Effective anti-tumor immunity is induced in mice utilizing RNA-pulsed epidermal cells (EC) for in vivo immunization or by injecting RNA intradermally into naïve mice. A vaccine comprising total cell RNA and a pharmaceutically acceptable carrier for inducing an immune response to reduce or prevent the occurrence of a tumor.

DETAILED DESCRIPTION OF THE INVENTION

The invention advantageously provides for modulating immune responses by administering antigen RNA. In one aspect, the invention provides immunotherapy against tumors and pathogens using antigen-specific RNA, particularly total cellular RNA or total cellular mRNA. Effective immunization using total cellular RNA can be effected through direct in vivo intradermal injection of RNA or through administration of epidermal cells which have been modified with total cellular RNA.

Alternatively, the invention provides for inducing immune tolerance to an antigen by administering RNA for the antigen via a tolerization route.

The present invention provides a method for preparing vaccines and compositions and medicaments based thereon advantageously using antigen-specific RNA, especially total cellular RNA, or total cellular mRNA from cells against which immunity or tolerization is desired, e.g., tumor cells or derived from pathogens (for immunity), or autoantigens, allergens, and transplant cells (for tolerance).

The present invention is based, in part on the unexpected discovery that epidermal cells can be used as effective antigen presenting cells in vivo and ex vivo. A murine model of human cancer suggests that immunization using total cellular RNA effectively inhibits tumor growth upon challenge with live tumor cells. In this model, total cellular RNA was isolated from the S1509a spindle cell tumor and used to pulse CAF, EC enriched for Langerhans cell content and pre-exposed to GM-CSF. These cells were then injected subcutaneously into naïve CAF, mice three times at weekly intervals, followed by challenge with living S1509a cells. Tumor growth was significantly less in vaccine-treated animals than in control animals immunized in an identical fashion but with irrelevant RNA, or in the case in which S1509a RNA was digested with RNase prior to pulsing of the EC. RNase treatment prevented the development of immunity.

It has also been found that direct administration of tumor RNA into the skin effectively induces anti-tumor immunity. In particular, evidence shows that intradermal injection of S1509a RNA into naïve mice three times at weekly intervals induced immunity to challenge with the tumor. Digestion of S1509a RNA with RNase prior to intradermal injection prevented development of immunity in this system. Thus, effective anti-tumor immunity can be induced utilizing RNA-pulsed EC for ex vivo immunization or by injecting RNA intradermally in vivo into naïve mice.

It is believed, without being bound to any particular theory, that using cutaneous antigen-presenting cells, RNA diffuses across the basement membrane zone of the skin, cutaneous antigen presenting, which may be LCs, take up RNA, translates it in situ and presents relevant antigens for induction of in vivo immunity. Thus, effective immunity is induced by intradermal injection of total cellular RNA from a tumor or pathogenic microorganism.

Ex vivo epidermal RNA vaccination provides the benefits obtained with RNA vaccination. Further, using epidermal cells as antigen presenting cells advantageously avoids the time-consuming and costly procedures involved in generating autologous dendritic cells, e.g., from blood or epidermis.

The present invention is further based on the observation that administration of total cellular RNA from the S1509a tumor intravenously induces tolerance to subsequent immunization with that tumor.

Induction of tolerance in this manner can be accomplished with total cellular RNA, for example, from an allogeneic cell or cells obtained from an organ to be transplanted to a subject; cells from an allergenic organism such as a stinging insect (specifically venom sac RNA), plant (specifically pollen RNA), animal (specifically saliva gland RNA), or mite; cells from an autoantigen or target tissue of an autoimmune response (e.g., nervous tissue in multiple sclerosis, chondrocytes in rheumatoid arthritis, etc); or specific messenger RNA that codes for a protein from one of the foregoing tissues or cells to which it is desirable to induce tolerance. The use of RNA, instead of protein antigens, offers a number of advantages. First, the use of total cellular RNA obviates the need to know the precise antigen or antigens that are relevant for induction of tolerance. Secondly, because RNA can be easily amplified, only a small sample of the material is necessary to obtain RNA for tolerization.

While in accordance with the present invention the intravenous (iv) route of administration effected tolerization, as exemplified, administration of RNA by other routes known to favor tolerance (i.e., intranasal or oral) is also expected to be effective.

Preferably, this technique allows for more profound induction of tolerance, compared to other techniques such as intravenous administration of protein antigen, exposure to antigen in the presence of agents known to block effective co-stimulation, etc. This can be shown by direct comparison.

