# Patent

## Title
RNA interference that blocks expression of pro-apoptotic proteins potentiates immunity induced by DNA and transfected dendritic cell vaccines

## Abstract

An immunotherapeutic strategy is disclosed that combines antigen-encoding DNA vaccine compositions combined with siRNA directed to pro-apoptotic genes, primarily Bak and Bax, the products of which are known to lead to apoptotic death. Gene gun delivery (particle bombardment) of siRNA specific for Bak and/or Bax to antigen-expressing DCs prolongs the lives of such DCs and lead to enhanced generation of antigen-specific CD8+ T cell-mediated immune responses in vivo. Similarly, antigen-loaded DC's transfected with siRNA targeting Bak and/or Bax serve as improved immunogens and tumor immunotherapeutic agents.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of Application No. PCT/US2005/047200, filed Dec. 30, 2005, which claims the benefit of U.S. Provisional Application No. 60/641,901, filed Jan. 6, 2005, and U.S. Provisional Application No. 60/738,900, filed Nov. 22, 2005, the contents of all of which are specifically incorporated by reference herein.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention in the fields of molecular biology, immunology and medicine relates to combinations or mixtures of nucleic acid molecules and chimeric nucleic acid molecules that encode an antigen and a small interfering RNA (siRNA). The expression of the siRNA blocks expression of one or more an anti-apoptotic protein in vivo. This results in prolonging the life of important antigen presenting cells, dendritic cells (DCs), and as a consequence, the more potent induction and enhancement immune responses, primarily cytotoxic T lymphocyte (CTL) responses to specific antigens such as tumor or viral antigens.
2. Description of the Background Art
Cytotoxic T lymphocytes (CTL) are critical effectors of anti-viral and antitumor responses (reviewed in Chen, C H et al., J Biomed Sci. 5: 231-252, 1998; Pardoll, D M. Nat Med. 4: 525-531, 1998; Wang, R F et al., Immunol Rev. 170: 85-100, 1999). Activated CTL are effector cells that mediate antitumor immunity by direct lysis of their target tumor cells or virus-infected cells and by releasing of cytokines that orchestrate immune and inflammatory responses that interfere with tumor growth or metastasis, or viral spread. Depletion of CD8+ CTL leads to the loss of antitumor effects of several cancer vaccines (Lin, K-Y et al., Canc Res 56: 21-26, 1996; Chen, C-H et al., Canc Res. 60: 1035-42, 2000). Therefore, the enhancement of antigen presentation through the MHC class I pathway to CD8+ T cells has been a primary focus of cancer immunotherapy.
Naked DNA vaccines have emerged recently as attractive approaches for vaccine development (reviewed in Hoffman, S L et al., Ann NY Acad Sci 772: 88-94, 1995; Robinson, H L. Vaccine 15: 785-787, 1997; Donnelly, J J et al., Annu Rev Immunol 15: 617-648, 1997; Klinman, D M et al., Immunity 11: 123-129, 1999; Restifo, N P et al., Gene Ther 7: 89-92, 2000; Gurunathan, S et al., Annu Rev Immunol 18: 927-974, 2000). DNA vaccines generated long-term cell-mediated immunity (reviewed in Gurunathan, S et al., Curr Opin Immunol 12: 442-447, 2000) and can generate CD8+ T cell responses in vaccinated humans (Wang, R et al. Science 282: 476-480, 1998).
However, one limitation of these vaccines is their lack of potency, since the DNA vaccine vectors generally do not have the intrinsic ability to be amplified and to spread in vivo as do some replicating viral vaccine vectors. Furthermore, some tumor antigens such as the E7 and E6 proteins of human papillomavirus-16 (“HPV-16”) are weak immunogens (Chen et al., 2000, supra). Therefore, there is a need in the art for strategies to enhance DNA vaccine potency, particularly for more effective cancer and viral immunotherapy.
The present inventors and their colleagues demonstrated that linkage of HPV-16 E7 antigen to a number of immunogenicity-potentiating polypeptides (Kim J W et al., Gene Ther. 11:1011-18, 2004,), such as Mycobacterium tuberculosis (Mtb) heat shock protein 70 (Hsp70) (Chen et al., supra; Wu et al., WO 01/29233) and CRT (Cheng W F et al., J Clin Invest, 2001, 108:669-78; WO/0212281) result in the enhancement of DNA vaccine potency. See, also Cheng W F et al., Vaccine 23:3864-74, 2005; Peng S et al., J Biomed Sci. 12:689-700, 2005; Peng S et al., J Virol. 2004, 78:8468-76; Peng S et al., Gene Ther. 2005 (Sep. 22; Epublished ahead of print)
Others have shown, using protein vaccines, as distinct from DNA immunogens, that immunization with HSP complexes isolated from tumor or virus-infected cells potentiated anti-tumor immunity (Janetzki, S et al., J Immunother 21:269-7, 1998) or antiviral immunity (Heikema, A et al., Immunol Lett 57:69-74, 1997). Immunogenic HSP-peptide complexes could be reconstituted in vitro by mixing the peptides with HSPs (Ciupitu, A M et al., 1998. J Exp Med 187:685-9, 1998). HSP-based protein vaccines have been created by fusing antigens to HSPs (Suzue, K et al., J Immunol 156:873-79, 1996). However, prior to the discoveries of the present inventors and their colleagues since about 1999 with DNA immunogens, HSP vaccines (and those employing other intracellular transport proteins or intercellular spreading proteins) were limited to peptide/protein molecules that were typically produced bacteria using bacterial expression vectors and purified therefrom. The present inventors and their colleagues were the first to provide naked DNA and self-replicating RNA vaccines that incorporated HSP70 and other immunogenicity-potentiating polypeptides. The present inventors and their colleagues were also the first to demonstrate that linking antigen to intracellular targeting moieties calreticulin (CRT), domain II of Pseudomonas aeruginosa exotoxin A (ETA(dII)), or the sorting signal of the lysosome-associated membrane protein type 1 (Sig/LAMP-1) enhanced DNA vaccine potency compared to compositions comprising only DNA encoding the antigen of interest. To enhance MHC class II antigen processing, one of the present inventors and colleagues (Lin, K Y et al., 1996, Canc Res 56: 21-26) linked the sorting signals of the lysosome-associated membrane protein (LAMP-1) to the cytoplasmic/nuclear human papilloma virus (HPV-16) E7 antigen, creating a chimera (Sig/E7/LAMP-1). Expression of this chimera in vitro and in vivo with a recombinant vaccinia vector had targeted E7 to endosomal and lysosomal compartments and enhanced MHC class II presentation to CD4+ T cells. This vector was found to induce in vivo protection against an E7+ tumor, TC-1 so that 80% of mice vaccinated with the chimeric Sig/E7/LAMP1 vaccinia remained tumor free 3 months after tumor injection. Treatment with the Sig/E7/LAMP-1 vaccinia vaccine cured mice with small established TC-1 tumors, whereas the wild-type E7-vaccinia showed no effect on this established tumor burden. These findings point to the importance of adding an immunopotentiating “element” (in the form of DNA encoding that “element”) to DNA encoding an antigen to enhance in vivo potency of a recombinant DNA vaccine for antigens that are presented as either MHC class I- or MHC class II-antigen complexes, such as by rerouting a cytosolic tumor antigen to the endosomal/lysosomal compartment.
Intradermal administration of DNA vaccines via gene gun can efficiently deliver genes of interest into professional antigen presenting cells (APCs) in vivo (Condon C et al., Nat Med, 2: 1122-28, 1996). The skin contains numerous bone marrow-derived APCs (called Langerhans cells) that are able to move through the lymphatic system from the site of injection to draining lymph nodes (LNs), where they can prime antigen-specific T cells (Porgador A et al., J Exp Med 188: 1075-1082, 1998). Powerful APCs in other sites, particularly in lymphatic tissue are dendritic cells (DC). Gene gun immunization therefore provides the opportunity to test vaccine strategies that require direct delivery of DNA or RNA to APCs.
Antigen presentation by DCs is a critical element for the induction of the cellular immune responses that mediate various types of immunotherapy, particularly tumor immunotherapy. Several studies demonstrated that immunization with tumor antigen-pulsed DCs could break the tolerance of the immune system against antigens expressed by tumor cells and in some cases generate appreciable clinical responses. Thus, DC-based vaccines represent a promising method for the treatment of malignancies. See, for example, Gunzer, M et al., Crit Rev Immunol 21: 133-45, 2001; Engleman, E G Dendritic cell-based cancer immunotherapy. Semin Oncol 30:23-29, 2003; Schuler, G et al., Curr Opin Immunol 15:138-147, 2003; Cerundolo, V et al., Dendritic cells: a journey from laboratory to clinic. Nat Immunol 5:7-10, 2004; Figdor, C G et al., Nat Med 10:475-480, 2004; Markiewicz, M A et al., Cancer Invest 22:417-434, 2004; Turtle, C J et al., Curr Drug Targets 5:17-39, 2004).
Dendritic cell-based vaccines have become an important approach for the treatment of malignancies. Numerous techniques have recently been designed to optimize dendritic cell activation, tumor antigen delivery to dendritic cells, and induction of tumor-specific immune responses in vivo. Dendritic cells, however, have a limited life span because they are subject to apoptotic cell death mediated by T cells, hindering their long-term ability to prime antigen-specific T cells.
DCs, however, have a limited life span that hinders their long-term ability to prime antigen-specific T cells (see Ronchese, F et al. J Exp Med 194:F23-26, 2001). A principal contributor to the shortened lifespan of DCs is CTL-induced apoptosis. After activation by DCs, CTLs that recognize epitopes can kill target cells expressing these epitopes, typically presented by MHC Class I proteins. Because DCs express MHC-I:antigen peptide complexes, newly primed CTLs can kill the very DCs that activated them (Medema, J P et al., J Exp Med 194:657-667, 2001). Thus, DC-based vaccination should be enhanced by inhibiting apoptosis and prolong survival of antigen-expressing DCs in vivo (Kim, T W et al., J Immunol 171:2970-2976, 2003a; Kim, T W et al., J Clin Invest 112:109-17, 2003(b); and a patent application by the present inventors and colleagues WO05/047501 (26 May 2005) incorporated herein by reference in its entirety.
The present inventors and their colleagues have used gene gun immunization of DNA compositions to test vaccine strategies that involve intracellular targeting strategies that direct delivery of DNA or RNA to APCs. The targeting molecules (using coding DNA linked to DNA encoding an antigen) that have shown potent effects include Mycobacterium tuberculosis heat shock protein 70 (HSP70) (Chen C H et al., 2000, Cancer Res 60:1035-42, 2000), calreticulin (CRT; Cheng W F, 2001, supra), and the sorting signal of the lysosome-associated membrane protein 1 (LAMP-1; Ji H et al., Hum Gene Therapy, 10:2727-40, 1999).
Vaccination with DNA vectors that encode such fusion proteins are able to route an antigen (generally exemplified with HPV-16 E6 and E7) to desired subcellular compartments, and enhance antigen processing and presentation to T cells. Therefore, direct delivery of DNA vaccines into DCs via gene gun provides an opportunity to modify the quality and quantity of DNA-transfected DCs and influence vaccine potency.
T cell-mediated apoptotic cell death can occur through two major pathways, the intrinsic and the extrinsic pathways. See, for example, Russell, J H et al., Annu Rev Immunol 20:323-370, 2002). In general, death domain-containing receptors such as CD95 (APO-1/Fas) can sense the external signal (such as Fas ligand) and activate the extrinsic apoptotic pathway through the Fas-associated death domain (Fadd). This pathway is mediated by recruitment and activation of caspase-8, an initiator caspase, in the death-inducing signaling complex (DISC) followed by direct cleavage of downstream effector caspases.
The intrinsic pathway (granzyme B/perforin-mediated apoptosis), important for T cell-mediated induction of apoptotic DC death, initiates from within the cell. The pore-forming protein perforin and the serine protease granzyme B secreted into cells by antigen-specific CD8+ T cells induce intracellular changes, such as DNA damage, resulting in the release of a number of pro-apoptotic factors from mitochondria, such as cytochrome c, leading to the activation of another initiator caspase, caspase-9 (Jacotot, E et al., Ann NY Acad Sci 887:18-30, 1999; Korsmeyer, S J et al., Cell Death Differ 7:1166-73, 2000; Degli Esposti, M et al., Dive, C. Biochem Biophys Res Commun 304:455-61, 2003; Opferman J T et al., Nat Immunol 4: 410-15, 2003). 5-61, 2003; Opferman J T et al., Nat Immunol 4: 410-15, 2003). Activated caspase-9 leads to the activation of effector caspases (caspase-3, -6, and -7) in a protein complex called the apoptosome (for review, see Johnson, C R et al., Apoptosis 9:423-27, 2004) leading to proteolysis of a cascade of substrates and apoptotic death.
Thus Bak, Bax, and caspase 9 are clearly important pro-apoptotic proteins for the intrinsic apoptotic pathway and caspases-8 and -3 are is an important pro-apoptotic proteins in the extrinsic apoptotic pathway. Because of the role of Bak and Bax as gatekeepers in the intrinsic apoptotic pathway, the present inventors have conceived of targeting these genes for inhibition by RNA interference (RNAi) to diminish DC apoptosis. This is disclosed in detail and exemplified below. However, the present inventors conception includes a similar targeting of caspase-9, caspase-3 and caspase-8.
RNA interference (RNAi) is a recently reported phenomenon that has developed into a new approach for elucidating and regulating gene function. RNAi is a sequence-specific, post-transcriptional, gene-silencing mechanism that is effected through double-stranded RNA (dsRNA) molecules homologous to a sequence of the target gene (Elbashir, S M et al., Nature 411:494-498, 2001; Fire, A et al., Nature 391:806-811, 1998; Tuschl, T et al., Genes Dev 13:3191-3197, 1999). Fragments of the dsRNA called “small interfering” RNAs (siRNAs) can rapidly induce loss of function, and only a few molecules are required in a cell to produce the effect (Fire et al., supra) through hybrid formation between a homologous siRNA and mRNA (Lin, S L et al., Curr Cancer Drug Targets 1:241-247, 2001). A member of the RNase III family of nucleases named dicer has been identified as being involved in processing (Bernstein, E et al., Nature 409:363-366, 2001). DNA vector-mediated RNAi technology has made it possible to develop therapeutic applications for use in mammalian cells (Sui, G et al., Proc Natl Acad Sci USA 99:5515-5520, 2002; McCaffrey, A P et al., Nature 418:38-39, 2002; Lee, N S et al., Nat Biotechnol 20:500-505, 2002). There have been several reports of delivery of siRNA by retroviral vectors for stable expression (Barton, G. M et al., Proc Natl Acad Sci USA 99:14943-14945, 2002; Paddison, P J et al., Cancer Cell 2:17-23, 2002; Rubinson, D A et al., Nat Genet 33:401-406, 2003; Tiscornia, G et al., Proc Natl Acad Sci USA 100:1844-1848, 2003) or by adenoviral vectors for transient expression (Xia, H et al., Nat Biotechnol 20:1006-1010, 2002). RNAi may be effected by small interfering RNA molecules (siRNA) that induce sequence-specific degradation of mRNA or by inhibiting translation of its complementary mRNA (see, for example, Mittal V. Nat Rev Genetics 5:355-65, 2004). Use of this approach to prolong the life of DCs by targeting pro-apoptotic proteins with the appropriate siRNAs is one of the objects of the present invention.
SUMMARY OF THE INVENTION
Partial List of Abbreviations Used


    
    
        APC, antigen presenting cell; BM, bone marrow; BM-DC, BM-derived dendritic cells; CMV, cytomegalovirus; CTL, cytotoxic T lymphocyte; CRT, calreticulin; DC, dendritic cell; E6, HPV oncoprotein E6; E7, HPV oncoprotein E7; ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; HPV, human papillomavirus; HSP, heat shock protein; Hsp70, mycobacterial heat shock protein 70; IFNγ, interferon-γ; i.m., intramuscular(ly); i.v., intravenous; IPP, immunogenicity-potentiating (or -promoting) polypeptide; LN, lymph node; MHC, major histocompatibility complex; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RNAi, RNA interference or interfering RNA; siRNA, small interfering RNA; siNA, small interfering nucleic acid; β-gal, β-galactosidase.
    
    


The present inventors have designed and disclose herein an immunotherapeutic strategy that combines antigen-encoding DNA vaccine compositions with siRNAs directed to pro-apoptotic genes, primarily Bak and Bax, the products of which are known to lead to apoptotic death of, inter alia, DCs. The present inventors conceived that gene gun delivery (particle bombardment) or delivery by other appropriate routes of siRNA specific for Bak and/or Bax to antigen-expressing (antigen-presenting) DCs would prolong the lives of such transfected DCs and lead to enhanced generation of antigen-specific T cell-mediated immune responses in vivo.
The present disclosure shows the impact of intradermal (gene gun) coadministration of DNA vaccines encoding HPV-16 E7 antigen with Bak and/or Bax siRNA. The present inventors chose HPV-16 E7 as a model antigen because HPVs, particularly HPV-16, are associated with a majority of cervical cancers, and E7 (and E6) is essential for oncogenic cell transformation. Use of constructs comprising DNA encoding HPV protein E6 would be expected to have the same activity (as supported by comparisons between the two using the present inventors' other immunopotentiating strategies). Minimally genetically modified E7 or E6 proteins (“detox”) which have been rendered incapable of oncogenic activity by between 1 and 3 point mutations may be used in place of wild-type E7 and E6, and are safer for human subjects.
Effective vaccines against E7 (and/or E6) can be used to control HPV infections and HPV-associated lesions. As disclosed herein, evaluation of E7-specific immune responses, antitumor effects, and survival of DNA-transfected DCs, confirmed the present inventors' conception that co-administration of (i) a DNA vaccine or immunogen comprising sequences encoding an antigen with Bak- and/or Bax-specific siRNA (which term is used interchangeably with “Bak- or Bax siRNA”) is a successful and innovative strategy for enhancing DNA vaccine potency.
As disclosed in herein (see Examples 8 et seq.) in DCs that are transfected with Bak/Bax siRNA Bak and Bax protein expression is abolished. According to the present invention, DCs transfected with Bak and Bax siRNA that are pulsed (loaded) with an antigenic peptide, so that they present that peptide, induce more potent antigen-specific CD8+ T cell immune responses and antitumor effects in vaccinated subject mice, compared to peptide-pulsed DCs transfected with control siRNA. Bak/Bax siRNA-transfected DCs survive better in vivo than do antigenic peptide-loaded DCs transfected with a control siRNA in mice into which antigen-specific CD8+ T cells (able to kill the antigen-presenting DCs) have been adoptively transferred. Bone marrow-derived DCs (BM-DCs) and long-term DC cell lines as shown to be useful cellular immunogens.
The foregoing conceptions and discoveries provide a basis for clinical therapy of pathologies associated with any antigen, such as an antigen from a pathogenic microorganism (virus, bacterium, parasite), and pathogenic “endogenous” cells such as a tumor or cancer cells. Examples of viral antigens against which this strategy is exemplified herein are the two oncoprotein antigens from HPV-16, namely E6 and E7.
The invention exploits siRNA-based strategies to manipulate the functions, primarily to promote the survival, of DCs exposed to the siRNA ex vivo and/or in vivo. The siRNA-encoding constructs described can be used in combination with the strategy of enhancing the presentation of antigen through the MHC class I pathway to CD8+ T cells by exploiting the features of certain polypeptides to target or translocate the antigenic polypeptide to which they are fused. Such polypeptide are referred to collectively herein as “immunogenicity-potentiating (or -promoting) polypeptide” or “IPP” to reflect this general property, even though these IPP's may act by any of a number of cellular and molecular mechanisms that may or may not share common steps. This designation is intended to be interchangeable with the term “targeting polypeptide.” Inclusion of nucleic acid sequences that encode polypeptides that modify the way the antigen encoded by molecular vaccine is “received” or “handled” by the immune system serve as a basis for enhancing vaccine potency. All of these polypeptides in some way, contribute to the augmentation of the specific immune response to an antigen to which they are linked by one or another means that these molecules “employ” to effect the way in which the cells of the immune system handle the antigen or respond with cell proliferation and/or survival. IPP's may be produced as fusion or chimeric polypeptides with the antigen, or may be expressed from the same nucleic acid vector but produced as distinct expression products.
In addition to the strategy of including DNA encoding such IPPs in their vaccine constructs, the present invention harnesses the additional biological mechanism of inhibiting apoptosis by employing the RNAi approach significantly enhances T cell responses to DNA vaccine comprising antigen-coding sequences (with or without linked sequences encoding such IPPs).
Intradermal vaccination by gene gun efficiently delivers a DNA vaccine into DCs of the skin, resulting in the activation and priming of antigen-specific T cells in vivo. DCs, however, have a limited life span, hindering their long-term ability to prime antigen-specific T cells. According to the present invention, a strategy that prolongs the survival of DNA-transduced DCs enhances priming of antigen-specific T cells and thereby, increase DNA vaccine potency. As described herein co-delivery of siRNA that suppresses the expression of apoptotic pathways via Bak and Bax, prolongs the survival of transduced DCs. More importantly, vaccinated subjects exhibited significant enhancement in antigen-specific CD8+ T cell immune responses, resulting in a potent antitumor effect against antigen-expressing tumors. In another embodiment, instead of delivering the siRNA directly via gene gun, DNA encoding the siRNA is delivered either as part of the same vector that encodes the antigen, or as a separate vector that is co-administered.
The combination of a strategy to prolong DC life (via siRNA) with intracellular targeting strategies afforded by certain IPPs produces a more effective DNA vaccine against E7, E6 or any antigen. Co-administration of siRNA (or DNA encoding siRNA) directed to Bak and/or Bax with DNA encoding antigen (exemplified as E7) linked to DNA encoding HSP70, CRT, or Sig/LAMP-1 results in further enhancement of the antigen (here E7)-specific CD8+ T cell response for all three types of constructs. This combination increases CD8+ T cell functional avidity, and increases the E7-specific CD4+ Th1 cell response, enhances tumor therapeutic effect, and will yield more durable tumor protection when compared with mice vaccinated without the siRNA. Therefore, DNA vaccines that combine strategies to enhance intracellular antigen processing and prolong DC life have clinical utility for control of viral infection and neoplasia, among other forms of pathology where immunotherapy is useful as an ameliorative or curative therapy.
Thus, the present invention is directed to a nucleic acid composition useful as an immunogen, comprising a combination of:

    (a) a first nucleic acid molecule comprising a first sequence encoding an epitope of an antigenic polypeptide or peptide; and optionally, linked to the first sequence, directly or via a linker, a second sequence that encodes an immunogenicity-potentiating polypeptide (IPP); and
    (c) a second nucleic acid molecule the activity or expression of which stimulates development of an immune response to the epitope, which second nucleic molecule is (i) a siNA or (ii) DNA that encodes the siNA, wherein the siNA has a sequence that is sufficiently complementary to, and thus targets, the sequence of mRNA that encodes a pro-apoptotic protein expressed in a dendritic cell (DC), such that the activity or expression of the siNA in the cell results in inhibition of or loss of expression of the mRNA, resulting in inhibition of apoptosis and increased survival of DCs,

wherein the development of the immune response is stimulated.



The IPP above is preferably fused in frame to the first sequence such that the first and the second sequences encode a fusion protein comprising the antigenic epitope and the IPP. The IPP acts in potentiating an immune response preferably by promoting:

    (a) processing of the linked antigenic polypeptide via the MHC class I pathway or targeting of a cellular compartment that increases the processing;
    (b) development, accumulation or activity of antigen presenting cells or targeting of antigen to compartments of the antigen presenting cells leading to enhanced antigen presentation;
    (c) intercellular transport and spreading of the antigen; or
    (d) any combination of (a)-(c).


In the above composition, the IPP is: preferably

    (a) the sorting signal of the lysosome-associated membrane protein type 1 (Sig/LAMP-1)
    (b) a mycobacterial HSP70 polypeptide, the C-terminal domain thereof, or a functional homologue or derivative of the polypeptide or domain;
    (c) a viral intercellular spreading protein selected from the group of herpes simplex virus-1 VP22 protein, Marek's disease virus UL49 protein or a functional homologue or derivative thereof;
    (d) an endoplasmic reticulum chaperone polypeptide selected from the group of calreticulin or a domain thereof, ER60, GRP94, gp96, or a functional homologue or derivative thereof.
    (e) domain II of Pseudomonas exotoxin ETA or a functional homologue or derivative thereof;
    (f) a polypeptide that targets the centrosome compartment of a cell selected from γ-tubulin or a functional homologue or derivative thereof; or
    (g) a polypeptide that stimulates DC precursors or activates DC activity selected from the group consisting of GM-CSF, Flt3-ligand extracellular domain, or a functional homologue or derivative thereof.


In the above composition the pro-apoptotic protein is preferably selected from the group consisting of one or more of (a) Bak, (b) Bax, (c) caspase-8, (d) caspase-9 and (e) caspase-3. Most preferably the siNA targets the encoding mRNA of anti-apoptotic protein Bak and/or Bax.
Preferably the siNA is an siRNA. The siRNA preferably targets SEQ ID NO:4 of Bak and/or SEQ ID NO:8 of Bax. The preferred siRNA is selected from the group consisting of (a) SEQ ID NO:1/SEQ ID NO:2; and (b) SEQ ID NO:5/SEQ ID NO:6.
The antigenic polypeptide or peptide of the above composition preferably comprises an epitope that binds to and is presented on surfaces of antigen-presenting cells by MHC class I proteins. The epitope may be between about 8 and about 11 amino acid residues in length.
The antigenic polypeptide or peptide of the above composition preferably (i) is derived from a pathogen such as a mammalian cell (e.g., specific or tumor-associated antigen), a microorganism or a virus; or (ii) cross-reacts with an antigen of the pathogen; or (iii) is expressed on the surface of a pathogenic cell. Preferred antigens are from a human papilloma virus, primarily the E7 and E6 polypeptide, including the “detox” forms of these polypeptides.
In the above composition, the first nucleic acid molecule is preferably an expression vector comprising a promoter operatively linked to the first and/or the second sequence; the promoter is preferably one that is expressed in an APC, most preferably in a DC.
Also provided herein are particles comprising a material suitable for introduction into a cell or an animal by particle bombardment to which particles is bound the above composition. Also intended is a combination of first and second particles each comprising a material is suitable for introduction into a cell or an animal by particle bombardment, and to which particles is bound the above composition. Wherein (a) the first nucleic acid molecules are bound to a first set of particles; and (b) the second nucleic acids (the siNA, preferably siRNA) are bound to a second set of particles. The preferred particles are gold particles.
This invention includes a pharmaceutical composition capable of inducing or enhancing an antigen specific immune response, comprising any of the above compositions or particles and a pharmaceutically acceptable carrier or excipient.
In another embodiment, the invention is directed to a method of inducing or enhancing an antigen specific immune response in a subject comprising administering to the subject an effective amount of the above composition or particles thereby inducing or enhancing the antigen specific immune response. The antigen specific immune response is preferably one mediated at least in part by CD8+ cytotoxic T lymphocytes (CTL). The method is preferably carried out on a mammalian, most preferably a human subject. In the method, the composition or particles are preferably administered intradermally by particle bombardment (gene gun). The composition may also be administered intratumorally or peritumorally.
One embodiment is directed to a method of increasing the numbers of CD8+ CTLs specific for a selected desired antigen in a subject comprising administering an effective amount of the above composition, particles or pharmaceutical compositions, wherein the antigenic peptide or polypeptide comprises an epitope that binds to and is presented on surfaces of APCs by MHC class I proteins.
Also provided is a method of inhibiting the growth of a tumor in a subject comprising administering an effective amount of the above composition, particles or pharmaceutical compositions, wherein the antigenic epitopes are those expressed by the tumor or ones cross reactive with those expressed by the tumor.
This invention is further directed to an immunogenic cellular composition, including a pharmaceutical composition thereof, comprising DCs which have been modified by:

    (a) loading the DCs with an antigen so that the antigen is expressed on the DC surface, or transducing or transfecting the DCs with DNA that encodes an antigen fused to an IPP; and
    (b) transfecting the DCs with a nucleic acid molecule that is (i) a siNA or (ii) DNA that encodes the siNA, preferably siRNA, wherein the siNA has a sequence that is sufficiently complementary to the sequence of, and thus targets, mRNA that encodes a pro-apoptotic protein expressed in the DC, such that expression or activity the siNA in the cell results in diminution or loss of expression of the mRNA, resulting in inhibition of apoptosis and prolonged survival of the DC.


