Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
2000-02-09
2004-05-11
Falk, Anne-Marie (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C536S023500
Reexamination Certificate
active
06734173
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to DNA vaccines.
Antigen-specific immunotherapy has recently emerged as an approach for controlling cancer because it is capable of developing specific immunity against neoplastic cells while not attacking normal cells. DNA vaccination differs from traditional vaccination in that DNA encoding an antigen (not the antigen itself) is injected into the subject. The production of the antigen, i.e., expression of the antigen encoded by DNA in the vaccine, takes place in the body of the vaccinated individual. However, conventional DNA vaccines have limitations. For example, a major drawback of most DNA vaccines is their potency.
SUMMARY OF THE INVENTION
The invention is based on the discovery that the immunogenicity of a target antigen encoded by a DNA vaccine is enhanced by the presence in the DNA vaccine construct of DNA encoding a carboxyterminal portion of
Mycobacterium tuberculosis
heat shock protein 70 (HSP70). Accordingly, the invention provides compositions and methods of vaccination that significantly enhance the potency, and thus clinical efficacy, of DNA vaccines.
An immunogenic composition contains a first DNA encoding a carboxyterminal fragment of a heat shock protein, e.g., HSP70, operably linked to a second DNA encoding a MHC class I restricted antigen. A carboxyterminal fragment of a protein is a polypeptide which is at least 10 amino acids in length and is derived from a half of a naturally-occurring protein that contains a COOH end. For example, a carboxyterminal fragment of HSP70 is a peptide the amino acid sequence of which is derived from a portion of HSP70 spanning residues 312-625 of SEQ ID NO:9 and is encoded by DNA spanning the coding region of those residues. Preferably, the carboxyterminal fragment contains the amino acid sequence of residues 517-625 of SEQ ID NO:9. For example, the immunogenic composition contains a first DNA encoding a polypeptide containing the amino acid sequence of residues 517 to 625 of SEQ ID NO:9 operably linked to a second DNA encoding a MHC class I restricted antigen. The invention therefore includes an E7-HSP70-CD fusion polypeptide as well as DNA encoding the fusion polypeptide. Optionally, the composition includes DNA encoding a polypeptide containing residues derived from the aminoterminal fragment of HSP70, e.g., residues 161-370 of SEQ ID NO:9. The order in which the DNA components, e.g, first and second (and optionally, third), DNAs are operably linked can be altered without affecting immunogenicity. For example, the HSP-encoding DNA sequences are located 5′ or 3′ to the target antigen-encoding sequences. Preferably, the DNA is operably linked so that the DNA construct encodes a recombinant polypeptide in which the MHC class I restricted antigen is located aminoterminal to the HSP-derived residues.
As is discussed below, the MHC class I restricted antigen is derived from a pathogen such as a virus or from a cancer tissue. The DNA vaccine of the invention contains a plasmid vector, which contains a first DNA encoding a carboxyterminal fragment of a heat shock protein operably linked to a second DNA encoding a MHC class I restricted antigen. Therapeutic methods include methods of inducing a cytotoxic T cell response to an antigen in a mammal by administering to the mammal the immunogenic compositions described herein.
Alternatively, an immunogenic composition of the invention contains the following components: (a) a first DNA containing a sequence encoding a polypeptide which binds to a professional antigen presenting cell, (b) a second DNA containing a sequence encoding a cytoplasmic translocator polypeptide, and (c) a third DNA containing a sequence encoding a major histocompatibility complex (MHC) class I restricted antigen. The first, second, and third DNAs are operably linked. By “operably linked” is meant that DNA encoding a polypeptide is joined in frame to another DNA encoding a another polypeptide. Translation of operably linked DNAs yields a chimeric polypeptide or fusion gene product. The order in which the first, second, and third DNAs are operably linked is irrelevant. The construct may also contain regulatory sequences. A polypeptide coding sequence and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).
The first DNA of the immunogenic composition encodes a fragment of a heat shock protein (HSP), e.g.,
Mycobacterium tuberculosis
heat shock protein 70 (HSP70). A “fragment” of a given protein is a polypeptide which is shorter in length than the reference polypeptide. For example, the polypeptide is at least 9 or 10 amino acids in length but less than the total number of residues of the mature reference protein or polypeptide. For example, a fragment of HSP70 is less than 70 kDa in molecular mass. A fragment of HSP70 has an amino acid sequence that is at least 50% identical to the amino acid sequence of a naturally-occurring HSP70. The length of an HSP70 fragment is less than 625 amino acids. Preferably, the fragment has a biological activity of the reference protein. For example, the fragment binds to a professional antigen presenting cell (APC) such as a dendritic cell (DC), or the fragment mediates translocation into the cytoplasm of the chimeric polypeptide encoded by the DNA of the immunogenic composition. In some embodiments, the first DNA or the second DNA (or both) encode a fragment of a heat shock protein.
The third DNA of the immunogenic composition encodes an antigenic epitope such as a MHC class I-restricted antigen. Preferably, the antigen is derived from a virus such as a human papovavirus, e.g., a cervical cancer-associated human papillomavirus (HPV). The viral antigen is all or a part of E6 or E7 antigen derived from HPV-16. Other viral antigens include the E2 antigen of bovine viral diarrhea virus; ppUL83 or pp89 of cytomegalovirus; prM/E of encephalitis virus SLE; HBV surface antigen or HBV core antigen of hepatitis B virus; HIV-1 antigens such as gp160; ICP27, gD2, glycoprotein B, or glycoprotein B of herpes simplex virus. Alternatively, the antigen is a cancer antigen such as a mutant p53; MAGE-1 or MAGE-3 associated with melanoma cells, e.g. a cancer-associated antigen expressed on the surface of a tumor cell. Other antigens include malaria peptide (NANP)40, HIV-1 p24, or influenza nucleoprotein.
In one example, the first DNA encodes granulocyte-macrophage colony stimulating factor (GM-CSF) or a fragment thereof. Preferably, a functional GM-CSF fragment contains a disulfide bridge, e.g., a disulfide bridge which spans Cys 51 and Cys93 of SEQ ID NO:1. Other biologically active fragments of GM-CSF are polypeptides which include residues 18-22, 34-41, 38-48, 52-61, 94-115, 95-111 of SEQ ID NO:1. Alternatively, the first DNA (encoding an APC-binding polypeptide) encodes Flt3 ligand (FL), CTLA-4, 4-1BB, CD40 ligand, or TNF receptor (or an APC-binding fragment thereof).
The second DNA may encode a translocation domain of a Pseudomonas exotoxin A (ETA), e.g,. domain II (dII) of ETA (spanning residues 253-364 of SEQ ID NO:3). A translocation domain is a polypeptide that induces translocation of protein or polypeptide to which it is linked into the cytosol of a cell. For example, the second DNA encodes a polypeptide derived from a Diphtheria, clostridial (Botulinum, Tetanus), Anthrax, Yersinia, Cholera, or
Bordetella pertussis
toxin. The presence of DNA encoding a translocation domain in the immunogenic composition enhances MHC class I presentation of the antigen encoded by the composition through translocation of antigen from the endosomal/lysosomal compartment to the cytosol. Preferably, the toxic domain of the gene encoding the toxin is mutated or deleted.
The immunogenic composition need not contain a first, second, and third DNA, as described above. In one alternative embodiment, the third DNA (encoding a target antigen to which immunity is desired) is operably linked to a DNA encoding a
Hung Chien-Fu
Wu Tzyy-Choou
Johns Hopkins University
Livnat Shmuel
Venable LLP
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