Introduction

I started a science degree course at the University of Sydney in 1940. In 1944, I began to work for an MSc degree. Part way through the year, Dr Bob Walsh, then Director of the New South Wales Blood Transfusion Service, approached the biochemistry department for some help. The problem was that serum sent to the front line for transfusion to wounded soldiers often became cloudy and, of course, there were no facilities in the trenches for testing serum for sterility! Amazing as it may seem now, I was asked by the head of the department if I would like to work on this topic. Of course, I said yes! Not too long after that, Major Val Bazeley, on Bob Walsh's advice, called in to see me. He had been seconded to the Commonwealth Serum Laboratories (CSL) from the Armed Services to establish a penicillin production plant, and he did a fantastic job in this respect. This achievement by some staff of the Serum Laboratories at that time was celebrated in 1998, which is the 100th anniversary of Howard Florey's birth.

Bazeley strongly recommended that I come to the CSL to work on my project, so I worked there for 2 years. I achieved some success by extracting serum with ether, but my efforts and those of workers at the Lister Institute in London on the same problem were completely overshadowed by the development by Harvard scientists of the ethanol fractionation of serum into discrete protein fractions. During those 2 years, I got to know one of the scientists at the Hall Institute quite well and by attending evening scientific meetings, I realized that I wanted to learn how to do 'real' medical research. So I asked for paid leave of absence from the CSL to work at the National Institute for Medical Research in London, learning new technologies such as moving boundary electrophoresis and ultracentrifugation to study the properties of proteins. These technologies were unavailable in Australia. But that was refused by the Head Office in Canberra just a few weeks before the first boat to carry academics to the UK after the war was due to sail from Sydney. So I resigned and set off for London. Fortunately, the UK Medical Research Council (MRC) gave me a salary a few months later. Approximately 1 year after that, I received an offer from Macfarlane Burnet to join the staff at the Hall Institute and, in association with Mr Henry Holden, to establish these techniques at the Hall Institute. So my gamble paid off, but for some years the Director of the CSL complained that Burnet had pinched one of his up-and-coming young staff.

In the 1980s I became a member of an advisory group to the Director of the CSL, but this ceased when the Laboratories were privatized later that decade.

In 1988 I gave the Plenary Lecture on the prospects for an HIV vaccine at the Fourth International Congress on AIDS held in Stockholm. In front of 8000 delegates, I listed many of the reasons why it could be difficult to develop an effective vaccine against HIV.¹ Because of the most recent information about the very great antigenic variation in the HIV envelope antigen, I stipulated that the vaccine must generate a strong memory CTL response and recommended using a chimeric live vector as the basis of a vaccine. This technology had been developed in the Department of Microbiology by a unit funded by the Commonwealth Scientific and Industrial Research Organization (CSIRO) and headed by David Boyle. (It took 7–8 years for that advice to be generally accepted.) After the end of that (the final day's sessions, in Stockholm), Ian Gust, who had by then joined the newly privatized CSL, shouted me to a glass or two of beer at one of the many sidewalk cafes. I can still remember the look on his face when he received the bill; alcoholic beverages in Sweden are very costly.

My interest in cancer was stimulated in the early 1970s when I became associated with the International Agency for Research on Cancer (IARC) in Lyon and, in due course, I became chairman of the Scientific Council. The main activity of the IARC was to identify, through epidemiological investigation, risk factors associated with the incidence of certain cancers. I learnt to appreciate the critical role of epidemiology in this field. In the next nearly 20 years, I became involved with nearly every group at the World Health Organization (WHO) headquarters in Geneva involved with different aspects of vaccine development, testing and use. Partly because of a period of nearly 4 years spent working abroad after retiring from the John Curtin School of Medical Research (JCSMR), I have been extremely fortunate to be able to retain that interest.

Cancer and the immune system

We are indebted to two great scientific minds for stimulating our interest in an immunotherapeutic approach to the control of cancer (Table 1).^{2, 3, 4} Initially, there wasn't much support for this concept, but it received more consideration as our understanding of the immune system grew.

Table 1 - Early concepts.

