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WO1998008947A1 - Amelioration de l'immunisation a l'adn obtenu a l'aide de cytokines - Google Patents

Amelioration de l'immunisation a l'adn obtenu a l'aide de cytokines Download PDF

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Publication number
WO1998008947A1
WO1998008947A1 PCT/US1996/020571 US9620571W WO9808947A1 WO 1998008947 A1 WO1998008947 A1 WO 1998008947A1 US 9620571 W US9620571 W US 9620571W WO 9808947 A1 WO9808947 A1 WO 9808947A1
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cancer
immunogen
nucleic acid
dna
disease
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PCT/US1996/020571
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English (en)
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Kari R. Irvine
Steven A. Rosenberg
Nicholas P. Restifo
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The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services
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Priority to AU55070/98A priority Critical patent/AU5507098A/en
Publication of WO1998008947A1 publication Critical patent/WO1998008947A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5412—IL-6
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA

Definitions

  • the present invention relates to immunization and vaccination using nucleic acid-based immunogens for the prevention or treatment of pathogenic diseases and cancer. More particularly, plasmid DNA comprising one or more genes encoding an antigen associated with a pathogenic disease or cancer is used in accordance with the invention for immunizing or vaccinating an animal via direct delivery of the DNA to cells and tissues.
  • the prophylactic and therapeutic effects of the nucleic acid-based vaccines of the invention are enhanced by the administration of cytokines and, optionally, costimulatory or immunomodulatory molecules.
  • TAAs tumor associated antigens
  • MHC major histocompatibility
  • TAAs tumor associated antigens
  • HTV vaccinia-human immunodeficiency virus
  • immune responses against heterologous protein may be reduced when the host has had previous exposure to the virus, such as has been observed with vaccinia virus (Graham, B.S., et al., 1993, Augmentation of human immunodeficiency virus type 1 neutralizing antibody by priming the gpl60 recombinant vaccinia and boosting with rgpl ⁇ O in vaccinia-naive adults.
  • vaccinia virus Graham, B.S., et al., 1993, Augmentation of human immunodeficiency virus type 1 neutralizing antibody by priming the gpl60 recombinant vaccinia and boosting with rgpl ⁇ O in vaccinia-naive adults.
  • immunodominant peptides that represent unique tumor antigens allows new avenues for immunization against cancer. Substantial evidence exists in animal models that immunization with immunodominant viral peptides can induce viral- specific CTL that can confer protection against viral infection. Although pure peptide alone is ineffective in stimulating T cell responses, peptides emulsified in adjuvants or complexed with lipids have been shown to prime mice against challenge with fresh virus and can induce virus specific CTL that protect mice against lethal viral inocula (Kast, W.M. et al. , 1991 , Proc. Nat'l Acad. Sci. U.S.A. 88:2283-2287; Deres, K. et al.
  • Peptides representing antigenic epitopes of HTV gpl20 and gpl60 emulsified in complete Freund's adjuvant can also prime specific CTL responses (Takahashi, H. et al. , 1988, Proc. Nat'l Acad. Sci. U.S.A. 85:3105- 3109; Hart, M.K. et al. , 1991 , Proc. Nat'l Acad. Sci. U.S.A. 88:9448-9452).
  • TEL tumor infiltrating lymphocytes
  • T Cells- 1) and GP-100 T Cells- 1) and GP-100.
  • the genes encoding both of the peptides have been cloned and sequenced.
  • the MART-1 gene encodes a 118 amino acid protein of 13 kD.
  • the GP-100 gene encodes a protein identical to that recognized by monoclonal antibody HMB-45.
  • MART-1 antigen was expressed on nine out of nine tissue culture lines that were established from melanomas in the Surgery Branch, NCI, and on all fresh melanomas tested. Studies by others have shown that the MART-1 (also called Melan-A) antigen was expressed on 26 out of 26 fresh melanomas (Coulie, P.G. et al., 1992, J. Exp. Med. 180:35-42). The GP-100 antigen is also widely expressed in melanomas.
  • TTL cells that were raised in the Surgery Branch, NCI from different HLA-A2 individuals, 13 out of 14 recognized MART-1 and 4 out of 14 recognized the GP-100 antigen. Because TIL cells that recognize these determinants have been shown to be capable of mediating cancer regression in vivo, it appears that these antigens are involved in cancer regression.
  • MAGE-1 Another gene coding for a human tumor-specific antigen on a human melanoma was cloned by Van der Bruggen et al (1991 , Science 254: 1643-1647). This antigen is encoded by a gene called MAGE-1 which spans five kilobases. A 2,419 base pair coding sequence produces a predicted protein product of 26 kD.
  • the MAGE antigen is HLA-A1 restricted and the nine amino acid fragment that represents the Al restricted immunodominant peptide has been defined as Glu-Ala- Asp-Pro-Thr-Gly-His-Ser-Tyr. This nine amino acid peptide is encoded by the third exon of the MAGE gene.
  • Transfection of a 500 base pair fragment of this gene can confer recognition by a MAGE-specific CTL clone.
  • Incubation of an EBV-infected cell line with the immunodominant peptide can confer sensitivity to lysis by a MAGE-1 specific CTL clone.
  • MAGE-1 does not appear to be expressed in normal cells with the possible exception of testis, but is expressed on approximately half of metastatic melanomas and on about 20% of breast cancer cells, as well as on other selected types of cancers.
  • DNA vaccination may be accomplished by the expression of an inoculated isolated nucleic acid molecule, i.e., bacterial plasmid DNA, encoding a foreign gene of interest accompanied by other genetic sequences which enable the gene to be expressed within mammalian cells utilizing host machinery (Wolff, J.A. , et al. , 1990, Direct transfer into mouse muscle in vivo.
  • an inoculated isolated nucleic acid molecule i.e., bacterial plasmid DNA
  • DNA-based immunization approaches have been shown to successfully induce both humoral and cellular immunity in many antigen systems (Fynan, E., et al. , 1995, DNA vaccines: protective immunizations by parental, mucosal, and gene-gun inoculations. Proc. Natl. Acad. Sci. USA 90:11478; Eisenbraum, M. D., et al., 1993, Examination of parameters affecting the elicitation of humoral immune response by particle bombardment-mediated genetic immunization. DNA and Cell. Bio. 12:791; Fuller, D.
  • hGH human growth hormone
  • plasmid constructs encoding either the full length cDNA for carcinoembryonic antigen (CEA) or HIV-1 envelope protein, gpl60, have been shown to protect mice from subsequent challenge with syngeneic tumors expressing these model antigens (Conry, R.M., et al., 1994, Immune response to a carcinoembryonic antigen polynucleotide vaccine. Cancer. Res. 54: 1164; Conry, R.M., et al. , 1995, A carcinoembryonic antigen polynucleotide vaccine has in vivo antitumor activity. Gene Ther. 2:59; and Wang, B., et al.
  • the present invention provides direct delivery of immunogens or vaccines comprising isolated nucleic acid sequences for the prevention and treatment of cancer and pathological diseases, including bacterial, parasitic, protozoan, or viral infection.
  • the nucleic acid-based vaccines may comprise DNA, cDNA, or RNA, for example, and preferably comprise eukaryotic and mammalian vectors for expression in mammalian cells.
  • the invention further provides such DNA-based vaccines enhanced with the administration of cytokines to promote the active immunotherapy of cancer and treatment of infection and other pathogenic diseases.
  • costimulatory molecules may also be administered to enhance the immune reponse and to augment immunity via the local expression of such molecules.
  • DNA encoding one or more cytokines and DNA encoding one or more co-stimulatory molecules may be contained within the vector comprising DNA encoding one or more tumor associated antigens or other disease-associated antigens to augment cellular and humoral immunity.
  • Nucleic acid which encodes the cytokine(s) of interest can be immunized at the same time as the DNA encoding antigen(s), e.g. , tumor associated antigen(s), thereby causing local secretion of cytokines and enhancement of therapeutic immune responses.
  • a reduction in toxicities frequently associated with such cytokines may also be provided by the invention.
  • Yet another object of the invention is to provide prophylactic and therapeutic methods of cancer treatment using small amounts of DNA to induce potent and consistent cellular and humoral immune responses.
  • It is another object of the invention to provide a vaccine against a disease-causing agent or cancer comprising an isolated nucleic acid sequence (for example, in a plasmid) encoding one or more disease or tumor-associated antigens or immunodominant epitopes thereof, wherein the nucleic acid vaccine is directly put into cells or tissues, to prevent or reduce a cancer or a disease or infection caused by a disease-causing agent or tumor.
  • nucleic acid-based vaccine in accordance with the invention by the administration of cytokines, particularly interleukins, as adjuvant to reduce an established cancer or to prevent or ameliorate pathogenic disease and cancer.
  • CMV cytomegalovirus
  • TAA tumor associated antigen
  • ⁇ -gal beta-galactosidase
  • NP nucleoprotein
  • CEA carcinoembryonic antigen
  • r recombinant murine
  • rh recombinant human
  • mAb monoclonal antibody.
  • Fig. 1 demonstrates the induction of humoral immunity elicited with gene gun vaccination of the nucleic acid-based immunogen, pCMV/0-gal.
