Introduction

Analysis of the process of drug resistance indicates that it appears to be only too easy for malignancies to generate many varieties of drug-resistance mechanisms. Once a tumour has reached a certain critical size a combination of large numbers of candidate cells together with genetic instability will ensure that the collective molecular heterogeneity of the neoplasm will be immense. In retrospect, the surprising thing is not that cancers are difficult to treat with drugs but that some are highly sensitive to drug therapy and indeed are potentially curable. Although the addition of new cytotoxic agents provides a reasonably steady incremental improvement in cancer therapy outcome, these increments are small and are for the most part confined to the classes of tumour that are known already to be drug responsive. Malignancies such as pancreatic carcinoma, renal cell cancer, melanoma and nonsmall cell lung cancer are only minimally responsive to cytotoxic agents and it seems unlikely that random drug searches will yield single agents that are going to be much more effective than the drugs we currently possess. A major problem with these types of cancer seems to be the large number of different multidrug resistant mechanisms that they express. Modulation of one or two of these mechanisms is unlikely to be sufficient to render the advanced forms of the tumour curable. The greater the number of discreet mechanisms that need to be disabled then, of course, the more logistically cumbersome the treatment protocol becomes.

Introduction

We are coming to what some readers may find to be the most difficult section of the book because we will attempt to synthesize a number of the mathematical developments we have described previously into a more complex model that is intended to conform more closely to the behaviour of clinical malignancies. The most important elements in this synthesis will be the basic random mutation model of resistance (Chapters 4 and 5) and the stem cell model of tumour growth (Chapter 2). We will describe in more detail the birth/death processes that were introduced in Chapter 2 and indicate how they impact on the issue of drug resistance and the more general question of tumour heterogeneity.

It should be kept in mind that birth/death events are more than just convenient mathematical abstractions for they can provide a mathematical description of the effects of molecular processes that regulate movement through the cell cycle or signal differentiation and apoptosis.

In Chapter 5 we introduced and discussed the random mutation model for resistance to an anticancer drug. This model predicted that tumours which start sensitive would, as they grow, convert to drug resistance by the spontaneous evolution of drug-resistant cells whose population expands at a rate that exceeds that of the tumour as a whole. This model was developed within a framework in which cells divide with unlimited potential (stem cells) forming new stem cells at each expansion. Comparison with data from in vivo tumour systems showed that this model accurately simulated and explained the pattern of animal survival seen in some experiments.

Introduction

Much of what has been learned concerning the properties of cancer cells has been developed from studies of a variety of cell lines that have been adapted for growth in tissue culture or in appropriate experimental animals. A variety of mammalian (chiefly rodent) and human cancer cell lines have been developed for this purpose. It has to be kept in mind that the properties of these highly selected experimental systems may differ from those that one might expect to find occurring in primary tumours in patients. Despite this caveat, it is apparent that many principles that have been learned from the experimental systems have been valuable in the understanding of human malignancy.

Any line of tumour cells that has adapted to growing in tissue culture or through serial transplantation in animals will have been subject to an extremely rigorous selection process. Normal (i.e. nonmalignant) fibroblasts in tissue culture will die out after they have undergone a number of sequential cell divisions (approximately 50). This appears to be the case no matter how carefully the culture conditions are established. This also appears to be true for many cancer cells in that there appears to be an upper limit to the number of sequential divisions they will undergo before becoming senescent and dying. Some tumour cells, however, become ‘immortalized’ and they will replicate indefinitely in the right type of environment. Recently, it has been suggested that an important step in the process whereby cells become immortal is related to the expression of the enzyme telomerase.

Introduction

There are approximately 60 different chemical compounds generally available for the treatment of cancer (not including hormones or biological response modifiers). They are of diverse structure and from a variety of sources. They do not readily fit into a single classification system and are usually described partly on the basis of their chemical structure, partly on their primary source (fungi, plant, etc.) and partly on what is thought to be their general mechanism of action (antimetabolite, alkylating agent, etc.) (Table 3.1). We can generalize by stating that all of the drugs appear to exert their therapeutic effect by interfering with the processes involved in cell division. This interference results in the cell being physically disrupted or rendered permanently sterile. We can further say that cancer cells have the potential to become resistant to any of the drugs in our inventory and the cell can, moreover, express resistance to a great many agents simultaneously. Although we are not aware of the experiment actually having been done, it seems more than probable that an individual cancer cell could display resistance to all 60 available drugs concurrently. As we will see, this capacity to express resistance to many agents is, in part, related to the fact that there are a large number of mechanisms that once expressed by the cell will generate broad degrees of cellular resistance.

