- Email: [email protected]
Breast cancer tumour vaccines
Cancer
Treatment
Breast David
Reviews
cancer
(1997)
23,
tumour
S77-S85
vaccines
Miles
Consultant Medical Oncologist, London SN 9RT: U.K.
Guy’s Hospital,
St, Thomas
Street,
Introduction As the millennium approaches, one might think that there is little cause for enthusiasm regarding the prospects for potential immunological therapies in breast cancer. It has been assumed that modulating the immune response to tumours is most likely to be effective in the setting of minimal residual disease yet non-specific immunotherapy approaches were not associated with any attributable survival benefit in the adjuvant setting in the Early Breast Cancer Trialist Group overview. It should also be remembered that patients who are immunosuppressed for long periods of time, either in the context of organ transplantation or immune deficiency syndromes often develop virally-induced cancers such as lymphomas or cutaneous neoplasms and not the more common epithelial malignancies such as breast, bowel or ovarian cancer. As our understanding of the immune response to novel antigens increases as well as defining at the molecular level, antigenic changes associated with malignancy, the prospects for inducing more potent and more specific immune responses to human cancers are improving
Some immunological Non-MHC
restricted
considerations
cytotoxicity
A proportion of circulating peripheral blood mononuclear cells (PBMCs) have the ability to kill a limited range of tumour targets and this has been termed natural killer cell (NK) activity. The range of targets and the degree of lysis can be increased by culture of PBMCs with interleukin-2 to generate what has been termed lymphokine activated killer cell (LAK) activity. Unfortunately the levels of IL-2 required cannot be achieved in vivo without considerable systemic toxicity. An alternative strategy has been to culture PBMCs ex vivo and re-infuse the LAK cells into patients (see below). NK and LAK activity are non-specific immunological 0305-7372/97/s77
+ 09 $12.00/O
0 1997 s77
W.B. Saunders
Company
Ltd
S78
D. MILES
reactivities which do not rely upon the expression of specific antigenic determinants presented by molecules of the major histocompatibility complex (MHC). MHC restricted
cytotoxicity
Tumour antigens are taken up by antigen presenting cells in association with molecules of the major histocompatability complex (MHC). These are large molecules on the cell surface that take up and ‘present’ peptides (antigens) via T-cell receptors to T-helper lymphocytes. Accessory molecules between the antigen presenting cell and the T-helper lymphocyte are very important in effective antigen presentation. Following presentation, the T-helper lymphocyte produces cytokines which stimulate the proliferation of a population of cytotoxic T-lymphocytes which in turn recognize antigen in conjunction with another class of MHC molecule. This type of killing is dependant upon cells of the immune system recognizing foreign antigen by the MHC complex and hence is termed ‘MHC-restricted. Cytokines produced by T-helper lymphocytes also expand B-lymphocyte populations which can then produce antibody to the original antigenic determinant. Production of such antibodies can mediate killing by complement mediated cytotoxicity, antibody dependent cell mediated cytoxicity (binding of Fc portion of antibody by killer cells and by the hyper-variable region recognizing antigen bearing tumour cells).
Historical The modulation lines.
of the immune
response
perspective to tumour
has developed
along several
Non-specific approaches At the turn of this century, an American surgeon, William Coley noted occasional tumour regressions in cases where erysipelas (local inflammation caused by streptococcus) developed adjacent to tumour. This lead to the development of Coley’s Toxins, bacterial filtrates, which when given systemically led to tumour regressions in some cases. It is probable that these reductions in tumour burden were mediated by cytokines, the signalling molecules between cells of the immune system, particularly tumour necrosis factor (TNF). Interest in this type of immunotherapy continues with the investigation of BCG in certain tumour types (e.g. bladder carcinoma) and the testing of recombinant cytokines such as interleukin-2 (IL-2) and the interferons, in the clinical setting. Passive immunotherapy Following the identification of antigenic determinants expressed preferentially by tumour cells, antibodies of increasing sophistication, which can recognize these determinants have been tested in the clinical setting. While one line of investigation uses such antibodies to target radio-isotopes or toxins to antigen bearing (tumour ) cells, it is also thought that administration of antibodies alone may mediate anti-tumour activity possibly through complement fixation, antibody dependent cellular cytotoxicity (conjugation of ‘killer’ cells and target cells by antibody) and the idiotype network (antibody responses to the hypervariable region of the administered antibody, which by reflecting the original
BREAST
CANCER
TUMOUR
VACCINES
579