The pro-apoptotic protein target is preferably one or more of (a) Bak, (b) Bax, (c) caspase-8, (d) caspase-9 and (e) caspase-3. Preferably, the siRNA targets Bak and/or Bax; preferred targeted sequences are SEQ ID NO:4 of Bak and SEQ ID NO:8 of Bax. Preferred siRNA is selected is (a) SEQ ID NO:1/SEQ ID NO:2; or b) SEQ ID NO:5/SEQ ID NO:6.
A method of inducing or enhancing an antigen specific immune response in a subject comprises administering to the subject an effective amount of the above DC composition thereby inducing or enhancing the antigen specific immune response.
A method of increasing the numbers of CD8+ CTLs specific for a selected desired antigen in a subject comprises administering an effective amount of the DC composition wherein the loaded antigen or the antigen expressed from the transduced DNA comprises an epitope that binds to and is presented on the DC surface by MHC class I proteins, thereby increasing the numbers of antigen-specific CD8+ CTLs.
A method of inhibiting the tumor growth in a subject comprises administering an effective amount of the DC composition, wherein the antigenic epitopes are those expressed by the tumor or ones cross reactive with those expressed by the tumor, thereby inhibiting growth of the tumor.
The invention is directed to use of a nucleic acid composition as defined above or particles as defined above or a DC composition as defined above in the manufacture of a medicament, preferably a vaccine, for inducing or enhancing an antigen specific immune response in a subject.
Also included is the use of a nucleic acid composition as defined above, particles as defined above, or a DC composition as defined above in the manufacture of a medicament for inhibiting the growth of a tumor or treating cancer in a subject wherein the antigenic epitopes are those expressed by the tumor or ones cross-reactive with those expressed by the tumor.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1-3. Detection of Bak and Bax expression and evaluation of resistance to apoptotic cell death after delivery of Bak and/or Bax siRNA. FIG. 1 is a Western blot analysis demonstrating expression of Bak and/or Bax protein in transfected cells. FIG. 2 is a Western blot demonstrating kinetics of Bak and Bax expression in siRNA-transfected DC-1 cells. β-actin was used as an internal control for quantification of protein expression. FIG. 3A-B is a graph showing the percentage of apoptotic cells in E7 peptide-pulsed DC1 cells (RAHYNIVTF, SEQ ID NO:46) transfected with either Bak+Bax siRNA or with control siRNA, after incubation for 4 hrs (FIG. 3A) or 20 hrs (FIG. 3B)) with an E7-specific CD8+ T cell line. DC-1 cells pulsed with HA peptide (IYSTVASSL, SEQ ID NO:47) was used as a control.
FIGS. 4-7. ICCS and flow cytometric analysis to determine the antigen-specific CD8+ T cell response to an immunogenic DNA vaccine coadministered with control or Bax and/or Bak siRNA. FIGS. 4 and 5 show results of mice vaccinated with pcDNA3-E7. FIGS. 6 and 7 shows results of mice vaccinated with pcDNA3-HA or -OVA. pcDNA3 encoding Bak+Bax siRNA served as a negative control. FIG. 4 shows representative flow cytometry results. FIG. 5 represents the number of IFN-γ-expressing E7-specific CD8+ T cells in a population of spleen cells from vaccinated mice. FIG. 6 shows representative flow cytometric data. FIG. 7 represents the number of IFN-γ-expressing HA- or OVA-specific CD8+ T cells in a spleen cell population from vaccinated mice.
FIGS. 8-10. In vivo tumor protection and therapy studies using E7-expressing TC-1 Tumor cells. FIG. 8 shows results of an in vivo tumor protection experiment. pcDNA3 encoding Bak+Bax siRNA was used as a negative control. FIG. 9 shows results of an in vivo antibody depletion experiments indicating the contribution of lymphocyte subsets to the observed protective effect above. FIG. 10 shows results of an in vivo tumor therapy experiment using the hematogenous spread lung metastasis model. pcDNA3 encoding Bak+Bax siRNA was used as a negative control.
FIGS. 11-14. ICCS and flow cytometric analysis of E7-specific CD4+ or CD8+ T cell responses in mice vaccinated with a DNA vaccine employing intracellular targeting strategies and siRNA. In the experiments of FIGS. 11 and 12, mice were vaccinated with pcDNA3-E7, pcDNA3-Sig/E7/LAMP-1, pcDNA3-E7/HSP70, or pcDNA3-CRT/E7 combined with Bak+Bax siRNA or control siRNA. In the experiments of FIGS. 13 and 14, mice were vaccinated with pcDNA3-Sig/E7/LAMP-1 combined with Bak+Bax siRNA or control siRNA. FIG. 13 is a representative example of a flow cytometric analysis. FIG. 12, represents the number of IFN-γ-expressing E7-specific CD8+ T cells in a spleen cell population from vaccinated mice. FIG. 13 represents the number of IFN-γ-expressing E7-specific CD4+ Th1 cells in a spleen cell population from vaccinated mice. FIG. 14 represents the number of IL-4-expressing E7-specific CD4+ Th2 cells in the spleen cell population from vaccinated mice.
FIGS. 15-18. Flow cytometric analysis of GFP-expressing DCs in draining lymph nodes of mice vaccinated with E7/GFP DNA combined with Bak+Bax siRNA. FIG. 15, is a representative sample of a flow cytometry analysis 2 and 5 days after intradermal administration of pcDNA3-E7/GFP. The numbers indicate the percentage of GFP-expressing cells out of the total of CD11c+ cells. FIG. 16 shows percentages of GFP-expressing cells out of the total of CD11c+ cells. FIG. 17 shows results of a representative in vivo antibody depletion experiment. FIG. 18 shows percentages of GFP+ cells (out of total CD11c+ cells) after antibody depletion.
FIGS. 19-20. ICCS with flow cytometric analysis to determine the effect of co-administration of Bak+Bax siRNA during priming and/or boosting. Mice were vaccinated with pcDNA3-Sig/E7/LAMP-1 combined with Bak+Bax siRNA (“B+B”) and/or control siRNA (“Con”) in the priming phase and/or the boosting phase of the vaccination protocol. FIG. 19 shows representative flow cytometric results. FIG. 20 shows the numbers of IFN-γ-expressing E7-specific CD8+ T cells in the spleen cell population from vaccinated mice.
FIG. 21. Western blot analysis of the expression of Bak and Bax protein in DC-1 cells transfected with various siRNA constructs. DC-1 cells were transfected with either Bak/Bax siRNA or control siRNA. Western blot analysis was performed with 50 μg of cell lysates 24 and 48 hours after transfection. β-actin was used as a control for loading. Lysates of untransfected DC-1 cells were used as negative controls.
FIGS. 22-23. ICCS and flow cytometric analysis to determine the number of IFN-γ-producing E7-specific CD8+ T cells in mice after immunization with E7 peptide-pulsed DCs transfected with various siRNA constructs. Mice (5/group) were vaccinated with E7 peptide-loaded DCs transfected with (i) Bak/Bax siRNA or (ii) control siRNA. Mice vaccinated with E7 peptide-loaded DCs (no transfection) were additional controls. FIG. 22 shows representative flow cytometric results for pooled spleen cells harvested from vaccinated mice that were either (i) stimulated with E7 aa49-57 peptide or (ii) unstimulated in culture. FIG. 23 shows the numbers of IFN-γ-secreting E7-specific CD8+ T cell precursors (per 3×105 spleen cells) from mice vaccinated with E7 peptide-loaded DCs that were transfected with (i) control siRNA, (i) Bak/Bax siRNA or (iii) untransfected. Results shown are means±SD; p<0.001; Student's t test).
FIGS. 24-25. Flow cytometric analysis (FIG. 24) and ICCS (FIG. 25) of spleen cells from mice immunized with E7-pulsed bone marrow-derived DCs (BM-DCs) transfected with the various siRNA constructs. Mice (5/group) were vaccinated with E7 peptide-loaded BM-DCs transfected with (i) Bak/Bax siRNA or (ii) control siRNA. FIG. 25 represents the number of IFN-γ-secreting E7-specific CD8+ T cell precursors (per 3×105 spleen cells) after immunization with E7 peptide-loaded BM-DCs transfected with siRNA or from non-immunized mice (mean±SD; p<0.001; Student's t-test).
FIGS. 26-27. In vivo tumor protection and treatment experiments. FIG. 26 shows results of a tumor protection experiment in which mice (5/group) were immunized with E7 peptide-loaded DCs transfected with either (i) control siRNA or (ii) Bak/Bax siRNA and boosted after 1 week. 7 days after the last immunization, each mouse was challenged with 5×104 TC-1 tumor cells (see Example I). Tumors were monitored twice a week. Unvaccinated mice served as negative controls. FIG. 27 shows results of an in vivo tumor therapy experiment in which mice were given E7 peptide-loaded DCs transfected with (i) Bak/Bax siRNA or (ii) control siRNA, three days after TC-1 tumor cell challenge (5×104 tumor cells). Mice were boosted with the same dose and regimen of E7 peptide-loaded DCs one week later and sacrificed 28 days after tumor challenge. Lung nodules (experimental metastases) were evaluated and the results expressed as the mean number of lung nodules±SD (p<0.001; Student's t-test).
FIGS. 28A-28B. Survival of E7 peptide-loaded BM-DCs transfected with Bak/Bax siRNA or control siRNA after administration of E7-specific CD8+ T cells in vivo. FIG. 28A presents flow cytometric results showing the different level of carboxyfluorescein (CFSE)-labeled E7 peptide-loaded BM-DCs transfected with either (i) Bak/Bax siRNA (“low CFSE”) or (ii) control siRNA (“high CFSE”). Bak/Bax-transfected BM-DCs were labeled with a lower concentration (0.5 μM) of CF SE, whereas control BM-DCs (transfected with control siRNA) were labeled with a higher concentration (5 μM) of CFSE. A representative graph shows the presence of similar numbers of “low CFSE”-labeled E7 peptide-loaded BM-DCs transfected with Bak/Bax siRNA and “high CFSE”-labeled E7 peptide-loaded control BM-DCs before i.v. injection. FIG. 28B shows flow cytometric results demonstrating the ratio of “low CFSE” to “high CFSE” E7 peptide-loaded BM-DCs that have localized to the spleen and lungs of mice 16 hrs after i.v. injection of a mixture equal numbers (2.5×105/mouse) of “low CFSE” E7 peptide-loaded BM-DCs transfected with Bak/Bax siRNA and “high CFSE” E7 peptide-loaded control BM-DCs. These CFSE-labeled BM-DCs were injected into mice 3 days after the administration of 106 E7-specific T cells/mouse. Contact with these T cells are the basis for DC apoptosis in this study. Note that the number of “low CFSE” cells was significantly higher than the number of “high CFSE” cells.
FIG. 29. Characterization of the surface molecules of E7 peptide-loaded DCs after transfection with Bak/Bax siRNA or control siRNA. Flow cytometry was used to determine the level of expression of CD11c, CD40, CD86, MHC I, and MHC II-molecules in E7 peptide-loaded murine DC-1 cells transfected with either (i) Bak/Bax siRNA or (ii) control siRNA. E7 peptide-loaded DCs that were not transfected at all served as negative controls.
FIG. 30. Western blot analysis to detect expression of Bak and Bax protein in DCs transfected with the various siRNA constructs. Various DCs were transfected with control or BAK/BAX siRNAs. Equal amounts of protein (50 μg) were loaded and separated by SDS-PAGE using a 15% polyacrylamide gel. Western blot analysis was performed with 50 μg of the cell lysate and anti-BAK or BAX mouse monoclonal antibody 3 days after transfection. β-actin was used as a control to indicate that equal amounts of cell lysates were loaded.
FIG. 31. In vitro resistance of the DCs transfected with the siRNAs to CD8 T cell-mediated CTL-killing. DCs were incubated with an E7-specific CD8+ T cell line at different E:T ratios (12.5:1, 2.5:1, 0.5:1 or 0.1:1) for 4 (A) or 18 hours (B). FITC-conjugated anti-CD8 antibody was used to stain for a CD8+ E7-specific T cell line, and then CD8-negative cells (DCs) were gated to identify the percentage of apoptotic dendritic cells. Detection of apoptotic cells in the DC cells was performed using PE-conjugated rabbit anti-active caspase-3 antibody (BD Bioscience). The percent of apoptotic cells was analyzed using flow cytometry.
FIG. 32. Intracellular cytokine staining and flow cytometry analysis to demonstrate the in vitro activation of E7-specific T cells by DCs transfected with siRNAs. DCs expressing E7 or Sig/E7/LAMP-1, transfected with control or BAK/BAX siRNAs, were incubated with an E7-specific CD8+ T cell line at a 1:10 mixture of DC:T cell ratios for 18 hours. Intercellular cytokine staining assay was performed to count activated IFN-γ-secreting CD8+ T cells using flow cytometry. The data presented in this figure are from one representative experiment of three performed (A). Bar graph depicting the % of E7 specific CD8+ T cell (B).
FIG. 33. Intracellular cytokine staining and flow cytometry analysis to determine the number (A) and functional avidity (B) of IFN-γ-producing E7-specific CD8+ T cells in mice after immunization with E7-expressing DCs transfected with various siRNA constructs. Mice (five per group) were vaccinated twice with E7-expressing DCs transfected with BAK/BAX siRNA or control siRNA. There was a 1-week interval between injections. Splenocytes were harvested one week after the last vaccination, stained for CD8+ and IFN-γ, and analyzed by flow cytometry to detect activated E7-specific CD8+ T cells. The bar graph depicts the number of IFN-γ-expressing E7-specific CD8+ T cells per 3×105 splenocytes from vaccinated mice (mean±SD) (A). For the determination of the avidity of E7-specific CD8+ T cells, pooled splenocytes were incubated overnight with different concentrations of E7 peptide. The number of E7-specific IFN-γ-secreting CD8+ T cells was determined using intracellular cytokine staining and FACScan analysis as described above. We defined the number of IFN-γ-expressing CD8+ T cells stimulated with E7 peptide (amino acids 49-57; 100 μ/ml) as the maximal response. The horizontal line allows comparison of E7 peptide concentrations needed for 50% of maximal E7-specific CD8+ T cell response in mice vaccinated using the two regimens. The data presented in this figure are from one representative experiment of two performed (B).
FIG. 34. In vivo tumor treatment experiments in mice vaccinated with DCs expressing E7 or Sig/E7/LAMP-1. An in vivo tumor treatment experiment was performed using a hematogenous spread lung model. Mice were inoculated with 5×105 TC-1 tumor cells via tail vein injection and then treated with DC-Sig/E7/LAMP-1 cells transfected with control or BAK/BAX siRNAs 3 days after inoculation. No treatment served as a negative control (A). In vivo antibody depletion experiments to determine the contribution of subsets of lymphocytes to the observed protective anti-tumor effect (B). Mice were challenged and vaccinated as described in (A). CD4, CD8, or NK1.1 depletion was initiated 1 week after the second vaccination. Mice were sacrificed 28 days after tumor challenge to examine the growth of pulmonary nodules. Data are expressed as the mean number of lung nodules. The data presented in this figure are from one representative experiment of two performed.
FIG. 35. Flow cytometry analysis to determine the survival of DC-Sig/E7/LAMP-1 transfected with control siRNA or BAK/BAX siRNA in draining lymph nodes. C57BL/6 mice (five per group) were first primed with DC-no insert or DC-Sig/E7/LAMP-1. Seven days later, the mice received CFSE-labeled DC-Sig/E7/LAMP-1 transfected with control siRNA or BAK/BAX siRNA. Unlabeled DCs transfected with BAK/BAX siRNA were used as a negative control. Two days after boosting with the DCs, popliteal lymph nodes were harvested and isolated lymphocytes were analyzed by flow cytometry. The monocyte-like cells with size and granular characteristics of DCs were gated. The percentage of CFSE+ cells among the gated CD80+ cells was analyzed. The data presented in this figure are from one representative experiment of two performed (A). Bar graph depicting percentages of CFSE-expressing cells out of total CD80+ cells (B).
FIG. 36 shows the sequence of the pcDNA3 plasmid vector (SEQ ID NO:14).
FIG. 37 shows the sequence of the pNGVL4a plasmid vector (SEQ ID NO:15).
FIG. 38 shows the nucleotide sequence of the pcDNA3 vector encoding E7 and HSP70 (pcDNA3-E7-Hsp70) (SEQ ID NO:36). The E7-Hsp70 fusion sequence is shown in upper case, underscored. Plasmid sequences are in lower case.
FIG. 39 shows the nucleic acid sequence of plasmid construct pcDNA3-ETA(dII)/E7 (SEQ ID NO:37). The nucleotides encoding ETA(dII)/E7 are shown in upper case and underscored. Plasmid sequence is lower case.
FIG. 40 shows the nucleotide sequence of plasmid pNGVL4a-CRT/E7(detox) (SEQ ID NO:44). The sequence is annotated to show plasmid-derived sequences (lower case), CRT-derived (bold, upper case) and HPV-E7-derived (detoxified by two amino acid substitutions as described above (upper case, italicized, underlined)) sequences.



DETAILED DESCRIPTION
The present invention is directed to one of two fundamental approaches to the improvement of molecular vaccine potency. As the present inventors discovered, in addition to DNA encoding an antigen, the concomitant administration of a second DNA molecule encoding an siRNA specific for an apoptotic protein, preferably Bak and/or Bax, termed “anti-apoptotic siRNA DNA” for simplicity), enhances the magnitude and/or duration of a T cell mediated immune response, and potentiates a desired clinical effect—such as eradication of an existing tumor or prevention of the spread or metastasis of a tumor.
The anti-apoptotic siRNA DNA may be physically linked to the antigen-encoding DNA. Alternatively, and preferably, the anti-apoptotic siRNA DNA may be administered separately from, but in combination with the antigen-encoding DNA molecule. Examples of the co-administration of these two types of vectors is provided.
This strategy may be combined with an additional strategy pioneered by the present inventors and colleagues, that involve linking DNA encoding another protein, generically termed a “immunogenicity-potentiating polypeptide” or “IPP” to the antigen-encoding DNA. Again, for the sake of simplicity, the DNA encoding such a targeting polypeptide will be referred to herein as a “IPP DNA.” That strategy has been shown to be effective in enhancing the potency of the vectors carrying only antigen-encoding DNA. See for example: Wu et al., WO 01/29233; Wu et al., WO 02/009645; Wu et al., WO 02/061113; Wu et al., WO 02/074920; Wu et al., WO 02/12281, all of which are incorporated by reference in their entirety.
The details of the various targeting polypeptide strategies will not be discussed in detail herein, although several such vectors are useful in the present invention and their sequences are provided below. The preferred IPPs include Sig/LAMP-1, the translocation domain, which is domain II (dII) of Pseudomonas aeruginosa exotoxin A (ETA(dII)) or from similar toxins from Diptheria, Clostridium, Botulinum, Bacillus, Yersinia, Vibrio cholerae, or Bordetella), an endoplasmic reticulum chaperone polypeptide exemplified by calreticulin (CRT) but also including ER60, GRP94 or gp96, well-characterized ER chaperone polypeptide that representatives of the HSP90 family of stress-induced proteins (see WO 02/012281), VP22 protein from herpes simplex virus and its homologues from other herpes viruses such as Marek's disease virus (see WO 02/09645), mycobacterial heat shock protein HSP70 (WO0129233, U.S. Pat. No. 6,734,173; WO02061113), and γ-tubulin (Hung C F et al., Canc Res 63:2393-98, 2003)
DNA encoding each of these polypeptides, or fragments or variants thereof with substantially the same biological activity, when linked to an antigen-encoding or epitope-encoding DNA molecule, result in more potent T cell mediate responses to the antigen compared to immunization with the antigen-encoding DNA alone. These polypeptide can be considered as “molecular adjuvants.” These effects are manifest primarily with CD8+ T cells, although some of these approaches induce potent CD4+ T cell mediated effects as well.
The results presented herein prove that molecular vaccination with

    (a) a combination of an antigen-encoding DNA and an anti-apoptotic siRNA DNA; or
    (b) a combination of a chimeric DNA encoding (i) antigen plus (ii) an IPP and an anti-apoptotic siRNA

or a combination of (a) and (b) above, will results in a stronger and more durable immune response which can be protective and/or therapeutic. A related embodiment that is expected to give similar results is:

    (c) a combination of a chimeric DNA comprising an antigen-encoding DNA sequence optionally linked to an IPP-encoding DNA and a DNA composition encoding an anti-apoptotic siRNA.


Also included within the scope of this invention (compositions and methods for inducing more potent immune responses, is a DC that is (i) loaded with, and therefore presenting, an antigen, and (ii) transfected with siRNA or with DNA encoding siRNA directed to Bak/Bax that suppress or reverse Bak/Bax protein expression. Such transfected DCs are preferably “pulsed” (or “loaded”) with an antigenic peptide. The DCs may be transfected ex vivo with anti-apoptotic siRNA or siRNA DNA, and loaded with antigen ex vivo. Alternatively, the transfection or loading or both may take place in vivo. If transfection or loading takes place ex vivo, the other may be conducted in vivo, either prior to removing the cells for ex vivo treatment or after the ex vivo treated cells have been administered to the subject.
Any one of the types of vectors may also comprise DNA encoding an immunostimulatory cytokine, preferably those that target APCs, preferably DC's, such as granulocyte macrophage colony stimulating factor (GM-CSF), or active fragments or domains thereof, and/or DNA encoding a costimulatory signal, such as a B7 family protein, including B7-DC (see U.S. patent application Ser. No. 09/794,210), B7.1, B7.2, soluble CD40, etc.).
The vectors used to deliver the foregoing DNA sequences include naked DNA vectors (plasmids), DNA-based alphaviral RNA replicons (“suicidal DNA vectors”) and self replicating RNA replicons.
The order in which the two (or more) components of a chimeric DNA vaccine construct (antigen and IPP) are arranged, and therefore, the order of the encoding nucleic acid fragments in the nucleic acid vector, can be altered without affecting immunogenicity of the fusion polypeptides proteins and the utility of the composition. As has been disclosed by the present inventors and their colleagues in other published patent applications cited herein, for some combinations of antigen/IPP, one order is preferred, e.g. E7 . . . HSP70 and CRT . . . E7 (indicating N- to C-terminal in the polypeptide).
The experiments described herein demonstrate that the methods of the invention can enhance a cellular immune response, particularly, tumor-destructive CTL reactivity, induced by a DNA vaccine encoding an epitope of a human pathogen. Human HPV-16 E7 was used as a model antigen for vaccine development because human papillomaviruses (HPVs), particularly HPV-16, are associated with most human cervical cancers. The oncogenic HPV proteins E7 and E6 are important in the induction and maintenance of cellular transformation and co-expressed in most HPV-containing cervical cancers and their precursor lesions. Therefore, cancer vaccines, such as the compositions of the invention, that target E7 can be used to control of HPV-associated neoplasms (Wu (1994) Curr. Opin. Immunol. 6:746-754). Similar DNA vaccines encoding E6 are also included herein and studies with E6 using the IPPs disclosed herein have shown successful potentiation of E6-specific immune responses.
Based on studies of simultaneous vaccination with both CRT/E6 and CRT/E7 DNA vaccines generated significant E6- and E7-specific T-cell immune responses and significantly better therapeutic antitumor effects against E6- and E7-expressing tumors than vaccination with either CRT/E6 DNA or CRT/E7 DNA alone.
In one embodiment, the present invention is directed to simultaneous vaccination with both E6 and E7 DNA immunogens, or IPP/E6 and IPP/E7 DNA immunogens, in combination with delivery of siRNA targeting mRNA encoding pro-apoptotic proteins, preferably Bak and/or Bax.
The present invention is not limited to the exemplified antigen(s). Rather, one of skill in the art will appreciate that the same results are expected for any antigen (and epitopes thereof) for which a T cell-mediated response is desired. The response so generated will be effective in providing protective or therapeutic immunity, or both, directed to an organism or disease in which the epitope or antigenic determinant is involved—for example as a cell surface antigen of a pathogenic cell or an envelope or other antigen of a pathogenic virus, or a bacterial antigen, or an antigen expressed as or as part of a pathogenic molecule.
Thus, in one embodiment, the antigen (e.g., the MHC class I-binding peptide epitope) is one that is derived from a pathogen, e.g., a peptide expressed by a pathogen. The pathogen can be a virus, such as, e.g., a papilloma virus, a herpesvirus, a retrovirus (including an immunodeficiency virus, such as HIV-1), an adenovirus, and the like. The papilloma virus can be a human papilloma virus, for which a preferred antigen (e.g., a MHC class I-binding peptide) can be the HPV-16 E6 or E7 polypeptide or an immunogenic fragment thereof. In one embodiment employing E6 and/or E7, the polypeptide is rendered substantially non-oncogenic by about 1 to about 3 amino acid substitutions that maintain immunogenicity while destroying oncogenicity, for example, by destroying the ability of the polypeptide to bind retinoblastoma polypeptide (pRB) or substantially lowering the affinity for pRB. As a result, the E7 polypeptide is effectively non-oncogenic when expressed in vivo or delivered in vivo.
In alternative embodiments, the pathogen is a bacteria, such as Bordetella pertussis; Ehrlichia chaffeensis; Staphylococcus aureus; Toxoplasma gondii; Legionella pneumophila; Brucella suis; Salmonella enterica; Mycobacterium avium; Mycobacterium tuberculosis; Listeria monocytogenes; Chlamydia trachomatis; Chlamydia pnemoniae; Rickettsia rickettsii; or, a fungus, such as, e.g., Paracoccidioides brasiliensis; or other pathogen, e.g., Plasmodium falciparum. 
In another embodiment, the MHC class I-binding peptide epitope is derived from a tumor cell. The tumor cell-derived peptide epitope can comprise a tumor associated antigen, e.g., a tumor specific antigen, such as, e.g., a HER-2/neu antigen, or one of a number of known melanoma antigens, etc.
In one embodiment, the isolated or recombinant nucleic acid molecule is operatively linked to a promoter, such as, e.g., a constitutive, an inducible or a tissue-specific promoter. The promoter can be expressed in any cell, including cells of the immune system, including, e.g., antigen presenting cells (APCs), e.g., in a constitutive, an inducible or a tissue-specific manner.
In alternative embodiments, the APCs are DCs, keratinocytes, astrocytes, monocytes, macrophages, B lymphocytes, a microglial cell, or activated endothelial cells, and the like.
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art of this invention. As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
The term “antigen” or “immunogen” as used herein refers to a compound or composition or cell comprising a peptide, polypeptide or protein which is “antigenic” or “immunogenic” when administered in an appropriate amount (an “immunogenically effective amount”), i.e., capable of inducing, eliciting, augmenting or boosting a cellular and/or humoral immune response and of being recognized by the products of that response (T cells, antibodies). A nucleic acid such as DNA that encodes an immunogen and is used as a vaccine is referred to as a “DNA immunogen” as the encoded polypeptide is expressed in vivo after administration of the DNA. An immunogen may be effective when given alone or in combination, or linked to, or fused to, another substance (which can be administered at one time or over several intervals). An immunogenic composition can comprise an antigenic peptide/polypeptide of at least about 5, or about 10 or about 15, or about 20 amino acids, etc. Smaller antigens may require presence of a “carrier” polypeptide e.g., as a fusion protein, aggregate, conjugate or mixture, preferably linked (chemically or otherwise) to the antigen to be immunogenic. The immunogen can be recombinantly expressed from a vaccine vector, which can be naked DNA which comprises the polypeptide immunogen's coding sequence operably linked to a promoter, e.g., an expression vector or cassette as described herein. The immunogen includes one or more antigenic determinants or epitopes which may vary in size from about 3 to about 15 amino acids.
The term “epitope” as used herein refers to an antigenic determinant or antigenic site that interacts with an antibody or a T cell receptor (TCR), e.g., the MHC class I-binding peptide compositions (or expressed products of the nucleic acid compositions of the invention) used in the methods of the invention. An “antigen” is a molecule or chemical structure that either induces an immune response or is specifically recognized or bound by the product or mediator of an immune response, such as an antibody or a CTL. The specific conformational or stereochemical “domain” to which an antibody or a TCR bind is an “antigenic determinant” or “epitope.” TCRs bind to peptide epitopes which are physically associated with a third molecule, a major histocompatibility complex (MHC) class I or class II protein.
The term “recombinant” refers to (1) a nucleic acid or polynucleotide synthesized or otherwise manipulated in vitro, (2) methods of using recombinant DNA technology to produce gene products in cells or other biological systems, or (3) a polypeptide encoded by a recombinant nucleic acid. For example, the ETA(dII)-encoding nucleic acid or polypeptide, the nucleic acid encoding an MHC class I-binding peptide epitope (antigen) or the peptide itself can be recombinant. “Recombinant means” includes ligation of nucleic acids having various coding regions or domains or promoter sequences from different sources into a single unit in the form of an expression cassette or vector for expression of the coding sequences in the vectors resulting in production of the encoded polypeptide.
The term “self-replicating RNA replicon” refers to a construct based on an RNA viruses, such as alphavirus genome RNAs (e.g., Sindbis virus, Semliki Forest virus, etc.) that have been engineered to allow expression of heterologous RNAs and proteins. These recombinant vectors are self-replicating (“replicons”) which can be introduced into cells as naked RNA or DNA, as described in detail in co-pending, commonly assigned U.S. and PCT patent applications by the present inventors (U.S. Ser. No. 10/060,274 and WO 02/061113).
siRNAs
The present inventors designed siRNA sequences that hybridize to, and block expression of the activation of Bak and Bax proteins that are central players in the apoptosis signalling pathway. The present invention is directed to the siRNA molecules (sequences), vectors containing or encoding the siRNA, expression vectors with a promoter operably linked to the siRNA coding sequence that drives transcription of siRNA sequences that are “specific” for sequences Bak and Bax nucleic acid. siRNAs may include single stranded “hairpin” sequences because of their stability and binding to the target mRNA.
Since Bak and Bax are involved, among other death proteins, in apoptosis of APCs, particularly DCs, the present siRNA sequences may be used in conjunction with a broad range of DNA vaccine constructs encoding antigens to enhance and promote the immune response induced by such DNA vaccine constructs, particularly CD8+ T cell mediated immune responses typified by CTL activation and action. This is believed to occur as a result of the effect of the siRNA in prolonging the life of antigen-presenting DCs which may otherwise be killed in the course of a developing immune response by the very same CTLs that the DCs are responsible for inducing.
In addition to Bak and Bax, additional targets for siRNAs designed in an analogous manner include caspase 8, caspase 9 and caspase 3. These proteins and their role in apoptosis was described above. The present invention includes compositions and methods in which siRNAs targeting any two or more of Bak, Bax, caspase 8, caspase 9 and caspase 3 are used in combination, optionally simultaneously (along with a DNA immunogen that encodes an antigen), to administer to a subject. Such combinations of siRNAs may also be used to transfect DCs (along with antigen loading) to improve the immunogenicity of the DCs as cellular vaccines by rendering them resistant to apoptosis.
siRNAs suppress gene expression through a highly regulated enzyme-mediated process called RNA interference (RNAi) (Sharp, P. A., Genes Dev. 15:485-90, 2001; Bernstein, E et al., Nature 409:363-66, 2001; Nykanen, A et al., Cell 107:309-21, 2001; Elbashir et al., Genes Dev. 15:188-200, 2001). RNA interference is the sequence-specific degradation of homologues in an mRNA of a targeting sequence in an siNA. As used herein, the term siNA (small, or short, interfering nucleic acid) is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi (RNA interference), for example short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), translational silencing, and others. RNAi involves multiple RNA-protein interactions characterized by four major steps: assembly of siRNA with the RNA-induced silencing complex (RISC), activation of the RISC, target recognition and target cleavage. These interactions may bias strand selection during siRNA-RISC assembly and activation, and contribute to the overall efficiency of RNAi (Khvorova, A et al., Cell 115:209-216 (2003); Schwarz, D S et al. 115:199-208 (2003)))
Considerations to be taken into account when designing an RNAi molecule include, among others, the sequence to be targeted, secondary structure of the RNA target and binding of RNA binding proteins. Methods of optimizing siRNA sequences will be evident to the skilled worker. Typical algorithms and methods are described in Vickers et al. (2003) J Biol Chem 278:7108-7118; Yang et al. (2003) Proc Natl Acad Sci USA 99:9942-9947; Far et al. (2003) Nuc. Acids Res. 31:4417-4424; and Reynolds et al. (2004) Nature Biotechnology 22:326-330, all of which are incorporated by reference in their entirety.
The methods described in Far et al., supra, and Reynolds et al., supra, may be used by those of ordinary skill in the art to select targeted sequences and design siRNA sequences that are effective at silencing the transcription of the relevant mRNA. Far et al. suggests options for assessing target accessibility for siRNA and supports the design of active siRNA constructs. This approach can be automated, adapted to high throughput and is open to include additional parameters relevant to the biological activity of siRNA. To identify siRNA-specific features likely to contribute to efficient processing at each of the steps of RNAi noted above. Reynolds et al., supra, present a systematic analysis of 180 siRNAs targeting the mRNA of two genes. Eight characteristics associated with siRNA functionality were identified: low G/C content, a bias towards low internal stability at the sense strand 3′-terminus, lack of inverted repeats, and sense strand base preferences (positions 3, 10, 13 and 19). Application of an algorithm incorporating all eight criteria significantly improves potent siRNA selection. This highlights the utility of rational design for selecting potent siRNAs that facilitate functional gene knockdown.
Candidate siRNA sequences against mouse and human Bax and Bak are selected using a process that involves running a BLAST search against the sequence of Bax or Bak (or any other target) and selecting sequences that “survive” to ensure that these sequences will not be cross matched with any other genes.
siRNA sequences selected according to such a process and algorithm may be cloned into an expression plasmid and tested for their activity in abrogating Bak/Bax function cells of the appropriate animal species. Those sequences that show RNAi activity may be used by direct administration bound to particles, or recloned into a viral vector such as a replication-defective human adenovirus serotype 5 (Ad5).
One advantage of this viral vector is the high titer obtainable (in the range of 1010) and therefore the high multiplicities-of infection that can be attained. For example, infection with 100 infectious units/cell ensures all cells are infected. Another advantage of this virus is the high susceptibility and infectivity and the host range (with respect to cell types). Even if expression is transient, cells would survive, possibly replicate, and continue to function before Bak/Bax activity would recover and lead to cell death. Preferred constructs described in the Examples are the following:










For Bak:









(SEQ ID NO:1)











5′ P-UGCCUACGAACUCUUCACCdTdT-3′ (sense)




 








(SEQ ID NO:2)











5′ P-GGUGAAGAGUUCGUAGGCAdTdT-3′ (antisense),







The nucleotide sequence encoding the Bak protein (including the stop codon) (GenBank accession No. NM—007523 is shown below (SEQ ID NO:3) with the targeted sequence in upper case, underscored.









atggcatctggacaaggaccaggtcccccgaaggtgggctgcgatgagtc



 


cccgtccccttctgaacagcaggttgcccaggacacagaggaggtctttc


 


gaagctacgttttttacctccaccagcaggaacaggagacccaggggcgg


 


ccgcctgccaaccccgagatggacaacttgcccctggaacccaacagcat


 


cttgggtcaggtgggtcggcagcttgctctcatcggagatgatattaacc


 


ggcgctacgacacagagttccagaatttactagaacagcttcagcccaca


 


gccgggaaTGCCTACGAACTCTTCACCaagatcgcctccagcctatttaa


 


gagtggcatcagctggggccgcgtggtggctctcctgggctttggctacc


 


gtctggccctgtacgtctaccagcgtggtttgaccggcttcctgggccag


 


gtgacctgctttttggctgatatcatactgcatcattacatcgccagatg


 


gatcgcacagagaggcggttgggtggcagccctgaatttgcgtagagacc


 


ccatcctgaccgtaatggtgatttttggtgtggttctgttgggccaattc


 


gtggtacacagattcttcagatcatga 637






The targeted sequence of Bak, TGCCTACGAACTCTTCACC is SEQ ID NO:4











For Bax:









(SEQ ID NO:5)











5′ P-UAUGGAGCUGCAGAGGAUGdTdT-3′ (sense)




 








(SEQ ID NO:6)











5′ P-CAUCCUCUGCAGCUCCAUAdTdT-3′ (antisense)







The nucleotide sequence encoding Bax (including the stop codon) (GenBank accession No. L22472 is shown below (SEQ ID NO:7) with the targeted sequence shown in upper case and underscored









atggacgggtccggggagcagcttgggagcggcgggcccaccagctctga



 


acagatcatgaagacaggggcctttttgctacagggtttcatccaggatc


 


gagcagggaggatggctggggagacacctgagctgaccttggagcagccg


 


ccccaggatgcgtccaccaagaagctgagcgagtgtctccggcgaattgg


 


agatgaactggatagcaaTATGGAGCTGCAGAGGATGattgctgacgtgg


 


acacggactccccccgagaggtcttcttccgggtggcagctgacatgttt


 


gctgatggcaacttcaactggggccgcgtggttgccctcttctactttgc


 


tagcaaactggtgctcaaggccctgtgcactaaagtgcccgagctgatca


 


gaaccatcatgggctggacactggacttcctccgtgagcggctgcttgtc


 


tggatccaagaccagggtggctgggaaggcctcctctcctacttcgggac


 


ccccacatggcagacagtgaccatctttgtggctggagtcctcaccgcct


 


cgctcaccatctggaagaagatgggctga 589






The targeted sequence of Bax, TATGGAGCTGCAGAGGATG is SEQ ID NO:8

In a preferred embodiment, the inhibitory molecule is a double stranded nucleic acid (preferably an RNA), used in a method of RNA interference. The following show the “paired” 19 nucleotide structures of the siRNA sequences shown above, where the symbol :




Other Pro-Apoptotic Proteins to be Targeted

1. Caspase 8: The nucleotide sequence of human caspase-8 is shown below (SEQ ID NO:9). GenBank Access. #NM—001228. One target sequence for RNAi is underscored. Others may be identified using methods such as those described herein (and in reference cited herein, primarily Far et al., supra and Reynolds et al., supra).











atg gac ttc agc aga aat ctt tat gat att ggg gaa




 



caa ctg gac agt gaa gat ctg gcc tcc ctc aag ttc



 



ctg agc ctg gac tac att ccg caa agg aag caa gaa



 



ccc atc aag gat gcc ttg atg tta ttc cag aga ctc



 



cag gaa aag aga atg ttg gag gaa agc aat ctg tcc



 



ttc ctg aag gag ctg ctc ttc cga att aat aga ctg



 



gat ttg ctg att acc tac cta aac act aga aag gag



 



gag atg gaa agg gaa ctt cag aca cca ggc agg gct



 



caa att tct gcc tac agg ttc cac ttc tgc cgc atg



 



agc tgg gct gaa gca aac agc cag tgc cag aca cag



 



tct gta cct ttc tgg cgg agg gtc gat cat cta tta



 



ata agg gtc atg ctc tat cag att tca gaa gaa gtg



 



agc aga tca gaa ttg agg tct ttt aag ttt ctt ttg



 



caa gag gaa atc tcc aaa tgc aaa ctg gat gat gac



 



atg aac ctg ctg gat att ttc ata gag atg gag aag



 



agg gtc atc ctg gga gaa gga aag ttg gac atc ctg



 



aaa aga gtc tgt gcc caa atc aac aag agc ctg ctg



 



aag ata atc aac gac tat gaa gaa ttc agc aaa ggg



 



gag gag ttg tgt ggg gta atg aca atc tcg gac tct



 



cca aga gaa cag gat agt gaa tca cag act ttg gac



 



aaa gtt tac caa atg aaa agc aaa cct cgg gga tac



 



tgt ctg atc atc aac aat cac aat ttt gca aaa gca



 



cgg gag aaa gtg ccc aaa ctt cac agc att agg gac



 



agg aat gga aca cac ttg gat gca ggg gct ttg acc



 



acg acc ttt gaa gag ctt cat ttt gag atc aag ccc



 



cac gat gac tgc aca gta gag caa atc tat gag att



 



ttg aaa atc tac caa ctc atg gac cac agt aac atg



 



gac tgc ttc atc tgc tgt atc ctc tcc cat gga gac



 



aag ggc atc atc tat ggc act gat gga cag gag gcc



 



ccc atc tat gag ctg aca tct cag ttc act ggt ttg



 



aag tgc cct tcc ctt gct gga aaa ccc aaa gtg ttt



 



ttt att cag gct tgt cag ggg gat aac tac cag aaa



 



ggt ata cct gtt gag act gat tca gag gag caa ccc



 



tat tta gaa atg gat tta tca tca cct caa acg aga



 



tat atc ccg gat gag gct gac ttt ctg ctg ggg atg



 



gcc act gtg aat aac tgt gtt tcc tac cga aac cct



 



gca gag gga acc tgg tac atc cag tca ctt tgc cag



 



agc ctg aga gag cga tgt cct cga ggc gat gat att



 



ctc acc atc ctg act gaa gtg aac tat gaa gta agc



 



aac aag gat gac aag aaa aac atg ggg aaa cag atg



 



cct cag cct act ttc aca cta aga aaa aaa ctt gtc



 



ttc cct tct gat tga 1491






The sequences of sense and antisense siRNA strands for targeting this sequence (including dTdT 3′ overhangs, are:









(SEQ ID NO:10)











5′-AACCUCGGGGAUACUGUCUGAdTdT-3′ (sense)




 



5′-UCAGACAGUAUCCCCGAGGUUdTdT-3′ (antisense)






2. Caspase 9: The nucleotide sequence of human caspase-9 is shown below (SEQ ID NO:12). See GenBank Access. #NM—001229. The sequence below is of “variant α” which is longer than a second alternatively spliced variant β, which lacks the underscored part of the sequence shown below (and which is anti-apoptotic). Target sequences for RNAi, expected to fall in the underscored segment, are identified using known methods such as those described herein and in Far et al., supra and Reynolds et al., supra). and siNAs, such as siRNAs, are designed accordingly.











