Bugs as drugs for cancer (2002)

A link between microbial infection and cancer was first postulated by A. R. Ferguson in 1911 who described Schistosoma as the causative agent for bladder cancer in Egyptian men. Then, in 1926 the Nobel Prize for Medicine was awarded to Johannes Fibiger who identified a causal relationship between the nematode Spiroptera neoplastica and stomach cancer in rats. Although Fibiger's hypothesis was later to be disproved, a causative link between cancer and a range of infectious agents has subsequently been established.1 Nowadays there are a wide range of viruses and bacteria directly implicated in the transformation of normal cells and the eventual establishment of malignancy (Table 1). However, the link between infection and cancer is not always causative and the subject of this review is to describe a number of key advances in cancer immunotherapy based upon microbes. Many of these have already been translated into significant benefit for the patient and it is hoped that the latest advances in research will yield more effective and safer therapies and vaccines. Furthermore a number of therapies not initially designed to activate immune responses now appear to have a strong immune component involved in their mechanism of action. Perhaps one of the first uses for microbes in the treatment of cancer was as adjuvant. Like whole microbes, adjuvants provide the necessary danger signals required to prime naïve immune responses. This is particularly important when eliciting immunity against tumour antigens that are self-derived and to which the immune response is already substantially tolerized.2 Adjuvants provide depots for gradual release of antigen, however, as CD8+ cytotoxic T lymphocyte (CTL) responses are key to anti-tumour immunity the ideal adjuvant also directs antigen to the major histocompatibility complex (MHC) class I processing pathways. Adjuvants employed in man include Freunds, Mycobacterium bovis bacillus Calmette–Guérin (BCG), quill-saponin and alum. To date, bacterial adjuvants, in line with other types of adjuvant, have had little beneficial effect for anti-tumour immunity as defined by partial response or complete response clinical criteria. A likely cause of this may be the particular choice of peptide epitopes used, which until recently have been those with high affinity and stability for class I, i.e. those to which tolerance is likely to have already been established.3 Future clinical trials with subdominant epitopes may fare better however, the affinity of subdominant tumour epitopes for MHC is likely to require modification.4 Current investigations are focusing on more closely defined bacterial adjuvants and one candidate is the p40 outer membrane protein of Klebsiella pneumoniae. Recombinant p40 protein functions as a potent immune adjuvant which notably induces CD8+ CTL independent of CD4+ T-cell help.5 This novel adjuvant is thought to bind preferentially to professional antigen-presenting cells (APC), including dendritic cells (DC), and thus may represent an important advance in adjuvant technology. The curative potential of bacterial infections for the treatment of cancer was demonstrated more than a century ago. However, more recently it has been proposed that bacterial DNA and viral RNA, rather than live organisms, may be responsible for many of these effects. Unmethylated CpG dinucleotides (CpG motifs) directly stimulate a variety of leucocytes, including B cells, DC, macrophages and natural killer (NK) cells, and ultimately influence the development of T-cell responses. There is also an additive effect when classical adjuvants such as incomplete Freunds' adjuvant or alum, are co-administered with CpG DNA. Indeed, recent data suggest that CpG motifs can be used to stimulate directly tumour-specific CTL6 and much of the current interest in CpG centres on its use as either a single-agent therapeutic or as an adjuvant to B- and T-cell vaccines.7 Although the mechanisms by which microbes aid the treatment of cancer are not fully understood there is a growing body of knowledge concerning how these organisms influence the immune system. Immune ‘sensing’ of microbes is now known to be attributable to a diverse range of pattern recognition receptors (PRR) expressed by many different cells. The PRRs, responsible for transducing signals from pathogen-associated molecular patterns (PAMPs), include members of the Toll-like receptor (TLR) family of proteins, and a wide range of other molecules such as CD14, the mannose receptor and complement receptor type 3 (CR3).8,9 It is not the purpose of this article to describe these in detail as several reviews have dealt in depth with this important subject.10–12 However, a useful summary of PRR and their ligands with regard to micro-organisms is provided in Fig. 1. Although TLR have the ability to transduce signals, the binding of microbial ligand can also be mediated by other molecules such as the glycosyl-phosphatidylinositol-linked CD14 protein. Association of PAMP to the respective PRR provides much of the necessary ‘danger’ so essential for immune priming by triggering a range of intracellular pathways. TLR are not only reactive to proteins and lipids, but they also have specificity for nucleic acids, as in the case of TLR9, and unmethylated CpG DNA motifs.13 The use of multiple PRR by whole micro-organisms is well