Immune intervention against virus-associated human cancers

Annals of Oncology 6 (Suppl. 1): S69-S71, 1995. © 1995 Kluwer Academic Publishers. Printed in the Netherlands.

Symposium article Immune intervention against virus-associated human cancers A. B. Rickinson CRC Institute for Cancer Studies, University of Birmingham, Edgbaston, Birmingham, BIS 2TJ, U.K. Summary Background: A number of viruses have been shown to be carcinogenic in humans, including Epstein-Barr virus (EBV), hepatitis B virus (HBV), human papillomavirus (HPV) types 16 and 18, and human T-lymphotrophic virus (HTLV) 1. Cancer results from viral transformation of a single progenitor cell; the pathogenesis is complex, and viral infection is only one of many factors involved. Taking EBV-associated tumours as an example, a number of potential immune interventions, aimed at preventing viral infection or targeting virus-positive tumour cells, have been investigated. Envelope glycoprotein-based vaccines: The gp340 glycoprotein is the principal target of the neutralizing antibody response to EBV. A vaccine based on purified gp340 has been shown to protect against EBV-associated lymphoproliferative disease (B-cell lymphoma) in an animal model. Phase I clinical trials are being established.

Cytotoxic T-lymphocyte (CTL) epitope-based vaccines: EBV infection provokes a powerful CTL-mediated immune response that is directed primarily against the EBNA 3A, 3B, 3C subset of lal viral antigens. Clinical trials are investigating the effect of immunization with synthetic peptides representing EBNA3-derived CTL epitopes. CTL-based immunotherapy: Administration of activated T-cells has been shown to reverse lymphoproliferative disease in patients undergoing bone marrow transplantation. This approach may also be useful in other forms of cancer.

Introduction

cells. The latter approach seems appropriate in cancers associated with HPV, EBV and HTLV1, in which viral DNA is retained and to some extent expressed in the malignant clone. By contrast, in the case of HBV, the virus appears to act as a mutagenic agent and, although viral DNA is integrated in the tumour cells, its expression may not be required for tumour growth. In this case, therefore, immunological intervention relies on primary prevention with the conventional hepatitis B vaccine; indeed, it would be anticipated that the hepatitis B vaccination programmes currently under way in a number of countries would result in a reduced incidence of hepatocellular carcinoma. There are three current approaches to immunological intervention: • vaccines based on envelope glycoproteins; • vaccines based on peptide epitopes for cytotoxic T-lymphocytes (CTLs); • immunotherapy based on CTL responses to viral antigens. These approaches have been investigated in context of EBV. This virus is normally acquired early in life, and infected individuals are usually asymptomatic; infection later in life may lead to mononucleosis. The virus first infects pharyngeal epithelium and then through virus-driven B-cell proliferation colonizes the B-lymphoid system [2]. Immunological intervention by vaccine therefore, is either aimed at preventing primary infection in these or by limiting the viral transformation ofB-cells.

Viruses play an important role in the aetiology of a number of human cancers. The best characterized examples include primary hepatocellular carcinoma linked to hepatitis B virus (HBV), ano-genital carcinomas associated with human papillomavirus (HPV), particularly types 16 and 18, nasopharyngeal carcinoma, certain B-cell lymphomas and Hodgkin's disease associated with the Epstein-Barr virus (HPV), and a form of adult T-cell leukaemia that is linked to human T-lymphotrophic virus (HTLV) 1 [1]. These virus-associated tumours share a number of characteristics. The viruses involved are often common, with a high prevalence in most populations, but only a small proportion of infected persons develop a virus-associated cancer. Cancer normally develops after a latent period of several years, highlighting the importance of factors other than viral infection in the pathogenesis. The tumours are monoclonal with respect both to their cellular origin and to the viral genetic information carried within the cell. Thus, such cancers are derived from a single progenitor cell, in which the virus was originally present. Recognition of these features raises the possibility of developing immunological interventions as novel therapies for virus-associated cancers. Possible strategies include the prevention of infection, thereby reducing the incidence of disease, or targeting the immune response against viral proteins expressed in tumour

Keywords: B-cell lymphoma, bone marrow transplantation, Epstein-Barr virus, T-cells, vaccination, viral transformation