  • BALB/c mice (three per group) were immunized two times at two week intervals in the epidermis in the abdomen; each immunization consisted of four shots of 0.25 mg of gold, thus delivering a total of either 1.0 / xg of control pCMV/NP, or 0.01 ⁇ g, O. l ⁇ g, 1.0 ⁇ g of pCMV/3-gal.
  • sera were tested by ELISA as described in Example 1 for the presence of antibodies reactive with recombinant -gal protein.
  • Fig. 2A-2D demonstrate that secondary in vivo T CDg+ cells were induced by immunization with gene gun vaccination of pCMV/
  • BALB/c mice were immunized one time in the epidermis; each immunization consisted of four shots of 0.25 mg of gold, thus delivering a total of 1.0 ⁇ g pCMV/NP control DNA (Fig. 2A), 0.01 ⁇ g pCMV//3-gal (Fig. 2B), 0.1 ⁇ g pCMV/ ⁇ -gal (Fig.
  • CT26.WT (/S-gal-), CT26.CL25 (0-gal- ⁇ -), or CT26.WT pulsed with 3-gal 876 . 884
  • Fig. 3 shows that immunization with a DNA vaccine in accordance with the invention prevents the growth of intravenous tumors.
  • Day 0 BALB/c mice were immunized one time in the epidermis, each immunization consisted of five shots of 0.25 mg of gold delivering 1.0, 0.10, or 0.01 ⁇ g of pCMV//3-gal or 1.0 ⁇ g of control DNA alone.
  • mice Five-10 per group) were challenged, iv. with 2.5 x 10 s CT26.CL25 (0-gaH-) or CT26.WT (/3-gal-) tumor cells.
  • the graph in Fig. 3 represents a summary of all of the data obtained from three separate experiments.
  • the control DNA utilized was pCMV/hGH; in the remaining experiments pCMV/NP was used as control DNA.
  • Fig. 4 shows that adoptive immunotherapy of tumor bearing mice with immune splenocytes was induced by gene gun vaccination.
  • CT26.WT 3-gal-, O
  • CT26.CL25 J-gal + , •
  • tumor cell lines were each injected i.v. into BALB/c mice to create lung metastases.
  • tumor bearing mice were treated with effector splenocytes from donor mice.
  • the donor cells were generated by prior gene gun immunization with 1 ⁇ g of either pCMV/0-gal or pCMV/NP followed 14 days later by in vitro incubation for six days with 1 ⁇ g/ml of either ⁇ - ga ⁇ w (SEQ ID NO: l) or NP 147 ., 55 (SEQ ID NO:2) peptide. On day seventeen, the number of pulmonary metastases was enumerated in a coded, blind fashion.
  • FIG. 5 demonstrates active immunotherapy of established pulmonary metastases with the pCMV/3-gal vaccine in conjunction with systemic administration of rhTL- 2, rmIL-6 or rhIL-7.
  • BALB/c mice were injected i.v. with 5 x 10 5 CT26.CL25 (
  • treated mice were immunized with 10 ⁇ g of pCMV/3-gal.
  • Each mouse received 10 shots of 0.5 mg of gold with each shot delivering 1 ⁇ g of DNA.
  • mice (5-10 mice per group) began regimens of i.p. cytokine injections as described in Example 1.
  • Fig. 6 demonstrates active immunotherapy of established pulmonary metastases with the pCMV/ 3-gal nucleic acid vaccine plus systemic administration of rmlL- 12.
  • BALB/c mice were injected i.v. with 5 x 10 5 CT26.CL25 ( 3-gal+) or CT26 (/3-gal-) tumor cells.
  • treated mice were immunized with 10 ⁇ g of pCMV//3-gal or 10 ⁇ g of pCMV/NP.
  • Each mouse received 10 shots of 0.5 mg of gold, with each shot delivering l ⁇ g of DNA immunogen.
  • mice began regimens of i.p. injections with rmIL-12 as described in Example 1.
  • the present invention provides direct nucleic acid-based immunization to induce antigen-specific cellular and humoral immune responses, as well as protective immunity for an active treatment immunotherapy against cancer and pathogenic diseases, including bacterial, parasitic, protozoan, and viral infections.
  • DNA-based immunization offers an attractive and safe non-viral alternative for immunotherapy against cancer and other disease-causing agents and organisms.
  • Nucleic acid-based immunogens for vaccines offer several advantages over the use of other immunizing agents, such as attenuated or recombinant viruses, for example.
  • Purified DNA is relatively safe compared with replication-competent viruses, which may result in disseminated viremia, especially in immunocompromised individuals.
  • Plasmid DNA can also be easily and rapidly purified compared with the production of live viruses, which involves time consuming homologous recombination and plaque purification steps.
  • the use of DNA vectors would also obviate the problems of anamnestic responses that can eliminate recombinant viruses more rapidly, thereby reducing immune responses against heterologous proteins expressed by viral carriers.
  • genetic vaccines comprising isolated nucleic acid sequences represent a safe, convenient, and efficient option to other types of viral-based vaccines for the immunotherapy of cancer and pathogenic diseases.
  • the nucleic acid-based immunogens for prophylactic and therapeutic vaccination in accordance with the invention comprise plasmid or vector or plasmid vector DNA (these terms are used synonymously herein) comprising the isolated nucleic acid of interest, e.g., DNA, cDNA, RNA, with cDNA preferred, usually heterologous (e.g. , a nucleic acid sequence (a gene) preferably double stranded, encoding a tumor-associated antigen, a nucleic acid sequence encoding an antigen of a pathogenic disease-causing microorganism or virus, and the like) accompanied by other sequences or control elements that are desired or required for proper expression of the plasmid in a cell.
  • heterologous e.g. , a nucleic acid sequence (a gene) preferably double stranded, encoding a tumor-associated antigen, a nucleic acid sequence encoding an antigen of a pathogenic disease-causing microorganism or virus, and the like
  • Plasmid vectors suitable for use in the present invention comprise at least one expression control element or promoter operationally linked to the nucleic acid sequence encoding the gene of interest and, if desired, to other nucleic acid sequences encoding cytokines, immunostimulatory or immunomodulatory molecules, or combinations thereof, as described herein.
  • the expression control elements are inserted into the vector to control and regulate the expression of the nucleic acid sequence in a mammalian cell system, in particular.
  • Examples of expression control elements include, but are not limited to, operator regions and promoters derived from polyoma virus, adenovirus, Rous sarcoma virus, cytomegalovirus, retroviruses, or SV40.
  • Other promoters may include thymidine kinase (TK), phosphoglycerol kinase (PGK) or the ⁇ -actin promoters.
  • tissue-specific promoters or regulatory elements may be used, non-limiting examples of which include the following: the N-CAM promoter (specific for brain and central nervous system); the PIT-1 promoter (pituitary specific transcription factor); the crystalline promoter (specific for protein expression in the lens of the eye); the keratin promoter (specific for protein expression in the skin); the albumin promoter (liver-specific); the alpha- or beta- globin promoters (specific for red blood cells); the Ig enhancer (specific for B lymphocytes); the T cell receptor - or ⁇ -promoters (specific for T cells); the insulin promoter (specific for pancreatic cells); the gastrin promoter (specific for cells of the stomach); the cardiac actin promoter (specific for heart); the tropomyosin promoter (specific for skeletal muscle); and the lactalbumin promoter
  • WAP whey acidic protein
  • 15 for immunization include, but are not limited to, enhancer sequence(s), leader sequence(s), termination codon(s), polyadenylation signals, and any other sequences necessary or preferred for the appropriate transcription and subsequent translation of the nucleic acid sequence in host cells. It will be understood by
  • plasmid vectors may contain enhancer elements to augment the amount of protein expressed intracellularly.
  • Such additional elements may enhance or increase the levels of antigen production or persistence, as well as the quality and amounts of antigenic protein expressed in mammalian cells, in particular.
  • regulatory response elements and enhancers include, but are not limited to, immunoglobulin enhancers, glucocorticoid response element (GRE), estrogen response element (ERE), metallothionein response element (MRE), heat shock response element (HSRE), and CMV intron A enhancer element (Chapman, B.S. , et al. , 1991 , Effect of intron A from human cytomegalovirus (Towne) immediate-early gene on heterologous expression in mammalian cells, Nuc. Acids Res.. 19:3979-3986).
  • the DNA-based vaccination method of the invention is effective in treating or preventing disease caused by disease causing agents or a disease state, including cancers and tumors.
  • Each disease causing agent or disease state frequently has associated with it an antigen or immunodominant epitope on the antigen which is crucial in immune recognition by a host, and the ultimate elimination or control of the disease causing agent in a mammal, sometimes referred to in the art as a protective antigen.
  • the mammalian immune system must come in contact with the antigen or immunodominant epitope on the antigen in order to mount a humoral and/or cellular immune response against the associated disease causing agent or cancer.
  • the nucleic acid-based immunogens of the invention comprises one or more isolated nucleic acid sequences encoding one or more isolated antigens or immunodominant epitopes on the antigens.
  • the immunogen also preferably comprises one or more nucleic acid sequences encoding one or more cytokines and/or one or more immunostimulatory molecules for the purpose of enhancing or augmenting the immune response against the disease causing agent or cancer. It is to be understood that a single plasmid or nucleic acid-based immunogen may contain the above-described nucleic acid sequences encoding one or more isolated antigens or immunodominant epitopes, along with the nucleic acid sequences encoding one or more cytokines and/or immunostimulatory molecules.