It would be well beyond the scope of this book to discuss in any detail the molecular changes that have been described for all of the various cytotoxic agents. However, we will mention the general processes involved in drug action and how these may be modified in the drug-resistant state.

It is interesting to reflect back on the literature written between 1980 and 1987 regarding the prospects and anticipated first applications of human gene therapy. What were predicted with a fair degree of accuracy were the specific gene transfer technologies that would first be utilized in the initial human gene therapy protocols: liposomal-mediated gene transfer and gene transfer via replication-defective retroviral and adenoviral vectors. The vast majority of human gene transfer protocols to date have used these transfer technologies. However, it was not originally anticipated that naked DNA would be efficacious in certain specific tissues, such as muscle, for gene transfer and expression.

What were neither anticipated nor predicted with any degree of accuracy were the target diseases for the initial human gene therapy protocols. These were originally predicted to be primarily monogenetic diseases, some of which are listed in Table 1.1. However, although the very first pioneering human gene therapy protocol, performed on September 14, 1990, was for correction of adenosine deaminase deficiency disease – a milestone event in the continual evolution of modern medicine – this prediction has proved to be incorrect. Instead, reflecting back over the last few years, it is clear that most of the initial applications have been in the oncology area, and it is interesting to examine the evolution of human gene therapy over this period.

Chemotherapy and therapeutic selectivity

In selected cases, chemotherapy targeted against many infectious agents has proven to be both extremely safe and efficacious. In these cases, therapeutic selectivity is achieved by exploiting qualitative differences in the structure, function, or intermediary metabolism between the target organism and the human cells.

Structural

Certain very safe and effective antibiotics therapeutically exploit the fact that bacteria require intact cell walls to maintain their integrity. Certain antibiotics can disrupt the formation or the integrity of the cell wall, causing the invading bacterium to become fragile and ultimately lyse. For example, peptidoglycan is a highly crosslinked, heteropolymeric component of the cell wall which is the molecular basis for its rigid structure. It is composed of linear chains of alternating units of N-acetylglucosamine and N-acetylmuramic acid which are linked by peptide crosslinks. The final phase of peptidoglycan synthesis is the crosslinking of the glycopeptide polymers by a transpeptidase reaction which occurs on the outside of the bacterial cell membrane. It appears that this transpeptidase step is inhibited by the beta-lactam antibiotics, such as the penicillins (i.e., penicillin G, V, ampicillin, oxacillin, etc.) and cephalosporins (i.e., cephapirin, cephalexin, cefaclor, cefotaxime, etc.). The beta-lactam antibiotics also bind to other bacteria-specific proteins (called penicillin-binding proteins, or PBP), which may significantly contribute to their antibacterial effects. Inhibition of the cell wall transpeptidase reaction and PBP binding causes the bacteria to lyse due to the autolytic activity of autolysins and murein hydrolases located in the cell wall.

Introduction

The application of efficient gene-transfer techniques to lymphocyte populations has opened new pathways in the therapy of neoplastic disease. Genetically engineered lymphocytes possess unique functional characteristics which can be exploited in novel treatment protocols. The ability of lymphocytes to traffic to tumor deposits can be harnessed to effect the delivery of therapeutically active molecules to the tumor microenvironment. Specific changes in the local tumor milieu may augment the host immune response, while avoiding the toxicity associated with high-dose systemic administration of cytokines. In addition, the construction of chimeric T cell receptors has led to the direct coupling of the recognition and effector phases of the immune response. In addition, advances in the isolation and propagation of specific hematopoietic stem cell (HSC) populations may enable researchers partially to reconstitute the hematolymphoid system with genetically engineered precursor cells possessing specific antitumor activity. Targeting of HSC may allow the indirect transfer of genes into lymphocytic effector populations, while overcoming some of the limitations associated with direct gene insertion into mature lymphocytes. This chapter reviews methods of gene transfer into lymphocytes and HSC and the in-vivo characteristics of these cells following adoptive transfer. Strategies for using genetic modification of lymphocytes and HSC to enhance the antitumor immune response are discussed, and the relative merits of using either the mature lymphocyte or HSC populations for gene therapy of cancer are examined.