illustrated with mycobacterial adjuvants such as BCG which act via TLR2 (LAM), TLR9 (CpG motifs) and CD206 (mannosylated glycoproteins). Schematic illustrating the interactions between various human pattern recognition receptors (PRR) and their ligands known as pathogen-associated molecular patterns (PAMPs). It is thought that microbial recognition involves the use of multiple PRR to detect simultaneously the different pathogen attributes. In the case of TLR4 the potential signalling complex is illustrated and the known intracellular mediators of TLR4 signalling in response to LPS are shown. Other bacterial products have also entered trials in man including modified exotoxin from Pseudomonas conjugated to cytokines. A truncated form of exotoxin fused with interleukin-4 (IL-4) has been used to treat high-grade glioma.14 As IL-4 receptor is highly expressed in glioma and astrocytoma this provides a degree of targeting, particularly when coupled with intratumoral delivery. In this study six of nine patients showed evidence of tumour necrosis after immunotherapy and one patient underwent complete response. This, coupled with the lack of neurotoxicity, has justified further clinical studies. Further experimental work has encoded as cDNA the translocation domain II of P. aeroginosa exotoxin A fused to tumour antigen.15 Domain II translocates to the cytosol from endosomal compartments, thus endowing it with the key ability to target the processing pathways for presentation by MHC class I. Vaccination with this construct resulted in a marked increase in human papillomavirus (HPV) E7-specific CTL, which in turn eradicated E7-expressing experimental tumours. Anecdotal reports of the use of Streptococci and the tubercle bacilli for cancer therapy date back as far the latter part of the nineteenth century.16,17 Although Coley's initial work resulted in some tumour regression the toxicity was life-threatening, leading Coley to devise a heat-attenuated mixture of Streptococci and Serratia.18 Important research was published in 1928 when Raymond Pearl recognized that it was unusual for malignancy and florid tuberculosis to exist in the same individual. However, the hazards associated with using live pathogenic mycobacteria for cancer therapy were only overcome in 1924 with the attenuation of ‘Nocard’, the virulent strain of M. bovis.19 In addition to providing life-long protection against leprosy and tuberculosis, the attenuated M. bovis BCG vaccine has also been successfully applied to the immunotherapy of cancer. A number of tumours, including malignant melanoma, prostate carcinoma and leukaemia, respond to treatment with BCG.20–22 However, amongst the many live bacterial agents explored in immunotherapy the use of M. bovis BCG for high-grade bladder cancer (notably carcinoma-in-situ) stands out. Indeed, BCG therapy of bladder cancer is the most ‘successful’ immunotherapy for solid malignancies and can give complete response in 80% of patients with the high-grade, aggressive and potentially lethal carcinoma-in-situ.23,24 Understanding the mode of action of BCG therapy is the goal of several groups world-wide. Clues to the mechanisms invoked include the lack of effects with non-viable BCG, peripheral administration of BCG or its actions in immunocompromised patients. Initially considered to be strictly T-cell dependent, BCG therapy is now thought to act via a number of effectors including NK cells.25 Local administration triggers vigorous inflammatory responses in the bladder wall, characterized by infiltration with a large number of helper and cytotoxic T cells, polymorphonuclear cells and NK cells and a ‘flood’ of cytokines excreted into the urine. Further changes include those observed on malignant cells per se, such as induction of CD54 and MHC class II expression. Current research is aimed at increasing the efficacy of BCG therapy and reducing the potential for toxicity. A number of approaches are under evaluation including engineering BCG to secrete cytokines26 and alternative non-pathogenic mycobacteria such as M. vaccae, M. phlei, or M. smegmatis.27 Although other mycobacteria are currently being investigated in cancer therapy they are in non-viable form. A heat-killed preparation of M. vaccae termed ‘SRL172’ and the product of SR Pharma plc is currently in phase I and II investigations for a variety of tumours including non-small-cell lung cancer, malignant mesothelioma, renal cell carcinoma and advanced prostate disease.28–30 Although still at the early stages in clinical trials there are a number of promising findings associated with SRL172, including changes in immune status (e.g. decreased numbers of IL-4-producing T cells) and possible therapeutic benefit. There is also little toxicity or other potential hazard associated with SRL172, making it a promising candidate for continued evaluation in cancer therapy. One of the main problems facing cancer therapy in particular is that of targeting, whether it be adjuvant, cytotoxic compound, or gene therapy. Research using non-pathogenic, anaerobic bacteria such as Clostridium suggests that they are ideal vectors as they preferentially localize to and proliferate in hypoxic regions within tumours.31 One of the benefits of these bacteria is that they can be administered as inactive spores. Further destruction of the tumour is achieved by increasing the hypoxic area