Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
1999-01-04
2001-12-11
Nguyen, Dave T. (Department: 1633)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C514S04400A, C435S320100, C435S455000, C435S458000
Reexamination Certificate
active
06328957
ABSTRACT:
The invention relates to expression vectors which express heat shock proteins or chaperones.
Conventionally, solid tumours are treated by a combination of surgery, chemotherapy and radiotherapy. Other neoplasms, such as leukaemia, are also treated by chemotherapy and radiotherapy. However, these methods of treatment are not ideal because radiotherapy and chemotherapy cause unpleasant side effects and some solid neoplasms, such as brain tumours, cannot be treated surgically. For these reasons new methods for treating and preventing tumours are constantly being sought. One method which is currently being investigated is the use of gene transfer of, for example, IL-2 or TNF encoding genes.
Tumours may be induced in mice by administering tumour cell lines. In particular, mice to which the murine macrophage tumour cell line J774 is administered develop large, intraabdominal, highly malignant lymphoreticular neoplasms. Surprisingly, it has been found that, when such tumour cells are transfected with the gene encoding the
Mycobacterium leprae
65 kD hsp and subsequently administered to mice, there is a decrease in or loss of tumourigenicity of the cells. Furthermore, when such transfected cells are administered to a host, such as a mouse, and the host is subsequently challenged with tumour cells from the same cell line which have not been transfected, no tumour develops. Therefore the technique has potential for an alternative or additional approach to the treatment of neoplasms.
The present invention provides a mammalian cell or an expression vector for use as a therapeutic agent, for example in the treatment of neoplasms comprising nucleic acid including at least one sequence which encodes in expressible form a polypeptide which is a heat shock polypeptide (hsp) or a chaperone.
A polypeptide includes, for example, a heat shock protein, a heat shock protein fragment, a heat shock protein analogue, a chaperone, a chaperone fragment or a chaperone analogue. A fragment will be at least 10, preferably at least 15, for example, 20, 25, 30, 40, 50 or 60 amino acid residues in length. An analogue will be generally at least 70%, preferably at least 80% or 90% and more preferably at least 95% homologous to the heat shock protein or chaperone over a region of at least 5, preferably at least 10, for example 20, 40, 60 or 100 or more contiguous amino acids. The amino acid sequence of an analogue may differ from that of the heat shock protein or chaperone when at least one residue is deleted, inserted or substituted.
A chaperone is a protein which mediates the folding of proteins into their active conformation. Polypeptides exist which are both chaperones and heat shock polypeptides. Heat shock polypeptides, such as hsp60, hsp65 and hsp70 are involved in the folding and translocation of other proteins, and are therefore also chaperones.
Fragments include polypeptides which are not glycosylated but which retain their anti-neoplasm activity. Analogues include proteins which have been engineered with a different amino acid sequence, but which retain their anti-neoplasm activity.
The nucleic acid of the expression vector of the present invention may be double or single stranded and may be RNA or DNA. Generally the nucleic acid includes only one sequence which encodes a polypeptide as defined herein, but the nucleic acid may include 2, 3, 4 or more sequences which separately encode a polypeptide as defined herein.
The nucleic acid comprised in the expression vector or cell line of the present invention encodes any suitable polypeptide as defined herein but preferably does not encode a polypeptide of the host. Preferably the polypeptide is a chaparonin and most preferably the polypeptide is bacterial hsp, such as Mycobacterium leprae 65 kD hsp or
Mycobacterium leprae
70 kD hsp.
The expression vector of the invention is generally a virus, plasmid, cosmid, episomal vector or naked nucleic acid. Generally the naked nucleic acid comprises the sequence encoding the polypeptide together with regulatory sequences. The naked nucleic acid generally comprises at least 100, for example at least 200, 400, 600, 800, 1000, 2000 or 4000 base pairs. The naked nucleic acid may be single or double stranded and may be DNA or RNA. Suitable plasmids include, for example, pZIPNeo, pLXSN and MFG.
Viruses which are conveniently used as the expression vector of the invention include viruses in which the nucleic acid of the vector is RNA, for example disabled retroviruses, and DNA viruses such as the vaccinia virus or an adenovirus. The vector or virus may be administered directly to the host, so as to achieve expression of the polypeptide in infected cells, or may first be incorporated into a cell line which is then administered to the host. When the vector or virus is administered directly to the host, the virus used is generally helper-free and replication-defective.
Preferably the expression vector comprises recombinant nucleic acid.
The present invention also provides a process for producing an expression vector other than naked nucleic acid, which process comprises cloning into a vector a gene encoding a polypeptide as defined herein. According to the process of the present invention the polypeptide-encoding gene may be cloned directly into an expression vector using known methods (Silva, Palacios, Colston and Lowrie, Microbiol Pathogenesis 12, 27-38 (1992)). The invention also provides a process for producing a cell which process comprises inserting heterologous DNA into the genome of the cell or transfecting an expression vector of the invention into a cell line. According to the present invention the heterologous DNA may be inserted into the genome using known methods (Molecular Cloning: A Laboratory Manual, T. Maniatis et al, Cold Spring Harbour, 1989).
Preferably a transfected cell line is prepared by cloning the gene encoding the polypeptide into a suitable vector, such as a retroviral shuttle vector, for example pZIPNeoSV(x), using conventional techniques and transfecting the vector into a cell line using known methods (Silva et al).
Cell lines into which expression vectors according to the present invention may be incorporated are generally mammalian and include neoplasm cell lines, MHC I and II negative cell lines, and murine and human cell lines. It is possible to use cell lines, for instance neoplasm cell lines, that originate from the host or patient to which they will be administered, particularly in order to treat established tumours. Most preferably, the cell line which is transfected is that of the neoplasm against which protection is sought.
The expression vector or cell of the invention may further include a sequence which encodes a neoplasm associated antigen or an interleukin. The expression vector or cell of the invention may include an expression vector having a sequence which encodes a neoplasm associated antigen or an interleukin. Several studies have shown that transfection of murine tumour cells with genes encoding cytokines results in the immunological rejection of the parent tumour. The cytokirne genes which have been used for this type of experiment include IL-4, IL-2, &ggr; IFN, TNF&agr;, IL-6, IL-7 and GM-CSF. One or more of is these genes may act synergistically with a gene encoding a polypeptide of the invention and hence maximise the chances of achieving tumour eradication.
Neoplasms caused by different and unrelated neoplasm cell lines may have common neoplasm associated antigens, in which case immunisation according to the invention with one cell line which expresses such a common antigen may give protection against other neoplasms which also have common antigens (but is unlikely to give much protection-against those neoplasms which do not). Accordingly, the present invention also provides a cell line which expresses a polypeptide as defined herein and a neoplasm associated antigen. Such cell lines may be transfected with an expression vector according to the invention together with a separate vector encoding a neoplasm associated antigen or they may be transfected with an expressi
Colston Michael J.
Lowrie Douglas
Lukacs Katalin V.
Medical Research Council
Nguyen Dave T.
Nixon & Vanderhye P.C.
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