Cell-mediated immunity in patients with cervical cancer.

In 16 patients with cervical carcinoma (stages II and III) tumor-specific and general immune reactivity was investigated. Cancer patients' skin reactivity was different from that of controls insofar as there was a decreased response to streptokinase-streptodornase as well as to DNCB. Absence of DNCB sensitization was mainly in stage III patients. Cell-mediated immunity to autologous and/or allogeneic tumor-associated antigens (TAA) was demonstrated in vivo and in vitro. 3 of 8 patients had positive skin reactions to autologous TAA and 8 of 14 reacted to allogeneic TAA. In the lymphocyte migration inhibition test, 8 of 14 patients reacted to autologous tumor membrane preparations and 3 of 10 to allogeneic pooled preparations. In addition, there was some indication of cross-reaction with antigens from a human squamous cell carcinoma of the lung. There was no reaction in vitro to normal cervical tissue and no association between tumor-specific reactions and herpes simplex virus type II antibodies could be demonstrated. Patients with DNCB sensitization and in vivo as well as in vitro lymphocyte reactivity to TAA had a better prognosis than nonreacting patients, indicating the value of combined immunological tests.     

CTL-defined Cancer Vaccines: Perspectives for Active Immunotherapeutic Interventions in Minimal Residual Disease

The characterization of tumor-associated antigens recognized by cellular or humoral effectors of the immune system has opened new perspectives for cancer therapy. Several categories of cancer-associated antigens have been described as targets for cytotoxic T lymphocytes (CTL) in vitro and in vivo: (1) Cancer-Testis (CT) antigens expressed in different tumors and normal testis, (2) melanocyte differentiation antigens, (3) point mutations of normal genes, (4) antigens that are overexpressed in malignant tissues, and (5) viral antigens. Clinical studies with peptides derived from these antigens have been initiated to induce specific CTL responses in vivo. Immunological and clinical parameters for the assessment of peptide-specific reactions have been defined, i.e. induction of DTH-, CTL-, autoimmune-, and tumor-regression responses. Preliminary results demonstrate that tumor-associated peptides alone elicit specific DTH- and CTL-responses leading to tumor regression after intradermal injection. GM-CSF was proven effective to enhance peptide-specific immune reactions by amplification of dermal peptide-presenting dendritic cells. Long lasting complete tumor regressions have been observed after induction of CTL by peptide immunization. Based on these results, active immunotherapy with tumor-associated antigens may be a promising approach for patients with minimal residual disease, who are at high risk for tumor recurrence. However, in single cases with disease progression after an initial tumor response either a loss of the respective tumor antigen targeted by CTL or of the presenting MHC class I molecule was detected as mechanisms of immune escape under immunization in vivo. Based on these observations, cytokines to enhance antigen- and MHC-class I expression in vivo are being evaluated to prevent immunoselection. Recently, a strategy utilizing spontaneous antibody responses to tumor-associated antigens (SEREX) has led to the identification of a new CT antigen, NY-ESO-1. In a melanoma patient with high titer antibody against NY-ESO-1 also a strong HLA-A2 restricted CTL reactivity against the same antigen was found. Clinical studies involving tumor antigens that induce both antibody- and CTL-responses will show whether these are better candidates for immunotherapy of cancer.     

Cancer immunotherapy in clinical oncology

The identification of tumor-associated antigens recognized by cellular or humoral effectors of the immune system has opened new perspectives for cancer therapy. Different groups of cancer-associated antigens have been described as targets for cytotoxic T lymphocytes (CTLs) in vitro and in vivo: 1) cancer-testis (CT) antigens, which are expressed in different tumors and normal testis; 2) melanocyte differentiation antigens; 3) point mutations of normal genes; 4) antigens that are overexpressed in malignant tissues; and 5) viral antigens. Clinical studies with peptides derived from these antigens have been initiated to induce specific CTL responses in vivo. Immunological and clinical parameters for the assessment of peptide-specific reactions have been defined, i.e., delayed-type hypersensitivity (DTH), CTL, autoimmmune, and tumor regression responses. Preliminary results demonstrate that tumor-associated peptides alone elicit specific DTH and CTL responses leading to tumor regression after intradermal injection. Granulocyte-macrophage colony-stimulating factor (GM-CSF) was proven effective in enhancing peptide-specific immune reactions by amplification of dermal peptide-presenting dendritic cells. Long-lasting complete tumor regressions have been observed after induction of peptide-specific CTLs. However, in single cases with disease progression after an initial tumor response, either a loss of the respective tumor antigen targeted by CTLs or of the presenting major histocompatibility complex (MHC) class I allele was detected as a mechanism of immune escape under immunization. Based on these observations, cytokines to enhance antigen and MHC class I expression in vivo are being evaluated to prevent immunoselection. Recently, a strategy utilizing spontaneous antibody responses to tumor-associated antigens (SEREX) has led to the identification of a new CT antigen, NY-ESO-1, which is regarded as one of the most immunogenic antigens known today inducing spontaneous immune responses in 50% of patients with NY-ESO-1-expressing cancers. Clinical studies involving antigenic constructs that induce both antibody and CTL responses will show whether these are more effective for immunotherapy of cancer.     