  • DNA-based immunogens for example, a plasmid comprising a nucleic acid sequence encoding one or more disease-causing antigens or an immunogenic portion thereof; a plasmid comprising a nucleic acid sequence encoding one or more cytokines; a plasmid comprising a nucleic acid sequence encoding immunostimulatory molecules, or mixtures thereof.
  • DNA immunogens may be delivered or administered to cells at the same time or at different times, as required or desired.
  • one or more cytokines and/or immunostimulatory molecules may be administered to a subject, preferably systemically, before, during, or following, preferably following, vaccination or immunization with the DNA-based immunogen to enhance the immune response to the disease causing agent.
  • disease causing agents include, but are not limited to, cancers of a variety of types, pathogenic microorganisms, parasites, viruses, or mammals (and parts thereof). Mammals include, but are not limited to, non-human primates, humans, rats, mice, guinea pigs, rabbits, horses, cows, sheep, pigs, goats, and the like.
  • Cancers of mammals which may be treated using the DNA-based immunotherapy in accordance with the invention include, but are not limited to, melanoma, carcinoma, metastases, adenocarcinoma, neuroblastoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, colon cancer, non-Hodgkins lymphoma, Hodgkins lymphoma, leukemias, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, and the like.
  • melanoma includes, but is not limited to, melanomas, metastatic melanomas, melanomas derived from either melanocytes or melanocyte- related nevus cells, melanocarcinomas, melanoepitheliomas, melanosarcomas, melanoma in sjtji, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma, invasive melanoma or familial atypical mole, and melanoma (FAM-M) syndrome.
  • melanomas metastatic melanomas
  • melanocarcinomas melanoepitheliomas
  • melanosarcomas melanoma in sjtji
  • superficial spreading melanoma nodular melanoma
  • Such melanomas in mammals may be caused by chromosomal abnormalities, degenerative growth and developmental disorders, mitogenic agents, ultraviolet radiation (UV), viral infections, inappropriate tissue expression of a gene, alterations in expression of a gene, altered or abnormal presentation on a cell, or carcinogenic agents.
  • UV ultraviolet radiation
  • a gene encoding an antigen associated with the cancer comprises the plasmid DNA used for immunization, and preferably, in conjunction with a gene encoding one or more cytokines. Genes encoding one or more immunostimulatory molecules may also be used for enhancement of the recipient's immune response.
  • the antigen associated with the cancer may be expressed on the surface of a cancer cell or may be an internal antigen. Additionally, the internal antigen, or portions thereof, may eventually be expressed or displayed on the cell surface.
  • the antigen associated with the cancer is a tumor associated antigen (TAA) or a portion thereof.
  • TAA tumor associated antigen
  • TAAs examples include, but are not limited to, melanoma TAAs which include, but are not limited to, MART-1 (Kawakami et al. , 1994, J. Exp. Med. 180:347-352), MAGE-1 , MAGE-3, GP-100, (Kawakami et al., 1994, Proc. Nat'l. Acad. Sci. U.S.A.
  • MAGE-3 The nucleotide sequence of the MAGE-3 gene is disclosed in Gaugler et al. , 1994, J. Exp. Med. 179:921-930. MAGE-3 is expressed on many tumors of several types, such as melanoma, head and neck squamous cell carcinomas, lung carcinoma and breast carcinoma, but is not expressed in normal tissues, except for testes.
  • the approximately 1.6 kilobase (kb) cDNA of MART- 1 was cloned into a vector and the resulting plasmid has been deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852 USA on April 14, 1994, and given ATCC Deposit Number 75738.
  • ATCC American Type Culture Collection
  • MART-1 The cloning of MART-1 is disclosed in Kawakami et al. , 1994, J. Exp. Med. 180:347-352 and in U.S. Serial No. 08/231 ,565, filed April 22, 1994.
  • the full length MART-1 nucleic acid sequence can be isolated from the pCRII plasmid by digestion with Malawi and Xhol restriction enzymes.
  • This l . ⁇ kb nucleic acid sequence, or portions thereof, can be used in the DNA immunogens as described herein along with a cytokine-encoding gene or genes and/or with an immunostimulatory molecule-encoding gene or genes.
  • the TAAs are CA-19-A (pancreatic cancer), CA-125 (ovarian cancer), PSA (prostate cancer), erb-2 (breast cancer), CA-171A and the like (Boon et al. , 1994, Ann. Rev. Immunol. 12:337).
  • TAAs the nucleic acid of which, may be identified, isolated and cloned by methods known in the art, such as those disclosed in U.S. Patent No. 4,514,506.
  • genes encoding an antigen of a disease causing agent in which the agent is a pathogenic microorganism in mammals may include viruses such as HIV (e.g., gpl20, pl7, gpl60 antigens), influenza (NP, HA and HI antigens), herpes simplex (HSVdD antigen), human papilloma virus, equine encephalitis virus, hepatitis (e.g., Hepatitis B Surface Antigen or HBSAg; HAV; HCV; HEV) feline leukemia virus, canine distemper, rabies virus, and the like.
  • Pathogenic bacteria include, but are not limited to, Chlamydia.
  • Pathogenic protozoans include, but are not limited to, malaria, Babesia. Schistosoma. Toxiplasma. Toxocara cams, and the like.
  • Pathogenic yeast include Aspergillus. invasive Candida, and the like.
  • the pathogenic microorganism is an intracellular organism that infects cells.
  • Immunostimulatory Molecules Cytokines and Costimulatory/ Accessory Molecules
  • one aspect of the invention includes a nucleic acid- based immunogen comprising a gene or genes encoding one or more costimulatory/accessory molecule(s) and/or a gene or genes encoding one or more cytokine(s) in combination with a gene encoding an antigen, or an immunogenic portion thereof, from a disease causing agent for immunization by direct DNA delivery to cells, preferably by particle-mediated introduction into cells and tissue, most preferably by means of a gene gun device, and the like.
  • costimulatory or immunostimulatory molecules include, but are not limited to, B7- 1 , B7-2, ICAM-1 , ICAM-2, LFA-1 , LFA-3, CD72 and the like.
  • cytokines encompassed by the present invention include, but are not limited to, IL- 1, IL-2, and IL-3 through IL-9, IL-10, IL-11 , IL-12, and IL-13 through IL-15, G- CSF, M-CSF, GM-CSF, TNF ⁇ , IFN ⁇ , IFN ⁇ , RANTES (Regulated upon Activation, Normal T Expressed and presumably Secrected cytokines, Promega, G5661), and the like.
  • Preferred cytokines include IL-2, IL-6, JL-7, and IL-12.
  • chemokines examples include, but are not limited to, CTAP m, ENA-78, GRO, 1-309, PF-4, IP-10, LD-78, MBSA, MlP-l , MIP-1B, and the like.
  • Costimulatory molecules of the B7 family represent a more recently discovered, but important group of cell regulatory or immunostimulatory or accessory molecules.
  • B7.1 and B7.2 are both members of the Ig gene superfamily. These molecules are present on macrophages, dendritic cells, and monocytes, i.e. , antigen presenting cells (APCs). If a lymphocyte encounters an antigen alone, without costimulation by B7.1 , it will respond with either anergy or apoptosis (programmed cell death); if the costimulatory signal is provided, it will respond with clonal expansion against the target antigen.
  • APCs antigen presenting cells
  • the B7.1 gene is present in a plasmid immunogen comprising the gene encoding the ⁇ -gal TAA under the control of the CMV promoter.
  • the construct for B7.2 and B7.1/B7.2 in conjunction with a tumor antigen is prepared in the same fashion as that for B7.1.
  • the gene encoding a cytokine, e.g. , IL- 12 is also present in the construct with the gene encoding ⁇ -gal and the gene encoding B7.
  • discrete plasmids containing the gene encoding ⁇ -gal, the gene encoding IL-12, and the gene encoding B7 are used in admixture as immunogen and delivered to the epidermis in the gene gun particle-mediated delivery system.
  • the gene encoding the TAA (e.g., ⁇ -gal) is delivered as the DNA immunogen by a hand-held gene gun device and cytokine (e.g., IL-12) is provided as adjuvant thereafter by systemic intraperitoneal administration.
  • cytokine e.g., IL-12
  • the present invention also encompasses methods of treatment or prevention of a disease caused by the disease causing agents disclosed herein.
  • direct gene delivery, e.g. , gene-gun administration, of the DNA immunogen of the invention may be either for prophylactic or therapeutic purposes.
  • the immunogen of the invention is provided in advance of any disease symptom, as a prophylactic vaccination.
  • a plasmid vaccine comprising one or more tumor antigen-encoding genes in combination with a cytokine and/or immunostimulatory molecule is a powerful system to elicit a specific immune response in terms of prevention in patient with an increased risk of cancer development (preventive immunization), prevention of disease recurrence after primary surgery (anti-metastatic vaccination), or as a tool to expand the number of CTL in vivo, thus improving the effectiveness in the eradication of diffuse tumors or infection (treatment or therapy of established disease).
  • the nucleic acid-based immunogens of the present invention can elicit an immune response in patients that is enhanced gx vivo prior to being transferred back to the tumor bearer (i.e. , adoptive immunotherapy).
  • unit dose refers to physically discrete units suitable as unitary dosages for mammals, each unit containing a predetermined quantity of plasmid or nucleic acid immunogen calculated to produce the desired immunogenic effect in association with the required diluent, carrier, or excipient.
