Adoptive transfer enables tumor rejection targeted against a self-antigen without the induction of autoimmunity

We have previously described a tumor model in which the influenza hemagglutinin protein (HA) expressed on the BALB/c-derived MT901 tumor line serves as an immunization-dependent tumor rejection antigen in normal syngeneic mice. Although the HA antigen in this model is clearly foreign to normal BALB/c mice, many tumor antigens recognized by T cells in humans and mice are nonmutated antigens that are expressed on normal tissues as well as on the tumor cells, thereby raising issues of self-tolerance and autoimmunity in attempts to use such antigens therapeutically. To examine these issues, we have applied our tumor model to syngeneic mice that broadly express HA at low levels as a "self"-transgene. Unlike the situation in normal BALB/c mice, immunization of HA-transgenic mice did not result in tumor protection nor did it generate cytotoxic T cell or IgG responses against the HA self-antigen. However, if immunization of HA-transgenic mice was preceded by adoptive transfer of spleen and lymph node cells from normal untreated BALB/c mice, then HA-specific tumor protective immune responses were generated. Despite the self-nature of the HA antigen, no obvious manifestations of autoimmunity were observed. The immunity established in the transgenic mice was notably different from that observed in normal mice in that it was considerably more transient and required CD4 T cells for both successful immunization and subsequent tumor protection, qualities that were not associated with the immunity established in normal BALB/c mice. Collectively our results suggest that transferred cells can be transiently and selectively directed against a tumor-expressed self-antigen before returning to a tolerant state.     

Serological identification and bioinformatics analysis of immunogenic antigens in osteosarcoma

In order to improve disease-free survival and potentially a cure, it is necessary to identify more potent osteosarcoma antigen. Here, we defined the osteosarcoma-associated antigen (OSAA) recognized by the humoral immune system for the first time. We have applied the method of serologic analysis of recombinant cDNA expression library (SEREX) on osteosarcoma, followed by DNA sequencing and analyzing of positive clones. Then, the reactivity of normal and other bone tumor sera with positive clones were performed. Seven distinct novel antigens reactive with IgG antibodies were identified by SEREX analysis on osteosarcoma patients and were characterized according to cDNA sequence and the reactivity with allogeneic sera. Five of the seven antigens identified in this study were recognized by IgG antibodies in normal sera, and the remaining two were recognized exclusively by sera from allogeneic osteosarcoma patients but not by normal donor sera, suggested that the immune response to the two antigens are osteosarcoma related. Crude lysate ELISA methodology indicated that the optical density value of OSAA-3 and OSAA-5 were significantly higher in OS patients than in healthy donors. The two immunogenic antigens detected by immune responses might facilitate the identification of epitopes recognized by antigen-specific cytotoxic T lymphocytes (CTL). In summary, the antigens identified in this study may be potential candidates for diagnosis and targets for immunotherapy in osteosarcoma (OS).     

Recombinant human leukocyte interferon induces alterations in the antigen phenotype of human breast carcinoma cells

Three tumor antigens, TAG-72, carcinoembryonic antigen and a 90 Kd antigen, recognized by monoclonal antibodies (MAbs) B72.3, B1.1 and B6.2, respectively, are differentially expressed on the surface of four human breast carcinoma cell lines. These cell lines, MCF-7, BT-20, MDA-MB-231 and ZR-75-1, also expressed normal surface antigens such as the class I major histocompatibility and a second antigen found on the surface of all human cells by the binding of MAb B139. Treatment of these cells with human recombinant clone A leukocyte interferon (IFN-aA) usually resulted in an enhanced expression of the surface antigens constitutively expressed. For example, the level of B6.2 reactivity to the surface of the ZR-75-1 cells was increased more than 3-fold following IFN-aA treatment. In contrast, ZR-75-1, BT-20 and MD-MB-231 were all negative for the TAG-72 antigen both before and after IFN-aA treatment. The IFN-aA induced tumor antigen expression on the MCF-7 cell surface was shown to be time and dose dependent and consisted of a heterogeneous population of cells that exhibit clonal diversity in their response to tumor augmentation by IFN-aA. The diversity among the MCF-7 clones could not be explained by differences in the surface IFN-aA receptor.     

