the T cell receptor of antigenic peptides bound to MHC class JJ molecules on the surface of an APC. Upon activation, na ⁇ ve CD4 T cells differentiate into one of at least two cell types, Thl cells and Th2 cells, each type being characterized by the cytokines it produces. "Thl cells” are primarily involved in activating macrophages with respect to cellular immunity and the inflammatory response, whereas “Th2 cells” or “helper T cells” are primarily involved in stimulating B cells to produce antibodies (humoral immunity). CD4 is the receptor for the human immunodeficiency virus (HIV).
  • HAV human immunodeficiency virus
  • Effector molecules for Thl cells include, but are not limited to, IFN- ⁇ , GM-CSF, TNF- ⁇ , CD40 ligand, Fas ligand, IL-3, TNF- ⁇ , and IL-2.
  • Effector molecules for Th2 cells include, but are not limited to, IL-4, IL-5, CD40 ligand, IL-3, GS-CSF, JX-10, TGF- ⁇ , and eotaxin.
  • Activation ofthe Thl type cytokine response can suppress the Th2 type cytokine response.
  • CD8 is a cell surface protein important for recognition by the T cell receptor of antigenic peptides bound to MHC class I molecules.
  • CD8 T cells usually become “cytotoxic T cells” or “killer T cells” and activate macrophages. Effector molecules include, but are not limited to, perform, granzymes, Fas ligand, JJFN- ⁇ , TNF- ⁇ , and TNF- ⁇ .
  • activity is the ability of immune cells to respond and exhibit, on a measurable level, an immune function. Measuring the degree of activation refers to a quantitative assessment ofthe capacity of immune cells to express enhanced activity when further stimulated as a result of prior activation, the enhanced capacity may result from biochemical changes occurring during the activation process that allow the immune cells to be stimulated to activity in response to low doses of stimulants.
  • Immune cell activity that may be measured include, but is not limited to, (1) cell proliferation by measuring the cell or DNA replication; (2) enhanced cytokine production, including specific measurements for cytokines, such as IFN- ⁇ , GM-CSF, or TNF- ⁇ ; (3) cell mediated target killing or lysis; (4) cell differentiation; (5) immunoglobulin production; (6) phenotypic changes; (7) production of chemotactic factors or chemotaxis, meaning the ability to respond to a chemotactin with chemotaxis; (8) immunosuppression, by inhibition ofthe activity of some other immune cell type; and, (9) apoptosis, which refers to fragmentation of activated immune cells under certain circumstances, as an indication of abnormal activation.
  • adjuvant is any substance capable of enhancing the immune response to an antigen with which it is mixed. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol, as well as BCG (bacilli Calmette-Guerin) and Corynabacterium parvum, which are often used in humans, and ligands of CCR6 and other chemokine receptors.
  • adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol, as well as BCG (bacilli Calm
  • a "chemokine” is a small cytokine involved in the migration and activation of cells, including phagocytes and lymphocytes, and plays a role in inflammatory responses.
  • Three classes of chemokines have been defined by the arrangement ofthe conserved cysteine (C) residues ofthe mature proteins: the CXC or ⁇ chemokines that have one amino acid residue separating the first two conserved cysteine residues; the CC or ⁇ chemokines in which the first two conserved cysteine residues are adjacent; the C or ⁇ chemokines which lack two (the first and third) ofthe four conserved cysteine residues.
  • the chemokines can be further divided into two groups.
  • CXC chemokines have the characteristic three amino acid sequence ELR (glutamic acid-leucine-arginine) motif immediately preceding the first cysteine residue near the amino terminus.
  • a second group of CXC chemokines lack such an ELR domain.
  • the CXC chemokines with the ELR domain (including IL-8, GRO ⁇ / ⁇ / ⁇ , mouse KC, mouse MIP-2, ENA-78, GCP-2, PBP/CTAPIII/ ⁇ -TG/NAP-2) act primarily on neutrophils as chemoattractants and activators, inducing neutrophil degranulation with release of myeloperoxidase and other enzymes.
  • the CXC chemokines without the ELR domain e.g., IP-10/mouse CRG, Mig, PBSF/SDF-1, PF4
  • the CC chemokines e.g., MlP-l ⁇ , MIP-1 ⁇ , RANTES, MCP-1 /2/3/4/mouse JE/mouse MARC, eotaxin, I- 309/TCA3, HCC-1, CIO
  • the C chemokines e.g., lymphotactin
  • monocytes dendritic cells
  • T-lymphocytes natural killer cells
  • B-lymphocytes basophils
  • eosinophils e.g., lymphotactin
  • cytokine is a protein made by a cell that affect the behavior of other cells through a "cytokine receptor” on the surface ofthe cells the cytokine effects. Cytokines manufactured by lymphocytes are sometimes termed “lymphokines.” Examples of cytokines include interleukins, interferons and the like.
  • immunologically effective an amount ofthe peptide or fragment thereof which is effective to activate an immune response to prevent or treat proliferative cell growth disorders, such as cancer. Obviously, such amounts will vary between species and individuals depending on many factors. For example, higher doses will generally be required for an effective immune response in a human compared with a mouse.
  • polypeptide encompasses amino acid chains of any length, including full length proteins containing the sequences recited herein.
  • a polypeptide comprising an epitope of a protein containing a sequence as described herein may consist entirely ofthe epitope, or may contain additional sequences.
  • the additional sequences may be derived from the native protein or may be heterologous, and such sequences may (but need not) possess immunogenic or antigenic properties.
  • epitope is a portion of a polypeptide that is recognized (i.e., specifically bound) by a B-cell and/or T-cell surface antigen receptor. Epitopes may generally be identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-24.7 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides derived from the native polypeptide for the ability to react with antigen-specific antisera and/or T-cell lines or clones.
  • An epitope of a polypeptide is a portion that reacts with such antisera and/or T- cells at a level that is similar to the reactivity ofthe full length polypeptide (e.g., in an ELISA and/or T-cell reactivity assay).
  • Such screens may generally be performed using methods well known to those of ordinary skill in the art, such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.
  • B-cell and T-cell epitopes may also be predicted via computer analysis.
  • Polypeptides comprising an epitope of a polypeptide that is preferentially expressed in a tumor tissue (with or without additional amino acid sequence) are within the scope ofthe present invention.
  • Ad.HER-2/neu is a non-replicating E1-, E3-deleted recombinant adenoviral vector expressing a non-functional rat HER-2/ neu oncogene (figure 1).
  • Plasmid pDC316 encodes the adenoviral 5'-inverted terminal repeat, a murine cytomegalovirus (MCMV) promoter and multicloning site.
  • Ad. ⁇ ER-2/neu vector was generated by homologous recombination of pDC316.rHER2/neu(ECDtm) with the adenoviral backbone plasmid pBGH.lox.deltaElE3Cre (MicrobixTM, Toronto, Ontario). Homologous recombination was accomplished by co-transfection ofthe two plasmids into 293 HEK cells by calcium phosphate precipitation. Ad.HER-2/ «e « was screened purified and expanded by standard techniques.
  • recombinant vectors provide specific anti-tumor effect for patients who have been diagnosed with Wer2lneu tumors such as breast, renal, prostate, and other HER2 tumors.
  • this antigen is merely an illustrative example and is not meant to be construed as limiting in any way.
  • other antigens that are useful for treating different types of cancers, include, but not limited to overexpressed or mutated forms of antigens.
  • CCA carcinogenic embryonic antigen
  • K-ras for lung, gastrointestinal and bladder cancers
  • p53 which affects a wide variety of neoplastic growth
  • SARDT3 in neck and head cancers.
  • dendritic cells of an individual are transduced in vivo with recombinant adenoviral vectors (rAds) expressing truncated HER-2/ «e « genes.
  • rAds recombinant adenoviral vectors
  • the most important advantage over previous treatments is that dendritic cells from patients had to be isolated, cultured ex vivo with a vector, and then re-infusing the cultured dendritic cells into the patient.
  • DCs Dendritic cells
  • peptide sequences of tumor antigens designed from analyses of peptide specific HLA-binding affinities.
  • the present invention also provides methods of testing any potential antitumor antigens (e.g., variants of HER-2) in a transgenic mouse assay.
  • Transgenic mice are produced that express a transforming agent (e.g., a growth factor receptor) under the control of a tissue specific promoter.
  • a transforming agent e.g., a growth factor receptor
  • Such mice develop carcinomas that have genetic and pathological features that closely resemble human cancers.
  • a MMTV-neu transgenic mouse lineage MMTV-neu transgenic mouse lineage (neuT)
  • 100% ofthe female mice develop mammary adenocarcinomas [Sacco et al, Gene Therapy, 2:493-497 (1995); Sacco et al, Gene Therapy, 5:383-393 (1998)].
  • the antigen presentation ability of HER-2 modified dendritic cells to inhibit tumor formation or growth is then ascertained.
  • the size ofthe tumor is monitored by determining the tumor size and/or weight.
