HK1093899A - Nucleotide and cellular vaccine composition - Google Patents

Description

Nucleic acid and cellular vaccine components

Technical Field

The present invention relates to vaccine components, particularly to antigen presenting cells containing nucleic acids and genetic modifications, and their uses.

Background

The use of nucleic acid sequences, including DNA or RNA sequences, as a means of vaccination has been developed during the last decade. DNA vaccines are relatively easy to manufacture, stable and low in cost. In addition, repeated use of DNA vaccines does not produce significant vector-specific immune responses. When naked plasmid DNA is driven into the skin and muscle of a small mouse, the DNA will be taken up by nearby cells. These non-lymphoid tissues express the proteins encoded by the plasmids, and the antigenic peptide is then presented on T lymphocytes upon binding to Major Histocompatibility Complex (MHC) class I or class II molecules (annu. rev. immunel.2000, 18: 927).

The ability of DNA immunization to combat different pathogen infections and malignant diseases has been studied in animal models (Annu Rev. Immunol.1997, 15: 617-648). DNA immunization can suppress autoimmune diseases as well as allergic reactions (nat. Med.19962: 899-. Recent research results have shown that DNA vaccines in humans can generate cellular immune responses against malaria infection and HIV polypeptides (Science1998, 282: 476-480, Lancet 1998, 351: 1320-1325).

Typically, plasmid DNA vectors have two main units: (1) a plasmid backbone for delivery of a cofactor; (2) a transcriptional unit comprising a promoter, an antigenic nucleotide sequence, and a polyadenylation addition sequence, which together direct the synthesis of the protein.

The major problem with DNA vaccines today is that they are not as effective as we imagine. Unsatisfactory results from preclinical work and clinical trials have raised serious doubts about the use of DNA vaccines. Therefore, improvement of vaccine efficiency is an important goal for the development of DNA vaccines.

Dendritic Cells (DCs) are specialized Antigen Presenting Cells (APCs) of hematopoietic origin. Although all DCs share common functions of antigen (Ag) processing and T cell activation, DCs cells contain multiple subtype cell types and a wide variety of functions.

Tumors express several protein antigens that are recognized by T cells and are potential targets for cancer immunotherapy. Dendritic Cells (DCs) have the unique ability to present antigens to T cells. This property has been exploited in the development of vaccines for the treatment of cancer. In clinical trials with DC vaccination against non-Hodgkin's lymphoma and melanoma, induction of anti-tumor immune responses and tumor regression has been observed (Annu Rev Med.1999, 50: 507-.

Disclosure of Invention

It is a general object of the present invention to provide a novel vaccine composition.

It is an object of the present invention to provide a method for producing said vaccine composition.

It is another object of the present invention to provide a pharmaceutical composition.

It is a further object of the present invention to provide a vaccine composition comprising a nucleic acid sequence encoding an antigenic molecule and modified antigen presenting cells, not limited to Dendritic Cells (DCs).

It is a particular object of the present invention to provide a vaccine composition which can be used for the prevention and/or treatment of cancer, infectious diseases, senile dementia, allergy, autoimmune diseases and haematological disorders.

It is a further particular object of the invention to provide a vaccine composition comprising a sub-class of dendritic cells, referred to as plasmacytic dendritic cells (pDCs) or Interferon Producing Cells (IPCs). This subclass of dendritic cells is genetically engineered to express immune modulatory molecules.

These and other objects are defined in the respective claims of the invention.

Briefly, the present invention provides a novel vaccine composition. The composition comprises a nucleotide sequence encoding an antigenic molecule and genetically modified Antigen Presenting Cells (APCs), preferably pre-cultured as a mixture.

The nucleotide sequence encoding the antigen may be a naked DNA or RNA sequence. In addition, the nucleotide sequence encoding the antigen is preferably inserted and contained in a vector, where the nucleotide sequence is placed under the transcriptional control of a promoter, enhancer and/or other regulatory sequences. The vector of the present invention is preferably a plasmid DNA vector comprising a gene encoding an antigen. The vector may also comprise other nucleotide sequences which modulate or control the immune response of the subject host. The subject receiving the vaccine composition is preferably a mammal, more preferably a human. Such immune response regulatory sequences may be unmethylated cytidine monophosphate guanosine (CpG) sequences or gene sequences encoding xenogenic molecules (proteins/polypeptides) involved in modulating the immune response in a subject.

The APCs cells used in the vaccine composition of the invention are of a cell type suitable for processing and presenting antigen to other cells, particularly CD4+ and CD8+ T cells of the immune system. Examples of preferred APCs include professional APCs such as DCs, IPCs, macrophages, monocytes and B cells. The more preferred APCs cell types are those with pDC/IPCs characteristics and functions, particularly those that express Toll-like receptor 9(TLR9) and P2X7 receptors, secrete cytokines and produce large amounts of type I interferon-alpha and interferon-beta under microbial stimulation and stimulate effector cells of the immune system.

In addition, the APCs should be genetically modified or otherwise modified to express immune response modulating molecules. Such molecules can enhance the immune response in a subject by enhancing antigen delivery, stimulating secretion of Th1 or Th2 cytokines, activating APCs, Langerhans cells and effector cells, and/or enhancing the immune response. In addition, especially in autoimmune diseases and allergies, immune response modulating molecules can help suppress immune responses or induce immune tolerance or anergy in a subject. Suitable genes for modifying APCs include: cytokine genes, interleukin genes, adhesion molecules, interferon genes, chemokine genes, and chemokine receptor genes and genes encoding heat shock proteins, Tumor Necrosis Factor (TNF), anti-apoptotic agents, apoptosis-inducing molecules, growth factors, and pharmaceutically acceptable carriers.

The vaccine composition of the present invention may also include additional molecules in addition to the nucleic acid sequence and APCs. These additional molecules may enhance or suppress an immune response in a subject, increase antigen presentation of APCs, stimulate secretion of Th1 or Th2 cytokines, activate APCs, Langerhans cells, effector cells, and/or modulate immune function of APCs.

The most effective components of the vaccine compositions of the invention include genetically modified pDCs or interferon type I (IFN) producing cells (IPCs) and plasmid DNA encoding MHC-bound antigen and containing CpG motifs (CpG motifs).

The invention also relates to a method for the treatment and prevention of diseases, i.e. the administration of the vaccine composition of the invention to a subject, preferably a mammal, preferably a human, and the use thereof. The vaccine components of the invention include nucleic acid sequences encoding antigenic molecules associated with disease. For example, for infectious diseases, the nucleic acid sequence encodes a protein or polypeptide associated with the disease and derived from an infecting microorganism, such as a viral, bacterial, fungal, protozoan, or parasitic polypeptide or protein. Vaccination of humans and animals with nucleic acid sequences encoding proteins or polypeptides infecting microorganisms in conjunction with modified APCs will induce an immune response that is specific against the encoded protein or polypeptide.

Cancers often have altered or abnormal gene expression. Aberrant levels of protein expression in cancer cells may be targeted for T cell attack. Also included in the vaccine compositions of the present invention are nucleic acid sequences encoding tumor-associated antigens.

The invention also includes a method of producing the vaccine composition. The method comprises designing and identifying antigenic molecules that bind to MHC and are associated with a disease or disorder when the vaccine composition is used to treat or prevent a disease.

In a more preferred embodiment, the nucleic acid sequence encoding the identified antigenic molecule is introduced into a plasmid DNA vector, preferably a plasmid vector containing unmethylated CpG sequences. The APCs are isolated and may be from the subject's (or recipient's) autologous APCs. APCs are preferably special dendritic cell subtypes, like pDCs types and (natural) interferons result in cell types (NIPCs) and have the ability to produce type I interferons under stimulation by e.g.plasmid DNA. APCs, after being modified by one or more genes encoding immune co-stimulatory molecules, modulate APC function and stimulate immune effector cells. Finally, the nucleotide sequence encoding the antigen and the genetically modified APC are mixed together, incubated, the manufacturing process is completed and the embodiment of the vaccine composition of the present invention is concluded.

Thus, a major feature of the positive results obtained in accordance with the present invention is the use of genetically modified APCs in conjunction with the nucleotide sequence encoding the antigen. An additional feature of the invention is the pre-incubation of the two main components of the vaccine prior to use.

Such preincubation allows the APCs to take up the nucleotide sequence. After entering APCs, the antigenic sequences will be processed and presented, and then the CpG sequences will bind to various receptors including TLR9, resulting in the activation of APCs and the production of immune modulatory molecules such as type I interferons and cytokines.

The invention has the following advantages:

the vaccine composition of the invention has a superior anti-tumor efficacy when applied to tumor-bearing mice compared to previous DNA vaccine compositions;

-the vaccine composition of the invention has a 5-fold greater effect on the treatment of cancer compared to a vaccine comprising a nucleotide sequence alone or a plasmid encoding a nucleotide sequence for an antigen;

-the vaccine components of the invention have more than 8-fold effect on the activation of tumor-specific Cytotoxic T Lymphocytes (CTLs) in vivo compared to the use of plasmid DNA containing a nucleotide sequence encoding an antigen or an empty plasmid vector as a vaccine;

compared with the only genetic modified APCs and MHC-I molecules thereof loaded with antigen polypeptides as vaccines, the vaccine components of the invention have more than 2 times effect on the activation of tumor-specific Cytotoxic T Lymphocytes (CTLs) in vivo;

-vaccination with the vaccine composition of the invention with Cytotoxic T Lymphocytes (CTLs) capable of activating and inducing tumor-specific cytotoxic T lymphocytes that recognize the tumor peptides contained in the vaccine composition;

-the risk of introducing pathogens which are not completely inactivated compared to weakened or inactivated pathogens is eliminated;

-the vaccine can also comprise nucleotide sequences of heterologous species in order to break tolerance to self-antigens and to direct an immune response against self-antigens in a subject;

by simply exchanging the nucleotide sequence encoding the antigen, can be used for the treatment and/or prevention of a wide range of diseases and disorders; and

-allowing the introduction of immune modulatory molecules by genetic engineering of antigen presenting cells of the vaccine composition.

Other advantages afforded by the invention will be described in more detail below.

Brief description of the drawings

Objects and advantages of the present invention will be further better understood by reference to the following description and drawings.

FIG. 1 is a flow chart of a method of producing or preparing a vaccine composition according to the present invention;

FIG. 2 is a flow chart of a method for providing nucleotides in the vaccine provided in FIG. 1;

FIG. 3 is a flow chart of a method of providing APC in the vaccine provided in FIG. 1;

fig. 4 is a flow diagram of additional steps of the vaccine production process of fig. 1.

FIG. 5 is a schematic diagram showing a retroviral vector (RVV-mCD40L) containing the murine CD40 ligand (CD40L) gene constructed from a murine leukemia virus vector of Moloney (Moloney);

FIG. 6 is a diagram illustrating the transduction of DCs with RVV-mCD 40L. After repeated transduction and screening, DCs readily expressed CD40L on their cell surface, and more than 96% of DCs expressed CD40L, which were used in one example of the vaccine composition of the present invention;

figure 7A illustrates the expression of immune response activating molecules on parental BM185wt tumor cells.

