Title:  DNA vaccine and methods for its use

United States Patent:  6,472,375

Issued:   October 29, 2002

Inventors:  Hoon; Dave S. B. (Los Angeles, CA); Kaneda; Yasufumi (Osaka, JP)

Assignee:  John Wayne Cancer Institute (Santa Monica, CA)

Appl. No.:  144837

Filed:  August 31, 1998

DNA cancer vaccines and methods for their use are described. The vaccines are comprised of viral liposomes comprising nucleic acid, preferably DNA, encoding a tumor-associated antigen. The viral liposomes may be formed by the fusion of HVJ reagents with nonviral reagents. The vaccine may be administered subcutaneously, intradermally, intramuscularly or into an organ. The vaccine may be administered to induce a host normal cell to express the tumor associated antigen.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The invention provides novel efficient approaches using a viral liposome system to deliver tumor antigen nucleic acids in vivo to immunize the host. The invention avoids many of the tedious processes involved in vaccine preparation. Quality control and purity of plasmid DNA or RNA can be more easily monitored. Fusigenic viral liposomes can provide an efficient vehicle to package, deliver and direct nucleic acid to specific targets and at the same time protect against nucleic acid degrading enzymes in body fluids and cytoplasmic organelles. Tumor antigen is expressed on a background of normal cell(s) as opposed to a tumor cell. Nucleic acid vaccination provides an opportunity for molecular immuno-physiologic manipulation of antigen expression that can be a useful tool in cancer vaccine design. The vaccine provided by this invention does not incorporate into hemopoietic-derived cells as do other liposome delivery systems.

The invention provides methods and compositions for immunizing against a tumor-associated antigen, suppressing or attenuating tumor growth, and treating cancer. The methods and compositions provided may induce an antibody response (IgG, IgM, IgA) or a cell-mediated immune response through CD4, CD8 or other lymphocyte subsets.

The compositions provided by the invention are comprised of viral liposomes comprising nucleic acid encoding a tumor-associated antigen. A preferred method of producing the viral liposomes is to fuse anionic or cationic liposomes with an inactive virus, preferably HVJ as described in Dzau, et al. and U.S. Pat. No. 5,631,237.   HVJ proteins are known for their fusing properties of cell membranes of nonhemopoietic nucleated cells. This hybrid vector allows targeting to normal nonhemopoietic cells and delivery of encapsulated nucleic acid into the cytoplasm of cells without lysosome-endosome degradation. The viral liposome system also can deliver nucleic acid and protein molecules together. In addition, purified or recombinant fusion polypeptides of HVJ rather than the entire viral envelope may be used.

Any number and combination of nucleic acid sequences encoding TAAs may be included in the vaccine. The MAGE gene family members are examples of TAAs that would be useful in the invention. MAGE-1, MAGE-2, and MAGE-3 antigens were first described in melanoma and subsequently demonstrated in various other cancers. MAGE-1 and MAGE-3 genes are expressed in greater than 30 percent of melanomas and carcinomas such as lung, breast, liver and gastrointestinal cancers, but not in normal tissues except testes. The MAGE-1 and MAGE-3 antigens have been shown to be immunogenic, expressed by a wide variety of human cancers and not expressed by normal tissues. These factors are important in the overall design of an effective vaccine against multiple cancers.

There are other MAGE gene family members with similar homologies. De Plaen, E, et al. The strategy of using two dominant immunogenic MAGE family antigens may be beneficial in that they can elicit immunity to a wide spectrum of MAGE antigens expressed by different human tumors. Vaccination with MAGE-1 and MAGE-3 could induce immune responses to tumors expressing either antigen because of the cross-reactivity between MAGE-1 and MAGE-3. This property is useful because MAGE-1 and MAGE-3 are not always co-expressed in the same tumor biopsy or cell lines.

Nucleic acid sequences encoding many other TAAs will be useful in the vaccines provided by the invention. B-catenin, TRP-2, TRP-1, gp100/pmel17, MART-1, GAGE-1, BAGE-1, HSP-70, gp43, .beta.-HCG, Ras mutation, MUC-1, 2, and 3, PSA, p53 mutation, HMW melanoma antigen, MUC-18, HOJ-1, tyrosinase, and carcinoembryonic antigen (CEA) are examples. In general, any antigen that is found to be associated with cancer tumors may be used. See Gomella, et. al., Gerhard, et al., Zhang, et al., Nollau, et al., Mivechi, et al., Ralhan, et al., Yoshino, et al, Shirasawa, et al., Cheung, et al., Sarantou, et al., Doi, et aL, Hoon, et al. (1997), Eynde, et al., Hoon, et al. (1996), Takahashi, et al., Kawakami, et al., Wolfel, et al., Vijayasaradhi, et al., Yokoyama, et al., Kwon, and Sensi, et al.

Multiple genes can be incorporated into the vaccine to produce a polyvalent antigen DNA cancer vaccine. Effective tumor vaccination is highly likely to require a polyvalent antigen vaccine to control human tumor progression effectively. Nucleic acids encoding these antigens can be incorporated into the vaccine provided by this invention.

