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Annals of Oncology 7: 227-232, 1996. O 1996 Kluwer Academic Publishers. Printed in the Netherlands. Review 1975—1995 Revised anti-cancer serological response: Biological significance and clinical implications S. Canevari, S. M. Pupa & S. Menard Division of Experimental Oncology E, lstituto Nazionale Tumori, Milan, Italy Summary In the 1970s a considerable amount of work was carried out in an attempt to identify an anti-tumor serological response in cancer patients. These analyses have not been very informative due to the complexity and heterogeneity of the response. More recently, the availability of recombinant molecules, synthetic peptides and analytic and semi-quantitative assays has enabled a better dissection of humoral immunity. Antibodies against intracellular antigens (c-myb, c-myc, p53 and p21 ras) have been found in a significant, albeit varying, proportion of patients bearing various tumors. Association with a poor prognosis is documented for anti-p53 antibodies in breast carcinoma patients. A number of cell surface antigens, including mucins, oncoproteins and carbohydrate antigens have been found to elicit a humoral immune response Background In animal models it has been convincingly demonstrated that the immune system can recognize tumor cells [1-3]. In contrast to animal models, the existence of tumor-specific antigens that are recognized by the autologous tumor-bearing host was not unequivocally demonstrated in human neoplasms until recently [4]. Whereas only a very limited number of tumor rejection antigens involved in animal model systems have been identified through a serological immune response, most of tumor-associated antigens (TAA) present on human cancer cells have been detected by using antibodies. These TAA, expressed by cancer cells either to greater extent that in normal cells or as new molecules completely absent in normal cell counterparts, appear as a result of aberrant gene or oncogene expression or as a consequence of errors in glycosylation [5,6]. As for other cellular antigens, the immune system can turn on against TAA either one of its arms: the one that relies on T lymphocytes, which directly or indirectly kill tumor cells also by recruitment of macrophages, polymorphonuclear cells and natural killer (NK) cells, or the one that depends on antibodies. Table 1 lists the possible mechanisms of action of antibodies. The majority of these mechanisms were demonstrated to be active in experimental models and some of them have been proven effective also in the clinic [7-9]. and, in some instances, circulating immune complexes were observed. A protective role for or, on the other hand, masking effects of such antibodies is still controversial. An indication that a serologkal response can be beneficial comes from vaccination studies. A significant association between the development of an anti-tumor antigen antibody response and prolonged survival was observed following vaccination of melanoma patients with GM2 or anti-idiotypic antibodies which molecularly mimic tumor-associated antigens. It is to be hoped that in the near future the numerous ongoing immunization trials and prognostic studies demonstrate whether antibody response can exert a protective role in vivo. Key words: prognosis, serological response, TAA, therapy, vaccination The purpose of this presentation is to review the data obtained in recent years regarding serological response of cancer patients, with special emphasis on its biological relevance and the possibility of exploiting it for therapeutic purposes. Antibody response in tumor-bearing patients In the 1970s, at the beginning of the development of tumor immunology, a considerable amount of work was carried out in an attempt to identify an anti-tumor humoral immune response, and the sera of a thousand Table 1. Mechanisms of anti-tumor antibodies. Activity Antibody region involved* Direct: • inhibition of proliferation • inhibition of metastatic spread Only variable Only variable Indirect • activation of idiotypic network • complement-mediated lysis • antibody-dependent cellular cytotoxicity • targeting of toxic agents Only variable Both constant and variable13 Both constant and variable11 Both constant and variable11 • For antibody structure [37]. b The variable region is involved in the antigen recognition. 