FIG. 7B illustrates the expression of B220 molecules on DCs;

FIG. 8 illustrates the expression of CD8, CD11c, MHC class II (I-A), B7.1, B7.2 and CD40L molecules on D2SC/wt, genetically modified D2SC/CD40L and D2SC/GM-CSF cells;

FIG. 9 illustrates detection of expression of TLR9 protein by DCs using Western blot.

FIG. 10 schematically shows a portion of an empty pVAX-1 vector and a portion of a pVAX-e1a2 vector containing a minigene (minigene) sequence spanning the human e1a2 fusion region. In addition, the figure shows that the protein product encoded by the minigene sequence was examined by translational analysis of the transcriptional binding of the pVAX-1 and pVAX-e1a2 plasmid vectors in vitro;

FIG. 11A: the ability of D2SC/wt and D2SC/CD40L cells to induce allogeneic T cell proliferation is shown. The genetically modified DCs induced more than 8-fold proliferation of allogeneic T cells compared to unmodified parental DCs;

FIG. 11B illustrates that D2SC/wt and D2SC/CD40L direct autologous T cell proliferation. The genetically modified DCs induced more than 4-fold autologous T cell proliferation compared to the unmodified parental DCs.

FIG. 12.A illustrates the optimal dose for titration when using genetically modified DCs loaded with tumor lysate prior to injection for vaccination;

FIG. 12B illustrates treatment of long tumor mice by vaccination with genetically modified DCs of pulsed tumor lysate antigens alone;

FIG. 13 shows that tumor-specific CTLs are induced in long tumor mice by treatment with tumor lysates alone, or by pulsed tumor lysate of genetically modified DCs alone.

(a) Inoculation with D2SC/CD40L or D2SC/GM-CSF cells alone loaded with tumor lysate yielded tumor CTLs that specifically killed the parental BM185wt tumor cells. (b) Notably, tumor CTLs do not kill the cognate a20 lymphoma.

FIG. 14 schematically illustrates an example of a mouse inoculation model used in the present invention;

FIG. 15 illustrates the percentage of mice with lost tumor after vaccination with different vaccine components on mice with bcr/abl positive tumors. In addition, to test the efficacy and specificity of the induced immunoprotection, mice with tumor disappearance were challenged again with live parental BM185wt tumor cells.

FIG. 16 illustrates the use of pVAX-e1a2 and DC/CD40L to inoculate induced tumor-specific and e1a 2-specific CD8+ T cells;

FIG. 17 illustrates a comparison of the therapeutic efficacy of the vaccine compositions of the present invention in mice bearing bcr/abl positive tumors compared to other vaccine compositions;

figure 18.a illustrates the generation of tumor specific T cell responses in vivo following immunization with different vaccine strategies. CTLs formed from tumor-free mice are specific for direct control of parental tumor cells, BM185wt cells.

Figure 18B illustrates the response of generating tumor-specific T cells in vivo after vaccination with different vaccine components. CTLs extracted from tumor-depleted mice, produced in vitro culture, recognize the e1a2 peptide loaded on TAP-deficient RMA-S cells;

FIG. 19 illustrates the percentage of CD8+ CTLs produced following in vitro T cell expansion;

FIG. 20 illustrates specific CD8+ T cell recognition at H-2L^d: e1a2 peptide loaded on an Ig complex; and

FIG. 21 illustrates a hypothesis of a mechanism that may direct the effect described in the present invention.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein are to be understood by one of ordinary skill in the art to which this invention belongs.

The following provides references to a number of general definitions used in the present invention: singleton P and Sainsbury D, Dictionary of microbiology and Molecular biology, 3rd ed., 2002, Walker, The Cambridge Dictionary of science and technology, 1988, Rieger R, et al, eds., Glossary of genetics, 5The D., 1991, Hale WG and Marham JP, Harper Collins Dictionary of biology, 1991, Abbas A, et al, Cellular and Molecular biology, 2003, Blood 2001: 1: 587-600. for a clear understanding of the invention, the invention relates to the following definitions.

Unless otherwise indicated, the definition of "nucleic acid sequence" includes: double-and single-stranded DNA, oligonucleotides, cDNA and RNA hybrids like DNA-RNA, DNA-DNA are also included in the definition. Base modifications known to those of skill in the art, such as naturally occurring structural variations and synthetic non-naturally occurring analogs, are also included within the meaning of a nucleotide sequence or nucleic acid sequence.

An "immune response" refers to a collective and synergistic response to the invasion of foreign substances that occurs on an individual mediated by cells and molecules of the immune system.

The "immune system" refers to molecules, cells, tissues and organs that provide collective functions of immunity or protection against foreign organisms.

"CpG motifs" (CpG-motifs) refer to unmethylated "CpG dinucleotides" or "CpG motifs" such as in bacterial, yeast, insect and neomatode DNA. CpG motifs have been identified on a number of bacterial plasmids and they are potential adjuvants for DNA vaccines (Immunol. today 1998, 19: 89-97). CpG-DNA is now known to be a potent Th1-like adjuvant which not only promotes cross-activation of proteins or polypeptides by MHC class I restricted CTLs (cross-priming), but also triggers a Th1 mediated antibody response (Immunity. 200114: 499-502). In other aspects, DNA sequences that do not contain CpG motifs have an immune system suppressive effect (Arthritis and rhematic. 2003, 48: 1701-1707).

"P2 receptors (P2 receptors)" refers to receptors for extracellular nucleotides. The P2receptor is divided into two subtypes: g protein-coupled receptors (P2Y) and ligand-gated ion channel receptors (P2X) (Curr Opin Cell biol.1996, 8: 474-483).

"Antigen Presenting Cell (APC)" refers to a cell having antigen processing and antigen presenting functions. These APCs display peptide fragments of the protein antigen bound to MHC molecules on their cell surface and activate antigen-specific T cells. In addition to showing peptide fragment-MHC binding, APCs also express costimulatory molecules that are optimized for T cell activation. In particular, APC refers specifically to professional APC, including DCs, IPCs, NIPCs, monocytes, macrophages, T cells and B cells, especially pDCs, IPCs, NIPCs, and professional APCs with pDCs/IPCs/NIPCs characteristics and functions, such as cells that express TLR9 and produce or secrete type I interferon and further secrete TNF-a upon microbial stimulation.

"modified APCs" refers to antigen presenting cells that acquire genetic or protein information by treatment with viral vectors or by non-viral vectors. The result of the genetic modification is that the gene or protein material enters or binds to the antigen presenting cell and is expressed there.

According to one aspect of the invention there is provided a vaccine composition comprising isolated or substantially purified heterologous nucleotide sequences or nucleic acid sequences encoding antigenic molecules and genetically modified Antigen Presenting Cells (APCs).

The vaccine composition is preferably provided as a mixture of the nucleotide sequence and the APCs. For example, the nucleotide sequence and APCs are preferably pre-mixed and incubated prior to introduction into the subject. The vaccine composition of the present invention achieves an enhanced immune response and superior therapeutic and protective effects compared to previous DNA vaccines, particularly in the re-challenge of destroying existing cancer cells and protecting the host against tumor cells.

The antigenic molecules, RNA or more preferably antigenic peptides/proteins bound to MHC encoded by the nucleotide sequences of the present invention, when introduced into a subject, generate an immune response to the antigen. The antigen is preferably an immunogenic molecule, an immunogenic fragment of a molecule, such as an immunogenic protein, peptide or ribonucleic acid molecule or fragment thereof.

The nucleic acid-based vaccines of the present invention may be monovalent or multivalent vaccines. In the construction of a monovalent vaccine, the nucleotide sequence encodes one antigenic molecule, while in the construction of a multivalent vaccine, the nucleotide sequence comprises at least one heterologous gene encoding a multiple antigen of a heterologous or homologous antigen. Thus, the introduction and presentation of multiple antigens by multivalent vaccine injection into a subject results in the activation and recognition of antigen-specific T cells of different antigens. The invention also includes several repeated copies of the antigen sequence, e.g., provided as a diploid or triploid, etc.

The nucleotide sequences of the vaccine components also include other immune co-stimulatory or regulatory sequences, such as gene sequences encoding protein or peptide molecules having an immune response modulating effect. Co-stimulatory DNA sequences, such as unmethylated CpG motifs may also be included in the nucleotide sequence.

The nucleotide sequences of the present invention are preferably, but not limited to, naked DNA or RNA that are input together, preferably in admixture with genetically modified APCs. If provided as an RNA sequence, the nucleic acid sequence also includes motifs that allow translation in the subject (recipient) cell. Likewise, if DNA is provided, the nucleic acid sequence includes motifs such as promoters, possible enhancers, and/or other elements that regulate transcription and translation of the nucleotide sequence.

However, the nucleotide sequence encoding the antigen is preferably included in a vector having transcriptional control of a promoter, for example, in an expression cassette of a vector containing an expression control sequence. In addition, the vector or vectors containing the expression control sequences preferably include other regulatory sequences necessary and desirable for transcription/translation of the nucleotide sequence, including but not limited to polyadenylation sequences, transcription sequences and enhancers. The promoter or enhancer contained in the vector may be cell-type-specific or tissue-specific. The promoter may be inducible or selectively activated depending on experimental design or vaccine construction. Examples of suitable promoters for inoculation into humans include viral promoters such as the Cytomegalovirus (CMV) promoter. The vectors of the present invention may be microbial vectors or non-microbial vectors.

An example of a carrier according to the present invention is a liposome. Various cationic lipid forms have been used to introduce DNA into cells. Insertion of derivatives of polyethylene glycol into lipid membranes or liposomes may increase the circulating half-life of the liposomes after intravenous infusion.

Another class of synthetic carriers that is being actively investigated are cationic polymers. The general principle is the formation of a complex between a positively charged polymer and a negatively charged DNA molecule. Cationic polymers are highly effective at coagulating DNA as compared to cationic lipids. Examples of polymers which are preferred for gene delivery are poly-L-lysine, Polyethylenimine (PEI), and polyglucosamines (polyglucosamines), and liposomes (polylipids).

Also, small particles, such as nanoparticles (nanoparticles), can be used as the carrier of the present invention.

More preferred vectors of the invention are DNA plasmid vectors encoding MHC-bound antigens. The backbone of the plasmid DNA preferably comprises an immunomodulatory sequence or cofactor having mitogenic activity. The use of the bacterial DNA plasmid vectors of the present invention, whether naked DNA or embedded in liposomes or cationic polymers, or small particles, provides further significant advantages. These plasmid DNA vectors also include immunostimulatory CpG nucleotide sequences. Thus, in addition to delivering and expressing the antigen of the invention on a subject, the plasmid vector can stimulate an immune response in the subject.