Rheumatoid factor in melanoma patients: alterations of humoral tumor immunity in vitro.

Rheumatoid factors (RF) were associated with alterations of antibody reactions to melanoma cells in vitro by two serologic assays. Removal of RF from melanoma patients' sera by absorption with Cohn's Fraction II coated latex particles enhanced seroreactivity in the Immune Adherence (IA) assay and diminished IgM detection by the Indirect Membrane Immunofluorescence (IMI) assay. The addition of serum with high titers of RF to these assay systems led to diminution of IA reactivity and enhancement of IgM detection by IMI. Since these factors are found in cancer patients' sera and can alter humoral immune reactions directed against antigens on the membranes of tumor cells, their presence should be recognized when performing assays with tumor target cells. RF may be of significance in the host-tumor relationship in vivo.     

Kinetics of responses to tumor antigens and mouse mammary tumor virus in BALB/cCrgl and BALB/cfC3H mice

Spleen cells from BALB/cCrgl mice responded to murine mammary tumor virus (MuMTV) in cell-mediated immune assays at higher levels than did the spleen cells from the syngeneic BALB/cfC3H mice. Implantation in BALB/cCrgl with a chemically induced mammary tumor and in BALB/cfCrgl mice with spontaneous mammary tumors (SMT) arising in the same strain resulted in sensitization of these animals to the antigens of their tumors. Reactivities peaked 3 weeks after transplantation, whereas no positive reactions could be detected when tumors reached maximum size. A kinetic study with the use of MuMTV antigen(s) showed that the responses of lymphocytes from BALB/cfC3H with SMT followed the same pattern as that obtained with tumor antigens, which indicated that this might be a de novo sensitization. In sharp contrast, a steady type of response to MuMTV was observed with the spleen cells from BALB/cCrgl mice; i.e., the levels of responsiveness to MuMTV did not significantly vary at any stage of tumor development. In vivo studies explored the possible relevance of the in vitro cell-mediated immunity to the host defenses. MuMTV-expressing mammary tumor cells were implanted in BALB/cCrgl and BALB/cfC3H mice. The total incidence of tumors was significantly reduced and a delay occurred in their time of appearance in BALB/cCrgl mice in relation to BALB/cfC3H animals. Thus the in vitro reactivity to MuMTV antigen(s) in BALB/cCrgl mice was found to be coincidental with a degree of protection against the development of MuMTV-expressing mammary tumors.     

Detection of private and common tumor-associated antigens in murine sarcomas induced by different chemical carcinogens.

Two fibrosarcomas of similar histological type, induced in C3Hf mice by either methylcholanthrene or 3,4-benz(a)pyrene, were shown to have individually unique tumor-rejection antigens in classical transplantation-type experiments. By contrast, sera of autochthonous mice, which resisted only transplants of the immunizing sarcoma, were found to contain complement-dependent cytotoxic antibodies, specific for both sarcomas, in vitro. The existence of individually unique as well as common tumor-associated antigens in chemically-induced murine sarcomas is suggested. The private "tumor transplantation-type" antigens elicited tumor rejection responses in vivo. The common tumor-associated antigens, although immunogenic in autochthonous hosts, inducing the production of tumor-specific antibodies, failed to induce transplantation cross-resistance in vivo. This study supports the contention that, in carcinogen-induced murine tumors, and perhaps in human neoplasms as well the evaluation of humoral (and cell-mediated) immune responses in vitro may not reflect tumor rejection-type immune responses in vivo.     