  • the specifications for the novel unit dose of an inoculum of this invention are dictated by and are dependent upon the unique characteristics of the nucleic acid-based immunogen and the particular immunologic effect(s) to be achieved.
  • the inoculum is typically prepared as DNA precipitated onto gold particles as described herein, or in solution in a tolerable (acceptable) diluent such as saline, phosphate-buffered saline or other physiologically tolerable diluent, and the like, to form an aqueous pharmaceutical composition or excipient.
  • a tolerable (acceptable) diluent such as saline, phosphate-buffered saline or other physiologically tolerable diluent, and the like, to form an aqueous pharmaceutical composition or excipient.
  • the route of inoculation is preferably epidermal, although intramuscular delivery may also be used. It will be appreciated that the intramuscular route is preferred for direct immunization. Other routes of immunization include, but are not necessarily limited to, subcutaneous (s.c), intradermal (i.d.), intrasternal, intracranial, parenteral, and the like, to result in eliciting a protective or therapeutic response against the disease causing agent. The dose is administered at least once. Immunizations employing a direct gene delivery system, such as the gene gun, may be shot into sereral tissue types, including skin, liver, spleen, and muscle, for example.
  • shots may be delivered in the abdomen, and also in the back, limbs (e.g. , upper arm or leg (thigh)), buttocks, or the cranium, as necessary or required.
  • Subsequent boosting doses may be administered as indicated.
  • the dosage of administered immunogen will vary depending upon such factors as the mammal's age, weight, height, sex, general medical condition, previous medical history, disease progression, tumor burden, and the like.
  • a DNA immunogen in the range of from about 0.001 to about 100 ⁇ g of DNA, and more preferably from about 0.01 to about 50 ⁇ g, although a lower or higher dose may be administered depending upon the size and body mass of the recipient mammal.
  • Each direct delivery shot from a gene gun delivers 1 ⁇ g of DNA; therefore, multiple immunizing shots will be given to a recipient.
  • the animal receives from about 1 to 10 shots.
  • about 5 to 25 ⁇ g of DNA is a preferred range for immunization, and more preferably about 10 to 20 ⁇ g.
  • an individual will receive about 10 to 20 shots.
  • Immunizations may be given about 2 to 25 times (average for humans, about 10-20 times; average for mice about 2-3 times) during a course of treatment, although the number of times may be modified as required by the dosage to be administered and as determined by treatment protocol(s).
  • a cytokine may be given two to five times, preferably three times, per day. The amount of cytokine administered each time may vary, depending upon the cytokine used; however, the amount and parameters of administration for a given cytokine can be routinely determined by the skilled practitioner.
  • a typical high dose of IL-2 constitutes about 120,000 cetus units (cU)/kg administered three times per day, while a low dose of IL-2 constitutes about 12,000 cU/kg administered three times per day.
  • the nucleic acid-based immunogen can be introduced into a mammal either prior to any evidence of cancer, such as melanoma, or to mediate regression of the disease in a mammal afflicted with a cancer, such as meianoma or genetically inherited cancers.
  • a hand-held helium powered gene gun device was used to achieve the intracellular delivery of DNA- coated gold particles to the epidermis.
  • the DNA redissolves in the aqueous environment of the cytoplasm or nucleus of cells and is then available for expression.
  • epidermal gene gun immunization of DNA appeared to be most efficient at eliciting humoral and cellular immune responses in the recipient.
  • the efficacy of the vaccine can be assessed by monitoring the production of antibodies or immune cells that recognize the antigen, as assessed by specific lytic activity or specific cytokine production or by tumor regression.
  • Those having skill in the art are familiar with the conventional methods used to assess the aforementioned parameters. If the mammal to be immunized is already afflicted with cancer or metastatic cancer, the vaccine can be administered in conjunction with other therapeutic treatments.
  • autologous cytotoxic lymphocytes or tumor infiltrating lymphocytes may be removed from the patient with cancer as disclosed in U.S. Patent No. 5,126,132 and U.S. Patent No. 4,690,915.
  • the ly ⁇ bhocytes are grown in culture and antigen specific lymphocytes are expanded by culturing with antigen or antigen bearing cells or by delivering the DNA constructs of the invention to the cells.
  • the antigen specific lymphocytes are then reinfused back into the patient.
  • the present invention also encompasses combination therapy.
  • combination therapy is meant that the DNA-based immunogen containing one or more genes encoding one or more antigens associated with one or more disease- causing agents along with DNA encoding cytokine, and optionally, if desired, one or more genes encoding one or more immunostimulatory molecules is/are administered to the patient in combination with other exogenous unmunomodulators or immunostimulatory molecules, chemotherapeutic drugs, antibiotics, antifungal drugs, antiviral drugs, and the like, alone or in combination.
  • the combination therapy includes exogenous cytokines such as IL-2, IL-6, IL-7, and IL-12.
  • the combination therapy includes other exogenously added agents in addition to IL-2, IL-6, IL-7 or IL-12, or combinations thereof, such as cyclophosphamide, and cisplatin, gancyclovir, amphotericin B and the like.
  • Another aspect of the invention is an antibody or antibodies elicited by immunization with the DNA-based immunogen the present invention.
  • the antibody has specificity for and reacts with or binds to the antigen or epitope thereof, of interest.
  • the antibodies are monoclonal or polyclonal in origin.
  • Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules, or those portions of immunoglobulin molecules that contain the antigen binding site, including those portions of immunoglobulin molecules known in the art as F(ab), F(ab'); F(ab') 2 and F(v).
  • Polyclonal or monoclonal antibodies may be produced by methods conventionally known in the art.
  • the antibodies or antigen binding fragments may also be produced by genetic engineering.
  • the technology for expression of both heavy and light chain genes in IL C_QH is the subject of the PCT patent applications: publication numbers WO 901443 and WO 9014424, and in Huse et al., 1989, Science 246: 1275-1281.
  • the antibodies are used in immunoassays to detect the novel antigen(s) of interest in biological samples.
  • MART-1 antibodies generated by immunization with the DNA encoding MART-1 and the administration of cytokines, and, optionally, costimulatory molecules are used to assess the presence of the MART-1 antigen from a tissue biopsy of a mammal afflicted with melanoma, using immunocytochemistry.
  • Such assessment of the delineation of the MART-1 antigen in a diseased tissue can be used to prognose the progression of the disease or cancer in a mammal afflicted with the disease or the efficacy of immunotherapy.
  • Conventional methods for immunohistochemistry are described in Harlow and Lane (eds). 1988.
  • the dosage of administered antibodies or antigen binding fragments will vary depending upon such factors as the mammal's age, weight, height, sex, general medical condition, previous medical condition and the like. In general, it is desirable to provide the recipient with a dosage of antibodies or antigen-binding fragments which is in the range of from about 1 mg/Kg to about 10 mg/Kg of body weight of the mammal, although a lower or a higher dose may be administered.
  • the antibodies or antigen-binding fragments of the present invention are intended to be provided to the recipient subject in an amount sufficient to prevent, lessen, or attenuate the severity, extent, or duration of the disease or infection.
  • cytokine(s) may be administered as DNA which is co-injected with DNA encoding the tumor or disease-associated antigen, or as an exogenous agent serving as adjuvant. In this way, cytokines involved in the activation and expansion of lymphocyte populations may improve the therapeutic effects of the DNA-based immunization.
  • immunizations were performed using a nucleic acid-based immunogen comprising an isolated nucleic acid sequence encoding a disease or cancer-associated antigen, e.g. , a tumor associated antigen, using direct delivery of the nucleic acid to cells and tissues.
  • the preferred mode of delivery of the nucleic acid-based immunogen is directly into cells and tissues using a mechanical delivery device such as a gene gun for the treatment of an established tumor or disease-causing agent or to prevent tl.e establishment of a tumor or disease-causing agent.
  • DNA-based immunization was successfully used to prime T lymphocytes reactive with tumor associated antigen prior to ex vivo expansion and adoptive immunotherapy.
  • a model system as described and exemplified hereinbelow was employed to assess the efficacy of DNA-based immunization as a potential cancer treatment strategy.
  • the model murine tumor was CT26, which expresses the tumor associated antigen (TAA), /3-galactosidase (/3-gal); the /3-gal- expressing tumor is designated CT26.CL25 (see Example 1).
  • the nucleic acid- based immunogen comprising a plasmid expressing 3-gal (i.e., pCMV//3-gal), was administered by particle-mediated gene delivery to the epidermis using a hand-held, helium driven "gene gun” and induced /3-gal-specific antibody and lytic responses.
  • Immunization with this construct prevented the growth of pulmonary metastatic tumor.
  • m (SEQ DD NO: 1) immunodominant peptide reduced the number of established pulmonary nodules in recipient mammals.
  • cytokines was added as adjuvants following DNA administration. The reduction in the number of established metastases was particularly significant when IL-2, D 6, IL-7 or IL-12 were given after DNA ino ⁇ lation, with IL-12 as an adjuvant having a most profound effect in the reduction of tumor burden.
  • the invention embraces both the immunization of DNA immunogens directly delivered intracellularly, for example, by particle- mediated gene gun administration and the administration of cytokines for the activation and expansion of lymphocyte populations to improve the therapeutic effects of DNA immunization.