In addition, viral systems can also be used to deliver and subsequently produce the antigens or immune modulatory molecules of the invention. Viruses are attractive vectors for delivering nucleotide sequences because they develop specific and efficient methods for entering host cells and expressing their genes. A major challenge in the development of viral vectors is the safety issue. Replication-defective viral vectors or replication-competent viral vectors are currently used for gene therapy. Gene delivery using viral vectors is referred to as transduction. There are at least four types of viral vectors in clinical trials to date: retroviruses, adenoviruses, herpes simplex viruses and adeno-associated viruses. Viruses in other studies include poxviruses, reoviruses (reoviruses), lentivirus, newcastle disease virus, alphaviruses and vesicular stomatitis virus, all of which can be used as vectors for use in the present invention.

The APCs of the present invention are cell types that are specialized in the processing and presentation of antigens by the immune system. The APCs of the present invention are preferably professional APCs, including but not limited to DCs, IPCs, NIPCs, macrophages, monocytes, B cells, Langherhans cells, mast cells, T cells, bone marrow derived cells, cells differentiated from stem cells, vascular endothelial cells and/or a wide variety of epithelial and bone marrow stromal cells. Combinations of at least two types of APCs may also be used in the present invention. The presently preferred types of APCs are pDCs, IPCs, NIPCs or have pDCs/IPCs/NIPCs characteristics, with the preferred characteristics of expressing TLR9 and inducible production of type I Interferon (IFN). These cells play an important role in combating microbes and they can cross-activate host T cells and act as a link to innate and adaptive immune responses.

The dendritic cell forms of the present invention are preferably pDCs/IPCs, have high capacity for processing and presenting MHC-bound antigens, produce high levels of type I interferon when loaded with viral or bacterial DNA, and express the P2X7 receptor and toll-like receptors, such as TLR 9.

TLRs play an important role in the defense of a host against infection. TLRs recognize pathogenic bio-related molecules and signals responsible for activating the host defense system, in particular pro-inflammatory cytokines. TLR9 is particularly important to the present invention because the cellular type response to CpG-DNA is regulated by TLR 9. TLR9 is on the Endoplasmic Reticulum (ER) of Dendritic Cells (DCs) and macrophages. TLR9 does not trigger the endocytosis of CpG-DNA but rather activates the downstream endocytosis of DCs.

Thus, the APCs of the present invention are preferably selected from pDCs or other APCs having the above-described characteristics and functions.

The APCs in the vaccination composition of the invention are genetically modified APCs. Thus, the APCs are modified to express regulatory molecules, such as molecules that enhance or inhibit or induce tolerance or hypersensitivity of the immune response, induce apoptosis, and/or other cell survival modulating responses according to design or vaccination strategies. APCs may also be modified to increase antigen processing and presentation, activate APCs and/or enhance effector cell immune function. Thus, the genetic material is introduced or transferred into the APCs, either transiently expressed from an episomal location, or stably expressed by integration into the host genome of the APCs, or provided as a stable extrachromosomal element. Suitable genes for modifying APCs include cytokine genes, interleukin genes, adhesion molecules, interferon genes (e.g., type I interferons I IFN-a and IFN-b), chemokine genes, chemokine receptor genes, anti-apoptotic genes, and genes encoding various immune co-stimulatory molecules, immune modulatory molecules, ligands (e.g., CD40L), and receptors and pharmaceutically acceptable carriers.

For example, CD40 ligand plays an important role in participating in the adaptive immune response. CD40 has become a key signal for B cell, monocyte and DCs cell function. Following antigen stimulation and co-activation with DCs, CD40L (CD154) is expressed in activated T cells. CD40-CD40L interactions result in activation and differentiation of DCs. Upon activation of CD40-CD40L, DCs acquired the ability to induce high levels of cytokine IL-12 production, polarizing CD4+ T cells to develop Th1 type, increasing CD8+ T cell proliferation and activating NK cells. Thus, the CD40-CD40L interaction acts as a trigger for co-agonist expression and efficient T cell activation in an adaptive immune response. Thus, the APCs of the invention can provide high efficiency CD40 expression or CD40L expression through genetic modification.

Suitable gene delivery protocols for modifying APCs include, but are not limited to, viral and non-viral methods. Examples of viral vectors that can be used include, but are not limited to, retroviruses, adenoviruses, papules and adeno-associated viruses, vaccinia viruses (vaccina viruses), herpes simplex viruses, and lentiviruses. Non-viral delivery of genes into APCs includes, but is not limited to, plasmid DNA transfer, liposomes, electropulse (electroporation) microinjection, and vectors of microbial origin and toxins obtained from microorganisms.

In addition, APCs better express other cell surface molecules including, but not limited to, adhesion and co-stimulatory molecules required for efficient activation of T cells and other types of immune cells. In addition, the APCs of the present invention better express chemokines and chemokine receptors and the FLIt3 ligand.

In particular, the APCs of the invention may be obtained from a subject and preferably, but not limited to, the same subject administered with the composition, e.g., using autologous APCs. Alternatively, allogeneic or syngeneic APCs (from the same twin of the subject) are also included.

In another embodiment, the APCs can optionally be enriched or purified and/or amplified by methods known in the art, either in vitro or in vivo. For example, without limitation, APCs are activated and differentiated from stem and progenitor cells of peripheral blood, cord blood, or bone marrow origin in the presence of cytokines.

In a further embodiment, the vaccine component is provided as a pre-treated mixture comprising a nucleotide sequence encoding the foreign antigen and the genetically modified APCs.

After the APCs take up or endocytose the nucleotide sequence, the nucleotide sequence is processed. The CpG motifs are preferably included in the nucleotide sequences that activate the toll-like receptor pathway in APCs, and stimulate the production of type I IFN-a and IFN-b, as well as the presentation of the encoded antigen. This event may occur during the incubation of the nucleotide sequence and modified APCs, prior to injection of the vaccine composition. Thus, after incubation, the modified APCs, presenting MHC-bound antigen and expressing or secreting immune co-stimulatory molecules, directly or indirectly activate the initial APCs when the subject is vaccinated. The modified APCs used in the present invention can then colonize lymphoid organs, directly or indirectly activating naive T cells, B cells and DCs, thereby achieving a more rapid, enhanced and efficient treatment against and protective immune response to foreign antigens.

Thus, the mixed nucleotide sequences and modified APCs of the present invention are the preferred and novel steps for achieving high efficiency of vaccines.

In addition, once injected into a subject, the nucleotide sequence or a vector containing the nucleotide sequence will be ingested by and expressed in the cells of the subject. Subsequently, the synthesized antigenic molecules are processed in the cytosol by the proteasome (proteosomes) into polypeptides.

In addition, after vaccine delivery, professional APCs can directly acquire antigen or release antigen from other transduced cells. Cytolysis of the transfection vector or nucleotide sequence of the present invention results in the release of the encoded antigen for uptake by APCs.

The nucleotide sequences encoding the antigenic molecules and the (genetically) modified APCs of the present vaccine composition are provided and introduced into an isotonic solution, preferably a buffered solution, or into a pharmaceutically acceptable solution, a gel, suitable for injection into a subject, preferably a human. An example of such a solution is phosphate-buffered saline (PBS).

The vaccination solution of the invention may comprise additional molecules in addition to the nucleotide sequence and the APCs. These additional molecules include regulatory molecules which can modulate (enhance or suppress) the immune response of a subject, increase the antigen presentation of APCs, stimulate the secretion of Th1 or Th2 cytokines, activate APCs, Langherhans cells, effector cells and/or enhance the immune function of APCs, cytokines, adhesion molecules, heat shock proteins and chemokines, such as interleukin 1(interleukin-1, IL-1), IL-2, IL-4, IL-6, IL-12, TNF α, granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), IFN gamma, type I IFN-alpha, IFN-beta, heat shock protein (hsp)70, hsp90, gp96, CD40L and B7, carriers and adjuvants.

The solution may also include carriers and adjuvants that modulate the immune response, increase antigen presentation, redirect the vaccine against the immune system and/or promote DNA entry into cells. Adjuvants include, but are not limited to, mineral salt adjuvants or mineral salt gel adjuvants, particulate adjuvants, toxins, particulate adjuvants, mucosal adjuvants, and immunostimulatory adjuvants. Examples of adjuvants include aluminium hydroxide, aluminium phosphate gel, Freund's complete adjuvant, Freund's incomplete adjuvant, bacterial superantigens, squalene or squalene oil-in-water (oil-in-water) adjuvant forms, biodegradable and biocompatible polyesters, polymeric liposomes, triterpenoid glycosides or saponins, N-acetyl-muramyl-L-threo-D-isoglutamine (N-acetyl-muramyl-L-threonyl-D-isoglumin), LPS and monophosphoryl lipid A and inactive microorganisms.

It is another object of the invention to generate an immune response in a subject using a vaccine. In this case, the vaccine composition, including the nucleotide sequence encoding the antigenic molecule and the genetically modified APCs, is administered to a subject, preferably a mammalian subject and more preferably a human subject.

In a further embodiment of the invention is a method of treating and/or preventing a disease in a subject, preferably a mammal, most preferably a human, by injecting into the subject an effective amount of a vaccine composition according to the invention, as required to treat and/or prevent the disease in the subject.

The invention is also directed to a vaccine composition comprising a nucleotide sequence encoding an antigen and genetically modified APCs for use as a medicament. In other embodiments, the invention teaches the use of a vaccine component for the manufacture of a medicament for the treatment or prevention of an infectious disease, wherein the nucleotide sequence encoding an antigen associated with an infectious agent is disease-intervening. Yet another embodiment is the use of a vaccine component for the manufacture of a medicament for the treatment or prevention of cancer, the nucleotide sequence encoding a tumor associated antigen expressed by a cancer cell.

The vaccine components of the present invention for treating or preventing infectious diseases are caused by infectious agents including, but not limited to, viruses, bacteria, fungi, protozoa, and parasites. Regardless, the vaccine component contains a nucleotide sequence that encodes an antigenic molecule associated with the pathogenic microbe. Furthermore, this antigenic molecule is better recognized as a non-self by the immune system of the vaccinated subject.

Viral diseases can be treated or prevented by the vaccines of the present invention, including diseases caused by the following viruses: adenovirus, arbovirus, coxsackievirus, cytomegalovirus, echinovirus (echinoviruses), echinovirus (echinovirus), hantavirus (hantavirus), hepatitis a virus, hepatitis b virus, hepatitis c virus, herpes simplex I virus, herpes simplex II virus, Alzheimer's Disease Virus (ADV), HIV types I and II (HIVenv protein can also be used as antigenic molecule), influenza virus (NP antigen can be used as antigenic molecule), measles virus, mumps virus, papilloma virus (papilloma), papova virus (papova), polio virus, respiratory syncytial virus (syncytial), rhinovirus (rhinovirus), bovine plague (rinpedersest), diarrhea-causing rotavirus (rotavirus), rubella virus and varicella.

Infectious diseases can also be prevented and/or treated with the vaccine composition of the present invention, and examples of infectious diseases are: legionella, mycobacteria (hsp65 antigen may be used as a mycobacterium tuberculosis antigen molecule), mycoplasm, neisseria and rickettsia.

