Therapies designed to cure or prevent the progression of cancer are often based on biological or metabolic differences between normal and malignant cells. Many cancers are characterized by unrestricted or abnormal growth of the cancer cell population at the expense of host cells and tissues. The relatively rapid cancer cell growth rate compared to normal cells requires high levels of metabolic activity on the part of the cancer cells. One activity associated with malignant cells, the high rate of deoxyribonucleic acid (DNA) replication as compared with that of most normal cells, has been extensively examined as a potential target for therapeutic approaches to treat cancer. Thus, there has been a great deal of effort directed toward the development of therapeutic agents or treatments that preferentially kill malignant cells by interfering with one or more aspects of their DNA replication. Such approaches have met with some success, but because such therapies are often nonspecific, normal host cells with high growth rates, such as bone marrow cells, are also susceptible to killing by the same agent or therapy. The toxicity of cancer therapies toward normal cells often leads to severe side effects and limits their ultimate efficacy.
Another difficulty related to cancer treatment centers on the characteristic spread or metastasis of cancer cells to sites distant from the origin of the initial cancer cell. Advanced stages of cancer are often associated with malignant growths at multiple sites. Such spreading often complicates or prevents the successful treatment of cancer by surgical means.
The difficulties related to the design of effective therapies for cancer has spurred intense research directed toward understanding the molecular basis of cancer and its development from normal cells. This research has led to an understanding of some of the biological events that lead to full-blown malignancy starting from a normal cell. Land et al., Science, 222, 771 (1983) summarized research findings that implicated cellular oncogenes in the development of cancer. Additional subsequent efforts have been directed toward understanding the function of oncogenes at a molecular level. Activated oncogenes have been used to construct animal models of cancer development, further substantiating their role in the etiology of cancer (Hanahan, Ann. Rev. Genet., 22, 479 (1988)). This knowledge has created an awareness that activated oncogenes appear to represent targets for therapeutic intervention in cancer therapy (Huber, Fed. Amer. Soc. Exper. Biol. J., 3, 5 (1989)). For example, Drebin et al., Cell, 41, 695 (1985) demonstrated that modulation of the neu oncogene protein by monoclonal antibodies to the surface of transformed cells reversed the transformed phenotype of tumor cells in vitro. However, it is becoming clear that while activated oncogenes represent potential therapeutic targets for cancer therapy, the differences between them and their normal cellular counterparts are often subtle. The subtle differences suggests that development of specific and effective therapies, without toxic side effects, will prove to be difficult. The fundamental similarity between malignant and normal cells renders malignant cells only somewhat more susceptible to killing based on therapies that affect targets or activities that both cell types share.
An alternate approach to the development of tumor-specific therapy has been adduced wherein a metabolic difference is artificially introduced into cancer cells that renders them susceptible to killing by a therapeutic agent (Moolten, Can. Res., 46, 5276 (1986)). This concept, the mosaic strategy, requires prophylactic generation of mosaicism in tissues that may in the future become cancerous (Moolten, Med. Hypotheses, 24, 43 (1987)). The success of the strategy relies on tumor cells differing from normal cells by carrying an inserted gene that confers either drug susceptibility to the malignant cells or drug resistance to the normal cells. The aim of creating mosaicism is to artificially create a significant metabolic difference between normal and malignant cells. This difference then serves as a therapeutic target for effective malignant cell killing with minimal toxicity to normal cells. Because cancer often results from proliferation of a single transformed cell, all of the cells in a given case of cancer will tend to be identical or clonal (Fialkow, Biochim. Biophys. Acta, 458, 283 (1976)). If, prior to the development of a malignant cell, a drug susceptibility gene was inserted into that same cell, then all of the cells associated with the ensuing cancer would be susceptible to the appropriate therapeutic agent. Cells in the subject that did not receive the drug sensitivity gene would be resistant to the drug. Thus, mosaic strategy clearly represents a prophylactic application of transduced cells for therapeutic use.