available for bacterial replication by treatment with antivascular agents.32 The demonstration that these bacteria localize to tumours and cause lysis led to clinical trials as early as the 1960s but with disappointing results. In recent years significant interest has developed in attenuated mutants of Salmonella typhimurium. Much of the work with tumour-targeted Salmonella was done in conjunction with Vion Pharmaceuticals Inc. whose attenuated bacterium, ‘VNP20009’, was generated by deletion of the msbB and purI genes.33,34 The targeting properties of VNP20009 are illustrated in rodent models reaching tumour to normal ratios of 25 000 : 1.35 Phase I studies have shown that VNP20009 persists in tumours for more than 2 weeks. Further studies have recently been completed in which VNP20009 was used to treat malignant melanoma,36 however, no detectable efficacy was observed. Clostridium and Salmonella can be used to deliver pro-drug activating genes [e.g. cytosine deaminase (CD) converting 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU)] only within the tumour.32,37 Early clinical trials are underway with VNP20029 (VNP20009 expressing CD) in conjunction with 5-FC38 or in conjunction with radiation-inducible promoters.39 Viruses have a number of features making them strong candidates for tumour immunotherapy. In addition to their relative ease of production they are potent stimulators of immune responses operating via TLR3, -4 and -7 whilst inducing production of type I interferons. Recently the potential to generate virus which specifically acts on tumour cells has been realized. The p53 and pRb proteins and the pathways surrounding them regulate the cell cycle, are frequently disrupted in malignancy, and define tumours at the molecular level. Degradation of p53 is mediated by MDM2, which in turn is regulated by p14ARF.40 Despite the high incidence of p53 mutation in many cancers it is not mutated in some tumours, e.g. melanoma.41 However, mutations in genes associated with the p53 regulation account for deregulation of this pathway.42,43 The protein pRb regulates progression from G1 to S-phase,44 and in turn is regulated by CDK4-cyclin D and p16INK4a and abnormalities in this pathway are common in cancer.45,46 Therapies aimed at these pathways are being exploited through use of selectively replicating viruses, two of which are described in further detail. Adenoviruses replicate by preventing infected cells from arresting cell cycle and apoptosing. Adenoviral E1A and E1B gene products bind to pRb and p53 allowing continued DNA synthesis and viral replication and mutations in E1A or E1B hamper viral replication in normal cells. The first mutated adenovirus, ONYX-015, employed deletions in E1B and E3.47 However, E1B is no longer required for viral replication in cells lacking p5348,49 and so E1B-deleted viruses undergo productive infection, resulting in lytic cell death. A similar approach was devised to target cells with pRb deficiency.44,50 The pRb checkpoint in normal cells resists S-phase induction, preventing viral replication. However, wild-type adenoviruses promote entry to S-phase by the E1A gene product binding pRb. ONYX has completed phase I and II studies in malignancies of the head and neck, pancreas, ovary, lung, and colo-rectal and oral tracts.51–55 It is safe and regression is observed in up to 25% of patients. However, the precise mechanism of action remains unknown. Therapy with such viruses does not require all tumour cells to be infected in the first instance. Rather, as a result of permissive conditions, virus spreads from cell to cell. In T-cell-deficient models, the anti-tumour activity of these viruses is primarily a result of the lysis of infected cells. However, in immunocompetent mice the additional of immune responses is and release of tumour antigens may priming of CTL as described in several immune responses may infection by infected cells. Adenoviruses strong and T-cell and this may immune responses against tumour antigens are Current research to increase the activity of or and thus promote more effective This may be achieved by ‘danger’ or immune into the virus or by viral known to influence immune i.e. by MHC or preventing signalling from tumour necrosis viruses have also been cells genes that complement deletions in the viral replication that in normal cells. Phase I studies of and viruses are underway in glioma with evidence of tumour regression and The immune component of remains to be demonstrated however, as with adenovirus, most normal have and have significant responses. key of the immune particularly those associated with of MHC class I under the action of the gene whilst binding of to of into the Current approaches to include mutations in the genes responsible for cells infected with are better recognized than those infected with findings were in therapeutic models was more than In the mechanisms of action of viruses not on tumour lysis but rather antigen available for priming the of ‘danger’ can be provided with viral associated with immune these agents for cancer therapy. The MHC class I pathway has been in recent years with the that a number of antigens from intracellular bacteria are and on MHC class This is in with that only antigens were and by MHC class different pathways have been postulated for the of bacterial epitopes to class I are up into a within the cell