antigenic determinant, may act as an immunogen itself). Phase III studies of antibody alone have been conducted in colon cancer (Reithmuller et a/.) and form the basis for ongoing phase III studies testing their contribution to current adjuvant therapies in this disease. Active immunotherapy The aim of active specific immunotherapy is to induce an immune response to a known or unknown antigenic determinants. Increased understanding of how antigens are presented to the immune system allows the prediction of epitopes which will be immunogenic in patients with a given HLA type and amino acid substitutions of such immunodominant peptides may further increase the binding to major histocompatibility complex. Adoptive irnrnunotherapy This approach involves the ex vivo stimulation of populations of immune effector cells with levels of cytokines which would not be achievable in vivo. Originally, work in this area by Rosenberg and colleagues involved the ex vivo expansion if natural killer cells from the peripheral circulation, by IL-2 to generate lymphokine activated killer cells (capable of killing a much greater range of tumour targets in vivo compared with NK cells) which were then re-infused into the patient together with systemic IL-2. Later work by the same group and others involved the expansion of populations of lymphocytes isolated from tumour tissue, tumour infiltrating lymphocytes or TIL. The aim of this approach was to expand populations of T-lymphocytes which already recognize tumour associated antigens. It is also possible that following immunization with known antigenic determinants, expansion of the cytotoxic lymphocyte precursors could be achieved ex vivo, for subsequent re-infusion into patients. The main problem with the approach of adoptive immunotherapy is that it is extremely labour-intensive and unlikely to be feasible on a large scale. What do we know about the natural history of early disease and the impact of the immune response on that? Tumour cells are good at evading the immune response. They may grow so quickly that they may out-pace the immune response. A more major consideration is the ability of tumours to alter their characteristics. Clones which fail to express MHC molecules are probably ‘selected for’. In addition, loss of MHC is very common in breast cancer and so losing these molecules may also be important. Finally, tumours may suppress the immune response through inappropriate signals to T-lymphocytes.
Cancer vaccines Cancer vaccines can be divided into categories depending on their origin. Although none currently apply to breast cancer treatment they include: Whole fumour cells. Tumour cells, allogeneic or autologous, are irradiated and either given with or without an immunological adjuvant or, more recently, transfected with cytokines or accessory molecules capable of augmenting the immune response (IL-2/GM-CSF/B7). The most feasible approach is to use allogeneic cell lines in which it is assumed that shared antigens, which are not of course defined, may
sao induce an immune
D. MILES
response
which will lead to rejection
of tumour.
Proteins. These are taken up by antigen presenting cells and cleaved into peptides, some of which may be presented on MHC molecules. Peptides. Following increased understanding of presentation of peptides by MHC molecules, it is becoming possible to predict peptide sequences which would be presented by particular MHC haplotypes. Putative MHC binding sequences of peptides of proteins overexpressed in malignancy or whose epitopes are normally cryptic are being investigated. An alternative approach is to define mutations in particular individuals and synthesize appropriate peptides for immunization. The use of proteins and peptides is dependent upon their uptake into antigen presenting cells and their entry into the endogenous pathway which processes peptides and incorporates them into MHC for subsequent presentation. Nucleic acids. The use of encode tumour associated munization since production cells themselves probably pathways.
naked DNA or viral vectors containing cDNA which antigens may well be a more efficient method of imof the immunogen of interest by the antigen presenting increases the likelihood of processing by the MHC
Carbohydrates. Although antibody responses towards carbohydrate antigens can be generated, the way in which they are processed and presented by the immune system is currently unclear.
lmmunogens
for breast
cancer
There are several potential immunogens for breast cancer that could be targeted such as erb-2, ~53. Other epitopes which could be targeted include the MAGE/MART antigens, which though originally identified in melanoma are also expressed in a proportion of breast tumours. Our particular area of interest has been the potential use of MUC-1, a large membrane bound mucin consisting of a peptide backbone and complex branching carbohydrate structures. It has a transmembrane domain with an extracellular domain made up of tandem repeats. In MUC-1 associated with malignancy, carbohydrate side chains are shorter than normal and some peptide epitopes which usually are not revealed to the immune system are exposed. MUC-1 is also upregulated in malignancy and its apical distribution is lost. For these reasons, MUC-1 may be a relevant immunogen in breast cancer. Both cellular and humoral immune responses to MUC-1 can be seen in breast cancer patients. Proliferative responses, as a measure of T-cell function, have been assigned to MUC-1 in women who are pregnant, and cytotoxic T-lymphocytes have been isolated. It is of particular interest that these T-lymphocytes may not be restricted to MHC expression by tumour cells. The T-cell receptor may be cross-linked as a result of the multiple tandem repeat epitopes on the extracellular domain.
BREAST
CANCER
TUMOUR
VACCINES
S81
A variety of epitopes (possible immunogens based on MUC-1) can be used; carbohydrates, a combination of carbohydrate and peptide (glycopeptide), and peptide alone (antibody, lipopeptide encapsulation or cDNA).