  • the potential tumor antigen encoded by a vector can be administered by a variety of ways including orally, subcutaneously, or intraperitoneally. Generally, at least two groups of animals are used in the assay, with at least one group being a control group which is administered the administration vehicle without the potential tumor antigen e.g. Ad.null. Detailed descriptions of experiments conducted in the newr-transgenic mouse model are in the Examples which follow.
  • the transduced dendritic cells present antigen to cells ofthe immune system and activate the immune system to recognize tumor antigen epitopes, such as for example a tumor cell expressing the HER-2 oncogene.
  • transduced dendritic cells present antigen, for example, peptide fragments ofthe HER-2 antigen on their surface.
  • Lymphocytes specific for the presented antigens, are activated, proliferate and recognize tumor cells expressing the HER-2 antigen. Lymphocytes include, B cells, T helper cells and cytotoxic T cells. Recognition, of any cell expressing antigenic epitopes by the immune cells, results in the destruction of a tumor cell.
  • cytotoxic T-cells While various procedures involving the use of antibodies have been applied in the treatment of tumors, few if any successful attempts using activated cytotoxic T-cells have been recorded. Theoretically, cytotoxic T-cells would be the preferable means of treating tumors. However, no procedures have been available to specifically activate cytotoxic T- cells. In contrast to antibodies, the T-cell receptors on the surface of CD8 cells cannot recognize foreign antigens directly. Antigen must first be presented to the T cell receptor, such as a dendritic cell.
  • MHC major histocompatibility complex
  • HLA human leukocyte antigen
  • MHC products are grouped into three major classes, referred to as I, II, and III.
  • T- cells that serve mainly as helper cells express CD4 and primarily interact with Class ⁇ molecules, whereas CD8-expressing cells, which mostly represent cytotoxic effector cells, interact with Class I molecules.
  • Class I molecules are membrane glycoproteins with the ability to bind peptides derived primarily from intracellular degradation of endogenous proteins. Complexes of MHC molecules with peptides derived from viral, bacterial and other foreign proteins comprise the ligand that triggers the antigen responsiveness of T-cells. In contrast, complexes of MHC molecules with peptides derived from normal cellular jsroducts play a role in "teaching" the T-cells to tolerate self-peptides, in the thymus. Class I molecules do not present entire, intact antigens; rather, they present peptide fragments thereof, "loaded” onto their "peptide binding groove".
  • the present invention also overcomes problems associated with the sequence of peptides which can be presented and recognized in the context of HLA-peptide complex, by cells ofthe immune system.
  • HLA haplotypes/allotypes vary from individual to individual and identification of peptides that are presented by certain HLA types, would require determining the individual's HLA type.
  • the HLA type may be determined via standard typing procedures and the peripheral blood lymphocytes (PBLs) purified by Ficoll gradients.
  • the peptide for example BER-2/neu
  • an antigen presenting cell for example dendritic cells
  • each individual's HLA type need not be determined.
  • the antigen presenting cells process and present antigen to the immune system.
  • the immune system will recognize whichever peptide fragment that is presented in the context of HLA molecules by engagement with the receptor of an immune effector cell such as, for example a T cell.
  • an immune effector cell such as, for example a T cell.
  • host compatible cells means cells that are ofthe same or similar haplotype as that ofthe subject or "host” to which the cells are administered.
  • the presentation of Class I MHC molecules bound to peptide alone has generally been ineffective in activating CD8 cells.
  • the CD8 cells are activated by antigen-presenting cells, such as, for example, dendritic cells, which present not only a peptide-bound Class I MHC molecule, but also a costimulatory molecule.
  • antigen-presenting cells such as, for example, dendritic cells, which present not only a peptide-bound Class I MHC molecule, but also a costimulatory molecule.
  • B7 which is now recognized to be two subgroups designated as B7.1 and B7.2. It has also been found that cell adhesion molecules such as integrins assist in this process.
  • Dendritic cells are antigen-presenting cells that are found in all tissues and organs, including the blood.
  • dendritic cells present antigens for T lymphocytes, i.e., they process and present antigens, and stimulate responses from naive and memory T cells. In addition to their role in antigen presentation, dendritic cells directly communicate with non-lymph tissue and survey non-lymph for an injury signal (e.g., ischemia, infection, or inflammation) or tumor growth. Once signaled, dendritic cells initiate the immune response by releasing IL-1 which triggers lymphocytes and monocytes.
  • an injury signal e.g., ischemia, infection, or inflammation
  • the CD8 T-cell When the CD8 T-cell interacts with an antigen-presenting cell, such as a dendritic cell, having the peptide bound by a Class I MHC and costimulatory molecule, the CD8 T-cell is activated to proliferate and becomes an effector T-cell. See, generally, Janeway and Travers, Immunobiology, published by Current Biology Limited, London (1994), incorporated by reference.
  • an antigen-presenting cell such as a dendritic cell, having the peptide bound by a Class I MHC and costimulatory molecule
  • T-cells so that they proliferate, become cytotoxic for cells expressing the desired antigen, such as for example, HER-2, and maintain memory cells specific for the administered antigen.
  • the immune system is primed against various tumor epitopes so if spontaneous tumors arise, a pool of primed immune cells exist which become activated to recognize and kill the tumor cells.
  • Memory cells express a different pattern of cell surface markers, and they respond in several ways that are functionally different from those of naive cells.
  • Human memory cells are CD45RA " , CD45RO + .
  • memory cells secrete a full range of T cell cytokines.
  • Chemokines and cytokines also play a powerful role in the development of an immune response.
  • the role of chemokines in leukocyte trafficking is reviewed by Baggiolini (1998) Nature 392:565-8, in which it is suggested that migration responses in the complicated trafficking of lymphocytes of different types and degrees of activation will be mediated by chemokines.
  • the use of small molecules to block chemokines is reviewed by Baggiolini and Moser (1997) J. Exp. Med. 186:1189-1191.
  • the role of various specific chemokines in lymphocyte homing has been previously described. For example, Campbell et al.
  • SDF-1 also called PBSF
  • 6-C-kine also called Exodus-2
  • MLP-3beta also called ELC or Exodus-3
  • ELC Enhanced circulating lymphocytes
  • MIP-3 alpha also called LARC or Exodus- 1
  • SLC secondary lymphoid-tissue chemokine
  • HEV high endothelial venule
  • Mature B cells can be measured in immunoassays, for example, by cell surface antigens including CD 19 and CD20 with monoclonal antibodies labeled with fluorochromes or enzymes may be used to these antigens.
  • B cells that have differentiated into plasma cells can be enumerated by staining for intracellular immunoglobulins by direct immunofluorescence in fixed smears of cultured cells.
  • phenotypes of immune cells and any phenotypic changes can be evaluated by flow cytometry after immunofluorescent staining using monoclonal antibodies that will bind membrane proteins characteristic of various immune cell types.
  • a second means of assessing cell differentiation is by measuring cell function. This may be done biochemically, by measuring the expression of enzymes, mRNA's, genes, proteins, or other metabolites within the cell, or secreted from the cell. Bioassays may also be used to measure functional cell differentiation or measure specific antibody production directed at a patient's tumor, tumor cell lines or cells from fresh tumors.
  • Immune cells express a variety of cell surface molecules which can be detected with either monoclonal antibodies or polyclonal antisera. Immune cells that have undergone differentiation or activation can also be enumerated by staining for the presence of characteristic cell surface proteins by direct immunofluorescence in fixed smears of cultured cells.
  • T cell cytotoxic assays are well known to those skilled in the art.
  • a preferred method is to measure cytotoxicity in a 5 hr 51 Sodium chromate ( 51 _Cr ) release assay.
  • 51 _Cr Sodium chromate
  • 20 hr 51 Cr-release assay is preferred.
  • Tumor cells also referred to herein as "target cells” are plated in flat-bottomed microtiter plates and incubated at 37°C overnight. The targets are washed and labeled the next day with 51 Cr at 37°C. 51 Cr is taken up by the target cells, either by endocytosis or pinocytosis, and is retained in the cytoplasm.
  • effector cells are plated at different E:T ratios and incubated overnight at 37°C. Cytolysis is a measure ofthe 51 Cr released from the target cells into the supernatant due to destruction ofthe target cells by the effector cells.
  • the microtiter plates are centrifuged at 1000 rpm for 10 minutes and an aliquot of about 50 ⁇ l to about 100 ⁇ l is removed and the level of radioactivity is measured the next day by a gamma counter and the percent specific lysis calculated.
  • Percent specific lysis is measured by using the formula: ( 51 Cr released from the target cells) - (spontaneous 51 Cr released from the target cells)/ (maximum 5l Cr released from the target cells) - (spontaneous 51 Cr released from the target cells) X 100
  • the spontaneous 51 Cr released from the target cells is measured with tumor cells to which no effector cells have been added.
  • Maximum 51 Cr released from the target cells is obtained by adding, for example, IM HCl and represents the total amount of 51 Cr present in the cytoplasm ofthe target cell.
  • 3 H-TdR tritiated thymidine
  • 3 H-TdR is taken up by target cells into the nucleus ofthe cell. Release of 3 H-TdR is a measure of cell death by DNA fragmentation.