A drug sensitive gene can be incorporated into the DNA vector to turn off protein expression at any time in vivo. Examples of drugs to which genes can be sensitive are Tetracycline, Ampicillin, Gentamycin, etc.

An immunogenic determinant, such as Diphtheria toxin, also may be included as a "helper" antigen on the TAA to improve its efficacy. Diphtheria toxin B fragment COOH-terminal region has been shown to be immunogenic in mice. Autran, B., et al. HSP70, in part or in whole, as well as other immunogenic peptides, such as influenza viral or immunogenic sequences peptide with an anchoring motif to HLA class I and class II molecules, also may be included in the vaccines of the invention.

The compositions may include other components to serve certain functions, for example, directing the nucleic acid to a certain location in the cell or directing transcription of the tumor-associated antigen. Compositions for transport to the nucleus may be included, particularly members of the high mobility group (HMG), more particularly HMG-1, which is a non-histone DNA-binding protein. In combination with antisense molecules, RNAses such as RNAseH, may be used, which degrade DNA-RNA hybrids. Other proteins which will aid or enhance the function of the TAA may be included, such as peptide sequences that direct antigen processing, particularly HLA presentation, or movement in the cytoplasm.

The vaccine provided by this invention may be administered subcutaneously, intramuscularly, intradermally, or into an organ. The vaccine also may be injected directly into the tumor to enhance or induce immunity. Intramuscular injection has been shown in the past to be an important delivery route for induction of immunity. Skeletal muscle has properties such as high vascularization and multi-nucleation. In addition, it is nonreplicating and capable of expressing recombinant proteins. These properties are advantageous for gene therapy. One theory of the mechanism of how muscle presents the protein and induces immune response is that recombinant protein is produced and released into the vascular network of the muscle and eventually presented by professional antigen-presenting cells such as dendritic cells, myoblasts, or macrophages infiltrating the muscle. Another suggestion is that at the injection site muscle injury induces myoblast proliferation and activation of infiltrating macrophages or dendritic-like cells, and they then present antigens through MHC class II antigen. Thus, other tissues which have similar qualities also would be good delivery sites for the vaccine.

The chosen route of administration will depend on the vaccine composition and the disease status of patients. Relevant considerations include the types of immune cells to be activated, the time which the antigen is exposed to the immune system and the immunization schedule. Although many vaccines are administered consecutively within a short period, spreading the immunizations over a longer time may maintain effective clinical and immunological responses.

In determining immunization scheduling for cancer vaccines, the following questions should be considered with regard to individualizing vaccine protocols:

Are multiple immunizations over a short period of time better than over a long period of time to induce long term effective immunity?

If the patient develops a tumor recurrence should the vaccination protocol be changed?

Does excessive immunization induce immune suppression or tolerance?

Should immunization schedules differ among individuals with different clinical stages of disease?

An example of an administration schedule is to administer vaccines by injection at weeks 0, 2, 4, 8, 12, 16, and every fourth week successively for 1 year. After that, patients are laced on a 3- to 6-month vaccine schedule for several years. A preventative immunization schedule may consist of three immunizations, one every three to four weeks. Treatment after removal of a tumor may consist of immunization every week for one month.

An example of when the cancer vaccines described herein may be useful follows. Typically, patients come to the clinic with early stage (AJCC I, II, III) melanoma which has a potential to spread in the body. The tumor is excised. The patient is free of disease. Based on historical prognostic factors often AJCC stage II and III patients (>40%) will have disease recurrence and eventually death within 5 years. Prophylactic vaccination would help in preventing disease recurrence in these patients to improve survival and control tumor progression. DNA vaccination can be applied in this situation. Vaccination has no major deleterious side effects as chemotherapy or radiation. Vaccines also may be given to high risk individuals likely to have cancer (based on congenital, family history of cancer, high frequency of nevi on the body, or other known indicators).

Unlike most non-neoplastic diseases treated with vaccines, an actively growing cancer is a dynamic biological entity that is genetically and phenotypically continuously evolving. A tumor that is allowed to evolve genetically and phenotypically will eventually become more difficult for the host immune system to control. However, cancer vaccines may be more effective when combined with other adjuvant therapies such as chemotherapies, or other immunotherapies such as monoclonal antibodies and cytokines. A more aggressive treatment regimen approach at early stages of tumor development may be more effective in preventing the evolution of escape mechanisms. A more aggressive treatment also may be necessary for cancers that are highly malignant versus relatively benign cancers with a low risk of recurrence. In general, if the host has a weak immune response to the vaccination, then a larger dose or a more frequent vaccination should be given.

The present invention allows repeated administration of the vaccine because injection of HVJ-liposomes produces levels of antibody not sufficient to neutralize ether vaccination by HVJ-liposomes. In addition, significant effective cytotoxic T-cells against HVJ are not generated.

Claim 1 of 33 Claims

What is claimed is:

1. A vaccine comprising, in an amount effective to suppress or attenuate melanoma growth upon administration to a human, viral liposomes comprising a fusigenic HVJ polypeptide and DNA encoding a melanoma-associated antigen, wherein said viral liposomes do not include live viral particles.
 

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