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There are two main reasons why we should be cautiously optimistic about developing a vaccine to control at least some cancers.The first and most important is the fact that the practice of vaccination to control and even to eradicate or eliminate from different world regions some infectious diseases has had some outstanding successes; and second, there have been great advances in our understanding of and ability to manipulate immunological processes.

The control of some infectious diseases by vaccination

Table 2 shows the incidence of some mainly childhood infectious diseases in a major epidemic in the USA prior to the introduction of vaccination, and the incidence of those diseases in 1997, some years after most of the various vaccines were first introduced. The achievements are quite spectacular. In the case of measles, which has a very high transmission rate, the latest data indicate that most, if not all of the few cases now occurring in the USA, are introduced from abroad; that is, transmission in the country may have been interrupted for the first time.⁵ The message is: with an effective vaccine, then excellent control of many infections, especially acute infections, is achievable. However, there are many infectious diseases for which, so far, attempts to make an effective vaccine have been unsuccessful. HIV is of course a well known example, because it has almost all the criteria that make it difficult to develop an effective vaccine. Some of those are shown in Table 3, and I have marked with an asterisk those criteria that I believe may also be relevant to developing a cancer vaccine, especially those which are associated with a viral infection.

Table 2 - Vaccine efficacy in the USA, as assessed by comparing maximum morbidity levels before vaccine availability, and the levels some years after vaccination became compulsory.

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Table 3 - Some difficulties associated with HIV vaccine development that are also relevant to tumour vaccine development.

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As mentioned earlier, I believe that many manufacturers got off to a wrong start in their approach to HIV vaccine development. Their goal was a genetically engineered subunit vaccine like the hepatitis B yeast-derived vaccine, which is very safe and reasonably effective. But I still believe that at least a reasonably effective vaccine will become available. Most agents that cause a chronic persisting infection in one way or another evade the cell-mediated immune (CMI) response, and this applies to HIV and, it seems, to most cancers.

There are three non-infectious situations where vaccination could be useful. They are the control of tumours, autoimmunity and fertility.⁶ Few would disagree that the former offers the greatest rewards in preventing or at least minimizing human misery and mortality.

Advances in our understanding of immunological processes

The second golden age of immunology began in the late 1950s when we really began to understand for the first time how the immune system worked. The enunciation of the clonal selection theory by Burnet was a tremendous stimulus, and scientists working in Australia helped to verify this concept, and made notable contributions to our understanding of some of the basic principles of immunology, some of which are listed in Table 4. They largely correspond to a list made by Gus Nossal who, in 1991, wrote a paper called 'Landmarks in Australian immunology'.⁷ It would be a challenge for younger colleagues to bring that list up to date. But there is one major difference between then and now. With the advent of cooperative research centres and much more competition for funds, there is now a greater emphasis in this country towards research leading to practical outcomes. I would like to believe that the earlier emphasis on basic immunological research helped to provide the scientific basis which has allowed recent great progress in vaccine development, including of course immunotherapy for cancer.

Table 4 - Some basic immunological contributions from Australia.

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A basic requirement of any immunotherapeutic (or immunoprophylactic) approach to cancer is that the tumour expresses one or more antigen(s) that has/have significant specificity for that tumour relative to other host cells. A straightforward and unambiguous identification of a specific antigen is if the tumour arises because of an initial infection by a virus. Table 5 lists the common human tumours and the associated tumours. Fortunately, however, antigens are being defined at an increasingly rapid rate on other tumours, especially melanoma,⁷ where there is no evidence of a viral infection.

Table 5 - Viral infections associated with malignant tumours in humans.

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There are two general types of targets. A specific antibody should recognize largely unmodified antigens expressed at the tumour cell surface. In contrast, T cells have the potential to recognize peptides derived from any protein synthesized by the cell, provided that the peptide can effectively associate with a class I or class II MHC antigen and the complex transported to the plasma membrane.

