Immunotherapy and breast cancer

CANCER

TREATMENT

REVIEWS

1998: 24: 55-67

ANTlTUNiOUR TREATMENT

Immunotherapy T. A. Plunkett KRF Immunotherapy

(zi&

and breast cancer

and D. W. Miles Laboratory,

Guys Hospital, London, U.K.

INTRODUCTION Reviews of cancer immunotherapy often begin with mention of Coley’s toxins, and this review is no exception. At the turn of the twentieth century, Coley noted significant regression of tumour in some patients with advanced sarcoma following severe infections (1). Mixed bacterial ‘toxins’ were tested in patients with a variety of cancers, and several instances of regression were reported. The effect was probably mediated by cytokines released in response to bacterial products such as lipopolysaccharide (2). The work has not been repeated successfully, and with the advent of radiotherapy and the development of systemic therapies for the treatment of cancer, such as cytotoxic drugs, tumour immunotherapy has been regarded with scepticism. However, in the last decade, there have been major advances in the understanding of tumour immunology including the identification of tumourassociated antigens. As a result, the prospects for inducing specific immune responses to human cancers are improving. This review will discuss general considerations in cancer immunotherapy, and examine polymorphic epithelial mucin specifically as a target for the immunotherapy of breast cancer.

ADVANCES IN THE UNDERSTANDING OF IMMUNE-MEDIATED CYTOTOXICITY Non-specific

cytotoxicity

Natural killer (NK) cells were originally described on a functional basis according to their ability to lyse tumour cell lines without prior immunization Correspondence: ory, Guy’s Hospital, 0305-7372/98/O

T. A. Plunkett, ICRF Immunotherapy London SE1 9m, U.K.

I0055 + I 3 $12.00/O

Laborat-

(3,4). This function is present in athymic mice and so involves a novel population of cells different from T-lymphocytes. The cytotoxic activity of NK cells against tumour or virally infected cells is non-MHC restricted (5, 6). The molecular basis for NK cell recognition and function has been elucidated recently, and while receptors on the NK cell membrane recognize MHC class I molecules expressed on normal cells, it is the lack of expression of one or more MHC class I alleles which leads to NK-mediated target cell lysis (7). The range of targets and degree of lysis of NK cells can be increased by the culture of peripheral blood mononuclear cells with IL-2 to generate socalled lymphokine-activated killer (LAK) cells.

Specific cytotoxicity An effective immune response depends on the interaction of three cell types: antigen-presenting cells (APCs), T-cells and B-cells. Antigen-presenting cells include macrophages, activated B-cells and dendritic cells. Although all these cells are capable of presenting antigen to T-cells, dendritic cells are: (i) the most efficient APCs in the activation of resting T-cells; (ii) the major APCs for the activation of naive T-cells in vivo (8); and (iii) the only APCs known to induce antigen-specific CTLs in vivo (9). Dendritic cells (DC) arise from a CD34+ precursor common to granulocytes and macrophages. In undamaged tissue, DCs present antigen poorly, but mature in response to a variety of stressors, resulting in the increased surface expression of MHC and co-stimulatory molecules such as B7.1 and B7.2. These effects can be reproduced in vitro by co-incubation with cytokines such as TNFcl and IL-1s (10). The maturation of dendritic cells is completed by interaction with T-cells, and is characterized by further expression of co-stimulatory 0

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molecules and the synthesis of cytokines (11-13). Dendritic cells are motile, an important attribute for their role as an APC, allowing migration from peripheral tissues to lymphoid organs. T-lymphocytes can be divided into two distinct populations on the basis of the T-cell receptor (TCR). The TCR consists of two disulphide-linked polypeptides. These heterodimers consist of c1 and 8 chains (a8 TCR) or y and 6 chains (~6 TCR). The y6 T-cells comprise 5-10% of circulating T-lymphocytes, and also infiltrate epithelial surfaces. The function of y6 T-cells is not well understood, but they are conserved in all vertebrate immune systems. They may play a role in neonatal and mucosal immunity, and may also have a regulatory role. The a8 Tcells provide pathogen-specific host protection to immunocompetent animals. The a8 T-cells are classified on the basis of cell membrane expression of CD8 or CD4, and recognize antigen bound to MHC class I or class II, respectively (14). Analysis of the structure of MHC class I (15), and more recently MHC class II (16), has revealed features governing functional antigen presentation. The molecules share a similar architecture with peptide binding occurring within a groove formed between two cl-helices on the outermost extracellular domains (17). The key immunoregulatory role of CD4+ cells is to secrete cytokines to promote cellular proliferation, cytotoxicity and antibody-mediated responses. CD8+ T-cells are specialized for cytotoxic killing of other cells, particularly virally infected cells and, at least experimentally, tumour cells. The distinction is not absolute, as CD4+ cytotoxic cells have also been identified (18). T-cells do not recognize antigen as intact protein, but rather as processed peptide or glycopeptide bound to MHC. The mechanisms for antigen processing and presentation are complex, but data suggest that in general, antigens synthesized within the cell are presented by MHC class I molecules, and foreign protein, internalized by APCs, are presented by class II MHC (19, 20). However, examples of endogenous antigen presentation on class II (21) and exogenous antigen expression on class I have been reported (22). The role of each T-cell subset is reflected in the cellular distribution of MHC class I and class II. MHC class I is expressed almost ubiquitously, so that theoretically, CD8+ cytotoxic T-cells could recognize and lyse virally infected cells or tumour cells expressing mutated or oncogenic proteins. MHC class II expression, however, is confined to APCs. Activation of aB T-cells requires the binding of antigen in association with MHC to the TCR. Costimulatory signals delivered to the T-cell via accessory molecules, which are receptors for specific ligands on APCs, are also important. The T-cell