The basic concept of the mosaic model approach has not yet been demonstrated in vivo because generation of malignant cells from normal transduced cells in vivo has not been observed. Insertion of the herpesvirus thymidine kinase (TK) gene into mammalian cells renders them sensitive to the nucleoside analog ganciclovir (GCV). GCV toxicity is conferred by enzymatic activity of the TK gene which metabolically activates GCV in cells (Nishiyama et al., J. Gen. Virol., 45, 227 (1979)). The activated GCV is toxic to cells and kills them. The TK gene by itself is not lethal to cells in the absence of GCV. Mouse tumor cell lines containing the TK gene were shown to give rise to tumors in vivo in mice. Tumor regression occurred if GCV was administered to the mice, while control mice that did not receive GCV failed to survive the progression of cancer (Moolten, Can. Res., 46, 5276 (1986)). Subsequent experiments with retroviral vectors carrying the TK gene were used to transfer TK enzyme activity to tumor cell lines in tissue culture. One mouse tumor, a sarcoma, was then shown to be sensitive to GCV therapy in vitro and in vivo (Moolten and Wells, J. Natl. Cancer Inst., 82, 297 (1990)). These experiments demonstrated the feasibility of killing tumor cells in vivo by using genetic engineering to create a target for cancer chemotherapy.
However, as pointed out by the authors of those experiments (Moolten, Can. Res., 46, 5276 (1986); Moolten, Med. Hypotheses, 24, 43 (1987); Moolten et al., J. Natl. Cancer Inst., 82, 297 (1990), the mosaic strategy suffers from a number of drawbacks and cannot be used in human subjects with the existing technology for genetically engineering cells. A current limitation associated with gene therapy using retroviral vectors is the inefficient transfer of genes to long-lived normal stem cells in humans (Eglitis et al., Biochem. Biophys. Res. Commun., 151, 201 (1988)). Retroviral vectors are the most efficient vehicles in current use for the transfer of exogenous genes into mammalian cells (Eglitis et al., Biotechniaues, 6, 608 (1988)). Another limitation associated with retroviral vectors when used in vivo as envisioned in the mosaic strategy, is their limited and transient expression of inserted genes after introduction of the genetically-engineered cells into animals (Anderson et al., Cold Spring Harbor Symp. Quant. Biol., 51, 1073 (1986)). Successful application of the mosaic model to cancer treatment requires that a gene inserted in vivo will continue to express at a later time, possibly years later, when tumors arise from mosaic tissues. Another requirement of the mosaic strategy is the efficient infection of a relatively high proportion of stem cells that may later give rise to a tumor. If stem cells are inefficiently infected, then the probability that a tumor will arise from a genetically-engineered cell is low. Current technologies for long-term in vivo gene expression of inserted genes and for efficient widespread gene transfer to cells in human subjects are not currently available.
Other disadvantages of the mosaic model include stringent safety requirements for vectors used for gene therapy in healthy human subjects (Moolten, Med. Hypotheses, 24, 43 (1987); Anderson, Science, 226, 401 (1984)) or the need to insert genes into stem cells, which cells are poorly defined and difficult to genetically manipulate (Anderson et al., Cold Spring Harbor Sym. Quant. Biol., 51, 1073 (1986)). These considerations prevent the application of the mosaic strategy for cancer chemotherapy in the near future as pointed out by Moolten et al., J. Natl. Cancer Inst., 82, 297 (1990).
Another approach to cancer therapy that relies on retroviral-mediated gene transfer is described in a patent application entitled "Gene Therapy," (U.S. Ser. No. 07/365,567, NTIS publication No. PB89-206155). This approach describes the use of retroviral vectors that carry either marker or therapeutic genes for genetically-engineering immune cells that fight against cancer in a subject. The genetically-engineered cells in this case are tumor infiltrating lymphocytes (TIL) which are believed to mediate cytotoxic immune responses against certain cancers (Rosenberg et al., New Engl. J. Med., 316, 889 (1987)). The TIL are explanted from tumors in human subjects and grown in tissue culture in vitro. Following growth in tissue culture, the cells are transduced with a retroviral vector, selected for growth of only those TIL that express vector genes and then reinfused back into patients. One aspect of the approach is the potential to use the genetically-engineered TIL to target or return to the tumors in vivo, after the TIL are returned to the subject.
Therefore, a need exists for effective methods of cancer therapy employing the techniques of genetic engineering to render populations of cancer cells susceptible to destruction with agents that do not adversely affect normal cells.