Carbohydrates The complex carbohydrate structure of MUC-1 is formed by carbohydrate chain extension. Enzyme deficiency in malignancy results in premature termination of these chains and, if sialic acid is added, tumour-associated sialated carbohydrate antigens (STn) can be produced. STn expression has been shown to be associated with poor prognosis in some adenocarcindmas and gastric carcinomas and more recently in breast cancer where expression may predict relative resistance to adjuvant chemotherapy. Studies in animal models suggested that immunotherapy using carbohydrate antigens may prolong survival and the use of low dose cyclophosphamide prior to immunotherapy potentiated the immune response and further increased survival. These results support the theory that T-suppressor cells which switch off immune responses are killed by relatively low doses of cyclophosphamide. Two randomized Phase II studies (U.K. and Canada) of STn were conducted to find out whether low dose intravenous cyclophosphamide modulates the immune response. The immunogen, STn, was linked to a protein carrier called keyhole limpet haemocyanin (KLH) and administered with the an immunological adjuvant, DETOX. Patients were randomized to intravenous cyclophosphamide versus no cyclophosphamide (U.K. study) or to intravenous cyclophosphamide versus oral cyclophosphamide (Canadian study). Immunotherapy was administered subcutaneously at 2, 3 and 4 week intervals and patients were assessed at week 12. Maintenance therapy was given every 4 weeks to stable or responding patients who were assessed at week 29. Systemic toxicity was mild, the main problem being the development of delayedtype hypersensitivity responses at the injection site which occasionally ulcerated. In the first 12 weeks, minor responses were seen in two patients, while 18 patients had stable disease. Nineteen of these patients were given maintenance therapy of which one had a partial response and two had minor responses. The single i.v. dose of cyclophosphamide was shown to have increase significantly IgM and IgG levels to STn compared with those who had been given oral cyclophosphamide or no cyclophosphamide treatment. Combining data from the two trials, the median survival time for the group given intravenous cyclophosphamide had not been reached, whereas median survival was about 12.5 months for those with oral or no cyclophosphamide pre-treatment. The mechanism of action may be related to antibody responses as there was an inverse correlation between antibody titres to STn and marker lesions. This may be explained by the selection of the subgroup of patients who have less aggressive disease and slower tumour growth. However, this is probably not the case as antibodies titres against carrier glycoprotein (KLH) could be measured and did not correlate with size of lesion. Patients who generated a greater than median STn antibody response had significantly better survival rate than those
S82
D. MILES
who developed a less than median response. These data are hypothesis generating and indicate the need for a randomized phase III study.
Peptides Another way in which we are targeting mucin, is be using antibodies to the peptide backbone (humanized HMFG-1 antibodies). A Phase I study in an adjuvant setting is currently looking at patients who have undergone surgery, are taking tamoxifen and have no measurable disease. The endpoints are toxicity and immunological responses. Although this type of immunotherapy can be passive, promoting conjugation of a killer T-cell with tumour cell, it can also be regarded as active through the idiotype network (antibodies generated to the hypervariable region of the administered antibody may resemble the original immunogen and themselves induce a further immune response). Two studies are proposed for 1997. Firstly, a study with BLP25 liposome - a 25 amino acid sequence from the MUC-1 peptide backbone. Liposome encapsulation may improve uptake by antigen presenting cells because, by being within a lipid bilayer, it can fuse with the cell membrane and aid antigen presentation and, the lipid bilayer can act as an immunological adjuvant. Endpoints include toxicity and immunological factors. Secondly, a gene therapy trial of a MUC-l-IL-2 vaccinia construct is to start. A vaccinia virus containing cDNA for MUC-1 and IL-2 can infect antigen presenting cells which subsequently can transcribe and translate the proteins, MUC-1 and IL-2. The main problem is that vaccinia virus is a replication competent virus and viral shedding is being examined. Toxicity, conventional response criteria and immunology will be explored to identify markers to predict benefits and patients that will benefit. Adjuvants injected under the skin are irritants and cause an influx of host immune cells including antigen presenting cells. Conventional adjuvants include DETOX, alum and Freund’s. There are other ways of attracting antigen presenting cells that involve enhancing ‘signals’ to cytotoxic T-cells and cytokines (IL-2) and growth promoters of subcutaneous dendritic cells (GM-CSF). Specific palindromic DNA sequences may also augment the immune response. Antigen presenting cells which express high levels of class 1 and 2 MHC also express the co-stimulatory molecules, B7. The importance of such ‘professional’ antigen presenting cells can be demonstrated in animal models. Mice immunized with peptide pulsed dendritic cells produce better responses than mice immunized with peptide alone. Similarly, in tumour protection studies, mice immunized with human peptide pulsed dendritic cells and challenged with tumour cells are able to control tumour growth. The tumour grows unchecked in those immunized with human peptide alone. In another tumour protection experiment, expression of B7.1 lead to significantly prolonged survival. It is possible to take peripheral blood from patients, culture it ex vivo and generate potent antigen presenting cells. The phenotype of peripheral blood, cultured with GM-CSF and IL-4, is made more CD14 negative and the 87 accessory molecule and class 1 and 2 MHC are upregulated. I hope by the year 2000 that we will have tested the feasibility of culturing patients dendritic cells ex vivo and
BREAST
CANCER
TUMOUR
VACCINES
using them as the definitive adjuvant following pulsing with appropriate
S83
immunogens.