Cancer initiation and progression

Pre-malignancy is the term for the state prior to neoplasia. Genetic events can occur that change this and initiate the formation and growth of tumours. Two things can happen: full-scale cancer can develop and/or a state of dormancy can occur. The latter is described as the formation of foci of fully malignant cells, each consisting of ~ 1 million cells. These colonies are kept under control in some way which is currently something of a mystery. The immune response is thought not to play a major role in this respect.⁸

Some factors will activate these colonies and neoplasia will occur, including metastasis. For example, 35 years ago Woodruff described a patient who developed a carcinoma of the breast 3 years after apparent complete excision of a melanoma on the foot, and was treated by radical mastectomy and postoperative radiotherapy.⁹ Six weeks later, melanomatous nodules appeared first in the field of irradiation and then elsewhere, and the patient died from generalized melanoma a few months later. Some cancers can currently be treated and long-term remission occur to the extent that patients may be classified as clinically cured. However, with some cancers such as melanomas and breast cancers, a secondary tumour of the same specificity can arise a decade or more later, and this is thought to be due to the persistence and, finally, activation of dormant colonies.

It could be a major step forward if it became possible to identify the most important factors that keep these colonies under control. It is not clear whether cell cycle arrest occurs or whether cell replication is balanced by cell death, and we know little about the role of different soluble factors.

Escape of tumours from immune control

In a fascinating but sober article, Ellem et al. have recently discussed three categories of important mechanisms whereby tumours escape immune control.¹⁰

1. Tumour epitope loss, or what might be called tumour epitope stability. This is the loss of tumour cell epitopes and/or down-regulation of expression of MHC antigens.
2. Immunosuppressive products produced by tumours. A remarkable variety of products can maim or kill activated T cells.
3. Defective tumour capillaries, affecting tumour cell accessibility. This raises the question of whether it is because of a defective neovascular system that immune cells or their products, from antibodies to cytokines, can gain effective entry into and access to a dormant or metastatic colony of cells.

In the view of this group, the task of making a vaccine that would generate an appropriate immune response is straightforward relative to solving the aforementioned three aspects. They make two claims: that a CMI response based on CD8+ CTL, and use of the whole tumour cell rather than isolated antigens or peptides, is likely to be the most effective approach to the former goal. Of course, this would increase the risk of enhancing autoimmunity, but that would be the lesser of two evils.

Approaches to developing a cancer vaccine

Animal models

The mouse is an incredibly convenient animal model for almost any aspect of immunology. The ability to genetically modify mice is making this host an even more valuable model for cancer research. A recent striking demonstration is the demonstration that mice transfected with genes coding for human CD4 as well as for the chemokine receptor, CCR5, become susceptible to HIV infection.¹¹ But there is sometimes a surprise in store when one attempts to extrapolate from mice to humans. Apostolopoulos et al. experienced such a surprise quite recently.¹² In mice, they obtained mainly a T1 response to mannan MUC1, whereas in patients, a T2 response predominated. They have established the reason for this.¹³ Working with viruses, we found another situation.¹⁴ In mice, antibody can clear infections of influenza (low dose, egg-grown), vaccinia, respiratory syncytial and Sindbis viral infections. In contrast, CTL are required to clear lymphocytic choriomeningitis (LCM), ectromelia and Theilers viral infections. Clearance of Sendai viral infections in CTL-negative mice is considerably delayed.

One interpretation of these findings is that LCM, ectromelia and Theilers viruses are natural pathogens for mice, whereas the other is not. (Sendai virus in mice behaves like a mouse pathogen in the sense that the infection is readily transmitted to other mice in the same cage.) Perhaps a full complement of immune responses is required to deal with natural pathogens. There are many other examples, especially with some bacterial infections where it has been found that CD4+ Th1 responses appear to be more effective than a CTL response in controlling the murine infection. Again, in many cases (tuberculosis, chlamydia), the agent being used is a human, not a mouse, pathogen.¹⁴

I am not suggesting that mice not be used as a model system; only that sometimes, simple extrapolation to the human situation may not be warranted.

Generation of CTL responses

Despite the comments of Ellem et al., I would like to say something about a general approach to developing a cancer vaccine. During the last 6 or so months, partly in preparation for this paper, I have collected ~ 100 papers featuring approaches to making cancer vaccines. Out of curiosity, I have categorized them under several headings (Table 6). I am of course not claiming that this is an accurate assessment of all the different approaches being used, but it does give some idea. Clearly, the main accent is on cell-mediated immunity, and culturing dendritic cells and pulsing them with different forms of cancer antigens has become very popular.Though I do not rule out a role for specific antibody, in this presentation I concentrate on a CMI response, specifically CD8+ CTL.