  • the assay is conducted as above except the incubation period is at least about 48 hours and 50 ⁇ l to about 100 ⁇ l ofthe supernatant is measured by a beta-counter in the presence of at least about 1 ml of scintillation fluid. Calculation of percent specific lysis is performed using the above formula.
  • vectors encoding the HER-2 as identified by SEQ ID NO: 1 are presented to cells ofthe immune system.
  • a polypeptide encoded by the nucleic acid molecule is detected by cells ofthe immune system.
  • the polypeptide is preferably detected in a subject sample, suffering from or susceptible to, any type of breast cancer, or other HER-2 type tumors such as for example renal, prostate, ovarian, gastric and the like.
  • any fragments or variants ofthe sequence identified by SEQ ID NO: 1 can be used and the polypeptide is encoded by a sequence comprising a sequence identified by SEQ ID NO: 1.
  • the polypeptide is encoded by a sequence having at least about 80% sequence identity to a molecule identified by SEQ ID NO: 1. More preferably the polypeptide is encoded by a sequence having at least about 90% sequence identity to a molecule by SEQ ID NO: 1, most preferred, the polypeptide is encoded by a sequence having at least about 95% sequence identity to a molecule identified by SEQ ID NO: 1.
  • nucleotide sequences, or portions thereof, other than SEQ ID NO: 1, that are useful for encoding HER-2/neu polypeptides include those found in the GenBank at reference numbers Ml 1730, X03363, and NM 004448.
  • peptides are administered to an individual suffering from or susceptible to cancers.
  • the peptides of ⁇ SER-2/neu are encoded by a sequence having at least about 80% sequence identity to a molecule identified by SEQ ID NO: 1. More preferably, the peptides of ⁇ ER-2/neu are encoded by a sequence having at least about 90% sequence identity to a molecule identified by SEQ ID NO: 1, most preferably, the peptides of ⁇ ER-2/neu are encoded by a sequence having at least about 95% sequence identity to a molecule identified by SEQ ID NO: 1.
  • the peptides are administered to an individual suffering from or susceptible to cancers.
  • Prophylactic applications are warranted in cases where individuals with familial history of disease and predicted to be at risk by reliable prognostic indicators could be treated prophylactically to interdict cancer prior to onset, such as breast cancer; or can be administered post operatively.
  • Peptide vaccines can be administered in many possible formulations, in pharmacologically acceptable mediums.
  • the peptide can be conjugated to a carrier, such as KLH, in order to increase its immunogenicity.
  • the vaccine can be administered in conjunction with an adjuvant, various of which are known to those skilled in the art.
  • a booster can be provided.
  • the vaccines are administered by conventional methods, in dosages which are sufficient to elicit an immunological response, which can be easily determined by those skilled in the art.
  • Efficacy ofthe peptide in the context of prevention is judged based on the following criteria: frequency of peptide reactive T cells determined by limiting dilution, proliferation response of peptide reactive T cell lines and clones, cytokine profiles of T cell lines and clones to the desired peptide established from patients. Efficacy is established by decrease in frequency of reactive cells, a reduction in thymidine incorporation with altered peptide compared to native, and a reduction in TNF and IFN- ⁇ .
  • Clinical measurements include the relapse rate in one and two year intervals, on a Kaplan-Meier curve, a delay in sustained cancer stage progression reduction in the number and size of tumors including a change in area and volume of T2 images on MRI, and the number and volume of lesions determined by gadolinium enhancedjmages.
  • compositions ofthe present invention may comprise one or more ofthe peptides, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like, carbohydrates such as glucose, marihose, sucrose or dextrans, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and preservatives.
  • pharmaceutical compositions ofthe present invention may also contain one or more additional active ingredients, such as, for example, cytokines like ⁇ -interferon.
  • compositions ofthe present invention may be formulated for the manner of administration indicated, including for example, for oral, nasal, venous, intracranial, intraperitoneal, subcutaneous, or intramuscular administration.
  • the compositions described herein may be administered as part of a sustained release implant.
  • compositions ofthe present invention may be formulized as a lyophilizate, utilizing appropriate excipients which provide stability as a lyophilizate, and subsequent to rehydration.
  • compositions ofthe present invention maybe administered in a manner appropriate to the disease to be treated (or prevented).
  • the quantity and frequency of administration will be determined by such factors as the condition ofthe patient, and the type and severity ofthe patient's disease.
  • the peptides, variants, or fragments thereof, or pharmaceutical compositions described herein may be administered at a dosage ranging from about 5 to 50 mg/kg, although appropriate dosages may be determined by clinical trials. Dosages of peptide analogue will be approximately 5-50 mg/kg, but are determined more accurately following trials. Patients may be monitored for therapeutic effectiveness by MRI, and signs of clinical exacerbation, as described above.
  • MRI can be used to measure active lesions using gadolinium-DTPA-enhanced imaging (McDonald et al. Ann. NeuroL 36:14, 1994) or the location and extent of lesions using T 2 -weighted techniques. Briefly, baseline MRIs are obtained. The same imaging plane and patient position are used for each subsequent study. Positioning and imaging sequences are chosen to maximize lesion detection and facilitate lesion tracing. The same positioning and imaging sequences are used on subsequent studies. The presence, location and extent of MS lesions are determined by radiologists. Areas of lesions are outlined and summed slice by slice for total lesion area.
  • any tumor antigen polypeptide can be administered to an individual diagnosed as suffering from or susceptible to cancers.
  • the polypeptides corresponding to identified tumor antigens can be used to stimulate the cells ofthe immune system to recognize and lyse tumor cells expressing tumor antigens, such as for example, CEA, p53, K-ras, and the like.
  • the tumor antigen and vector is not limited to HER- 2 or adenoviral vectors but, can include vectors such as plasmids, viral vectors, naked DNA and the like. Any antigen that is oncogenic may be used such as for example, CEA, K-ras, p53 and the like.
  • the polypeptide is expressed at least at a higher level in a patient with cancer as compared to expression levels in normal individuals, preferably the polypeptide is expressed at least about 5 to about 10 fold higher in a patient with cancer as compared to expression in a normal individual.
  • the cancer is a breast cancer and the subject sample is obtained from a mammalian patient, including a primate such as a human patient.
  • variants ofthe nucleic acid molecule as identified by SEQ ID NO: 1 can be used to transduce immune cells for the detection and lysing of, for example, HER-2 positive cancers.
  • An "allele” or “ variant” is an alternative form of a gene.
  • variants ofthe genes encoding any potential breast tumor markers identified by the methods of this invention may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms.
  • Common mutational changes that give rise to variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • compositions and methods ofthe present invention also encompass variants ofthe above polypeptides and nucleic acid sequences encoding such polypeptides.
  • a polypeptide "variant,” as used herein, is a polypeptide that differs from the native polypeptide in substitutions and/or modifications, such that the antigenic and/or immunogenic properties ofthe polypeptide are retained. Such variants may generally be identified by modifying one ofthe above polypeptide sequences and evaluating the reactivity ofthe modified polypeptide with antisera and/or T-cells as described above.
  • Nucleic acid variants may contain one or more substitutions, deletions, insertions and/or modifications such that the antigenic and/or immunogenic properties ofthe encoded polypeptide are retained.
  • One preferred variant ofthe polypeptides described herein is a variant that contains nucleotide substitutions, deletions, insertions and/or modifications at no more than 20% ofthe nucleotide positions.
  • a variant contains conservative substitutions.
  • a "conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature ofthe polypeptide to be substantially unchanged.
  • Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenic or antigenic properties, secondary structure and hydropathic nature ofthe polypeptide.
  • a polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-franslationally or post-translationally directs transfer ofthe protein.
  • the polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification ofthe polypeptide (e.g., poly-His), or to enhance binding ofthe polypeptide to a solid support.
  • a polypeptide may be conjugated to an immunoglobulin Fc region.
  • nucleotide sequences encoding all or a portion ofthe polypeptides described herein may be prepared using any of several techniques.
  • cDNA molecules encoding such polypeptides may be cloned on the basis ofthe breast tumor- specific expression ofthe corresponding mRNAs, using differential display PCR. This technique compares the amplified products from RNA template prepared from normal and breast tumor tissue.
  • cDNA may be prepared by reverse transcription of RNA using a random primer, such as for example, (dT) 12 AG primer.
  • a band corresponding to an amplified product specific to the tumor RNA may be cut out from a silver stained gel and subcloned into a suitable vector, such as the adenovirus vector described in the examples which follow.
  • a suitable vector such as the adenovirus vector described in the examples which follow.
  • Nucleotide sequences encoding all or a portion ofthe breast tumor-specific polypeptides disclosed by SEQ ID NO: 1 and variants thereof may be amplified from cDNA prepared as described above using any random primers.
  • a gene encoding a polypeptide as described herein may be amplified from human genomic DNA, or from breast tumor cDNA, via polymerase chain reaction.
  • the presence ofthe one or more nucleic acid molecules is correlated to a sample of a normal subject.