Although not intending to be limited to any particular theory or mechanism, the molecular mechanisms by which tolerance to antigens or tissues delivered by RNA may result from uptake of the RNA by appropriate cells, probably in the spleen, with translation and presentation of peptides derived from the RNA for induction of tolerogenic mechanisms.

Total Cellular and Antigen-Specific RNA

The total cellular RNA to be used in the present invention may be obtained using a variety of methods e.g., as described in U.S. Pat. No. 5,853,719. It is not necessary that the RNA be in purified form. Preferably the RNA sample is at least 80% and most preferably at least 90% RNA (wt/wt or by mole). The term total cellular RNA includes total messenger RNA (mRNA) or poly-A RNA. To be most effective, total cellular RNA contains mRNA encoding antigens against which an immune response (or, alternatively, immune tolerance) is desired.

Total cell RNA may be obtained, for example, by lysing the cells, e.g., tumor or transplant organ cells, or pathogenic bacteria, or cells containing a pathogenic virus, by homogenization or sonication in suitable buffers, extracting and precipitating the RNA fraction from the cell homogenate. Alternatively, the RNA can be prepared utilizing RNA purification methods known in the art such as guanidinium isothiocyanate methods and/or oligo dT chromatography methods for isolating poly A⁺ RNA. The RNA containing preparation can be fractionated to decrease the concentration of other components in the preparation for example, lipids, proteins or DNA to enrich the concentration of RNA in the preparation. The preparation can also be treated with proteases or RNase-free Dnases. Total mRNA can be obtained by isolating it on a poly-T column. The dose of total cellular RNA will be in an amount effective to induce an immune response or immune tolerance. Such amount can be readily determined by a skilled physician and will vary with the nature and severity of the condition to be treated. Typical dosage ranges of cellular RNA are from about 1 μg to about 100 μg and depending on the nature and severity of the condition being treated can be adjusted to a range from about 0.1 μg to about 10 mg RNA. Typically a total of 20 μg RNA, in two (2) 10 μg/flank doses can be administered in each animal.

Although it can vary with cell types, 10⁶ cells provide approximately 10 μg RNA. Thus dosages can range from about 10⁶ to about 10⁷ cell equivalents, or can be adjusted for example to about 10⁶ to about 10⁸ cell equivalents per administration.

In order to obtain amounts of RNA sufficient for use in the methods described herein, conventional amplification techniques may be used. Cellular RNA can be reverse transcribed in vitro to produce cDNA for amplification by PCR. The cDNA then is transcribed in vitro to produce tumor RNA.

The RNA may be obtained by isolating RNA from a cultured cell line, for example, S1509a methylcholanthrene-induced fibrosarcoma cell line. The cells are lysed by adding a lysing buffer such as TRIzol Reagent (GIBCO-BRL), and homogenizing by passing the mixture through a pipette several times. Chloroform is added the mixture is shaken and centrifuged. Phase separation of the mixture provides the RNA in the aqueous phase which is removed and the RNA is precipitated with isopropyl alcohol.

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant nucleic acid techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds. (1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins, eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

Delivering RNA

The epidermal cells can be "transfected" by exogenous or heterologous RNA when such RNA has been introduced inside the cell. The RNA can be introduced into the cells by "pulsing", i.e., incubating the cells with the total cell RNA. Alternatively, the RNA can be introduced in vivo by lipofection, as naked RNA, or with other transfection facilitating agents (peptides, polymers, etc.). Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Felgner, et. al., Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417, 1987; Felgner and Ringold, Science 337:387-388, 1989; see Mackey, et al., Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031, 1988; Ulmer, et al., Science 259:1745-1748, 1993). Useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications WO95/18863 and WO96/17823, and in U.S. Pat. No. 5,459,127. Suitable lipids include DODC, DOPE, CHOL, DMEDA, DDAB, DODAC, DOTAP and DOTMA. Lipids may be chemically coupled to other molecules for the purpose of targeting (see Mackey, et al., supra). Targeted peptides, e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.

Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., International Patent Publication WO95/21931), peptides derived from DNA binding proteins (e.g., International Patent Publication WO96/25508), or a cationic polymer (e.g., International Patent Publication WO95/21931).