Serological identification of immunogenic antigens in acute monocytic leukemia

In order to improve disease-free survival and potentially a cure, it is necessary to identify more potent leukemia antigen. Here, we defined the acute monocytic leukemia-associated antigen (LAA) recognized by the humoral immune system for the first time. We have applied the method of serologic analysis of recombinant cDNA expression library (SEREX) on acute monocytic leukemia (FAB M5), followed by DNA sequencing and analyzing of positive clones. Then, the reactivity of normal and other leukemia sera with positive clones were performed. Thirty-five distinct novel antigens reactive with autologous IgG were identified by SEREX analysis on an acute monocytic leukemia patient and were characterized according to cDNA sequence and the reactivity with allogeneic sera. Twenty of the 35 antigens identified in this study were recognized by IgG antibodies in normal sera, and the remaining 15 were recognized exclusively by sera from allogeneic leukemia patients but not by normal donor sera, suggested that the immune response to these 15 antigens are leukemia related. The 15 immunogenic antigens detected by immune responses in the autologous host facilitate the identification of epitopes recognized by antigen-specific cytotoxic T lymphocytes (CTL) and are potential candidates for diagnosis and immunotherapy in acute myeloid leukemia (AML).     

Immune response of athymic nude mice to papovavirus SV40 tumor-associated antigens.

The requirement of T cell functions in the induction of immune response to SV40-specific transplantation rejection antigen and intranuclear tumor antigen was studied using athymic nude mice. The results obtained indicate that virus-immunized athymic nude mice were unable to reject SV40 tumor cell challenge, and sensitized lymphocytes capable of inhibiting tumor growth in vivo could not be demonstrated in the spleens of virus-immunized mice. Athymic nude mice bearing tumor induced by virus-free SV40-transformed BALB/c cells failed to develop antibodies to intranuclear T antigen. Athymic nude mice also failed to respond to viral antigens. Thus it can be concluded that T cell functions are required in the induction of cellular immune response to SV40-specific transplantation rejection antigen and in humoral immune response to SV40-specific T antigen and virion antigen.     

Generation of monoclonal antibodies against human lung squamous cell carcinoma and adenocarcinoma using mice rendered tolerant to normal human lung

Four murine monoclonal antibodies against human lung carcinoma were generated using a novel immunization procedure. BALB/c mice were rendered neonatally tolerant to normal human lung tissues and subsequently immunized with human lung tumor tissues. The lower level of antibody-reactivity to the tolerogen was seen in the sera of mice rendered neonatally tolerant as compared with the level of reactivity in the sera of nontolerant mice. The mice which maintained a sufficiently tolerant state were selected for hybridoma production. Two monoclonal antibodies, KM-32 and KM-34, were developed from mice immunized with lung squamous cell carcinoma tissues and two other monoclonal antibodies, KM-52 and KM-93, were developed from mice immunized with lung adenocarcinoma tissues. Distribution of antigens detected by the monoclonal antibodies were investigated by enzyme-linked immunosorbent assay using membrane fractions prepared from a number of tumorous and normal tissues and various human normal and tumor cell lines. KM-32 recognized a carbohydrate antigen expressed predominantly on lung squamous cell carcinoma cells and its carbohydrate structure appeared to be associated with blood group A antigen. KM-93 recognized a sialylated carbohydrate epitope on the antigen expressed on lung adeno-carcinoma cells and a few other tumor cells. KM-34 and KM-52 detected protein or glycoprotein antigens and they showed predominant reactivity to lung squamous cell carcinoma and lung adenocarcinoma, respectively. KM-32 and KM-34 antibodies showed complement-dependent cytotoxicity against a lung tumor cell line. These results suggest that the tolerance technique is useful for efficient screening of murine monoclonal antibodies specific to human tumors. The efficacy of these four monoclonal antibodies in diagnosis and therapy of lung cancer will be the subject of a sequential study.     