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Envelope glycoprotein-based vaccines The EBV capsid is surrounded by a lipid membrane, derived from the cell that originally shed the virus, and carrying a number of virus-encoded glycoproteins. The most important envelope glycoprotein is gp340, which binds to the CRZ, the cellular receptor for the C3d component of complement, thereby enabling the virus to enter target B cells [3]. gp340 is the principal target for the host antibody response, which protects against reinfection by a second EBV isolate [4]. Other forms of cell-mediated immune response may also be directed against this antigen. A vaccine based on purified gp340 has been tested in tamarins, a primate in which high doses of EBV produce a lymphoproliferative disease similar to that seen in immunosuppressed patients. In this animal model, vaccination against gp340 has been shown to reduce the viral load to levels below those necessary to cause disease [5]. Phase I clinical trials with this vaccine are currently being established. CTL epitope-based vaccines In vitro, EBV transforms B-lymphocytes, with expression of both viral and membrane proteins. This transformation is mediated by a series of six nuclear antigens (EBNAs 1, 2, 3A, 3B, 3C, -LP) and two membrane-derived proteins, (LMP1 and LMP2) [6]. In the presence of T-lymphocytes from individuals infected with EBV, however, outgrowth of the transformed B-cells is inhibited by a T-cell-mediated cytotoxic response. This cell-mediated immunity provides a potential means of controlling both viral infection and the development of virus-associated malignancies [7]. The CTL response appears to be directed primarily against latently-infected B-cells, with the 3A, 3B and 3C subgroups of nuclear antigens being the major targets [8,9]. This appears to create a paradox, as nuclear antigens would seem to be inappropriate targets for extracellular T-cells. It is now clear, however, that antigenic proteins are not recognized in native form, but are broken down in the cytoplasm to form peptide fragments that bind to HLA molecules and are transported to the cell membrane, where they become accessible to T-cells. CTL activation occurs in primary infection. Activated T-cells are detectable in the blood of patients with acute mononucleosis, whereas in healthy carriers they exist in dormant form; such cells can, however, be readily reactivated in vitro. The activated cells are specific for EBV, and show HLA class I-restriction. Thus, they recognize complexes of cognate EBV peptide and self HLA antigens [11-13]. CTL-mediated immunity is, therefore, a specific and potent response to EBV infection. This raises the possibility of priming the T-cells by peptide immunization before infection occurs, accelerating the CTL response

to primary infection and thereby limiting subsequent colonization of the B-lymphocyte pool. Ideally, such a strategy should use synthetic peptides representing the major CTL target epitopes presented by common HLA alleles. For example, in individuals bearing the HLAB8 antigen, which is present in approximately 20% of the population, a large proportion of the CTL response to EBV will be directed against a 9-mer peptide from the EBNA 3A nuclear protein [11]. A Phase I trial of immunization with this peptide is currently in progress in Australia. Similarly, one of the most common alleles is A2, which is present in up to 50% of the population. The response to this is directed against a ninemer peptide derived from the LMP2 protein [14]. CTL-based immunotherapy This approach seeks to exploit the CTL response as an immune therapy for malignancies associated with EBV. Two such conditions are commonly seen in this country: Hodgkin's disease and lymphoproliferative disease (B-cell lymphoma) in immunosuppressed patients. The CTL response is attenuated or abolished in patients receiving immunosuppressant treatment. When this inhibition is profound, as in patients undergoing bone marrow transplantation, infection with EBV (especially primary infection) can lead to lymphoproliferative disease [15, 16]. In patients undergoing bone marrow transplantation [17], lymphoproliferative disease usually originates from the donor's B-cells. Thus, lymphocytes can be harvested from the donor a few weeks before transplantation, the CTLs reactivated in tissue culture, and the reactivated CTLs administered to the patient. This approach is being used in a number of trials in the U.S.A. following earlier work reinforcing uncultured CD2 and donor T-cells [18]. The results to date indicate that administration of reactivated CTLs reverses severe lymphoproliferative disease [19]. In principle, therefore, such treatment may also be effective against other EBV-associated tumours. A potential problem, however, is the differential expression of viral antigens in other EBV-associated tumours. In the lymphoproliferative disease described above, ah1 eight of the target antigens are expressed, and antigen presentation by the cells is highly effective. By contrast, in conditions such as Burkitt's lymphoma and Hodgkin's disease, not all of the latent proteins are expressed; in particular, the EBNA 3A, 3B and 3C antigens, that are the principal targets of the CTL response, are absent [7]. Although CTL responses can be elicited against the remaining antigens, these constitute only a minor proportion of the total response. Thus, for immune therapy to be effective in these conditions, these minor responses would need to be selectively activated and amplified. There are some parallels between this situation in respect of virus-positive tumours and that of certain other potentially antigenic tumours,

71 such as melanomas. In vivo, melanoma cells appear to provoke a CTL response against normal cellular proteins that are inappropriately expressed in the malignant cells [20]. Thus, melanoma represents a situation in which relatively minor CTL responses are being induced; immune therapy, amplifying these responses and redelivering the activated T-cells to the patient, could provide a new approach to the treatment of melanoma. Conclusion A number of possible immune interventions are available for preventing or limiting infection with potentially tumourigenic viruses, or for targeting the immune response against viral antigens expressed in tumour cells. These are now being studied in preliminary clinical trials. The experience to date suggests that immune interventions will be a useful approach to the treatment of virus-associated cancers, and possibly to other forms of cancer.

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