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activation threshold (number of TCRs that need to be triggered for T-cell activation) can be lowered by the presence of co-stimulatory signals (23). The costimulatory signals to the T-cell also dictate its response to antigen binding. In the presence of costimulation, antigen binding to the TCR results in a productive immune response. Antigen binding in the absence of appropriate co-stimulation results in anergy and antigenic tolerance (24, 25). Cytokines produced by CD4+ T-cells also stimulate the clonal expansion of antigen-specific B-cells. B-cells secrete antibody that binds to the target antigen and can mediate cell killing by complement fixation, opsonization or antibody-dependent cellmediated cytotoxicity. B-cells recognize native antigen, and IgM secretion may be T-cell-independent. However, long-lasting immunity, involving IgG, IgA or IgE, requires activated T-cells (26). The T-cell stimulates the B-cell not only by secreting cytokines which activate and aid its multiphcation and differentiation, but also by means of cognate co-stimulatory interactions (27).

ADVANCES IN TUMOUR IMMUNOTHERAPY Non-specific

immune

enhancement

Cytokines have been used to enhance MHC-restricted and -unrestricted tumour cytotoxicity. Interferons have shown potent immunomodulatory effects on the expression of MHC class I and class II, as well as tumour-associated antigens, resulting in increased NK and T-cell cytotoxicity (28). In a randomized controlled trial, adjuvant therapy with interferon-a (IFNa) significantly increased diseasefree and overall survival in patients with high-risk resected cutaneous malignant melanoma (29). It is the first adjuvant treatment shown to have such an effect in melanoma, and although it was associated with considerable toxicity, the side-effects were deemed to have been outweighed by the clinical benefits in a quality-of-life analysis (30). A confirmatory study is in progress, as are studies to assess the benefits of intermediate doses of IFNw In patients with renal cancer, prolonged stabilization of advanced disease and rare spontaneous regressions in the absence of systemic treatment suggested that host immune reponses are important in regulating tumour growth, and have led to the study of immunotherapy for this malignancy. The overall proportion of responses to IFNu in over 1000 patients with metastatic renal cancer was 12% (31), although in patients whose predominant site of metastatic disease was pulmonary, the response rate

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has been reported to be as high as 30% (32). Comparisons between treatment with interferon and drugs such as medroxyprogesterone acetate are about to be published. Interleukin-2 (IL-2) affects tumour growth by activating lymphoid cells in vim without affecting tumour growth directly. The intravenous infusion of IL-2 combined with autologous LAK cells resulted in objective responses in over 30% of cancer patients (33). Subsequent studies showed that LAK cells could be omitted from the treatment schedule without affecting the response rate (34). In a multicentre study, 255 patients with metastatic renal cancer were treated with high-dose IL-2 (35, 36): 14% of these patients had complete or partial responses lasting for a median of 23 months. The rate and duration of these responses led the Food and Drug Administration in the U.S.A. to approve the regimen as standard therapy for metastatic renal cell carcinoma. However, inpatient monitoring is required, often in an intensive-care unit, and there is a 4% incidence of treatment-related death. More recently, similar response rates were obtained with lower dose IL-2 given subcutaneously (34). The toxicity was considerably less, but the duration of the responses is not yet known. New combinations of IL-2, IFN~L and fluorouracil; IFN~L and 13-cis-retinoic acid have been tested and shown promising results (37).

Antigen-specific

immunotherapy

The objective of active-specific immunotherapy is to produce tumour-specific immunity by combining a non-specific immune adjuvant with a tumourassociated antigen. In the past, irradiated tumour cells have been used as immunogens in the hope that an immune response to putative tumour antigens might result. This approach has been most widely used in the treatment of malignant melanoma. Both autologous and allogeneic tumour cells have been utilized, with variable results (38). Some investigators have sought to enhance the immunogenicity of tumours by producing cells that express both tumour antigens and co-stimulatory molecules. 87.1 (CD80) and B7.2 (CD86) are important co-stimulatory molecules expressed on the surface of activated B-cells, monocytes and dendritic cells (39). Both bind to CD28 (CTLA-4) on T-cells. Co-stimulation of CD80 and CD86, in the presence of a suboptimal TCR signal, results in increased transcription and translation of multiple cytokines as well as T-cell clonal expansion and effector function. The biological importance of this pathway has been demonstrated in vivo. Non-immunogenic murine tumour cell lines have been engineered to express