Table 6 - A random assessment of the relative importance of different approaches to cancer vaccine development.

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What is the best way to generate a strong CTL response to tumour antigens? Until quite recently, it was standard practice in a vaccination schedule to administer repeated doses of the same product. Some years ago, it was found that when using different versions of an HIV antigen to prime (a chimeric live vector) and boost (the isolated env antigen, rgp120), a higher antibody response occurred.¹⁵

Several groups (e.g. Leong et al. and Richmond et al. with viral antigens,^{16, 17} Richmond et al. with SIV antigens,¹⁸ Hanke et al. with HIV antigens¹⁹ and Schneider et al. with malaria antigens²⁰) have reported that priming with a plasmid DNA preparation and boosting with a chimeric live vector gave an enhanced humoral or CTL response to the foreign antigen. This suggests that immunization with DNA is particularly effective at priming for an immune response, whether it is a humoral or CMI response, because the same enhancement did not occur if the regimen was reversed.^{19, 20} The question of whether it is a humoral or a CMI response that mainly prevails may depend upon several factors, including the nature of the antigen.

Kent et al. have now reported that immunizing Macaque nemestrina monkeys with chimeric DNA (HIV gag, pol) followed by chimeric fowl pox (gag, pol) cleared the virus after challenge with HIV-1.²¹ This sequence induced a very strong CTL response but only a rather low antibody response to these antigens. Although HIV-1 is only weakly pathogenic in these monkeys, this is an encouraging finding. At the recent International Congress of Immunology in New Delhi (November 1998), Harriett Robinson reported that monkeys immunized with a similar sequence of retroviral antigens, first as DNA and then as a chimeric fowl pox construct, and challenged with a pathogenic SHIV preparation (SIV modified to express some HIV antigens), were able to clear the challenge virus (I. Ramshaw, pers. comm., 1998).

Regarding DNA administration, the usual ways are to inject intramuscularly or by gene gun into the dermis.²² There are other possibilities however. One is to use attenuated Salmonella bacteria (licensed for human use) which contain chimeric plasmids originally produced in Escherichia coli. When administered orally to mice,²³ the bacteria reach the draining lymph nodes where they perish and then are phagocytosed. This seems an ideal way to direct the DNA to APC, and it resulted in a particularly strong CTL response. If a strong CTL response to immunization is the primary aim of a project to prevent, control or eliminate a tumour, it would seem worthwhile to investigate different approaches. One possibility would be to prime with DNA either in the usual way or preferably as a Salmonella construct, followed by boosting with an antigen-pulsed dendritic cell preparation.

Towards elimination of cancers associated with viral infections?

The classical examples are the tumours associated with hepatitis B viral infections, and these take 20 or more years to occur following infection. Because vaccination (at least in industrialized countries) with this vaccine began in the early 1980s, early in the new millennium should see the accumulated data showing a trend to a decrease in hepatocellular carcinoma. All of the other viruses in Table 5 are the subject of vaccine development efforts, especially HIV.

The interaction between tumour cells and CTL

The final aspect of my presentation concerns the question: how can we study more closely the in vivo interaction between effector CTL and the target tumour cells? This would be possible if there was a system where dormant cancer colonies and their activation in a murine model was available, or, say, other tumour cells were transferred that formed small metastases in a convenient location, such as the lung. Initially at least, in selecting the most appropriate tumour model, the tumour cells could be transfected with DNA coding for an antigen with a nonapeptide known to bind strongly to the class I MHC antigens of the mouse strain being used. Alternatively, the peptide(s) employed would be major determinants of the cancer-specific antigen for that MHC specificity.⁷

It is possible now to quantify in tissue extracts and probably to locate in tissue sections, the number of CTL of a given receptor specificity (say for the aforementioned antigen) by using fluorescently labelled tetrameric formations of specific peptide/MHC class I complexes.²⁴ This technique, when applied to tissues following a viral infection, has shown that the number of CTL of that specificity are much greater than was previously thought to be the case. Most of the CTL so observed secrete IFN- but are non-cytotoxic. Possibly the first group to have observed such non-cytotoxic CTL was Walker et al. back in 1991.²⁵ They showed that such cells secreted HIV suppressor factors.