The vaccine compositions of the present invention may also be used to treat protozoan diseases including those caused by kokzidioa, leishmania and trypanosoma (trypanosoma). However, the corresponding parasitic diseases can be caused by chlamydia, malarial parasites, rickettsia and leishmania major murine infections (the antigenic molecule may be the LACK antigen).

The vaccine composition of the present invention is also well suited for the prevention and/or treatment of cancer. The nucleotide sequence in the vaccine encodes an antigenic molecule that is associated with a tumor in a particular cancer type.

Many cancers are characterized by gene or chromosome translocations in cancer cells. Such translocation causes the joining of two or more coding sequences or portions, which then form a hybrid or fusion protein or polypeptide. Such hybrid proteins may be recognized by the patient's immune system as non-self and thus antigenic. A typical example of a most distinctive feature is the hybridization of cancer genes caused by translocations, derived from Philadelphia (pH)¹) Chromosomal translocations occur in patients with Chronic Myelogenous Leukemia (CML) and Acute Lymphoblastic Leukemia (ALL). A translocation between chromosome 22 and chromosome 9 forms the philadelphia chromosome of the synthetic bcr-abl fusion transcript. In ALL, the breakpoint occurs at the first intron of the bcr gene to form the e1a2 fusion gene and to generate a 185kDa tyrosine kinase with oncogenic activity.

In addition, many cancers are characterized by abnormal expression of specific genes and gene products. Tumor-associated antigens are expressed not only in tumors but also in normal tissues of the host. In order to induce an effective anti-tumor immune response, vaccination must overcome immune tolerance to self-antigens. An alternative method of the invention is to select antigens of heterologous origin, for example, by providing a DNA plasmid vector containing a nucleotide sequence encoding a tumor associated antigen to break immune tolerance to the autoantigen and induce anti-tumor immunity. In addition, the immune system contains autoreactive T cells and B cells that are not removed from the development of the immune system. Autoreactive lymphocytes can be triggered by cross-reactivity between species. Cross-reactive immunity to mouse self-antigens can be induced by immunization with heterologous DNA followed by recognition of the corresponding human protein. Thus, to obtain an immune response in a subject, the nucleotide sequence encoding the autoantigen is preferably provided in a heterologous vector. For example, heterologous DNA plasmid vectors (containing heterologous sequences, heterologous gene sequences, foreign gene sequences) are preferred to gene sequences encoding heterologous proteins and polypeptides in order to break tolerance to autoantigens.

In non-limiting examples of tumor-specific or tumor-associated antigens, coding sequences useful in the vaccine compositions of the present invention include KS 1/4 whole cancer antigen, ovarian cancer antigen (CA125), prostatic acid phosphate, prostate-specific antigen, melanoma-associated antigen p97, melanoma antigen gp75, heavy polymer melanoma antigen, the MAGE family of antigens, T cell receptor gamma chain selectable reading frame protein (TARP) antigen, prostate-specific membrane antigen and e1a2 fusion protein antigen and bcr/abl fusion protein.

Vaccines for the treatment of melanoma, pancreatic cancer, breast cancer and prostate cancer are currently in use in clinical trials. Superior results are obtained with the vaccine compositions of the present invention for these cancer types compared to previous cancer vaccines. Further examples of non-limiting cancers to be treated and/or prevented with the vaccine composition of the invention are the following types of cancers: human sarcomas and carcinomas such as, for example, fibroma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, hemangioma, sarcomatosis, lymphangioma, lymphangiosarcoma, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenoma, medullary carcinoma, bronchial cancer, renal cell carcinoma, hepatocellular carcinoma, choriocarcinoma, seminoma, embryonic carcinoma, Wilms' tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioblastoma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, angioma, neuroblastoma, adenocarcinomas, neuroblastoma, neuro, Oligodendroglioma, meningioglioma, melanoma, neuroblastoma, and retinoblastoma; leukemias, for example, Acute Lymphoblastic Leukemia (ALL), and acute myelogenous leukemia (myeloblasts, promyelocytes, myelomonocytic cells, monocytes and erythrocytes), chronic leukemia (chronic monocytic leukemia, chronic intragranular leukemia and chronic intralymphocytic leukemia), essential erythrocytosis, lymphomas (hodgkin's and non-hodgkin's disease), multiple myeloma, Waldenstr (Waldenstr * m's) macroglobulinemia, heavy chain disease; cancer caused by viruses.

The vaccine composition of the present invention can be used for eliminating existing tumors or pathogenic organisms, and treating cancers or infectious diseases. However, the vaccine composition may also or alternatively be used to protect a subject, preferably a mammal, most preferably a human, from disease encounter or from recurrence of a challenge or tumor cell or recurrence of a pathogenic infectious agent (microbe). In this case, the vaccine is used to prevent cancer or infectious diseases, for example, having a disease-preventing property.

Various forms of allergy and hypersensitivity can be treated by the present invention by using the appropriate nucleotide sequence encoding the antigen in a carrier. The use of the sequence depends on the type of allergy and can be chosen by the person skilled in the art. For example, for peanut allergy, the vector may comprise a nucleotide sequence encoding the Arah 2 antigen.

The vaccine components of the present invention may be modified to treat type I diabetes, but are not limited to, comprising all or part of the nucleotide sequence encoding insulin. The vaccine composition can also be used for treating patients with senile dementia, different blood diseases, hereditary diseases, diseases accompanied or obtained by transplantation and acquired immunodeficiency thereof.

The vaccine compositions of the present invention may be introduced into a subject, preferably a mammal, and most preferably a human, using an appropriate and clinically acceptable route of administration, including, but not limited to, subcutaneous, intramuscular, intraarterial, intravenous, intravascular, oral, intradermal, intraperitoneal, direct injection into lymph nodes and injection into tumors. The nucleotide sequence encoding the antigenic molecule and the genetically modified APCs are preferably pre-mixed prior to introduction, e.g., a mixture of the two components of the vaccine composition of the invention.

Vaccines can be introduced by all conventional means, including, but not limited to, syringes, tube trocars, catheters, electroporation, needleless introduction, and the like.

The dosage introduced depends on the condition and size of the subject to be treated or vaccinated and on the number of vaccine components introduced, the frequency of introduction, the route of introduction, the type of therapy, such as treatment and/or prevention, the type of disease to be treated or vaccinated. The continued treatment regimen, including site, dose and frequency, may be determined by the subject's initial response and clinical assessment. However, in the treatment of cancer, it is better to administer at least three repetitive vaccinations. The amount of vaccine component used can be determined by methods well known in the art using the results of dose response tests on animals.

Yet another aspect of the invention is to provide a kit prepared by the vaccination method of the invention. The first embodied kit comprises a container containing a mixture of nucleotide sequences encoding antigenic molecules and genetically modified APCs. The antigenic molecules are preferably associated with a disease, such as an infectious disease or cancer, and the subject is treated or prevented by injecting the contents of the kit, preferably the subject is a mammal and most preferably the subject is a human.

Other embodied kits comprise a first container comprising a nucleotide sequence encoding an antigenic molecule and a second container comprising an article of manufacture of genetically modified APCs. It is preferred that the contents of the first and second containers are intermixed prior to introduction into the subject, i.e. either the contents of the first container are added to the contents of the second container, or the contents of the second container are added to the contents of the first container or the contents of the first and second containers are added separately to a third container provided for mixing. The mixture may be incubated together prior to injection into a subject, preferably a mammal and most preferably a human.

The kits of the invention comprise a vaccine component formulated (to treat or prevent a disease or disorder (disorder), such as an infectious disease, cancer, autoimmune disease, allergy, or diabetes). The kit may include at least one container, and more preferably a container containing genetically modified APCs, and may also include additional substances and adjuvants to enhance the antigen presentation of the APCs, activate the APCs, and/or enhance the immune function of the APCs.

It is a further object of the invention to provide a method for producing a vaccine composition. As shown in the flowchart of FIG. 1, at step S1, a nucleotide sequence encoding an antigenic molecule is provided (to induce a desired immune response). Genetically modified APCs, such as DCs, pDCs, IPCs, macrophages, monocytes and/or B cells, are provided at step S2. At step S3, the nucleotide sequences and APCs are mixed to complete the method and to end the production of the vaccine composition of the invention.

FIG. 2 more specifically illustrates the provision of the nucleotide sequence of step S1 in FIG. 1. In optional step S11, a nucleotide sequence encoding an antigenic molecule associated with a disease or disorder that is treated or prevented by the vaccine component is identified and isolated. This identification, and at least part of the identification, can be queried using MHC-binding peptide motifs as known in the art. Methods known in the art, including chemical synthesis of DNA sequences and PCR (polymerase chain reaction), can be used to obtain the relevant nucleotide sequences. The identified and isolated nucleotide sequence is then cloned into a suitable vector at step S12. The vector is selected to be suitable for introduction into a subject and once inside the subject, to express the nucleotide sequence and subsequently obtain the desired antigenic molecule. The resulting vector is then propagated in step S13, i.e., within the host cell, in vitro (in vitro), etc.

Step S2 of providing APC in FIG. 1 is illustrated in more detail in FIG. 3. In step S21, the APCs are preferably isolated from the subject to which the vaccine composition is administered or from another source as previously described. At this step S21, a particularly advantageous APC subclass, such as the DCs or DC subclasses, can be selected and isolated. In step S22, the APCs are (genetically) modified. In this case, one or more genes, such as the CD40L gene, encoding molecules that modulate immune response, are introduced into the antigen presenting cells as extra chromosomal elements or inserted into the chromosomes of APCs.

In a further step S31 in fig. 4, additional substances are added to the carrier-APCs mixture. These agents include at least cytokines, adhesion molecules, chemokines, heat shock proteins and the aforementioned adjuvants that generally modulate the immune response and immune cells of a subject receiving the vaccine composition and/or enhance the immune effects of effector cells and APCs. The mixture of nucleotide sequences and modified APCs may also be supplemented with additional substances, preferably by pre-incubation prior to use as a vaccine in step S32, the physical conditions of incubation being determined by one skilled in the art. In order to increase the endocytosis of the nucleotide sequence, the incubation preferably lasts at least 5 minutes, more preferably at least several hours. The incubation time is typically dictated by other physiological factors, such as incubation temperature. Generally, overnight, for example to 24 hours, is appropriate, but not limited to, until the time of incubation is limited. The temperature during incubation is preferably at least 4 deg.C, more preferably room temperature (about 20-25 deg.C) or about 37 deg.C. The pH of the incubation solution is preferably neutral or slightly acidic. It is preferable to force the combination of the nucleotide sequences (plasmids) and APCs by co-centrifugation to enhance endocytosis. Or by other techniques, or by addition of other techniques, such as lipofection agents.

The following is an example of vaccination against acute leukemia with the vaccine composition of the present invention. Thus, the nucleotide sequence encodes an antigenic molecule that is associated with this type of cancer. However, it will be appreciated by those skilled in the art that the invention is not limited to this particular disease or antigen and may be used to treat and/or prevent any of the diseases or disorders mentioned above.