Lymphoid cell subpopulations infiltrating into autologous rat tumors undergoing rejection

Lymphoid cell subpopulations infiltrating into autografts of methylcholanthrene-induced sarcomas in rats immunized with autologous tumor cells were identified in terms of immunohistochemical and cytofluorographic techniques using various monoclonal antibodies raised against different classes of rat lymphohemopoietic cells. These antibodies included in this study directed to rat T-cell antigens corresponding to mouse Lyt-1 (RLyt-1) and Lyt-2,3 antigens (RLyt-2) and to W3/25 antigen expressed on a particular subset of rat T-cells with helper function, as well as to rat granulocyte-macrophage-specific antigen (RGM-1). Histological studies demonstrated that the autografts of highly antigenic tumors introduced to the primary hosts were completely rejected following massive immigration of lymphoid cells into the tumor sites, which was not observed in progressively growing, minimally antigenic tumors. These lymphoid cells found within regressing highly antigenic tumor autografts were identified mostly to be T-cells bearing RLyt-1 (approximately 70%), and more than two-thirds of these T-cells expressed RLyt-2 antigen. In contrast to T-cells, macrophages and B-cells, each of which could be recognized by the presence of either RGM-1 antigen or immunoglobulin on their cell surfaces, appeared to have a minimal role in the rejection of autochthonous tumors, as reflected by their less frequent appearance within the tumor tissues during the rejection process.     

CD4+ T cell-mediated HER-2/neu-specific tumor rejection in the absence of B cells

HER-2/neu (HER-2) is a cell surface proto-oncogene that is often overexpressed in carcinomas. Passive administration of anti-HER-2 antibodies in breast cancer patients has achieved promising results, but less is known about the role of antibodies in active immunization. We asked whether B cells/antibodies are needed for tumor immunity induced by plasmid (HER-2 and GM-CSF) immunization. HER-2 specific tumor immunity relied completely on both CD4+ and CD8+ T cells. IFN-gamma, and to a lesser extent IL-4, seemed to be crucial cytokines during tumor rejection. Protection was associated with production of anti-HER-2 IgG antibodies in B cell competent mice. After immunization, however, B cell-deficient mice rejected HER-2-expressing tumors as efficiently as control littermates. We conclude that T cells are the main effector cells in DNA vaccine induced immunity against HER-2 and that anti HER-2 antibodies are not necessary to elicit a protective anti tumor immune response in this model.     

Tumor microenvironment: what have we learned studying the immune response in this puzzling battlefield?

Recent developments hallmark the progress in the understanding of tumor immunology and related therapeutic strategies. The administration of interleukin-2 (IL-2) to patients with cancer has shown that immune manipulation can mediate the regression of established cancers. The identification of the genes encoding cancer antigens and the development of means for effectively immunizing against these antigens has opened new avenues for the development of active immunization of patients with cancer. However, an efficient immune response against tumor comprises an intricate molecular network still poorly understood. Only when the code governing immune responsiveness of cancer will be deciphered, new therapeutic strategies could be designed to fit biologically defined mechanisms of immune rejection of cancer. In this review, we propose that the mechanisms regulating tumor rejection in response to vaccination will be more efficiently identified by following the evolution of treatment induced events within the tumor microenvironment taking advantage of recently developed technological tools. As a model, we will discuss the observed immune response to tumor antigen -specific immunization and its relationship with the systemic administration of IL-2.     

Membrane-associated antigens on tumor cells from transitional-cell carcinoma of the human urinary bladder. I. Immunological characterization by xenogeneic antisera