  • the sample is preferably obtained from a mammal suspected of having a proliferative cell growth disorder, in particular, a breast cancer.
  • a nucleic acid molecule that is indicative of a breast cancer comprises a sequence having at least about 80% sequence identity to a molecule identified by SEQ ID NO: 1, more preferably the nucleic acid molecule comprises a sequence having at least about 90% sequence identity to a molecule identified by SEQ ID NO: 1, most preferably the nucleic acid molecule comprises a sequence having at least about 95% sequence identity to a molecule identified in any of by SEQ ID NO: 1.
  • Percent identity and similarity between two sequences can be determined using a mathematical algorithm (see, e.g., Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).
  • ⁇ ER-2lneu fragments and derivatives ofthe invention are of a sufficient length such that they activate the immune system resulting in the lysing of cancer cells, such as, for example cells expressing HER-2/neu, but these fragments or derivatives do not posses kinase signaling activity. That is these fragments or truncated HER-2/neu fragments lack an intracellular domain.
  • ⁇ ER-2/neu fragments and derivatives lacking an intracellular domain, thus preferably comprise at least about 90% nucleotides as compared to the sequence identified by SEQ ID NO: 1, usually at least about 80% nucleotides as compared to the sequence identified by SEQ ID NO: 1, more usually at least about 70% nucleotides as compared to the sequence identified by SEQ ID NO: 1, even more typically at least about 40% or 50% nucleotides.
  • Preferred ER-2/neu fragments or derivatives ofthe invention include those that have at least about 70 percent homology (sequence identity) to the ⁇ ER-2/neu of SEQ ID NO:l, more preferably about 80 percent or more homology to the HER-2//zew of SEQ JD NO:l, still more preferably about 85 to 90 percent or more homology to the BER-2/neu ofSEQ JD NO.T.
  • the ⁇ ER-2/neu expressed in the vector may code for 10% ofthe intracellular domain and 100% ofthe extracellular .domain.
  • the ⁇ ER-2/neu expressed in the vector may code for 100% ofthe intracellular domain and 10% ofthe exfracellular domain, or 50% ofthe intracellular domain and 50% ofthe extracellular domain, as long as the truncated ⁇ ER-2/neu is able to transduce antigen presenting cells and activate an immune response resulting in the lysis of cancer cells expressing, for example HER-2/neu.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% ofthe length ofthe reference sequence.
  • the nucleotides at corresponding nucleotide positions are then compared.
  • nucleic acid “identity” is equivalent to nucleic acid "homology”
  • percent identity between the two sequences is a function ofthe number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment ofthe two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at online through the Genetics Computer Group), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • GAP program in the GCG software package (available at online through the Genetics Computer Group), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • parameters to be used in conjunction with the GAP program include a Blosum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of Meyers and Miller (Comput. Appl. Biosci. 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0 or version 2.0U), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • neoplastic disease or neoplastic cells refers to an amount ofthe vectors and/or peptides, described throughout the specification and in the Examples which follow, capable of invoking one or more ofthe following effects: (1) inhibition, to some extent, of tumor growth, including, (i) slowing down and (ii) complete growth arrest; (2) reduction in the number of tumor cells; (3) maintaining tumor size; (4) reduction in tumor size; (5) inhibition, including (i) reduction, (ii) slowing down or (iii) complete prevention, of tumor cell infiltration into peripheral organs; (6) inhibition, including (i) reduction, (ii) slowing down or (iii) complete prevention, of metastasis; (7) enhancement of anti-tumor immune response, which may result in (i) maintaining tumor size, (ii) reducing tumor size, (iii) slowing the growth of a tumor, (iv) reducing, slowing or preventing invasion or (v) reducing, slowing or preventing metastasis; and/or
  • any variant, fragment, mutant can be used to transduce immune cells, such as for example dendritic cells, for the treatment of an individual suffering from, or, prophylactically to an individual susceptible to cancer.
  • immune cells such as for example dendritic cells
  • a preferred use of nucleic acid sequences identified in the present invention is for the generation of treatments that lyse for example, breast cancer cells.
  • the nucleic acid molecules can be expressed by a vector containing a DNA segment encoding the wild-type, alleles, variants, mutations or fragments ofthe genes. Mutations and alleles ofthe nucleic acid molecules are also preferably used in the construction of a vector for use in treatment.
  • the vector comprising the desired nucleic acid sequence for conferring resistance to, for example, breast cancer preferably has at least one such nucleic acid sequence.
  • the vector may be comprised of more than one such nucleic acid sequence, or combinations of allelic variants.
  • the vector can also be comprised of cassettes of different allelic variants or wild type nucleic acid molecules.
  • Vectors include chemical conjugates such as described in WO 93/04701, which has a targeting moiety (e.g. a ligand to a cellular surface receptor), and a nucleic acid binding moiety (e.g. polylysine), viral vector (e.g. a DNA or RNA viral vector), fusion proteins such as described in PCT/US95/02140 (WO 95/22618) which is a fusion protein containing a target moiety (e.g. an antibody specific for a target cell) and a nucleic acid binding moiety (e.g.
  • the vectors can be chromosomal, non-chromosomal or synthetic. It is a preferred embodiment of this invention that the choice of cells for delivery ofthe nucleic acid molecules include embryonic stem cells, hematopoietic cells which can be differentiated into monocytes/macrophages using cytokines known in the art and delivery of these cells into an individual. Most preferably, the cells are dendritic cells.
  • the vector is a recombinant adenovirus vector with deletions in the El- and E3-regions ofthe adenovirus vector as described in detail in the examples which follow.
  • Other vectors may also be used.
  • Preferred vectors include viral vectors, fusion proteins and chemical conjugates.
  • Retroviral vectors include moloney murine leukemia viruses. DNA viral vectors are preferred.
  • Viral vectors can be chosen to introduce the genes to cells of choice.
  • Such vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as herpes simplex I virus (HSV) vector (Geller et al., 1995, J. Neurochem.
  • HSV herpes simplex I virus
  • Pox viral vectors introduce the gene into the cells cytoplasm.
  • Avipox virus vectors result in only short term expression ofthe nucleic acid.
  • Adenovirus vectors, adeno-associated virus vectors and herpes simplex virus vectors are preferred for introducing the nucleic acid into neural cells.
  • the adenovirus vector results in a shorter term expression (about 2 months) than adeno-associated virus (about 4 months), which in turn is shorter than HSV vectors.
  • the vectors can be introduced by standard techniques, e.g. infection, transfection, transduction or transformation. Examples of modes of gene transfer include for example, naked DNA calcium phosphate precipitation, DEAE dexfran, electroporation, protoplast fusion, lipofection, cell microinjection and viral vectors.
  • the vector can be employed to target essentially any desired target cell.
  • stereotaxic injection can be used to direct the vectors (e.g. adenovirus, HSV) to a desired location.
  • Other methods that can be used include catheters, intravenous, parenteral, intraperitoneal, and subcutaneous injection, and oral or other known routes of administration.
  • DNA immunization employs the subcutaneous injection of a plasmid DNA (pDNA) vector encoding a tumor marker.
  • the pDNA sequence is taken up by antigen presenting cells (APC), preferably by dendritic cells. Once inside the cell, the DNA encoding protein is transcribed and translated and presented to lymphocytes.
  • APC antigen presenting cells
  • Genetic constructs comprise a nucleotide sequence that encodes the nucleic acid sequence of choice and preferably includes an intracellular trafficking sequence operably linked to regulatory elements needed for gene expression.
  • the genetic construct(s) When taken up by a cell, the genetic construct(s) may remain present in the cell as a functioning exfrachromosomal molecule and/or integrate into the cell's chromosomal DNA.
  • DNA may be introduced into cells where it remains as separate genetic material in the form of a plasmid or plasmids.
  • linear DNA which can integrate into the chromosome may be introduced into the cell.
  • reagents which promote DNA integration into chromosomes may be added. DNA sequences which are useful to promote integration may also be included in the DNA molecule.
  • RNA may be administered to the cell.
  • Gene constructs may remain part of_the genetic material in attenuated live microorganisms or recombinant microbial vectors which live in cells. Gene constructs may be part of genomes of recombinant viral vaccines where the genetic material either integrates into the chromosome ofthe cell or remains exfrachromosomal. Genetic constructs include regulatory elements necessary for gene expression of a nucleic acid molecule. The elements include: a promoter, an initiation codon, a stop codon, and a polyadenylation signal.
  • enhancers may be required for gene expression ofthe sequence of choice, for example, the nucleic acid sequences identified by SEQ ID NO: 1, gene, alleles or fragments thereof. It is necessary that these elements be operably linked to the sequence that encodes the desired proteins and that the regulatory elements are operable in the individual to whom they are administered.
  • Initiation codons and stop codons are generally considered to be part of a nucleotide sequence that encodes the immunogenic target protein. However, it is necessary that these elements are functional in the individual to whom the gene construct is administered. The initiation and termination codons must be in frame with the coding sequence.
  • Promoters and polyadenylation signals used must be functional within the cells of the individual.