Conclusion Systems are now established where a variety of immunogens in differing formulations, (carbohydrates, peptides, glycoproteins) can be studied, in mouse models and in human antigen presenting cells for testing of immunogenicity. From a combination of these approaches, it is hoped that the appropriate immunogens can be selected and progressed to clinical trials.
Suggested
reading
Rosenberg, S. (1997) Cancer vaccines based on the identification of genes encoding cancer regression antigens. Immunol. Today 18: 175-182. Boon, T., Coulie, P G. & Van den Eynde, B. (1997) Tumour antigens recognised by T cells. Immunol. Today 18: 267-268. Hsu, F., Casper, C. B., Czerkinski D., et al. (1997) Tumour-specific idiotype vaccines in the treatment of patients with B-cell lyphoma. Blood 89: 3129-3135. Riethmuller, G., Schneidergadicke, E., Schlimok, G., et al. (1994) Randomized trial of monoclonal-antibody for adjuvant therapy of resected dukes-C colorectal-carcinoma. Lancet 343: 1177-l 183. Burchell, J., Graham, R. & Taylor-Papadimitriou J. (1993) Active specific immunotherapy - PEM as a potential target molecule. Cancer Surv. 18: 135-148. Longernecker, B. M., Reddish, M., Miles, D. W. & MacLean, G. D. (1993) Synthetic tumour associated -antigens as immunotherapeutic cancer vaccines. Vaccine Res. 2: 151-i62. Miles, D. W.. Towlson. K. E.. Graham, R., et al. A randomized Dhase II studv of sialvl-Tn and DETOX-B adjuvant with or without cyclophosphamide pre-treatment for the active specific immunotherapy of breast cancer.
Questions Question:
Dr A. Howell,
Christie
and Answers
Hospital
NHS Trust, Manchester,
U.K.
What has been deduced from the clinical trial where one arm had intravenous cyclophosphamide and the other arm nothing or oral cyclophosphamide? Answer: The study was designed to assess how cyclophosphamide would influence the immune response and our conclusion was that intravenous cyclophosphamide appeared to augment the antibody response. Concerning survival it is noteworthy that the two intravenous groups on separate sides of the Atlantic were superimposable, as were the oral and the no cyclophosphamide groups. I would emphasize that the survival differences observed were by randomization, though the differences observed in survival by ability to generate antibody responses to STn are necessarily more speculative. It is possible that i.v. cyclophosphamide increased the immune response to an undefined antigen but I would argue against that as the KLH data did not provide the same split in survival estimates.
584
Question:
D. MILES
Dr A. Howell,
Christie
Why is oral cyclophosphamide
Hospital
NHS
Trust, Manchester,
U.K.
not immunomodulatory?
Answer: I think that our experience from adjuvant chemotherapy with cyclophosphamide regimens suggests that prolonged oral CTX may be immunosupressive.The reasons for these different effects is unclear. Question:
Professor
You mentioned as KLH?
R. Rubens,
adjuvants:
Guy’s Hospital,
what is the necessity
London,
U.K.
for large carrier molecules
such
Answer: KLH is used because it is a large protein to which STn can be conjugated. It is a way of attaching 5 STns to 1 KLH molecule to allow presentation of multiple epitopes. Question:
Dr R. Coleman,
Weston
Park Hospital,
Sheffield,
U.K.
Is the standard phase II trial approach the way forward (you presumably would have thrown out the phase II approach if you had not seen the survival data). or is some sort of randomized design more appropriate? Answer: That is a question that goes for all biological approaches. A randomized phase II may give you an angle on biological responsiveness to demonstrate that you are indeed having an effect on the immune response. Obviously people are looking for surrogate markers (circulating tumour associated antigens or markers of immune response and hoping to identify those which will predict subsequent clinical response. Comment:
Dr A. Howell,
Christie
Hospital
NHS
Trust, Manchester,
If you are using surrogate markers then you need a controlled any sense out of the results.
population
U.K. to make
Reply: I agree. Biological approaches are going to be high risk strategy for companies because, with a cytotoxic agent, a product licence can be given on the response rate of a group of 50 patients. Taxol and Taxotere bear witness to that. This can not be achieved with many of these biological approaches.
BREAST
Comment:
Dr A, Howell,
CANCER
Christie
TUMOUR
Hospital
VACCINES
NHS Trust, Manchester,
S85
U.K.
We should not be so worried. Both John Robertson and I are great proponents of stabilization of disease and John Robertson is also a great proponent of tumour markers. Also, recently there has been work published stating that you do not want to kill tumour cells but maintain them and not let them grow. Thus, stabilization or no increase in tumour disease could be monitored by measuring changes in levels of tumour markers. If you can show differences between two groups then randomization is extremely important.