Examples

The vaccine compositions of the present invention are compared to a wide variety of other vaccination methods, including vaccination with antigenic peptides and the encoded antigenic peptide plasmid DNA.

Cell lines

A20(H-2^d) Is a B cell lymphoma cell line obtained from BALB/c mice and used as a CTL target in the present study. A20 cells express B220, MHC-I, MHC-II and CD19 molecules.

Tumor cells (BM185wt, H-2)^d) Is a mouse acute leukemia cell line (pre-B ALL). It was established by transducing BALB/c mouse bone marrow cells with a retroviral vector encoding a 185kDa bcr/abl oncoprotein from humans (Cell 1995, 82: 981-988). BM185wt cells express B220, CD19 and MHC-I molecules on their cell surface.

D2SC/wt cells (or D2SC/1) were provided by the heart of Dr. Paola Paglia. D2SC/wt, (H-2) used in the present invention^d) Is obtained by immortalizing a spleen dendritic cell of a BALB/c mouse by a retrovirus. (J.Immunol. methods 1994, 174: 269-279) D2SC/wt cells mostly showed immature DC morphology, immunophenotypic and functional attributes, including constitutive expression of MHC group II molecules (low), costimulatory molecules B7/BB1, thermostable Ag, and ICAM-1, and potent Ag presentation functions (J.Immunol methods 1994, 174: 269-279, J.Immunol.1999, 162 (7): 3757-3760).

RMA-S is a TAP2 deficient tumor cell line. It was established from T-cell lymphoma RBL-5 induced by the Lousser leukemia virus from B6 mice (Nature (London)1986, 319: 675-678).

Cell culture

DCs cells were cultured in IMDM medium and supplemented with 10% heat-inactivated Fetal Calf Serum (FCS) (GibcoBRL, Life Technologies Ltd., Scotland, UK), 2mM L-glutamate, 100IU/ml penicillin/streptomycin, 10mM hepes and 5X10^-5M2-mercaptoethanol. Cells were incubated at 37 ℃ in humidified air with 7% carbon dioxide.

Tumor cells were cultured in RPMI-1640 medium (ICN Biomedicals, Inc. Costa Mesa, CA) supplemented with heat-inactivated Fetal Calf Serum (FCS) (GibcoBRL, Life Technologies Ltd. Scotland, UK), 2mM L-glutamate, 100IU/ml penicillin/streptomycin, 10mM hepes, 0.1mM sodium pyruvate, 5X10-5M 2-mercaptoethanol, and 10mM non-essential amino acids. Cells were incubated at 37 ℃ in humidified air with 5% carbon dioxide.

Search Synthesis of MHC-binding peptide motifs

Amino acid sequence of ALL-specific e1a2 fusion protein for screening against mouse MHC-I antigen (H-2K)^d) Binding capacity of (table I) (HLA polypeptide binding prediction program developed by doctor Kenneth Parker, http: v/bimas.dcrt.nih.gov/molbio/hla _ bind). A nine amino acid sequence, AFHGDAEAL, located in the junction region of the e1a2 fusion protein (Table I and FIG. 10), for binding mouse H-2K^dA high score is displayed. The e1a 2-covering mini-protein polypeptide was synthesized by standard methods and purified by High Pressure Liquid Chromatography (HPLC). High scoring binding peptides (AFHGDAEAL, SEQ ID NO: 5, considered as e1a2 peptide) and low scoring binding peptides (HGDAEALQR, SEQ ID NO: 6, considered as peptide 8) were used in the experiments. Peptides K (ATGFKQSSK, SEQ ID NO: 7) and H-2K^dNot bound and used as control peptides.

Table I below illustrates the results of using peptide binding prediction programs to search for HLA-peptide motifs, and using parameters to score binding above 500, which is considered to be a very high score.

TABLE 1

Usage parameters and scoring information
					Method for limiting the selection of numerical results the numerical selection of HLA molecule typesSelection of column score for length of input sequence selected echogenic Format of echogenic user input peptide sequence length subsequences score calculated number first subsequence scored number reported on score output table			Line 1799 to define the numerical 20Kd9Y numbering
Scored results
					Grade	Starting position	List of subsequence residues		Scoring device
123456789	379285146	AFHGDAEALDAEALQRPVEALQRPVASGAFHGDAEAAEALQRPVAHGDAEALQREGAFHGDAEFHGDAEALQGDAEALQRP		1152.00014.4002.0001.4400.1200.1200.1000.0120.012

Genetically modified DCs

Retrovirus-based gene transduction systems have been developed. The retrovirus provirus was treated to remove all gag, pol and env genes, leaving only the 3 '-and 5' -LTRs. Defective retroviral vectors can be obtained from the supernatant of a packaging cell line.

In the present invention, retroviral vectors (see FIG. 5), (obtained from MFG-mGMCSF packaging cell line (Dr. Richard Muligan gift, Children's Hospital, Harvard Medical School, USA)) and Moloney murine leukemia viral vector (MoMLV), (PG1a. mu. CD40L (Dr. M. Brenner gift, Baylor College of medicine, USA)) were used to modify DCs.

DCs used in the present invention are modified to release GM-CSF or express CD40L molecule on their cell surface. CD40L gene or GM-CSF was transferred to DCs using either mucD40L or MFG-mGMCSF retroviral vectors. DCs used in the present invention are modified to release GM-CSF or express CD40L molecule on their cell surface. D2SC/wt cells were transduced by repeated spin centrifugation in the presence of polybrene (10. mu.g/ml, Sigma). Briefly, 10⁵The individual cells were suspended in 0.5ml of virus supernatant and in the coagulant amine, and the cells and virus were co-centrifuged at 10000rpm for 60 minutes at room temperature. After centrifugation, the supernatant was removed and the cells were suspended in fresh medium and incubated in humidified air at 37 ℃ and 7% carbon dioxide for a second transduction after 24 hours. The transduction efficiency of DCs was significantly improved by repeated centrifugation. The corresponding percentage of cells expressing the CD40L molecule after multiple rounds of transduction is illustrated in figure 6.

After repeated transduction, approximately 70-80% of the DCs expressed the CD40L transgene. D2SC/CD40L cells were further screened for expression of the transgene. And approximately 96% of the DCs expressed the CD40L gene product. CD40L gene expression remained stable for many years and stable even after repeated thawing-freezing procedures. DCs express CD40L specifically on their cell surface. See figure 6 below.

According to the manufacturer's instructions (Cytoscreen^TMImmuneasay kit, Biosource int., California, USA, BD Bioscience, U.S.A.), production of IL-12, GM-CSF, IFN- γ in DCs cell culture supernatants after D2SC/wt and CD40L and GM-CSF gene transduction was assessed by ELISA methods. GM-CSF secretion in cell culture supernatants of D2SC/GM-CSF cells was 11, 370pg/ml/106 cells/24 h.

Immunophenotypic characterization of D2SC/wt and genetically modified D2SC/CD40L and D2SC/GM-CSF DCs tumor cells

Cells andmonoclonal antibodies against surface molecules from mice were cultured together (2X 10)⁵Cells/0.5. mu.g mAbs, BD, Pharmingen, San Diego, Calif.). The following mAbs were used: CD40-FITC (fluorescein isothiocyanate conjugated mAB), CD40L-PE (thiolated phycoerythrin), I-A-FITC, H-2K^dFITC, B7.1-FITC, B7.2-PE, CD11 c-FITC, CD8 α -PE, B220-FTIC, B220-PE, Thy1.2-FITC, Thy1.1-PE, IgG1-FITC, IgG 2-PE. Cell phenotype was analyzed on a FACSCalibur flow cytometer (Becton Dickinson).

The immunophenotype of tumor cells (BM185/wt) is illustrated in FIG. 7A. BM185 cells express B220, CD19 and MHC-I, I-A molecules on their cell surface. However, BM185wt cells lacked expression of costimulatory and CD40 molecules (data not shown).

The expression of the B220 molecule in D2SC/wt cells can be seen in FIG. 7B. Thus, the D2SC/wt cells expressed markers (markers), CD8a +, CD11c + (see FIG. 8); b220+, is considered a mouse DCs subtype, a typical marker of plasma cellular dendritic cells (pDCs).

A comparison of the parental D2SC/wt cells and the genetically modified dendritic cell immunophenotype (D2SC/CD40L) and their descriptions can be found in FIG. 8. Parental D2SC/wt cells had an immature DC phenotype and expressed MHC-1, I-a, CD8 α, cd11.c, B7.1 and B7.2 on their cell surface as shown, but lacked expression of CD40 ligand. In contrast, CD40L was clearly expressed in D2SC/CD40L cells. However, IL-12 secretion was not detected in the culture of D2SC/CD40L cells. The in vitro (inv itro) growth kinetics of D2SC/CD40L cells were similar to their parental cells (data not shown).

Western blotting (Western Blot)

DCs were dissolved in buffer containing 1% NP40, 0.1% SDS, TBS and protease inhibitor cocktail (cocktail) according to manufacturer's instructions (Sigma). Cells were sonicated in buffer at 4 ℃. After centrifugation at 5000rpm for 5 minutes at 4 ℃, soluble proteins were collected and analyzed by western blotting. Proteins were isolated in 12% SDS polyacrylamide gels and transferred onto PVDF filter paper according to the manufacturer's instructions (BioRad, USA, Amersham Bioscience, Uppsala, Sweden). The primary polyclonal antibody, rabbit anti-mouse TLR9(Cat. No. IMG-431, Imgenex, San Diago, USA), was diluted 1: 1000 in TBS buffer before use (10mM Tris-Cl and 150mM NaCl, pH 8.0). The secondary antibody is a goat anti-rabbit antibody conjugated with horseradish peroxidase (HRP). The DCs of the present invention express the mouse TLR9 receptor as illustrated in FIG. 9.

D2SC/wt cells were fully functional and immature dendritic cells isolated from the spleen. D2SC/wt cells express several well-known markers, such as CD11c +, B220+, CD8 α +, MHC-II, found on a subset of DCs, pDCs^lowTLR9+ marker. In addition, D2SC/wt cells released much type I interferon- α and interferon- β when interacting with viral, bacterial and CpG DNA. It was possible to check that D2SC/wt cells expressed a very high copy number of type I interferon mRNA (Scand. J. Immunol.1997, 46: 235-41; ElorantaML., et al unpublished data) from in situ hybridization studies, leading to the conclusion that D2SC/wt cells are a pDC/IPCs subclass that corresponds to putative human natural interferon-producing cells in mice and are dendritic cells.