  • promoters useful to practice the present invention include but are not limited to promoters from Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters from human genes such as human Actin, human Myosin, human Hemoglobin, human muscle creatine and human metallothionein.
  • SV40 Simian Virus 40
  • MMTV Mouse Mammary Tumor Virus
  • HIV HIV Long Terminal Repeat
  • ALV Moloney virus
  • CMV Cytomegalovirus
  • EBV Epstein Barr Virus
  • RSV Rous Sarcoma Virus
  • polyadenylation signals useful to practice the present invention include but are not limited to SV40 polyadenylation signals and LTR polyadenylation signals.
  • additional elements include enhancers.
  • the enhancer may be selected from the group including but not limited to: human Actin, human Myosin, human Hemoglobin, human muscle creatine and viral enhancers such as those from CMV, RSV and EBV.
  • Genetic constructs can be provided with mammalian origin of replication in order to maintain the construct extrachromosomally and produce multiple copies ofthe construct in the cell.
  • plasmids pCEP4 and pREP4 from Invitrogen contain the Epstein Barr virus origin of replication and nuclear antigen EBNA-1 coding region which produces high copy episomal replication without integration.
  • regulatory sequences may be selected which are well suited for gene expression in the cells the construct is administered into. Moreover, codons may be selected which are most efficiently transcribed in the cell.
  • codons may be selected which are most efficiently transcribed in the cell.
  • the method ofthe present invention comprises the steps of administering nucleic acid molecules to tissue ofthe individual.
  • the nucleic acid molecules are administered intramuscularly, intranasally, intraperitoneally, subcutaneously, intradermally, or topically or by lavage to mucosal tissue selected from the group consisting of vaginal, rectal, urethral, buccal and sublingual.
  • the nucleic acid molecule is delivered to the cells in conjunction with administration of a facilitating agent.
  • Facilitating agents are also referred to as polynucleotide function enhancers or genetic vaccine facilitator agents.
  • Facilitating agents are described in e.g. International Application No. PCT/US94/00899 filed Jan. 26, 1994 and International Application No. PCT/US95/04071 filed Mar. 30, 1995, both incorporated herein by reference.
  • Facilitating agents which are administered in conjunction with nucleic acid molecules may be administered as a mixture with the nucleic acid molecule or administered separately simultaneously, before or after adminisfration of nucleic acid molecules.
  • the genetic constructs of the invention are formulated with or administered in conjunction with a facilitator selected from the group consisting of, for example, benzoic acid esters, anilides, amidines, urethans and the hydrochloride salts thereof such as those ofthe family of local anesthetics.
  • a facilitator selected from the group consisting of, for example, benzoic acid esters, anilides, amidines, urethans and the hydrochloride salts thereof such as those ofthe family of local anesthetics.
  • the facilitating agent is administered prior to, simultaneously with or subsequent to the genetic construct.
  • the facilitating agent and the genetic construct may be formulated in the same composition.
  • the individual is first subject to injection ofthe facilitator prior to adminisfration ofthe genetic construct. That is, for example, up to a about a week to ten days prior to administration ofthe genetic construct, the individual is first injected with the facilitator. In some embodiments, the individual is injected with the facilitator about 1 to 5 days; in some embodiments 24 hours, before or after administration ofthe genetic construct. Alternatively, if used at all, the facilitator is administered simultaneously, minutes before or after administration ofthe genetic construct. Accordingly, the facilitator and the genetic construct may be combined to form a single pharmaceutical composition.
  • the genetic constructs are administered free of facilitating agents, that is in formulations free from facilitating agents using administration protocols in which the genetic constructions are not administered in conjunction with the administration of facilitating agents.
  • Nucleic acid molecules which are delivered to cells according to the invention may serve as genetic templates for proteins that function as prophylactic and/or therapeutic immunizing agents.
  • the nucleic acid molecules comprise the necessary regulatory sequences for transcription and translation ofthe coding region in the cells ofthe animal.
  • the compounds described herein may be used for the immunotherapy of breast cancer.
  • the compounds (which may be polypeptides, antibodies or nucleic acid molecules) are preferably incorporated into pharmaceutical compositions or vaccines.
  • Pharmaceutical compositions comprise one or more such compounds and a physiologically acceptable carrier.
  • Vaccines may comprise one or more polypeptides and an immune response enhancer, such as an adjuvant or a liposome (into which the compound is incorporated).
  • Pharmaceutical compositions and vaccines may additionally contain a delivery system, such as biodegradable microspheres which are disclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109.
  • Pharmaceutical compositions and vaccines within the scope of the present invention may also contain other compounds, including one or more separate polypeptides.
  • the type of carrier will vary depending on the mode of administration.
  • the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer.
  • any ofthe above carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed.
  • Biodegradable microspheres e.g., polylactate polyglycolate
  • adjuvants may be employed in the vaccines of this invention to nonspecifically enhance the immune response.
  • Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins.
  • Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.), Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.), alum, biodegradable microspheres, monophosphoryl lipid A and quil A.
  • Cytokines such as GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.
  • autologous dendritic cells can be isolated from a patient, transduced with the vectors, described in detail herein, cultured, and re- infused into the patient.
  • substantially free or substantially purified APCs is meant at least 50% ofthe population are APCs, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% free of non-APCs cells with which they are associated in nature.
  • Dendritic cells of different maturation stages can be isolated based on the cell surface expression markers. For example, mature dendritic cells are less able to capture new proteins for presentation but are much better at stimulating resting T cells to grow and differentiate. Thus, mature dendritic cells can be of importance. Mature dendritic cells can be identified by their change in morphology; by their nonadherence; and by the presence of various markers. Such markers include, but are not limited to, cell surface markers such as B7.2, CD40, CDI lc + , and MHC class II. Alternatively, maturation can be identified by observing or measuring the production of pro-inflammatory cytokines.
  • Dendritic cells can be collected and analyzed using typical cytofluorography and cell sorting techniques and devices, such as a fluorescence-activated cell sorter (FACS).
  • FACS fluorescence-activated cell sorter
  • Antibodies specific to cell surface antigens of different stages of dendritic Gell maturation are commercially available.
  • TUBO cells were derived from a breast cancer of a BALB-neu ⁇ transgenic mouse, and N202.1A cells were isolated from a breast cancer of a FVB-raewN transgenic mouse (both cell lines were the kind gift of Dr. Patrizia Nanni, University of Bologna, Italy).
  • TUBO and N202.1 A constitutively express HER-2/neu and were grown in Dulbecco's modified Eagle's medium (DMEM; BioSource, Rockville, MD) supplemented with 10% heat inactivated fetal bovine serum (FBS; Gemini, Woodland, CA).
  • DMEM Dulbecco's modified Eagle's medium
  • FBS heat inactivated fetal bovine serum
  • TS/A also a gift of Dr.
  • Patrizia Nanni was established from a spontaneous breast cancer from a BALB/c mouseand was grown in RPMI- 1640 (BioSource, Rockville, MD) with 10% heat-inactivated FBS.
  • Human embryonic kidney 293 cells were obtained from ATCC (Manassas, VA) and grown in DMEM with 10% heat-inactivated FBS. All cells were maintained in an incubator at 37°C and 5% CO 2 .
  • Ad. ⁇ ER-2/neu is a non-replicating E1-, E3-deleted recombinant adenoviral vector expressing a non-functional rat HER-2/ neu oncogene (figure 1). El, E3-deleted adenoviruses expressing ⁇ ER-2lneu (Ad.HER2), and a control vector (Ad.null) were generated using the AdMax ® system (Microbix, Toronto, Canada).
  • Plasmid pDC316 encodes the adenoviral 5 '-inverted terminal repeat, a murine cytomegalovirus (MCMV) promoter and multicloning site.
  • the Ad.HER-2/new vector was generated by homologous recombination of ⁇ DC316.rHER2/new(ECDtm) with the adenoviral backbone plasmid pBGH.lox.deltaElE3Cre (MicrobixTM, Toronto, Ontario). Homologous recombination was accomplished by co-fransfection ofthe two plasmids into 293 HEK cells by- calcium phosphate precipitation.
  • Ad.HER-2/ ⁇ zeM was screened purified and expanded by standard techniques, for example, all viruses were double plaque-isolated, expanded on 293 cells, purified on a CsCl gradient, titrated as plaque forming units (pfu)/mL and -stored at - 70oC. (See figures 1 and 10).
  • Ad.GFP a recombinant El, E3-deleted adenovirus-5 expressing green fluorescent protein was purchased from Quantum Biotechnologies (Toronto, Canada).
  • BALB-neuT transgenic mice express the rat ⁇ ER-2/neu oncogene under the control of a cliimeric mouse mammary tumor virus promoter. These mice provide an aggressive model of mammary carcinogenesis as the transgene is over expressed in rudimentary mammary glands of 3-week-old mice. Atypical lobular hyperplasia is seen at 6-weeks of age that progresses to multiple in-situ carcinomas that enlarge and converge to form rapidly growing, invasive and metastasizing tumors in all ten glands by 25-weeks of age.