228 cancer patients were analyzed for the presence of antibodies reacting with their own or with allogenic tumors [10]. However, since the patient's humoral immune response, as identified by the methodologies utilized at that time, was likely to be polyclonal, it soon became evident from the complexity of the immune response that molecular techniques were needed to identify 'altered' molecules expressed by tumor cells. Although antibodies were quite common, an extensive survey of sera from normal as well as non-cancer and cancer patients indicated that only a small proportion of them were restricted to TAA, whereas the majority of serological responses were against artifactual antigens (Fetal Calf Serum, HLA antigens) or undefined molecules. As reviewed by L. J. Old [11], two major problems limited the development of tumor immunology: the need to identify cancer-associated antigens and the need for means by which to monitor immune responses. More recently, new approaches, such as molecular biology and hybridoma technology, made possible to identify a series of molecules, all of them having the characteristic of behaving as 'tumor antigens'. At that point, become easier to evaluate the possible presence in cancer patients' sera of antibodies specifically directed against these altered molecules. The availability of recombinant molecules, synthetic peptides and analytic and semi-quantitative immunochemical assays guarantees the specificity of observed reactions. More convincing evidence of anti-tumor serological response in cancer patients is listed in Table 2. Note that the majority of them were obtained after 1990, when new biotechnological tools, previously mentioned, became widely available. Antibodies against intracellular antigens such as c-myb, c-myc, p53 and p21 ras proteins have been found in a significant, although varying, proportion of patients with different tumors. The greater incidence of antibody in cancer patients compared to normal sera suggests that immunization to intracellular proteins occurred as a result of the malignancy. However the issue of controls representative of tissue destruction and inflammation was probably not enough critically dealt with and further studies are needed to exclude the role of the surrounding tissues in the release of intracellular components. Forthy-three percent of breast carcinoma patients were found positive for the presence of anti-c-myb antibodies [12]. p53 was also found to be immunogenic even though the patients' antibodies recognized both the wild type and the mutated proteins. The release of intracellular molecules by necrotic tumor cells seems the most likely explanation of how such proteins become immunogenic. A possible role for these antibodies in the control of tumor progression is highly unlikely, since they do not react against live tumor cells. Detection of anti-p53 antibodies at the time of diagnosis in breast and colorectal carcinoma patients appears to be associated with a poor prognosis [13,14], and it has been suggested that this plasma assay be used for prognostic purposes [13]. The finding that anti-p53 antibodies are mainly elicited in patients with an altered p53 in their tumors indicates that detection of such humoral immune response might be a simpler assay than p53 primary tumor characterization by molecular sequencing. In lung carcinoma development, alteration of p53 was found to be a preneoplastic event which occurs already in the normal bronchus epithelium. Detection of anti-p53 antibodies in a population at high risk for lung cancer might be a useful indicator for early diagnosis [15]. Moreover, an antibody response to wild type and mutated p21 ras proteins even occurs in 32% of the examined colon cancer patients. Whether humoral immune response to ras p21 protein in cancer patients correlates with clinical or pathological variables is still under investigation [16]. Antibodies against cell-surface proteins might have a completely different impact on the control of tumor growth. Therefore, much more attention has been paid to the identification of a humoral immune response to cell-surface TAA. As shown in Table 2, a number of cell-surface antigens, including mucins, oncoproteins and carbohydrate antigens have been found to elicit a humoral immune response in cancer-bearing patients. Mucins, like PEM/MUC1, were found to induce antibodies, and in early-stage breast carcinomas these antibodies were observed as immune complexes with the circulating mucins [17]. The finding that patients with mucin/antibody complexes have a better prognosis than patients with circulating free mucins suggests a protective role for the anti-mucin antibodies against tumor progression. In breast cancer patients, a humoral Table 2. Antibody response in cancer patients. Antigen Tlimor Intracellular localization c-myb Breast, colorectal, ovarian c-myc Colorectal, breast, ovarian, osteosarcoma p53 Breast, lung, colorectal, prostate, bladder, ovarian, thyroid, pancreatic, B-cell lymphoma, multiple-myeloma p21 ras Colorectal Cell-surface localization HER2/neu Breast PEM/MUC1 Anonymous proteins T, Tn, sialyl Tn Gangliosides (GM1, GM2, GD2) Melanoma antigen Ovarian, breast, colorectal, pancreatic Lung Breast, lung, pancreatic Melanoma Melanoma References [121 [38, 39) (13-15, 40-50] (161 [18', 51, 52) (53b-551 (20] [21] (211 [561 EBV-immortalized B-cell clones from "breast and bovarian cancer patients. 