General considerations

In the USA the authority to regulate gene therapy products rests with the Center for Biologies Evaluation and Research (CBER) in the Food and Drug Administration (FDA). At present, all gene therapy products are at the investigational stage and are regulated under the standards for Investigational New Drugs (INDs) in 21 Code of Federal Regulations (CFR) part 312 (Taylor, 1993). Certain existing guidelines for the regulation of biologic therapeutics (21 CFR parts 210, 211, and 600) apply to the production of gene therapy products. Specifically, since biologicals cannot be completely defined, quality control on the starting materials and production process is equally as important as on the finished product to avoid the introduction of harmful contaminants. To outline the requirements for production of gene therapy products and to address issues that are more specific to the concerns unique to gene therapy approaches (e.g., genetic consequences), CBER has issued a series of ‘Points to Consider’ (PTC) documents to aid investigators with IND submissions.

Points to consider in human somatic cell therapy and gene therapy

At the time this document was drafted (Center for Biologies Evaluation and Research Staff, 1991) the majority of the protocols being submitted were ex-vivo cell therapies or cell-marking studies, thus its emphasis is on characterization of modified cell populations.

After much public, scientific, and regulatory debate, the first clinical trial involving gene marking was initiated on May 22, 1989. Less than seven years later, over 600 patients had been enrolled in clinical gene transfer/gene therapy protocols at numerous institutions around the world. About 70% of these trials have studied oncology patients, but trials treating inborn genetic disorders such as adenosine deaminase deficiency, cystic fibrosis, and famial hypercholesterolemia, and acquired diseases such as AIDS, are also under way (Table 9.1; for review see Miller, 1992; Morgan and Anderson, 1993; Crystal, 1995). The lack of any unexpected or untoward problems in the early trials has led to a profusion of gene marking/gene therapy protocols. In addition, the trend over time has been a shift from ex-vivo gene marking toward in-vivo gene therapy protocols. New protocols are now being approved more quickly by the appropriate regulatory agencies as the public, scientific, and regulatory communities have become more comfortable with this emerging technology. In fact the need for any special regulatory overview of gene therapy protocols is currently under review (Marshall, 1995).

Current clinical gene marker/gene therapy protocols in oncology can be divided into four broad groups based on the target cell population – that is, the type of cells being genetically modified (Table 9.2). The first group of protocols involves the genetic modification of tumor-infiltrating lymphocytes (TIL). Initial marking studies are addressing questions about TIL trafficking and the general safety of gene marking/gene therapy protocols.

The concept of gene replacement for disease logically followed from the observation that certain diseases are caused by the inheritance of a single functionally defective gene. Diseases caused by a known monogenic defect, such as adenosine deaminase deficiency or Gaucher's disease, could be treated and potentially cured by insertion and expression of a normal copy of the mutant or deleted gene in host cells. This can be conceptualized as ‘gene replacement therapy’ and represents the basic framework for the development of gene therapy approaches to monogenic diseases.

The identification of specific genes that contribute to the development of the cancer cell presents an opportunity to use these genes and their products as prevention and treatment targets. This is a considerably more complex situation than the monogenic diseases as the development of cancer is associated with multiple genetic abnormalities. The gene families implicated in carcinogenesis include dominant oncogenes and tumor suppressor genes (Bishop, 1991; Weinberg, 1992). An example of the diversity of genes implicated in a common cancer, lung cancer, is shown in Table 5.1. Protooncogenes (normal genes that are the homologs of oncogenes) participate in critical cell functions, including signal transduction and transcription. Only a single mutant allele is required for malignant transformation. Primary modifications in the dominant oncogenes that confer gain of transforming function include point mutation, amplification, translocation, and rearrangement. Tumor suppressor genes appear to require homozygous loss of function by either mutation or deletion or by a combination of these.