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CD80 (40). CD80 expression resulted in their rejection in vim which was mediated by cytotoxic Tcells.. A variety of new adjuvants have also been advocated, including cytokine gene transfer into tumour cells to activate or by-pass T-cell helper function. In animal studies, such cellular vaccines have eradicated established tumours (41), and are now being employed in humans (42). The advances in the understanding of immunology have coincided with the identification of tumour-associated antigens (TAAs) (43, 44). These TAAs are potential targets for specific immunotherapy and include viral antigens, mutated proteins and oncogene products. Many of these TAAs have now been cloned. As a result, instead of using tumour cells as immunogens, peptides can be synthesized whose sequences correspond to known epitopes of TAAs recognized by T-cells. The elucidation of the structure of class I and class II MHC has also made it possible to identify putative MHCbinding regions from the DNA sequence of known TAAs. These peptides, with an appropriate adjuvant, could be used as a vaccine. ‘Cellular adjuvants’ have been advocated for use with peptide vaccines. Studies in animals and in man (45) have shown that autologous DCs pulsed with antigen in vitro and then re-infused, can induce immune responses that led to inhibition of tumour growth in vivu. A limitation to these studies had been the generation of sufficient DCs. It is now possible to generate DCs from peripheral blood by using GM-CSF and IL-4 (46,47). In animals, dendritic cells derived from peripheral blood have been shown to migrate from the site of injection to draining lymph nodes (48). These findings suggest that DCs differentiated in vitro from peripheral blood may be useful vehicles for immunotherapy. The use of peptides to pulse DCs may have limited clinical application because of MHC restriction. As a result, different peptides would be needed for different MHC hapiotypes. An alternative strategy is to harness the antigen-presenting machinery of the DC and allow the DC to identify epitopes on the TAA that bind to host MHC. This could be attempted by transfecting DCs with cDNA encoding the tumour antigen. In this way, the DC would cleave the TAA intracellularly, and peptide sequences that bind to MHC would be selected for presentation at the cell surface. A similar approach, particularly if a tumour antigen has not been cloned, is to fuse tumour cells with dendritic cells. Viruses transfected with cDNA encoding the tumour antigen have also been developed. Ideally, the viral vector should be safe, non-oncogenic and not immunosuppressive. It should be easy to engineer and be genetically stable. The virus acts as

58 an adjuvant by altering the intra- and extracellular trafficking of antigen, and provides an additional substrate for specific and non-specific immune recognition. In animals, vaccination with tumour cells expressing a model antigen resulted in a negligible immune response. Vaccination with recombinant viral vectors expressing the same antigen resulted in specific cell-mediated immunity and caused tumour regression (49). It is also possible to engineer recombinant viral immunogens that express immunomodulatory molecules (such as CD80) together with a TAA, and this can enhance their immunogenicity (50, 51). Immunization and repetitive boosting with the same recombinant virus can induce a strong immune response to the viral vector itself (52). These responses limit the immunogenicity of the TAA, perhaps by rapidly eliminating the recombinant virus (53). Alternatively, the response to immunodominant epitopes on the vector may suppress those to the weaker determinants of the TAAs (54). This problem can be overcome by using different viral vectors that express the same TAA. A potentially safer and easier method of vaccination is to use naked cDNA without a viral carrier system as an immunogen. The intramuscular injection of naked cDNA resulted in foreign protein expression in mice (55) and non-human primates (56). In mice, immunization with cDNA encoding the influenza A nucleoprotein led to the generation of specific cellular and humoral responses, and protected the mice from subsequent challenge with the virus (57). Immunization with cDNA encoding the car&-to-embryonic antigen (CEA) showed antitumour activity (58). Monoclonal antibodies were developed in the 1970s and those developed against TAAs are being tested in a variety of forms in different clinical settings. Initial studies were hampered by the development of human antibody against the constant regions of xenogeneic monoclonal antibodies. The availability of human and humanized antibodies has revolutionized their use. These antibodies are produced by molecular engineering, including grafting antibody genes (59) and transgenic mice that produce human IgG in response to immunization (60). Antibodies can activate immune effector functions as outlined above. Concomitant treatment with cytokines can enhance the effector function. Bispecific antibodies have been generated that bind a TAA at one antigen-binding site and T-cells at the other antigen-binding site. These antibodies can activate T-cells adjacent to tumours and enhance cytotoxicity (61). Monoclonal antibodies directed against TAAs when conjugated with a therapeutic agent, such as

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a radio-isotope, can deliver therapy selectively to cancer cells. A novel means of drug delivery is by antibody-directed enzyme prodrug therapy (ADEPT), in which an antitumour antibody conjugated to an enzyme is administered intravenously (62). The antibody binds to tumour cells, and as a result, the enzyme concentration is increased around the tumour compared to normal tissues. A prodrug is then given, and is converted to an active cytotoxic agent around the tumour by the enzyme. A phase I trial using such a system is planned in patients with advanced colorectal cancer (62). The high molecular weight of antibodies (approx. mol. wt 150 000 kDa) can impair their passage into cell aggregates. Single-chain Fv antibodies comprise the variable heavy and variable light regions of an IgG molecule linked by a short peptide (approx. mol. wt 27 kDa). Studies in patients with advanced colorectal cancer demonstrated that single-chain Fv antibody specific for CEA can be more sensitive for tumour imaging than computed tomography (63). These antibodies may have great potential for the delivery of antitumour therapy. Antibodies can also be used as active specific immunotherapy by utilizing the anti-idiotype antibody network. A monoclonal antibody specific for a known TAA (Abl) is administered intravenously. The host generates an immune response to Abl, including anti-idiotype antibody against the variable region (Ab2). The anti-idiotype antibody (Ab2), if properly selected, would have a three-dimensional conformation similar to the TAA and could be used in its place. An increasing understanding of the immune system and the identification and cloning of TAAs has opened new avenues for cancer immunotherapy. The following part of this review will detail how these new approaches have been applied to the immunotherapy of breast cancer.