Treatment
Breast David
Reviews
cancer
(1997)
23,
tumour
S77-S85
vaccines
Miles
Consultant Medical Oncologist, London SN 9RT: U.K.
Guy’s Hospital,
St, Thomas
Street,
Introduction As the millennium approaches, one might think that there is little cause for enthusiasm regarding the prospects for potential immunological therapies in breast cancer. It has been assumed that modulating the immune response to tumours is most likely to be effective in the setting of minimal residual disease yet non-specific immunotherapy approaches were not associated with any attributable survival benefit in the adjuvant setting in the Early Breast Cancer Trialist Group overview. It should also be remembered that patients who are immunosuppressed for long periods of time, either in the context of organ transplantation or immune deficiency syndromes often develop virally-induced cancers such as lymphomas or cutaneous neoplasms and not the more common epithelial malignancies such as breast, bowel or ovarian cancer. As our understanding of the immune response to novel antigens increases as well as defining at the molecular level, antigenic changes associated with malignancy, the prospects for inducing more potent and more specific immune responses to human cancers are improving
Some immunological Non-MHC
restricted
considerations
cytotoxicity
A proportion of circulating peripheral blood mononuclear cells (PBMCs) have the ability to kill a limited range of tumour targets and this has been termed natural killer cell (NK) activity. The range of targets and the degree of lysis can be increased by culture of PBMCs with interleukin-2 to generate what has been termed lymphokine activated killer cell (LAK) activity. Unfortunately the levels of IL-2 required cannot be achieved in vivo without considerable systemic toxicity. An alternative strategy has been to culture PBMCs ex vivo and re-infuse the LAK cells into patients (see below). NK and LAK activity are non-specific immunological 0305-7372/97/s77
+ 09 $12.00/O
0 1997 s77
W.B. Saunders
Company
Ltd
S78
D. MILES
reactivities which do not rely upon the expression of specific antigenic determinants presented by molecules of the major histocompatibility complex (MHC). MHC restricted
cytotoxicity
Tumour antigens are taken up by antigen presenting cells in association with molecules of the major histocompatability complex (MHC). These are large molecules on the cell surface that take up and ‘present’ peptides (antigens) via T-cell receptors to T-helper lymphocytes. Accessory molecules between the antigen presenting cell and the T-helper lymphocyte are very important in effective antigen presentation. Following presentation, the T-helper lymphocyte produces cytokines which stimulate the proliferation of a population of cytotoxic T-lymphocytes which in turn recognize antigen in conjunction with another class of MHC molecule. This type of killing is dependant upon cells of the immune system recognizing foreign antigen by the MHC complex and hence is termed ‘MHC-restricted. Cytokines produced by T-helper lymphocytes also expand B-lymphocyte populations which can then produce antibody to the original antigenic determinant. Production of such antibodies can mediate killing by complement mediated cytotoxicity, antibody dependent cell mediated cytoxicity (binding of Fc portion of antibody by killer cells and by the hyper-variable region recognizing antigen bearing tumour cells).
Historical The modulation lines.
of the immune
response
perspective to tumour
has developed
along several
Non-specific approaches At the turn of this century, an American surgeon, William Coley noted occasional tumour regressions in cases where erysipelas (local inflammation caused by streptococcus) developed adjacent to tumour. This lead to the development of Coley’s Toxins, bacterial filtrates, which when given systemically led to tumour regressions in some cases. It is probable that these reductions in tumour burden were mediated by cytokines, the signalling molecules between cells of the immune system, particularly tumour necrosis factor (TNF). Interest in this type of immunotherapy continues with the investigation of BCG in certain tumour types (e.g. bladder carcinoma) and the testing of recombinant cytokines such as interleukin-2 (IL-2) and the interferons, in the clinical setting. Passive immunotherapy Following the identification of antigenic determinants expressed preferentially by tumour cells, antibodies of increasing sophistication, which can recognize these determinants have been tested in the clinical setting. While one line of investigation uses such antibodies to target radio-isotopes or toxins to antigen bearing (tumour ) cells, it is also thought that administration of antibodies alone may mediate anti-tumour activity possibly through complement fixation, antibody dependent cellular cytotoxicity (conjugation of ‘killer’ cells and target cells by antibody) and the idiotype network (antibody responses to the hypervariable region of the administered antibody, which by reflecting the original
BREAST
CANCER
TUMOUR
VACCINES
579