  • Female BALB/c mice (5 to 6 weeks old) were obtained from the Division of Cancer Treatment, NCI (Fredrick, MD).
  • DCs Bone-Marrow-derived Dendritic Cells
  • Erythrocytes were lysed with ACK buffer (BioWhittker, Walkersville, MD) and nucleated cells were plated in RPMI-1640 with 10% heat- inactivated FBS (GIBCO-Invitrogen, Grand Island, NY) and 20 ng mL murine granulocyte/macrophage colony-stimulating factor (mGM-CSF; PeproTec, JRocky Hill, NJ) in Falcon plastic bacteriological dishes (BD Biosciences, San Jose, CA).
  • ACK buffer BioWhittker, Walkersville, MD
  • FBS heat- inactivated FBS
  • mGM-CSF murine granulocyte/macrophage colony-stimulating factor
  • DCs non-adherent cells
  • DCs were incubated with fluorescein isothiocyanate (FITC) or phycoerytherin (PE)-labeled anti-mouse CDI lc, CDI lb, CD40, CD80, CD86, H-2Kd, I-Ad, CD8a (BD Pharmingen, San Diego, CA) and analyzed on a FACSort flow cytometer (Becton Dickinson, San Jose, CA).
  • Day 10 Ad.HER2 infected DCs were incubated with anti-rat HER-2/neu monoclonal antibody (Oncogene Research, La Jolla, CA) followed by incubation with secondary FITC-labeled rabbit anti-mouse immunoglobulin and analyzed by FACScan. To detect cytoplasmic HER-2/neu, the DCs were permeabilized using Cytofix/Cytoperm (BD Pharmingen, San Diego, CA) and similarly stained.
  • FITC fluorescein isothiocyanate
  • PE
  • Dendritic cell cultures were infected with Ad.rHER-2/ «ew(ECDtm), Ad.rHER- 2/neu (ECD) or Ad.null at multiplicities of infection (m.o.i.) of 30.
  • Splenocytes obtained from 8-10 week old na ⁇ ve female BALB-neuT mice were incubated with irradiated unmodified DCs or DCs infected with either ofthe rAd vectors. Four days later, 3 H- thymidine was added and its incorporation was measured by a beta-counter.
  • BALB/c-neuT transgenic mice express the rat HER-2/neu oncogene under the control ofthe MMTV promoter. These mice progressively develop cancers in all 10 mammary glands between 15 and 25 weeks of age ( Figure 2). Five to six week old BALB/c-neuT ⁇ ce were vaccinated sub-cutaneously on a weekly basis for 3 weeks with 1 x 10 6 dendritic cells that were infected with one ofthe recombinant adenoviral" vectors (rAd).
  • the mice were followed twice weekly for the development of tumors.
  • Mice that were free of tumor at 28 weeks were challenged with a s.c. injection of 1 x 10 5 HER-2/ «ew-expressing TUBO cells.
  • Another group of 3-4 week old female BALB-neuI mice were s.c.
  • mice were s.c. injected with Ad.null 1 x 10 8 pfu weekly for 4 weeks.
  • Ad.null groups of mice were vaccinated with either 1 x 10 6 DC AC I. HER2 S.C, or directly with 1 x 10 8 pfu Ad.HER2 s.c. weekly for two weeks.
  • the mice were challenged with 5 x 10 5 TUBO cells and observed for tumor growth. Tumor volumes were calculated using the formula for the estimate of a rotational ellipse.
  • mice vaccinated with DCA C I.HE R2 were challenged with 5 x 10 5 ⁇ ER-2/neu expressing TUBO cells. Animals free of tumor at 37 days were re-challenged with an injection of 5 x 10 5 TUBO cells, or HER-2/r ⁇ ew-negative TS/A cells and followed for development of tumor.
  • mice Five to six week old female BALB/c mice were intraperitoneally injected daily for three days, then every three days until the mice were challenged with tumor with 200 mg of anti-CD4, or anti-CD8 purified from the supernatants of hybridomas GK1.5 (ATCC) and 2.43 (ATCC), respectively, or with anti-asialo-GMl (WAKO, Richmond, VA). Five days after initiation of antibody treatment, the mice were vaccinated as described above. One week following the last vaccination, the mice received a challenge of 5 x 10 5 TUBO cells and were monitored for tumor growth.
  • N202.1A cells (HER-2/ ⁇ ew-positive) were used to detect and quantify anti- HER2/neu antibodies. Briefly, 2 x 10 5 N202.1A cells were incubated with the sera diluted 1 :10 in 2% BSA in PBS at 4 °C for one hour. The cells were washed and incubated with FITC-labeled rabbit anti-mouse immunoglobulin antibody (DAKO, Carpanteria, CA) for one hour and submitted to flow cytometry. Ten thousand cells were examined.
  • DAKO FITC-labeled rabbit anti-mouse immunoglobulin antibody
  • the specific N202.1 A binding potential ofthe sera was calculated using the following equation: [(%positive cells with test serum)(fluorescence mean)]-[(%positive cells with control serum)(fluorescence mean)] x serum dilution. Pooled sera obtained from untreated age-matched mice were used as controls.
  • ELISPOTAssay Spleens were removed from mice one week after the last vaccination. Splenocytes were mechanically dissociated and the erythrocytes were lysed with ACK buffer. One hundred thousand spleen cells were plated in a 96-well dish coated with anti- mouse interferon- ⁇ (IFN- ⁇ ) antibody (R&D systems, Minneapolis, MN) and incubated for 48 hours at 37 °C. The plates were developed in accordance wi ⁇ rthe manufacturer's protocol and the spots counted.
  • IFN- ⁇ anti- mouse interferon- ⁇
  • IFN- ⁇ secreting cells were detected using Cytokine Secretion Assay kit (Miltenyi, Auburn, CA) in accordance with the manufacturer's protocol. Briefly, re-stimulated cells were harvested and stained with PE-labeled anti-mouse IFN- ⁇ antibody together with PreCP-labeled CD45RA/B220 and FITC-labeled anti-CD4 or FITC-labeled anti-CD8.
  • the stained cells were incubated with anti-PE antibody conjugated with magnetic beads for 15 min at 4°C. Cells with magnetic beads were captured with a MS column followed by elution with 1% BSA-containing PBS. Eluted cells were incubated with 7-AAD for 10 min and were analyzed on the FACScan gating out the dead cells and CD45R/B220-positive cells. The IFN- ⁇ positive CD4 or CD8 cells were calculated and reported as percentage.
  • Mammary glands from 8-9 week old BALB-neuT mice and TUBO tumors grown in BALB/c mice were removed, frozen in OCT compound (SAKURA-Finetek U.S. A. Inc., Torrance, CA), and sectioned by cryostat. Endogeneous peroxidase activity was blocked with 0.3% hydrogen peroxide followed by blocking of avidin and biotin using the Avidin Biotin Blocking Kit (Zymed Laboratories Inc., South San Francisco, CA). After washing with PBS, the tissues were stained with either rabbit anti-mouse CD4 (BD Pharmingen) or anti-mouse CD8 (BD Pharmingen).
  • OCT compound SAKURA-Finetek U.S. A. Inc., Torrance, CA
  • Endogeneous peroxidase activity was blocked with 0.3% hydrogen peroxide followed by blocking of avidin and biotin using the Avidin Biotin Blocking Kit (Zymed Laboratories Inc., South San Francisco, CA). After washing with PBS, the
  • the slides were washed and then incubated with biotinylated anti-rabbit antibody (Vector Laboratory, Burlingame, CA), stained with Streptavidin-Horseradish Peroxidase (DAKO, Carpinteria, CA) and counterstained with hematoxylin.
  • biotinylated anti-rabbit antibody Vector Laboratory, Burlingame, CA
  • Streptavidin-Horseradish Peroxidase DAKO, Carpinteria, CA
  • Murine bone marrow cells cultured for 8-10 days under the conditions described above generated a CDI lc + , CDI lb “ , CDBa " myeloid dendritic cell phenotype as shown in figure 3.
  • the cells also expressed CD80, CD86, CD40, as well as MHC class I and class II.
  • Surface marker expression on DC infected with Ad.HER2 (DCA ⁇ I.HER2) > or Ad.null (DC Ad .nui ⁇ ) was examined.
  • DCAd.m ⁇ i ⁇ and DCAd.HER2 expressed higher levels of CD80, CD86, CD40, and MHC classes I and II indicating DC maturation (Figure 19B).
  • dendritic cells were infected with Ad.EYFP at varying m.o.i.'s.
  • Ad.EYFP Ad.EYFP at varying m.o.i.'s.
  • DC Ad .HER2 showed the expected 2.1 kb truncated HER-2/neu mRNA transcript which was not detected in DC A d.nui ⁇ ( Figure 20A).
  • HER-2/neu transcripts were absent in uninfected BALB-H ⁇ wT bone marrow-derived DC, even when analyzed by reverse transcriptase polymerase chain reaction, indicating tissue- specific expression in the transgenics.
  • Example 3 Phenotypic Change of Dendritic Cells Following rAd Infection.