BREAST CANCER AND IMMUNOTHERAPY USING POLYMORPHIC EPITHELIAL MUCIN Breast cancer and the immune

system

Breast cancer has a long natural history and the influence of the immune system on pre-clinical disease is unknown. Nevertheless, there is evidence of an immune response in some breast cancers. A mononuclear cell infiltrate consisting predominantly of T-cells is found within many established solid tumours (64), and has been taken as evidence of a host immune response. In breast cancer, tumourinfiltrating lymphocytes (TILs) have been reported

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to be associated with a better prognosis (65). It is not known whether these TILs are directed at specific antigens or whether it is a non-specific infiltrate in response to cellular damage. Although these TILs express activation markers, they ultimately fail to impede tumour growth. Freshly isolated TIL demonstrate limited cytotoxicity against autologous and allogeneic tumour cells. The cytotoxicity of TIL can be enhanced in vitro by stimulation with IL-2 (66), suggesting that the differential expression of cytokines by tumour cells or TILs may impair the antitumour immune response in vivo. In recent studies of breast cancer using mRNA detection by reverse-transcriptase polymerase chain reaction (67) and by in situ hybridization (68,69), TILs from breast cancers did not secrete IL-2 or IL-4, suggesting a downregulation of cellular immunity. IL-10 has been detected in primary breast cancers. This cytokine is associated with the induction of T-cell anergy (70), as a result of inhibition of T-cell proliferation and function, reduced IL-2 production and antigen presentation (71). Therefore, there is evidence that cells of the immune system infiltrate breast cancers, and the aim of immunotherapy is to enhance this response to effect cytotoxicity of malignant cells. Polymorphic epithelial mucin (now termed MUCl) has been utilized as a target for the specific immunotherapy of breast cancer.

Molecular

structure

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and distribution

of MUCI

MUCl is a high molecular mass mucin-like glycoprotein and was the first mucin gene to be cloned (72). Unlike other mucins, it is an integral membrane protein. The transmembrane and cytoplasmic tail show a high degree of homology between species, which suggests that it may have a basic function. The extracellular domain consists of tandemly repeated sequences of up to 20 amino acids rich in serine, threonine and proline. The number of tandem repeats varies as a result of genetic polymorphism and, as a consequence, the length of the extracellular domain can vary between about 600 and 2200 amino acids (73). Each tandem repeat has five potential 0-glycosylation sites, and two or possibly three of these are utilized. As an Olinked glycosylated peptide of 28 amino acids is thought to be about 7nm long, the extracellular domain of MUCl could extend 150-500nm above the cell membrane.

Possible roles of MUC I. in tumourigenesis The over-expression of MUCl on malignant cells suggests that it may confer an advantage to the

tumour. MUCl expression reduces intercellular adhesion and participates in epithelial sheet differentiation and lumen formation during organogenesis (74). Its overexpression decreases E-cadherin-mediated cell-cell and integrin-mediated cell-matrix interactions, especially when the normal apical distribution is lost (75). As a consequence, the overexpression of MUCl may increase cellular metastatic potential. The physical size of MUCl may prevent immune effector cells from binding to the tumour cell membrane. A murine mammary carcinoma cell line was rendered allotransplantable by transfection with MUCl; it was suggested that MUCl had effectively masked the MHC and other cell surface molecules that would have been recognized as foreign by the recipient (76).

MUCI

as an immunogen

As a result of its physical size, the MUCl molecule may be among the first cellular structures encountered by the immune system. There are a number of features that may enhance the immunogenicity of MUCl on the surface of malignant cells. In normal epithelial cells, MUCl is expressed only on the apical surface and, consequently, is not exposed to the immune system. In carcinomas that arise from such cells, its expression is upregulated and it is found on the entire plasma membrane (77).

Glycosylation

of MUCI

in malignant

cells

There is evidence of aberrant MUCl glycosylation in malignant cells that may expose antigens on the peptide backbone and also create novel carbohydrate antigens. The first sugar added to serine or threonine in mu&-type 0-glycosylation is N-acetylgalactosamine (GalNAc). In normal mammary epithelial cells, galactose (Gal) is added to form the core 1 structure. pl-6-N-acetylglucosamine transferase @1-6GlcNAc-T) then catalyses the addition of Nacetylglucosamine (GlcNAc). The structure is then further extended to form polyactosamine side chains. In breast cancers, truncated side chains are found. The enzyme c&3-sialyl transferase (c&3SA-T) competes with l31-6GlcNAc-T for the core 1 structure as substrate. u2-3SA-T adds sialic acid instead of GlcNAc and thereby inhibits chain extension. The activity of P1-6GlcNAc-T has been shown to be reduced in some breast cancer cell lines compared with cell lines derived from normal epithelial cells; conversely, the activity of the cQ-3-SA-T enzyme is increased as much as lo-fold (78). Such differences

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may explain the truncated carbohydrate found in tumour-associated MUCl.