Plasmid DNA vector

Recent studies have shown that the empty plasmid pcDNA3 vector backbone contains a certain number of unmethylated CpG motifs. (Science 1996, 273: 352-354) high levels of interferon-. alpha.and IL-6 production were induced in porcine leukocytes by incubation of the porcine leukocytes with plasmid pcDNA3. Methylation of all cytidine at the CpG dinucleotide of pcDNA3 abolishes the ability to induce interferon-. alpha.production (Vet immunological Immunopathol.200, 78: 45-56).

pVAX-1 * was constructed modified from the pcDNA3.1 vector according to FDA recommendations (Invitrogen, CA, USA). pVAX-1 * is a 3.0kb plasmid vector designed for the development of human cancer vaccines.

Cloning of a minigene encoding e1a2 fusion peptide into a plasmid vector containing CpG motifs

PCR primers were designed to amplify a nucleotide sequence encoding a predetermined fusion peptide of e1a2 that binds to MHC-I type. The production of the minigenes was started with a fill-in reaction (Pharmacia), using the following overlapping primers: 5'-TGCTAGCATGATCTGGCCCAACGACGGCGAGGGCGCCTTCCACGGCGACGCCGAGGCCCTGCAGCGC-3' (see SEQ ID NO: 1) and 5'-AATCGATCACAGGCCCTGGGGCTCGAAGTCGCTGGCCACGGGGCGCTGCAGGGC-3' (see SEQ ID NO: 2). According to the manufacturer's recommendation, in addition to the two primers, reaction mixture including Klenow fragment (Escherichia coli DNA polymerase I), dNTPs and enzyme buffer. The reaction was carried out at room temperature for 1 hour, followed by PCR reaction using the same primers, adding PCR buffer, Taq DNA polymerase, dNTPs, and operating at 95 ℃ for 1 minute, followed by 35 cycles of 95 ℃ for 30 seconds, 65 ℃ for 30 seconds, and 72 ℃ for 30 seconds. The resulting PCR fragment was isolated and initially cloned into the pUC19 plasmid vector at Nhe I/Cla I position (NewEngland BioLabs). The Nhe I/Cla I fragment was then inserted into the pBK-CMV plasmid vector (Stratagene). The Nhe I/Kpn I fragment containing the e1a2 fusion minigene in pBK-CMV was finally cloned into the Nhe1 and Kpn1 positions of the pVAX-1 plasmid vector (Invitrogen, CA, USA). Once inserted into the vector, the e1a2 fusion minigene is placed under the control of the CMV promoter. The pVAX-e1a2 structure was confirmed by DNA digestion and DNA sequencing. The Plasmid was amplified with the Qiagen EndoFree Plasmid Maga Maga kit (Qiagen, Santas Clarita, Calif., USA) according to the manufacturer's instructions. Plasmid DNA purity was determined by UV spectrophotometry and agarose gel electrophoresis. Purified DNA is available with an OD 260nm/OD 280nm absorbance ratio of greater than 1.9.

A diagram of the empty pVAX-1 vector and the vector pVAX-e1a2 containing the nucleotide sequence encoding the mini-e 1a2 fusion protein is shown in FIG. 10. The mini e1a2 fusion gene sequence and the corresponding polyamino acid sequence are shown in SEQ ID NO: 3 and SEQ ID NO: 4.

the size of the product produced by the plasmid construct pVAX-e1a2 was tested by an in vitro transcription coupled translation assay (TNT, Promega). Approximately 1. mu.g of plasmid DNA was incubated in a volume of 50. mu.l for 2 hours, 30 ℃ and the mixture contained 25. mu.l of rabbit reticulocyte lysate, 2. mu.l of TNT reaction buffer, 1. mu.l of TNT T T7RNA polymerase, 1. mu.l of a mixture of 1mM amino acid minus methionine, 4. mu.l (35S) of methionine 10mCi/ml and 1. mu.l of 40U/. mu.l of RNA RNAsin ribonuclease inhibitor. A3. mu.l volume of the reaction was loaded on a 16% SDS polyacrylamide gel. After drying, the gel was exposed to autoradiography. The results are shown in FIG. 10, where the e1a2 fusion protein is present only in pVAX-e1a2 and not in empty pVAX-1. In addition, pVAX-e1a2 transcription was detected in the COS-7 cell line following transfection with Lipofectin reagent (data not shown). In summary, pVAX-e1a2 plasmid DNA, containing a 96bp nucleotide sequence spanning the fusion region of the e1a2 gene, was transcribed and expressed in transfected cells.

Activation of naive T cells

A suspension of single cells was prepared from the spleen. T cells were isolated from spleens of BALB/C mice and C57BL/6J mice. T cells enriched with a Percoll gradient from the spleen of C57BL/6J mice were used as a source of allogeneic T cells. CD4+ or CD8+ T cells were isolated and purified from BALB/c mice using MACS CD4 or CD8 microBeads according to the manufacturer's instructions (Miltenyi Biotec, Germany). Stimulators (DCs) were added to human responder cells (spleen or T cells, 2X 10) in fractionated amounts after irradiation (50Gy, from 137Cs)⁵Cells/well) were performed on 96-well round bottom microplates (Becton, Dickinson). Experiments were performed in triplicate at a final capacity of 200. mu.l/well. The proliferation is measured by adding on the third day³H]Thymidine (1. mu. Ci/well) (Amersham International, Amersham, UK), incubated for 6 hours, followed by harvesting of the glass-fiber filter paper and loading into a scintillation counter (Beta counter, Pharmacia) for performance.

The genetic modification elicits the ability of immature DCs to stimulate T cells

As an immature DCs, unmodified D2SC/wt cells were poor T cell stimulators. Thus, the ability of D2SC/wt and D2SC/CD40L to stimulate proliferation of native allogeneic T cells was compared. FIG. 11A illustrates that CD40L genetically modified DCs (D2SC/CD40L) stimulated allogeneic T cell proliferation 8-fold higher compared to non-modified parent DCs (D2 SC/wt).

D2SC/wt was compared to the ability of D2SC/CD40L to induce proliferation of natural autologous T cells. FIG. 11B illustrates that the CD40L gene modified DCs (D2SC/CD40L) stimulated 4-fold higher proliferation of autologous T cells compared to the unmodified parent DCs (D2 SC/wt). DCs were modified to release GM-CSF (D2SC/GM-CSF) and also resulted in strong autologous and autologous T cell proliferation (data not shown). Thus, CD40 ligand expression on DCs is functionally active and involved in T cell stimulation. Clearly, D2SC/CD40L cells acquired the ability to sensitize autologous T cells directly after in vivo injection. Following injection under the skin, DCs were found to migrate into the spleen and lymph nodes (data not shown).

Preparation of tumor antigens

Autologous tumor lysates were prepared from BM185/wt cells. Tumor cells were treated with several repeated cycles of freezing (liquid nitrogen) -thawing (37C hot water bath). The tumor cell lysate was further sonicated at 4 ℃ for 30 minutes. The cell debris was removed by centrifugation at 2500rpm for 10 minutes at 4 ℃. Soluble proteins were collected and Protein content determined by the Bradford method (Protein Assay, Bio-Rad, CA, U.S.A.).

Pulsing DCs with tumor antigens or tumor antigen peptides

DCs and tumor cell lysates (100. mu.g/106 cells) were preincubated overnight at 37 ℃ in 7% carbon dioxide. DCs were detached from the flask with PBS/5mM EDTA and washed carefully prior to injection into mice. In the peptide pulse experiment, 106 cells were pulsed with 10. mu.M peptide in FCS-free medium at 37 ℃ in 5% carbon dioxide for 2 hours.

Immunization with vaccine Components

Female BALB/c mice (H-2d), 6-8 weeks of age, purchased from M & B (Denmark), and maintained in an animal house under standard conditions at the university of Uppsala. All animal experiments were performed as required by the animal care committee (ref. nr. C63/97, C36/1).

Immunocompetent syngeneic mice can be injected subcutaneously (s.c) with increasing doses of BM185/wt cells on a tumorigenic study. Injection of 500 tumor cells into aged mice at 6-8 weeks resulted in 100% death within 3-4 weeks (see FIG. 14 above). Lethal doses for injection (800 tumor cells/mouse) are commonly used in current studies.

Minimal doses of genetically modified DCs required for effective elimination of tumor outgrowth were sought. DCs were loaded with tumor cell lysate overnight prior to immunization. By 10⁶A single inoculation of D2SC/CD40L cells was found to be effective in treating tumor-bearing mice, see fig. 12. a.

Tumor-bearing mice were treated once (at the tumor site or at a distant location) with tumor cell lysate pulses D2SC/CD40L (p < 0.001) or D2SC/GM-CSF cells (p < 0.001). Immunization with DCs modified with CD40L or the GM-CSF gene induced a potent anti-tumor immune response. In fig. 12B, it is shown that a single vaccination has sufficiently eradicated pre-existing tumor cells and all treated mice have tumor eliminated (100%). This result represents one of four replicates.

The anti-tumor immune response induced is associated in vivo with T Cells (CTLs) that produce tumor-specific cytotoxins. CTLs proliferating in vitro killed parental BM185 tumor cells exclusively (fig. 13A), but did not kill the homologous a20 tumor cell line (fig. 13B).

These studies indicate that genetically modified DCs, D2SC/CD40L cells, have the ability to stimulate an effective therapeutic immune response in tumor-bearing mice.

An example of a tumor-bearing mouse model for therapeutic and prophylactic vaccination used in the present invention is illustrated in FIG. 14.

Pre-incubation of plasmid DNA and DCs

To improve plasmid binding to DCs and to promote endocytosis of plasmid DNA into DCs, co-centrifugation was developed. Briefly, genetically modified DCs were grown at the appropriate density in cell culture flasks and dissociated with PBS/5mM EDTA at 37 ℃ for 20 minutes. DCs were collected and washed with PBS (Ca +, Mg + free) and suspended in PBS. Plasmid DNA (1mg/ml) to 10⁷DCs/ml. They were co-centrifuged at 10000rpm for 30 minutes at room temperature. The plasmid DNA/DCs mixture was incubated for an additional 3 hours at 37 ℃ in 7% carbon dioxide prior to injection into mice after centrifugation.

CTL assay

After vaccination treatment, spleen and Lymph Nodes (LN) were isolated from normal or vaccinated mice in order to investigate the development of cytotoxic T Cells (CTLs) in vivo. Purified T cells were isolated by Percoll gradient method or magnetic activated cell sorting according to the manufacturer's instructions (Miltenyi Biotec, Germany) with MACS CD4 or CD8 MicroBeads. The samples were analyzed by BM185/wt (200Gy,¹³⁷cs γ — radiation) or BM185/CD40L (200Gy) restimulated T cells for 5-7 days. In the conventional 4 hours⁵¹Cr release cytotoxicity assay, dead cells were removed and live cells were used as effectors. Briefly, the target cells are first coated⁵¹Cr designation (25. mu. Ci/10)⁶Cells) were at 37 ℃ for 2 hours. Viable effector cells were incubated with pre-labeled target cells for 4 hours at different effector-to-target ratios. By⁵¹Cr was released into the culture broth to measure cell lysis, and the results represent triplicate samples.