In this proposed experimental approach, one could either look for CTL induced in the host following introduction of the melanoma, or following the transfer from another host of CTL generated to and potentially capable of clearing this tumour. In the latter case, effector CTL of the appropriate specificity might be separated by FACS and then transferred, or such cells could be obtained by further activation and replication in tissue culture. If sections of the tissue containing the tumour were 'stained' in this way, it might be possible to determine the precise location of the CTL relative to the tumour. If CTL were found closely associated with the tumour, they could be further investigated by additional staining. For example, do they express the genes active in target cell lysis or cytokines such as IFN-?

An additional point which may not be generally considered is the migration patterns of effector T cells. Many years ago, delayed-type hypersensitivity reactions were sometimes evoked by injecting the antigen directly into the footpad (or later into the ear pinna) and measuring the swelling 24–48 h later. Identifying the nature of the effector cell was done by cell transfer. It was noted that although transferred primary lymphocytes induced a good response at this site, transfer of cultured effector T cells did not, and one of the possibilities was that the cultured cells did not migrate to the injection site. Altered migration patterns of cloned T cells have been noted in other situations.

Thus, one of the three challenging situations discussed by Ellem et al. might be directly investigated in this way.

Vaccine trials

There will be, from now on, increasing numbers of trials of candidate immunotherapeutic preparations. For a variety of reasons, melanoma has been a favourite target for the study of such preparations. One recent report refers to the first phase III clinical trials in melanoma patients, involving immunization with an antigen, the ganglioside GM2 present on melanoma cells. This was identified on the cell surface, using monoclonal antibodies raised to tumour cells.²⁶ There are many more trials at earlier stages, including some in this country.

Vaccination against many acute infectious diseases 'came of age' many years ago, as you have seen. We have still some way to go to control many persistent infections, and they remain a real challenge.

What distinguishes the current cancer protocols from earlier attempts at immunotherapy is that they are based on more sound immunological principles, including the known specificity of the targeted antigen(s), and invoke a well- characterized specific immune response. This surely is an indication of the increased maturity of the current approaches, indicating a 'coming of age' of this technology.

A final comment is that the goal of developing effective immunotherapy for human tumours is a major challenge, even more so than developing an effective HIV vaccine. Possibly, a combination of two approaches, such as immunotherapy and antiangiogenesis therapy, could be worthwhile to attempt. Controlling cancer has such a high public profile that, although it may be very difficult to achieve such collaboration, it is worth the try.