An antiserum was raised in rabbits by immunization with a human tumor cell line, T-24, derived from transitional cell carcinoma (TCC) of the urinary bladder. The specificity of the IgG fraction was assessed by antibody-dependent cellular cytotoxicity (ADCC), using purified blood lymphocytes from healthy human donors as effector cells and seven human cell lines as target cells (T-24 and two additional TCC-lines, two normal urothelial lines, one colon carcinoma and one malignant melanoma). The IgG induced strong lysis of all seven target cell types. However, lysis of the five urothelial lines was significantly stronger than that of the control tumors. Conversely, antisera to either of the control tumors induced a significantly stronger lysis of the homologous tumors than of the urothelial cells. All antisera contained antibodies to fetal bovine serum but removal of these by absorption did not change the specificity of the ADCC reactions. When the anti T-24 serum was exhaustively absorbed with glutaraldehyde-fixed spleen homogenate, the cytotoxicity to the control tumor targets was abolished. Although absorption reduced the antibody titer of the IgG preparation, ADCC to the TCC targets remained at a high level. There was no difference in the degree of lysis of the three TCC targets. Lysis of the two target lines from normal urothelium was slightly lower than that of the tumor cells. The results indicate that the anti-TCC serum contained antibodies to one or several antigens shared by five urothelial cell lines but not by the control tumors. Whether or not it also contained antibodies to TCC-associated antigens remains to be established. The molecular basis for these findings will be given in the accompanying paper.     

Identification of tumor associated antigens recognized by IgG from tumor-infiltrating B cells of lung cancer: correlation between Ab titer of the patient's sera and the clinical course

We previously demonstrated that TIB recognize tumor antigens and produce antibodies against them. In the present study, we identified three tumor antigens recognized by TIB in lung cancer and evaluated whether changes in the antibody titer against these antigens correlated with the patient's clinical course. A lung cancer cell line, G603L, was established from a primary lung tumor of a patient, G603. Seven months later, adrenal metastasis was detected and surgically resected. The latter tumor was mildly infiltrated with B cells and xenotransplanted into SCID mice to obtain human IgG. A cDNA library was constructed from G603L and SEREX was carried out using TIB-derived IgG. The sero-reactive clones were sequenced and one of these antigens was revealed to be MAGE-B2 whereas the others were novel antigens. In the immuno-monitoring of the patient's sera, high antibody titer against MAGE-B2 was observed before operation and the titer decreased after resection of the primary tumor. It was elevated again at the time of adrenal metastasis, but then decreased after resection. The change in antibody titer against the second antigen was similar to MAGE-B2, and the antibody titer against the third antigen was low before the primary operation but increased at the time of recurrence. Our results suggest that TIB recognized tumor antigens and the antibody titers against these antigens were changed along with the patient's clinical course. Therefore, these antibodies could be used as tumor markers for the patient.     

Inhibition of tumor cell growth by antibodies induced after vaccination with peptides derived from the extracellular domain of Her-2/neu

The anti Her-2/neu monoclonal antibody Trastuzumab has strong inhibiting effects on tumor growth in vitro and in vivo and is therefore used for immunotherapy in breast cancer patients. Due to necessity of frequent applications, however, cost intensiveness of Trastuzumab treatment and its limited duration of affectivity, an active immunization inducing a perhaps preventive and long-term immunity to Her-2/neu remains a desirable goal. We attempted to induce anti Her-2/neu antibodies by peptide vaccination and to test their efficacy in inhibiting tumor cell growth in vitro. By computer aided analyses, 7 putative B cell epitopes of Her-2/neu were defined and synthesized. These peptide epitopes were coupled to tetanus toxoid and used for immunization in BALB/c mice. Among these peptides, immunizations with 2 single peptides or a combination of 2 peptides induced anti-peptide antibody levels, primarily of the IgG1 isotype. These antibodies were also directed against the native Her-2/neu antigen, as shown in precipitation assays and ELISA with cell lysates of the Her-2/neu overexpressing breast cancer cell line SK-BR-3. Isolated IgG fractions from immune sera incubated with SK-BR-3 cells led to a moderate inhibition of the tumor cell growth in vitro, as well as to complement dependent cell lyses comparable to that achieved by incubation with Trastuzumab. Moreover, peptide immunization in rabbits generated anti-Her 2-neu IgG that, in contrast to mouse sera, were able to mediate a 31-46% lysis of SK-BR-3 cells in ADCC experiments. We conclude from our data that immunization with Her-2/neu peptides successfully induced humoral immune response with anti-tumor activity in an animal model.     