Immune

responses

side chains

to MUC I in malignancy

Cellular and humoral immune responses to tumourassociated MUCl have been demonstrated in cancer patients. T-lymphocytes isolated from patients with breast or ovarian cancer specifically killed tumour cells expressing MUCl(79,80). The cellular response was inhibited by antibodies to the al3 TCR and by antibodies specific for tumour-associated MUCl, but not by antibodies to the MHC. These results demonstrated that the cellular response was specific for tumour-associated MUCl and TCR-dependent but was MHC-unrestricted (81). MHC-unrestricted cytotoxicity against MUCl has also been demonstrated in a patient with multiple myeloma (82). The most plausible explanation for this is that TCR binding to the tandem repeat sequences of MUCl resulted in TCR cross-linking, obviating the need for for peptide-MHC presentation. In other studies, hapten-specific, MHC-restricted murine and human T-cell clones lost the requirement for MHC presentation when the hapten was made multivalent by polymerization (83). These investigators speculated that although the affinity of individual epitopes for the specific TCR was low, a polyvalent antigen may bind multiple TCRs, increasing the overall avidity of the interaction and triggering Tcell function. The structural analysis of an unglycosylated synthetic peptide corresponding to three tandem repeats, demonstrated an ordered rod-shaped structure in solution (84). A short sequence (APDTRP) protrudes from each repeat and is the sequence recognized by several MUCl-specific antibodies (85). The APDTRP sequence is also the epitope that binds to the TCR of MUCl-specific Tcells (81). Tumour-associated MUCl could therefore behave as a multi-valent antigen and activate Tcells as described above. The concept of non-MHC restricted cytotoxicity in a malignancy that frequently fails to express MHC is appealing, but the issue of MI-K dependency remains controversial. Serum IgM antibodies specific for MUCl have been found in approximately lO-20% of patients with breast, pancreatic or colonic cancer (86). The antibody response was directed against the same epitope on the mucin polypeptide core tandem repeat as the cellular response. Circulating immune complexes (CIC) of IgM and IgC with MUCl have been detected in sera from patients with benign breast disease or breast cancer, but rarely in normal individuals (87). A more recent study in breast cancer patients has demonstrated a negative correlation

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between positivity for MUCl-CIC and the extent of disease (88). The data have been interpreted as showing that a humoral response against MUCl slows disease progression, but an alternative explanation may be that patients with more advanced disease are less able to mount an immune response because of the immunosuppressive effects of cancer. The high incidence of MUCl expression in primary breast cancer and the possible MHC-unrestricted response suggest that most patients should be expected to generate an immune response to the tumour-associated antigen. However, MUCl-specific immunity is found in a minority of patients. Soluble antigen is thought to induce tolerance (89), possibly by binding to the TCR without appropriate co-stimulation. In mouse models, low doses of intravenous epiglycanin have been used to mimic tumour shedding of mu&-t, and resulted in specific immunosuppression (90). In man, increased serum levels of circulating mucin correlated with a poorer prognosis (87). As well as simply reflecting disease burden, it is possible that the poor outcome was partly a result of the secreted mucin leading to immunological anergy.

MUCI

as a target for immunotherapy

Tumour-associated MUCl has both tumourigenic and immunogenic potential. It may be an effective immunogen for several reasons: l l l

l

l

l

Expression is upregulated in tumour cells; Normal apical distribution is lost in cancer cells; Aberrant glycosylation exposes peptide epitopes and novel carbohydrate antigens on cancer cells; The extracellular domain consists of tandem repeats so that epitopes are repeated up to 100 times; The immune response to tumour-associated MUCl may be MHC-unrestricted; and there is evidence from patients with cancer that an immune response to tumour-associated MUCl may be associated with a better prognosis.

Mouse

models

of MUCI

immunity

Mouse models have been developed to investigate the immune response to MUCl. For example, the human MUCl gene was transfected into a mouse mammary tumour cell line 410.4, which has the same MHC haplotype as Balb/C mice (91). The injection of a low dose of MUCl-expressing transfectants resulted in a lower tumour incidence or delayed tumour growth compared to the injection of 410.4 cells that did not express MUCl. Pre-immunization with a low dose of MUCl-expressing

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tumour cells or with peptides derived from the tandem repeat sequence also inhibited tumour growth. In a different murine system, cytotoxic Tcells specific for MUCl were derived from mice that rejected low doses of MUCl-expressing tumour cells (92). These findings suggested that an immune response to MUCl-expressing tumour cells may result in inhibition of tumour growth. However, when higher doses of MUCl-expressing tumour cells were used, no inhibitory effects were seen. Therefore, even in the murine model, where MUCl is a foreign antigen, the immune response to MUCl-expressing tumour cells does not always lead to tumour rejection.