References

1. Ada GL. Prospects for HIV vaccines. J. AIDS 1988; 1: 295–303. | ChemPort |
2. Burnet FM. Cancer: A biological approach. BMJ 1957; 1: 779–86. | PubMed |
3. Thomas L. Quoted in Fenner F. Frank Macfarlane Burnet, 1899–1985. Hist. Rec. Aust. Sci. 1987; 7: 39–77. | PubMed |
4. Burnet FM. Immunological aspects of malignant disease. Lancet 1967; 1: 1171–1174. | Article | PubMed | ISI | ChemPort |
5. Advances in global measles control and elimination. Summary of the 1997 international meeting. Morb. Mortal. Wkly Rep. 1998; 24: 1–23.
6. Ada G, Ramsay A. Vaccines, Vaccination and the Immune Response. Philadelphia: Lippincott-Raven, 1997.
7. Nossal GJV. Some landmarks in Australian immunology. Immunol. Cell Biol. 1991; 69: 327–36. | PubMed |
8. Uhr JW, Scheuermann RH, Street NE, Vitetta ES. Cancer dormancy: Opportunities for new therapeutic approaches. Nature Med. 1997; 3: 505–9. | Article |
9. Woodruff M. Interaction of cancer and host [The Walter Hubert Lecture]. Br. J. Cancer 1982; 46: 313–22. | PubMed | ISI | ChemPort |
10. Ellem KAO, Schmidt CW, Li C-L et al. The labyrinthine ways of cancer immunotherapy: T cell, tumour cell encounter: 'How do I lose thee? Let me count the ways'. Adv. Cancer Res. 1998; 75: 204–42.
11. Browning J, Horner JW, Pettoello-Mantovani M et al. Mice transgenic for human CD4 and CCR5 are susceptible to HIV infection. Proc. Natl Acad. Sci. 1988; 94: 14 637–41.
12. Apostolopoulos V, Osinski C, McKenzie IFC. MUC1 cross- reactive Gal (1,3) Gal antibodies in humans switch immune response from cellular to humoral. Nature Med. 1988; 4: 315–20.
13. Apostolopoulos V, Pietersz G, Gordon S et al. Aldehyde–mannan antigen complexes target the MHC class I pathway. J. Immunol. 1999; in press.
14. Ada GL. The immunology of vaccination. In: Plotkin SE, Orenstein W (eds). Vaccines, 3rd edn. Philadelphia: W. Saunders and Co., 1999; in press.
15. Graham BS, Matthews TJ, Belshe RB et al. Augmentation of human immunodeficiency virus type-1 neutralising antibody by priming with gp160 recombinant vaccinia virus and boosting with rpg160 in vaccinia-naive adults. J. Infect. Dis. 1993; 167: 533–7. | PubMed | ChemPort |
16. Leong KH, Ramsay AJ, Morin MJ et al. Generation of enhanced immune responses by consecutive immunisation with DNA and recombinant fowlpox virus. In: Brown F, Chanock R, Ginsberg H, Norrby E (eds). Vaccines 95. New York: Cold Spring Harbor Laboratory Press, 1995; 327–31.
17. Ramsay AJ, Leong KH, Ramshaw IA. DNA vaccination against virus infection and enhancement of antiviral immunity following consecutive immunisation with DNA and viral vectors. Immunol. Cell Biol. 1997; 75: 382–8. | PubMed | ChemPort |
18. Richmond JFL, Mustafa F, Lu S et al. Screening of HIV-1 env glycoproteins for the ability to raise neutralising antibody using DNA immunization and recombinant vaccinia virus boosting. Virology 1997; 230: 265–74. | Article | PubMed | ChemPort |
19. Hanke T, Blanchard TJ, Schneider J et al. Enhancement of MHC class I-restricted peptide-specific T cell induction by a DNA prime/MVA boost vaccination regime. Vaccine 1988; 16: 439–45.
20. Schneider J, Gilbert SC, Blanchard TJ et al. Enhanced immunogenicity for CD8+T cell induction and complete protective efficacy of malaria DNA vaccination by boosting with modified vaccinia virus Ankara. Nature Med. 1988; 4: 397–402.
21. Kent SJ, Zhao A, Best SJ et al. Enhanced T-cell immunogenicity and protective efficacy of a human immunodeficiency virus type-1 vaccine regimen consisting of consecutive priming with DNA and boosting with recombinant fowlpox virus. J. Virol. 1998; 72: 10 180–8.
22. Robinson HL, Ginsburgh HS, Davis HL et al. The Scientific Future of DNA for Immunization. Washington: American Academy of Microbiology, 1997; 1–31.
23. Darji A, Guzman CA, Gerstel B et al. Oral somatic transgene vaccination using attenuated S. typhimurium. Cell 1997; 91: 765–75. | ChemPort |
24. Altman JD, Moss PAH, Goulder PJR et al. Phenotypic analysis of antigen-specific T lymphocytes. Science 1996; 274: 94–6. | Article | PubMed | ISI | ChemPort |
25. Walker CM, Erikson AL, Hseuh FC, Levy J. Inhibition of human immunodeficiency virus replication in acutely infected CD4+ cells by CD8+ cells involves a non-cytotoxic mechanism. J. Virol. 1991; 65: 5921–7. | PubMed | ISI | ChemPort |
26. Pardoll D. Maturation of cancer vaccines. A meeting report on 'Cancer vaccines 1996' sponsored by the Cancer Research Institute, held 7–9 October 1996. Biochim. Biophys. Acta 1997; 1332: R33–6. | PubMed | ChemPort |