they are either e.g. or they of the with the and for of e.g. M. bacteria such as and the the of infected cells they which in the thus allowing its proteins to the cytosol and MHC class I bacteria such as have also been shown to deliver antigens to the MHC class I pathway in a variety of such as and The presentation of epitopes from these bacteria by class I by the pathway and by a This has led to of an alternative pathway in the It is thought that the epitopes generated either bind to class I molecules or are from the cell they bind to class I on the cell from other bacteria such as mycobacteria are also thought to be via this alternative the that bacteria be used as to deliver tumour antigens to MHC class I there has been much interest in their use as anti-tumour of their additional and ability to helper epitopes to MHC class Although there are no published bacterial in clinical trials there have been promising in models and it is hoped that clinical trials may and alternative MHC class I processing pathways. proteins are by the and into the they are on MHC class I molecules and for at the cell proteins are in the resulting in of MHC class I binding epitopes which are from the cell they MHC class I molecules by already through the classical pathway these epitopes can and bind MHC class I molecules to and from the cell in The first that be used as an vaccine in when it was published that a protection against lethal with and renal cancer cells expressing and tumour regression when administered The bacterial vaccine was also when administered and regression of the highly be however, when as when they two one of which the protein fused to a same as for and the other which the protein It was that only the first vaccine tumour The lack of tumour regression was mediated by CD4+ T cells with a major in the of tumour The intracellular Salmonella is a particularly candidate for oral of its ability to cells such as the The is by macrophages and dendritic cells it MHC class I and class II The type that the uses to can also be for the entry of antigens to the Although not used for tumour this effective protection against lethal virus The use of Salmonella against cancer possible with the development of attenuated bacteria tumour and are more effective when the tumour antigen is expressed from a that is upon with Salmonella expressing was also shown to be in a and Perhaps the most important advance in the of Salmonella is the development of Salmonella DNA vaccines. are they are to target DNA to which is for immune responses. mutants of Salmonella antigen under the of the were demonstrated to the tumour antigens in infected and give tumour The has also been shown to be a candidate for the of DNA into Despite the use of BCG as a tumour immunotherapy there has been little with mycobacteria as tumour so The only published data of an anti-tumour M. bovis BCG vaccine in a was in protection against an E7-expressing This may be the used a which expressed the tumour antigen in the bacterial it has been shown that for effective immunity this case against antigens be or membrane The only tumour protection so far demonstrated has been with the administration of a protein between BCG protein and to the of safe use of BCG as a vaccine and the that its further research is to the of mycobacteria as cancer vaccines. Current work in has on of tumour antigens from several of notably BCG and M. To have and processing of antigens into epitopes have to the epitopes are to MHC class I molecules with that is their for within the endosomal the and of to class I may not the classical pathways described for antigens or for other bacteria such as and in represent promising candidates for tumour gene therapy as they can be to a variety of genes in such as tumour antigens that are on MHC class I and class II in infected cells, such as To be vaccine viral vectors be that have a safe for use in cancer patients who will be this attenuated virus such as are under One of the problems with viruses is the strong immune responses in the which the number of on which it can be In this case prime and with different vectors a more adenoviruses are also available but they strong immune responses so be vectors have been to be replication in cell so they safe whilst the family have a range and not replicate in human cells. This, and the that they can be them ideal vectors for prime and viruses to molecules as well as tumour antigens as this can overcome the danger and properties of many Many viral vectors to tumour antigen are already in clinical and some have been most studies are only in phase I. 2 some of these and further are in the reviews by and As with many similar studies CTL are in response to the vaccine the between their and clinical response is The for this are at not fully understood but may in the patterns generated by for and the as in most tumour cells. Further to in the choice of which are those which with and with stability with To date, cancer has been by and However, it is that microbes and their products have much to in of and the suggests that bacteria and viruses can be to in the against cancer. first showed that bacteria be employed to stimulate immune bacteria and viruses have treatment for some cancers and current research in this area will one yield some of the for and cancer of the The to the to them by Research and the of to for this