Cellular adjuvants B-cells have been used as cellular adjuvants to enhance the immune response to MUCl. EBV-immortalized B-cells are effective APCs and express a variety of co-stimulator-y molecules. EBV-immortalized B-cells from patients with cancer have been tranfected to express MUCl. If mucin glycosylation in these cells is inhibited, then they become effective targets for autologous cytotoxic Tcells in vitro (93), emphasizing the importance of aberrant glycosylation in MUCl-expressing cancers. These findings led to experimental studies using chimpanzees (94). The chimpanzees were immunized subcutaneously with MUCl-transfected immortalized autologous B-cells. Prior to the immunization, no MUCl-specific CTLs were detectable. Following a single injection, MUCl-specific CTL activity was demonstrable in peripheral blood from both animals. The CTLs were expanded in vitro and lysed cells from a MUCl-expressing human breast cancer cell line and two MUCl-expressing pancreatic cancer cell lines. No antibody response was detectable in these animals. There was no evidence of an autoimmune response; chimpanzee and human MUCl genes are thought to be similar as there is complete homology of the tandem repeat sequences. There are no data currently available on tumour rejection or regression, but the approach has clear potential. Purified human dendritic cells have been produced that express high levels of MUCl following transduction with a retroviral vector containing MUCl cDNA (95). These cells are being used to prime naive autologous human T-cells, and similar studies are being planned in chimpanzees (96). In an effort to enhance the immunogenicity of tumour cells, murine adenocarcinoma MC38 cells

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expressing MUCl were fused with bone-marrowderived dendritic cells (97). The fusion cells expressed MUCl as well as MHC class I, MHC class II, CDSO, CD86 and the intercellular adhesion molecule ICAM-1. They were not tumourigenic but did stimulate naive T-cells in the primary mixed lymphocyte reaction. Therefore, the fusion cells expressed MUCl and co-stimulatory molecules, and were capable of antigen presentation. Pre-immunization of mice with fusion cells prevented subsequent tumour growth, whereas pre-immunization with irradiated tumour cells had no effect. Cytotoxic T-cells specific for tumour cells were isolated from mice pre-immunized with fusion cells. The protective effect was dependent on both CD4+ and CDB+ lymphocytes, as antibodies to either of these increased tumour incidence. A treatment model was also developed. Mice with established metastases were injected with fusion cells. In contrast with control mice that developed more than 250 pulmonary metastases, 90% of experimental mice had no detectable metastases. While it is not clear from these studies whether the T-cell response was directed at processed antigen or native antigen on the cell surface, these results suggest that autologous tumour/dendritic cell hybrids may have potential in immunotherapy of cancer.

Peptide vaccines Some of the humoral and cytotoxic immune responses in cancer patients are known to be directed at specific sequences in the tandem repeat. As a result, peptide sequences derived from MUCl have been used in animal and human studies of immunotherapy. Structural studies demonstrated that peptides consisting of one to three tandem repeats have a configuration identical to MUCl (84). This suggested that in synthetic tandem repeat sequences, the peptide epitopes would be the same as those found in the native protein. Mice immunized with a 20 amino acid synthetic peptide (derived from the MUCl tandem repeat region) coupled to KLH in Ribi adjuvant (98) produced antibodies specific for MUCl, and also developed a DTH reaction in response to re-challenge with antigen. Following immunization, the growth of implanted MUClexpressing tumours was impaired and the survival of tumour-bearing mice was prolonged (98). In a different study, mice were immunized with a 24 amino acid sequence spanning the tandem repeat region of MUCl. T-cell proliferation to the antigen was demonstrated in vitro (77). Others have found that immunization with synthetic antigens resulted in a predominant humoral response with little antitumour activity (99). The

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same investigators subsequently linked a peptide of five MUCl tandem repeats to oxidized mannan, and found that it is a potent immunogen in mice. Immunization with the fusion protein prevented the subsequent growth of MUCl-expressing tumour cells and eradicated disease in tumour-bearing mice (100). The tumour rejection was associated with an increase in the frequency of cytotoxic T-lymphocyte precursors specific for MUCl. Mannan (a polysaccharide also known as polymannose) may target the antigen to mannose receptors on APCs, thereby increasing its immunogenicity. The mannan oxidization state modulated the imoxidized mannan resulting in a mune response; predominantly cytotoxic response and production of IFN-)I (a Thl response), whereas reduced mannan in the fusion protein resulted in a strong antibody response and secretion of IL-4 (Th2 response). The reasons for this apparent difference are unclear. These studies have been extended to clinical trials in cancer patients. In a phase I trial, 13 patients were injected with synthetic peptide from the human MUCl tandem repeat conjugated to diphtheria toxoid (101). Six patients generated antibody responses to both the peptide and MUCl, two developed a DTH response on re-challenge, and in three patients, proliferative T-cell responses to MUCl were detected. Stable disease was reported in six of 12 evaluable patients during the study period. No long-term follow-up data are available. The same group conducted a trial in patients with advanced breast or colorectal cancer using peptides from the tandem repeat conjugated to mannan and results from these studies are awaited. Others have used a 105 amino acid peptide (corresponding to five tandem repeats) admixed with BCG as an immunogen in cancer patients (102). Most patients developed a DTH reaction to re-challenge with the peptide; biopsy from these sites showed variable degrees of T-cell infiltration. In one-third of patients tested, the frequency of cytotoxic T-cell precursors specific for MUCl rose more than twofold. Prior to immunization, most T-cells in these patients lacked the CD3 c chain, a key component for T-cell activation. A similar defect has been reported in tumour-infiltrating T-cells from patients with colorectal cancer (103). Following administration of the vaccine, this defect was reversed in 20% of patients, and suggests that vaccination could be associated with restitution of normal T-cell function. In mouse studies, the cytotoxic T-cell response response to both peptides and MUCl-expressing cells was MI-K-restricted (104). In non-immunized cancer patients, the MUCl-specific cytotoxic T-cells are MHC-unrestricted; it is not yet known whether

