Optimizing cancer immunotherapy trials: Back to basics


  • Freda K Stevenson Prof.,

    Corresponding author
    1. Molecular Immunology Group, Cancer Sciences Division, Southampton University Hospitals Trust, Southampton, UK
    • Molecular Immunology Group, Cancer Sciences Division, Southampton University Hospitals Trust, Southampton, SO16 6YD, UK, Fax: +44-23-8070-1385
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  • Jason Rice

    1. Molecular Immunology Group, Cancer Sciences Division, Southampton University Hospitals Trust, Southampton, UK
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Attempts to raise effective immunity against cancer are benefiting from information on the nature of the immunity involved and its regulation and, perhaps, now it is time to step back and define our approach in molecular terms prior to clinical testing. Although there are immunological differences between mice and patients, results from murine studies are encouraging early ‘translation’ of concepts to the clinic and it is vital to take immunological principles emerging from mice into clinical vaccine design. One is the requirement to break tolerance against over-expressed self-antigens, a potentially risky procedure but necessary for several cancer targets. A study in this issue of the European Journal of Immunology attempts to do this by using xenogeneic antigens, albeit with variable outcome. The unstated goal is to activate T-cell help but this can be achieved more effectively by harnessing a predictable anti-microbial repertoire. The second issue lies in the delivery of antigen. One strategy is "prime/boost" using DNA priming and boosting with a viral vector; however, this induces blocking immunity against viral proteins, and must be used judiciously. There are other physical methods to increase immunity such as electroporation, which can itself be used in ‘prime/boost’ sequence. These twin problems of engagement of T-cell help and delivery of adequate antigen can now be addressed by applying immunological logic to cancer vaccines.

See accompanying article http://dx.doi.org/10.1002/eji.200535514


fragment C

There is considerable interest in harnessing the immune response to attack cancer cells in patients. A basic tenet of vaccinology is the induction of immunological memory and, once induced, immune effector pathways should continuously survey the host for re-emergent tumor cells, thereby providing the much needed long term protection against relapse; however, the difficulty with this approach has always been how to induce immunity in patients where antigens are weakly immunogenic, and in whom, if spontaneous immunity has developed, the tumor is likely to have led to immune tolerance. A further problem is that drug treatment, used to induce remission, could have damaged the immune capacity. Fortunately, recent developments in our understanding of the immune response, coupled with new strategies for delivering tumor antigens, are beginning to reveal ways to circumvent these problems.

The expression of tumor antigens in patients can generate either central tolerance, with deletion of responding T cells from the repertoire, or peripheral tolerance. The latter is mediated by a complex range of regulatory pathways, including regulatory T cells, now a focus of much investigation. The fact that cytotoxic T cells (CTL) can be generated in vitro against a wide range of peptide antigens encourages us to conclude that central tolerance in human subjects may not be total, leaving some repertoire available for activation; however, our vaccine strategies still have to reverse peripheral tolerance, and the key to this is activation of T-cell help.

Importance of T-cell help

The pivotal cell of the immune response is the CD4+ helper T cell (Th), without which only low levels of immunity can be induced, and the maintenance of the response is poor 1, 2. The requirement for foreign sequences to induce Th for the B-cell response has been known for many years, and is the basis for conjugate vaccines 3. It is clear that there is generally a similar requirement to help the CTL response 4, 5. This applies also to adoptive transfer of CTL to attack viruses such as CMV, where the transferred CTL only survive and function efficiently with co-transferred Th cells 6.

Since Th cells control responses to vaccination, it is unsurprising that self-antigens such as Ep-CAM, which either do not contain epitopes likely to be recognized by available Th cells or where responses to such antigens are suppressed by regulatory T cells 7, are incapable of inducing immunity. One reason for the ability of xenogeneic antigen to break tolerance, as described in the paper in this issue of the European Journal of Immunology by Leonardo et al. 8, is likely to be the presence of some foreign sequence in the xenogeneic antigen that is able to activate Th cells; however, this is rather a ‘hit and miss’ approach to breaking tolerance for therapeutic purposes since the degree of difference between self and xenogeneic antigen is often small and might be sufficient to induce an immune response only in a minority of patients with a suitable MHC Class II background. The variable performance of the mouse Ep-CAM vaccine in outbred mice described in the study by Lenardo et al. 8 could reflect this heterogeneity.