  • DNA immunization administered together with cytokine adjuvant, and optionally, costimulatory molecules offers an attractive alternative to other types of vaccines, such as recombinant or attenuated viral vaccine preparations.
  • the invention further provides the novel use of gene gun immunization of nucleic acid-based immunogens for the prevention and treatment of cancer and pathogenic disease and/or infection.
  • a preferred aspect is immunization by gene gun delivery of plasmid DNA encoding tumor associated antigen for the prevention and treatment of cancers and established tumors.
  • Plasmid DNA delivered by particle- mediated delivery induced potent humoral and CTL lytic immune responses that likely afforded a protective immunity from tumor challenge (see Figs. 1 and 2A-2D).
  • Other modes of DNA delivery such as particle bombardment or direct intradermal or intramuscular inoculation of DNA, may also be suitable for use in the invention; however, the gene gun method offers potent and consistent antibody and CTL responses to antigen(s) using small quantities of nucleic acid as immunogen (e.g. , as little as 0.1 ⁇ g to 0.01 ⁇ g of DNA).
  • the invention also encompasses beads that are coated with DNA encoding immune enhancing and regulating molecules, such as cytokines and costimulatory molecules, for immunomodulating, augmenting and improving anti-tumor and anti- disease causing antigen immune effects.
  • immune enhancing and regulating molecules such as cytokines and costimulatory molecules
  • non-self antigens do bear similarities to some potential tumor antigens such as viral antigens expressed in virally-induced tumors, mutated tumor suppressor genes (Hollstein, M., et al., 1991, p53 mutations in human cancers. Science 253:49), fusion proteins resulting from translocations (Gribben, J.G.
  • Bone marrows of non-Hodgkin's lymphoma patients with a bcl-2 translocation can be purged of polymerase chain reaction-detectable lymphoma cells using monoclonal antibodies and immunomagnetic bead depletion. Blood 80: 1083), frame-shifts, and loss of stop codons.
  • DNA encoding the foreign ⁇ -gal antigen administered via the gene gun technique in conjunction with the effects of exogenously administered cytokines stimulated potent immune responses and protection in animals in vivo.
  • the invention provides not only potent humoral and cellular immune responses, but also therapeutic responses such that established tumors were treated in active immunotherapy, as demonstrated by the observed impact on tumor burden in a tumor bearing animal model system.
  • This aspect of the invention was achieved by the use of one or more cytokines (particularly the interleukins, such as recombinant human interleuldn-2 (rhIL-2), recombinant murine interleukin-6 (rmIL-6), recombinant human interleukin-7 (rhIL-7), and recombinant murine interleukin-12 (rmIL-12), administered following DNA administration.
  • cytokines and the DNA encoding the cytokines are commercially available.
  • immunstimulatory molecules suitable for combined use in the invention include, but are not limited to, TNF- ⁇ , IFN- ⁇ , G-CSF, GM-CSF, and IL-1 to IL-15.
  • IL-2, H.-6, IL-7 and IL-12 were optimally able to enhance the therapeutic responses, with IL-12 having a most profound adjuvant effect.
  • IL-2, EL-7 and IL-12 have all been reported to stimulate or activate T cell populations as well as natural killer (NK) cells.
  • IL-12 also functions to regulate immune responses by directing a Th2 to a Thl phenotype (Hsieh, C.S., et al., 1993, Development of Thl CD4-I- T cells through IL-12 produced Listeria-induced macrophages.
  • IL-6 known as B cell stimulatory factor-2, is a pleiotropic cytokine that can enhance CTL function, NK activity, LAK and ⁇ L activity (Mule, J.J. , et al. 1992. Cellular mechanisms of the antitumor activity of recombinant IL-6 in mice. J_ Immunol. 148:2622; Takai, Y., et al.
  • cytotoxic T lymphocytes J. Immunol. 140:508
  • the above-mentioned cytokines have been reported to have anti-tumor effects when administered as single agent therapy.
  • the doses of the cytokines used in the present invention had little or no effect on the growth of CT26.CL25 cells.
  • IL-4 may steer the T helper T cell population toward a Th2 phenotype responsible for the enhancement of humoral responses, but not of cell mediated immunity.
  • IFN- ⁇ which upregulates the production of key molecules involved in antigen processing and presentation of intracellular antigens, may also have an anti-proliferative effect on T cells.
  • cytokines to serve as effective adjuvants resulting in active treatment to mediate tumor reduction in accordance with the present invention, they should function to cause the proliferation and activation of primed lymphocytes, particularly CTLs.
  • increasing the concentrations of cytokines used, or employing combinations of different cytokines, including those mentioned above may achieve an enhanced immune response against pathogenic diseases and cancer when used in conjuction with DNA-based immunization.
  • DNA encoding one or more cytokines may be contained in the plasmid DNA comprising the immunizing DNA encoding the tumor or pathogenic disease-associated antigen, or immunodominant portion thereof, and under the control of the same or a different promoter and/or regulatory or control sequences.
  • DNA encoding one or more costimulatory agents may be contained in the plasmid DNA comprising the immunizing DNA encoding the tumor or pathogenic disease-associated antigen and under the control of the same or a different promoter or regulatory or control sequences.
  • a mammal is immunized not only with DNA which encodes proteins from the disease causing agent or tumor, but also with DNA which encodes molecules that stimulate the activation and proliferation of cytolytic T lymphocytes, which are needed to reduce or eradicate the disease or tumor.
  • the effect of antigen-specific DNA immunization using cytokine DNA (and/or costimulatory molecule DNA) is similar to another of the invention's aspects which includes the administration of exogenous cytokines as adjuvants (and/or the ex- administration of exogenous costimulatory molecules) following (or at the time of) DNA immunization.
  • Cytokines as adjuvants may be administered by several modes and routes, such as parenterally, including intraperitoneal (i.p.), subcutaneous (s.c), intradermal (i.d.), intramuscular (i.m.), intrathecal, and intrasternal, for example.
  • the cytokines are administered in a sterile solution or suspension, such as sterile physiological saline or other injectable aqueous liquids, or may be emulsified in pharmaceutically- and physiologically-acceptable aqueous or oleaginous vehicles, which may contain preservatives and material for rendering the solution or suspension isotonic with body fluids (e.g. , blood) of the recipient.
  • Excipients suitable for use are water, phosphate buffered saline, pH 7.4, aqueous sodium chloride solution (0.15 M) dextrose, glycerol, dilute ethanol, and the like, and mixtures thereof. If oral administration is desired or necessary, the composition, including cytokine, may be presented as a draught in water or in a synip, in capsules, cachets, boluses, or tablets, as will be known to those having skill in the art.
  • the present invention which preferably comprises double stranded DNA as immunogen, promises to be safe and is not expected to generate adverse antibody responses, but rather to result in the generation of heightened antibody and cell-mediated responses that are specific for the disease causing antigens, as demonstrated by the experimental evidence herein.
  • Example 1 describes the materials and methods employed in the further examples presented in Examples 2-6 hereinbelow. Tumor Cell Lines and Animals
  • the murine colon adenocarcinoma, CT26.WT is a clone of the N- nitroso-N-methylurethane induced BALB/c (H-2 d ) undifferentiated colon carcinoma (Brattin, M. G., et al., 1980, Establishment of mouse colonic carcinoma cell lines with different metastatic properties. Cancer Res. 40:2142).
  • CT26.WT was subcloned to generate the /3-gal-expressing cell line CT26.CL25 (Wang, M., et al., 1995, Active immunotherapy of cancer with a nonreplicating recombinant fowlpox virus encoding a model tumor-associated antigen. J. Immunol. 154 (9):4685). All tumor cell lines were grown and maintained in complete medium (CM) containing RPMI 1640, 10% heat inactivated FCS (both from Biofluids, Rockville, MD), 0.03% fresh L-glutamine, and 100 ⁇ g/ml gentamicin sulfate.
  • CM complete medium
  • CT26.CL25 was grown in the presence of 400 ⁇ g/ml G418 (GIBCO, Grand Island, N.Y.).
  • G418 G418
  • CMV human cytomegalovirus
  • pCMV//3-gal Agracetus, Middleton, WI
  • plasmids containing the LacZ gene and suitable promoters such as the CMV immediate-early promoter, which is commercially available from Clontech, Palo Alto, CA
  • CMV immediate-early promoter which is commercially available from Clontech, Palo Alto, CA
  • the CMV- ⁇ - galactosidase portion of a CMV/ ⁇ -gal plasmid obtained from Clontech was produced by digestion of the CMV/ ⁇ -gal plasmid with EcoRl and Hindm, thereby creating a fragment containing the CMV promoter and the LacZ coding region. This fragment was ligated into the PGEM 4Zf vector, commercially available from Promega Corp., Madison, WI, which had been restricted with £ ⁇ >Rl and Hindm.
  • Plasmids expressing either human growth hormone (pCMV/hGH) (provided by Agracetus, Middleton, WI or constructed employing commercially available or published nucleic acid sequences) or the nucleoprotein from influenza A (A/PPJ8/34) (pCMV/NP) under the control of the CMV intermediate-early promoter were used as control vectors.