The percentage of specific lysis was calculated according to the following formula:

concanavalin a (con a) -activated T blasts

In control experiments, from normalSpleen cells isolated from mice at 2X 10⁶Cells/ml were incubated for 48 hours in RPMI1640 medium with penicillin/streptomycin, 10% FCS, 10mM Hepes, 3X 10^-5M2-mercaptoethanol and 3. mu.g/ml ConA (Sigma). Con A-activated T blasts were used as effector cells against tumor cells (negative control).

Statistical analysis

All animal experiments had at least five or ten animals per group. All studies were repeated at least three times to approximate the results. Analysis of survival differences is the Chi-square test (Chi-square test) to explain the significance of differences between experimental groups.

Investigation of DNA and DCs inoculation efficiency

On day 0, each mouse was injected subcutaneously on the right side (5 mice/group) with a lethal dose of parental BM185/wt tumor cells. Mice were immunized at tumor sites on days 7, 14, and 21 with (1) PBS; (2) empty pVAX-1 vector (100. mu.g/mouse); (3) pVAX-e1a2 (100. mu.g/mouse); (4) d2SC/CD40L alone had no antigen; (5) preincubated pVAX-e1a2 plasmid DNA and D2SC/CD40L cells (10)⁶Cells/100. mu.g plasmid DNA/mouse). Tumor size was measured twice weekly. When tumors were detected, mice were considered to be at the survival endpoint and sacrificed.

Although the plasmid vector contains potentially immunostimulatory CpG motifs in the backbone, tumor-bearing mice were treated with either empty pVAX-1 vector or pVAX-e1a2 vector alone, resulting in no elimination of existing tumors. Tumor-bearing mice were treated with D2SC/CD40L cells in the absence of tumor antigen, resulting in no tumor elimination. In contrast, co-delivery of pVAX-e1a2 vector and D2SC/CD40L cells showed superior anti-tumor effects. Inoculation with pVAX-e1a2 and D2SC/CD40L cells resulted in 100% high efficiency elimination of the original tumor cells in all treated mice (p < 0.01).

The ability of the induced anti-tumor immune response to protect mice from re-challenge by parental tumor cells was further investigated. One week after the final inoculation, a lethal dose of parental tumor cells is injected at a distant site into tumor-depleted mice. FIG. 15 shows that multiple treatments of tumor-bearing mice with pVAX-e1a2 vector and D2SC/CD40L cells resulted in the development of a highly potent anti-tumor immune response sufficient to protect the mice from re-challenge with tumor cells, with approximately 80% of the mice being protected and remaining tumor-free (results representing one of three replicates).

Immunization with the vaccine of the present invention, pVAX-e1a2 and DC/CD40L resulted in the generation of tumor-specific T cells in vivo that killed the tumor cells. T cells isolated from mice with tumor ablated were analyzed. CTLs cultured in vitro specifically kill parental BM185 tumor cells. These tumor-specific CTLs only recognized the e1a2 peptide, but not the MHC low-avidity binding peptide 8 loaded RAM-s cells (fig. 16).

Comparing the composition of different inoculations

The efficacy of the vaccine compositions of the invention will, in comparison to other vaccination methods, treat mice bearing bcr/abl positive tumors and protect mice against re-challenge of parental tumor cells. On day 0, mice were injected subcutaneously on the right side (5 mice/group) with a lethal dose of parental viable BM185/wt tumor cells. Mice were immunized at the tumor site with (a) PBS on days 7, 14, and 21; (b) pVAX-e1a2 (100. mu.g/mouse); (c) e1a2 peptide (AFHGDAEAL, 10 mM/mouse, SEQ ID NO: 5); (d) d2SC/wt cells pulsed with e1a2 peptide (106 cells/10. mu.M/mouse); (e) d2SC/CD40L cells pulsed with e1a2 peptide (10)⁶Cells/10 μ M/mouse); (f) d2SC/CD40L cells pulsed with low-bound MHC peptides (HGDAEALQR, 10)⁶Cells/10 μ M/mouse); (g) pulse of D2SC/CD40L cells with tumor lysate obtained from BM185 cells; (h) preincubated D2SC/CD40L cells and pVAX-e1a2 plasmid DNA (10)⁶Cells/100. mu.g/mouse). In the immunoprotection study, one week after the last vaccination, mice with tumor eliminated were re-challenged with a lethal dose of parental viable BM185wt tumor cells on the left flank. Tumor size was measured twice weekly. When tumors were detectable, mice were considered to beAt the end of survival and sacrificed. The experiments were repeated three times and the results represent one of these experiments.

The vaccine components of the invention (pre-incubated D2SC/CD40L cells and pVAX-e1a2 plasmid DNA, filled with dots) were the most effective treatment for eliminating the original tumor (p < 0.01), all treated mice were tumor-free (100%). The efficacy and capacity of the immune response elicited by vaccination to protect the host from tumor challenge was investigated. A lethal dose of parental live tumor cells is injected at a distant site into tumor-depleted mice. Multiple treatments of tumor-bearing mice with pVAX-e1a2 vector and D2SC/CD40L cells resulted in the development of a highly potent anti-tumor immune response sufficient to protect mice from tumors, with about 80% of mice being protected and remaining tumor-free (p < 0.01) for several months after re-challenge of tumor cells, see FIG. 17.

Consistent with previous studies, the mice were not eradicated by vaccination with pVAX-e1a2, or e1a2 peptide, or D2SC/CD40L cells, pulsed with the peptide, having low affinity for MHC-I.

Pulsing of D2SC/CD40L cells with MHC class I binding peptides significantly increased tumor antigen presentation. Treatment of tumor-bearing mice with D2SC/CD40L loaded with the e1a2 peptide resulted in induction of anti-tumor immunity, however, this method was less than 2-fold more effective than treatment of tumor-bearing mice with the vaccine of the present invention.

The superior efficacy of the present vaccine components pVAX-e1a2 and DC/CD40L also underscores the key elements of the present vaccine components. In addition to the antigen, pVAX-e1a2 plasmid DNA contains CpG motifs. CpG motifs are known to stimulate and activate DCs via the TLR9 pathway, resulting in the production of type I interferons. D2SC/wt and D2SC/CD40L cells were found to contain TLR 9. Furthermore, early studies showed that D2SC/wt cells released type I interferon- α and interferon- β after stimulation by viruses and bacteria.

Thus, treatment of tumor-bearing mice with the vaccine composition has the potential to produce type I interferon in vivo and stimulate immune responses with CpG-motifs in the plasmid backbone, contributing to superior efficacy compared to the pulse method using D2SC/CD40L injected cells and tumor antigen peptides (FIG. 17).

Priming of tumor-specific and tumor antigen peptide-specific CTLs after different vaccination protocols

Immunization with the vaccine of the invention results in tumor-specific and e1a2 peptide-specific T cells that kill tumor cells in vivo. For in vitro stimulation of T cells, Cs or tumor cells were first pulsed with e1a2 peptide and then irradiated as stimulators. T cells were isolated from tumor-free mice after tumor challenge and were co-stimulated with these DCs or tumor cells for seven to ten days. Cell culture supernatants were collected and stored at-80 ℃ for analysis of cytokine secretion. Live cells (80% CD8+ T cells) were used as effector cells in CTL assays.

CTLs formed in vitro were tested for their ability to lyse tumor cells. Mice treated for long tumors (a) PBs; (b) e1a2 peptide; (c) pVAX-e1a 2; (d) d2SC/CD40L cells pulsed with e1a2 peptide; (e) pulsing D2SC/CD40L cells with tumor lysate; (f) previously incubated D2SC/CD40L cells and pVAX-e1a2 plasmid DNA. T cells were then isolated from mice after challenge with parental tumor cells. Anti-tumor immunity was elicited in tumor-depleted mice following treatment with DC/CD40L loaded with tumor antigen (lysate or e1a2 peptide) or pre-incubated D2SC/CD40L cells and pVAX-e1a2 plasmid (FIG. 18. A). In addition, IFN-secretion was examined in CTLs culture supernatants (at 6 days) and is shown in Table II.

TABLE II

Vaccine compositions	IFN-γ(ng/ml)
		Normal (control) PBSe1a2 peptide pVAX-e1a2D2SC/CD40L/e1a2 peptide D2SC/CD40L/lysate (lysate) D2SC/CD40L/pVAX-e1a2	1.52.96.14.515.22321.5

Treatment of mice with tumor peptides and plasmids containing tumor peptides did not result in the production of tumor-specific CTLs. In contrast, repeated vaccination with pVAX-e1a2 plasmid DNA vector and DC/CD40L resulted in a strong tumor-specific CTL response, with a therapeutic effect.

Tumor-specific CTLs recognize tumor peptides presented by the vaccine compositions of the present invention

The ability of tumor-specific CTLs to kill peptide-pulsed target cells was investigated. The e1a2 peptide was loaded with TAP-deficient RMA-S cells. RMA-S cells lack the ability to load their own MHC class I. Thus, after pulsing RAM-S cells with the e1a2 peptide, the e1a2 peptide was the only peptide present on the cell surface. After loading of DC/CD40L and incubated D2SC/CD40L cells and pVAX-e1a2 with tumor antigen (tumor lysate or e1a2 peptide), CTLs generated on tumor-free mice recognized the e1a2 peptide presented by RAM-S cells (fig. 18B). Tumor peptide-specific CTLs did not kill parent RMA-S cells (no peptide loaded, data not shown). It was concluded that the anti-tumor immunity elicited in vivo by vaccination was tumor antigen specific and directed against the parent BM185 tumor cells.

Frequency of E1a2 peptide-specific CTLs elicited after vaccination

The e1a2 peptide-specific CTLs were expanded in the presence of rhIL-2(25ng/ml, R & D, UK) by co-stimulating purified T cells (containing 90% CD8+ cells and 10% CD4+ cells) with e1a2 peptide-pulsed D2SC/CD40L cells (50 Gy). After seven days of stimulation, approximately 85% of T cells were CD8+ T cells (fig. 19).

The frequency of CTLs specific for the e1a2 peptide, an in vitro tumor peptide, was studied using the DimerX peptide presentation method (BD Bioscience, u.s.a). Briefly, H-2L^dIg was incubated overnight at 37 ℃ in 160 molar excess in combination with peptide (e1a2 peptide or peptide 8). CD8+ T cells suspended at 10⁶Cells/50. mu.l. From peptidesLoading with H-2L^dMu.g of the Ig protein complex was added to T cells and incubated at 4 ℃ for 60 minutes. The bound protein complexes were detected using PE-conjugated rat anti-mouse IgG1 antibody and flow cytometry.

E1a2 peptide-specific CD8+ T cells that recognized tumor cells and e1a2 peptide-pulsed target cells showed binding to the e1a2 peptide-loaded H-2Ld: Ig complex (fig. 20). Consistent with CTL assay analysis, treatment of mice with the DC/CD40L and pVAX-e1a2 vaccines elicited over 8-fold more production of CTLs specific for the e1a2 peptide than vaccination with pVAX-e1a2 alone. Treatment of mice with the DC/CD40L and pVAX-e1a2 vaccines elicited more than 2-fold greater production of CTLs specific for the e1a2 peptide, compared to vaccination with DC/CD40L loaded with the e1a2 peptide.