A more predictable strategy to activate Th cells for inducing anti-tumor immunity is to engage a repertoire against non-tolerized antigens. We have focused on the anti-microbial repertoire, known to be large and conserved. Specifically we have designed DNA fusion gene vaccines encoding the tumor antigen linked to an antigen derived from tetanus toxin 9. Fusion of the Fragment C (FrC) of tetanus toxin amplifies the immune response against a range of tumor antigens, leading to suppression of tumor growth 9. This design is in clinical trial for patients with lymphoma, and modified designs using the same principle can be used to amplify CTL responses against antigens from prostate and other cancers 10. The principle is illustrated in Fig. 1 where CD4+ T cells against FrC are induced. The dendritic cell has taken up the fusion gene or protein and is presenting peptides derived from both FrC and tumor sequences. Importantly the interaction between the dendritic cell and the CD4+ T cells responding to the FrC peptides stimulates and maintains the presentation of tumor peptides, thereby engaging anti-tumor effector T cells. This ‘back door’ route to activate immunity evades toleragenic pressure operating on anti-tumor CD4+ T cells and is simple and effective. It can even generate immunity against highly tolerizing antigens such as male minor histocompatibility antigens in male mice 11. It is likely that the strategy can activate whatever repertoire remains after central tolerance has operated.

Figure 1.

A ‘back door’ route to activation of immunity against weak antigens by harnessing T-cell help from the anti-microbial repertoire. Injection of DNA vaccines encoding fusion proteins leads to uptake and presentation of antigen (Ag) by dendritic cells (DC). Fused Fragment C (FrC) of tetanus toxin engages CD4+ Th cells that are able to ‘licence’ and support DC through interacting co-stimulatory molecules. The activated DC are then able to present weak tumor antigen more effectively, activating specific T-cell responses against poorly immunogenic tumor antigens.

Delivery of antigen

In taking immunological principles from mice to patients, the devil is in the detail. For DNA vaccines it is known that the volume injected has a critical influence on performance 12, 13, and it is impossible to scale up from 50μl in a small mouse muscle to the corresponding volume in human muscle. Other means of expressing sufficient antigen and inducing some level of inflammation are required. One of these is electroporation, which has been shown to increase performance in large animals, including rhesus macaques 14. Leonardo et al.8 use electroporation but combine it with a boost using a viral vector. The latter, however, introduces the problem of immunity against viral proteins which may be pre-existing and can block any boosting 15. There is the additional possibility of competition from viral epitopes which can suppress anti-tumor responses in patients 16. We found that priming with DNA followed by boosting with DNA plus electroporation gives a dramatic boost of immunity. This physical procedure removes the concerns of a viral vector and leads to antibody levels similar to those induced by protein vaccines plus adjuvant 13. Levels of CTL are also boosted significantly. Currently we are testing electroporation with our DNA fusion gene vaccine in patients with prostate cancer, with an encouraging acceptance of the procedure in our cohort so far.

Matching immune pathways to the target antigen

Leonardo et al. 8 have selected Ep-CAM as a target antigen. This is expressed at the cell surface and therefore should be susceptible to antibody attack. To generate antibodies in our system, we would incorporate the full protein sequence into the DNA vaccine and fuse it to the FrC sequence as described 9. Hopefully a continuous production of antibody might attack the tumor cells specifically, although reactivity with normal cells is always a concern; however, if the antibody fails to eliminate the tumor cells, or if the antigen is derived from an intracellular protein with peptides displayed at the cell surface only in association with MHC Class I or II, a T-cell response is desired.

For induction of CTL, the immunological principles shift slightly. The CTL response tends to be focused on a limited number of epitopes, a phenomenon termed ’immunodominance’ 17. Attempts to activate Th therefore must ensure that there is not a parallel induction of competitive CTL and that the CTL response remains focused on vaccine-specific epitopes that are tumour-derived. For our DNA vaccines we have minimized the FrC sequence to a single domain in order to remove potentially competitive FrC-derived CTL epitopes, while retaining the ability to induce Th. Another modification is to place the tumor-derived epitope sequences at the C-terminus of the FrC domain. This promotes processing and presentation of the epitope and provides a competitive advantage 18. The outcome is induction of high levels of CTL against specific epitopes which are able to kill target tumor cells efficiently 19, 20. It is this design which is in trial for patients with prostate cancer 21. Clearly combinations of single epitope vaccines can be used for attack on multiple epitopic targets.

Data from inbred mouse models have the advantage of being able to predict responses restricted by defined HLA molecules. Similar influences will inevitably apply to human subjects, but are more difficult to determine in the outbred setting. Howewer, there are two questions: one in terms of the available repertoire of CTL, and another of the ability to break peripheral tolerance by inducing Th responses. Unfortunately, outbred mice do not easily allow separation of these two factors. In the light of the many currently known factors operating on central and peripheral tolerance perhaps the best way forward is to assess the performance of vaccines designed to break peripheral tolerance in the clinic, in the anticipation that at least some of the CTL repertoire will still be present.

In conclusion, the opportunities provided by genetic vaccines are allowing us to take immunological principles into vaccine design. The scepticism which greeted the first attempts to take DNA vaccines into human subjects is now being overcome by new delivery technologies. Both immunological knowledge and genetic technology are advancing rapidly and will underpin future clinical trials. Another area of development is the immune monitoring of patients’ responses, so that these can be used as a surrogate for clinical effect. This early evaluation is essential to maintain a flexible learning approach to defeating the cancer cell by activating the power of immunity as fully as possible.


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