atg gac gaa gcg gat cgg cgg ctc ctg cgg cgg tgc




 



cgg ctg cgg ctg gtg gaa gag ctg cag gtg gac cag



 



ctc tgg gac gcc ctg ctg agc cgc gag ctg ttc agg



 



ccc cat atg atc gag gac atc cag cgg gca ggc tct



 



gga tct cgg cgg gat cag gcc agg cag ctg atc ata



 



gat ctg gag act cga ggg agt cag gct ctt cct ttg



 



ttc atc tcc tgc tta gag gac aca ggc cag gac atg



 



ctg gct tcg ttt ctg cga act aac agg caa gca gca



 



aag ttg tcg aag cca acc cta gaa aac ctt acc cca



 



gtg gtg ctc aga cca gag att cgc aaa cca gag gtt



 



ctc aga ccg gaa aca ccc aga cca gtg gac att ggt



 



tct gga gga ttt ggt gat gtc ggt gct ctt gag agt



 



ttg agg gga aat gca gat ttg gct tac atc ctg agc



 



atg gag ccc tgt ggc cac tgc ctc att atc aac aat



 



gtg aac ttc tgc cgt gag tcc ggg ctc cgc acc cgc



 



act ggc tcc aac atc gac tgt gag aag ttg cgg cgt



 



cgc ttc tcc tcg ctg cat ttc atg gtg gag gtg aag



 



ggc gac ctg act gcc aag aaa atg gtg ctg gct ttg



 



ctg gag ctg gcg cag cag gac cac ggt gct ctg gac



 



tgc tgc gtg gtg gtc att ctc tct cac ggc tgt cag



 



gcc agc cac ctg cag ttc cca ggg gct gtc tac ggc



 



aca gat gga tgc cct gtg tcg gtc gag aag att gtg



 



aac atc ttc aat ggg acc agc tgc ccc agc ctg gga



 



ggg aag ccc aag ctc ttt ttc atc cag gcc tgt ggt



 



ggg gag cag aaa gac cat ggg ttt gag gtg gcc tcc



 



act tcc cct gaa gac gag tcc cct ggc agt aac ccc



 



gag cca gat gcc acc ccg ttc cag gaa ggt ttg agg



 



acc ttc gac cag ctg gac gcc ata tct agt ttg ccc



 



aca ccc agt gac atc ttt gtg tcc tac tct act ttc



 



cca ggt ttt gtt tcc tgg agg gac ccc aag agt ggc



 



tcc tgg tac gtt gag acc ctg gac gac atc ttt gag



 



cag tgg gct cac tct gaa gac ctg cag tcc ctc ctg



 



ctt agg gtc gct aat gct gtt tcg gtg aaa ggg att



 



tat aaa cag atg cct ggt tgc ttt aat ttc ctc cgg



 



aaa aaa ctt ttc ttt aaa aca tca taa 1191






3. Caspase 3: The nucleotide sequence of human caspase-3 is shown below (SEQ ID NO:13). See GenBank Access. #NM—004346. The sequence below is of “variant α” which is the longer of two alternatively spliced variants, all of which encode the full protein. Target sequences for RNAi are identified using known methods such as those described herein and in Far et al., supra and Reynolds et al., supra) and siNAs, such as siRNAs, are designed accordingly.











atg gag aac act gaa aac tca gtg gat tca aaa tcc




 



att aaa aat ttg gaa cca aag atc ata cat gga agc



 



gaa tca atg gac tct gga ata tcc ctg gac aac agt



 



tat aaa atg gat tat cct gag atg ggt tta tgt ata



 



ata att aat aat aag aat ttt cat aaa agc act gga



 



atg aca tct cgg tct ggt aca gat gtc gat gca gca



 



aac ctc agg gaa aca ttc aga aac ttg aaa tat gaa



 



gtc agg aat aaa aat gat ctt aca cgt gaa gaa att



 



gtg gaa ttg atg cgt gat gtt tct aaa gaa gat cac



 



agc aaa agg agc agt ttt gtt tgt gtg ctt ctg agc



 



cat ggt gaa gaa gga ata att ttt gga aca aat gga



 



cct gtt gac ctg aaa aaa ata aca aac ttt ttc aga



 



ggg gat cgt tgt aga agt cta act gga aaa ccc aaa



 



ctt ttc att att cag gcc tgc cgt ggt aca gaa ctg



 



gac tgt ggc att gag aca gac agt ggt gtt gat gat



 



gac atg gcg tgt cat aaa ata cca gtg gag gcc gac



 



ttc ttg tat gca tac tcc aca gca cct ggt tat tat



 



tct tgg cga aat tca aag gat ggc tcc tgg ttc atc



 



cag tcg ctt tgt gcc atg ctg aaa cag tat gcc gac



 



aag ctt gaa ttt atg cac att ctt acc cgg gtt aac



 



cga aag gtg gca aca gaa ttt gag tcc ttt tcc ttt



 



gac gct act ttt cat gca aag aaa cag att cca tgt



 



att gtt tcc atg ctc aca aaa gaa ctc tat ttt tat



 



cac taa 834






Long double stranded interfering RNAs, such a miRNAs, appear to tolerate mismatches more readily than do short double stranded RNAs. In addition, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, or an epigenetic phenomenon. For example, siNA molecules of the invention can be used to epigenetically silence genes at both the post-transcriptional level or the pre-transcriptional level. In a non-limiting example, epigenetic regulation of gene expression by siNA molecules of the invention can result from siNA mediated modification of chromatin structure and thereby alter gene expression (see, for example, Allshire Science 297:1818-19, 2002; Volpe et al., Science 297:1833-37, 2002; Jenuwein, Science 297:2215-18, 2002; and Hall et al., Science 297, 2232-2237, 2002.)
An siNA can be designed to target any region of the coding or non-coding sequence of an mRNA. An siNA is a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary. The siNA can be assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s). The siNA can be a polynucleotide with a hairpin secondary structure, having self-complementary sense and antisense regions. The siNA can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siNA molecule capable of mediating RNAi. The siNA can also comprise a single stranded polynucleotide having nucleotide sequence complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof (or can be an siNA molecule that does not require the presence within the siNA molecule of nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein the single stranded polynucleotide can further comprise a terminal phosphate group, such as a 5′-phosphate (see for example Martinez et al. (2002) Cell 110, 563-574 and Schwarz et al. (2002) Molecular Cell 10, 537-568), or 5′,3′-diphosphate.
In certain embodiments, the siNA molecule of the invention comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, Van der Waal's interactions, hydrophobic interactions, and/or stacking interactions. Some preferred siRNAs are discussed above and in the Examples.
As used herein, siNA molecules need not be limited to those molecules containing only ribonucleotides but may also further encompass deoxyribonucleotides (as in the preferred siRNAs which each include a dTdT dinucleotide) chemically-modified nucleotides, and non-nucleotides. In certain embodiments, the siNA molecules of the invention lack 2′-hydroxy (2′-OH) containing nucleotides. In certain embodiments, siNAs do not require the presence of nucleotides having a 2′-hydroxy group for mediating RNAi and as such, siNAs of the invention optionally do not include any ribonucleotides (e.g., nucleotides having a 2′-OH group). Such siNA molecules that do not require the presence of ribonucleotides within the siNA molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. If modified, the siNAs of the invention can also be referred to as “short interfering modified oligonucleotides” or “siMON.” Other chemical modifications, e.g., as described in Int'l Patent Publications WO 03/070918 and WO 03/074654, can be applied to any siNA sequence of the invention.
Preferably a molecule mediating RNAi has a 2 nucleotide 3′ overhang (dTdT in the preferred sequences disclosed herein). If the RNAi molecule is expressed in a cell from a construct, for example from a hairpin molecule or from an inverted repeat of the desired sequence, then the endogenous cellular machinery will create the overhangs.
Methods of making siRNAs are conventional. In vitro methods include processing the polyribonucleotide sequence in a cell-free system (e.g., digesting long dsRNAs with RNAse III or Dicer), transcribing recombinant double stranded DNA in vitro, and, preferably, chemical synthesis of nucleotide sequences homologous to Bakor Bax sequences. See, e.g., Tuschl et al., Genes & Dev. 13:3191-3197, 1999. In vivo methods include

    (1) transfecting DNA vectors into a cell such that a substrate is converted into siRNA in vivo. See, for example, Kawasaki et al., Nucleic Acids Res 31:700-07, 2003; Miyagishi et al., Nature Biotechnol 20:497-500, 2003; Lee et al., Nature Biotechnol 20:500-05, 2002; Brummelkamp et al., Science 296:550-53, 2002; McManus et al., RNA 8:842-50, 2002; Paddison et al., Genes Dev 16:948-58, 2002; Paddison et al., Proc Natl Acad Sci USA 99:1443-48, 2002; Paul et al., Nature Biotechnol 20:505-08, 2002; Sui et al., Proc Natl Acad Sci USA 99:5515-20, 2002; Yu et al., Proc Natl Acad Sci USA 99:6047-52, 2002)
    (2) expressing short hairpin RNAs from plasmid systems using RNA polymerase III (pol III) promoters. See, for example, Kawasaki et al., supra; Miyagishi et al., supra; Lee et al., supra; Brummelkamp et al., supra; McManus et al., supra), Paddison et al., supra (both); Paul et al., supra, Sui et al., supra; and Yu et al., supra; and/or
    (3) expressing short RNA from tandem promoters. See, for example, Miyagishi et al., supra; Lee et al., supra).


When synthesized in vitro, a typical micromolar scale RNA synthesis provides about 1 mg of siRNA, which is sufficient for about 1000 transfection experiments using a 24-well tissue culture plate format. In general, to inhibit Bak or Bax expression in cells in culture, one or more siRNAs can be added to cells in culture media, typically at about 1 ng/ml to about 10 μg siRNA/ml.
For reviews and more general description of inhibitory RNAs, see Lau et al., Sci Amer August 2003: 34-41; McManus et al., Nature Rev Genetics 3, 737-47, 2002; and Dykxhoom et al., Nature Rev Mol Cell Bio 4:457-467, 2003. For further guidance regarding methods of designing and preparing siRNAs, testing them for efficacy, and using them in methods of RNA interference (both in vitro and in vivo), see, e.g., Allshire, Science 297:1818-19, 2002; Volpe et al., Science 297:1833-37, 2002; Jenuwein, Science 297:2215-18, 2002; Hall et al., Science 2972232-37, 2002; Hutvagner et al., Science 297:2056-60, 2002; McManus et al. RNA 8:842-850, 2002; Reinhart et al., Genes Dev. 16:1616-26, 2002; Reinhart et al., Science 297:1831, 2002; Fire et al. (1998) Nature 391:806-11, 2002; Moss, Curr Biol 11:R772-5, 2002: Brummelkamp et al., supra; Bass, Nature 411 428-9, 2001; Elbashir et al., Nature 411:494-8; U.S. Pat. No. 6,506,559; Published US Pat App. 20030206887; and PCT applications WO99/07409, WO99/32619, WO 00/01846, WO 00/44914, WO00/44895, WO01/29058, WO01/36646, WO01/75164, WO01/92513, WO 01/29058, WO01/89304, WO01/90401, WO02/16620, and WO02/29858.
Ribozymes and siNAs can take any of the forms, including modified versions, described for antisense nucleic acid molecules; and they can be introduced into cells as oligonucleotides (single or double stranded), or in the form of an expression vector.
In a preferred embodiment, an antisense nucleic acid, siNA (e.g., siRNA) or ribozyme comprises a single stranded polynucleotide comprising a sequence that is at least about 90% (e.g., at least about 93%, 95%, 97%, 98% or 99%) identical to a target segment (such as those indicted for Bak and Bax above) or a complement thereof. As used herein, a DNA and an RNA encoded by it are said to contain the same “sequence,” taking into account that the thymine bases in DNA are replaced by uracil bases in RNA.
Active variants (e.g., length variants, including fragments; and sequence variants) of the nucleic acid-based inhibitors discussed herein are also within the scope of the invention. An “active” variant is one that retains an activity of the inhibitor from which it is derived (preferably the ability to inhibit expression). It is routine to test a variant to determine for its activity using conventional procedures.
As for length variants, an antisense nucleic acid or siRNA may be of any length that is effective for inhibition of a gene of interest. Typically, an antisense nucleic acid is between about 6 and about 50 nucleotides (e.g., at least about 12, 15, 20, 25, 30, 35, 40, 45 or 50 nt), and may be as long as about 100 to about 200 nucleotides or more. Antisense nucleic acids having about the same length as the gene or coding sequence to be inhibited may be used. When referring to length, the terms bases and base pairs (bp) are used interchangeably, and will be understood to correspond to single stranded (ss) and double stranded (ds) nucleic acids. The length of an effective siNA is generally between about 15 bp and about 29 bp in length, preferably between about 19 and about 29 bp (e.g., about 15, 17, 19, 21, 23, 25, 27 or 29 bp), with shorter and longer sequences being acceptable. Generally, siNAs are shorter than about 30 bases to prevent eliciting interferon effects. For example, an active variant of an siRNA having, for one of its strands, the 19 nucleotide sequence of any of SEQ ID NO: 1, 2, 5 and 6 herein can lack base pairs from either, or both, of ends of the dsRNA; or can comprise additional base pairs at either, or both, ends of the ds RNA, provided that the total of length of the siRNA is between about 19 and about 29 bp, inclusive. One embodiment of the invention is an siRNA that “consists essentially of” sequences represented by SEQ ID NO:1, 2, 5 or 6 or complements of these sequence. The term “consists essentially of” is an intermediate transitional phrase, and in this case excludes, for example, sequences that are long enough to induce a significant interferon response. An siRNA of the invention may consist essentially of between about 19 and about 29 bp in length.
As for sequence variants, it is generally preferred that an inhibitory nucleic acid, whether an antisense molecule, a ribozyme (the recognition sequences), or an siNA, comprise a strand that is complementary (100% identical in sequence) to a sequence of a gene that it is designed to inhibit. However, 100% sequence identity is not required to practice the present invention. Thus, the invention has the advantage of being able to tolerate naturally occurring sequence variations, for example, in human c-met, that might be expected due to genetic mutation, polymorphism, or evolutionary divergence. Alternatively, the variant sequences may be artificially generated. Nucleic acid sequences with small insertions, deletions, or single point mutations relative to the target sequence can be effective inhibitors.
The degree of sequence identity may be optimized by sequence comparison and alignment algorithms well-known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). At least about 90% sequence identity is preferred (e.g., at least about 92%, 95%, 98% or 99%), or even 100% sequence identity, between the inhibitory nucleic acid and the targeted sequence of targeted gene.
Alternatively, an active variant of an inhibitory nucleic acid of the invention is one that hybridizes to the sequence it is intended to inhibit under conditions of high stringency. For example, the duplex region of an siRNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript under high stringency conditions (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C., hybridization for 12-16 hours), followed generally by washing.
DC-1 cells or BM-DCs presenting a given antigen X, when not treated with the siRNAs of the invention, respond to sufficient numbers X-specific CD8+ CTL by apoptotic cell death. In contrast, the same cells transfected with the siRNA or infected with a viral vector encoding the present siRNA sequences survive better despite the delivery of killing signals.
Delivery and expression of the siRNA compositions of the present invention inhibit the death of DCs in vivo in the process of a developing T cell response, and thereby promote and stimulate the generation of an immune response induced by immunization with an antigen-encoding DNA vaccine vector. These capabilities have been exemplified by showing that:

    (1) co-administration of DNA vaccines encoding HPV-16 E7 with siRNA targeted to Bak and Bax prolongs the lives of antigen-presenting DCs in the draining lymph nodes, thereby enhancing antigen-specific CD8+ T cell responses, and eliciting potent antitumor effects against an E7-expressing tumor in vaccinated subjects.
    (2) DCs transfected with siRNA targeting Bak and Bax resist killing by T cells in vivo. E7-loaded DCs transfected with Bak/Bax siRNA so that Bak and Bax protein expression is down-regulated resist apoptotic death induced by T cells in vivo. When administered to subjects, these DCs generate stronger antigen-specific immune responses and manifest therapeutic effects (compared to DCs transfected with control siRNA).


Thus the siRNA constructs of the present invention are useful as a part of nucleic acid vaccination and immunotherapy regimen.
Vectors, Antigen, and IPP Nucleic Acids and Polypeptides
Plasmid Sequences
The sequence of the pcDNA3 plasmid vector (SEQ ID NO:14) is shown in FIG. 36, and the sequence of the pNGVL4a plasmid vector (SEQ ID NO:15) is shown in FIG. 37.
pNGVL4a, a preferred plasmid backbone for the present invention was originally derived from the pNGVL3 vector, which has been approved for human vaccine trials. The pNGVL4a vector includes two immunostimulatory sequences (tandem repeats of CpG dinucleotides) in the noncoding region. Whereas any other plasmid DNA that can transform either APCs, preferably DC's or other cells which, via cross-priming, transfer the antigenic moiety to DCs, is useful in the present invention, pNGFVLA4a is preferred because of the fact that it has already been approved for human therapeutic use.
Antigen Polypeptide Sequences
Thus using either CRT or any of the other IPPs, the present invention includes a combined DNA vaccine composition that includes a DNA immunogen encoding E6 with a DNA immunogen encoding E7 along with the delivery of siRNA targeting Bak and/or Bak (or several other pro-apoptotic proteins as described above). The siRNA may be delivered directly (e.g., bound to particles delivered via gene gun) or in the form of a DNA vector that encodes this siRNA.
The E7 nucleic acid sequence (SEQ ID NO:16) and amino acid sequence (SEQ ID NO:17) from HPV-16 are shown below (see Accession Number NC—001526)









atg cat gga gat aca cct aca ttg cat gaa tat atg tta gat ttg caa cca gag aca act
60



Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr
20


 


gat ctc tac tgt tat gag caa tta aat gac agc tca gag gag gag gat gaa ata gat ggt
120


Asp Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly
40


 


cca gct gga caa gca gaa ccg gac aga gcc cat tac aat att gta acc ttt tgt tgc aag
180


Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys
60


 


tgt gac tct acg ctt cgg ttg tgc gta caa agc aca cac gta gac att cgt act ttg gaa
240


Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu
80


 


gac ctg tta atg ggc aca cta gga att gtg tgc ccc atc tgt tct cag gat aag ctt
297


Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser Gln Asp Lys Leu
99






In single letter code, the wild type E7 amino acid sequence is









(SEQ ID NO:17 above))











MHGDTPTLHE YMLDLQPETT DLYCYEQLND SSEEEDEIDG




 



PAGQAEPDRA HYNIVTFCCK CDSTLRLCVQ STHVDIRTLE



 



DLLMGTLGIV CPICSQDKL 99






In another embodiment (See GenBank Accession No. AF125673, nucleotides 562-858 and the E7 amino acid sequence) the C-terminal four amino acids QDKL (and their codons) above are replaced with the three amino acids QKP (and the codons cag aaa cca yielding a protein of 98 residues.
When an oncoprotein or an epitope thereof is the immunizing moiety, it is preferable to reduce the tumorigenic risk of the vaccine itself. Because of the potential oncogenicity of the HPV E7 protein, the E7 protein is preferably used in a “detoxified” form
To reduce oncogenic potential of E7 in a construct of this invention, one or more of the following positions of E7 is mutated:















Original
Mutant
Preferred
nt Position (in
Amino acid (in



residue
residue
codon mutation
SEQ ID NO:16)
SEQ ID NO:17)




















Cys
Gly (or Ala)
TGT→GGT
70
24



 


Glu
Gly (or Ala)
GAG→GGG
77
26




(or GCG)


 


Cys
Gly (or Ala)
TGC→GGC
271
91









The preferred E7 (detox) mutant sequence has the following two mutations:
a TGT→GGT mutation resulting in a Cys→Gly substitution at position 24 of SEQ ID NO:17 a and GAG→GGG mutation resulting in a Glu→Gly substitution at position 26 of SEQ ID NO:17. This mutated amino acid sequence is shown below with the replacement residues underscored.








(SEQ ID NO:18)











MHGDTPTLHE YMLDLQPETT DLYGYEGLND SSEEEDEIDG




 



PAGQAEPDRA HYNIVTFCCK CDSTLRLCVQ STHVDIRTLE



 



DLLMGTLGIV CPICSQKP 97






These substitutions completely eliminate the capacity of the E7 to binding capacity to Rb, and thereby nullify its transforming activity.
Any nucleotide sequence that encodes encoding the above E7 or E7(detox) polypeptide, or an antigenic fragment or epitope thereof, can be used in the present compositions and methods, though the preferred E7 and E7(detox) sequences are shown above.
E6 Protein from HPV-16
The wild type HPV E6 amino acid sequence (see GenBank Accession Number NC—001526) (SEQ ID NO:4) is shown below. This sequence has 158 amino acids.
The wild type E6 nucleotide (SEQ ID NO:19) and amino acid (SEQ ID NO:20) sequences are shown below (see GenBank accession #'s K02718 and NC—001526)):









atg cac caa aag aga act gca atg ttt cag gac cca cag gag cga ccc aga aag tta cca
60



 


Met His Gln Lys Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Arg Pro Arg Lys Leu Pro
20


 


cag tta tgc aca gag ctg caa aca act ata cat gat ata ata tta gaa tgt gtg tac tgc
120


 


Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp Ile Ile Leu Glu Cys Val Tyr Cys
40


 


aag caa cag tta ctg cga cgt gag gta tat gac ttt gct ttt cgg gat tta tgc ata gta
180


 


Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Phe Arg Asp Leu Cys Ile Val
60


 


tat aga gat ggg aat cca tat gct gta tgt gat aaa tgt tta aag ttt tat tct aaa att
240


 


Tyr Arg Asp Gly Asn Pro Tyr Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile
80


 


agt gag tat aga cat tat tgt tat agt ttg tat gga aca aca tta gaa cag caa tac aac
300


 


Ser Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu Gln Gln Tyr Asn
100


 


aaa ccg ttg tgt gat ttg tta att agg tgt att aac tgt caa aag cca ctg tgt cct gaa
360


 


Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile Asn Cys Gln Lys Pro Leu Cys Pro Glu
120


 


gaa aag caa aga cat ctg gac aaa aag caa aga ttc cat aat ata agg ggt cgg tgg acc
420


 


Glu Lys Gln Arg His Leu Asp Lys Lys Gln Arg Phe His Asn Ile Arg Gly Arg Trp Thr
140


 


ggt cga tgt atg tct tgt tgc aga tca tca aga aca cgt aga gaa acc cag ctg taa
474


 


Gly Arg Cys Met Ser Cys Cys Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Leu stop
158






This polypeptide has 158 amino acids and is shown below in single letter code:









[SEQ ID NO:20, above]











MHQKRTAMFQ DPQERPRKLP QLCTELQTTI HDIILECVYC




 



KQQLLRREVY DFAFRDLCIV YRDGNPYAVC DKCLKFYSKI



 



SEYRHYCYSL YGTTLEQQYN KPLCDLLIRC INCQKPLCPE



 



EKQRHLDKKQ RFHNIRGRWT GRCMSCCRSS RTRRETQL 158






E6 proteins from cervical cancer-associated HPV types such as HPV-16 induce proteolysis of the p53 tumor suppressor protein through interaction with E6-AP. Human mammary epithelial cells (MECs) immortalized by E6 display low levels of p53. HPV-16 E6 as well as other cancer-related papillomavirus E6 proteins also binds the cellular protein E6BP (ERC-55). As with E7, it is preferred to used a non-oncogenic mutated form of E6, referred to as “E6(detox).” Several different E6 mutations and publications describing them are discussed below.
The preferred amino acid residues to be mutated are underscored in the E6 amino acid sequence above. Some studies of E6 mutants are based upon a shorter E6 protein of 151 nucleic acids, wherein the N-terminal residue was considered to be the Met at position 8 in SEQ ID NO:20 above. That shorter version of E6 is shown below as SEQ ID NO:21.










MFQDPQERPR KLPQLCTELQ TTIHDIILEC VYCKQQLLRR




 



EVYDFAFRDL CIVYRDGNPY AVCDKCLKFY SKISEYRHYC



 



YSLYGTTLEQ QYNKPLCDLL IRCINCQKPL CPEEKQRHLD



 



KKQRFHNIRG RWTGRCMSCC RSSRTRRETQ L






To reduce oncogenic potential of E6 in a construct of this invention, one or more of the following positions of E6 is mutated:














Original
Mutant
aa position in
aa position in



residue
residue
SEQ ID NO:20
SEQ ID NO:21



















Cys
Gly (or Ala)
70
63



 


Cys
Gly (or Ala)
113
106


 


Ile
Thr
135
128









Nguyen M et al., J Virol. 6:13039-48, 2002, described a mutant of HPV-16 E6 deficient in binding α-helix partners which displays reduced oncogenic potential in vivo. This mutant, that involves a replacement of Ile with Thr as position 128 (of SEQ ID NO:21), may be used in accordance with the present invention to make an E6 DNA vaccine that has a lower risk of being oncogenic. This E6(I128T) mutant is defective in its ability to bind at least a subset of α-helix partners, including E6AP, the ubiquitin ligase that mediates E6-dependent degradation of the p53 protein,
Cassetti M C et al., Vaccine 22:520-52, 2004, examined the effects of mutations four or five amino acid positions in E6 and E7 to inactivate their oncogenic potential. The following mutations were examined: E6-C63G and E6 C106G (positions based on SEQ ID NO:21); E7-C24G, E7-E26G, and E7 C91G (positions based on SEQ ID NO:17). Venezuelan equine encephalitis virus replicon particle (VRP) vaccines encoding mutant or wild type E6 and E7 proteins elicited comparable CTL responses and generated comparable antitumor responses in several HPV16 E6(+)E7(+) tumor challenge models: protection from either C3 or TC-1 tumor challenge was observed in 100% of vaccinated mice. Eradication of C3 tumors was observed in approximately 90% of the mice. The predicted inactivation of E6 and E7 oncogenic potential was confirmed by demonstrating normal levels of both p53 and Rb proteins in human mammary epithelial cells infected with VRPs expressing mutant E6 and E7 genes.
The HPV16 E6 protein contains two zinc fingers important for structure and function; one cysteine (C) amino acid position in each pair of C-X-X-C (where X is any amino acid) zinc finger motifs are preferably was mutated at E6 positions 63 and 106 (based on SEQ ID NO:21). Mutants are created, for example, using the Quick Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.). HPV16 E6 containing a single point mutation in the codon for Cys106 in SEQ ID NO:21 (=Cys 113 in SEQ ID NO:20). Cys106 neither binds nor facilitates degradation of p53 and is incapable of immortalizing human mammary epithelial cells (MEC), a phenotype dependent upon p53 degradation. A single amino acid substitution at position Cys63 of SEQ ID NO:21 (=Cys70 in SEQ ID NO:20) destroys several HPV16 E6 functions: p53 degradation, E6TP-1 degradation, activation of telomerase, and, consequently, immortalization of primary epithelial cells.
Any nucleotide sequence that encodes this E6 polypeptide, or preferably, one of the mutants thereof discussed below, or an antigenic fragment or epitope thereof, can be used in the present invention. Other mutations can be tested and used in accordance with the methods described herein including those described in Cassetti et al., supra. These mutations can be produced from any appropriate starting sequences by mutation of the coding DNA.
The present invention also includes the use of a tandem E6-E7 vaccine, using one or more of the mutations described herein to render the oncoproteins inactive with respect to their oncogenic potential in vivo. VRP vaccines (described in Cassetti et al., supra) comprised fused E6 and E7 genes in one open reading frame which were mutated at four or five amino acid positions (see below). Thus, the present constructs may include one or more epitopes of E6 and E7, which may be arranged in their native order or shuffled in any way that permits the expressed protein to bear the E6 and E7 antigenic epitopes in an immunogenic form. DNA encoding amino acid spacers between E6 and E7 or between individual epitopes of these proteins may be introduced into the vector, provided again, that the spacers permit the expression or presentation of the epitopes in an immunogenic manner after they have been expressed by transduced host cells.
Influenza Hemagglutinin (HA)
A nucleic acid sequence encoding HA [SEQ ID NO:22] is shown below.








atgaaggcaaacctactggtcctgttaagtgcacttgcagctgcagatgc



 


agacacaatatgtataggctaccatgcgaacaattcaaccgacactgttg


 


acacagtactcgagaagaatgtgacagtgacacactctgttaacctgctc


 


gaagacagccacaacggaaaactatgtagattaaaaggaatagccccact


 


acaattggggaaatgtaacatcgccggatggctcttgggaaacccagaat


 


gcgacccactgcttccagtgagatcatggtcctacattgtagaaacacca


 


aactctgagaatggaatatgttatccaggagatttcatcgactatgagga


 


gctgagggagcaattgagctcagtgtcatcattcgaaagattcgaaatat


 


ttcccaaagaaagctcatggcccaaccacaacacaaacggagtaacggca


 


gcatgctcccatgaggggaaaagcagtttttacagaaatttgctatggct


 


gacggagaaggagggctcatacccaaagctgaaaaattcttatgtgaaca


 


aaaaagggaaagaagtccttgtactgtggggtattcatcacccgcctaac


 


agtaaggaacaacagaatatctatcagaatgaaaatgcttatgtctctgt


 


agtgacttcaaattataacaggagatttaccccggaaatagcagaaagac


 


ccaaagtaagagatcaagctgggaggatgaactattactggaccttgcta


 


aaacccggagacacaataatatttgaggcaaatggaaatctaatagcacc


 


aatgtatgctttcgcactgagtagaggctttgggtccggcatcatcacct


 


caaacgcatcaatgcatgagtgtaacacgaagtgtcaaacacccctggga


 


gctataaacagcagtctcccttaccagaatatacacccagtcacaatagg


 


agagtgcccaaaatacgtcaggagtgccaaattgaggatggttacaggac


 


taaggaacactccgtccattcaatccagaggtctatttggagccattgcc


 


ggttttattgaagggggatggactggaatgatagatggatggtatggtta


 


tcatcatcagaatgaacagggatcaggctatgcagcggatcaaaaaagca


 


cacaaaatgccattaacgggattacaaacaaggtgaacactgttatcgag


 


aaaatgaacattcaattcacagctgtgggtaaagaattcaacaaattaga


 


aaaaaggatggaaaatttaaataaaaaagttgatgatggatttctggaca


 


tttggacatataatgcagaattgttagttctactggaaaatgaaaggact


 


ctggatttccatgactcaaatgtgaagaatctgtatgagaaagtaaaaag


 


ccaattaaagaataatgccaaagaaatcggaaatggatgttttgagttct


 


accacaagtgtgacaatgaatgcatggaaagtgtaagaaatgggacttat


 


gattatcccaaatattcagaagagtcaaagttgaacagggaaaaggtaga


 


tggagtgaaattggaatcaatggggatctatcagattctggcgatctact


 


caactgtcgccagttcactggtgcttttggtctccctgggggcaatcagt


 


ttctggatgtgttctaatggatctttgcagtgcagaatatgcatctga






The amino acid sequence of HA [SEQ ID NO:23; immunodominant epitope underscored, is:











MKANLLVLLS ALAAADADTI CIGYHANNST DTVDTVLEKN




 



VTVTHSVNLL EDSHNGKLCR LKGIAPLQLG KCNIAGWLLG



 



NPECDPLLPV RSWSYIVETP NSENGICYPG DFIDYEELRE



 



QLSSVSSFER FEIFPKESSW PNHNTNGVTA ACSHEGKSSF



 



YRNLLWLTEK EGSYPKLKNS YVNKKGKEVL VLWGIHHPPN



 



SKEQQNIYQN ENAYVSVVTS NYNRRFTPEI AERPKVRDQA



 



GRMNYYWTLL KPGDTIIFEA NGNLIAPMYA FALSRGFGSG



 



IITSNASMHE CNTKCQTPLG AINSSLPYQN IHPVTIGECP



 



KYVRSAKLRM VTGLRNTPSI QSRGLFGAIA GFIEGGWTGM



 



IDGWYGYHHQ NEQGSGYAAD QKSTQNAING ITNKVNTVIE



 



KMNIQFTAVG KEFNKLEKRM ENLNKKVDDG FLDIWTYNAE



 



LLVLLENERT LDFHDSNVKN LYEKVKSQLK NNAKEIGNGC



 



FEFYHKCDNE CMESVRNGTY DYPKYSEESK LNREKVDGVK



 



LESMGIYQIL AIYSTVASSL VLLVSLGAIS FWMCSNGSLQ



 



CRICI






Other Antigens Associated with Pathogens

A major use for the present invention is as a therapeutic vaccine for cancer and for major chronic viral infections that cause morbidity and mortality worldwide. Such vaccines are designed to eliminate infected cells—this requires T cell responses as antibodies are often ineffective. The vaccines of the present invention are designed to meet these needs.
Preferred antigens are epitopes of pathogenic microorganisms against which the host is defended by effector T cells responses, including CTL and delayed type hypersensitivity. These typically include viruses, intracellular parasites such as malaria, and bacteria that grow intracellularly such as Mycobacteria and Listeria species. Thus, the types of antigens included in the vaccine compositions of this invention are any of those associated with such pathogens (in addition, of course, to tumor-specific antigens). It is noteworthy that some viral antigens are also tumor antigens in the case where the virus is a causative factor in cancer.
In fact, the two most common cancers worldwide, hepatoma and cervical cancer, are associated with viral infection. Hepatitis B virus (HBV) (Beasley, R. P. et al., Lancet 2:1129-1133 (1981) has been implicated as etiologic agent of hepatomas. 80-90% of cervical cancers express the E6 and E7 antigens (discussed above and exemplified herein) from one of four “high risk” human papillomavirus types: HPV-16, HPV-18, HPV-31 and HPV-45 (Gissmann, L. et al., Ciba Found Symp. 120:190-207, 1986; Beaudenon, S., et al. Nature 321:246-9, 1986). The HPV E6 and E7 antigens are the most promising targets for virus associated cancers in immunocompetent individuals because of their ubiquitous expression in cervical cancer. In addition to their importance as targets for therapeutic cancer vaccines, virus associated tumor antigens are also ideal candidates for prophylactic vaccines. Indeed, introduction of prophylactic HBV vaccines in Asia have decreased the incidence of hepatoma (Chang, M H et al. New Engl. J. Med. 336, 1855-1859 (1997), representing a great impact on cancer prevention.
Among the most important viruses in chronic human viral infections are HPV, HBV, hepatitis C Virus (HCV), human immunodeficiency virus (HIV-1 and HIV-2), herpesviruses such as Epstein Barr Virus (EBV), cytomegalovirus (CMV) and HSV-1 and HSV-2 and influenza virus. Useful antigens include HBV surface antigen or HBV core antigen; ppUL83 or pp89 of CMV; antigens of gp120, gp41 or p24 proteins of HIV-1; ICP27, gD2, gB of HSV; or influenza hemagglutinin or nucleoprotein (Anthony, L S et al., Vaccine 1999; 17:373-83). Other antigens associated with pathogens that can be utilized as described herein are antigens of various parasites, includes malaria, preferably malaria peptide based on repeats of NANP.
In addition to its applicability to human cancer and infectious diseases, the present invention is also intended for use in treating animal diseases in the veterinary medicine context. Thus, the approaches described herein may be readily applied by one skilled in the art to treatment of veterinary herpesvirus infections including equine herpesviruses, bovine viruses such as bovine viral diarrhea virus (for example, the E2 antigen), bovine herpesviruses, Marek's disease virus in chickens and other fowl; animal retroviral and lentiviral diseases (e.g., feline leukemia, feline immunodeficiency, simian immunodeficiency viruses, etc.); pseudorabies and rabies; and the like.
As for tumor antigens, any tumor-associated or tumor-specific antigen that can be recognized by T cells, preferably by CTL, can be used. These include, without limitation, mutant p53, HER2/neu or a peptide thereof, or any of a number of melanoma-associated antigens such as MAGE-1, MAGE-3, MART-1/Melan-A, tyrosinase, gp75, gp100, BAGE, GAGE-1, GAGE-2, GnT-V, and p15 (see, for example, U.S. Pat. No. 6,187,306).
DNA Encoding Immunogenicity-Potentiating Polypeptides (IPPs)
The present inventors and their colleagues have described a number of IPPs and their use in DNA vaccines, in the following publications, all of which are incorporated by reference in their entirety: Kim T W et al., J Clin Invest 112: 109-117, 2003; Cheng W F et al., J Clin Invest 108: 669-678, 2001; Hung C F et al., Cancer Res 61:3698-3703, 2001; Chen C H et al., 2000, supra; U.S. Pat. No. 6,734,173; published patent applications WO05/081716, WO05/047501, WO03/085085, WO02/12281, WO02/074920, WO02/061113, WO02/09645, and WO01/29233. Recently, they have described comparative studies of these IPPs using HPV E6 as the antigen in Peng, S. et al., J Biomed Sci. 12:689-700 2005
The DNA sequence encoding the E7 protein fused to the translocation Signal sequence and LAMP-1 domain (Sig-E7-L1) [SEQ ID NO:24] is:








ATGGCGGCCCCCGGCGCCCGGCGGCCGCTGCTCCTGCTGCTGCTGGCAGG



 