  • pCMV/hGH human growth hormone
  • A/PPJ8/34 the nucleoprotein from influenza A
  • the control plasmid vectors are constructed using routine methods and reported nucleic acid sequence information for hGH and influenza A NP, for example. Constructs were transformed into R ccji DH5 alpha competent cells (GIBCO/BRL,
  • influenza NP I47 [55 peptide, TYQRTRALV (SEQ ID NO: 2) , presented by H-2K d were synthesized by Peptide Technologies (Washington, DC) to a purity of greater than 99 % as assessed by HPLC and amino acid analysis.
  • Plasmid DNA was affixed to gold particles by adding 10-50 mg of 0.95 ⁇ m gold powder (Agracetus, Middleton, WI) to 1.5 ml centrifuge tubes containing 100 ⁇ l of 0.1 M spermidine (Sigma Chemical Co., St. Louis, MO). Plasmid DNA and gold were coprecipitated by the addition of 200 ⁇ l of 2.5 M CaClj during vortex mixing as previously described (Fuller, D.H., et al., 1995, A qualitative progression in HIV type 1 glycoprotein 120-specif ⁇ c cytotoxic cellular and humoral immune response in mice receiving a DNA-based glycoprotein 120 vaccine. AIDS Res. Hum. Retrovir. 10 (11): 1433).
  • Accell ® gene delivery system (Agracetus, Middleton, WI, and commercially available from BioRad, Hercules, CA). Each animal received 2-10 non- overlapping deliveries per immunization (as designated below), at a pressure of 400 psi of helium.
  • Enzyme Linked Immunosorbant Assay (ELISA)
  • BALB/c mice were immunized two times at two week intervals with 0.001-1.0 ⁇ g of either pCMV//3-gal or pCMV/NP using the gene gun. Serum samples were collected two weeks following the second immunization and analyzed for the presence of anti-/3-gal antibodies by ELISA. Specifically, microtiter plates were dried overnight at 37°C in a non-humidified incubator with 200 ng/well/50 ⁇ l of either purified /3-gal or control antigen, ovalbumin (both obtained from Sigma Chemical Company, St. Louis, MO). One hundred ⁇ l of 5% bovine serum albumin (BSA) in PBS were incubated on each well for 1 hour to prevent nonspecific Ab binding.
  • BSA bovine serum albumin
  • mice Primary lymphocyte populations were generated by immunization with different amounts of purified pCMV/j3-gal or control pCMV/NP. Secondary in vitro effector populations were generated by harvesting spleens of mice 14 days after immunization and culturing single cell suspensions of splenocytes in T-75 flasks (Nunc, Roskilde, Denmark) at a density of 3.0 x IO 6 cells/ml with 1 ⁇ g/ml antigenic peptide at a total volume of 30 ml of CM containing 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate (both from Biofluids) and 5 x 10 "3 M 2- meitaptoethanol (GIBCO BRL, Rockville, MD) in the absence of IL-2. Six days later, splenocytes were harvested and washed in CM before testing in a 5l Cr release assay or for transfer to tumor bearing animals. sl Cr release assay
  • mice were immunized with different amounts of pCMV/3-gal or with either control pCMV/hGH or pCMV/NP. Fourteen days later, mice were challenged i.v. with 2 x 10 5 tumor cells (Mule, J.J. , et al., 1987, Identification of cellular mechanisms operational in vivo during the regression of established pulmonary metastases by the systemic administration of high-dose recombinant interleukin-2. J. Immunol. 139:285). Mice were sacrificed on day 17 and randomized before counting lung metastases in a blinded fashion as previously described (Ibid.). Adoptive Immunotherapy experiments
  • mice were infected i.v. with 2 x 10 s CT26.WT (0-gal -) or CT26.CL25 (/3-gal +) cultured tumor cells in 0.5 ml of HBSS to induce pulmonary metastases.
  • tumor bearing mice were treated with an i.v. injection of various effector cells at 5 x 10 6 cells/dose.
  • mice were immunized with 1 ⁇ g of pCMV//3-gal or pCMV/NP. Each mouse received two 0.25 mg shots of gold loaded with 0.5 ⁇ g DNA.
  • mice were challenged with 5 x 10 5 CT26.WT or CT26.CL25 tumor cells i.v. to establish pulmonary metastases.
  • mice were immunized with 10 ⁇ g of either pCMV//3-gal, pCMV/NP DNA or no DNA.
  • Each mouse received ten non -overlapping shots of gold (0.5 mg each) delivering 1 ⁇ g of DNA.
  • the mice were killed and metastatic lung modules were enumerated in a randomized, blinded manner.
  • mice As a positive control, a group of mice was included that received a recombinant vaccinia virus encoding /3-gal (VJS6) plus the exogenous administration of the cytokine IL-2 (15,000U, BID, for 5 days) as previously reported (Bronte, V., et al. , 1995, IL-2 enhances the function of recombinant poxvirus-based vaccines in the treatment of established pulmonary metastases. J. Immunol. 154 (10):5282).
  • VJS6 recombinant vaccinia virus encoding /3-gal
  • IL-2 enhances the function of recombinant poxvirus-based vaccines in the treatment of established pulmonary metastases. J. Immunol. 154 (10):5282).
  • Intraperitoneal treatments of various cytokines began 18-24 hours following DNA administration and continued daily for 3-7 days depending on the cytokine. Specifically, one group of mice received 15,000 cetus units of recombinant human IL-2 (rhIL-2) twice daily (BUD) for five days (Chiron Corp., Emeryville, CA) (Ibid.). A second group of mice received treatments of 0.5 ⁇ g of recombinant mouse IL-6, BID, for three days (Peprotech, Inc., Rocky Hill, NJ). A third group received 5 ⁇ g of rhIL-7 for seven days (Peprotech, Inc., Rocky Hill, NJ).
  • a fourth group received 1.0 ⁇ g of rmIL-12 once daily (QD) for five days (Genetics Institute, Boston, MA).
  • QD rmIL-12 once daily
  • rmIL-4 5 ⁇ g, BID, 7 days
  • rmIL-10 1.0 ⁇ g, QD, 7 days
  • GM-CSF 1.0 ⁇ g
  • mice were immunized with a plasmid cDNA encoding the model TAA /3-gal (pCMV//3-gal).
  • Mice immunized and boosted with gold particles coated with as little as 0.01 ⁇ g of pCMV//S-gal developed /3-gal specific antibody responses (Fig. 1).
  • gold particles coated with 1.0 ⁇ g of the control plasmid pCMV/NP failed to elicit a S-gal specific antibody response.
  • no reactivity was observed against a control antigen, ovalbumin, confirming the specificity of the humoral immune response.
  • mice were inoculated one time with varying quantities of pCMV//3-gal or control vector, pCMV/NP.
  • CT26.WT cells pulsed with the immunodominant peptide /3-galg 76 .gg 4 was also recognized by the pCMV//3-gal immune splenocytes generated with at little as 0.01 ⁇ g of DNA. Pulsed cells in this case appeared to be more sensitive to lytic cells than the transfected cells, CT26.CL25. Unpulsed CT26.WT cells were not significantly lysed, thus demonstrating the specificity of the lytic response.
  • Prophylactic DNA vaccine protects mice from intravenous tumor challenge
  • mice were immunized with the
  • DNA vaccines of the invention and assayed for the growth of a subsequent intravenous tumor challenge. Only the mice that had received pCMV//3-gal as immunogen showed significant responses when compared with the mice that were inoculated with control DNA (Fig. 3). In pooled results from three experiments, virtually complete protection was observed with 1.0 ⁇ g of pCMV//3-gaI immunogen; 19 out of 20 pairs of lungs from immunized mice were devoid of any detectable tumor. At a 10-fold lower dose, 16 out of 20 pairs of lungs were completely free of disease. With a dose of 0.01 ⁇ g of DNA immunized, the protective effects began to wane, with only 6 out of 20 mice remaining disease free.
  • tumor bearing mice were treated with splenocytes from donor mice that had received gene gun immunization with either pCMV//3-gal DNA as immunogen or pCMV/NP DNA as immunogen, and then subsequent ex vivo incubation of the cells with either 3-gal g76 .gg 4 (SEQ ID NO: l) or control NP 147 . 15J
  • gg4 (SEQ ID NO: l) peptide completely cleared the lungs of mice bearing 3 day old pulmonary metastases (Fig. 4). By contrast, lungs from
  • NP, 47 .uj SEQ ID NO: 2
  • the splenocytes induced by pCMV//3-gal DNA and stimulated with /3-gal g76 . gg4 (SEQ ID NO:l) peptide were not effective at eliminating metastases in mice bearing the antigen-negative tumor, CT26.WT (Fig. 4), thus demonstrating the in vivo specificity for the /S-gal antigen. Similar results were obsSrved in a repeat experiment.
  • mice bearing two day established pulmonary metastases were immunized with pCMV//3-gal to evaluate the ability of gene gun immunization of
  • IL-2 enhances the function of recombinant poxvirus-based vaccines in the treatment of established pulmonary metastases. J. Immunol. 154 (10):5282).
  • DNA immunogen i.e. , > lOO ⁇ g reduce tumor burden, in the absence of cytokine co-administration following inoculation with immunogen.
  • Cytokine administration following DNA vaccine leads to treatment of established pulmonary metastases
  • rhIL-2, rmIL-6 and rhIL-7 have been reported to activate or proliferate antigen-specific CTL populations (Mul ⁇ , J.J., et al., 1992, Cellular mechanisms of the antitumor activity of recombinant IL-6 in mice. J. Immunol. 148:2622; Jicha, D.L., et al., 1991 , Interleukin 7 generates antitumor cytotoxic T lymphocytes against murine sarcomas with efficacy in cellular adoptive immunotherapy. J. Exp. Med. 174: 1511).