Alternatively, non-viral DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., electroporation, microinjection, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun (ballistic transfection; see, e.g., U.S. Pat. No. 5,204,253, U.S. Pat. No. 5,853,663, U.S. Pat. No. 5,885,795, and U.S. Pat. No. 5,702,384 and see Sanford, TIB-TECH, 6:299-302, 1988; Fynan et al., Proc. Natl. Acad. Sci. U.S.A., 90:11478-11482, 1993; and Yang et al., Proc. Natl. Acad. Sci. U.S.A., 87:1568-9572, 1990), or use of a DNA vector transporter (see, e.g., Wu, et al., J. Biol. Chem. 267:963-967, 1992; Wu and Wu, J. Biol. Chem. 263:14621-14624, 1988; Hartmut, et al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990; Williams, et al., Proc. Natl. Acad. Sci. USA 88:2726-2730, 1991). Receptor-mediated DNA delivery approaches can also be used (Curiel, et al., Hum. Gene Ther. 3:147-154, 1992; Wu and Wu, J. Biol. Chem. 262:4429-4432, 1987). U.S. Pat. Nos. 5,580,859 and 5,589,466 disclose delivery of exogenous DNA sequences, free of transfection facilitating agents, in a mammal. Recently, a relatively low voltage, high efficiency in vivo DNA transfer technique, termed electrotransfer, has been described (Mir, et al., C. P. Acad. Sci., 321:893, 1998; WO 99/01157; WO 99/01158; WO 99/01175).

A "tumor-associated antigen"preparation (TAA) used herein is obtained by solubilizing tumor cells. In this unfractionated RNA preparation the tumor antigens may not be specifically identified.

Anti-Tumor and Anti-Microbial Therapy

The present invention provides an immunogenic composition capable of inducing an immune response to challenge by tumor or a microorganism. The immunogenic molecule is total cellular RNA. As used herein, the term immunogenic means that the RNA is capable of eliciting a humoral or cellular immune response, and preferably elicits responses from both facets of the immune system.

The present treatment is suitable for application to a variety of conditions, which benefit from the stimulation and/or inhibition of the immune system. As used herein, the term "pathogen infection" includes diseases and disorders that result from a tumor, or from viral or bacterial infection. It further includes parasite infestation. In addition, for purposes of the present invention as it relates to immunotherapy, the term "infectious disease" includes cancer.

The vaccine of the present invention may be utilized to treat or protect subjects afflicted with conditions manifesting tumors such as but not limited to melanomas, breast cancers, bladder cancers, colon cancers, ovarian cancers, pancreatic cancers, prostate cancers, etc., as set forth above. The method of the present invention may be used to eliminate reduce preexisting mestastases in subjects whose primary tumor has been removed (surgically) or destroyed (chemo/radio therapy); prevent the occurrence of primary tumor growth; treat existing tumor to inhibit reduce growth; and induce regression of existing tumors.

Tumor Cells

The compositions of the present invention are prepared using RNA from tumor cells, e.g., cells obtained from tumors surgically resected in the course of a treatment for a cancer. The tumor cells for use in the present invention may be prepared as follows. Tumors may be processed as described by Berd et al., Cancer Res., 46:2572, 1986, Sato, et al., Cancer Invest., 15:98, 1997, U.S. Pat. No. 5,290,551, or corresponding PCT application WO96/40173, each of which is incorporated herein by reference in its entirety. Briefly, the cells are extracted by dissociation, such as by enzymatic dissociation with collagenase and DNase, by mechanical dissociation in a blender, by teasing with tweezers, using mortar and pestle, cutting into small pieces using a scalpel blade, and the like. With respect to liquid tumors, blood or bone marrow samples may be collected and tumor cells isolated by density gradient centrifugation.

Preferably the cells originate from the type of cancer which is to be treated, and more preferably, from the same patient who is to be treated. The tumor cells may be, and are not limited to, autologous cells dissociated from biopsy or surgical resection specimens, or from tissue culture of such cells. Nonetheless, allogeneic cells and stem cells are also within the scope of the present invention. In either case, amplification procedures permit preparation of large amounts of tumor cell RNA for vaccination immunotherapy of the tumor.

The ability of the tumor RNA vaccine to reduce or inhibit the formation of tumors in a subject can be determined by measuring the tumor growth over a period of time before during and after immunization, as applicable. Such measurements can be made after surgical excision of the tumor, e.g., by CAT scan, MRI, PET scan, and the like.