CCTTGCACATGGCGCCTCAGCACTCTTTGAGGATCTAATCATGCATGGAG


 


ATACACCTACATTGCATGAATATATGTTAGATTTGCAACCAGAGACAACT


 


GATCTCTACTGTTATGAGCAATTAAATGACAGCTCAGAGGAGGAGGATGA


 


AATAGATGGTCCAGCTGGACAAGCAGAACCGGACAGAGCCCATTACAATA


 


TTGTTACCTTTTGTTGCAAGTGTGACTCTACGCTTCGGTTGTGCGTACAA


 


AGCACACACGTAGACATTCGTACTTTGGAAGACCTGTTAATGGGCACACT


 


AGGAATTGTGTGCCCCATCTGTTCTCAGGATCTTAACAACATGTTGATCC


 


CCATTGCTGTGGGCGGTGCCCTGGCAGGGCTGGTCCTCATCGTCCTCATT


 


GCCTACCTCATTGGCAGGAAGAGGAGTCACGCCGGCTATCAGACCATCTA


 


G






The amino acid sequence of Sig-E7-L1 [SEQ ID NO:25] is:











MAAPGARRPL LLLLLAGLAH GASALFEDLI MHGDTPTLHE




 



YMLDLQPETT DLYCYEQLND SSEEEDEIDG PAGQAEPDRA



 



HYNIVTFCCK CDSTLRLCVQ STHVDIRTLE DLLMGTLGIV



 



CPICSQDLNN MLIPIAVGGA LAGLVLIVLI AYLIGRKRSH



 



AGYQTI






The nucleotide sequence of the immunogenic vector pcDNA3-sigE7-L1 [SEQ ID NO:26] is shown below with the SigE7-L1 coding sequence in lower case and underscored:








GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATC



 


TGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTT


 


GGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAG


 


GCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCG


 


CTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGAC


 


TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATA


 


TGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCG


 


CCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGT


 


AACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGT


 


AAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCC


 


CCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTA


 


CATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCA


 


TCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGA


 


TAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA


 


TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTA


 


ACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAG


 


GTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTG


 


GCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGC


 


GTTTAAACGGGCCCTCTAGACTCGAGCGGCCGCCACTGTGCTGGATATCT


 


GCAGAATTCatggcggcccccggcgcccggcggccgctgctcctgctgct


 


gctggcaggccttgcacatggcgcctcagcactctttgaggatctaatca


 


tgcatggagatacacctacattgcatgaatatatgttagatttgcaacca


 


gagacaactgatctctactgttatgagcaattaaatgacagctcagagga


 


ggaggatgaaatagatggtccagctggacaagcagaaccggacagagccc


 


attacaatattgttaccttttgttgcaagtgtgactctacgcttcggttg


 


tgcgtacaaagcacacacgtagacattcgtactttggaagacctgttaat


 


gggcacactaggaattgtgtgccccatctgttctcaggatcttaacaaca


 


tgttgatccccattgctgtgggcggtgccctggcagggctggtcctcatc


 


gtcctcattgcctacctcattggcaggaagaggagtcacgccggctatca


 


gaccatctagGGATCCGAGCTCGGTACCAAGCTTAAGTTTAAACCGCTGA


 


TCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTC


 


CCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCT


 


AATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATT


 


CTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAA


 


TAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAA


 


GAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCA


 


TTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGC


 


CAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCA


 


CGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCATCCCTTTAGGG


 


TTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGG


 


TGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTT


 


TGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGA


 


ACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTT


 


GGGGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTA


 


ACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCC


 


CAGGCTCCCCAGGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTC


 


AGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGC


 


AAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCG


 


CCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGG


 


CTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTG


 


AGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGC


 


AAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGAC


 


AGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTT


 


CTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAG


 


ACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCG


 


CCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGC


 


AGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGC


 


GCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATT


 


GGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCG


 


AGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGAT


 


CCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGC


 


ACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAG


 


AGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGC


 


ATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCC


 


GAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCC


 


GGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGAT


 


ATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTA


 


CGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTG


 


ACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGA


 


CGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAA


 


AGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCA


 


GCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTG


 


CAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAAT


 


AAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAA


 


TGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGT


 


AATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATT


 


CCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTA


 


ATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCC


 


AGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCG


 


GGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGA


 


CTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAA


 


AGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAAC


 


ATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTT


 


GCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATC


 


GACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAG


 


GCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCC


 


GCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTT


 


CTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCC


 


AAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTT


 


ATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGC


 


CACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGC


 


GGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAG


 


GACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAA


 


GAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGT


 


TTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGA


 


AGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACT


 


CACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAG


 


ATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGA


 


GTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCT


 


CAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTG


 


TAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAAT


 


GATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACC


 


AGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCC


 


TCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCC


 


AGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGT


 


CACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCA


 


AGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTT


 


CGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCA


 


TGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGA


 


TGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTG


 


TATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCG


 


CGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCG


 


GGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTA


 


ACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCG


 


TTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATA


 


AGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTA


 


TTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAAT


 


GTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAA


 


GTGCCACCTGACGTC






HSP70 from M. tuberculosis 

The nucleotide sequence encoding HSP70 (SEQ ID NO:27) is shown below (nucleotides 10633-12510 of the M. tuberculosis genome in GenBank NC—000962);










atggctcg tgcggtcggg atcgacctcg ggaccaccaa




 



ctccgtcgtc tcggttctgg aaggtggcga cccggtcgtc



 



gtcgccaact ccgagggctc caggaccacc ccgtcaattg



 



tcgcgttcgc ccgcaacggt gaggtgctgg tcggccagcc



 



cgccaagaac caggcagtga ccaacgtcga tcgcaccgtg



 



cgctcggtca agcgacacat gggcagcgac tggtccatag



 



agattgacgg caagaaatac accgcgccgg agatcagcgc



 



ccgcattctg atgaagctga agcgcgacgc cgaggcctac



 



ctcggtgagg acattaccga cgcggttatc acgacgcccg



 



cctacttcaa tgacgcccag cgtcaggcca ccaaggacgc



 



cggccagatc gccggcctca acgtgctgcg gatcgtcaac



 



gagccgaccg cggccgcgct ggcctacggc ctcgacaagg



 



gcgagaagga gcagcgaatc ctggtcttcg acttgggtgg



 



tggcactttc gacgtttccc tgctggagat cggcgagggt



 



gtggttgagg tccgtgccac ttcgggtgac aaccacctcg



 



gcggcgacga ctgggaccag cgggtcgtcg attggctggt



 



ggacaagttc aagggcacca gcggcatcga tctgaccaag



 



gacaagatgg cgatgcagcg gctgcgggaa gccgccgaga



 



aggcaaagat cgagctgagt tcgagtcagt ccacctcgat



 



caacctgccc tacatcaccg tcgacgccga caagaacccg



 



ttgttcttag acgagcagct gacccgcgcg gagttccaac



 



ggatcactca ggacctgctg gaccgcactc gcaagccgtt



 



ccagtcggtg atcgctgaca ccggcatttc ggtgtcggag



 



atcgatcacg ttgtgctcgt gggtggttcg acccggatgc



 



ccgcggtgac cgatctggtc aaggaactca ccggcggcaa



 



ggaacccaac aagggcgtca accccgatga ggttgtcgcg



 



gtgggagccg ctctgcaggc cggcgtcctc aagggcgagg



 



tgaaagacgt tctgctgctt gatgttaccc cgctgagcct



 



gggtatcgag accaagggcg gggtgatgac caggctcatc



 



gagcgcaaca ccacgatccc caccaagcgg tcggagactt



 



tcaccaccgc cgacgacaac caaccgtcgg tgcagatcca



 



ggtctatcag ggggagcgtg agatcgccgc gcacaacaag



 



ttgctcgggt ccttcgagct gaccggcatc ccgccggcgc



 



cgcgggggat tccgcagatc gaggtcactt tcgacatcga



 



cgccaacggc attgtgcacg tcaccgccaa ggacaagggc



 



accggcaagg agaacacgat ccgaatccag gaaggctcgg



 



gcctgtccaa ggaagacatt gaccgcatga tcaaggacgc



 



cgaagcgcac gccgaggagg atcgcaagcg tcgcgaggag



 



gccgatgttc gtaatcaagc cgagacattg gtctaccaga



 



cggagaagtt cgtcaaagaa cagcgtgagg ccgagggtgg



 



ttcgaaggta cctgaagaca cgctgaacaa ggttgatgcc



 



gcggtggcgg aagcgaaggc ggcacttggc ggatcggata



 



tttcggccat caagtcggcg atggagaagc tgggccagga



 



gtcgcaggct ctggggcaag cgatctacga agcagctcag



 



gctgcgtcac aggccactgg cgctgcccac cccggcggcg



 



agccgggcgg tgcccacccc ggctcggctg atgacgttgt



 



ggacgcggag gtggtcgacg acggccggga ggccaagtga






The amino acid sequence of HSP70 [SEQ ID NO:28] is:










MARAVGIDLG TTNSVVSVLE GGDPVVVANS EGSRTTPSIV




 



AFARNGEVLV GQPAKNQAVT NVDRTVRSVK RHMGSDWSIE



 



IDGKKYTAPE ISARILMKLK RDAEAYLGED ITDAVITTPA



 



YFNDAQRQAT KDAGQIAGLN VLRIVNEPTA AALAYGLDKG



 



EKEQRILVFD LGGGTFDVSL LEIGEGVVEV RATSGDNHLG



 



GDDWDQRVVD WLVDKFKGTS GIDLTKDKMA MQRLREAAEK



 



AKIELSSSQS TSINLPYITV DADKNPLFLD EQLTRAEFQR



 



ITQDLLDRTR KPFQSVIADT GISVSEIDHV VLVGGSTRMP



 



AVTDLVKELT GGKEPNKGVN PDEVVAVGAA LQAGVLKGEV



 



KDVLLLDVTP LSLGIETKGG VMTRLIERNT TIPTKRSETF



 



TTADDNQPSV QIQVYQGERE IAAHNKLLGS FELTGIPPAP



 



RGIPQIEVTF DIDANGIVHV TAKDKGTGKE NTIRIQEGSG



 



LSKEDIDRMI KDAEAHAEED RKRREEADVR NQAETLVYQT



 



EKFVKEQREA EGGSKVPEDT LNKVDAAVAE AKAALGGSDI



 



SAIKSAMEKL GQESQALGQA IYEAAQAASQ ATGAAHPGGE



 



PGGAHPGSAD DVVDAEVVDD GREAK






The E7-Hsp70 Chimera/Fusion Polypeptide (Nucleotide sequence SEQ ID NO:29 and amino acid sequence SEQ ID NO:30) are provided below. The E7 coding sequence is shown in upper case and underscored.









1/1                                     31/11



ATG CAT GGA GAT ACA CCT ACA TTG CAT GAA TAT ATG TTA GAT TTG CAA CCA GAG ACA ACT


Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr


 


61/21                                   91/31


GAT CTC TAC TGT TAT GAG CAA TTA AAT GAC AGC TCA GAG GAG GAG GAT GAA ATA GAT GGT


Asp Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly


 


121/41                                  151/51


CCA GCT GGA CAA GCA GAA CCG GAC AGA GCC CAT TAC AAT ATT GTA ACC TTT TGT TGC AAG


Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys


 


181/61                                  211/71


TGT GAC TCT ACG CTT CGG TTG TGC GTA CAA AGC ACA CAC GTA GAC ATT CGT ACT TTG GAA


Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu


 


241/81                                  271/91


GAC CTG TTA ATG GGC ACA CTA GGA ATT GTG TGC CCC ATC TGT TCT CAA GGA TCC atg gct


Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser Gln Gly Ser Met Ala


 


301/101                                 331/111


cgt gcg gtc ggg atc gac ctc ggg acc acc aac tcc gtc gtc tcg gtt ctg gaa ggt ggc


Arg Ala Val Gly Ile Asp Leu Gly Thr Thr Asn Ser Val Val Ser Val Leu Glu Gly Gly


 


361/121                                 391/131


gac ccg gtc gtc gtc gcc aac tcc gag ggc tcc agg acc acc ccg tca att gtc gcg ttc


Asp Pro Val Val Val Ala Asn Ser Glu Gly Ser Arg Thr Thr Pro Ser Ile Val Ala Phe


 


421/141                                 451/151


gcc cgc aac ggt gag gtg ctg gtc ggc cag ccc gcc aag aac cag gca gtg acc aac gtc


Ala Arg Asn Gly Glu Val Leu Val Gly Gln Pro Ala Lys Asn Gln Ala Val Thr Asn Val


 


481/161                                 511/171


gat cgc acc gtg cgc tcg gtc aag cga cac atg ggc agc gac tgg tcc ata gag att gac


Asp Arg Thr Val Arg Ser Val Lys Arg His Met Gly Ser Asp Trp Ser Ile Glu Ile Asp


 


541/181                                 571/191


ggc aag aaa tac acc gcg ccg gag atc agc gcc cgc att ctg atg aag ctg aag cgc gac


Gly Lys Lys Tyr Thr Ala Pro Glu Ile Ser Ala Arg Ile Leu Met Lys Leu Lys Arg Asp


 


601/201                                 631/211


gcc gag gcc tac ctc ggt gag gac att acc gac gcg gtt atc acg acg ccc gcc tac ttc


Ala Glu Ala Tyr Leu Gly Glu Asp Ile Thr Asp Ala Val Ile Thr Thr Pro Ala Tyr Phe


 


661/221                                 691/231


aat gac gcc cag cgt cag gcc acc aag gac gcc ggc cag atc gcc ggc ctc aac gtg ctg


Asn Asp Ala Gln Arg Gln Ala Thr Lys Asp Ala Gly Gln Ile Ala Gly Leu Asn Val Leu


 


721/241                                 751/251


cgg atc gtc aac gag ccg acc gcg gcc gcg ctg gcc tac ggc ctc gac aag ggc gag aag


Arg Ile Val Asn Glu Pro Thr Ala Ala Ala Leu Ala Tyr Gly Leu Asp Lys Gly Glu Lys


 


781/261                                 811/271


gag cag cga atc ctg gtc ttc gac ttg ggt ggt ggc act ttc gac gtt tcc ctg ctg gag


Glu Gln Arg Ile Leu Val Phe Asp Leu Gly Gly Gly Thr Phe Asp Val Ser Leu Leu Glu


 


841/281                                 871/291


atc ggc gag ggt gtg gtt gag gtc cgt gcc act tcg ggt gac aac cac ctc ggc ggc gac


Ile Gly Glu Gly Val Val Glu Val Arg Ala Thr Ser Gly Asp Asn His Leu Gly Gly Asp


 


901/301                                 931/311


gac tgg gac cag cgg gtc gtc gat tgg ctg gtg gac aag ttc aag ggc acc agc ggc atc


Asp Trp Asp Gln Arg Val Val Asp Trp Leu Val Asp Lys Phe Lys Gly Thr Ser Gly Ile


 


961/321                                 991/331


gat ctg acc aag gac aag atg gcg atg cag cgg ctg cgg gaa gcc gcc gag aag gca aag


Asp Leu Thr Lys Asp Lys Met Ala Met Gln Arg Leu Arg Glu Ala Ala Glu Lys Ala Lys


 


1021/341                                1051/351


atc gag ctg agt tcg agt cag tcc acc tcg atc aac ctg ccc tac atc acc gtc gac gcc


Ile Glu Leu Ser Ser Ser Gln Ser Thr Ser Ile Asn Leu Pro Tyr Ile Thr Val Asp Ala


 


1081/361                                1111/371


gac aag aac ccg ttg ttc tta gac gag cag ctg acc cgc gcg gag ttc caa cgg atc act


Asp Lys Asn Pro Leu Phe Leu Asp Glu Gln Leu Thr Arg Ala Glu Phe Gln Arg Ile Thr


 


1141/381                                1171/391


cag gac ctg ctg gac cgc act cgc aag ccg ttc cag tcg gtg atc gct gac acc ggc att


Gln Asp Leu Leu Asp Arg Thr Arg Lys Pro Phe Gln Ser Val Ile Ala Asp Thr Gly Ile


 


1201/401                                1231/411


tcg gtg tcg gag atc gat cac gtt gtg ctc gtg ggt ggt tcg acc cgg atg ccc gcg gtg


Ser Val Ser Glu Ile Asp His Val Val Leu Val Gly Gly Ser Thr Arg Met Pro Ala Val


 


1261/421                                1291/431


acc gat ctg gtc aag gaa ctc acc ggc ggc aag gaa ccc aac aag ggc gtc aac ccc gat


Thr Asp Leu Val Lys Glu Leu Thr Gly Gly Lys Glu Pro Asn Lys Gly Val Asn Pro Asp


 


1321/441                                1351/451


gag gtt gtc gcg gtg gga gcc gct ctg cag gcc ggc gtc ctc aag ggc gag gtg aaa gac


Glu Val Val Ala Val Gly Ala Ala Leu Gln Ala Gly Val Leu Lys Gly Glu Val Lys Asp


 


1381/461                                1411/471


gtt ctg ctg ctt gat gtt acc ccg ctg agc ctg ggt atc gag acc aag ggc ggg gtg atg


Val Leu Leu Leu Asp Val Thr Pro Leu Ser Leu Gly Ile Glu Thr Lys Gly Gly Val Met


 


1441/481                                1471/491


acc agg ctc atc gag cgc aac acc acg atc ccc acc aag cgg tcg gag act ttc acc acc


Thr Arg Leu Ile Glu Arg Asn Thr Thr Ile Pro Thr Lys Arg Ser Glu Thr Phe Thr Thr


 


1501/501                                1531/511


gcc gac gac aac caa ccg tcg gtg cag atc cag gtc tat cag ggg gag cgt gag atc gcc


Ala Asp Asp Asn Gln Pro Ser Val Gln Ile Gln Val Tyr Gln Gly Glu Arg Glu Ile Ala


 


1561/521                                1591/531


gcg cac aac aag ttg ctc ggg tcc ttc gag ctg acc ggc atc ccg ccg gcg ccg cgg ggg


Ala His Asn Lys Leu Leu Gly Ser Phe Glu Leu Thr Gly Ile Pro Pro Ala Pro Arg Gly


 


1621/541                                1651/551


att ccg cag atc gag gtc act ttc gac atc gac gcc aac ggc att gtg cac gtc acc gcc


Ile Pro Gln Ile Glu Val Thr Phe Asp Ile Asp Ala Asn Gly Ile Val His Val Thr Ala


 


1681/561                                1711/571


aag gac aag ggc acc ggc aag gag aac acg atc cga atc cag gaa ggc tcg ggc ctg tcc


Lys Asp Lys Gly Thr Gly Lys Glu Asn Thr Ile Arg Ile Gln Glu Gly Ser Gly Leu Ser


 


1741/581                                1771/591


aag gaa gac att gac cgc atg atc aag gac gcc gaa gcg cac gcc gag gag gat cgc aag


Lys Glu Asp Ile Asp Arg Met Ile Lys Asp Ala Glu Ala His Ala Glu Glu Asp Arg Lys


 


1801/601                                1831/611


cgt cgc gag gag gcc gat gtt cgt aat caa gcc gag aca ttg gtc tac cag acg gag aag


Arg Arg Glu Glu Ala Asp Val Arg Asn Gln Ala Glu Thr Leu Val Tyr Gln Thr Glu Lys


 


1861/621                                1891/631


ttc gtc aaa gaa cag cgt gag gcc gag ggt ggt tcg aag gta cct gaa gac acg ctg aac


Phe Val Lys Glu Gln Arg Glu Ala Glu Gly Gly Ser Lys Val Pro Glu Asp Thr Leu Asn


 


1921/641                                1951/651


aag gtt gat gcc gcg gtg gcg gaa gcg aag gcg gca ctt ggc gga tcg gat att tcg gcc


Lys Val Asp Ala Ala Val Ala Glu Ala Lys Ala Ala Leu Gly Gly Ser Asp Ile Ser Ala


 


1981/661                                2011/671


atc aag tcg gcg atg gag aag ctg ggc cag gag tcg cag gct ctg ggg caa gcg atc tac


Ile Lys Ser Ala Met Glu Lys Leu Gly Gln Glu Ser Gln Ala Leu Gly Gln Ala Ile Tyr


 


2041/681                                2071/691


gaa gca gct cag gct gcg tca cag gcc act ggc gct gcc cac ccc ggc tcg gct gat gaA


GLU ALA ALA GLN ALA ALA SER GLN ALA THR GLY ALA ALA HIS PRO GLY SER ALA ASP GLU


 


2101/701


AGC a


Ser






ETA(dII) from Pseudomonas aerginosa 

The complete coding sequence for Pseudomonas aeruginosa exotoxin type A (ETA)-SEQ ID NO:31-GenBank Accession No. K01397, is shown below:








ctgcagctgg tcaggccgtt tccgcaacgc ttgaagtcct



 


ggccgatata ccggcagggc cagccatcgt tcgacgaata


 


aagccacctc agccatgatg ccctttccat ccccagcgga


 


accccgacat ggacgccaaa gccctgctcc tcggcagcct


 


ctgcctggcc gccccattcg ccgacgcggc gacgctcgac


 


aatgctctct ccgcctgcct cgccgcccgg ctcggtgcac


 


cgcacacggc ggagggccag ttgcacctgc cactcaccct


 


tgaggcccgg cgctccaccg gcgaatgcgg ctgtacctcg


 


gcgctggtgc gatatcggct gctggccagg ggcgccagcg


 


ccgacagcct cgtgcttcaa gagggctgct cgatagtcgc


 


caggacacgc cgcgcacgct gaccctggcg gcggacgccg


 


gcttggcgag cggccgcgaa ctggtcgtca ccctgggttg


 


tcaggcgcct gactgacagg ccgggctgcc accaccaggc


 


cgagatggac gccctgcatg tatcctccga tcggcaagcc


 


tcccgttcgc acattcacca ctctgcaatc cagttcataa


 


atcccataaa agccctcttc cgctccccgc cagcctcccc


 


gcatcccgca ccctagacgc cccgccgctc tccgccggct


 


cgcccgacaa gaaaaaccaa ccgctcgatc agcctcatcc


 


ttcacccatc acaggagcca tcgcgatgca cctgataccc


 


cattggatcc ccctggtcgc cagcctcggc ctgctcgccg


 


gcggctcgtc cgcgtccgcc gccgaggaag ccttcgacct


 


ctggaacgaa tgcgccaaag cctgcgtgct cgacctcaag


 


gacggcgtgc gttccagccg catgagcgtc gacccggcca


 


tcgccgacac caacggccag ggcgtgctgc actactccat


 


ggtcctggag ggcggcaacg acgcgctcaa gctggccatc


 


gacaacgccc tcagcatcac cagcgacggc ctgaccatcc


 


gcctcgaagg cggcgtcgag ccgaacaagc cggtgcgcta


 


cagctacacg cgccaggcgc gcggcagttg gtcgctgaac


 


tggctggtac cgatcggcca cgagaagccc tcgaacatca


 


aggtgttcat ccacgaactg aacgccggca accagctcag


 


ccacatgtcg ccgatctaca ccatcgagat gggcgacgag


 


ttgctggcga agctggcgcg cgatgccacc ttcttcgtca


 


gggcgcacga gagcaacgag atgcagccga cgctcgccat


 


cagccatgcc ggggtcagcg tggtcatggc ccagacccag


 


ccgcgccggg aaaagcgctg gagcgaatgg gccagcggca


 


aggtgttgtg cctgctcgac ccgctggacg gggtctacaa


 


ctacctcgcc cagcaacgct gcaacctcga cgatacctgg


 


gaaggcaaga tctaccgggt gctcgccggc aacccggcga


 


agcatgacct ggacatcaaa cccacggtca tcagtcatcg


 


cctgcacttt cccgagggcg gcagcctggc cgcgctgacc


 


gcgcaccagg cttgccacct gccgctggag actttcaccc


 


gtcatcgcca gccgcgcggc tgggaacaac tggagcagtg


 


cggctatccg gtgcagcggc tggtcgccct ctacctggcg


 


gcgcggctgt cgtggaacca ggtcgaccag gtgatccgca


 


acgccctggc cagccccggc agcggcggcg acctgggcga


 


agcgatccgc gagcagccgg agcaggcccg tctggccctg


 


accctggccg ccgccgagag cgagcgcttc gtccggcagg


 


gcaccggcaa cgacgaggcc ggcgcggcca acgccgacgt


 


ggtgagcctg acctgcccgg tcgccgccgg tgaatgcgcg


 


ggcccggcgg acagcggcga cgccctgctg gagcgcaact


 


atcccactgg cgcggagttc ctcggcgacg gcggcgacgt


 


cagcttcagc acccgcggca cgcagaactg gacggtggag


 


cggctgctcc aggcgcaccg ccaactggag gagcgcggct


 


atgtgttcgt cggctaccac ggcaccttcc tcgaagcggc


 


gcaaagcatc gtcttcggcg gggtgcgcgc gcgcagccag


 


gacctcgacg cgatctggcg cggtttctat atcgccggcg


 


atccggcgct ggcctacggc tacgcccagg accaggaacc


 


cgacgcacgc ggccggatcc gcaacggtgc cctgctgcgg


 


gtctatgtgc cgcgctcgag cctgccgggc ttctaccgca


 


ccagcctgac cctggccgcg ccggaggcgg cgggcgaggt


 


cgaacggctg atcggccatc cgctgccgct gcgcctggac


 


gccatcaccg gccccgagga ggaaggcggg cgcctggaga


 


ccattctcgg ctggccgctg gccgagcgca ccgtggtgat


 


tccctcggcg atccccaccg acccgcgcaa cgtcggcggc


 


gacctcgacc cgtccagcat ccccgacaag gaacaggcga


 


tcagcgccct gccggactac gccagccagc ccggcaaacc


 


gccgcgcgag gacctgaagt aactgccgcg accggccggc


 


tcccttcgca ggagccggcc ttctcggggc ctggccatac


 


atcaggtttt cctgatgcca gcccaatcga atatgaattc 2760






The amino acid sequence of ETA (SEQ ID NO:32), GenBank Accession No. K01397, is:











MHLIPHWIPL VASLGLLAGG SSASAAEEAF DLWNECAKAC




 



VLDLKDGVRS SRMSVDPAIA DTNGQGVLHY SMVLEGGNDA



 



LKLAIDNALS ITSDGLTIRL EGGVEPNKPV RYSYTRQARG



 



SWSLNWLVPI GHEKPSNIKV FIHELNAGNQ LSHMSPIYTI



 



EMGDELLAKL ARDATFFVRA HESNEMQPTL AISHAGVSVV



 



MAQTQPRREK RWSEWASGKV LCLLDPLDGV YNYLAQQRCN



 



LDDTWEGKIY RVLAGNPAKH DLDIKPTVIS HRLHFPEGGS



 



LAALTAHQAC HLPLETFTRH RQPRGWEQLE QCGYPVQRLV



 



ALYLAARLSW NQVDQVIRNA LASPGSGGDL GEAIREQPEQ



 



ARLALTLAAA ESERFVRQGT GNDEAGAANA DVVSLTCPVA



 



AGECAGPADS GDALLERNYP TGAEFLGDGG DVSFSTRGTQ



 



NWTVERLLQA HRQLEERGYV FVGYHGTFLE AAQSIVFGGV



 



RARSQDLDAI WRGFYIAGDP ALAYGYAQDQ EPDARGRIRN



 



GALLRVYVPR SSLPGFYRTS LTLAAPEAAG EVERLIGHPL



 



PLRLDAITGP EEEGGRLETI LGWPLAERTV VIPSAIPTDP



 



RNVGGDLDPS SIPDKEQAIS ALPDYASQPG KPPREDLK 638






Residues 1-25 (italicized) above represent the signal peptide. The first residue of the mature polypeptide, Ala, is bolded/underscored. The mature polypeptide is residues 26-638 of SEQ ID NO:32.

Domain II (ETA(II)), translocation domain (underscored above) spans residues 247-417 of the mature polypeptide (corresponding to residues 272-442 of SEQ ID NO:32) and is presented below separately as SEQ ID NO:33.










RLHFPEGGSL AALTAHQACH LPLETFTRHR QPRGWEQLEQ




 



CGYPVQRLVA LYLAARLSWN QVDQVIRNAL ASPGSGGDLG



 



EAIREQPEQA RLALTLAAAE SERFVRQGTG NDEAGAANAD



 



VVSLTCPVAA GECAGPADSG DALLERNYPT GAEFLGDGGD



 



VSFSTRGTQN W 171






The construct in which ETA(dII) is fused to HPV-16 E7 is shown below (nucleotides; SEQ ID NO:34 and amino acids; SEQ ID NO:35). The ETA(dII) sequence appears in plain font, extra codons from plasmid pcDNA3 are italicized. Nucleotides between ETA(dII) and E7 are also bolded (and result in the interposition of two amino acids between ETA(dII) and E7). The E7 amino acid sequence is underscored (ends with Gln at position 269).









1/1                                     31/11



atg cgc ctg cac ttt ccc gag ggc ggc agc ctg gcc gcg ctg acc gcg cac cag gct tgc


Met arg leu his phe pro glu gly gly ser leu ala ala leu thr ala his gln ala cys


 


61/21                                   91/31


cac ctg ccg ctg gag act ttc acc cgt cat cgc cag ccg cgc ggc tgg gaa caa ctg gag


His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu Glu


 


121/41                                  151/51


cag tgc ggc tat ccg gtg cag cgg ctg gtc gcc ctc tac ctg gcg gcg cgg ctg tcg tgg


Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp


 


181/61                                  211/71


aac cag gtc gac cag gtg atc cgc aac gcc ctg gcc agc ccc ggc agc ggc ggc gac ctg


Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu


 


241/81                                  271/91


ggc gaa gcg atc cgc gag cag ccg gag cag gcc cgt ctg gcc ctg acc ctg gcc gcc gcc


Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala


 


301/101                                 331/111


gag agc gag cgc ttc gtc cgg cag ggc acc ggc aac gac gag gcc ggc gcg gcc aac gcc


Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Ala


 


361/121                                 391/131


gac gtg gtg agc ctg acc tgc ccg gtc gcc gcc ggt gaa tgc gcg ggc ccg gcg gac agc


Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser


 


421/141                                 451/151


ggc gac gcc ctg ctg gag cgc aac tat ccc act ggc gcg gag ttc ctc ggc gac ggc ggc


Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly


 


481/161                                 511/171


gac gtc agc ttc agc acc cgc ggc acg cag aac gaa ttc atg cat gga gat aca cct aca


Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Glu Phe Met His Gly Asp Thr Pro Thr


 


541/181                                 571/191


ttg cat gaa tat atg tta gat ttg caa cca gag aca act gat ctc tac tgt tat gag caa


Leu His Glu Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln


 


601/201                                 631/211


tta aat gac agc tca gag gag gag gat gaa ata gat ggt cca gct gga caa gca gaa ccg


Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro


 


661/221                                 691/231


gac aga gcc cat tac aat att gta acc ttt tgt tgc aag tgt gac tct acg ctt cgg ttg


Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu


 


721/241                                 751/251


tgc gta caa agc aca cac gta gac att cgt act ttg gaa gac ctg tta atg ggc aca cta


Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr Leu


 


781/261                                 811/271


gga att gtg tgc ccc atc tgt tct caa gga tcc gag ctc ggt acc aag ctt aag ttt aaa


Gly Ile Val Cys Pro Ile Cys Ser Gln Gly Ser Glu Leu Gly Thr Lys Leu Lys Phe Lys


 


841/281


ccg ctg atc agc ctc gac tgt gcc ttc tag


Pro Leu Ile Ser Leu Asp Cys Ala Phe AMB






The nucleotide sequence of the pcDNA3 vector encoding E7 and HSP70 (pcDNA3-E7-Hsp70) (SEQ ID NO:36) is shown in FIG. 38. The E7-Hsp70 fusion sequence is shown in upper case, underscored. Plasmid sequences are in lower case.
The nucleic acid sequence of plasmid construct pcDNA3-ETA(dII)/E7 (SEQ ID NO:37) is shown in FIG. 39. ETA(dII)/E7 is ligated into the EcoRI/BamHI sites of pcDNA3 vector. The nucleotides encoding ETA(dII)/E7 are shown in upper case and underscored. Plasmid sequence is lower case.
Calreticulin (CRT)
“Calreticulin” or “CRT” describes the well-characterized  ̃46 kDa resident protein of the ER lumen that has lectin activity and participates in the folding and assembly of nascent glycoproteins. CRT acts as a “chaperone” polypeptide and a member of the MHC class I transporter TAP complex; CRT associates with TAP1 and TAP2 transporters, tapasin, MHC Class I heavy chain polypeptide and β2 microglobulin to function in the loading of peptide epitopes onto nascent MHC class I molecules (Jorgensen, Eur. J. Biochem. 267:2945-54, 2002. The term “calreticulin” or “CRT” refers to polypeptides and nucleic acids molecules having substantial identity (defined herein) to the exemplary CRT sequences as described herein. A CRT polypeptide is a polypeptides comprising a sequence identical to or substantially identical (defined herein) to the amino acid sequence of CRT. An exemplary nucleotide and amino acid sequence for a CRT used in the present compositions and methods are presented below. The terms “calreticulin” or “CRT” encompass native proteins as well as recombinantly produced modified proteins that induce an immune response, including a CTL response. The terms “calreticulin” or “CRT” encompass homologues and allelic variants of CRT, including variants of native proteins constructed by in vitro techniques, and proteins isolated from natural sources. The CRT polypeptides of the invention, and sequences encoding them, also include fusion proteins comprising non-CRT sequences, particularly MHC class I-binding peptides; and also further comprising other domains, e.g., epitope tags, enzyme cleavage recognition sequences, signal sequences, secretion signals and the like.
The term “endoplasmic reticulum chaperone polypeptide” as used herein means any polypeptide having substantially the same ER chaperone function as the exemplary chaperone proteins CRT, tapasin, ER60 or calnexin. Thus, the term includes all functional fragments or variants or mimics thereof. A polypeptide or peptide can be routinely screened for its activity as an ER chaperone using assays known in the art, such as that set forth in Example 1. While the invention is not limited by any particular mechanism of action, in vivo chaperones promote the correct folding and oligomerization of many glycoproteins in the ER, including the assembly of the MHC class I heterotrimeric molecule (heavy (H) chain, β2m, and peptide). They also retain incompletely assembled MHC class I heterotrimeric complexes in the ER (Hauri FEBS Lett. 476:32-37, 2000).
The sequences of CRT, including human CRT, are well known in the art (McCauliffe, J. Clin. Invest. 86:332-5, 1990; Burns, Nature 367:476-80, 1994; Coppolino, Int. J. Biochem. Cell Biol. 30:553-8, 2000). The nucleic acid sequence appears as GenBank Accession No. NM 004343 and is SEQ ID NO:38.