  • GM-CSF promotes the differentiation of hematopoietic precursors to dendritic cells that function to present antigen to prime naive lymphocytes (Inaba, K. , et al. , 1992, Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 176: 1693).
  • rmIL-12, rmIL-4 and rmIL-10 have been shown to direct T helper populations to different Thl or Th2 phenotypes resulting in shifts to either humoral or cell mediated responses, respectively (Mehrona, P.T.
  • rhIL-2, rmIL-4, rmIL-6, rhIL-7, rmIL-10, rmIL-12 or rmGM-CSF administered exogenously.
  • rhIL-2, rmIL-6, rhIL-7 and rmIL-12 were found to specifically and optimally induce antigen-specific active immunotherapy.
  • Fig. 5 illustrates the average number of pulmonary metastases on day 12 following tumor challenge for those mice that received the DNA vaccine followed by either rhIL-2, rmIL-6 or rhIL-7 administration.
  • Fig. 5 represents the pooled averages of two separate experiments performed using the same protocol. Mice that were injected with pCMV//3-gal DNA immunogen alone demonstrated no significant reduction in the number of metastases (215), compared with control non-immunized mice ( > 250).
  • rmIL-12 used alone demonstrated only a modest reduction in the tumor burden with an average of 177 ⁇ 45 metastatic nodules (mets) compared with > 250 in the untreated group.
  • patients with advanced melanoma are immunized against MART-1 , an immunodominant protein from a cancer antigen, in conjunction with cytokine as adjuvant as described herein.
  • costimulatory /accessory molecules such as B7-1 , also as described herein, may be used in combination with DNA immunization and cytokine administration.
  • B7-1 costimulatory /accessory molecules
  • Patients must have evidence of measurable or evaluable metastatic melanoma that has failed standard effective therapy. Patients must have tumors that express the MART-1 antigen as evidenced by PCR or Northern Blot analysis of tumor cell RNA.
  • the DNA-based vaccine/immunogen used in this protocol is a PUC-1 -based plasmid containing the gene coding for the MART-1 protein under the control of the CMV promoter, and also having the CMV intron A enhancer region and splice donor/acceptor sites.
  • the DNA-based immunogen is produced in a Food and Drug Administration (FDA) approved facility for the manufacture of GMP grade clinical material.
  • FDA Food and Drug Administration
  • Vaccination procedure The DNA for vaccination will be precipitated onto gold pellets which are coated and dried onto the interior of Teflon tubing (e.g. , Teszel
  • Tubing 1/8 inches OD; 3/32 inches ID; McMasters Carr). Approximately half- inch pieces of the gold-coated tubing are used as "bullets". Within each bullet is approximately 0.25 to 0.5 mg of gold which carries from 0.001 ⁇ g to as much as 1 ⁇ g of DNA. The size of the gold pellets are determined according to need and use and can range from about 0.95 microns to about 1 to 3 microns.
  • Melanoma patients receive epidermal immunization with plasmid cDNA encoding MART-1 driven by the CMV promoter as described above and approved by the FDA. Patients receive three successive immunizations, each separated by three to four weeks with the same dose of DNA. Cohorts of patents are treated at each dose. Patients will be observed for 24 hours after immunization. Body temperature will be measured at 12 hours and at 24 hours and any adverse reactions noted.
  • the dose escalation schedule for the MART-1 DNA C immunization ( ⁇ g DNA) is approximately as follows:
  • the adjuvant cytokine is administered 18 to 24 hours following DNA vaccination.
  • Doses of 0.001-10 ⁇ g are well tolerated by a 20 gram mouse and are thus 1/30000 of the dose by weight tolerated by mice. Patients are carefully evaluated for toxicity. When three patients at each dose level have been followed for at least two weeks after the first immunization without achieving grade DJ or IV toxicity, not easily reversible by standard measures, then the dose in that patient category will be escalated to the next dose level. Patients are also monitored for anti-DNA antibodies. In addition, cells at the site of injection are assayed for potential DNA integration, a potential problem, but one that has not previously been observed in the mouse model system when DNA was directly injected into muscle. Expected accruel is 15-20 patients a year.
  • IL-2 DNA immunogen
  • IL-2 or IL-.2 administration in each of the following groups: a) IL-2, subcutaneous injection of 250, IU/kg daily for 5 days and repeated once after a 2 day rest. b) IL-2, intravenous infusion of 720,000 IU/kg every 8 hours o for 7 days, not to exceed 21 doses. c) IL-2, intravenous doses of 720,000 IU/kg every 8 hours for 5 days not to exceed 15 doses. d) IL-12, intravenous doses in amounts determined in consideration of murine studies in which mice were given 0.5 ⁇ g/mouse once a day for 5 days.
  • IL-2 is administered at a dose of 720,000 IU/kg as an intravenous bolus over a 15 minute period every eight hours beginning on the day after DNA immunization and continuing for a total of four days. Doses may be skipped depending on patient tolerance. For example, doses are skipped if patients reach grade DJ or IV toxicity. If this toxicity is easily reversed by supportive measures, then additional doses may be given. No more than 12 doses of IL-2 are administered after each immunization. IL-2 is administered as an inpatient.
  • IL-2 (Chiron), NSC #373364, is provided as a lyophilized powder.
  • Each 5 cc vial has a labeled strength of 1.3 mg (22 Million IU).
  • the vial is reconstituted with 1.2 ml of sterile water for injection, USP, and the resultant concentration is 18 million IU/ml.
  • Diluent should be directed against the side of the vial to avoid excess foaming. Vial contents are swirled, not shaken, gently until completely dissolved. Since vials contain no preservative, the reconstituted solution should be used within 8 hours.
  • Intact vials are stored in the refrigerator (i.e. , 2°C-8°C) with protection from light. Each vial bears an expiration date.
  • Reconstituted IL-2 is further diluted with 5 % Dextrose, USP and is not mixed with saline-containing solutions.
  • Reconstituted IL-2 may be diluted as necessary in volumes of 50 ml to 500 ml with 5 % Dextrose, USP plus 0.1 % Albumin Human, USP.
  • the Albumin Human USP should be added to the 5 % Dextrose Injection, USP prior to the addition of the IL-2.
  • a plastic bag e.g. Viaflex, manufactured by Travenol Laboratories, Inc. ,
  • IL-2 is chemically stable for 48 hours at both refrigerated and room temperatures, (i.e., 2°C-30°C).
  • IL-2 is provided as lyophilized powder to be reconstituted by the patient with 1.2 cc of sterile water (U.S. P.). All reconstituted vials must be kept refrigerated (5°C) and must be discarded after 24 hours. All patients receive tylenol 650 mg p.o., every 4-6 hours while on therapy and indocin 50 mg p.o. every 6 hours is used on a PRN basis to treat constitutional symptoms.
  • a Durable Power of Attorney is signed by patients receiving IL-2 to identify a surrogate to make decisions, if a patient becomes unable to make decisions.
  • patients are treated as in-patients and have vital signs monitored every 4 hours and are seen by physicians at least twice per day to monitor for confusion.
  • Concomitant therapy All patients receiving IL-2 receive concomitant medications to relieve side effects, as is routine in high-dose IL-2 protocols.
  • acetaminophen 650 mg every 4 hours
  • indomethacin 50-75 mg every 6 hours
  • ranitidine 150 mg every 12 hours
  • Patients may receive intravenous meperidine (25 to 50 mg) to control chills when they occur, although chills are unusual after the first one to two doses of IL-2.
  • Steroids are not used in these patients and if steroids are required, then the patient is removed from the protocol.
  • Immunologic assessment will be made of the patient's response to the MART-1 antigen, as follows: a) Serum samples will be tested for anti-MART-1 antibody, e.g., by ELISA. b) Cryopreserved lymphocytes will be tested for response to the MART-1 associated antigen (or GP-100 associated antigen, if applicable; see below) using limiting dilution analysis of precursor CTL frequency using the method of Coulie, P. et al., 1992, International Journal of Cancer 50:289-297. c) Patients will easily accessible disease may have biopsy under local anesthesia of accessible tumor to study the histopathologic nature of the tumor, as well as the isolation of tumor infiltrating lymphocytes (JTL) for jn vitro growth. TIL will be tested for specific reactivity and specific cytokine release against the MART-1 associated antigen. Assessment of response
  • a complete response is defined as the disappearance of all clinical evidence of disease that lasts at least four weeks.
  • a partial response is a 50% or greater decrease in the sum of the products of the perpendicular diameter of all measurable lesions for at least four weeks, with no appearance of new lesions or increase in any lesions.
  • Minor responses are defined as about a 25% to 49% decrease in the sum of the products of the perpendicular diameters of all measurable lesions with no appearance of new lesions and no increase in any lesions. Any patient with less than a partial response is considered a non- responder.
  • the appearance of new lesions, or greater than 25 % increase in the product of perpendicular diameters of prior lesions following a partial or complete response, will be considered as a relapse.
  • Vaccines to be tested include, but are not limited to, immunogens comprising DNA encoding MART-1 , followed by the systemic administration of one or more cytokines, preferably IL-6, IL-7, or EL-12, or combinations thereof; immunogens compnsing DNA encoding MART-1 and
  • ADDRESSEE MORGAN AND FINNEGAN, L.L.P.