antigenic determinant, may act as an immunogen itself). Phase III studies of antibody alone have been conducted in colon cancer (Reithmuller et a/.) and form the basis for ongoing phase III studies testing their contribution to current adjuvant therapies in this disease. Active immunotherapy The aim of active specific immunotherapy is to induce an immune response to a known or unknown antigenic determinants. Increased understanding of how antigens are presented to the immune system allows the prediction of epitopes which will be immunogenic in patients with a given HLA type and amino acid substitutions of such immunodominant peptides may further increase the binding to major histocompatibility complex. Adoptive irnrnunotherapy This approach involves the ex vivo stimulation of populations of immune effector cells with levels of cytokines which would not be achievable in vivo. Originally, work in this area by Rosenberg and colleagues involved the ex vivo expansion if natural killer cells from the peripheral circulation, by IL-2 to generate lymphokine activated killer cells (capable of killing a much greater range of tumour targets in vivo compared with NK cells) which were then re-infused into the patient together with systemic IL-2. Later work by the same group and others involved the expansion of populations of lymphocytes isolated from tumour tissue, tumour infiltrating lymphocytes or TIL. The aim of this approach was to expand populations of T-lymphocytes which already recognize tumour associated antigens. It is also possible that following immunization with known antigenic determinants, expansion of the cytotoxic lymphocyte precursors could be achieved ex vivo, for subsequent re-infusion into patients. The main problem with the approach of adoptive immunotherapy is that it is extremely labour-intensive and unlikely to be feasible on a large scale. What do we know about the natural history of early disease and the impact of the immune response on that? Tumour cells are good at evading the immune response. They may grow so quickly that they may out-pace the immune response. A more major consideration is the ability of tumours to alter their characteristics. Clones which fail to express MHC molecules are probably ‘selected for’. In addition, loss of MHC is very common in breast cancer and so losing these molecules may also be important. Finally, tumours may suppress the immune response through inappropriate signals to T-lymphocytes.
Cancer vaccines Cancer vaccines can be divided into categories depending on their origin. Although none currently apply to breast cancer treatment they include: Whole fumour cells. Tumour cells, allogeneic or autologous, are irradiated and either given with or without an immunological adjuvant or, more recently, transfected with cytokines or accessory molecules capable of augmenting the immune response (IL-2/GM-CSF/B7). The most feasible approach is to use allogeneic cell lines in which it is assumed that shared antigens, which are not of course defined, may
sao induce an immune
D. MILES
response
which will lead to rejection
of tumour.
Proteins. These are taken up by antigen presenting cells and cleaved into peptides, some of which may be presented on MHC molecules. Peptides. Following increased understanding of presentation of peptides by MHC molecules, it is becoming possible to predict peptide sequences which would be presented by particular MHC haplotypes. Putative MHC binding sequences of peptides of proteins overexpressed in malignancy or whose epitopes are normally cryptic are being investigated. An alternative approach is to define mutations in particular individuals and synthesize appropriate peptides for immunization. The use of proteins and peptides is dependent upon their uptake into antigen presenting cells and their entry into the endogenous pathway which processes peptides and incorporates them into MHC for subsequent presentation. Nucleic acids. The use of encode tumour associated munization since production cells themselves probably pathways.
naked DNA or viral vectors containing cDNA which antigens may well be a more efficient method of imof the immunogen of interest by the antigen presenting increases the likelihood of processing by the MHC
Carbohydrates. Although antibody responses towards carbohydrate antigens can be generated, the way in which they are processed and presented by the immune system is currently unclear.
lmmunogens
for breast
cancer
There are several potential immunogens for breast cancer that could be targeted such as erb-2, ~53. Other epitopes which could be targeted include the MAGE/MART antigens, which though originally identified in melanoma are also expressed in a proportion of breast tumours. Our particular area of interest has been the potential use of MUC-1, a large membrane bound mucin consisting of a peptide backbone and complex branching carbohydrate structures. It has a transmembrane domain with an extracellular domain made up of tandem repeats. In MUC-1 associated with malignancy, carbohydrate side chains are shorter than normal and some peptide epitopes which usually are not revealed to the immune system are exposed. MUC-1 is also upregulated in malignancy and its apical distribution is lost. For these reasons, MUC-1 may be a relevant immunogen in breast cancer. Both cellular and humoral immune responses to MUC-1 can be seen in breast cancer patients. Proliferative responses, as a measure of T-cell function, have been assigned to MUC-1 in women who are pregnant, and cytotoxic T-lymphocytes have been isolated. It is of particular interest that these T-lymphocytes may not be restricted to MHC expression by tumour cells. The T-cell receptor may be cross-linked as a result of the multiple tandem repeat epitopes on the extracellular domain.
BREAST
CANCER
TUMOUR
VACCINES
S81
A variety of epitopes (possible immunogens based on MUC-1) can be used; carbohydrates, a combination of carbohydrate and peptide (glycopeptide), and peptide alone (antibody, lipopeptide encapsulation or cDNA).