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the immune responses generated in humans by immunization are MHC-restricted. In the mouse model, the immune response is to a foreign antigen (there is no homology between human and murine PEM), so the process in man may well be different. A human MUCl transgenic mouse (in which the human MUCl is regarded as a self-antigen) has been developed which will allow this to be explored further (77). These preliminary studies suggest that peptide vaccination in man can result in specific cellular and humoral immune responses. Importantly, immunization was not associated with significant toxicity or evidence of autoimmunity.

h-al

vectors

MUCl cDNA cloned in to the vaccinia virus (W) genome (W-MUCl) has been tested as an immunogen in animal models and in man. In a rat model, W-MUCl was used for immunization prior to tumour challenge with a fibroblast cell line transfected with MUCl (105). Immunization inhibited tumour growth in 82% of animals. Although there were high titres of MUCl-specific antibody, no cytotoxic T-cell responses were detected. Murine and human cells infected with W-MUCl express the MUCl molecule, with three to four tandem repeats per molecule and with exposure of tumour-associated epitopes (106). In DBA mice, immunization with W-MUCl resulted in 30% rejection of tumourigenic P815 cells transfected with MUCl. In Balb/c mice, immunization with WMUCl delayed tumour growth of MUCl-transfected 3T3 cells rather than eliciting complete rejection. No MUCl-specific CTLs were detected in either of these model systems. Anti-MUCl antibodies were observed, but the titres did not correlate with antitumour response. The immune response to viral vaccines can be increased by the co-expression of tumour-associated antigen and an adjuvant cytokine. A recombinant W carrying cDNA for MUCl and for IL-2 (WMUCl/IL2) has been constructed (107) and has been used in a phase I study of patients with advanced breast cancer. Immunization was not associated with significant toxicity, and immune responses were detected in some patients (108). A phase II multicentre trial using the W-MUCl/IL-2 construct in patients with metastatic breast cancer has been planned for 1998.

cDNA

vaccines

A syngeneic mouse model using C57 mice and RMA tumour cells expressing MUCl has been used to

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examine the use of immunization with MUCl cDNA as a cancer vaccine (92). Immunization resulted in significant tumour protection, with 82% of mice tumour-free compared to 41% of controls. Tumour protection was dose-dependent. Anti-MUCl antibodies were detected after immunization, indicating that the injected cDNA was expressed. The humoral response did not correlate with tumour rejection. MUCl-specific CTLs were detected, but only after tumour challenge. CTL lines from immunized mice that did not develop tumours survived longest in vitro, specifically killing MUCl-expressing targets. Therefore, in this mouse model, immunization with naked MUCl cDNA resulted in a hurnoral response and may have augmented the cellular response to MUCl. Following cDNA vaccination, the mice were capable of resisting tumour growth. This is the subject of further research.

Carbohydrate

antigen

MUCl expressed by malignant cells at the plasma membrane is aberrantly glycosylated, and novel carbohydrate antigens, such as sialyl-Tn (STn), are present. Expression of STn is associated with a worse prognosis in colonic (109) and gastric cancer (110). Circulating antigen has been detected in gastrointestinal and ovarian malignancies, and raised levels have been associated with a worse prognosis (111). In breast cancer, STn expression has been demonstrated in 16-80% of breast cancers and may be associated with a poor prognosis (112). The variation probably reflects the use of different antibodies, tissue fixation and whether the tissue is from a primary or metastatic site. In a recent study, STn expression was of borderline prognostic significance for the entire group (p = 0.07), but was highly significant for patients with node-positive disease (p
63 thought to be exposed by aberrant glycosylation, were tested as immunogens in this system (115,116). The survival of mice with TA3-Ha tumours was prolonged by treatment with synthetic carbohydrate antigen conjugated to keyhole limpet haemocyanin (KLH) and emulsified in DETOX adjuvant. Approximately 30% of the animals were able to resist and sustain long-term survival when re-challenged with turnour cells (115). Immunization of mice with desialylated ovine submaxillary mucin, which expresses large amounts of Tn, carbohydrate antigen protected mice against TA3-Ha tumour challenge and produced a high antibody titre to Tn (116). A prospective, randomized clinical trial using STn as a target for active specific immunotherapy in patients with breast cancer has been reported recently (117). The patients had histologically proven breast cancer, with locoregional relapse after appropriate primary therapy, or metastatic disease. They were Eastern Co-operative Oncology Group Performance Status O-2, and their disease did not require conventional therapy and they did not require management of symptoms caused by the cancer. All patients were immunized subcutaneously with STn conjugated to KLH, with DETOX adjuvant on weeks 0, 2, 5 and 9. STn has been detected in the circulation, and soluble antigens have been demonstrated to induce tolerance or anergy, rather than an effective immune response. In mice, this apparent ‘suppressor’ activity can be overcome by pre-treatment with cyclophosphamide to allow active specific immunotherapy (90, 115). To determine its effectiveness in man, patients were randomized to receive before the first immunization either cyclophosphamide 300 mg intravenously on day -3 or cyclophosphamide 50 mg orally days - 14 to - 3, or no pre-treatment with cyclophosphamide. The treatment had minimal toxicity. All patients generated an antibody response to STn, STn-positive mucin and KLH. The highest antibody titres were in patients pre-treated with intravenous cyclophosphamide. The median survival for the group pre-treated with i.v. cyclophosphamide was significantly longer than that for the other groups (19.7 ZIS 12.6 months, p =0.0176). The patients receiving i.v. cyclophosphamide were less likely to have progressive disease, and there was a negative correlation between the growth of measurable tumours and antibody titre to STn. There was no correlation between progression and antibody titres to KLH. As there were no differences between the groups in terms of the natural history of their disease or the number and type of previous treatments, the results suggest a therapeutic effect for pre-treatment with i.v. cyclophosphamide followed by immunization