229 immune response directed against the HER2/neu oncoprotein was found [18]. The presence of circulating antibodies correlates with oncoprotein overexpression on primary tumors and with advanced-stage disease. The finding that the soluble extracellular domain of the oncoprotein is also detectable in the sera of advanced-stage patients [19] raises the question of whether immune complexes are formed and if so what their relevance is to tumor progression. In small-cell lung cancer patients, the presence of antibodies directed against autologous tumor cell proteins was found to be associated with improved survival but the actual target molecule recognized by these antibodies is still unknown [20]. Finally, it has been reported that antibodies to carbohydrate structures are quite commonly found in human sera. In particular, in many melanoma patients humoral immune response to gangliosides (GM1, GM2 and GD2) is present either as 'natural' antibodies or as a result of immunogenic stimulation by the tumor [21]. Very recently the expression of cDNA libraries from human rumors of different oncotypes in Escherichia coli and screening for clones reactive with high-titer IgG antibodies in autologous patient serum lead to the discovery of several antigens with a restricted expression pattern in each tumor [22]. Antibody response upon vaccination Approaches to the control of cancer by manipulating the host immune response date back to the turn of this century [23, 24]. 'Active specific immunotherapy' was the term originally used to describe the approaches which involve immunization with a specified antigen to induce a specific immune response. The field, now termed tumor vaccinology, was the object of a renewed surge of interest based on recent advances in biotechnology and the much more detailed understanding of cancer immunology [4]. About one half of human cancer vaccines on trial used purified, synthetic or recombinant TAA [25], and the majority of these studies relied on the possibility of inducing or increasing a preexisting antibody response. Table 3 lists the more relevant ongoing studies, some of which have already reached the phase HI level. Two pilot studies were conducted in ovarian and breast cancer patients with T or sialyl-Tn. All patients developed antigen specific antibodies of both IgM and IgG isotypes. Complement mediated cytotoxic antibodies for cancer cells were also induced, which were partially inhibited by the immunizing carbohydrate. However, no correlation between antibody titers and clinical outcome was found. Over 300 patients with melanoma have been immunized with purified gangliosides. The antibody response induced by these immunizations was primarily an IgM response of moderate titer and short duration. A double blind randomized Table 3. Ongoing clinical trials in which antibody response becomes evident upon vaccination. Immunizing antigen Tumor References TAA Synthetic T, sialyl-Tn Purified gangliosides (GM2, GD2, GD3) Ovarian, breast Melanoma [57, 58] [26, 59] Melanoma Colorectal Colorectal [8] [601 [61] Colorectal [31] Anti-idiotypic antibody (Ab2fi)' Mouse Ab2p anti-HMW-MAA mAb Mouse Ab2p" anti-CEA mAb Polyclonal goat Ab2p" anti-CCAA mAb Human Ab2p" anti-CCAA mAb * See Figure 1 for the rationale and terminology of the approach. Abbreviations: HMW-MAA - high molecular weight melanomaassociated antigen; CEA - carcinoembryonic antigen; CCAA colon carcinoma-associated antigen. trial of vaccination with GM2 plus BCG was conducted in 122 tumor-free patients after surgery. Anti-GM2 antibody-positive patients showed a highly significant increase in disease-free survival and a 17% increase in overall survival [26]. Three approaches to augment the immunogenicity of gangliosides and carbohydrate TAA in general have been tested: 1) the use of immunological adjuvants; 2) the synthesis of derivates (congeners) of these saccharides; and 3) the chemical coupling to immunogenic carrier proteins [27]. An attractive alternative to the TAA for efficient immunization is represented by ann'-idiotypic antibodies (Ab2P) which molecularly mimic TAA (for the rationale and the terminology of the approach see legend of Figure 1). Anti-idiotype vaccination has already been used successfully to prevent or suppress the growth of experimental tumors [28, 29], and it has the advantage of not being genetically restricted. Moreover, by presenting the antigen in a different molecular environment it is possible to break immune tolerance [30]. Several clinical studies, listed in Table 3, are underway. A study in melanoma patients aimed to evaluate the relationship between the development of humoral immune response following Ab2 p1 vaccination and survival time was conducted in 58 sequential tumorbearing patients. Kaplan-Meier survival analysis showed that the Ab3 antibody level (in 60% of cases) and development of anti-TAA antibodies (in 35% of cases) displayed a significant association with survival prolongation [8]. Various approaches to enhancing cellular and humoral responses to Ab2 vaccines have been tested, such as the use of different carriers, adjuvants and immunomodulators