Introduction

Antisense RNA and DNA techniques have been developed as a relatively recent approach to the specific modulation of gene expression. The development of antisense RNA techniques began with observations from the laboratories of J. Tomizawa and N. Kleckner that bacteria can regulate gene replication and transcription by the elaboration of small complementary or ‘antisense’ RNA molecules (for a review see Weintraub, 1990; or Inouye, 1988). Evidence also now exists that eukaryotic cells elaborate antisense RNA transcripts (Kochbin and Lawrence, 1989; Krystal, Armstrong and Battey, 1990), but the physiologic role of such molecules remains controversial. Nevertheless, in 1985, Izant and Weintraub reported that a transfected expression vector could generate RNA capable of modulating eukaryotic gene expression in a target-specific manner. Actually, prior to the discovery of regulatory RNAs, several laboratories had demonstrated that synthetic oligodeoxynucleotides complementary to mRNA sequences could downregulate mRNA translation in vitro and in cells (Zamecnik and Stephenson, 1978). A profusion of reports has followed, describing the modulation of various oncogenes by both DNA and RNA antisense techniques. Such manipulations offer the possibility of combining the specificity of genetic approaches with the reversibility and temporal control of more pharmacologic approaches. The continued development of antisense technology and related delivery systems has led to initial explorations of the in-vivo utility of antisense, particularly as it relates to oligonucleotides.

However, the excitement generated by initial in-vitro successes has been tempered by the dawning realization that antisense technology is not as ‘clean’ as previously thought.

Introduction

Recombinant DNA technology has allowed for the efficient introduction of defined genes into mammalian cells. Utilizing this technology, it has been feasible to express a variety of immunoregulatory proteins in tumor cells in order to modulate the host's immune response to native tumor antigens. The components of the immune response necessary to generate immunity to tumors consist of: (a) emigration of inflammatory cells to the site of tumor growth; (b) processing of tumor antigens by antigen-presenting cells; (c) sensitization of lymphoid cells; and (d) amplification and/or suppression of mature effector cells. Thus, there is a variety of avenues to modulate an immune response by specifically adding defined immune regulatory genes in this process.

Transplantable animal tumors have provided a wealth of information concerning the host antitumor immune response. Depending upon the inherent immunogenicity of the tumor, experimental methods are capable of eliciting systemic immunity to a variety of tumors in naive hosts. Many of the initial studies with genetically modified tumor cells have focused on the ability of the host to reject an inoculum of modified tumor cells, with the induction of systemic immunity to a subsequent challenge of the parental tumor. Hence, the inherent immunogenicity of the tumor being examined is important in interpreting the significance of genetic modification. Tumor immunogenicity has been traditionally defined by transplantation procedures. Table 6.1 presents the framework for discussing immunogenicity that is utilized in this chapter and is based on the ability to immunize animals to resist tumor challenge by various manipulations.

In recent years there has been a dramatic increase in developing gene therapy approaches for the treatment of cancer. In principle, gene therapy involves the transfer of foreign genetic material into a patient in an effort to treat a particular disease (Mulligan, 1993). The two events which have permitted the formulation of the concept of cancer gene therapy are the new understanding of the molecular mechanisms underlying oncogenesis and the development of the DNA-delivery vehicles, or vectors. It was initially thought that successful gene therapy would require that pre-existing defects be corrected in every cancer cell. However, in recent years it has become clear that a high level of transient gene expression in even a limited number of the target cells could also be potentially beneficial. Many approaches to cancer gene therapy have been proposed, and many viral and nonviral vectors have been utilized. The purpose of this chapter is to describe the biology, construction and unique characteristics of some of the commonly used vectors, and some of the preclinical studies that have been performed with them. The problems associated with these vectors, and future considerations pertaining to vector development for the gene therapy of cancer are also discussed. The clinical protocols utilizing these vectors are discussed in various chapters throughout the book.