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64

with STn-KLH. A similar association between antibody titres and survival has been demonstrated in patients with colorectal cancer immunized with STnKLH (118). A large multicentre trial comparing i.v. cyclophosphamide and STn-KLH/DETOX-B with i.v. cyclophosphamide and KLH/DETOX-B in the treatment of patients with breast cancer is set to begin in 1998.

Monoclonal

antibodies

to MUC I

There are no published data on the use of antibodies to MUCl in the treatment of breast cancer. However, trials have been reported on their use in ovarian cancer. Radiolabelled murine antibody to tumourassociated MUCl (HMFG-1) was administered via peritoneal catheter to 52 women with ovarian cancer (119). All the women had undergone cytoreductive surgery and subsequently received platinum chemotherapy. Patients with no residual disease at laparoscopy prior to administration of the antibody were regarded as receiving the treatment in an adjuvant setting (n = 21). The actuarial survival of these patients was significantly better when compared with a historical control group, though such data must necessarily be viewed with caution, and confirmatory prospective randomized studies are required. It is not clear whether the possible therapeutic effect was due to the radiolabel or whether the antibody itself may be immunogenic. Subsequent studies have demonstrated that intracavitary monoclonal antibody can activate cellular immunity (120). Part of the cellular response was directed at epitopes in the constant region of the murine antibody, but repeated administration of the antibody resulted in an anti-idiotypic response. Since the antibody was bound to tumour cells, the authors suggest that this may result in an enhanced immune response to the tumour with both specific and possibly bystander effects.

CONCLUSION Recent scientific advances in the understanding of immunology have led to a renewed interest in immunotherapy. The use of systemically administered cytokines to enhance the immune response nonspecifically may be regarded as standard adjuvant treatment for high-risk malignant melanoma, and is employed by some for the treatment of metastatic renal cell cancer. The MHC class I and class II structures have been elucidated, and the processes of antigen processing are being unravelled. The mechanisms for T-cell recognition and activation are

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also becoming clearer. These developments have been paralleled by the identification of tumourassociated antigens and, together, have the potential to produce specific cancer immunotherapies. Early studies suggest that such approaches may be of clinical value not only in breast cancer but also in other tumours; for example, in a randomized trial, 189 colorectal cancer patients who had undergone curative resection for Dukes C cancer received either monoclonal antibody to the tumour-associated antigen 17-lA, or were assigned to an observation arm (121). At a median follow-up of 5 years, the mortality rate and recurrence were reduced by 30 and 27%, respectively, in the treatment group compared to controls. This effect was comparable to that of adjuvant leucovorin-primed 5-fluorouracil, but was associated with minimal toxicity. The two treatments alone or in combination are now being tested in a randomized trial. Encouraging results have also been demonstrated with antibody-based therapy in leukaemias and lymphomas (122). In patients with metastatic breast cancer who had received extensive prior anti-cancer therapy, treatment with anti-pl85produced response rates comparable to third- or fourth-line chemotherapy, but with minimal toxicity (123). Cancer vaccines have also been developed. It is unlikely immunization with peptides, even with carrier molecules and/or adjuvants, will generate a sufficiently potent and diverse immune response to induce tumour regression. Indeed, in some mouse models, vaccination with peptide in the absence of appropriate T-cell co-stimulation increased tumour growth rate (124). By contrast, the parent proteins incorporate multiple antigenic peptides with differing MHC requirements. These tumour-associated antigens can now be presented in a variety of ways to the immune system in an effort to enhance their immunogenicity and promote tumourolysis. In malignant melanoma, vaccination with ganglioside GM2 tended to increase survival (125) and other studies of active specific immunization are encouraging. In breast cancer, a variety of MUCl-based immunogens are entering clinical trials. It is likely that cancer immunotherapy will be most useful as an adjuvant treatment in the setting of minimal residual disease (126). Immunotherapy is unlikely to produce effects equivalent to the current criteria for complete or partial response to treatment in patients with metastatic cancer (127), although long-term stabilization of growth may be achievable. This is partly due to the non-specific immunosuppression associated with disseminated malignancy, prior chemotherapy or radiotherapy, and downregulation or loss of expression of MHC class I by tumour cells (128), as well as impaired antigen

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processing (129). Surrogates for the efficacy of immunotherapy need to be defined, probably utilizing immunological assays in order to prevent overlooking potentially useful treatments.

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