   1 gtccgtactg cagagccgct gccggagggt cgttttaaag ggccgcgttg ccgccccctc



 


  61 ggcccgccat gctgctatcc gtgccgctgc tgctcggcct cctcggcctg gccgtcgccg


 


 121 agcccgccgt ctacttcaag gagcagtttc tggacggaga cgggtggact tcccgctgga


 


 181 tcgaatccaa acacaagtca gattttggca aattcgttct cagttccggc aagttctacg


 


 241 gtgacgagga gaaagataaa ggtttgcaga caagccagga tgcacgcttt tatgctctgt


 


 301 cggccagttt cgagcctttc agcaacaaag gccagacgct ggtggtgcag ttcacggtga


 


 361 aacatgagca gaacatcgac tgtgggggcg gctatgtgaa gctgtttcct aatagtttgg


 


 421 accagacaga catgcacgga gactcagaat acaacatcat gtttggtccc gacatctgtg


 


 481 gccctggcac caagaaggtt catgtcatct tcaactacaa gggcaagaac gtgctgatca


 


 541 acaaggacat ccgttgcaag gatgatgagt ttacacacct gtacacactg attgtgcggc


 


 601 cagacaacac ctatgaggtg aagattgaca acagccaggt ggagtccggc tccttggaag


 


 661 acgattggga cttcctgcca cccaagaaga taaaggatcc tgatgcttca aaaccggaag


 


 721 actgggatga gcgggccaag atcgatgatc ccacagactc caagcctgag gactgggaca


 


 781 agcccgagca tatccctgac cctgatgcta agaagcccga ggactgggat gaagagatgg


 


 841 acggagagtg ggaaccccca gtgattcaga accctgagta caagggtgag tggaagcccc


 


 901 ggcagatcga caacccagat tacaagggca cttggatcca cccagaaatt gacaaccccg


 


 961 agtattctcc cgatcccagt atctatgcct atgataactt tggcgtgctg ggcctggacc


 


1021 tctggcaggt caagtctggc accatctttg acaacttcct catcaccaac gatgaggcat


 


1081 acgctgagga gtttggcaac gagacgtggg gcgtaacaaa ggcagcagag aaacaaatga


 


1141 aggacaaaca ggacgaggag cagaggctta aggaggagga agaagacaag aaacgcaaag


 


1201 aggaggagga ggcagaggac aaggaggatg atgaggacaa agatgaggat gaggaggatg


 


1261 aggaggacaa ggaggaagat gaggaggaag atgtccccgg ccaggccaag gacgagctgt


 


1321 agagaggcct gcctccaggg ctggactgag gcctgagcgc tcctgccgca gagcttgccg


 


1381 cgccaaataa tgtctctgtg agactcgaga actttcattt ttttccaggc tggttcggat


 


1441 ttggggtgga ttttggtttt gttcccctcc tccactctcc cccaccccct ccccgccctt


 


1501 tttttttttt tttttaaact ggtattttat cctttgattc tccttcagcc ctcacccctg


 


1561 gttctcatct ttcttgatca acatcttttc ttgcctctgt gccccttctc tcatctctta


 


1621 gctcccctcc aacctggggg gcagtggtgt ggagaagcca caggcctgag atttcatctg


 


1681 ctctccttcc tggagcccag aggagggcag cagaaggggg tggtgtctcc aaccccccag


 


1741 cactgaggaa gaacggggct cttctcattt cacccctccc tttctcccct gcccccagga


 


1801 ctgggccact tctgggtggg gcagtgggtc ccagattggc tcacactgag aatgtaagaa


 


1861 ctacaaacaa aatttctatt aaattaaatt ttgtgtctc 1899






Human CRT protein (GenBank Accession No. NM 004343), (SEQ ID NO:39) is shown below:









  1 MLLSVPLLLG LLGLAVAEPA VYFKEQFLDG DGWTSRWIES KHKSDFGKFV LSSGKFYGDE



 


 61 EKDKGLQTSQ DARFYALSAS FEPFSNKGQT LVVQFTVKHE QNIDCGGGYV KLFPNSLDQT


 


121 DMHGDSEYNI MFGPDICGPG TKKVHVIFNY KGKNVLINKD IRCKDDEFTH LYTLIVRPDN


 


181 TYEVKIDNSQ VESGSLEDDW DFLPPKKIKD PDASKPEDWD ERAKIDDPTD SKPEDWDKPE


 


241 HIPDPDAKKP EDWDEEMDGE WEPPVIQNPE YKGEWKPRQI DNPDYKGTWI HPEIDNPEYS


 


301 PDPSIYAYDN FGVLGLDLWQ VKSGTIFDNF LITNDEAYAE EFGNETWGVT KAAEKQMKDK


 


361 QDEEQRLKEE EEDKKRKEEE EAEDKEDDED KDEDEEDEED KEEDEEEDVP GQAKDEL 417






For the generation of plasmid encoding the full length of rabbit calreticulin (there is more than 90% homology between rabbit, human, mouse, and rat calreticulin), pcDNA3-CRT, the DNA fragment encoding this protein was first amplified with PCR using conditions as described in Chen, Cancer Res., 2000, supra, using rabbit calreticulin cDNA template (Michalak, Biochem J. 344 Pt 2:281-292, 1999), provided by Dr. Marek Michalak, University of Alberta, Edmonton, Canada, and a set of the following primers:









5′-ccggtctagaatgctgctccctgtgccgct-3′
(SEQ ID NO:40)



and


 


5′-ccggagatctcagctcgtccttggcctggc-3′
(SEQ ID NO:41)






The amplified product was then digested with the restriction digest enzymes XbaI and BamHI and further cloned into the XbaI and BamHI cloning sites of pcDNA3 vector (Invitrogen, Carlsbad, Calif.). For the generation of pcDNA3-CRT/E7, the E7 DNA was amplified by PCR using pcDNA3-E7 as a DNA template and a set of primers:










5′-ggggaattcatggagatacaccta-3′
(SEQ ID NO:42)



and


 


5′-ggtggatccttgagaacagatgg-3′
(SEQ ID NO:43)






The amplified E7 DNA fragment was then digested with BamHI and further cloned into the BamHI cloning sites of pcDNA3-CRT vector. The orientation and accuracy of these constructs was confirmed by DNA sequencing.

Plasmid DNA with CRT, E7 or CRT/E7 gene insert and the “empty” plasmid vector were transfected into subcloning-efficient DH5 TM cells (Life Technologies, USA). The DNA was then amplified and purified using double CsCl purification (BioServe Biotechnologies, Laurel, Md.). The integrity of plasmid DNA and the absence of E. coli DNA or RNA were verified by agarose gel electrophoresis, and the presence of the inserted E7 fragment was confirmed by restriction enzyme digestion and gel electrophoresis.
The present inventors and their colleagues have found that DNA vaccines encoding CRT linked either to E6 or to E7 both of generate significant antitumor effects against E6- and E7-expressing tumors, respectively. Moreover, simultaneous vaccination with both CRT/E6 and CRT/E7 DNA vaccines generated significant E6- and E7-specific T-cell immune responses and significantly better therapeutic antitumor effects against E6- and E7-expressing tumors than vaccination with either CRT/E6 DNA or CRT/E7 DNA alone.
The three domains of CRT also produce E7-specific antitumor immunity and antiangiogenic effects (Cheng W F et al., Vaccine. 23:3864-74, 2005). DNA vaccines encoding each of N, P, and C domains of CRT linked to E7 antigen produced significant stimulation of E7-specific CD8+ T cell precursors and antitumor effects against E7-expressing tumors. The N domain of CRT also showed antiangiogenic properties that might have contributed to the antitumor effect. Thus, the present invention includes DNA immunogens in which the IPP is the N, P, or C domain of CRT.
The nucleotide sequence of plasmid pNGVL4a-CRT/E7(detox) (SEQ ID NO:44) is shown in FIG. 40. The sequence is annotated to show plasmid-derived sequences (lower case), CRT-derived (bold, upper case) and HPV-E7-derived (detoxified by two amino acid substitutions as described above (upper case, italicized, underlined) sequences.
General Recombinant DNA Methods
Basic texts disclosing general methods of molecular biology, all of which are incorporated by reference, include: Sambrook, J et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989; Ausubel, F M et al. Current Protocols in Molecular Biology, Vol. 2, Wiley-Interscience, New York, (current edition); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); Glover, D M, ed, DNA Cloning: A Practical Approach, vol. I & II, IRL Press, 1985; Albers, B. et al., Molecular Biology of the Cell, 2nd Ed., Garland Publishing, Inc., New York, N.Y. (1989); Watson, J D et al., Recombinant DNA, 2nd Ed., Scientific American Books, New York, 1992; and Old, R W et al., Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2nd Ed., University of California Press, Berkeley, Calif. (1981).
Techniques for the manipulation of nucleic acids, such as, e.g., generating mutations in sequences, subcloning, labeling probes, sequencing, hybridization and the like are well described in the scientific and patent literature. See, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Tijssen, ed. Elsevier, N.Y. (1993).
Nucleic acids, vectors, capsids, polypeptides, and the like can be analyzed and quantified by any of a number of general means well known to those of skill in the art. These include, e.g., analytical biochemical methods such as NMR, spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography, various immunological methods, e.g. fluid or gel precipitin reactions, immunodiffusion, immuno-electrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immunofluorescence assays, Southern analysis, Northern analysis, dot-blot analysis, gel electrophoresis (e.g., SDS-PAGE), RT-PCR, quantitative PCR, other nucleic acid or target or signal amplification methods, radiolabeling, scintillation counting, and affinity chromatography.
Amplification of Nucleic Acids
Oligonucleotide primers can be used to amplify nucleic acids to generate fusion protein coding sequences used to practice the invention, to monitor levels of vaccine after in vivo administration (e.g., levels of a plasmid or virus), to confirm the presence and phenotype of activated CTLs, and the like. The skilled artisan can select and design suitable oligonucleotide amplification primers using known sequences. Amplification methods are also well known in the art, and include, e.g., polymerase chain reaction, PCR (PCR Protocols, A Guide to Methods and Applications, ed. Innis, Academic Press, N.Y. (1990) and PCR Strategies (1995), ed. Innis, Academic Press, Inc., N.Y., ligase chain reaction (LCR) (Wu (1989) Genomics 4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117); transcription amplification (Kwoh (1989) Proc. Natl. Acad. Sci. USA 86:1173); and, self-sustained sequence replication (Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Qβ replicase amplification (Smith (1997) J. Clin. Microbiol. 35:1477-1491; Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerase mediated techniques (NASBA, Cangene, Mississauga, Ontario; Berger (1987) Methods Enzymol. 152:307-316; U.S. Pat. Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology 13:563-564).
Unless otherwise indicated, a particular nucleic acid sequence is intended to encompasses conservative substitution variants thereof (e.g., degenerate codon substitutions) and a complementary sequence. The term “nucleic acid” is synonymous with “polynucleotide” and is intended to include a gene, a cDNA molecule, an mRNA molecule, as well as a fragment of any of these such as an oligonucleotide, and further, equivalents thereof (explained more fully below). Sizes of nucleic acids are stated either as kilobases (kb) or base pairs (bp). These are estimates derived from agarose or polyacrylamide gel electrophoresis (PAGE), from nucleic acid sequences which are determined by the user or published. Protein size is stated as molecular mass in kilodaltons (kDa) or as length (number of amino acid residues). Protein size is estimated from PAGE, from sequencing, from presumptive amino acid sequences based on the coding nucleic acid sequence or from published amino acid sequences.
Specifically, cDNA molecules encoding the amino acid sequence corresponding to the fusion polypeptide of the present invention or fragments or derivatives thereof can be synthesized by the polymerase chain reaction (PCR) (see, for example, U.S. Pat. No. 4,683,202) using primers derived the sequence of the protein disclosed herein. These cDNA sequences can then be assembled into a eukaryotic or prokaryotic expression vector and the resulting vector can be used to direct the synthesis of the fusion polypeptide or its fragment or derivative by appropriate host cells, for example COS or CHO cells.
This invention includes isolated nucleic acids having a nucleotide sequence encoding the novel fusion polypeptides that comprise a translocation polypeptide and an antigen, fragments thereof or equivalents thereof. The term nucleic acid as used herein is intended to include such fragments or equivalents. The nucleic acid sequences of this invention can be DNA or RNA.
A cDNA nucleotide sequence the fusion polypeptide can be obtained by isolating total mRNA from an appropriate cell line. Double stranded cDNA is prepared from total mRNA. cDNA can be inserted into a suitable plasmid, bacteriophage or viral vector using any one of a number of known techniques.
In reference to a nucleotide sequence, the term “equivalent” is intended to include sequences encoding structurally homologous and/or a functionally equivalent proteins. For example, a natural polymorphism in a nucleotide sequence encoding an anti-apoptotic polypeptide according to the present invention (especially at the third base of a codon) may be manifest as “silent” mutations which do not change the amino acid sequence. Furthermore, there may be one or more naturally occurring isoforms or related, immunologically cross-reactive family members of these proteins. Such isoforms or family members are defined as proteins that share function amino acid sequence similarity to the reference polypeptide.
Fragment of Nucleic Acid
A fragment of the nucleic acid sequence is defined as a nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the full length translocation polypeptide, antigenic polypeptide or the fusion thereof. This invention includes such nucleic acid fragments that encode polypeptides which retain (1) the ability of the fusion polypeptide to induce increases in frequency or reactivity of T cells, preferably CD8+ T cells, that are specific for the antigen part of the fusion polypeptide.
For example, a nucleic acid fragment as intended herein encodes an antigen or an IPP that that retains the ability to improve the immunogenicity of an antigen vaccine when administered as a chimeric DNA with antigen-encoding sequence, or when co-administered therewith.
Generally, the nucleic acid sequence encoding a fragment of an anti-apoptotic polypeptide comprises of nucleotides from the sequence encoding the mature protein (or an active fragment thereof).
Nucleic acid sequences of this invention may also include linker sequences, natural or modified restriction endonuclease sites and other sequences that are useful for manipulations related to cloning, expression or purification of encoded protein or fragments. These and other modifications of nucleic acid sequences are described herein or are well-known in the art.
The techniques for assembling and expressing DNA coding sequences for translocation types of proteins, and DNA coding sequences for antigenic polypeptides, include synthesis of oligonucleotides, PCR, transforming cells, constructing vectors, expression systems, and the like; these are well-established in the art such that those of ordinary skill are familiar with standard resource materials, specific conditions and procedures.
Expression Vectors and Host Cells
This invention includes an expression vector comprising a nucleic acid sequence encoding (a) an antigen, optionally linked to (b) an IPP or (c) an siRNA operably linked to at least one regulatory sequence, which includes a promoter that is expressable in a eukaryotic cell, preferably in a mammalian cells, more preferably in a human cell.
The term “expression vector” or “expression cassette” as used herein refers to a nucleotide sequence which is capable of affecting expression of a protein coding sequence in a host compatible with such sequences. Expression cassettes include at least a promoter operably linked with the polypeptide coding sequence; and, optionally, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression may also be included, e.g., enhancers.
“Operably linked” means that the coding sequence is linked to a regulatory sequence in a manner that allows expression of the coding sequence. Known regulatory sequences are selected to direct expression of the desired protein in an appropriate host cell. Accordingly, the term “regulatory sequence” includes promoters, enhancers and other expression control elements. Such regulatory sequences are described in, for example, Goeddel, Gene Expression Technology. Methods in Enzymology, vol. 185, Academic Press, San Diego, Calif. (1990)).
Thus, expression cassettes include plasmids, recombinant viruses, any form of a recombinant “naked DNA” vector, and the like. A “vector” comprises a nucleic acid which can infect, transfect, transiently or permanently transduce a cell. It will be recognized that a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid. The vector optionally comprises viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.). Vectors include replicons (e.g., RNA replicons), bacteriophages) to which fragments of DNA may be attached and become replicated. Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA, e.g., plasmids, viruses, and the like (U.S. Pat. No. 5,217,879), and includes both the expression and nonexpression plasmids. Where a recombinant cell or culture is described as hosting an “expression vector” this includes both extrachromosomal circular and linear DNA and DNA that has been incorporated into the host chromosome(s). Where a vector is being maintained by a host cell, the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome.
Those skilled in the art appreciate that the particular design of an expression vector of this invention depends on considerations such as the host cell to be transfected and/or the type of protein to be expressed.
The present expression vectors comprise the full range of nucleic acid molecules encoding the various embodiments of the fusion polypeptide and its functional derivatives (defined herein) including polypeptide fragments, variants, etc., as well as those encoding siRNA or other siNAs of the present invention.
Such expression vectors are used to transfect host cells (in vitro, ex vivo or in vivo) for expression of the DNA and production of the encoded proteins which include fusion proteins or peptides. It will be understood that a genetically modified cell expressing the fusion polypeptide may transiently express the exogenous DNA for a time sufficient for the cell to be useful for its stated purpose.
The present in invention provides methods for producing the fusion polypeptides, fragments and derivatives. For example, a host cell transfected with a nucleic acid vector that encodes the fusion polypeptide or an siRNA is cultured under appropriate conditions to allow expression of the polypeptide or siRNA.
Host cells may also be transfected with one or more expression vectors that singly or in combination comprise (a) DNA encoding at least a portion of the fusion polypeptide and (b) DNA encoding at least a portion of a second protein, preferably an antigen, or (c) DNA encoding an siRNA, so that the host cells produce yet further fusion polypeptides or siRNAs
A culture typically includes host cells, appropriate growth media and other byproducts. Suitable culture media are well known in the art. The fusion polypeptide can be isolated from medium or cell lysates using conventional techniques for purifying proteins and peptides, including ammonium sulfate precipitation, fractionation column chromatography (e.g. ion exchange, gel filtration, affinity chromatography, etc.) and/or electrophoresis (see generally, “Enzyme Purification and Related Techniques”, Meth Enzymol, 22:233-577 (1971)). Once purified, partially or to homogeneity, the recombinant polypeptides or siRNAs of the invention can be utilized in pharmaceutical compositions as described in more detail herein.
The term “isolated” as used herein, when referring to a molecule or composition, such as a translocation polypeptide or a nucleic acid coding therefor, means that the molecule or composition is separated from at least one other compound (protein, other nucleic acid, etc.) or from other contaminants with which it is natively associated or becomes associated during processing. An isolated composition can also be substantially pure. An isolated composition can be in a homogeneous state and can be dry or in aqueous solution. Purity and homogeneity can be determined, for example, using analytical chemical techniques such as polyacrylamide gel electrophoresis (PAGE) or high performance liquid chromatography (HPLC). Even where a protein has been isolated so as to appear as a homogenous or dominant band in a gel pattern, there are trace contaminants which co-purify with it.
Host cells transformed or transfected to express the fusion polypeptide or a homologue or functional derivative thereof are within the scope of the invention. For example, the fusion polypeptide may be expressed in yeast, or mammalian cells such as Chinese hamster ovary cells (CHO) or, preferably human cells. Preferred cells for expression of the siRNA of the present invention are APCs most preferably, DCs. Other suitable host cells are known to those skilled in the art.
Expression in eukaryotic cells leads to partial or complete glycosylation and/or formation of relevant inter- or intra-chain disulfide bonds of the recombinant protein.
Although preferred vectors are described in the Examples, other examples of expression vectors are provided here. Examples of vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari et al., EMBO J. 6:229-34, 1987), pMFa (Kurjan et al., Cell 30:933-43, 1982), pJRY88 (Schultz et al., Gene 54:113-23, 1987), and pYES2 (Invitrogen Corp.). Baculovirus vectors available for expression of proteins in cultured insect cells (SF 9 cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-65, 1983) and the pVL series (Lucklow, V A et al., Virology 170:31-9, 1989). Generally, COS cells (Gluzman, Y., Cell 23:175-82, 1981) are used in conjunction with such vectors as pCDM 8 (Aruffo A et al., supra, for transient amplification/expression in mammalian cells, while CHO (dhfr-negative CHO) cells are used with vectors such as pMT2PC (Kaufman et al. EMBO J. 6:187-95, 1987) for stable amplification/expression in mammalian cells. The NS0 myeloma cell line (a glutamine synthetase expression system.) is available from Celltech Ltd.
Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the reporter group and the target protein to enable separation of the target protein from the reporter group subsequent to purification of the fusion protein. Proteolytic enzymes for such cleavage and their recognition sequences include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase, maltose E binding protein, or protein A, respectively, to the target recombinant protein. Inducible non-fusion expression vectors include pTrc (Amann et al., Gene 69:301-15, 1988) and pET 11d (Studier et al., Gene Expression Technology: Meth Enzymol 185:60-89, Academic Press, 1990).
Vector Construction
Construction of suitable vectors comprising the desired coding and control sequences employs standard ligation and restriction techniques which are well understood in the art. Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and re-ligated in the form desired. The sequences of several preferred plasmid vectors, with and without inserted coding sequences, have been disclosed above.
The DNA sequences which form the vectors are available from a number of sources. Backbone vectors and control systems are generally found on available “host” vectors which are used for the bulk of the sequences in construction. For the pertinent coding sequence, initial construction may be, and usually is, a matter of retrieving the appropriate sequences from cDNA or genomic DNA libraries. However, once the sequence is disclosed it is possible to synthesize the entire gene sequence in vitro starting from the individual nucleotide derivatives. The entire gene sequence for genes of sizeable length, e.g., 500-1000 bp may be prepared by synthesizing individual overlapping complementary oligonucleotides and filling in single stranded nonoverlapping portions using DNA polymerase in the presence of the deoxyribonucleotide triphosphates. This approach has been used successfully in the construction of several genes of known sequence. See, for example, Edge, Md., Nature 292:756, 1981; Nambair, K P, et al., Science 223:1299, 1984; Jay, E, J Biol Chem 259:6311, 1984).
Synthetic oligonucleotides are prepared by either the phosphotriester method as described by references cited above or the phosphoramidite method (Beaucage, S L et al., Tet Lett 22:1859, 1981; Matteucci, M D et al., J Am Chem Soc 103:3185, 1981) and can be prepared using commercially available automated oligonucleotide synthesizers. Kinase treatment of single strands prior to annealing or for labeling is by conventional methods.
Once the components of the desired vectors are thus available, they can be excised and ligated using standard restriction and ligation procedures. Site-specific DNA cleavage is performed by treating with the suitable restriction enzyme (or enzymes) under conditions which are generally understood in the art, and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes. See, e.g., New England Biolabs, Product Catalog. A general description of size separations is found in Methods in Enzymology (1980) 65:499-560.
Restriction cleaved fragments may be blunt ended by treating with the large fragment of E. coli DNA polymerase I (Klenow) in the presence of the four deoxynucleotide triphosphates (dNTPs) using conventional methods and conditions. Ligations are performed using conventional methods. In vector construction employing “vector fragments”, the fragment is commonly treated with bacterial or mammalian alkaline phosphatase to remove the 5′ phosphate and prevent self-ligation. Alternatively, re-ligation can be prevented in vectors which have been double digested by additional restriction enzyme and separation of the unwanted fragments.
Any of a number of methods are used to introduce mutations into the coding sequence to generate the variants of the invention. These mutations include simple deletions or insertions, systematic deletions, insertions or substitutions of clusters of bases or substitutions of single bases.
For example, modifications of DNA sequences are created by site-directed mutagenesis, a well-known technique for which protocols and reagents are commercially available (Zoller, M J et al., Nucleic Acids Res 10:6487-500, 1982; Adelman, J P et al., DNA 2:183-193, 1983). Using conventional methods, transformants are selected based on the presence of the ampicillin-, tetracycline-, or other antibiotic resistance gene (or other selectable marker) depending on the mode of plasmid construction. Plasmids are then prepared from the transformants with optional chloramphenicol amplification (Clewell, D B et al., Proc Natl Acad Sci USA 62:1159, 1969); Clewell, D B, J Bacteriol 110:667, 1969)). Several mini DNA preps are commonly used. See, e.g., Anal Biochem 114:193-7, 1981; Nucleic Acids Res 7:1513-23, 1979). The isolated DNA is analyzed by restriction and/or sequenced by the dideoxy nucleotide method of Sanger (Proc Natl Acad Sci USA 74:5463, 1977; Messing, et al., Nucleic Acids Res 9:309, 1981), or by the method of Maxam et al., Meth Enzymology 65:499, 1980.
Vector DNA can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming host cells can be found in Sambrook et al. supra and other standard texts. In fusion expression vectors, a proteolytic cleavage site may be introduced at the junction of two sequences (such as a reporter group and the target protein to enable separation of the target protein from the reporter group subsequent to purification of the fusion protein). Proteolytic enzymes for such cleavage and their recognition sequences include Factor Xa, thrombin and enterokinase.
Promoters and Enhancers
A promoter region of a DNA or RNA molecule binds RNA polymerase and promotes the transcription of an “operably linked” nucleic acid sequence. As used herein, a “promoter sequence” is the nucleotide sequence of the promoter which is found on that strand of the DNA or RNA which is transcribed by the RNA polymerase. Two sequences of a nucleic acid molecule, such as a promoter and a coding sequence, are “operably linked” when they are linked to each other in a manner which permits both sequences to be transcribed onto the same RNA transcript or permits an RNA transcript begun in one sequence to be extended into the second sequence. Thus, two sequences, such as a promoter sequence and a coding sequence of DNA or RNA are operably linked if transcription commencing in the promoter sequence will produce an RNA transcript of the operably linked coding sequence. In order to be “operably linked” it is not necessary that two sequences be immediately adjacent to one another in the linear sequence.
The preferred promoter sequences of the present invention must be operable in mammalian cells and may be either eukaryotic or viral promoters. Although preferred promoters are described in the Examples, other useful promoters and regulatory elements are discussed below. Suitable promoters may be inducible, repressible or constitutive. A “constitutive” promoter is one which is active under most conditions encountered in the cell's environmental and throughout development. An “inducible” promoter is one which is under environmental or developmental regulation. A “tissue specific” promoter is active in certain tissue types of an organism. An example of a constitutive promoter is the viral promoter MSV-LTR, which is efficient and active in a variety of cell types, and, in contrast to most other promoters, has the same enhancing activity in arrested and growing cells. Other preferred viral promoters include that present in the CMV-LTR (from cytomegalovirus) (Bashart, M. et al., Cell 41:521, 1985) or in the RSV-LTR (from Rous sarcoma virus) (Gorman, C. M., Proc. Natl. Acad. Sci. USA 79:6777, 1982). Also useful are the promoter of the mouse metallothionein I gene (Hamer, D, et al., J. Mol. Appl. Gen. 1:273-88, 1982; the TK promoter of Herpes virus (McKnight, S, Cell 31:355-65, 1982); the SV40 early promoter (Benoist, C., et al., Nature 290:304-10, 1981); and the yeast gal4 gene promoter (Johnston, S A et al., Proc. Natl. Acad. Sci. USA 79:6971-5, 1982); Silver, P A, et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5, 1984)). Other illustrative descriptions of transcriptional factor association with promoter regions and the separate activation and DNA binding of transcription factors include: Keegan et al., Nature 231:699, 1986; Fields et al., Nature 340:245, 1989; Jones, Cell 61:9, 1990; Lewin, Cell 61:1161, 1990; Ptashne et al., Nature 346:329, 1990; Adams et al., Cell 72:306, 1993. The relevant disclosure of all of these above-listed references is hereby incorporated by reference.
The promoter region may further include an octamer region which may also function as a tissue specific enhancer, by interacting with certain proteins found in the specific tissue. The enhancer domain of the DNA construct of the present invention is one which is specific for the target cells to be transfected, or is highly activated by cellular factors of such target cells. Examples of vectors (plasmid or retrovirus) are disclosed in (Roy-Burman et al., U.S. Pat. No. 5,112,767). For a general discussion of enhancers and their actions in transcription, see, Lewin, B M, Genes IV, Oxford University Press pp. 552-576, 1990 (or later edition). Particularly useful are retroviral enhancers (e.g., viral LTR) that is preferably placed upstream from the promoter with which it interacts to stimulate gene expression. For use with retroviral vectors, the endogenous viral LTR may be rendered enhancer-less and substituted with other desired enhancer sequences which confer tissue specificity or other desirable properties such as transcriptional efficiency.
Nucleic acids of the invention can also be chemically synthesized using standard techniques, including solid-phase synthesis which, like peptide synthesis, has been fully automated with commercially available DNA synthesizers (Itakura U.S. Pat. Nos. 4,598,049, 4,401,796 and 4,373,071; Caruthers et al. U.S. Pat. No. 4,458,066.
Proteins and Polypeptides
The terms “polypeptide,” “protein,” and “peptide” when referring to compositions of the invention are meant to include variants, analogues, and mimetics with structures and/or activity that substantially correspond to the polypeptide or peptide from which the variant, etc., was derived.
The present invention includes an “isolated” fusion polypeptide comprising a targeting polypeptide linked to an antigenic polypeptide.
The term “chimeric” or “fusion” polypeptide or protein refers to a composition comprising at least one polypeptide or peptide sequence or domain that is chemically bound in a linear fashion with a second polypeptide or peptide domain. One embodiment of this invention is an isolated or recombinant nucleic acid molecule encoding a fusion protein comprising at least two domains, wherein the first domain comprises an IPP and the second domain comprises an antigenic epitope, e.g., an MHC class I-binding peptide epitope. The “fusion” can be an association generated by a peptide bond, a chemical linking, a charge interaction (e.g., electrostatic attractions, such as salt bridges, H-bonding, etc.) or the like. If the polypeptides are recombinant, the “fusion protein” can be translated from a common mRNA. Alternatively, the compositions of the domains can be linked by any chemical or electrostatic means. The chimeric molecules of the invention (e.g., targeting polypeptide fusion proteins) can also include additional sequences, e.g., linkers, epitope tags, enzyme cleavage recognition sequences, signal sequences, secretion signals, and the like. Alternatively, a peptide can be linked to a carrier simply to facilitate manipulation or identification/location of the peptide.
Also included is a “functional derivative” of an IPP (or of its coding sequence) which refers to an amino acid substitution variant, a “fragment,” or a “chemical derivative” of the protein, which terms are defined below. A functional derivative of an IPP retains measurable activity, preferably that is manifest as promoting immunogenicity of one or more antigenic epitopes fused thereto or co-administered therewith. “Functional derivatives” encompass “variants” and “fragments” regardless of whether the terms are used in the conjunctive or the alternative herein.
A functional homologue must possess the above biochemical and biological activity. In view of this functional characterization, use of homologous proteins including proteins not yet discovered, fall within the scope of the invention if these proteins have sequence similarity and the recited biochemical and biological activity.
To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, 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). In a preferred method of alignment, Cys residues are aligned.
In a preferred embodiment, the length of a sequence being compared 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% of the length of the reference sequence. The amino acid residues (or nucleotides) at corresponding amino acid (or nucleotide) positions are then compared. When a position in the first sequence is occupied by the same amino acid residue (or nucleotide) as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the 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 of the two sequences.
The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970) algorithm which has been incorporated into the GAP program in the GCG software package (available at the GCG website on the world wide web), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at the GCG website on the world wide web), 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. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases, for example, to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to a reference nucleic acid molecules. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to HVP22 protein molecules. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See the NCBI website on the world wide web.
Thus, a homologue of a particular IPP as described herein is characterized as having (a) functional activity of the native IPP and (b) sequence similarity to a native IPP when determined as above, of at least about 20% (at the amino acid level), preferably at least about 40%, more preferably at least about 70%, even more preferably at least about 90%, 95%, 97%, 98% or 99%.
It is within the skill in the art to obtain and express such a protein using DNA probes based on the disclosed sequences.
Then, the chimeric DNA construct or fusion protein's biological activity can be tested readily using art-recognized methods such as those described herein in the Examples. A biological assay of the stimulation of antigen-specific T cell reactivity will indicate whether the homologue has the requisite activity to qualify as a “functional” homologue.
A “variant” of a protein, e.g., an antigen or an IPP, refers to a molecule substantially identical to either the full protein or to a fragment thereof in which one or more amino acid residues have been replaced (substitution variant) or which has one or several residues deleted (deletion variant) or added (addition variant). A “fragment” of the IPP refers to any subset of the molecule, that is, a shorter polypeptide of the full-length protein.
A preferred group of conservative variants are those in which at least one amino acid residue and preferably, only one, has been substituted by different residue. For a detailed description of protein chemistry and structure, see Schulz, G E et al., Principles of Protein Structure, Springer-Verlag, New York, 1978, and Creighton, T. E., Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 1983, which are hereby incorporated by reference. The types of substitutions that may be made in the protein molecule may be based on analysis of the frequencies of amino acid changes between a homologous protein of different species, such as those presented in Table 1-2 of Schulz et al. (supra) and FIG. 3-9 of Creighton (supra). Based on such an analysis, conservative substitutions are defined herein as exchanges within one of the following five groups:

