In one aspect, the present invention is directed for use in the preparation of tumor cell vaccines for treating cancer, including metastatic and primary cancers. Cancers treatable with the present invention include solid tumors, including carcinomas, and non-solid tumors, including hematologic malignancies. Examples of solid tumors that can be treated according to the invention include sarcomas and carcinomas such as, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. Hematologic malignancies include leukemias, lymphomas, and multiple myelomas. The following are non-limiting preferred examples of the cancers treatable with the composition and methods of the present invention: melanoma, including stage-4 melanoma; ovarian, including advanced ovarian; leukemia, including and not limited to acute myelogenous leukemia; colon, including colon metastasized to liver; rectal, colorectal, breast, lung, kidney, and prostate cancers. In a specific example, the tumor is a fibrosarcoma.

RNA Vaccines

As used herein, the term "RNA vaccine" refers to a vaccine comprising RNA. It can further include an adjuvant. The term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response (Hood, et al., Immunology, Second Ed., 1984, Benjamin/Cummings: Menlo Park, Calif., p. 384). Adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, and potentially useful pharmaceutically acceptable human adjuvants such as BCG (bacille Calmetle-Guerin) and Corynebacterium parvum.

Alternatively, or in addition, immunostimulatory proteins, as described below, can be provided as an adjuvant or to increase the immune response to a vaccine. Preferably, the adjuvant is pharmaceutically acceptable.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Sterile water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

Certain adjuvants mentioned above, particularly mineral oils and adjuvants containing mineral oils (e.g., Freund's adjuvant) are not acceptable for use in humans.

Vaccination effectiveness may be enhanced by co-administration of an immunostimulatory molecule (Salgaller and Lodge, J. Surg. Oncol., 1988, 68:122), such as an immunostimulatory, immunopotentiating, or pro-inflammatory cytokine, lymphokine, or chemokine with the vaccine, particularly with a vector vaccine. For example, cytokines or cytokine genes such as interleukin (IL)-1, IL-2, IL-3, IL-4, IL-12, IL-13, granulocyte-macrophage (GM)-colony stimulating factor (CSF) and other colony stimulating factors, macrophage inflammatory factor, Flt3 ligand (Lyman, Curr. Opin. Hematol., 1998, 5:192), CD40 ligand, as well as some key costimulatory molecules or their genes (e.g., B7.1, B7.2) can be used. These immunostimulatory molecules can be delivered systemically or locally as proteins or by expression of a vector that codes for expression of the molecule. The techniques described above for delivery of the immunogenic polypeptide can also be employed for the immunostimulatory molecules.

Tolerization

As noted above, the present invention provides isolated or total cellular RNA for eliciting immune tolerance to an antigen, e.g., an autoantigen, allergen, or transplant tissue.

Examples of sources of autoantigens include, but are by no means limited to, thyroid (associated with various thyroiditises, such as Hashimoto's disease), pancrease (especially beta cells of the islets of Langerhans; associated with insulin-dependent diabetes mellitus), heart tissue (associated with rheumatic fever), nerve tissue (associated with multiple sclerosis), joint tissue/chondrocytes (associated rheumatoid arthritis), to mention a few sources of total or antigen-specific RNA (and the autoimmune diseases with which they are associated).

Examples of allergens include ragweed pollen, tree pollen, vespid (wasp) venom, apid (bee) venom, dust mites, cat dander, dog dander, and the like. (Note that animal dander typically causes allergy because of the presence of saliva allergens). Whole allergens (insects, mites, plants), or specific cells or tissues from allergens (pollen, venom sacs, salivary cells) can be used to generate total cell RNA or antigen-specific RNA. The use of total cellular RNA ensures that the allergen-specific RNA (or RNAs, as often there are multiple allergenic components from a single source) will be present in the preparation. Moreover, induction of tolerance to total cellular RNA ensures that new allergens will not elicit allergic responses.

Examples of transplant tissues include organs, such as heart, lungs, liver, kidney, pancreas (especially beta islet cells); tissues, such as skin; and blood cells, such as platelets, lymphocytes, leukocytes, and red blood cells (which can be tolerized with reticulocyte RNA).

Tolerization to an antigen (or total RNA antigens) can depend on the route of administration of the tolerigen. Intravenous administration is preferred, but other routes of administration are available. For example, oral and intranasal administration of protein antigens elicits immune tolerance. By appropriate formulation of the antigen RNAs, these routes can also be used with RNA.
 

Claim 1 of 4 Claims

1. A method of inducing an immune response to a tumor in a subject, which method comprises intradermally or subcutaneously administering total tumor cell RNA to cutaneous cells of the subject in vivo, in an amount effective to elicit an immune response against the tumor, wherein the total tumor cell RNA is from tumor cells from the subject, and the immune response reduces or inhibits growth of the tumor.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.