Tumor-infiltrating B lymphocytes as a potential source of identifying tumor antigen in human lung cancer

Functional studies using freshly isolated tumor-infiltrating B lymphocytes(TIB) are difficult to perform and interpret. Here we document a novel function of TIB using fresh human lung cancer tissues engrafted in SCID mice; they are at activated state and produce tumor-specific antibodies in tumor microenvironment: (a) TIB engrafted in SCID mice produced human IgG; (b) IgG derived from TIB highly bound intracellular and membrane-bound antigens of autologous cancer cells; and (c) less recognition of autoantigens expressed on normal lymphocytes by IgG derived from TIB compared with IgG from the serum of the patient. On the basis of the novel findings presented in this study, we modified the original serological analysis of antigens by recombinant cDNA expression cloning design in a patient with lung cancer who expressed unusually favorable clinical evolution and analyzed humoral immunity against identified mutated p53 antigen. This study provides the first demonstration that antibodies derived from TIB recognize tumor antigens by serological analysis of antigens by recombinant cDNA expression cloning methodology and circulating anti-p53 antibodies in sera derived from TIB in tumor microenvironment. Our approach using TIB may allow the identification of key antigens in the humoral cancer-related immune system.     

Evidence of determinant spreading in the antibody responses to prostate cell surface antigens in patients immunized with prostate-specific antigen.

PURPOSE: Prostate cancer consistently remains a difficult clinical problem. The development of novel therapy strategies for effective control and treatment of prostate cancer is essential. The prostate represents a unique site for immunotherapy, in part because prostate-specific immunity would most probably be without significant long-term sequellae. Antibodies and cell-mediated immunity, induced by either active or passive immunization, represent potential means to specifically target prostate tumor cells. EXPERIMENTAL DESIGN: The serum IgG response to cell surface antigens expressed on LNCAP [prostate-specific antigen (PSA)-positive] and PC-3 (PSA-negative) were analyzed in individuals with advanced disease receiving vaccinia- or fowlpox-expressed PSA (v-PSA or f-PSA, respectively) by flow cytometry. RESULTS: Sera from all seven patients in a Phase I study of v-PSA, collected prior to the third immunization, reacted with both prostate tumor cell lines. The majority of individuals (n = 12) in a Phase II trial of v-PSA and f-PSA developed sustainable antibody responses to cell surface antigens on the prostate tumor cell lines. The magnitude and kinetics of these responses were dependent on the immunization schedule. Of importance, the baseline serum of only one of nine patients tested had reactivity with nonprostate tumor cell lines. Sera from three normal males also lacked reactivity with prostate tumor cells. CONCLUSIONS: PSA vaccine constructs are immunogenic and induce antibody responses to a multitude of surface antigens on prostate tumor cell lines by epitope or determinant spreading after stimulation of the immune system by PSA immunization.     

Crypt cell antigen expression in human colon tumor cell lines: analysis with a panel of monoclonal antibodies to CaCo-2 luminal membrane components

Twenty BALB/c mouse monoclonal antibodies to surface membrane components of confluent CaCo-2 cells were used to study the expression of small intestine markers in cultured human colon tumor cells. The antigens recognized by 15 of these antibodies were identified as membrane proteins of relatively high molecular weight, one of them corresponding to the single-chain precursor of sucrase-isomaltase. By immunofluorescence, 14 antibodies were found to stain the small intestine epithelial cells, and various distinctive patterns of antigen distribution in human jejunum were observed. Five antibodies stained exclusively the crypt cells, 3 the luminal membranes of the absorptive villus cells, and 4 the entire epithelium. In contrast, only 4 antibodies stained normal human colon tissue. The different antigens defined by this panel of monoclonal antibodies showed great variability in their expression in 14 human intestinal tumor cell lines tested. These results demonstrated a surprising similarity between many colon tumor cell lines and intestinal crypt cells and identified a set of new intestinal cell surface markers that can be used to identify and characterize intestinal tumor cells in vivo and in culture.     