1
Small aliphatic, nonpolar or
Ala, Ser, Thr (Pro, Gly);




slightly polar residues



2
Polar, negatively charged
Asp, Asn, Glu, Gln;




residues and their amides



3
Polar, positively charged
His, Arg, Lys;




residues



4
Large aliphatic, nonpolar
Met, Leu, Ile, Val (Cys)




residues



5
Large aromatic residues
Phe, Tyr, Trp.










The three amino acid residues in parentheses above have special roles in protein architecture. Gly is the only residue lacking a side chain and thus imparts flexibility to the chain. Pro, because of its unusual geometry, tightly constrains the chain. Cys can participate in disulfide bond formation, which is important in protein folding.
More substantial changes in biochemical, functional (or immunological) properties are made by selecting substitutions that are less conservative, such as between, rather than within, the above five groups. Such changes will differ more significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Examples of such substitutions are (i) substitution of Gly and/or Pro by another amino acid or deletion or insertion of Gly or Pro; (ii) substitution of a hydrophilic residue, e.g., Ser or Thr, for (or by) a hydrophobic residue, e.g., Leu, Ile, Phe, Val or Ala; (iii) substitution of a Cys residue for (or by) any other residue; (iv) substitution of a residue having an electropositive side chain, e.g., Lys, Arg or H is, for (or by) a residue having an electronegative charge, e.g., Glu or Asp; or (v) substitution of a residue having a bulky side chain, e.g., Phe, for (or by) a residue not having such a side chain, e.g., Gly.
Most acceptable deletions, insertions and substitutions according to the present invention are those that do not produce radical changes in the characteristics of the wild-type or native IPP in terms of its intracellular processing, intercellular translocation, or other activity that is responsible for its ability to stimulate antigen specific T cell reactivity to an antigenic epitope or epitopes that are fused to the IPP. However, when it is difficult to predict the exact effect of the substitution, deletion or insertion in advance of doing so, one skilled in the art will appreciate that the effect can be evaluated by routine screening assays such as those described here, without requiring undue experimentation.
The term “chemically linked” refers to any chemical bonding of two moieties, e.g., as in one embodiment of the invention, where a translocation polypeptide is chemically linked to an antigenic peptide. Such chemical linking includes the peptide bonds of a recombinantly or in vivo generated fusion protein.
Therapeutic Compositions and their Administration
A vaccine composition comprising the nucleic acid encoding the antigen or the antigen in a fusion polypeptide with an IPP, a particle comprising the nucleic acid or a cell expressing this nucleic acid, is administered to a mammalian subject, preferably a human together with an siNA, preferably an siRNA, that targets mRNA for a pro-apoptotic protein, preferably Bak and/or Bax. Another embodiment is a vaccine composition comprising DCs that are loaded with the antigen and transfected with the above siNA. The vaccine composition and siNA or the modified DCs are administered in a pharmaceutically acceptable carrier in a biologically effective or a therapeutically effective amount.
Certain preferred conditions are disclosed in the Examples. The composition may be given alone or in combination with another protein or peptide such as an immunostimulatory molecule. Treatment may include administration of an adjuvant, used in its broadest sense to include any nonspecific immune stimulating compound such as an interferon. Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether.
A therapeutically effective amount is a dosage that, when given for an effective period of time, achieves the desired immunological or clinical effect.
A therapeutically active amount of a nucleic acid encoding the fusion polypeptide may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the peptide to elicit a desired response in the individual. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A therapeutically effective amounts of the protein, in cell associated form may be stated in terms of the protein or cell equivalents.
Thus an effective amount of the vaccine and the siNA are between about 1 nanogram and about 1 gram per kilogram of body weight of the recipient, more preferably between about 0.1 μg/kg and about 10 mg/kg, more preferably between about 1 μg/kg and about 1 mg/kg. Dosage forms suitable for internal administration preferably contain (for the latter dose range) from about 0.1 μg to 100 μg of active ingredient per unit. The active ingredient may vary from 0.5 to 95% by weight based on the total weight of the composition. Alternatively, an effective dose of DCs loaded with the antigen and expressing siRNA is between about 104 and 108 cells. Those skilled in the art of immunotherapy will be able to adjust these doses without undue experimentation.
The composition may be administered in a convenient manner, e.g., injection by a convenient and effective route. Preferred routes for the DNA/siRNA combination include intradermal “gene gun” delivery or intramuscular injection. The modified DCs are preferably administered by subcutaneous, intravenous or intramuscular routes. Other possible routes include oral administration, intrathecal, inhalation, transdermal application, or rectal administration. For the treatment of existing tumors which have not been completely resected or which have recurred, direct intratumoral injection is also intended.
Depending on the route of administration, the composition may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound. Thus it may be necessary to coat the composition with, or co-administer the composition with, a material to prevent its inactivation. For example, an enzyme inhibitors of nucleases or proteases (e.g., pancreatic trypsin inhibitor, diisopropylfluorophosphate and trasylol) or in an appropriate carrier such as liposomes (including water-in-oil-in-water emulsions as well as conventional liposomes (Strejan et al., J. Neuroimmunol 7:27, 1984).
As used herein “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
Preferred pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride may be included in the pharmaceutical composition. In all cases, the composition should be sterile and should be fluid. It should be stable under the conditions of manufacture and storage and must include preservatives that prevent contamination with microorganisms such as bacteria and fungi. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
Prevention of the action of microorganisms in the pharmaceutical composition can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
Compositions are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form refers to physically discrete units suited as unitary dosages for a mammalian subject; each unit contains a predetermined quantity of active material (e.g., the nucleic acid vaccine) calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of, and sensitivity of, individual subjects
For lung instillation, aerosolized solutions are used. In a sprayable aerosol preparations, the active protein may be in combination with a solid or liquid inert carrier material. This may also be packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant. The aerosol preparations can contain solvents, buffers, surfactants, and antioxidants in addition to the protein of the invention.
Other pharmaceutically acceptable carriers for the nucleic acid vaccine compositions according to the present invention are liposomes, pharmaceutical compositions in which the active protein is contained either dispersed or variously present in corpuscles consisting of aqueous concentric layers adherent to lipidic layers. The active protein is preferably present in the aqueous layer and in the lipidic layer, inside or outside, or, in any event, in the non-homogeneous system generally known as a liposomic suspension. The hydrophobic layer, or lipidic layer, generally, but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surface active substances such as dicetylphosphate, stearylamine or phosphatidic acid, and/or other materials of a hydrophobic nature. Those skilled in the art will appreciate other suitable embodiments of the present liposomal formulations.
Delivery of Vaccine Nucleic Acid to Cells and Animals
The following references set forth principles and current information in the field of basic, medical and veterinary virology and are incorporated by reference: Fields Virology, Fields, B N et al., eds., Lippincott Williams & Wilkins, NY, 1996; Principles of Virology: Molecular Biology, Pathogenesis, and Control, Flint, S. J. et al., eds., Amer Soc Microbiol, Washington D.C., 1999; Principles and Practice of Clinical Virology, 4th Edition, Zuckerman. A. J. et al., eds, John Wiley & Sons, NY, 1999; The Hepatitis C Viruses, by Hagedorn, C H et al., eds., Springer Verlag, 1999; Hepatitis B Virus: Molecular Mechanisms in Disease and Novel Strategies for Therapy, Koshy, R. et al., eds, World Scientific Pub Co, 1998; Veterinary Virology, Murphy, F. A. et al., eds., Academic Press, NY, 1999; Avian Viruses: Function and Control, Ritchie, B. W., Iowa State University Press, Ames, 2000; Virus Taxonomy: Classification and Nomenclature of Viruses: Seventh Report of the International Committee on Taxonomy of Viruses, by M. H. V. Van Regenmortel, M H V et al., eds., Academic Press; NY, 2000.
The Examples below describe certain preferred approaches to delivery of the vaccines and combinations of the present invention. A broader description of other approaches including viral and nonviral vectors and delivery mechanisms follow.
DNA delivery involves introduction of a “foreign” DNA into a cell ex vivo and ultimately, into a live animal or directly into the animal. Several general strategies for gene delivery (=delivery of nucleic acid vectors) for purposes that include “gene therapy” have been studied and reviewed extensively (Yang, N-S., Crit. Rev. Biotechnol. 12:335-356 (1992); Anderson, W F, Science 256:808-13, 1992; Miller, A S, Nature 357:455-60, 1992; Crystal, R G, Amer. J. Med. 92(suppl 6A):44-52S, 1992; Zwiebel, J A et al., Ann NY Acad Sc. 618:394-404, 1991; McLachlin, J R et al., Prog. Nucl. Acid Res. Molec. Biol. 38:91-135, 1990; Kohn, D B et al., Cancer Invest. 7:179-92, 1989), which references are herein incorporated by reference in their entirety).
One approach comprises nucleic acid transfer into primary cells in culture followed by autologous transplantation of the ex vivo transformed cells into the host, either systemically or into a particular organ or tissue.
The term “systemic administration” refers to administration of a composition or agent such as a molecular vaccine as described herein, in a manner that results in the introduction of the composition into the subject's circulatory system or otherwise permits its spread throughout the body. “Regional” administration refers to administration into a specific, and somewhat more limited, anatomical space, such as intraperitoneal, intrathecal, subdural, or to a specific organ. The term “local administration” refers to administration of a composition or drug into a limited, or circumscribed, anatomic space, such as intratumoral injection into a tumor mass, subcutaneous injections, intramuscular injections. One of skill in the art would understand that local administration or regional administration may also result in entry of a composition into the circulatory system.
For accomplishing the objectives of the present invention, nucleic acid therapy would be accomplished by direct transfer of a the functionally active DNA into mammalian somatic tissue or organ in vivo. DNA transfer can be achieved using a number of approaches described below. These systems can be tested for successful expression in vitro by use of a selectable marker (e.g., G418 resistance) to select transfected clones expressing the DNA, followed by detection of the presence of the antigen-containing expression product (after treatment with the inducer in the case of an inducible system) using an antibody to the product in an appropriate immunoassay. Efficiency of the procedure, including DNA uptake, plasmid integration and stability of integrated plasmids, can be improved by linearizing the plasmid DNA using known methods, and co-transfection using high molecular weight mammalian DNA as a “carrier”.
The DNA molecules encoding the fusion polypeptides of the present invention may be packaged into retrovirus vectors using packaging cell lines that produce replication-defective retroviruses, as is well-known in the art (see, for example, Cone, R. D. et al., Proc. Natl. Acad. Sci. USA 81:6349-6353 (1984); Mann, R. F. et al., Cell 33:153-159 (1983); Miller, A. D. et al., Molec. Cell. Biol. 5:431-437 (1985),; Sorge, J., et al., Molec. Cell. Biol. 4:1730-1737 (1984); Hock, R. A. et al., Nature 320:257 (1986); Miller, A. D. et al., Molec. Cell. Biol. 6:2895-2902 (1986). Newer packaging cell lines which are efficient an safe for gene transfer have also been described (Bank et al., U.S. Pat. No. 5,278,056.
This approach can be utilized in a site specific manner to deliver the retroviral vector to the tissue or organ of choice. Thus, for example, a catheter delivery system can be used label, E G et al., Science 244:1342 (1989)). Such methods, using either a retroviral vector or a liposome vector, are particularly useful to deliver the nucleic acid to be expressed to a blood vessel wall, or into the blood circulation of a tumor.
Other virus vectors may also be used, including recombinant adenoviruses (Horowitz, M S, In: Virology, Fields, B N et al., eds, Raven Press, NY, 1990, p. 1679; Berkner, K L, Biotechniques 6:616-29, 1988; Strauss, S E, In: The Adenoviruses, Ginsberg, H S, ed., Plenum Press, NY, 1984, chapter 11), herpes simplex virus (HSV) for neuron-specific delivery and persistence. Advantages of adenovirus vectors for human gene delivery include the fact that recombination is rare, no human malignancies are known to be associated with such viruses, the adenovirus genome is double stranded DNA which can be manipulated to accept foreign genes of up to 7.5 kb in size, and live adenovirus is a safe human vaccine organisms. Adeno-associated virus is also useful for human therapy (Samulski, R J et al., EMBO J. 10:3941, 1991) according to the present invention.
Another vector which can express the DNA molecule of the present invention, and is useful in the present therapeutic setting is vaccinia virus, which can be rendered non-replicating (U.S. Pat. Nos. 5,225,336; 5,204,243; 5,155,020; 4,769,330; Sutter, G et al., Proc Natl Acad Sci USA 89:10847-51, 1992; Fuerst, T R et al., Proc. Natl. Acad. Sci. USA 86:2549-53, 1992; Falkner F G et al.; Nucl. Acids Res 15:7192, 1987; Chakrabarti, S et al., Mol Cell Biol 5:3403-9, 1985). Descriptions of recombinant vaccinia viruses and other viruses containing heterologous DNA and their uses in immunization and DNA therapy are reviewed in: Moss, B, Curr Opin Genet Dev 3:86-90, 1993; Moss, B, Biotechnol. 20:345-62, 1992); Moss, B, Curr Top Microbiol Immunol 158:25-38, 1992; Moss, B, Science 252:1662-7, 1991; Piccini, A et al., Adv. Virus Res 34:43-64, 1988; Moss, B et al., Gene Amplif Anal 3:201-13, 1983).
In addition to naked DNA or RNA, or viral vectors, engineered bacteria may be used as vectors. A number of bacterial strains including Salmonella, BCG and Listeria monocytogenes(LM) (Hoiseth et al., Nature 291:238-239, 1981; Poirier, T P et al., J. Exp. Med. 68:25-32, 1988); Sadoff, J C et al., Science 240:336-8, 1988; Stover, C K et al., Nature 351:456-60, 1991; Aldovini, A et al., Nature 351:479-82, 1991; Schafer, R, et al., J Immunol 149:53-9 (1992); Ikonomidis, G et al., J Exp Med 180:2209-18, 1994). These organisms display two promising characteristics for use as vaccine vectors: (1) enteric routes of infection, providing the possibility of oral vaccine delivery; and (2) infection of monocytes/macrophages thereby targeting antigens to professional APCs.
In addition to virus-mediated gene transfer in vivo, physical means well-known in the art can be used for direct transfer of DNA, including administration of plasmid DNA (Wolff et al., 1990, supra) and particle-bombardment mediated gene transfer (Yang, N-S, et al., Proc Natl Acad Sci USA 87:9568, 1990; Williams, R S et al., Proc Natl Acad Sci USA 88:2726, 1991; Zelenin, A V et al., FEBS Lett 280:94, 1991; Zelenin, A V et al., FEBS Lett 244:65, 1989); Johnston, S A et al., In Vitro Cell Dev Biol 27:11, 1991). Furthermore, electroporation, a well-known means to transfer genes into cell in vitro, can be used to transfer DNA molecules according to the present invention to tissues in vivo (Titomirov, A V et al., Biochim Biophys Acta 1088:131, 1991).
“Carrier mediated gene transfer” has also been described (Wu, C H et al., J Biol Chem 264:16985, 1989; Wu, G Y et al., J Biol Chem 263:14621, 1988; Soriano, P et al., Proc Nat. Acad Sci USA 80:7128, 1983; Wang, C-Y et al., Pro. Natl Acad Sci USA 84:7851, 1982; Wilson, J M et al., J Biol Chem 267:963, 1992). Preferred carriers are targeted liposomes (Nicolau, C et al., Proc Natl Acad Sci USA 80:1068, 1983; Soriano et al., supra) such as immunoliposomes, which can incorporate acylated mAbs into the lipid bilayer (Wang et al., supra). Polycations such as asialoglycoprotein/polylysine (Wu et al., 1989, supra) may be used, where the conjugate includes a target tissue-recognizing molecule (e.g., asialo-orosomucoid for liver) and a DNA binding compound to bind to the DNA to be transfected without causing damage, such as polylysine. This conjugate is then complexed with plasmid DNA of the present invention.
Plasmid DNA used for transfection or microinjection may be prepared using methods well-known in the art, for example using the Quiagen procedure (Quiagen), followed by DNA purification using known methods, such as the methods exemplified herein.
Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.
EXAMPLE 1
Materials and Methods
Plasmid DNA Constructs and DNA Preparation:
The production of the following vectors have been described: pcDNA3-E7, pcDNA3-Sig/E7/LAMP-1, pcDNA3-E7/HSP70, pcDNA3-CRT/E7, pDNA3-E7/GFP and pcDNA3-OVA (Kim T W et al., J Clin Invest 112: 109-117, 2003; Cheng W F et al., J Clin Invest 108: 669-678, 2001; Hung C F et al., Cancer Res 61: 3698-3703, 2001; Chen C H et al., Cancer Res 60: 1035-1042, 2000; see also U.S. Pat. No. 6,734,173 and published patent applications WO05/081716, WO05/047501, WO03/085085, WO02/12281C2, WO02/074920, WO02/061113, WO02/09645, and WO01/29233. The plasmid containing a sequence encoding influenza hemagglutinin (HA), pcDNA3-HA, was provided by Dr. Drew Pardoll, Johns Hopkins School of Medicine. The accuracy of these constructs was confirmed by DNA sequencing. DNA was amplified in E. coli DH5α and purified as described in Chen, C H et al., supra).
Preparation (Synthesis) of siRNAs and Transfection:
siRNAs were synthesized using 2′-O-ACE-RNA phosphoramides (Dharmacon, Lafayette, Colo.). The sense and anti-sense strands of siRNA were:















SEQ ID



Gene targeted
siRNA Sequence
NO:







Bak,
5′-UGCCUACGAACUCUUCACCdTdT-3′(sense)
1



beginning
5′-GGUGAAGAGUUCGUAGGCAdTdT-3′(antisense)
2


at nt 310,


 


Bax,
5′-UAUGGAGCUGCAGAGGAUGdTdT-3′(sense)
5


beginning
5′-CAUCCUCUGCAGCUCCAUAdTdT-3′(antisense)
6


at nt 217,


 


Non-specific
5′-NNATTGTATGCGATCGCAGAC-3′
45 


ctrl siRNA









RNAs were deprotected and annealed according to the manufacturer's instruction. Non-specific control siRNA was acquired from Dharmacon.

Dendritic cells—either DC-1 cells or bone marrow-derived DCs (BM-DCs) incubated for 6 days were transfected with Bak and Bax siRNA or control siRNA using Oligofectamine (Invitrogen, Carlsbad, Calif.). 24 to 48 hours later, the transfected cells were used.
Cells:
The HPV-16 E7-expressing murine tumor model, TC-1, has been described previously. In brief, HPV-16 E6, E7, and the ras oncogene were used to transform primary C57BL/6 mouse lung epithelial cells to generate TC-1.
DC-1 cells were generated from the dendritic cell line provided by Dr. Kenneth Rock, University of Massachusetts. With continued passage, subclones of DCs (DC-1) have been generated that can be easily transfected (Kim et al., 2004, supra).
Cells were maintained in RPMI medium (Invitrogen, Carlsbad, Calif.) supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 100 μM non-essential amino acids, 20 mM HEPES, 50 μM β-mercaptoethanol, 100 IU/ml penicillin, 100 μg/ml streptomycin and 10% fetal bovine serum (Gemini Bio-Products, Woodland, Calif.).
An H-2 Db-restricted HPV-16 E7-specific T cell line has also been described previously (Wang, T L et al., Gene Ther 7:726-733, 2000). These cell lines were stimulated weekly with irradiated TC-1 cells and 20 U/ml murine rIL-2 weekly.
Generation of Bone Marrow-Derived DCs (BM-DCs):
BM-DCs were generated from bone marrow (BM) progenitor cells generally as described by Inaba et al. (J Exp Med 176:1693-1702, 1992) with a modification. Briefly, BM cells were flushed from femurs and tibias of 5-8-wk old C57BL/6 mice. Cells were washed twice with RPMI-1640 after lysis of red blood cells, and were resuspended at a density of 106/ml in RPMI-1640 medium supplemented as above, although with 5% fetal bovine serum, and further with 20 ng/ml recombinant murine GM-CSF (PeproTech, Rock Hill, N.J.). The cells were cultured in 24-well plates (1 ml/well) at 37° C. in 5% humidified CO2. Wells were replenished on days 2 and 4 with fresh medium supplemented GM-CSF as above. Cells were harvested after 6 days and subjected to transfection with siRNA.
Western Blot Analysis:
2×105 DC-1 cells were transfected with 300 pmol of the Bak+Bax siRNA or control siRNA in a final volume of 2 ml using Oligofectamine® (Invitrogen, Carlsbad, Calif.) according to vendor's instructions. Fluorescein-labeled siRNA was used to assess the transfection efficiency of DC-1 cells by flow cytometric analysis. Virtually 100% of DC-1 cells were successfully transfected with siRNA. The expression of Bak and Bax pro-apoptotic proteins in DC-1 cells transfected with Bak and/or Bax siRNA was characterized by Western blot analysis using 50 μg of cell lysate from transfected DC-1 cells and anti-Bak and/or anti-Bax mouse mAb (Cell Signaling Technology, Inc., Beverly, Mass.) using a protocol similar to that described previously (Hung et al., 2001, supra).
Measurement of Apoptotic Cells:
As described, 2×105 DC-1 cells were transfected with Bak+Bax siRNA or control siRNA. Two days after transfection, the cells were pulsed with 10 μg/ml E7 peptide (RAHYNIVTF; SEQ ID NO:46) or HA peptide (IYSTVASSL; SEQ ID NO:47) for 2 hours and subsequently incubated with an E7-specific CD8+ T cell line (Wang T L et al., 2000, supra) at different E:T ratios (5, 1, 0.5 and 0.1) for 4 or 20 hrs. Apoptotic DC-1 cells were detected using PE-conjugated rabbit anti-active caspase-3 mAb (BD Pharmingen San Diego Calif.) according to the vendor's protocol. Briefly, cells were harvested and stained with FITC-conjugated anti-CD8 antibody as described previously. The cells were subsequently fixed and permeabilized using the Cytofix/CytopermTM Kit (BD Pharmingen) for 20 minutes at room temperature, and stained with PE-conjugated rabbit-anti-active caspase-3 monoclonal antibody using 20 μl per 106 cells for 60 minutes at room temperature. Following incubation with the antibodies, the cells were washed, resuspended and analyzed by flow cytometric analysis. Analysis was performed on a Becton-Dickinson FACScan with CELLQuest software (Becton Dickinson Immunocytometry System, Mountain View, Calif.). CD8neg cells were gated and active caspase-3-positive DC-1 cells were analyzed to determine the percentage of apoptotic DC-1 cells.
Mice:
C57BL/6 mice (6- to 8-week-old) were purchased from the National Cancer Institute (Frederick, Md.) and maintained under specific pathogen-free conditions in the oncology animal facility of the Johns Hopkins Medical Institutions (Baltimore, Md.). All procedures were performed according to approved protocols and in accordance with recommendations for the proper use and care of laboratory animals.
DNA/siRNA Vaccination:
Gene gun particle-mediated DNA/siRNA vaccination was performed using a helium-driven gene gun (Bio-Rad, Hercules, Calif.) according to the protocol for RNA vaccination provided by the manufacturer, with a slight modification. Briefly, DNA/siRNA-coated gold particles were prepared by combining 25 mg of 1.6 μm gold microcarriers (Bio-Rad), 50 μg of plasmid DNA (50 μl), 5 μg of siRNA (50 μl), and 10 μl of 3M sodium acetate. Isopropyl alcohol (200 μl) was added to the mixture drop-wise while mixing by vortex. The mixture was allowed to precipitate at room temperature for 10 min. The suspension of microcarrier/DNA/siRNA was centrifuged 10,000 rpm for 30 s and washed 3 times in fresh absolute ethanol before resuspending in 3 ml of polyvinylpyrrolidone (0.1 mg/ml; Bio-Rad) in absolute ethanol. The solution was then loaded into 2.5 ft. of Gold-CoatedTM tube (Bio Rad) and allowed to settle for 10 min. The ethanol was gently removed, and the microcarrier/DNA/siRNA suspension was evenly attached to the inside surface of the tube by rotation. The tube was then dried using flowing nitrogen gas at a rate of 0.4 liters/min. The dried tube coated with microcarrier/DNA/siRNA was then cut into 0.5 inch cartridges and stored in a capped dry bottle at 4° C. The DNA/siRNA-coated gold particles (1 μg of DNA and 0.1 μg of siRNA/bullet) were delivered to the shaved abdomens of mice using a helium-driven gene gun (supra) with a discharge pressure of 400 psi. Mice were immunized with 2 μg of the desired pcDNA3 plasmid, including those encoding E7, Sig/E7/LAMP-1, E7/HSP70, CRT/E7, HA, or OVA, mixed with 0.2 μg of Bak+Bax siRNA or control siRNA. The mice were boosted with the same dose 1 wk later.
To determine the effect of Bak+Bax siRNA and/or control siRNA administered during priming and/or boosting phases, mice were primed with 2 μg of pcDNA3-Sig/E7/LAMP-1 co-administrated with 0.2 μg of Bak+Bax siRNA or with control siRNA. Mice were then boosted with 2 μg of pcDNA3-Sig/E7/LAMP-1 co-administrated with 0.2 μg of Bak+Bax siRNA or control siRNA.
DC Immunization:
DC-1 cells or BM-DCs were transfected with the Bak+Bax siRNA or control siRNA as above. Two days later, DC-1 cells or BM-DCs transfected with Bak/Bax siRNA or with control siRNA were incubated with E7 aa49-57 peptide (RAHYNIVTF; SEQ ID NO:46) (10 μg/ml) at 37° C. for 2 hours. The cells were then washed with RPMI-1640/10% FCS and HBBS, and resuspended in HBBS at the final concentration of 5×106/ml (DC-1 cells) or 2×106/ml (BM-DCs). DC-1 cells or BM-DCs were injected s.c. into footpads of mice (100 μl/mouse). One week later, the mice were boosted once with the same dose and immunization regimen.
Intracellular Cytokine Staining (ICCS) and Flow Cytometric Analysis:
Spleen cells were harvested from mice one week after the last vaccination. Prior to ICCS, 4×106 (or 3.5×105) pooled spleen cells from each treatment group were incubated overnight or for about 16 hours with

    (a) 1 μg/ml of E7 peptide (RAHYNIVTF; SEQ ID NO:46), HA (IYSTVASSL; SEQ ID NO:47) (underscored in SEQ ID NO:23), or OVA peptide (SIINFEKL; SEQ ID NO:48), each of which includes an MHC class I epitope, to detect antigen-specific CD8+ T cell precursors; or
    (b) 1 μg/ml of E7 peptide (aa 30-67) containing an MHC class II epitope—DSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRL (SEQ ID NO:49)—for detection of antigen-specific CD4+ T cell precursors. Intracellular IL-4 and IFN-γ staining and flow cytometric analysis were performed as described previously.