Carbohydrates The complex carbohydrate structure of MUC-1 is formed by carbohydrate chain extension. Enzyme deficiency in malignancy results in premature termination of these chains and, if sialic acid is added, tumour-associated sialated carbohydrate antigens (STn) can be produced. STn expression has been shown to be associated with poor prognosis in some adenocarcindmas and gastric carcinomas and more recently in breast cancer where expression may predict relative resistance to adjuvant chemotherapy. Studies in animal models suggested that immunotherapy using carbohydrate antigens may prolong survival and the use of low dose cyclophosphamide prior to immunotherapy potentiated the immune response and further increased survival. These results support the theory that T-suppressor cells which switch off immune responses are killed by relatively low doses of cyclophosphamide. Two randomized Phase II studies (U.K. and Canada) of STn were conducted to find out whether low dose intravenous cyclophosphamide modulates the immune response. The immunogen, STn, was linked to a protein carrier called keyhole limpet haemocyanin (KLH) and administered with the an immunological adjuvant, DETOX. Patients were randomized to intravenous cyclophosphamide versus no cyclophosphamide (U.K. study) or to intravenous cyclophosphamide versus oral cyclophosphamide (Canadian study). Immunotherapy was administered subcutaneously at 2, 3 and 4 week intervals and patients were assessed at week 12. Maintenance therapy was given every 4 weeks to stable or responding patients who were assessed at week 29. Systemic toxicity was mild, the main problem being the development of delayedtype hypersensitivity responses at the injection site which occasionally ulcerated. In the first 12 weeks, minor responses were seen in two patients, while 18 patients had stable disease. Nineteen of these patients were given maintenance therapy of which one had a partial response and two had minor responses. The single i.v. dose of cyclophosphamide was shown to have increase significantly IgM and IgG levels to STn compared with those who had been given oral cyclophosphamide or no cyclophosphamide treatment. Combining data from the two trials, the median survival time for the group given intravenous cyclophosphamide had not been reached, whereas median survival was about 12.5 months for those with oral or no cyclophosphamide pre-treatment. The mechanism of action may be related to antibody responses as there was an inverse correlation between antibody titres to STn and marker lesions. This may be explained by the selection of the subgroup of patients who have less aggressive disease and slower tumour growth. However, this is probably not the case as antibodies titres against carrier glycoprotein (KLH) could be measured and did not correlate with size of lesion. Patients who generated a greater than median STn antibody response had significantly better survival rate than those
S82
D. MILES
who developed a less than median response. These data are hypothesis generating and indicate the need for a randomized phase III study.
Peptides Another way in which we are targeting mucin, is be using antibodies to the peptide backbone (humanized HMFG-1 antibodies). A Phase I study in an adjuvant setting is currently looking at patients who have undergone surgery, are taking tamoxifen and have no measurable disease. The endpoints are toxicity and immunological responses. Although this type of immunotherapy can be passive, promoting conjugation of a killer T-cell with tumour cell, it can also be regarded as active through the idiotype network (antibodies generated to the hypervariable region of the administered antibody may resemble the original immunogen and themselves induce a further immune response). Two studies are proposed for 1997. Firstly, a study with BLP25 liposome - a 25 amino acid sequence from the MUC-1 peptide backbone. Liposome encapsulation may improve uptake by antigen presenting cells because, by being within a lipid bilayer, it can fuse with the cell membrane and aid antigen presentation and, the lipid bilayer can act as an immunological adjuvant. Endpoints include toxicity and immunological factors. Secondly, a gene therapy trial of a MUC-l-IL-2 vaccinia construct is to start. A vaccinia virus containing cDNA for MUC-1 and IL-2 can infect antigen presenting cells which subsequently can transcribe and translate the proteins, MUC-1 and IL-2. The main problem is that vaccinia virus is a replication competent virus and viral shedding is being examined. Toxicity, conventional response criteria and immunology will be explored to identify markers to predict benefits and patients that will benefit. Adjuvants injected under the skin are irritants and cause an influx of host immune cells including antigen presenting cells. Conventional adjuvants include DETOX, alum and Freund’s. There are other ways of attracting antigen presenting cells that involve enhancing ‘signals’ to cytotoxic T-cells and cytokines (IL-2) and growth promoters of subcutaneous dendritic cells (GM-CSF). Specific palindromic DNA sequences may also augment the immune response. Antigen presenting cells which express high levels of class 1 and 2 MHC also express the co-stimulatory molecules, B7. The importance of such ‘professional’ antigen presenting cells can be demonstrated in animal models. Mice immunized with peptide pulsed dendritic cells produce better responses than mice immunized with peptide alone. Similarly, in tumour protection studies, mice immunized with human peptide pulsed dendritic cells and challenged with tumour cells are able to control tumour growth. The tumour grows unchecked in those immunized with human peptide alone. In another tumour protection experiment, expression of B7.1 lead to significantly prolonged survival. It is possible to take peripheral blood from patients, culture it ex vivo and generate potent antigen presenting cells. The phenotype of peripheral blood, cultured with GM-CSF and IL-4, is made more CD14 negative and the 87 accessory molecule and class 1 and 2 MHC are upregulated. I hope by the year 2000 that we will have tested the feasibility of culturing patients dendritic cells ex vivo and
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using them as the definitive adjuvant following pulsing with appropriate
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immunogens.