Two new tumor-specific antigenic peptides encoded by gene MAGE-C2 and presented to cytolytic T lymphocytes by HLA-A2

We have identified 2 antigens recognized by several melanoma-specific cytolytic T lymphocyte clones isolated from a melanoma patient with a clinical history of tumor regression after immunotherapy. Both antigens are presented by HLA-A2 and encoded by gene MAGE-C2, a cancer-germline gene shown previously to be silent in normal somatic tissues and expressed in 40% of melanomas and in other tumor types. One antigen corresponds to peptide ALKDVEERV(336-344), whereas the other corresponds to peptide LLFGLALIEV(191-200). The CTL clones recognizing these 2 peptides also recognized allogeneic tumor cell lines expressing MAGE-C2 and HLA-A2. These 2 new peptides are the first known MAGE-C antigens and represent promising targets for cancer immunotherapy.     

Mimicry of a carcinoembryonic antigen epitope by a rat monoclonal anti-idiotype antibody

Anti-idiotype antibodies (Ab2) that immunologically mimic tumor antigens are auspicious agents for the active immunization of cancer patients. We have developed W12, a rat monoclonal IgG₁ Ab2 to MN -14, a murine anti-carcinoembryonic antigen (CEA) monoclonal antibody. W12 is specific for MN-14 and does not react with other isotype-matched anti-CEA monoclonal antibodies. Moreover, W12 inhibits the binding between MN-14 and CEA. Anti-CEA antibodies can be induced by immunization with W12 (but not with control rat IgG) in xenogenic animals (mice or rabbits). Immunoblotting studies indicate that the internal image determinant borne by W12 is conformational and requires the association of the heavy and light chains of the Ab2 molecule. This study indicates that W12 is a potential idiotype vaccine in patients with CEA-producing cancers.     

Antibodies to a surface membrane marker from human mammary carcinoma cell line

A tumor surface antigen (BTA-BT20-68K) was isolated from a human mammary carcinoma cell line (BT-20). The antigen (Mr 68,000) induced the formation of high titer antibodies which recognized BTA-BT20-68K as a cell surface marker by the immune adherence hemagglutination test and recognized the soluble antigen by solid phase radioimmunoassay. The antibodies which failed to recognize human beta-2-microglobulin, alpha-fetoprotein, and carcinoembryonic antigen were cytotoxic to the parent BT-20 tumor cells at high serum dilutions. The antibodies recognized a similar tumor surface marker isolated directly from human breast adenocarcinomas, but failed to recognize human lymphocyte antigens isolated from BT-20 cells or bound to human lymphocytes bearing human lymphocyte antigen markers in common with those of BT-20 cells. Added to BT-20 tumor cells in culture and in the absence of complement, antibody-dose-related inhibition of tumor cell growth was documented. In the presence of complement, the antibodies were highly cytotoxic to the parent cells. These results demonstrate the presence of a unique tumor surface marker with chemical and immunological properties in common with that isolated directly from human breast adenocarcinomas.     

Active induction of tumor-specific IgE antibodies by oral mimotope vaccination

A role of IgE antibodies in cancer surveillance has been implicated for a long time. Studies dealing with IgE antibodies directly targeted to tumor antigens have shown marked anticancer effects mediated by this antibody class. Thus, the basic function of IgE antibodies may be to control tumor growth. Thus far, cancer-specific IgE has only been applied passively. Consequently, the aim of this study was to establish an active vaccination protocol to induce tumor antigen-specific IgE antibodies, and to evaluate functional properties. We previously generated epitope mimics, so-called mimotopes, for the epitope recognized by the anti-HER-2 antibody trastuzumab. Upon i.p. immunizations, IgG antibodies with trastuzumab-like properties could be elicited. In the present study, we immunized BALB/c mice via the oral route with these trastuzumab mimotopes, under simultaneous neutralization and suppression of gastric acid. As shown in preceding experiments, this feeding regimen effectively induces Th2 immune responses. Oral immunizations with trastuzumab mimotopes under hypoacidic conditions indeed resulted in the formation of IgE antibodies towards the HER-2 antigen. Moreover, anti-HER-2 IgE-sensitized effector cells mediated SK-BR-3 target cell lysis in an antibody-dependent cytotoxicity assay. We conclude that directed and epitope-specific induction of IgE against tumor antigens is feasible with an oral mimotope vaccination regimen, and that these antibodies mediate anticancer effects.