In studies of DC-1 or BM-DC vaccination, 3.5×105 pooled spleen cells from each group (see above) were used. GolgiPlug (BD Pharmingen) was added to the culture, and incubated at 37° C. overnight. Cells were then washed once with FACScan® buffer and stained with phycoerythrin-conjugated monoclonal rat antimouse CD8a antibody (clone 53.6.7). Cells were subjected to ICCS using the Cytofix/Cytoperm kit according to the manufacturer's instructions (BD Pharmingen). Intracellular IFN-γ was stained with FITC-conjugated rat antimouse IFN-γ. Analysis of surface markers of untransfected or siRNA-transfected DCs was performed on FACS Calibur and analyzed using CellQuest software (BD Bioscience, San Jose, Calif.). FITC-conjugated mouse mAbs specific for the surface markers CD11c, CD40, CD86, I-Ab, or H-2 Kb/Db (BD Pharmingen) were used.
In Vivo Tumor Protection and Tumor Treatment Experiments:
For tumor protection studies, C57BL/6 mice (5/group) were challenged s.c., with 5×104 TC-1 tumor cells/mouse in the right leg one week after the last vaccination. Mice were monitored for evidence of tumor growth by palpation and inspection twice a week. To evaluate lymphocytes subsets responsible for antitumor effects, in vivo antibody depletion studies were performed using standard methods (e.g., Lin K Y et al., Canc Res 56:21-6, 1996).
For tumor therapy studies, mice were challenged with 1 or 5×104 TC-1 tumor cells/mouse i.v., in the tail vein to simulate hematogenous spread of tumors (Ji et al., supra). Mice were treated three days after tumor challenge with (a) DNA vaccine mixed with siRNA, boosted once after 1 wk and sacrificed on day 42 after the last vaccination or (b) 5×105 E7 peptide-pulsed siRNA-transfected DC-1, boosted once after 1 wk and sacrificed on day 28 after the last immunization. The mean number of pulmonary nodules in each mouse was evaluated by experimenters blinded to sample identity. In vivo tumor protection, antibody depletion, and tumor therapy experiments were performed at least two times to generate reproducible data.
Preparation of CD11c+ Cells from Inguinal Lymph Nodes of Vaccinated Mice:
C57BL/6 mice (5/group) were first primed with pcDNA3-Sig/E7/LAMP1 or control pcDNA3 DNA via gene gun at a dose of 2 μg/mouse. Seven days later, mice received 16 inoculations of non-overlapping gene gun intradermal administration on their abdomens. Gold particles used for each inoculation were coated with 1 μg of pcDNA3-E7/GFP DNA mixed with 0.1 μg of Bak+Bax siRNA or control siRNA. pcDNA3 mixed with Bak+Bax siRNA was used as a negative control.
Inguinal lymph nodes (LN) draining the inoculation site were harvested from vaccinated mice 2 or 5 days after vaccination. CD11c+ cells were enriched from a single cell suspension of isolated LN cells using CD11c (N418) microbeads (Miltenyi Biotec, Auburn, Calif.). Enriched CD11c+ cells were analyzed by forward and side scatter and gated around a population of cells with size and granular characteristics of DCs. GFP+ cells were analyzed by flow cytometry using a protocol described previously (Lappin M B et al., Immunology 98:181-8, 1999). Results are expressed as percent of GFP+ CD11c+ cells among gated monocytes. The percent of GFP+ cells among the gated CD11c+ cells was analyzed by flow cytometry.
In vivo antibody depletion studies were performed using conventional methods as noted above. Depletion was initiated 5 days after priming and terminated at time of LN harvest.
Adoptive Transfer of T Cells and Rapid DC Elimination Assay:
To create two distinctly labeled populations of BM-DCs, different concentrations of the dye carboxyfluorescein (CFSE) were used to label cells. E7-peptide-loaded BM-DCs transfected with either Bax/Bax siRNA or control siRNA were prepared using methods described above. The E7-peptide loaded BM-DCs transfected with control siRNA were labeled with 5 μM CFSE (“high-CFSE”), whereas Bak/Bax siRNA-transfected DCs were labeled with 10-fold lower concentration, 0.5 μM CFSE (“low-CFSE”). A 1:1 mixture of 2.5×105 low CFSE-labeled E7-peptide loaded BM-DCs and 2.5×105 high CFSE-labeled E7-peptide loaded BM-DCs was administered i.v. to C57BL/6 mice three days after adoptive transfer i.v. of 106 E7-specific T cells into the mice. Sixteen hours later, single cell suspensions from the lung and spleen were prepared and analyzed for CFSE content by flow cytometry.
Statistical Analysis:
All results expressed as means±standard errors (SE) are representative of at least two experiments. Results of ICCS with flow cytometric analysis and tumor treatment experiments were evaluated by analysis of variance (ANOVA). Comparisons between individual data points were made using Student's t-test. In tumor protection experiments, the principal outcome of interest was time to tumor development. The event time distributions for different mice were compared using the Kaplan and Meier method and the log-rank statistic. All p values <0.05 were considered significant.
EXAMPLE 2
Transfection with Bak and/or Bax siRNA Leads to Downregulation of Bak and Bax, and Resistance to Apoptotic Cell Death
To determine if the expression of Bak and/or Bax was downregulated in a DC cell line (DC-1) transfected with Bak and/or Bax siRNA, western blot analysis was performed using cell lysate from DC-1 cells, transfected with the various siRNAs. As shown in FIG. 1, the expression of Bak and/or Bax proteins was undetectable in DC-1 cells transfected with Bak and/or Bax siRNA. In contrast, expression of Bak and Bax proteins was detected in DC-1 cells after transfection with control siRNA, the levels of expression being similar to the levels in nontransfected DC-1 cells. The expression of β-actin protein was consistent among all DC-1 cell groups. The kinetics of inhibition of Bak and Bax protein expression by DC-1 cells transfected with Bak+Bax siRNA were examined. As shown in FIG. 2, significant downregulation of Bax and Bak expression was observed 1 day after transfection. No Bak or Bax expression was detectable at days 3, 5, and 7 and some expression was detected at by day 9 (below-normal levels). Expression returned to normal levels by day 11 after transfection.
To determine if DC-1 cells transfected with Bak and/or Bax siRNA could resist CTL-induced apoptosis, E7 peptide-loaded, siRNA-transfected DC-1 cells were incubated with an E7-specific CD8 T cell line and the percentages of apoptotic cells was measured. As shown in FIGS. 3A and 3B, 80-90% of E7 peptide-loaded DC-1 cells transfected with control siRNA were apoptotic by 20 hrs. In comparison, fewer DC-1 cells transfected with Bak+Bax siRNA were apoptotic, particularly at low E:T ratios (T cells to DC-1 cells).
These results show that transfection of DC-1 cells with Bak and/or Bax siRNA downregulates Bak and Bax protein expression, resulting in resistance to the apoptotic effects of activated, antigen-specific CD8+ T cells on the DCs.
EXAMPLE 3
Coadministration of Bax+Bak siRNA with Antigen-Specific DNA Vaccines Significantly Enhances Numbers of Antigen-Specific CD8+ T Cell Precursors in Vaccinated Mice
To determine if the anti-apoptotic action of Bak+Bax siRNA observed in DCs in vitro can be manifest in vivo, Bak+Bax siRNA was coadministered with pcDNA3-E7 intradermally via gene gun. As shown in FIGS. 4 and 5, coadministration of pcDNA3-E7 with Bak and/or Bax siRNA significantly enhanced the E7-specific CD8+ T cell response (by at least 10-fold) in vaccinated mice, compared to coadministration of pcDNA3-E7 with control siRNA.
To determine if this result was obtained using other antigens, pcDNA3-HA and pcDNA3-OVA plasmids were coadministered with Bak+Bax siRNA. As shown in FIGS. 6-7, the coadministration of pcDNA3-HA or pcDNA3-OVA with Bak+Bax siRNA significantly enhanced the HA- and OVA-specific CD8+ T cell response in vaccinated mice, compared to coadministration of the antigen vectors with control siRNA. Thus Bak and/or Bax siRNA significantly enhance antigen-specific CD8+ T cell-mediated immune responses when coadministered with antigen-encoding DNA vaccines.
EXAMPLE 4
Co-Administration of Bak+Bax siRNA with an E7-Specific DNA Vaccine Significantly Enhances Antitumor Effects Against an E7-Expressing Target Tumor Cell Line
To determine if the observed enhancement of E7-specific T cell-mediated immunity described above can manifest itself in E7-specific antitumor effects, an in vivo tumor protection experiment was performed using E7-expressing TC-1 tumor cells. As shown in FIG. 8, all mice receiving E7 DNA mixed with Bak+Bax siRNA remained tumor-free for 35 days after TC-1 challenge. In contrast, all of the mice receiving E7 DNA with control siRNA or pcDNA3 (negative control for antigen) combined with Bak+Bax siRNA developed tumors by day 10.
An in vivo antibody depletion experiment was conducted to determine which subsets of lymphocytes were responsible for the anti-tumor effects. As shown in FIG. 9, 100% of mice depleted of CD8+ T cells grew tumors within 10 days after TC-1 challenge. In contrast, 100% of the mice depleted of CD4+ T cells or NK cells remained tumor-free 35 days after TC-1 challenge (as with the “non-depleted mice discussed above). It was concluded that CD8+ T cells are needed for the antitumor effects induced by the combination of a DNA vaccine and Bak+Bax siRNA.
An in vivo tumor therapy experiment was performed using a model of hematogenous spread of tumors to the lungs (Ji et al., supra). As shown in FIG. 10, mice immunized with E7 DNA combined with Bak+Bax siRNA exhibited the fewest pulmonary tumor nodules (p<0.005) compared to mice vaccinated with E7 DNA combined with control siRNA, or pcDNA3 (no antigen) combined with Bak+Bax siRNA. Taken together, these results indicate that vaccination with the combination of E7 DNA with Bak+Bax siRNA leads to potent protective and therapeutic effects against E7-expressing TC-1 tumor cells.
EXAMPLE 5
Combined Application of Anti-Apoptotic Bak+Bax SiRNA and an Intracellular Targeting Strategy Enhances Antigen-Specific T Cell-Mediated Immune Responses
To assess the effect of coadministration of Bak+Bax siRNA with DNA encoding E7 linked to an DNA encoding an PP such as an intracellular targeting molecule, mice were vaccinated with either Sig/E7/LAMP-1 DNA, HSP70/E7 DNA, or CRT/E7 DNA each combined with either (i) Bak+Bax siRNA or (ii) control siRNA. As shown in FIGS. 11-12, coadministration of Bak+Bax siRNA with pcDNA3 encoding Sig/E7/LAMP-1, HSP70/E7, or CRT/E7 resulted in increased numbers of IFN-γ-expressing E7-specific CD8+ T cell precursors compared to coadministration of each of these three constructs with control siRNA. Among these intracellular targeting strategies, mice vaccinated with pcDNA3-Sig/E7/LAMP-1 combined with Bak+Bax siRNA displayed the greatest increase in the number E7-specific CD8+ T cell precursors (about a 19-fold). Thus, administration of Bak+Bax siRNA can be combined with any of the intracellular targeting strategies (using any IPP that itself can potentiate responses over those of DNA encoding antigen alone) to further enhance the potency of a DNA vaccine. Of the Antigen/IPP fusions tested, immunity was enhanced the most when pcDNA3-Sig/E7/LAMP-1 was combined with Bak+Bax siRNA.
The ability of the Sig/E7/LAMP-1 targeting strategy to enhance antigen presentation to CD4+ T lymphocytes is achieved through targeting of expressed antigen to endosomal/lysosomal compartments, important loci for the MHC class II antigen presentation pathway (Wu T C et al., Proc Natl Acad Sci USA 92:11671-5, 1995). As shown in FIGS. 13-14, vaccination with pcDNA3-Sig/E7/LAMP-1 combined with Bak+Bax siRNA generated significantly more E7-specific CD4+ Th1 cells and similar numbers of E7-specific CD4+ Th2 cells when compared to vaccination with the same immunogen plus control siRNA. These results show that coadministration of Sig/E7/LAMP-1 DNA with Bak+Bax siRNA elicits an immune response mediated predominantly by E7-specific CD4+ Th1 cells.
EXAMPLE 6
Co-Administration with Anti-Apoptotic Bax+Bak siRNA Improves Survival of DNA-Transfected DCs in Inguinal Lymph Nodes of Mice Vaccinated with E7/GFP DNA
Mice were primed with pcDNA3-Sig/E7/LAMP-1 to generate sufficient E7-specific CD8+ T cells for testing of the anti-apoptotic ability of Bak+Bax siRNA in E7/GFP-expressing DCs. pcDNA3 (plasmid only) was the negative control. One week later, mice were treated via gene gun with pcDNA3-E7/GFP DNA plus either Bax+Bak siRNA or control siRNA. As shown in FIGS. 15-16, two days after vaccination. Control groups primed with pcDNA3 showed no significant difference in the percentages of GFP+ CD11c+ DCs between mice that received Bak+Bax siRNA and in that received siRNA. In comparison, in mice primed with the DNA immunogen pcDNA3-Sig/E7/LAMP-1, a significant decrease was observed in the percentage of GFP+ CD11c+ DCs detected in mice receiving control siRNA vs. the percentage of GFP CD11c+ DCs in mice administered Bak+Bax siRNA. Five days after vaccination with pcDNA3-E7/GFP, a similar, albeit weaker trend was observed in mice primed with Sig/E7/LAMP-1.
Assays for apoptotic GFP+ CD11c+ DCs were performed by staining cells for activated caspase-3 followed by flow cytometry. More than 90% of GFP+ CD11c+ DCs were caspase-3 negative, indicating that these cells were not apoptotic (not show). Thus, these results show that co-administration of anti-apoptotic Bak+Bax siRNA with the DNA immunogen E7/GFP protects DNA-transfected DCs from being killed by E7-specific CD8+ T cells generated as a result of antigen-specific priming (by pcDNA3-Sig/E7/LAMP-1).
An antibody depletion experiment confirmed that CD8+ T cells were responsible for the induction of apoptosis in GFP+ CD11c+ DCs. As shown in FIG. 17, the percentages of GFP+ CD11+ DCs in the inguinal LNs of mice depleted of CD8+ T cells were similar in mice administered Bak+Bax siRNA compared to mice administered control siRNA. In comparison, percentages of GFP+ CD11 cells in the inguinal LNs of mice depleted of CD4+ T cells, NK cells, or control mice (no depletion) were significantly lower in mice receiving control siRNA compared to mice receiving Bak+Bax siRNA (p<0.005). Thus, CD8+ T cells are responsible for the induction of apoptosis in antigen-expressing DCs in the draining LNs of vaccinated mice.
EXAMPLE 7
Co-Administration of Bak+Bax siRNA with DNA Vaccines During Boosting Elicits a Stronger Antigen-Specific CD8+ T Cell Response than Co-Administration During Priming
The results shown in FIGS. 15-18, indicate that the anti-apoptotic siRNA strategy is most critical for prolonging DC life when a pre-existing active antigen-specific CD8+ T cell population is present; this occurs in the boosting phase of the DNA vaccination protocols used here.
To determine whether prolonging the life of antigen-presenting DCs has more of an impact during the priming or the boosting phases of the vaccination process, pcDNA3-Sig/E7/LAMP-1 was co-administered with Bak+Bax siRNA or control siRNA during the priming or during the boosting phases. As shown in FIGS. 19-20, mice administered Bak+Bax siRNA during the priming and boosting phases generated the greatest number of E7-specific CD8+ T cell precursors when compared to the other vaccination groups. Administration of Bak+Bax siRNA during the boosting phase resulted in a markedly higher number of E7-specific CD8+ T cells than administration during the priming phase (p=0.002). These results show that prolonging the life of antigen-expressing DCs via administration of siRNA during the boosting phase has a greater impact on the (clonal) expansion of antigen-specific T cells.
EXAMPLE 8
Discussion of Examples 2-7
In vivo delivery of siRNA to target cells represents a significant challenge. Considerable endeavors have been devoted to efficient delivery of siRNA to specific cell types or organs in vivo (Song E et al., Nat Med 9:347-51, 2003). So far, these endeavors have met with only limited success (for a review, see Wall N R et al., Lancet 362:1401-3, 2003). The present inventors have shown that intradermal delivery to APCs via gene gun is an effective system for delivery of siRNA into professional antigen-presenting cells, allowing evaluation of siRNA-based strategies to modify DCs. Thus, the present work is the first to use intradermal delivery of siRNA to DCs and permits investigation of the properties of antigen-expressing DCs in vivo.
The encouraging results reported above indicate that modifying the function of DCs in vivo using siRNA technology targeting other key pro-apoptotic proteins, such as caspases 3, 6, 7, 8, or 9, should also enhance DNA vaccine potency. Furthermore, according to this invention, a combination of more than one type of siRNA targeting multiple pro-apoptotic proteins within the extrinsic and intrinsic apoptotic pathways is useful to induce even greater resistance to apoptotic stress in transfected DC-1 cells. This should result in greater numbers of viable, functional antigen-expressing DCs in the LNs draining a site of immunization in effectively primed mice. Other cell surface molecules such as PD-L1 and PD-L2 (Khoury S J et al., Immunity 20:529-38, 2004; Carreno B M et al., Annu Rev Immunol 20:29-53, 2002) and/or cytokines, such as IL4 and IL-10 (Li-Weber M et al., Nat Rev Immunol 3:534-43, 2003; Moore K W et al., Annu Rev Immunol 19 683-765, 2001) expressed by DCs cells suppress T cell responses. Expression of these molecules can be silenced by the siRNA technology to enhance antigen specific immune responses and the resultant antitumor effects.
Disclosed above is a significant increase in the number of GFP-positive DCs in the draining LNs of vaccinated mice after coadministration of pcDNA3-E7/GFP with Bak+Bax siRNA, compared to coadministration of pcDNA3-E7/GFP and control siRNA. This increase is likely due to enhanced DC survival mediated by Bak+Bax siRNA, rather than an influence on migration of DCs cells due to some nonspecific siRNA effect. This is so because coadministration of pcDNA3-E7/GFP with control siRNA did not produce similar effects. Previous observations by the present inventors and colleagues using DNA-encoding anti-apoptotic proteins (Kim T W et al., J Clin Invest, 2003, supra) support such a notion. DNA vaccines encoding antigen were coadministered with DNA encoding BCL-xL to prolong the lives of transfected DCs. While co-administration to mice of DNA encoding antigen with DNA encoding BCL-xL led yielded increased number of antigen-expressing DCs in the draining LNs, coadministration of the same immunogen with DNA encoding mutant BCL-xL with minimal mutations in a region critical to anti-apoptotic function, failed to lead to such an increase. Thus, the increase in GFP-positive DCs in the draining LNs after co-administration of Bak+Bax siRNA discussed above can be ascribed to changes in survival of DCs.
The increased number of antigen-expressing DCs in the LNs following the coadministration of Bak+Bax siRNA can contribute to increased numbers of E7-specific CD8+ T cells through multiple mechanisms. Not only do antigen-expressing DCs provide signals to trigger proliferation and expansion of antigen-specific T cells, but they also can provide necessary signals that reduce T cell apoptotic death. Normally, DC death leads to decreasing interaction between APCs and lymphocytes, causing T cells to downregulate anti-apoptotic molecules and potentially upregulate pro-apoptotic molecules (Opferman et al., supra). This process would naturally lead to a decline in number of activated antigen-specific CD8+ T cells. The continued survival of antigen-expressing DCs thanks to siRNA-mediated silencing of pro-apoptotic molecules would provide the necessary signals to prevent this decline. Other explanations for enhanced T cell responses include qualitative changes in antigen-expressing DCs as a result of vaccination together with Bak+Bax siRNA administration. The present inventors have observed that antigen-expressing DCs transfected with Bak+Bax siRNA could activate antigen-specific CD8+ T cells more efficiently than DCs transfected with control siRNA (not shown). Thus, the anti-apoptotic function mediated by Bak+Bax siRNA may modify the quantity and quality of DCs, thereby leading to enhanced T cell activation.
The present results show that prolonging the life of antigen-expressing DCs during the boosting phase is important for clonal expansion of antigen-specific T cells. Killing of antigen-expressing DCs is a natural process that regulates clonal expansion of antigen-specific CD8+ T cells. Pre-existing antigen-specific CD8+ T cells in draining LNs can lyse antigen-expressing DCs, limiting clonal expansion (Ritchie D S et al., J Immunol Meth 246:109-17, 2000; Hermans I F et al., J Immunol 164:3095-3101, 2000). Such CD8+ T cell-mediated lysis of DCs is more significant during the boosting phase of vaccination than during the priming phase, due to the increased number of antigen-specific CD8+ T cells that were elicited by the priming. Therefore, while prolonging the lives of antigen-expressing DCs during priming and boosting leads to the strongest clonal expansion of antigen-specific CD8+ T cells, it is during the boosting phase that this effect on DCs contributes most to T cell expansion.
The Bak and Bax siRNA technology can also be extended to the treatment of DCs ex vivo for subsequent in vivo use. As disclosed above, E7 peptide-pulsed DC-1 cells transfected with Bak+Bax siRNA were more resistant to killing by E7-specific CD8+ T cells than were DC-1 cells transfected with control siRNA.
Furthermore, as described in the Examples below, vaccination with E7 peptide-pulsed DC-1 cells transfected with Bak+Bax siRNA leads to significantly higher numbers of E7-specific CD8+ T cells compared to vaccination with antigen-pulsed DC-1 cells transfected with control siRNA. Thus, the potency of DC-based vaccines prepared ex vivo can be further enhanced by the specific targeting of key pro-apoptotic proteins, such as Bak and Bax, using siRNA.
In summary, the targeting of Bak+Bax siRNA with DNA vaccines (encoding antigen) to DCs in vivo represents an innovative approach to enhancing DNA vaccine potency. In addition, the use of siRNA alleviates safety concerns associated with the use of DNA vaccines encoding anti-apoptotic proteins. Not only does gene gun delivery of siRNA to DCs result in prolonged DC life, but it also avoids concerns for oncogenicity associated with DNA encoding anti-apoptotic proteins. Further safety is achieved by using detoxified (mutant) forms of the HPV antigens E7 or E6 as disclosed above. Indeed no gross anatomical or histological changes were observed in the vital organs of vaccinated mice compared to non-vaccinated mice, alleviating concerns about the induction of autoimmunity that as a sequela of prolonging DC life. Thus, the strategy of using siNA to silence pro-apoptotic proteins, as exemplified with siRNA targeting Bak+Bax useful in the clinical arena where enhanced DNA vaccine potency is a desirable goal in improving the immunologic control of cancer or infectious disease.
EXAMPLE 9
Examples 9-15 Incorporate by Reference Peng S et al., Hum Gene Ther 16:584-93 (2005 May)
Transfected of Dendritic Cells with Bak/Bax siRNA Abolishes Expression of Bak and Bax Proteins
Western blot analysis was performed to examine in DC-1 cells (a murine DC line) the effects of transfection with Bak/Bax siRNA on expression of Bak and Bax proteins. As shown in FIG. 21, lysates from DC-1 cells transfected with Bak/Bax siRNA showed significant reduction in the expression of Bak and Bax proteins 24 and 48 hrs after transfection. In contrast, when transfection with control siRNA was done, the expression of Bak and Bax did not differ from that in non-transfected DC-1 cells. Analysis of β-actin expression in transfected DCs confirmed that equal amounts of cell lysates had been loaded in all the Western blots. These results indicate that transfection of DC-1 cells with Bak/Bax siRNA abolishes Bak and Bax protein expression during the intervals examined.
DC-1 cells transfected with Bak and/or Bax siRNA can resist CTL-induced apoptosis. E7-loaded, siRNA-transfected, DC-1 cells were incubated with an E7-specific CD8+ T cell line. These DC-1 cells resisted killing by E7-specific CD8+ T cells in vitro. Taken together, these results show that transfection of DC-1 cells with Bak and/or Bax siRNA downregulates Bak and Bax protein expression, a consequence of which is resistance to apoptosis caused by activated antigen-specific CD8+ T cells in DCs.
EXAMPLE 10
Vaccination with E7 Peptide-Loaded DCs Transfected with Bak/Bax siRNA Leads to a Significant Increase in E7-Specific IFN-γ+ CD8+ T Cell Precursors
To determine whether vaccination with E7 peptide-loaded DCs transfected with Bak/Bax siRNA could enhance the generation of E7-specific IFN-γ+ CD8+ T cell precursors in mice, ICCS and flow-cytometry analysis was performed on spleen cells from mice vaccinated with the various DC-1 cells. As shown in FIGS. 22-23, mice vaccinated with E7-loaded DCs transfected with Bak/Bax siRNA exhibited an  ̃5.4-fold increase in the number of E7-specific IFN-γ+ CD8+ T cells (655±21) compared to mice vaccinated with E7-loaded DCs transfected with control siRNA (121±5) (which were similar to the number of E7-specific CD8+ T cells induced by E7-loaded DC-1 that remained untransfected). Thus, administration of DCs that are transfected with Bak/Bax siRNA is markedly more immunogenic than the use of control DCs when measured by the number of E7-specific IFN-γ+ CD8+ T cells that are generated in vivo.
EXAMPLE 11
Vaccination with E7 Peptide-Loaded BM-DCs Transfected with Bak/Bax siRNA Increases E7-Specific IFN-γ+ CD8+ T Cell Precursors
It was important to determine if the Bak/Bax siRNA technology also works with a more “physiological” source of DCs, not derived from an immortalized cell line, since the former would be a more appropriate source of cells for clinical use. For this purpose bone marrow-derived DCs (BM-DCs) were tested—after loading with E7 peptide and transfection with either Bak/Bax siRNA or control siRNA. To determine E7-specific CD8+ T cell precursors in vaccinated mice, ICCS followed by flow cytometry analysis was performed. As shown in FIGS. 24-25, mice vaccinated with E7-peptide-loaded BM-DCs transfected with Bak/Bax siRNA exhibited a  ̃2.2-fold increase in the number of E7-specific IFN-γ+ CD8+ T cells (4706±78.5) compared to mice vaccinated with E7 peptide-loaded DCs transfected with control siRNA (2210±134.3) (p≦0.002). Thus, the Bak/Bax siRNA technology can also be applied to BM-DCs to enhance their potency as immunogens.
EXAMPLE 12
Vaccination with E7-Loaded DCs Transfected with Bak/Bax siRNA Generates Stronger Antitumor Effects than E7-Loaded DCs Transfected with Control siRNA
To determine whether the observed increase in the number of E7-specific CD8+ T cell precursors translated into a stronger E7-specific antitumor effect, an in vivo tumor protection experiment was carried out using the TC-1 system (supra). As shown in FIG. 26, 100% of mice receiving E7 peptide-loaded DCs transfected with either control siRNA or Bak/Bax siRNA remained tumor-free for 30 days after a s.c. challenge with TC-1 cells, whereas non-vaccinated mice developed tumors within 10 days of tumor challenge. Therefore, vaccination with E7 peptide-loaded DC-1 transfected with either Bak/Bax siRNA or control siRNA elicited protective antitumor immunity against challenge by an E7-expressing tumor. The in vivo tumor protection model failed to distinguish between the use of Bak/Bax targeted and control siRNA.
To extend the comparison, an in vivo tumor trial was performed using a more stringent lung tumor metastasis model in which TC-1 tumor cells were delivered i.v. Thus, mice were first challenged with the TC-1 tumor cells i.v. (tail vein) followed by treatment with E7-peptide loaded DC-1 cells transfected either with Bak/Bax siRNA or with control siRNA. Mice were sacrificed 28 days after the tumor challenge and the growth of pulmonary nodules was examined. As shown in FIG. 27, mice treated with E7-peptide loaded DCs transfected with Bak/Bax siRNA demonstrated the lowest number of pulmonary nodules (2.2±0.84) compared to mice treated with E7-peptide loaded DCs transfected with control siRNA (24.8±5.89), or the naïve control group (103±12.29; p<0.001; Student's t test). Thus, vaccination with E7-loaded DCs transfected with Bak/Bax siRNA generates a markedly better highly significant therapeutic effect than vaccination with E7-loaded DCs transfected with control siRNA.
EXAMPLE 13
E7 Peptide-Loaded DCs Transfected with Bak/Bax siRNA Survive Longer In Vivo than E7 Peptide-Loaded DCs Transfected with Control siRNA
To determine if transfection with Bak/Bax siRNA improves the survival of E7-peptide loaded DCs in vivo, two distinct groups of BM-DC cells loaded with carboxyfluorescein (CFSE)-labeled E7 peptide and transfected with different siRNAs were first created. E7 peptide-loaded BM-DCs transfected with control siRNA were labeled with a higher concentration of CFSE (5 μM), while Bak/Bax siRNA-transfected BM-DCs were labeled with a lower concentration of CFSE (0.5 μM). The relative levels of CFSE in these two distinctly CFSE-labeled E7 peptide-loaded BM-DCs were characterized by flow cytometry (FIG. 28A). Mice were then challenged with 106 E7-specific T cells/mouse i.v. Three days later, a mixture of 2.5×105 low CFSE-labeled BM-DCs and 2.5×105 of high CFSE-labeled BM-DCs were injected i.v. into each challenged mouse. Sixteen hours later, flow-cytometry analysis was performed to characterize the ratio of low CFSE-labeled BM-DCs to high CFSE-labeled BM-DCs using cells collected from the spleen and lungs of challenged mice. As shown in FIG. 28B, a significantly higher number of low CFSE-labeled BM-DCs was observed ( ̃3.7-fold), compared to the number of high-CFSE-labeled BM-DCs. These results show that transfection of E7 peptide-loaded BM-DCs with Bak/Bax siRNA can prolong DC life in vivo, and resulting in a higher number of E7-peptide loaded BM-DCs.
EXAMPLE 14
E7 Peptide-Loaded DC-1 Cells Transfected with Bak/Bax or Control siRNA Express Similar Levels of CD11c, CD40, CD86, MHC I and MHC II
The significant therapeutic effect generated by vaccination with E7-peptide-loaded DCs transfected with Bak/Bax siRNA could have been due to changes in the expression of molecules important for antigen presentation in DCs, such as CD11c, CD40, CD86, MHC I, and MHC II. Flow cytometric analyses were done to determine the expression levels of these molecules in cells of an E7 peptide-loaded DC-1 cell line transfected with Bak/Bax siRNA, control siRNA or in non-transfected DC-1 cells. As shown in FIG. 29, there was no significant change in the expression of any of the cell surface molecules evaluated among the E7 peptide-loaded DC-1 cells. A similar study was done with BM-DCs. Again, no significant changes in the expression of these molecules were observed among the E7 peptide-loaded BM-DCs transfected with the various siRNA constructs (not shown). Taken together, these results indicated that the expression of CD11c, CD40, CD86, MHC class I, and MHC class II proteins on the surface of DCs that has been E7-peptide loaded were are not affected by Bak/Bax siRNA.
EXAMPLE 15
Discussion of Examples 9-14
This set of studies demonstrated that vaccination with E7 peptide-loaded DCs transfected with Bak/Bax siRNA generated enhanced E7-specific T cell-mediated immune responses and antitumor effects in vivo. Transfection of DCs with Bak/Bax siRNA inhibited apoptotic cell death of DCs mediated by T cells, leading to prolongation of DC survival and resulting in an improved DC-based vaccine.
Previous studies showed that DC life can be efficiently prolonged in vivo through transfection of DCs with DNA encoding antiapoptotic proteins (Kim, T W et al., J Clin Invest 112:109-17, 2003b). This technique, however, has raised concerns regarding potential oncogenic transformation as a result of overexpression of these antiapoptotic proteins. Antiapoptotic proteins such as the Bcl-2 family are known to be over-expressed in some cancers and therefore have been implicated as contributors to cellular immortalization (Lebedeva, I., Cancer Res. 60:6052-60, 2000). The modification of DCs using siRNA targeting Bak and Bax proteins alleviates many of these concerns. Due to the transient nature of siRNA-mediated silencing of target genes as well as the fact that RNA-based strategies carry no concerns for integration and permanent genetic change, transfection of DCs with Bak/Bax siRNA represent a potentially safe and effective method for enhancing DC-based vaccine potency by prolonging DC life without risk of DC immortalization.
Results employing this DC-based vaccine prepared ex vivo using siRNA technology targeting Bak and Bax are consistent with results of modifying DCs using Bak/Bax siRNA vaccination in vivo. Examples 2-8 describe intradermal gene-gun co-administration of DNA encoding antigen with Bak/Bax siRNA to prolong the life of antigen-expressing DCs in vivo. Mice vaccinated with DNA coadministered with Bak/Bax siRNA manifest significantly enhanced antigen-specific CD8+ T cell-mediated immune responses and antitumor effects compared to mice vaccinated with DNA coadministered with control siRNA. Taken together, these results indicate that siRNA technology as described herein can be used to modify DCs either ex vivo or in vivo to improve vaccine potency.
Modification of a DC-based vaccine with Bak/Bax siRNA as well as siRNA targeting other key pro-apoptotic proteins will further enhance DC-based vaccine potency. Since Bak/Bax siRNA only affects the intrinsic granzyme B/perforin-mediated apoptotic pathway, a combination of siRNAs targeting key pro-apoptotic proteins in the intrinsic granzyme B/perforin pathway along with siRNAs targeting other key pro-apoptotic proteins in the extrinsic Fas-mediated apoptotic pathway will likely result in stronger resistance to killing of the transfected DCs by T cells in vivo. As discussed above, caspase-8, a caspase that induces the proteolysis of a cascade of effector caspases leading to apoptotic cell death, is an excellent candidate protein to target for RNAi. Other caspases involved in cell apoptosis that could serve as targets for siRNA include caspase 9 and caspases 3, 6, and 7. Thus, a DC-based vaccination strategy employing siRNAs targeting key pro-apoptotic proteins in both the intrinsic and extrinsic apoptotic pathways, for example, antigen-loaded DCs transfected with Bak/Bax siRNA and caspase-8 siRNA, are expected to result in even greater enhancement of DC resistance to endogenous T cell-mediated killing, and this will result in improved T cell immune response and antitumor effects in vivo.
In the present study, antigen was loaded onto DCs by pulsing DCs with antigenic peptides. This Bak/Bax siRNA technology could also be applied to DCs prepared through other antigen-loading strategies, including viral vector-mediated, protein-mediated, RNA-mediated, and DNA-mediated transfection strategies. Viral vector-mediated strategies show highly efficient transfection of DCs, but have a limited “life expectancy”, whereas DNA-mediated strategies are easily prepared but have a lower transfection efficiency in DCs. Thus, both viral vector-mediated and DNA-mediated strategies to deliver antigens to DCs benefit from the use of Bak/Bax siRNA technology. It will be possible to further enhance the potency of DC-based vaccines through the combined use of Bak/Bax siRNA as an antiapoptotic strategy with other vaccine enhancement strategies, such as the intracellular targeting of antigen inside DCs using various IPPs for more efficient intracellular processing. According to the present invention DNA-mediated strategies of DC-based vaccination employ DCs transfected with Bak/Bax siRNA co-administered with DNA plasmids comprising a DNA sequence encoding an antigen peptide linked to DNA encoding an IPP such as HSP70. The IPP targets the antigen for intracellular processing within the DCs, thereby resulting in increased expression/presentation of the antigen on the DC surface, while transfection by Bak/Bax siRNA would prolong the life of the DCs. The combination of these effects will increase T cell activation and result in an enhanced antigen-specific immune response.
In summary, antigen-loaded DCs transfected with Bak/Bax siRNA as a DC-based vaccine strategy offers an effective and potentially safer approach for prolonging the life of DCs and increasing the potency of DC-based vaccines than transfection of DCs in vivo with DNA encoding antiapoptotic proteins. Administering antigen-peptide loaded DCs transfected with Bak/Bax siRNA prolongs the life of transfected DCs and enhances antigen-specific CD8+ T cell activity, as well as eliciting strong antitumor effects in vivo. Thus, a DC-based vaccine strategy incorporating antigen-loaded DCs transfected with Bak/Bax siRNA shows potential is readily adaptable to clinical use with DC-based vaccines for the control of cancer and infectious disease.
EXAMPLE 15
Enhancing DC Vaccine Potency by Combining a BAK/BAX siRNA-Mediated Antiapoptotic Strategy to Prolong Dendritic Cell Life with an Intercellular Strategy to Target Antigen to Lysosomal Compartments
(This Example incorporates by reference Kang et al. (January 2007) Int. J. Cancer 120:1696)
This study is the new vaccine strategy combining retrovirally-transduction of endogenously expressing Sig/E7/LAMP-1 and transfection with BAK/BAX siRNA in DCs. Our study may offer a promising strategy for improving DC vaccine potency.
Abstract
Dendritic cell (DC)-based vaccines have become important in immunotherapeutics as a measure for generating anti-tumor immune responses. We have previously demonstrated that linkage of the antigen gene to a lysosomal targeting signal, a sorting signal of the lysosome-associated membrane protein type 1 (LAMP-1), enhances the potency of DC-based vaccines. DCs have a limited life span, hindering their long-term ability to prime antigen-specific T cells. In this study, we attempted to further improve the potency of a DC vaccine that targets human papilloma virus 16 (HPV16) E7 to a lysosomal compartment (DC-Sig/E7/LAMP-1) by combining a strategy to prolong DC life. We show that small interfering RNA-targeting Bak and Bax proteins can be used to allow transfected DCs to resist being killed by T cells. This is done by downregulating these proapoptotic proteins, which have been known as so-called gate keepers in mitochondria-mediated apoptosis. DCs expressing intact E7 or Sig/E7/LAMP-1 became resistant to attack by CD8+ T cells after transfection with BAK/BAX siRNA, leading to enhanced E7-specific T cell activation in vitro and in vivo. More importantly, vaccination with E7-presenting DCs transfected with BAK/BAX siRNA generated a strong therapeutic effect against an E7-expressing tumor in vaccinated mice, compared to DCs transfected with control siRNA. Our data indicate that a combination of strategies to enhance intracellular Ag processing and to prolong DC life may offer a promising strategy for improving DC vaccine potency.
Introduction
Antigen presentation by dendritic cells (DCs) is a critical element in the induction of the cellular immune responses necessary for tumor immunotherapy. DCs have an intrinsic ability to prime immune responses.1 Because of this, there has been a great deal of interest in the use of these cells for cancer therapy.2-6 However, clinical results have not been very promising. One of the limitations in the use of DC vaccines for clinical applications is their low potency. In particular, the generation of cellular immunity using a DC vaccine against low immunogenic tumor-specific antigens, such as human papillomavirus (HPV) E7, has been difficult. This problem presents a major hurdle in the goal of controlling cervical cancer. In a previous study, we reported that only 60% of mice who received intramuscular immunization with DC-E7 survived following a challenge with a low number of E7-expressing tumor cells (TC-1) (1×104/mouse).7 The development of strategies for improvement of DC vaccine potency is indispensable if we are to use DC-mediated cancer immunotherapy. To compensate for the weak immune response generated by DCs expressing wild-type E7 antigen, we have developed intracellular targeting strategies which increase MHC class I and class II presentation of E7 antigen by DCs.8-11 Recently, we found that linking the sorting signal of the lysosome-associated membrane protein 1 (Sig/LAMP-1)-targeted E7 to endosomal and lysosomal compartments enhanced MHC class I presentation to CD8+ T cells, as well as MHC class II presentation of E7 to CD4+ T cells.12 More importantly, immunization of mice with DC-Sig/E7/LAMP-1 led to more effective anti-tumor protection and treatment against a TC-1 cervical tumor model than did DC-E7. Despite these efforts, the potency of DC-base vaccines still needs to be improved to treat a large tumor. After administration, the life span of antigen-presenting DCs is limited in various ways, which hinders their ability to prime the immune response.13 A principal contributor to the short life of these DCs is cytotoxic cell-induced apoptosis.14,15 After activation by DCs, cytotoxic T lymphocytes (CTLs) recognize antigens and kill the cells that express them via apoptosis.16 Because DCs express MHC-I:antigen peptide complexes on their surface, newly primed CTLs can kill the very DCs that activated them.17 From these observations, we reasoned that an intracellular targeting strategy, employing an approach to inhibit apoptosis and prolong the survival of antigen-expressing DCs in vivo, could work better. In previous reports, we also found that a variety of antiapoptotic factors can enhance DC survival and the antigen-specific CD8+ T cell immune responses induced by various DNA vaccines.15,17 Since antiapoptotic proteins, such as Bcl-xL, raise significant concerns related to oncogenicity, there are practical limitations in introducing them to DC-based vaccines for clinical trials. To overcome this problem, we have tried to introduce RNA interference (RNAi) technology to DC vaccine systems, using small interference RNA (siRNA) for targeting and silencing key pro-apoptotic proteins, such as Bax and Bak. They are members of the Bcl-2 family and play key gatekeeping roles in the mitochondria-mediated intrinsic apoptotic pathway.18-21 By using siRNA, concerns related to biohazards are alleviated since RNA-based strategies avoid problems of integration and permanent genetic change. In this study, we show that the delivery of BAK and BAX siRNA to antigen-expressing DCs prolongs the lives of transfected DCs and DCs-Sig/E7/LAMP-1 transfected with BAK and BAX siRNA are capable of generating strong antigen-specific CD8+ T cell immune responses and anti-tumor effects in vaccinated mice.
Materials and Methods
Preparation of siRNAs and Transfection
siRNAs were synthesized using 2′-O-ACE-RNA phosphoramides (Dharmacon, Lafayette, Colo.). The sense and anti-sense strands of siRNA were: Bak, beginning at nt 310, 5′P-UGCCUACGAACUCUUCACCdTdT-3′ (sense), 5′P-GGUGAAGAGUUCGUAGGCAdTdT-3′ (antisense); Bax, beginning at nt 217, 5′P-UAUGGAGCUGCAGAGGAUGdTdT-3′ (sense), 5′P-CAUCCUCUGCAGCUCCAU

AdTdT-3′ (antisense); P represents 5′ phosphate. RNAs were deprotected and annealed according to the manufacturer's instruction. Non-specific Control siRNA (Target: 5′-NNATTGTATGCGATCGCAGAC-3′) was also acquired from Dharmacon (Lafayette, Colo.). Two hundred thousand recombinant DCs on a 6-well vessel were transfected with 300 pmol of the synthesized siRNAs using Oligofectamine (Invitrogen, Carlsbad, Calif.) according to the manufacturer's instructions. The transfected cells were used for subsequent experiments 3 days later. We used FITC-labeled siRNA to document transfection efficiency of the DCs, using flow cytometry analysis. Virtually 100% of the DCs were successfully transfected with siRNA (data not shown).

Construction of the DC Vaccines

The immortalized DC line, kindly provided by Dr. Kenneth Rock (University of Massachusetts, Worcester, Mass.), was genetically manipulated using a retroviral system.22 Briefly, Bone marrow cells flushed from the femurs and tibias of C57BW6 mice were infected with a retrovirus encoding murine GM-CSF and myc and raf oncogene. This DC line was used for construction of the DC vaccines expressing no insert, E7, or Sig/E7/LAMP-1 as described previously.12 For this, phoenix (φNX) packaging cells were transfected with a retroviral vector plasmid (pMSCV, pMSCV-E7, or pMSCV-Sig/E7/LAMP-1) using Lipofectamine 2000. Retroviral supernatant from the transfected phoenix cells was incubated with 50% confluent DC in the presence of polybrene (8 ug/ml; Sigma). Following transduction, the retroviral supernatants were removed, and DCs were grown in a culture medium containing 7.5 ug/ml of puromycin for selection. The expression of E7 antigens was confirmed by Western blot analysis.12 

Western Blot Analysis

The expression of E7 and Sig/E7/LAMP-1 proteins in the DCs and the expression of Bak and Bax pro-apoptotic proteins in DC cells transfected with BAK and/or BAX siRNA was characterized by Western blot analysis as described previously.23 Cells were lysed with protein extraction reagent (Pierce, Rockford, Ill.). Equal amounts of protein (50 μg) were loaded and separated by SDS-PAGE using a 15% polyacrylamide gel. The gels were electroblotted onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, Calif.). Blots were then blocked for 2 hr at room temperature with phosphate buffered saline (PBS)/0.05% Tween 20 (TTBS) containing 5% nonfat milk. Membranes were probed with E7-specific Ab (Zymed, San Francisco, Calif.) and anti-BAK or BAX mouse monoclonal antibody (Cell Signaling Technology, Inc., Beverly, Mass.), at 1:1000 dilution in TTBS for 2 hr. They were washed four times with TTBS, then incubated with goat anti-mouse IgG conjugated with horseradish peroxidase (Zymed, San Francisco, Calif.) in a 1:1000 dilution in TTBS containing 5% nonfat milk. Membranes were washed four times with TTBS and developed using Hyperfilm-enhanced chemiluminescence (Amersham, Piscataway, N.J.).

Determination of Apoptotic Cells after CTL Assay

DCs expressing no insert, E7, or Sig/E7/LAMP-1 transfected with control siRNA or Bak/Bax siRNA, were incubated for 4 or 18 hours with an E7-specific CD8+ T cell line at different E:T ratios (12.5:1, 2.5:1, 0.5:1 or 0.1:1).7 We used FITC-conjugated anti-CD8 antibody to stain for CD8+ E7-specific T cells. We gated CD8-negative cells (DCs) for activated caspase-3 analysis in order to characterize the percentage of apoptotic dendritic cells. Detection of apoptotic cells in the DCs was accomplished using PE-conjugated rabbit anti-active Caspase-3 antibody (BD Bioscience, San Diego, Calif.) according to the manufacturer's instructions. The percent of apoptotic cells was analyzed using flow cytometry.

In Vitro Activation of E7-Specific CD8+ T Cells by DCs

For assessing if the recombinant DCs transfected with the siRNAs were capable of presenting the E7 antigen to CD8+ T cells, 5×104 DCs were incubated for 18 hours with 5×105 of the E7-specific CD8+ T cell line.7,24 Activated IFN-γ-secreting E7-specific CD8+ T cells were identified by staining for both surface CD8+ and intracellular IFN-γ, and analyzed by flow cytometry analysis as described above.

Immunization with DCs

Six- to eight-week-old female C57BL/6 mice were purchased from Daehan Biolink (Chungbuk, Korea). All animal procedures were performed according to approved protocols and in accordance with recommendations for the proper use and care of laboratory animals. For the obvious down-regulation of Bax and Bak expression, DCs transfected with BAK and/or BAX siRNA were used 3 days after transfection as described previously.25 After two washes in phosphate-buffered saline, 1×106 of the retrovirus-transduced DCs in 0.1 ml of phosphate-buffered saline were injected intramuscularly into mice twice, with one week interval between the injections as described previously.7 

Intracellular Cytokine Staining and Flow Cytometry Analysis

Splenocytes were harvested from mice (3 mice per group) one week after the last vaccination. Prior to intracellular cytokine staining, 4×106 pooled splenocytes from each vaccination group were incubated overnight with 1 μg/ml of E7 (RAHYNIVTF) peptide containing an MHC class I epitope (aa 49-57) for detection of E7-specific CD8+ T cell precursors.25 IFN-γ staining and flow cytometry analysis were performed as described previously. Analysis was performed on a Becton-Dickinson FACScan with CELLQuest software (Becton Dickinson Immunocytometry System, Mountain View, Calif.). For the determination of the avidity of E7-specific CD8+ T cells in mice vaccinated with DC-Sig/E7/LAMP-1 transfected with the siRNAs, the pooled splenocytes were incubated overnight with different concentrations of E7 peptide (aa 49-57; 1, 10−1, 10−2, 10−3, 10−4, 10−5, 10−6, 10−7, or 10−8 μg/ml). The number of E7-specific IFN-γ-secreting CD8+ T cells was determined using intracellular cytokine staining and FACScan analysis as described above.

In Vivo Tumor Treatment

The HPV-16 E7-expressing murine tumor model, TC-1, has been described previously.11 In brief, HPV-16 E6, E7 and ras oncogene were used to transform primary C57BL/6 mouse lung epithelial cells to generate TC-1. Mice (5 per group) were challenged by intravenous injection of 5×105 TC-1 tumor cells/mouse into the tail, in order to simulate hematogenous spread.11 Mice were treated with DCs three days after the tumor challenge. Mice were monitored twice a week and sacrificed on day 42 after the last vaccination. The mean number of pulmonary nodules in each mouse was evaluated by experimenters blinded as to sample identity. To study the subsets of lymphocytes that are important for the anti-tumor effects, a tumor protection experiment was performed, coupled with in vivo antibody depletion that used a protocol similar to one previously described.11 Briefly, antibody depletion was initiated 1 week after the second vaccination and continued until sacrifice. mAb GK1.5 was used for CD4 depletion. mAb 2.43 was used for CD8 depletion. mAb PK136 was used for NK depletion. In vivo tumor treatment and antibody depletion experiments were performed at least twice to generate reproducible data.