Conclusion Systems are now established where a variety of immunogens in differing formulations, (carbohydrates, peptides, glycoproteins) can be studied, in mouse models and in human antigen presenting cells for testing of immunogenicity. From a combination of these approaches, it is hoped that the appropriate immunogens can be selected and progressed to clinical trials.
Suggested
reading
Rosenberg, S. (1997) Cancer vaccines based on the identification of genes encoding cancer regression antigens. Immunol. Today 18: 175-182. Boon, T., Coulie, P G. & Van den Eynde, B. (1997) Tumour antigens recognised by T cells. Immunol. Today 18: 267-268. Hsu, F., Casper, C. B., Czerkinski D., et al. (1997) Tumour-specific idiotype vaccines in the treatment of patients with B-cell lyphoma. Blood 89: 3129-3135. Riethmuller, G., Schneidergadicke, E., Schlimok, G., et al. (1994) Randomized trial of monoclonal-antibody for adjuvant therapy of resected dukes-C colorectal-carcinoma. Lancet 343: 1177-l 183. Burchell, J., Graham, R. & Taylor-Papadimitriou J. (1993) Active specific immunotherapy - PEM as a potential target molecule. Cancer Surv. 18: 135-148. Longernecker, B. M., Reddish, M., Miles, D. W. & MacLean, G. D. (1993) Synthetic tumour associated -antigens as immunotherapeutic cancer vaccines. Vaccine Res. 2: 151-i62. Miles, D. W.. Towlson. K. E.. Graham, R., et al. A randomized Dhase II studv of sialvl-Tn and DETOX-B adjuvant with or without cyclophosphamide pre-treatment for the active specific immunotherapy of breast cancer.
Questions Question:
Dr A. Howell,
Christie
and Answers
Hospital
NHS Trust, Manchester,
U.K.
What has been deduced from the clinical trial where one arm had intravenous cyclophosphamide and the other arm nothing or oral cyclophosphamide? Answer: The study was designed to assess how cyclophosphamide would influence the immune response and our conclusion was that intravenous cyclophosphamide appeared to augment the antibody response. Concerning survival it is noteworthy that the two intravenous groups on separate sides of the Atlantic were superimposable, as were the oral and the no cyclophosphamide groups. I would emphasize that the survival differences observed were by randomization, though the differences observed in survival by ability to generate antibody responses to STn are necessarily more speculative. It is possible that i.v. cyclophosphamide increased the immune response to an undefined antigen but I would argue against that as the KLH data did not provide the same split in survival estimates.
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Question:
D. MILES
Dr A. Howell,
Christie
Why is oral cyclophosphamide
Hospital
NHS
Trust, Manchester,
U.K.
not immunomodulatory?
Answer: I think that our experience from adjuvant chemotherapy with cyclophosphamide regimens suggests that prolonged oral CTX may be immunosupressive.The reasons for these different effects is unclear. Question:
Professor
You mentioned as KLH?
R. Rubens,
adjuvants:
Guy’s Hospital,
what is the necessity
London,
U.K.
for large carrier molecules
such
Answer: KLH is used because it is a large protein to which STn can be conjugated. It is a way of attaching 5 STns to 1 KLH molecule to allow presentation of multiple epitopes. Question:
Dr R. Coleman,
Weston
Park Hospital,
Sheffield,
U.K.
Is the standard phase II trial approach the way forward (you presumably would have thrown out the phase II approach if you had not seen the survival data). or is some sort of randomized design more appropriate? Answer: That is a question that goes for all biological approaches. A randomized phase II may give you an angle on biological responsiveness to demonstrate that you are indeed having an effect on the immune response. Obviously people are looking for surrogate markers (circulating tumour associated antigens or markers of immune response and hoping to identify those which will predict subsequent clinical response. Comment:
Dr A. Howell,
Christie
Hospital
NHS
Trust, Manchester,
If you are using surrogate markers then you need a controlled any sense out of the results.
population
U.K. to make
Reply: I agree. Biological approaches are going to be high risk strategy for companies because, with a cytotoxic agent, a product licence can be given on the response rate of a group of 50 patients. Taxol and Taxotere bear witness to that. This can not be achieved with many of these biological approaches.
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Comment:
Dr A, Howell,
CANCER
Christie
TUMOUR
Hospital
VACCINES
NHS Trust, Manchester,
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U.K.
We should not be so worried. Both John Robertson and I are great proponents of stabilization of disease and John Robertson is also a great proponent of tumour markers. Also, recently there has been work published stating that you do not want to kill tumour cells but maintain them and not let them grow. Thus, stabilization or no increase in tumour disease could be monitored by measuring changes in levels of tumour markers. If you can show differences between two groups then randomization is extremely important.