and there appear to have been interesting results in colorectal and melanoma patients from the association of Ab2 vaccines with IL-2 (M. Maio, personal communication). In a preliminary series of 6 colorectal cancer patients it was recently demonstrated that a human Ab2 vaccine induces both 230 Ab1 Ab2p Ab3 = Ab1' (anti-TAA) (anti-idiotype) (anti-anti-idiotype) tin TAA tumor cell Figure 1. The idiotypic network and the molecular mimicry of antigen. According to the network hypothesis [62] immunization with a specific TAA gives rise to antibody against this TAA, termed Abl; Abl, which, in turn, induces a series of anti-idiotypic antibodies, termed Ab2. Some of these Ab2 molecules can effectively mimic the three-dimensional structure of the TAA identified by the Abl. These particular anti-idiotypes called Ab28, due to the antigen molecular mimicry can induce specific antibody responses (Ab3) similar to those induced by the original TAA. Thus Ab3 are also termed Abl-like or Abl'. Idiotype - collection of the unique antigenic sites (idiotopes) of an antibody. a cellular and a humoral immune response against the nominal TAA [31]. Since the entire idiotypic network can be mounted in immunocompetent cancer patients and murine monoclonal antibodies (mAb) to several TAA have been administered in a thousand patients for diagnostic and therapeutic studies over the past decade, a reevaluation of the possible beneficial effect of the human anti-mouse antibody (KAMA) response is warranted. Indeed, a large survey of literature data [32] indicated that following just a single injection of murine mAb, more than 60% of patients are able to develop a humoral immune response against the foreign protein. The specificity of HAMA response in regard to reactivity with different regions of the murine immunoglobulin molecule was analyzed in less than one fourth of the study. A consistent proportion (from 30% to 60%) of cases with positive HAMA was found to produce anti-idiotypic antibodies, however only in few instances the development of Ab2 p anti-idiotypic antibodies which mimic TAA was sought. Up to now, there have been only anecdotal reports of positive influence of HAMA response on the disease course [33]. In contrast, shortening of mAb pharmacokinetics and interference in in vitro tumor marker evaluation in patient sera are well documented [32,34,35]. Concluding remarks On the basis of the data reported, it is not yet possible to assert that humoral immune response to any immunogenic tumor antigen truly represents a protective immune response in vivo. The finding that seropositivity in tumor-bearing patients is frequently compatible with progressive growth of immunogenic tumors can be explained, assuming that the mechanism of antibodydependent epitope masking, a phenomenon envisaged in experimental models, is operating and therefore, indicative of a population with poor prognosis [36]. However, it is not possible at present, to rule out the possibility that its association with poor prognosis is an epi-phenomenon. Some investigators suggest that alteration of TAA (i.e., p53, HER2/neu) becomes evident only in advanced disease and evokes a humoral response which, in turn, is too late to control the growth of very aggressive tumor cells. Moreover, an indication that a serological response can be beneficial comes from the vaccination studies. The majority of the antibodies against TAA were of the IgG subclass, implying that cognate helper T-cell immunity was present and operative in patients with a B-cell response. While much emphasis has been placed on the role of CD 8+ T-cells in the TAA recognition and the effector phase of the immune response against tumors, evidence has accumulated that CD4+ T-cells also play a crucial role in the anti-tumor response by mediating critical priming and effector functions. The recent evidence that tumor antigens defined by T-cell responses such as MAGE-1 and tyrosinase can also be detected by a serological approach [22] suggests that an integrated immune response against TAA may exist that involves both CD8+ and CD4+ T-cells as well as B-cells. There are numerous immunization trials in progress which offer virtually unlimited opportunities to test for 231 the presence of anti-tumor antibodies. These studies as well as the numerous prognostic studies which have accumulated in recent years, are expected to provide an answer in the near future to the question of whether anti-tumor antibodies are protective per se or their presence simply reflects the development of a specific CD4+ T-cell response. Acknowledgements Partially supported by CNR-ACRO and AIRC/FIRC. We are grateful to Dr. Jose Garcia Puche for his constructive suggestions, Laura Mameli for manuscript preparation and Mario Azzini for figure preparation. References 1. Prehn RT, Main JM. 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Division of Experimental Oncology E Istituto Nazionale Tumori Via Venezian 1 20133 Milan Italy