In Vivo Clearance of DCs in the Primed Mouse

C57BL/6 mice (five per group) were first primed with DC-no insert or DC-Sig/E7/LAMP-1. Seven days later, the mice were boosted with carboxyfluorescein (CFSE)-labeled DC-Sig/E7/LAMP-1 transfected with control siRNA or BAK/BAX siRNA. To create CFSE positive DCs transfected with different siRNA, DCs were labeled with 5 μM CF SE for 10 min. Unlabeled DCs transfected with BAK/BAX siRNA were used as a negative control. Two days after boosting with the DCs, popliteal lymph nodes were harvested from the vaccinated mice. Isolated lymphocytes were analyzed by forward and side scatter and gated around a population of CD80 cells with the size and granular characteristics of DCs. The percentage of CFSE+ cells among the gated CD80 cells was analyzed using a protocol described previously.26 

Statistical Analysis

All data expressed as means □standard deviation (S.D.) are representative of at least two different experiments. Data for intracellular cytokine staining with flow cytometry analysis and tumor treatment experiments were evaluated by analysis of variance (ANOVA). Comparisons between individual data points were made using Student's t-test. In the tumor protection experiment, the principal outcome of interest was time to tumor development. The event time distributions for different mice were compared using the method of Kaplan and Meier and the log-rank statistic. All p values <0.05 were considered significant.

Results

Transfection of DC Cell Lines with BAK/BAX siRNA Silences the Expression of Bak and Bax Proteins.

To examine if E7 proteins were translated in DCs, Western blot analysis was performed with an E7-specific antibody. In DC-E7 and DC-Sig/E7/LAMP-1, the bands of E7 and Sig/E7/LAMP-1 at their corresponding molecular weight were detected. In contrast, there was no band in the DC-no insert. This result is the same as that previously described.12 We also performed Western blot analysis to determine whether transfection of recombinant DCs expressing no insert, E7 or Sig/E7/LAMP-1 with BAK/BAX siRNA would down-regulate the expression of Bak and Bax proteins in transfected cells. As shown in FIG. 1, the expression of Bak and Bax proteins was abolished 3 days after transfection in lysates from the DCs transfected with BAK/BAX siRNA. No expression of Bak or Bax was identified up to 7 days after transfection. Expression of Bak and Bax was detected at below-normal levels by day 9, and this expression returned to normal levels by day 11 after transfection as our previous report.25 In contrast, expression of Bak and Bax proteins was detected in the DCs after transfection with control siRNA, and the levels of expression were similar to the expression of Bak and Bax proteins by non-siRNA-transfected DC cells (data not shown). We also analyzed β-actin expression in transfected DCs in order to demonstrate that equal amounts of cell lysates were loaded for Western blot analysis. These results indicate that BAK/BAX siRNA abolish Bak and Bax protein expression during the period of immune priming by the transfected DCs.
DCs Transfected with BAK/BAX siRNA are More Resistant to CTL Killing than Those with Control siRNA.
To determine if the DCs expressing E7 or Sig/E7/LAMP-1 transfected with BAK and BAX siRNA could resist a CTL-induced apoptosis, we incubated the DCs transfected with the siRNAs with an E7-specific CD8+ T cell line and determined the percentages of apoptotic cells in DC populations 4 or 18 hours after incubation. DC-no insert was used as a negative control. As shown in FIG. 2, the significant difference in apoptotic cell percentages between the E7-presenting DCs transfected with control siRNA and those with BAK/BAX siRNA was observed 4 hours after incubation. The difference was greater at 18 hour. Although 80-90% of DC-E7 and DC-Sig/E7/LAMP-1 cells transfected with control siRNA were apoptotic, the DCs transfected with BAK/BAX siRNA generated less than 15% apoptotic cells 18 hours after incubation with the CD8+ T cell line, particularly at low E:T ratios (0.1:1). These data suggest that transfection of DCs with BAK/BAX siRNA leads to down-regulation of BAK and BAX protein expression, resulting in resistance to apoptosis induced by antigen-specific CD8+ T cells.
E7-Expressing DCs Transfected with BAK/BAX siRNA Increase the Number of Activated E7-Specific IFN-γ+ CD8+ T Cell Lines In Vitro.
We determined whether the transfection of E7-expressing DCs with the siRNAs influenced the ability of the DCs to activate an E7-specific CD8+ T cell line in vitro. For this, the transfected DCs were co-cultured with an MHC class I-restricted E7-specific T cell line at a DC:T cell ratio of 1:10 in vitro.24 As shown in FIG. 3, the DC-no insert transfected with control or BAK/BAX siRNAs failed to induce significant IFN-γ production in E7-specific CD8+ T cells. After 18 h, IFN-γ-secreting CD8+ T cells were counted using FACS analysis. FIGS. 3a and b demonstrate a more than 10 fold increase in the number of activated T cells following co-incubation of the E7-specific T cell line with the DCs transfected with BAK/BAX siRNA, when compared to DCs transfected with control siRNA. Taken together, these data suggest that the treatment of BAK/BAX siRNA may influence the ability of DCs to activate antigen-specific CD8+ T cells by prolonging DC life span during in vitro activation of an E7-specific IFN-γ+ CD8+ T cell line.
Vaccination with the E7-Expressing DCs Transfected with BAK/BAX siRNA Leads to a Significant Increase in the Number and Avidity of E7-Specific CD8+ T Cells in the Immune Response
To determine whether vaccination with E7-expressing DCs transfected with the siRNAs could enhance the generation of E7-specific IFN-γ+ CD8+ T cell precursors in vaccinated mice, we performed an intracellular cytokine staining and flow-cytometry analysis using splenocytes from mice vaccinated with the various DCs. As shown in FIG. 4a, mice vaccinated with DC-Sig/E7/LAMP-1 cells transfected with Bak/Bax siRNA exhibited an approximately 5 fold increase in the number of E7-specific IFN-γ+ CD8+ T cells, compared with mice vaccinated with DCs transfected with control siRNA. DC-E7 cells transfected with BAK/BAX siRNA also generated more E7-specific T cell precursors than those with control siRNA. In contrast, the DC-no insert transfected with control or BAK/BAX siRNAs failed to induce significant IFN-γ production in E7-specific CD8+ T cells. Our results demonstrate that immunization with E7-expressing DCs transfected with BAK/BAX siRNA can significantly increase the number of E7-specific IFN-γ+ CD8+ T cells generated in vaccinated mice. In previous studies, the duration of dendritic cell and T cell interaction has been felt to be important for the generation of high avidity T cells.27,28 In addition, we have shown that high-avidity CTLs provide better protection against a tumor challenge than low-avidity CTLs.27 Therefore, we performed a functional avidity assay to determine the avidity of E7-specific CD8+ T cells generated by vaccination with DC-Sig/E7/LAMP-1 transfected with control or BAK/BAX siRNAs. We defined the number of IFN-γ-secreting CD8+ T cells stimulated by 1 μg/ml of E7 peptide (aa 49-57) as a maximum response and compared the functional avidity of T cells from mice vaccinated with DC-Sig/E7/LAMP-1 transfected with control or BAK/BAX siRNAs at 50% of the maximum. We found that the concentration of E7 peptide required to achieve 50% of the maximum IFN-γ+ CD8+ T cell response was about 8×10−3 μg/ml for mice vaccinated with the DCs transfected with BAK/BAX siRNA, and about 9×10−2 μg/ml for mice vaccinated with DCs transfected with control siRNA (FIG. 4b). Transfection of DC-Sig/E7/LAMP-1 with BAK/BAX siRNA generates higher avidity E7-specific CD8+ T cells in vaccinated mice than transfection of DC-Sig/E7/LAMP-1 with control siRNA.
Vaccination with DC-Sig/E7/LAMP-1 Cells Transfected with (BAK/BAX) siRNA Generates Better Anti-Tumor Effects than Controls.
To determine whether the observed increase in the number and functional avidity of E7-specific CD8+ T cell precursors could translate into a better E7-specific anti-tumor effect, we performed an in vivo tumor treatment experiment using a previously characterized E7-expressing tumor model, TC-1.11 DC-Sig/E7/LAMP-1 cells, which exhibited better immunogenicity than DC-E7 cells in FIG. 4, were selected for this experiment. To compare the anti-tumor effects generated by vaccination with DC-Sig/E7/LAMP-1 transfected with Bak/Bax siRNA or control siRNA, we performed an in vivo tumor treatment experiment in a lung tumor metastasis model at a high lethal dose. In this experiment, 5×105 TC-1 cells were injected via the tail vein. This number was 50 times more TC-1 cells than that commonly used in our studies.7-10-12,15,17,29 Mice were first challenged with the tumor cells, followed 3 days after tumor challenge by treatment with DC-Sig/E7/LAMP-1 cells transfected with control or BAK/BAX siRNAs. Mice were sacrificed 28 days after challenge, and the number of pulmonary tumor nodules was counted. As shown in FIG. 5a, mice treated with the DCs transfected with BAK/BAX siRNA demonstrated the lowest number of pulmonary nodules, compared to mice treated with the DCs transfected with control siRNA (P<0.007), or the naïve control group. These results show that vaccination with DCs transfected with BAK/BAX siRNA generates a significantly better therapeutic anti-tumor effect than vaccination with DCs transfected with control siRNA.
We also performed a tumor treatment experiment with antibody depletion to determine the subset of T lymphocytes responsible for the anti-tumor response. Mice were challenged with TC-1 and subsequently vaccinated with the DCs as described above. Antibody depletion was initiated 1 week after the booster vaccination and continued for 28 days following the challenge. Mice depleted of CD8+ T cells displayed nearly the same degree of tumor growth as naïve mice, and mice depleted of CD4+ T cells displayed slightly increased tumor growth compared to non-depleted mice. Mice depleted of NK cells did not generate a significantly different number of tumor nodules compared to mice with no depletion (FIG. 5b). These data suggest that CD8+ T cells are essential for the anti-tumor effect and that CD4+ T cells may also contribute to the observed anti-tumor effect, though not as strongly as CD8+ T cells.

E7 Peptide-Loaded DCs Transfected with BAK/BAX siRNA Survive Longer In Vivo than E7 Peptide-Loaded DCs Transfected with Control siRNA.

To determine if transfection with BAK/BAX siRNA improves the survival of DC-Sig/E7/LAMP-1 during in vivo conditions, mice were first primed with DC-no insert or DC-Sig/E7/LAMP-1. One week later, the mice were boosted with CFSE-labeled DC-Sig/E7/LAMP-1 transfected with control siRNA or BAK/BAX siRNA. Unlabeled DCs transfected with BAK/BAX siRNA were used as a negative control. Two days after boosting with the DCs, popliteal lymph nodes were harvested from the vaccinated mice. Isolated lymphocytes were further analyzed using flow cytometry. The percentage of CFSE+ cells among the gated CD80+ monocyte-like cells with size and granular characteristics of DC was measured, using a protocol described previously.26 As shown in FIGS. 6a and b, at 2 days after vaccination with DC-no insert, there was no significant difference between the percentage of CFSE+ cells among the gated CD80+ monocyte-like cells in mice given BAK/BAX siRNA from mice given control siRNA. In comparison, in mice primed with DC-Sig/E7/LAMP-1, we detected a significant decrease in the percentage of CFSE+ CD80+ DCs in mice that received cells with control siRNA, compared to that of CFSE+ CD80+ DCs in mice that received cells with BAK/BAX siRNA. More than 90% of CFSE+ CD80+ DCs were caspase-3 negative, indicating that these cells were not apoptotic (data not shown). Our data suggest that transfection of E7-expressing DCs with BAK/BAX siRNA may protect DCs from being killed by E7-specific immunity, thus prolonging DC life in vivo.
Discussion
In this study, we have shown that vaccination with HPV16 E7-expressing DCs transfected with siRNA targeting BAK and BAX increased E7-specific anti-tumor immune responses. These siRNA transfected DCs were highly resistant to apoptotic cell death mediated by E7-specific CD8+ T cells, leading to prolonged DC survival. This resulted in a further increase in DC-mediated vaccine potency. In our previous studies, we demonstrated that retrovirally-transduced DCs endogenously expressing Sig/E7/LAMP-1 (the linkage of a Sig/LAMP-1 molecule to E7) increased the presentation of antigenic E7 peptides to E7-specific T cells in the context of MHC class I and class II, and enhanced the potency of DC vaccines.12 This enhancement of dendritic cell-based vaccine potency might be explained by qualitative changes in antigen-expressing DCs that lead to enhanced activation of E7-specific T cells. DCs, however, have a limited life span that hinders their long-term ability to prime antigen-specific T cells. DCs that present the relevant MHC-peptide complexes qualify as potential targets and are at risk of being eliminated by the CTLs they have activated. This would seriously limit the capacity of DCs to prime CTL immunity.14,25 During CTL-killing process, as shown in FIGS. 2 and 6, highly immunogenic DCs having more antigenic peptides on their surface such as DC-Sig/E7/LAMP-1 might be better targets for E7-specific CD8+ T cells, compared to less immunogenic DCs such as DC-E7. Thus, modulation of apoptosis in DCs using siRNA technology is necessary to prolong DC survival and further enhance the potency of DC-based vaccines.
The BAK and BAX siRNA technology can be extended to the preparation of antigen-specific T cells ex vivo. We have shown that E7-expressing DCs transfected with BAK/BAX siRNA were capable of resisting being killed by E7-specific CD8+ T cells, compared to DC cells transfected with control siRNA, leading to an increase in the number of activated CD8+ T cells (FIG. 3). It would be possible that the significant increase in activated CD8+ T cells might be due to change in the expression of molecules important for antigen presentation in DCs, such as CD11c, CD40, CD80, CD86, MHC I, and MHC II and pro-inflammatory cytokines, such as IFN-β, IFN-α, and TNF-α.30,31 We therefore performed flow cytometry to determine the expression levels of them important for antigen presentation in DC-Sig/E7/LAMP-1 transfected with BAK/BAX siRNA or control siRNA in non-transfected DC cell line. There were no significant changes in the expression of the tested molecules among the DC cell lines. In our previous report, we observed a similar result in an E7 peptide-loaded DC cell line transfected with BAK/BAX siRNAs or control siRNA and in non-transfected DC cell line.26 Recently, it has been reported that synthetic siRNAs complexed with liposomes can be potent inducers of pro-inflammatory cytokines.30,31 To confirm the induction of the IFN-β, IFN-α, and TNF-α, we performed RT-PCR to determine the mRNA expression levels of them in these DC cell lines transfected with or without the BAK/BAX siRNA or control siRNA/Oligofectamine complexes. Unexpectedly, we found that there were no significant changes in the mRNA levels of the cytokines among these DC cell lines (data not shown). One of plausible explanations about this discrepancy may be a point of time to assess the level of cytokines. Most literatures have shown the induction of cytokines within 1 or 2 days after transfection. Notably, no expression of Bak or Bax was detected in the siRNA-treated DCs we used at days 3 after transfection. This silence of Bak and Bax was lasted by day 9, and the expression of them returned to normal levels by day 11 after transfection.25 From this reason, in this study, the siRNA-treated DCs were used for in vitro and in vivo experiments 3 days after transfection. Similarly, the levels of the immune-modulating molecules were also determined 3 days after transfection of the DCs with the siRNAs. It would be possible that the pro-inflammatory molecules were induced within 1 day after transfection. Subsequently, the levels of the molecules could return to normal levels 3 days after treatment of siRNAs. We could not rule out, however, other possibility that transfection of DCs with siRNA/liposome complexes may affect immune-priming capacity of DCs through other cytokines we did not test such as IL-12, IL-6, IL-10 and INF-γ or other factors such as chemokines that influence DC homing to the draining lymph nodes. Despite a bundle of possible factors to affect DC capacity of priming an immune response, most important thing is that only difference between control siRNA and BAK/BAX siRNAs is a sequence. In some literatures, base sequence motifs including 5′-GUCCUUCAA-3′, which can induce inflammatory cytokines, were identified.31,32 It is worth to take a notice that the siRNAs we used do not contain these sequence motifs. Thus, the enhanced E7-specific T cell-mediated immune response may not be due to changes in the expression of the molecules important for antigen presentation in DCs and the pro-inflammatory cytokines in the DC cell lines transfected with BAK/BAX siRNA or control siRNA. Therefore, the increase in activated CD8+ T cells might be due to enhanced survival of dendritic cells mediated by BAK and BAX siRNA. Considering the difficulty in gathering blood from patients for ex vivo T cell preparation, the application of BAK/BAX siRNA technology might be promising for tumor immunotherapy using adoptive transfer of T cells.
We have shown DC survival can be prolonged by their transfection with DNA encoding antiapoptotic proteins.17 Among them, the antiapoptotic Bcl-2 family, such BCL-xL and BCL-2, were most effective in increasing the life span of DCs transfected with a gene gun via an intradermal route. However, since these molecules have been implicated as contributors to oncogenic transformation, utilization of the antiapoptotic DNA encoding Bcl-2 family for clinical applications might be limited.33 We have recently demonstrated that peptide-pulsed BM-DCs transfected with BAK/BAX siRNA were capable of resisting being killed by antigen-specific CD8+ T cells in vivo.29 RNA interference (RNAi) using siRNA targeting proapoptotic proteins provided similar effects, while alleviating oncogenic concerns associated with the use of DNA encoding antiapoptotic proteins.
The discovery of defined tumor antigens and their application in therapeutic cancer vaccines has not yet resulted in a successful therapy for cancer patients. One of the reasons is most of tumor antigens are self ones. There are simple evidences in mouse and man that most cancers, similar to normal somatic cells, do not directly prime self-tumor-antigen-specific T cells very efficiently.34-36 Like this, self tolerance that normally exists to prevent autoimmune disease may preclude the development of an adequate anti-tumor response and thwart the development of effective immune responses against tumors.37 Thus, breaking tolerance in tumor-bearing hosts has been seen as a primary requirement for cancer immunotherapy. In this aspect, it is worth to notify that DCs expressing the baculoviral caspase inhibitor, p35, display defective apoptosis, resulting in their accumulation and, in turn, chronic lymphocyte activation and systemic autoimmune manifestations.38 Considering the number of well characterized self-tumor antigens, it will be an interesting challenge to check whether antiapoptotic DCs that present a self tumor antigen break self-tolerance and induce a therapeutic immune response against various tumors. In this study, we further increased the potency of a Sig/E7/LAMP-1 expressing DC vaccine by prolonging DC life with BAK/BAX siRNAs. The DCs transfected with BAK/BAX siRNA enhanced E7-specific CD8+ T cell activation in vitro. They elicited stronger anti-tumor effects in vivo, compared with DCs transfected with control siRNA. Thus, a DC-based vaccine strategy incorporating Sig/E7/LAMP-1 DCs transfected with BAK/BAX siRNA may be a promising strategy for tumor immunotherapy.
REFERENCES


    1. Cerundolo V, Hermans I F, Salio M. Dendritic cells: a journey from laboratory to clinic. Nat Immunol 2004; 5:7-10.
    2. Engleman E G. Dendritic cell-based cancer immunotherapy. Semin Oncol 2003; 30: 23-9.
    3. Gunzer M, Grabbe S. Dendritic cells in cancer immunotherapy. Crit Rev Immunol 2001; 21:133-45.
    4. Schuler G, Schuler Thurner B, Steinman R M. The use of dendritic cells in cancer immunotherapy. Curr Opin Immunol 2003; 15:138-47.
    5. Turtle C J, Hart D N. Dendritic cells in tumor immunology and immunotherapy. Curr Drug Targets 2004; 5:17-39.
    6. Figdor C G, de VRIES I J, Lesterhuis W J, Melief C J. Dendritic cell immunotherapy: mapping the way. Nat Med 2004; 10:475-80.
    7. Wang T L, Ling M, Shih I M, Pham T, Pai S I, Lu Z, Kurman R J, Pardoll D M, Wu T C. Intramuscular administration of E7-transfected dendritic cells generates the most potent E7-specific anti-tumor immunity. Gene Ther 2000; 7:726-33.
    8. Moniz M, Ling M, Hung C F, Wu T C. HPV DNA vaccines. Frontiers Biosci 2003; 8: D55-68.
    9. Wu T C, Guamieri F G, Staveley-O'Carroll K F, Viscidi R P, Levitsky H I, Hedrick L, Cho K R, August J T, Pardoll D M. Engineering an intracellular pathway for major histocompatibility complex class II presentation of antigens. Proc Natl Acad Sci USA 1995; 92:11671-5.
    10. Cheng W F, Hung C F, Hsu K F, Chai C Y, He L, Ling M, Slater L A, Roden R B, Wu T C. Enhancement of sindbis virus self-replicating RNA vaccine potency by targeting antigen to endosomal/lysosomal compartments. Hum Gene Ther 2001; 12:235-52.
    11. Lin K Y, Guamieri F G, Staveley-O'Carroll K F, Levitsky H I, August J T, Pardoll D M, Wu T C. Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen. Cancer Res 1996; 56:21-6.
    12. Kang T H, Lee J H, Bae H C, Noh K H, Kim J H, Song C K, Shin B C, Hung C F, Wu T C, Park J S, Kim T W. Enhancement of dendritic cell-based vaccine potency by targeting antigen to endosomal/lysosomal compartments. Immunol lett. In press.
    13. Ronchese F, Hermans I F. Killing of dendritic cells: a life cut short or a purposeful death? J Exp Med 2001; 194:F23-6.
    14. Medema J P, Schuurhuis D H, Rea D, Van Tongeren J, de Jong J, Bres S A, Laban S, Toes R F, Toebes M, Schumacher T N, Bladergroen B A, Ossendorp F, Kummer J A, Melief C J, Offring a R. Expression of the serpin serine protease inhibitor 6 protects dendritic cells from cytotoxic T lymphocyte-induced apoptosis: differential modulation by T helper type 1 and type 2 cells. J Exp Med 2001; 194:657-67.
    15. Kim T W, Hung C F, Boyd D, Juang J, He L, Kim J W, Hardwick J M, Wu T C. Enhancing DNA vaccine potency by combining a strategy to prolong dendritic cell life with intracellular targeting strategies. Immunol 2003; 171:2970-6.
    16. Russell J H, Ley T J. Lymphocyte-mediated cytotoxicity. Annu Rev Immunol 2002; 20:323-70.
    17. Kim T W, Hung C F, Ling M, Juang J, He L, Hardwick J M, Kumar S, Wu T C. Enhancing DNA vaccine potency by coadministration of DNA encoding antiapoptotic proteins. J. Clin. Invest 2003; 112:109-17.
    18. Jacotot E, Costantini P, Laboureau E, Zamzami N, Susin S A, Kroemer G. Mitochondrial membrane permeabilization during the apoptotic process. Ann NY Acad Sci 1999; 887:18-30.
    19. Korsmeyer S J, Wei M C, Saito M, Weiler S, Oh K J, Schlesinger P H. Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c. Cell Death Differ 2000; 7:1166-73.
    20. Degli Esposti M, Dive C. Mitochondrial membrane permeabilisation by Bax/Bak. Biochem Biophys Res Commun 2003; 304:455-61.
    21. Opferman J T, Korsmeyer S J. Apoptosis in the development and maintenance of the immune system. Nat Immunol 2003; 4:410-5.
    22. Shen Z, Reznikoff G, Dranoff G, Rock K L. Cloned dendritic cells can present exogenous antigens on both MHC class I and class II molecules. J Immunol 1997; 158:2723-30.
    23. Hung C F, Cheng W F, Hsu K F, Chai C Y, He L, Ling M, Wu T C. Cancer immunotherapy using a DNA vaccine encoding the translocation domain of a bacterial toxin linked to a tumor antigen. Cancer Res 2001; 61:3698-703.
    24. Kim T W, Hung C F, Kim J W, Juang J, Chen P J, He L, Boyd D A, Wu T C. Vaccination with a DNA vaccine encoding herpes simplex virus type 1 VP22 linked to antigen generates long-term antigen-specific CD8-positive memory T cells and protective immunity. Hum Gene Ther 2004; 15:167-77.
    25. Kim T W, Lee J H, He L, Boyd D A, Hardwick J M, Hung C F, Wu T C. Modification of professional antigen-presenting cells with small interfering RNA in vivo to enhance cancer vaccine potency. Cancer Res 2005; 65:309-16.
    26. Peng S, Kim T W, Lee J H, Yang Hu, He L, Hung C F, Wu T C. Vaccination with Dendritic Cells Transfected with BAK and BAX siRNA Enhances Antigen-Specific Immune Responses by Prolonging Dendritic Cell Life. Human Gene Therapy 2005; 16: 584-93.
    27. Cheng W F, Hung C F, Pai S I, Hsu K F, He L, Ling M, Wu T C. Repeated DNA vaccinations elicited qualitatively different cytotoxic T lymphocytes and improved protective antitumor effects. J Biomed Sci 2002; 9:675-87.
    28. Langenkamp A, Casorati G, Garavaglia C, Dellabona P, Lanzavecchia A, Sallusto F. T cell priming by dendritic cells: thresholds for proliferation, differentiation and death and intraclonal functional diversification. Eur J Immunol 2002; 32:2046-54.
    29. Ji H, Wang T L, Chen C H, Pai S I, Hung C F, Lin K Y, Kuman R J, Pardoll D M, Wu T C. Targeting human papillomavirus type 16 E7 to the endosomal/lysosomal compartment enhances the antitumor immunity of DNA vaccines against murine human papillomavirus type 16 E7-expressing tumors. Hum Gene Ther 1999; 10:2727-40.
    30. Kariko K, Bhuyan P, Capodici J, Weissman D. Small interfering RNAs mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor 3. J Immunol 2004; 172:6545-9.
    31. Judge A D, Sood V, Shaw J R, Fang D, McClintock K, MacLachlan I. Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat Biotechnol 2005; 23:457-62.
    32. Hornung V, Guenthner-Biller M, Bourquin C, Ablasser A, Schlee M, Uematsu S, Noronha A, Manoharan M, Akira S, de Fougerolles A, Endres S, Hartmann G. Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat Med. 2005; 11:263-70
    33. Lebedeva I, Rando R, Ojwang J, Cossum P, Stein C A. Bcl-xL in prostate cancer cells: Effects of overexpression and down-regulation on chemosensitivity. Cancer Res 2000; 60:6052-60.
    34. van Mierlo G J, den Boer A T, Medema J P, van der Voort E I, Fransen M F, Offringa R, Melief C J, Toea R E. CD40 stimulation leads to effective therapy of CD40(−) tumors through induction of strong systemic cytotoxic T lymphocyte immunity. Proc Natl Acad Sci USA 2002; 99:5561-6.
    35. Overwijk W W, Theoret M R, Finkelstein S E, Surman D R, De Jong L A, Vyth-Dreese F A, Dellemijn T A, Antony P A, Spiess P J, Palmer D C, Heimann D M, Klebanoff C A, Yu Z, Hwang L N, Feigenbaum L, Kruisbeek A M, Rosenberg S A, Restifo N P. Tumor Regression and Autoimmunity after Reversal of a Functionally Tolerant State of Self-reactive CD8+ T Cells. J Exp Med 2003; 198:569-80.
    36. Ochsenbein A F, Sierro S, Odermatt B, Pericin M, Karrer U, Hermans J, Hemmi S, Hengartner H, Zinkenagel R M. Roles of tumour localization, second signals and cross priming in cytotoxic T-cell induction. Nature 2001; 411:1058-64.
    37. Mapara M Y, Sykes M. Tolerance and cancer: mechanisms of tumor evasion and strategies for breaking tolerance. J Clin Oncol 2004; 22:1136-51.
    38. Chen M, Wang Y H, Wang Y, Huang L, Sandoval H, Liu Y J, Wang J. Dendritic cell apoptosis in the maintenance of immune tolerance. Science 2006; 311:1160-4.


The references cited above are all incorporated herein by reference, whether specifically incorporated or not. All publications, patents, patent applications, GenBank sequences and ATCC deposits, cited herein are hereby expressly incorporated by reference for all purposes. When definitions of terms in documents that are incorporated by reference herein conflict with those used herein, the definitions used herein govern. Citation of the documents herein is not intended as an admission that any of them is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents. Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

## Claims

1. A nucleic acid composition useful as an immunogen, comprising a combination of:
(a) a first nucleic acid molecule comprising a first sequence encoding an epitope of an antigenic polypeptide or peptide; and optionally, linked to the first sequence, directly or via a linker, a second sequence that encodes an immunogenicity-potentiating polypeptide (IPP); and
(b) a second nucleic acid molecule the activity or expression of which stimulates development of an immune response to said epitope, which second nucleic molecule is (i) a siRNA or (ii) DNA that encodes said siRNA, wherein said siRNA has a sequence that is sufficiently complementary to and thus targets the sequence of mRNA that encodes a pro-apoptotic protein expressed in a dendritic cell (DC), such that the activity or expression of said siRNA in the cell results in inhibition of or loss of expression of said mRNA, resulting in inhibition of apoptosis and increased survival of DCs,

wherein the first and second nucleic acid molecules are bound to particles comprising a material suitable for introduction into a cell or an animal by particle bombardment and wherein the development of said immune response is stimulated.

2. The composition of claim 1 that includes said second nucleic acid sequence encoding said IPP which is fused in frame to said first sequence such that said first and said second sequence encode a fusion protein comprising said antigenic epitope and said IPP.

3. The composition of claim 1, wherein the IPP acts in potentiating an immune response by promoting:
(a) processing of the linked antigenic polypeptide via the MHC class I pathway or targeting of a cellular compartment that increases said processing;
(b) development, accumulation or activity of antigen presenting cells or targeting of antigen to compartments of said antigen presenting cells leading to enhanced antigen presentation;
(c) intercellular transport and spreading of the antigen; or
(d) any combination of (a)-(c).

4. The composition of claim 3 wherein the IPP is:
(a) the sorting signal of the lysosome-associated membrane protein type 1 (Sig/LAMP-1)
(b) a mycobacterial HSP70 polypeptide, the C-terminal domain thereof, or a functional homologue or derivative of said polypeptide or domain;
(c) a viral intercellular spreading protein selected from the group of herpes simplex virus-1 VP22 protein, Marek's disease virus UL49 protein or a functional homologue or derivative thereof;
(d) an endoplasmic reticulum chaperone polypeptide selected from the group of calreticulin or a domain thereof, ER60, GRP94, gp96, or a functional homologue or derivative thereof
(e) domain II of Pseudomonas exotoxin ETA or a functional homologue or derivative thereof;
(f) a polypeptide that targets the centrosome compartment of a cell selected from γ-tubulin or a functional homologue or derivative thereof; or
(g) a polypeptide that stimulates DC precursors or activates DC activity selected from the group consisting of GM-CSF, Flt3-ligand extracellular domain, or a functional homologue or derivative thereof.

5. The composition of claim 1, wherein said pro-apoptotic protein is selected from the group consisting of one or more of (a) Bak, (b) Bax, (c) caspase-8, (d) caspase-9 and (e) caspase-3.

6. The composition of claim 1 wherein the antigenic polypeptide or peptide comprises an epitope that binds to and is presented on surfaces of antigen-presenting cells by MHC class I proteins.

7. The composition of claim 1, wherein the antigenic polypeptide or peptide:
(i) is derived from a pathogen selected from the group consisting of a mammalian cell, a microorganism or a virus;
(ii) cross-reacts with an antigen of the pathogen; or
(iii) is expressed on the surface of a pathogenic cell.

8. The composition of claim 7, wherein the antigenic polypeptide or peptide is derived from a human papilloma virus.

9. The composition of claim 7, wherein the antigen is an HPV-16 E7, E7(detox), E6 or E6(detox) polypeptide or peptide.

10. The composition of claim 7, wherein antigenic polypeptide or peptide cross-reacts with an antigen of a pathogen that is a bacterium.

11. The composition of claim 7, wherein the antigenic polypeptide or peptide is a tumor-specific or tumor-associated antigen.

12. The composition of claim 1 wherein the first nucleic acid molecule is an expression vector comprising a promoter operatively linked to said first and/or said second sequence.

13. The composition of claim 12, wherein the promoter is one which is expressed in an antigen presenting cell (APC).

14. The composition of claim 13, wherein the APC is a DC.

15. A combination of first and second particles each comprising a material that is suitable for introduction into a cell or an animal by particle bombardment, and to which particles is bound the composition of claim 1, wherein
(a) the first nucleic acid molecules are bound to said first particles; and
(b) the second nucleic acids are bound to said second particles.

16. The particle of claim 1, wherein the particles are gold particles.

17. A pharmaceutical composition capable of inducing or enhancing an antigen specific immune response, comprising the composition of claim 1 and a pharmaceutically acceptable carrier or excipient.

18. A pharmaceutical composition capable of inducing or enhancing an antigen specific immune response, comprising the particle of claim 1, and a pharmaceutically acceptable carrier or excipient.

19. A method of inducing or enhancing an antigen specific immune response in a subject comprising administering to the subject an effective amount of the composition of claim 1, thereby inducing or enhancing the antigen specific immune response.

20. A method of inducing or enhancing an antigen specific immune response in a subject, comprising administering to the subject an effective amount of the particle of claim 1, thereby inducing or enhancing the antigen specific immune response.

21. A method of inducing or enhancing an antigen specific immune response in a subject, comprising administering to the subject an effective amount of the particles of claim 15, thereby inducing or enhancing the antigen specific immune response.

22. The method of claim 19, wherein the composition is administered intradermally by particle bombardment, intratumorally or peritumorally.

23. A method of increasing the numbers of CD8+ CTLs specific for a selected desired antigen in a subject comprising administering an effective amount of the composition of claim 1, wherein the antigenic peptide or polypeptide comprises an epitope that binds to and is presented on surfaces of antigen-presenting cells by MHC class I proteins, thereby increasing the numbers of antigen-specific CD8+ CTLs.

24. A method of inhibiting the growth of a tumor in a subject comprising administering an effective amount of the composition of claim 1, wherein the antigenic epitopes are those expressed by the tumor or ones cross reactive with those expressed by the tumor, thereby inhibiting growth of the tumor.

25. A method of inhibiting the growth of a tumor in a subject comprising administering an effective amount of the particle of claim 1, wherein the antigenic epitopes are those expressed by the tumor or ones cross reactive with those expressed by the tumor, thereby inhibiting growth of the tumor.

26. An immunogenic cellular composition, comprising dendritic cells (DCs) which have been modified by:
(a) loading the DCs with an antigen so that the antigen is expressed on the DC surface, or transducing or transfecting the DCs with DNA that encodes an antigen fused to an IPP, wherein the DNA is bound to particles comprising a material suitable for introduction into a cell or an animal by particle bombardment; and
(b) transfecting the DCs with a nucleic acid molecule that is (i) a siRNA or (ii) DNA that encodes said siRNA, wherein said siRNA has a sequence that is sufficiently complementary to and thus targets the sequence of mRNA that encodes a pro-apoptotic protein expressed in the DC and wherein the nucleic acid molecule is bound to particles comprising a material suitable for introduction into a cell or an animal by particle bombardment, such that expression or activity said siRNA in the cell results in diminution or loss of expression of said mRNA, resulting in inhibition of apoptosis and prolonged survival of the DC.

27. The composition of claim 26 wherein said pro-apoptotic protein is selected from the group consisting one or more of (a) Bak, (b) Bax, (c) caspase-8, (d) caspase-9 and (e) caspase-3.

28. A pharmaceutical composition capable of inducing or enhancing an antigen specific immune response, comprising the composition of claim 26 and a pharmaceutically acceptable carrier or excipient.

29. A method of inducing or enhancing an antigen specific immune response in a subject comprising administering to the subject an effective amount of the composition of claim 26, thereby inducing or enhancing the antigen specific immune response.

30. A method of increasing the numbers of CD8+ CTLs specific for a selected desired antigen in a subject comprising administering an effective amount of the composition of claim 26 wherein the loaded antigen or the antigen expressed from said transduced DNA comprises an epitope that binds to and is presented on the DC surface by MHC class I proteins, thereby increasing the numbers of antigen-specific CD8+ CTLs.

31. A method of inhibiting the growth of a tumor in a subject comprising administering an effective amount of the composition of claim 26, wherein the antigenic epitopes are those expressed by the tumor or ones cross reactive with those expressed by the tumor, thereby inhibiting growth of the tumor.