In summary, a novel composition and method of vaccination against tumors that enhance resistance to existing bcr/abl tumors is presented.

In this protocol DCs were used to genetically modify the subclasses of DCs, pDCs/IPCs, and plasmid vectors containing genes encoding e1a2 tumor-specific peptides and CpG. Repeated vaccination with DC/CD40L and pVAX-e1a2 plasmid containing CpG motifs to treat long tumor mice elicited tumor-specific and e1a2 peptide-specific T cell responses.

It further demonstrates that e1a2 peptide was identified by CTL detection and indirect immunofluorescence staining on the surface of these T cells. CTLs specific for the e1a2 peptide were induced in vivo. Apparently seeding induced T cells specific for the e1a2 peptide which were able to recognize tumor cells and kill them in situ. Newly primed e1a2 peptide-specific T cells may play an important role in protecting the host against re-challenge from parental tumors. The vaccine compositions of the invention are effective in eliminating the original bcr/abl positive tumors and in protecting animals against parental tumor challenge. Therefore, the vaccine components of the present invention are well applicable to the treatment and drug composition of philadelphia chromosome positive tumors.

Suggested mechanism for vaccine components

Based on the experimental results of the present invention and studies by others in the field of tumor immunology, the following and FIG. 21 show hypotheses that contribute to the possible mechanisms and key factors for the success of the present invention.

After forcing, plasmid DNA encoding tumor peptide (CpG-DNA-e1a2) was bound to DCs. Most CpG-DNA-e1a2 plasmid DNA is endocytosed and the peptide is subsequently presented on MHC molecules. CpG motifs on plasmid DNA bind to TLR9 and activate the toll-like receptor pathway within DCs.

Activation of the TLR9 pathway can result in secretion of type I interferon-alpha and interferon-beta. This event may occur during the incubation of CpG-DNA-e1a2 with DCs. Thus, it is better to enhance the binding of plasmid DNA to DCs by means of co-centrifugation at the time of pre-incubation, in advance, simultaneously and/or afterwards. DCs cells were genetically modified to express CD 40L. Thus DCs express B7 and CD40L, the two most important T cell activation signals. Genetically modified DCs present tumor peptides to immune cells, such as naive T cells, in the presence of CD40L, B7, and type I interferon and activate naive DCs through CD40-CD40L interaction. In vivo, DCs can take up unbound antigen and/or the product of CpG-DNA-e1a 2.

Both types of dendritic cells, pDCs/IPCs/CD40L/B7 and activated DCs load tumor antigen to migrate to lymphoid tissues and activate immune cells including naive T cells, B cells and DCs. Cytokines such as IL12, IL15, IFN-. gamma., TNF-. alpha.type I and other Th1 cytokines can be produced. CD40L modifies pDCs, expressing B7 molecules, possibly presenting tumor peptides directly to naive CD8+ T cells and activating tumor-specific CTLs, thus obtaining a faster, enhanced and more efficient therapeutic and protective immune response. The end result of immunization with the present invention is priming of e1a 2-specific CTLs and elimination of existing tumors. Anti-tumor immunity induced in vivo protects the host against tumor challenge.

Therefore, our novel vaccine strategy emphasizes the unique combination of activating innate immunity and adaptive immunity. The novel vaccine compositions are suitable for use in the design of pharmaceutical vaccine compositions for the treatment or prophylaxis of diseases in humans and animals. It will be understood by those skilled in the art that various modifications and changes may be made to the present invention without departing from the scope of the invention as defined in the appended claims.

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Claims (43)

1. A nucleic acid vaccine component comprising:

-a nucleotide sequence encoding an antigen; and

-a modified antigen presenting cell expressing an immune response modulating molecule.

2. The nucleic acid vaccine component comprises:

-a nucleotide sequence encoding an antigen; and

-a modified antigen presenting cell expressing a cell survival modulating molecule.

3. The vaccine composition according to claim 2, characterized in that: the cell survival regulating molecule is an apoptosis inducing molecule.

4. The vaccine composition according to any one of claims 1 to 3, characterized in that: the vaccine component is a mixture of the nucleotide sequence and the modified antigen presenting cells.

5. The vaccine component of claim 4, wherein: the vaccine component is a pre-incubated mixture of the nucleotide sequence and the modified antigen presenting cells.

6. The vaccine component of claim 1 or 5, characterized in that: the antigen presenting cell is a professional antigen presenting cell.

7. The vaccine composition according to claim, wherein: the professional antigen presenting cell is a dendritic cell.

8. The vaccine component of claim 7, wherein: the dendritic cells are highly efficient antigen processing and antigen presenting cells.

9. The vaccine component of claim 7 or 8, characterized in that: the dendritic cells are plasma cell dendritic cells.

10. The vaccine composition according to any one of claims 1 to 9, characterized in that: the antigen presenting cells are a human dendritic cell subset comparable to mouse dendritic cell subsets expressing CD8 α, B220, CD11C, and B7 molecules.

11. The vaccine composition according to any one of claims 1 to 10, characterized in that: the antigen presenting cells express Toll-like receptor 9.

12. The vaccine composition according to any one of claims 1 to 11, characterized in that: the antigen presenting cells express P2X 7.

13. The vaccine composition according to any one of claims 1 to 12, characterized in that: the antigen presenting cell can induce the production of type I interferon-alpha and/or interferon-beta.

14. The vaccine component of claim 13, wherein: when interacting with a microorganism, the antigen presenting cells are induced to produce type I interferon-alpha and/or interferon-beta.

15. The vaccine composition according to any one of claims 1 to 14, characterized in that: the antigen presenting cells serve as a link between the innate and adaptive immune responses of the subject.

16. The vaccine composition according to any one of claims 1 to 5, characterized in that: the antigen presenting cells are selected from at least one of the following:

-dendritic cells;

-plasma cellular dendritic cells;

-an interferon producing cell;

-a natural interferon producing cell;

-a monocyte;

-macrophages;

-bone marrow cells;

-cells differentiated from stem cells

-a B cell;

-a T cell; and

-mast cells.

17. The vaccine composition of claim 1, wherein: the immune response-modulating molecule is encoded by a nucleotide sequence that is transferred to the antigen-presenting cell, the gene sequence being selected from at least one of:

-a cytokine gene;

-an interleukin gene;

-an adhesion molecule gene;

-an interferon gene;

-chemokine genes and chemokine receptor genes.

18. The vaccine component of claim 17, wherein: the immune response regulating molecule is selected from CD40 ligand and GM-CSF.

19. The vaccine component of claim 2 or 3, characterized in that: the cell viability regulatory molecule is encoded by a nucleotide sequence transferred into the antigen presenting cell, the nucleotide sequence being selected from at least one of the following:

-an anti-apoptotic gene; and

-an apoptosis-inducing gene.

20. The vaccine composition according to any one of claims 1 to 19, characterized in that: the vector containing the nucleotide sequence is at least one of the following:

-a viral vector;

-a non-viral vector;

-a plasmid;

-a microbial carrier;

-a liposome;

-a small molecule carrier.

21. The vaccine component of claim 20, wherein: the vector comprises an immune response modulating nucleotide sequence.

22. The vaccine component of claim 21, wherein: the immune response regulating nucleotide sequence is an unmethylated cytidine-guanosine phosphate (CpG) sequence.

23. The vaccine composition according to any one of claims 1 to 22, characterized in that: the antigen comprises an amino acid sequence of SEQ ID NO: 5 e1a2 fusion polypeptide.

24. The vaccine composition according to any one of claims 1 to 22, characterized in that: the nucleotide sequence comprises a miniature e1a2 fusion gene, and the nucleotide sequence is SEQ ID NO: 3.

25. the vaccine composition according to any one of claims 1 to 22, characterized in that: the nucleotide sequence comprises a nucleotide sequence encoding SEQ ID NO: 4, the nucleotide sequence of the mini e1a2 fusion protein.

26. A method for producing nucleotide and cellular vaccine components comprising the steps of:

-providing a nucleotide sequence encoding an antigen;

-providing modified antigen presenting cells expressing an immune response modulating molecule; and

-mixing said antigen-encoding nucleotide sequence and said modified antigen-presenting cell.

27. The method of claim 26, wherein: further comprising the step of incubating said nucleotide sequence and said modified antigen presenting cell to enhance binding thereof.

28. The method of claim 26 or 27, wherein: said step of providing a nucleotide sequence comprises:

-providing an MHC-bound antigenic protein or polypeptide;

-cloning a nucleotide sequence encoding said antigenic protein or polypeptide into said vector; and

-amplifying said vector in an amplification system.

29. Method according to one of claims 26 to 28, characterized in that: said step of providing antigen presenting cells comprises:

-isolating said antigen presenting cells from the subject; and

-engineering an antigen presenting cell to express said immune response modulating molecule.

30. The method of claim 29, wherein: the isolating step comprises isolating a subset of dendritic cells that express Toll-like receptor 9 and are capable of producing interferon-alpha and/or interferon-beta.

31. The method of claim 30, wherein: the subclass of dendritic cells plays an important role in innate and adaptive immune responses in a subject.

32. The method of claim 30 or 31, wherein: the dendritic cell subclass is a plasma cell dendritic cell.

33. Use of a vaccine component according to claim 1 or 2 as a medicament.

34. Use of a vaccine component according to claim 1 or 2 for the preparation of a medicament for the treatment or prevention of infectious diseases, characterized in that: the nucleotide sequence encodes an antigen associated with an infectious agent in the disease.

35. Use of a vaccine component according to claim 1 or 2 for the preparation of a medicament for the treatment or prevention of cancer, characterized in that: the nucleotide sequence encodes a tumor associated antigen expressed in cancer.

36. A method of inducing an immune response comprising the step of introducing into a subject a vaccine composition according to claim 1 or 2.

37. A method of treating or preventing a disease in a subject, comprising the step of introducing into the subject the vaccine composition of claim 1 or 2, said antigen being an antigen associated with a pathogenic agent of said disease.

38. The method of claim 36 or 37, wherein: said antigen presenting cells are adapted to present at least one fragment of said antigen to cells of the immune system of a subject.

39. The method according to one of claims 36 to 38, wherein: the subject is a mammal.

40. The method of claim 39, wherein: the mammal is a human.

41. The method of claim 36, wherein the disease is at least one selected from the group consisting of:

-an infectious disease;

-cancer;

-a leukemia;

-lymphoma;

autoimmune diseases/disorders

-inflammation;

-a blood disease;

-allergy;

-a genetic disease;

-a disease required for transplantation; and

-diabetes mellitus.

42. A kit, comprising:

-a nucleotide sequence encoding an antigen; and

-a modified antigen presenting cell expressing an immune response modulating molecule.

43. The kit of claim 42, wherein: the nucleotide sequence and the modified antigen presenting cell are in admixture.