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Abstract

Cette invention a trait à une substance immunogène à base d'acide nucléique, administrée, de préférence, à un receveur à l'aide d'un dispositif d'administration génique direct, un fusil génétique par exemple, l'objectif étant l'atténuation de troubles d'origine pathogène, de cancers notamment, ou leur traitement. Plus précisément, une immunisation par administration directe de substances immunogènes à base d'acide nucléique en association avec un adjuvant, la cytokine en l'occurrence, et, si on le souhaite, avec une ou plusieurs molécules immunostimulatrices, possède, tout d'abord, une action prophylactique et thérapeutique dans le cas de cancer ou de tumeur. La substance immunogène à base d'acide nucléique sous la forme d'ADN plasmidique, par exemple, inoculée par administration particulaire, déclenche de puissantes réactions immunologiques humorales et à médiation cellulaire se soldant par une immunité protectrice contre les risques d'apparition de tumeurs. Des réactions thérapeutiques actives chez des mammifères porteurs de tumeurs sont élicitées lorsque des cytokines (IL-2, IL-6, et IL-12) sont administrées en tant qu'adjuvant à la suite d'une immunisation par substances immunogènes à base d'ADN. L'invention porte également sur l'utilisation d'une technique d'administration génique directe à médiation cellulaire ainsi que sur une nouvelle stratégie d'emploi de l'ADN comme substance immunogène de concert avec des cytokines et/ou des agents co-stimulateurs aux fins du traitement de tumeurs installées. Elle permet également d'enrober chaque particule de plusieurs gènes codant différentes molécules, le résultat étant que chaque cellule bombardée peut élaborer plus d'une protéine, ce qui contribue à stimuler la réaction immunitaire et à la renforcer.
PCT/US1996/020571 1995-12-19 1996-12-18 Amelioration de l'immunisation a l'adn obtenu a l'aide de cytokines WO1998008947A1 (fr)

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Cited By (15)

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WO1999030734A1 (fr) * 1997-12-18 1999-06-24 G.D. Searle & Co. Vecteurs viraux de l'encephalite equine venezuelienne exprimant des antigenes associes aux tumeurs, suscitant une immunite contre le cancer
WO1999036089A1 (fr) * 1998-01-16 1999-07-22 The Johns Hopkins University Immunisation genetique par l'administration conjointe d'acides nucleiques et de cytokines dans un excipient unique
WO1999040936A3 (fr) * 1998-02-12 1999-10-28 American Cyanamid Co Vaccins pneumococciques ou meningococciques formules avec l'interleukine-12
WO1999056784A3 (fr) * 1998-05-06 2000-02-17 Transgene Sa Utilisation d'un inhibiteur de nuclease ou de l'interleukine 10 (il-10) dans la preparation d'une composition therapeutique visant a ameliorer la transfection d'un polynucleotide dans une cellule, compositions utilisees en therapie genique
WO2000041679A1 (fr) * 1999-01-13 2000-07-20 Johns Hopkins University School Of Medicine Immunisation genetique avec administration conjointe d'acide nucleique et de cytokines
WO1999040938A3 (fr) * 1998-02-12 2000-08-24 American Cyanamid Co Vaccins comprenant l'interleukine 12 et antigene viral de l'herpes
WO2000029582A3 (fr) * 1998-11-18 2000-10-12 Pacific Northwest Research Ins Vaccins a base d'antigenes recepteurs de surface
WO2001025792A1 (fr) * 1999-10-01 2001-04-12 Glaxo Group Limited Dosage de reponse de cellules t in vivo suite a une vaccination par adn
WO2001046697A3 (fr) * 1999-12-21 2002-01-10 Millennium Pharmaceuticals, Inc. Compositions, kits et procedes pour l'identification, le diagnostic, la prevention et le traitement du cancer du sein
WO2002060921A3 (fr) * 2000-11-09 2003-09-25 Univ Illinois Facilitation de la reponse immunitaire au vaccin au moyen de l'interferon alpha
WO2005014642A3 (fr) * 2003-07-21 2005-05-26 Transgene Sa Nouvelles cytokines multifonctionnelles
US7268120B1 (en) 1997-11-20 2007-09-11 Vical Incorporated Methods for treating cancer using cytokine-expressing polynucleotides
US7608267B2 (en) 2003-07-21 2009-10-27 Transgene S.A. Multifunctional cytokines
AU2011203139B2 (en) * 1998-02-27 2014-06-19 The Trustees Of The University Of Pennsylvania Vaccines, immunotherapeutics and methods for using the same
WO2022099022A1 (fr) * 2020-11-05 2022-05-12 Neoimmunetech, Inc. Procédé de traitement d'une tumeur au moyen d'une combinaison d'une protéine il-7 et d'un vaccin nucléotidique

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US7268120B1 (en) 1997-11-20 2007-09-11 Vical Incorporated Methods for treating cancer using cytokine-expressing polynucleotides
US7470675B2 (en) 1997-11-20 2008-12-30 Vical Incorporated Methods for treating cancer using interferon-ω-expressing polynucleotides
WO1999030734A1 (fr) * 1997-12-18 1999-06-24 G.D. Searle & Co. Vecteurs viraux de l'encephalite equine venezuelienne exprimant des antigenes associes aux tumeurs, suscitant une immunite contre le cancer
WO1999036089A1 (fr) * 1998-01-16 1999-07-22 The Johns Hopkins University Immunisation genetique par l'administration conjointe d'acides nucleiques et de cytokines dans un excipient unique
WO1999040936A3 (fr) * 1998-02-12 1999-10-28 American Cyanamid Co Vaccins pneumococciques ou meningococciques formules avec l'interleukine-12
WO1999040938A3 (fr) * 1998-02-12 2000-08-24 American Cyanamid Co Vaccins comprenant l'interleukine 12 et antigene viral de l'herpes
AU2011203139B2 (en) * 1998-02-27 2014-06-19 The Trustees Of The University Of Pennsylvania Vaccines, immunotherapeutics and methods for using the same
AU752658B2 (en) * 1998-05-06 2002-09-26 Association Francaise Contre Les Myopathies Use of a nuclease inhibitor or interleukin-10 (IL-10) for the preparation of a therapeutic composition for improving transfection of a polynucleotide into a cell and compositions useful in gene therapy
EP1254961A1 (fr) * 1998-05-06 2002-11-06 Transgene S.A. Améliorations de la thérapie génique en utilisant des compositions qui contiennent un polynucléotide et de l'interleukin-10
US6528312B1 (en) 1998-05-06 2003-03-04 Transgene S.A. Use of G-actin for improving transfection of a polynucleotide into a cell
WO1999056784A3 (fr) * 1998-05-06 2000-02-17 Transgene Sa Utilisation d'un inhibiteur de nuclease ou de l'interleukine 10 (il-10) dans la preparation d'une composition therapeutique visant a ameliorer la transfection d'un polynucleotide dans une cellule, compositions utilisees en therapie genique
US7547681B2 (en) 1998-11-18 2009-06-16 University Of Washington Surface receptor antigen vaccines
WO2000029582A3 (fr) * 1998-11-18 2000-10-12 Pacific Northwest Research Ins Vaccins a base d'antigenes recepteurs de surface
US6734172B2 (en) 1998-11-18 2004-05-11 Pacific Northwest Research Institute Surface receptor antigen vaccines
WO2000041679A1 (fr) * 1999-01-13 2000-07-20 Johns Hopkins University School Of Medicine Immunisation genetique avec administration conjointe d'acide nucleique et de cytokines
WO2001025792A1 (fr) * 1999-10-01 2001-04-12 Glaxo Group Limited Dosage de reponse de cellules t in vivo suite a une vaccination par adn
WO2001046697A3 (fr) * 1999-12-21 2002-01-10 Millennium Pharmaceuticals, Inc. Compositions, kits et procedes pour l'identification, le diagnostic, la prevention et le traitement du cancer du sein
WO2002060921A3 (fr) * 2000-11-09 2003-09-25 Univ Illinois Facilitation de la reponse immunitaire au vaccin au moyen de l'interferon alpha
US7388087B2 (en) 2000-11-09 2008-06-17 The Board Of Trustees Of The University Of Illinois Enhancement of immune response to vaccine by interferon alpha
US7767653B2 (en) * 2000-11-09 2010-08-03 The Board Of Trustees Of The University Of Illinois Enhancement of immune response to vaccine by interferon alpha
US7534585B2 (en) 2003-07-21 2009-05-19 Transgene S.A. Multifunctional cytokines
US7608267B2 (en) 2003-07-21 2009-10-27 Transgene S.A. Multifunctional cytokines
AU2004263274B2 (en) * 2003-07-21 2009-11-05 Transgene S.A. Novel multifunctional cytokines
US7947288B2 (en) 2003-07-21 2011-05-24 Transgene S.A. Viral particles encoding multifunctional cytokines
WO2005014642A3 (fr) * 2003-07-21 2005-05-26 Transgene Sa Nouvelles cytokines multifonctionnelles
WO2022099022A1 (fr) * 2020-11-05 2022-05-12 Neoimmunetech, Inc. Procédé de traitement d'une tumeur au moyen d'une combinaison d'une protéine il-7 et d'un vaccin nucléotidique

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