  • Dendritic cells infected with Ad. ⁇ ER-2/neu (ECDtm) or Ad.rHER-2/wew (ECD) at an m.o.i. of 30 showed enhanced expression ofthe dendritic cell maturation markers CD80, CD86, MHC-class I, MHC-class II and CD40, as shown in figure 5.
  • Example 4 Expression of Rat HER-2/neu RNA in Dendritic Cells Infected with HER-2/neu-expressing rAds.
  • RNA isolated from dendritic cells infected with Ad.rHER-2/wew(ECDtm) or Ad.rHER-2/wew(ECD) and probed with 32 P-labeled fragment of rat HBR-2/neu cDNA showed the expected HER-2/neu RNA's, as shown in figure 6.
  • Example 5 Infection of Dendritic Cells with Ad.HER-2/neu Stimulated Lymphocyte Proliferation.
  • Example 6 Prophylaxis of Breast Cancer by Transfer of Genetically Modified DCs using Adenoviral-mediated Gene Transfer.
  • mice with allogeneic DCs infected with Ad.rHER2/new(ECDtm) or Ad.rHER2/new(ECD) beginning at 5-6 weeks of age improved the tumor-free survival ofthe mice, as shown in Figure 8.
  • Vaccination with DCs infected with the rAd expressing the longer HER2/ «ew gene sequence conferred greater protection to the mice.
  • the first mouse developing breast cancer in this group was at 27.5 weeks.
  • Ad.rHER2/ «ew(ECDtm) conferred significant protection over all other treatment groups, as shown in Figure 9.
  • 82.5% ofthe mammary glands of animals in the control groups had developed tumors compared to 60% ofthe breasts of animals treated with DCs + and the shorter HER2lneu oncogene expressing vector, Ad.rHER2/ «e «(ECD).
  • Various approaches to vaccination included subcutaneous, intraperitoneal, and intramuscular injection ofthe vector, as well as vaccination with bone marrow-derived dendritic cells transduced with the Ad.HER-2/wew vector.
  • vaccination with Ad.BER-2/neu resulted in superior disease free survival and a marked reduction in the number of tumors arising compared to unvaccinated control animals, animals vaccinated with an adenovirus expressing truncated ⁇ ER-2/neu gene (ECD only), a recombinant adenovirus encoding no transgene (Ad.null), or in the case of dendritic cells, unmodified dendritic cells.
  • Ad. ⁇ ER-2/neu afforded superior protection to the mice against the development of spontaneous breast cancers and improved survival (figures 8, 9 and 11).
  • Ad.HER-2/neu or its human derivatives can be an effective anticancer vaccine against human breast cancer and other HER-2/neu expressing tumors.
  • TUBO is a BER-2/neu expressing breast cancer cell line established from a spontaneous tumor from a BA Blc-neuT fransgenic mouse.
  • TUBO cells are tumorigenic in BALB/c-neuI transgenic mice as well as normal BALB/c mice.
  • BA B/c-neuT mice vaccinated with DCs + Ad.rHER2/»ew(ECDtm) and tumor free at 28 weeks were challenged with a subcutaneous injection of 1 x 10 5 TUBO cells.
  • the TUBO cells failed to form tumors.
  • the TUBO cells formed a tumor after a latencyTTable 2).
  • Example 8 Titration of Ad-Her2/neu vaccine dosage to induce protective immune responses.
  • BALBlneu T mice were intraperitoneally immunized with 10 5 , 10 6 , 10 7 or 10 8 pfu of Ad-Her2/neu vaccine at the age of 6, 9, 12 and 15 weeks. Tumor development was measured twice a week by palpating mammary glands. Three mice were used for each group. Ad-Null, not expressing Her2/neu protein, was used for control group.
  • mice immunized with 10 5 or 10 6 pfu dose of vaccine tumor development was delayed compared to the control group.
  • 10 7 pfu dosage was not sufficient to protect against mammary gland tumor development in BALB-neu T, Her2/ « ⁇ ?w-transgenic mice.
  • 10 8 pfu of Ad-Her2/rae « vaccine was needed and 3-4 times immunization with the same dosage was effective.
  • Example 9 CD4 T cells, but not CD8 T cells, are needed for the protective immune responses.
  • Lymphocytes were depleted with anti-CD4 (Figure 13D.), anti-CD8 ( Figure 13E.) or anti-CD4/anti-CD8 ( Figure 13F.) treatment (0.5mg/injection, i.p.) on days 0, 1, 2, 5 of immunization with Ad-Her2/wew.
  • Each group was immunized with 10 8 pfu adenovirus two times from the age of 6-8 weeks at the interval of 3 weeks, except for the group shown in Figure 13A (control group; no immunization).
  • Rat Ig (Figure 13C.)was used as an antibody control.
  • Example 10 Protection of tumor-injected mice with Ad-Her2/nea vaccine ; in vitro model.
  • mice were subcutaneously injected with TUBO cells(10 6 cells/injection.), established cell lines from mammary gland tumors in BALB-neu T mice, and intraperitoneally immunized with 10 8 pfu of Ad-He ⁇ 2/neu. Mice were immunized on days -11 ( Figure 14C) or +5 ( Figure 14D) relative to TUBO injection. Tumor size was measured twice a week. Ad-Null was used as a confrol. Four mice were used for each group.
  • Example 11 Therapeutic effects of Ad-Her2/neu vaccine.
  • BALB/c mice were immunized with Ad-Her2/neu (10 8 pfu, intraperitoneally, once) on days: 2, 4, 7, 10 or 15 ( Figure 15), respectively, after subcutaneous (s.c:)- injection with TUBO cells (10 6 cells, s.c). Mice were arbitrarily distributed to each group after tumor injection. Four mice were used for each group. Tumor size was measured twice a week.
  • Na ⁇ ve control mice have tumors larger than 250 mm 2 around day 30 of TUBO injection (see Figure 15). However, immunized mice (day 2, 4, 7, 10 - see Figure 15) were completely cured of TUBO growth around day 30. Mice immunized on day 10 ( Figure 15) had tumors which were about 50 mm 2 . Moreover mice on day 15 ( Figure 15) had tumors, 60-80 mm 2 . By immunization Ad- ⁇ er2/neu vaccine, tumors larger than 100 mm 2 were therapeutically cured.
  • Example 12 Therapeutic effects of Ad-Her2/neu vaccine ; Repeat under more severe conditions.
  • mice were subcutaneously injected with TUBO cells (10 6 cells/injection) and were grouped by their tumor sizes after day 17 of TUBO cell injection. Mice having the smallest tumors ( Figure 16 A) or Ad-Null treated group ( Figure 16B) were used as na ⁇ ve controls. Others were immumzed once with Ad-Her2/neu, 10 7 pfu ( Figure 16C) or 10 s pfu ( Figures 16D, 16E, and 16F).
  • Example 13 CD4 + T cells are needed for protective immune responses against tumor growth in mammary tumor-injected mice model.
  • BALB/c mice were immunized with Ad- ⁇ Ler2/neu (i.p.,10 8 pfu/injection, once) 7 days before they were injected with TUBO cells (s.c.,10 6 cells/injection).
  • T cells were depleted with anti-CD4 or/and anti-CD8 (i.p., 0.5 mg/injection) from the day of immunization. Tumor size was measured twice a week.
  • Rat IgG was used as a control antibody and 4 mice were used for each group. Depletion of T lymphocytes ' were confirmed as less than 1% on day 10 of tumor injection by FACS analysis.
  • Example 14 Ad-Her2/neu vaccination induced serum antibodies against Her2/neu by a CD4 T cell-dependent mechanism.
  • BALB-neu T mice were immunized with 10 pfu of Ad- ⁇ Rer2lneu vaccine at the age of 8 weeks and 11 weeks.
  • CD4 + T cells Figure 18B.
  • CD8 + T cells Figure 18C
  • Serum was collected from each mouse at the age of 34 weeks when most na ⁇ ve BALB-neu T mice had mammary gland tumors, but immunized mice did not.
  • Her2/neu-specific antibodies were detected by FACS using N202.1, a cell line expressing Her2/neu on their surfaces (expressed as MFI, mean fluorescence intensity).
  • Example 15 DCAd.HER2 Stimulates Proliferation of Na ⁇ ve Splenocytes.
  • DCAd.HER2 irradiated DCAd.HER2, DCAd.nuU, or DC were cultured in various ratios with splenocytes from non-immunized BALB-neuT mice and pulsed with [3H]-thymidine.
  • a 3 to 4-fold greater stimulation was seen with DCAd.HER2 compared to DCAd.null or unmodified DC ( Figure 21) when the ability ofthe DC to stimulate na ⁇ ve BALB-neuT splenocytes was examined in mixed lymphocyte culture.
  • Example 16 Vaccination of BALB-neuT Mice with DC A HEIU Prevented Spontaneous Autochthonous Breast Cancers.
  • BALB-newT develop progressive lesion in mammary glands till all mammary glands are involved in carcinoma as a consequence of rat HER-2/neu expression in mammary gland-specific manner.
  • Female BNLB-neuT at the age of 5 to 6 weeks old were vaccinated weekly for 3 weeks and monitored for palpable breast cancer.
  • Vaccination with DCAd.HER2 significantly improved tumor-free survival ofthe mice compared to mice receiving DCAd.nui ⁇ or unmodified DC ( Figure 22A).
  • Example 17 Vaccination of BALB-neuT Transgenic and BALB/c mice with DCAd.HER2 Inhibited Growth of a Transplantable HER-2/neu-Expressing Carcinoma.
  • DC A[J .H ER2 vaccination protected BALB-neuT mice from challenge with HER-2/ ⁇ ew-expressing TUBO breast cancer cells.
  • mice which were vaccinated with DC modified by Ad.HER-2/neu and remained tumor free 37 days after TUBO cells injection, were challenged with TUBO cells or TS/A cells subcutaneously.
  • the numbers in table indicate ratio of mice which developed tumor * * 121 days or ***10 days after re-challenge.
  • Example 18 DC Ad . HER2 -vaccination Induced Production of Serum Anti-HER- 2/neu Antibodies, Interferon (IFN)- ⁇ Secretion by Splenocytes.
  • IFN Interferon
  • SBP serum specific binding potential
  • IFN- ⁇ production by spleen cells from vaccinated and control mice was examined by ELISPOT assay (Table 5). Production of IFN- ⁇ was increased in the splenocytes of mice vaccinated with DCAd.HER2 compared to mice receiving either DCA d . nu i ⁇ or unmodified DCs alone. These results were confirmed by cytokine secretion assay.
  • DCAd.HER2-vaccinated BALB- neuT mice exhibited a 2-fold higher frequency of CD4 + IFN- ⁇ expressing splenic lymphocytes than mice vaccinated with DCAd.nuii, and 4-fold higher levels than the animals freated with unmodified DCs ( Figure 23B).
  • the population of CD8 + IFN- ⁇ producing splenic lymphocytes population was also increased by 2-fold in DC Ad .
  • the numbers of IFN- ⁇ producing CD8 + lymphocytes were also increased 2 to 5-fold in the spleens of DC A d. HER 2 vaccinated mice compared to animals receiving DCAd.nuU or unmodified DC ( Figure 23B).
  • Example 19 Infiltration of Mammary Glands with CD4+ and CD8+ cells.
  • Mammary glands were removed from mice at the age of 9 weeks old, one week after the last vaccination. Immv ohistochemical staining of mammary glands from BALB-newT mice vaccinated with DC d. H E 2 showed modestly increased numbers of CD4 + and CD8 + cells infiltrating hyperplastic and dysplastic mammary glands. In contrast, the breast tissue of mice receiving the control vaccines showed little cellular infiltrate (Figure 24).
  • DCAU.HER2 To explore the mechanism of DC Ad . HER2 -mediated antitumor vaccination, anti- CD4, anti-CD8 or anti-asialo-GMl antibodies were administered to groups of normal BALB/c mice prior to and during DCAd.H ER 2 vaccination. One week after the last vaccination, the mice were injected with 5 x 10 5 TUBO cells. Control animals vaccinated with DC A d.HER2 > but not treated with antibodies, were protected from tumor growth (Figre 25). Mice receiving anti-CD8 or anti-asialo GM1 antibodies were also protected by the vaccine; however, mice depleted with anti-CD4 antibody developed tumors indicating that CD4 + T cells are required for generation of antitumor immunity by DC A d. HER2 - Treatment with anti-CD4 monoclonal antibodies after completion ofthe DC A d. HER2 vaccination had no effect on the protection afforded by vaccination.
  • Example 21 Efficacy ofDCAd.HER2 Vaccination is Unaffected by Pre-existing Immunity to Adenovirus.
  • mice 3-week-old BALB-raeuT mice were s.c. injected with Ad.null 1 x 10 8 pfu to generate anti-adenovirus immunity. Three weeks later, the mice were divided into two groups and vaccinated s.c. with the Ad.HER2 vector (1 x 10 s pfu) itself or with 1 x 10 6 DC Ad . HER2 - Direct injection of Ad.HER2 is also protective in this model. Four out of seven mice receiving DC A d.
  • HER2 remained free of tumor at 28 weeks. These results were similar to our results in the mice treated with DC d . HER2 not undergoing pre-vaccination with Ad.null ( Figure 26, Figure 22A). In contrast, pre-existing immunity to adenovirus abrogated any protection afforded by direct injection of Ad.HER2. All mice pre- vaccinated with Ad.null and treated with s.c. injections of Ad.HER2 developed breast tumors by 24.5 weeks ( Figure 26).
  • mice were injected weekly for 4 weeks with Ad.null 1 x 10 8 pfu. Two weeks later the mice were treated with s.c. injections of either 1 x 10 8 pfu Ad.HER2 or 1 x 10 6 DC A d. HE 2 - All mice receiving the Ad.null injections showed significant increases in serum anti-adenovirus antibody titers, from a mean of less than 1 :2 prior to injection to greater than 1 :256 one week after the last injection. Untreated mice had a mean baseline titer of less than 1 :2 that remained unchanged over 6 weeks. One week later the mice were injected with 5 x 10 5 TUBO cells. Twenty-one days after challenge, all mice treated directly with Ad.HER2 had developed tumors, whereas six of seven mice receiving DC Ad . HER2 remained tumor-free ( Figure 26).

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Abstract

L'invention concerne des procédés de vaccination de cellules dendritiques, modifiées par des vecteurs adénoviraux de recombinaison (rAds) exprimant des gènes HER2/neu tronqués. L'infection de cellules dendritiques par des vecteur adénoviraux de recombinaison augmente l'expression des marqueurs de maturation superficielle CD80, CD86, MHC-classe I, II, et CD40 des CD et les cancers spontanés du sein sont retardés ou empêchés par vaccination avec des cellules dendritiques génétiquement modifiées par des adénovirus de recombinaison exprimant un antigène de HER2/neu de non signalisation.
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* Cited by examiner, † Cited by third party
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WO2006040660A3 (fr) * 2004-10-15 2006-06-15 Augusto Amici Plasmides codant pour des variantes de sequences de proteines p185neu et leurs utilisations therapeutiques
US7662586B2 (en) 2003-07-21 2010-02-16 Instituto Di Ricerche Di Biologia Molecolare P. Angeletti S.P.A. Synthetic gene encoding human epidermal growth factor 2/neu antigen and uses thereof
WO2017075570A1 (fr) 2015-10-30 2017-05-04 The United States Of America, As Represented By Secretary, Department Of Health And Human Sevices Compositions et méthodes pour le traitement de tumeurs solides à surexpression de her

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US7625859B1 (en) 2000-02-16 2009-12-01 Oregon Health & Science University HER-2 binding antagonists

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US20020039573A1 (en) * 2000-01-21 2002-04-04 Cheever Martin A. Compounds and methods for prevention and treatment of HER-2/neu associated malignancies

Cited By (10)

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Publication number Priority date Publication date Assignee Title
US7662586B2 (en) 2003-07-21 2010-02-16 Instituto Di Ricerche Di Biologia Molecolare P. Angeletti S.P.A. Synthetic gene encoding human epidermal growth factor 2/neu antigen and uses thereof
AU2004261724B2 (en) * 2003-07-21 2010-06-17 Msd Italia S.R.L. Synthetic gene encoding human epidermal growth factor 2/neu antigen and uses thereof
WO2006040660A3 (fr) * 2004-10-15 2006-06-15 Augusto Amici Plasmides codant pour des variantes de sequences de proteines p185neu et leurs utilisations therapeutiques
EP1939217A3 (fr) * 2004-10-15 2008-09-03 Augusto Amici Plasmides codant pour des variantes de séquence de la protéine p185neu et utilisations thérapeutiques correspondantes
RU2383620C2 (ru) * 2004-10-15 2010-03-10 Аугусто АМИЧИ ПЛАЗМИДЫ, КОДИРУЮЩИЕ ВАРИАНТЫ ПОСЛЕДОВАТЕЛЬНОСТИ БЕЛКА p185neu, И ИХ ТЕРАПЕВТИЧЕСКОЕ ПРИМЕНЕНИЕ
US7985582B2 (en) * 2004-10-15 2011-07-26 Augusto Amici Plasmids coding for p185neu protein sequence variants and therapeutic uses thereof
CN101886090B (zh) * 2004-10-15 2012-09-26 奥古斯托·阿米其 编码p185neu蛋白质序列变体的质粒及其治疗用途
KR101337706B1 (ko) * 2004-10-15 2013-12-06 아우구스토 아미치 ｐ185ｎｅｕ 단백질 서열 변이체를 암호화하는플라스미드와 그의 치료 용도
WO2017075570A1 (fr) 2015-10-30 2017-05-04 The United States Of America, As Represented By Secretary, Department Of Health And Human Sevices Compositions et méthodes pour le traitement de tumeurs solides à surexpression de her
US11266726B2 (en) 2015-10-30 2022-03-08 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Compositions and methods for the treatment of HER2-expressing solid tumors

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