Comparison of the techniques used for monitoring humoral immunity in cancer patients

The humoral immune response to tumor-associated antigens (TAA) in cancer patients undergoing immunotherapy can vary considerably. This variability is inherent from patient to patient and is also dependent on the type of assay used to measure patient antibody response to tumors. In this investigation four serological techniques, complement-dependent antibody-mediated cytotoxicity, immune adherence, complement fixation, and indirect membrane immunofluorescence, were used to assess the humoral immune response in melanoma patients receiving specific immunization with cultured allogeneic melanoma cell vaccine. Two different patterns of antibody response were elicited. One was directed against TAA and oncofetal antigens (OFA) and the other was against HLA antigens. The sensitivity and reactivity varied among the different techniques suggesting that one test or technique may not be sufficient to evaluate critically a patient's response to immunotherapy.     

Common antigens found on fetal cells and viral transformed adult prostatic tissue.

In vivo and in vitro immunologic cross reactivity between SV40 virus transformed prostatic tissue and fetal antigens of LVG/LAK strain hamsters has been studied. The hamsters immunized with fetal antigens demonstrated significant resistance to tumor growth. Complement-dependent humoral cytotoxicity of target SV40 tumor cells in microtest plates was used to demonstrate the presence of significant antibody titer in fetal-immunized hamsters. When the immune sera were absorbed with SV40 transformed prostatic tissue, cytotoxicity indices were markedly lower than with the unabsorbed immune sera. The transformed prostatic tissue was found to have an elevated tartrate inhibited acid phosphatase indicative of the presence of epithelial components in the transformed tissue. These experiments demonstrate in vivo and in vitro cross reactviity between fetal antigens and tumor associated antigens of transformed prostatic tissue. This study suggests a similarity between fetal antigens and antigens of a specific organ tissue (prostate) transformed by a DNA oncogenic virus.     

Immunosuppressive activity of human neuroblastoma tumor gangliosides

Gangliosides are shed in substantial amounts by some tumors, including human neuroblastoma, and these molecules modulate experimental tumor formation in vivo. We now demonstrate that neuroblastoma tumor gangliosides have potent immunoregulatory activity. Gangliosides of every one of 17 tumors studied were highly inhibitory for the normal in vitro human lymphoproliferative responses to the soluble antigen, tetanus toxoid; 30 nmol ganglioside/ml caused 43% to greater than 99% inhibition and the mean concentration causing 50% inhibition was only 17.3 nmol/ml. Furthermore, gangliosides isolated from clinically more aggressive tumors (Stage III or IV) were up to twice as immunosuppressive as those of the generally less aggressive tumors (Stage I or II) (p less than 0.05). Taken together with the lack of immunosuppressive activity of normal plasma gangliosides, the potent activity of neuroblastoma gangliosides supports the hypothesis that one mechanism by which these shed molecules may act to enhance tumor formation in vivo is through abrogation of the host cellular immune response at the site of tumor formation.     

Ability of delayed-type hypersensitivity reactions to distinguish tumor-associated antigens and histocompatibility antigens in soluble extracts from murine fibrosarcomas.

The footpad swelling (FPS) test for delayed-type hypersensitivity in the mouse was evaluated for its ability to measure both tumor-associated antigens (TAA) and histocompatibility (H) antigens solubilized from methylcholanthrene (MCA) induced fibrosarcomas of C57B1/6 (B6) mice. Tests for TAA were performed in mice immune to syngeneic tumors while H-antigens were assayed in mice immunized with skin allografts. FPS was most intense in B6 mice challenged with TAA from the immunizing B6 tumor, but also occurred in response to cross-reactive TAA solubilized from another B6 fibrosarcoma. Tests for tumor-associated H-antigens in allograft immune mice were strongly positive in response to donor/recipient H-antigen differences and proved sensitive to shared third-party H-antigen differences. Comparison of soluble antigens from the same tumor maintained in vitro and in vivo revealed that, while both TAA and H-antigens could be detected in preparations from the in vivo tumor line, only TAA and not H-antigens could be detected by RPS in extracts prepared from the in vitro tumor line. These experiments have demonstrated that the mouse FPS test can distinguish both TAA and H-antigen specificities persent in the same complex mixture of tumor-cell antigens.     

Equivalent induction of telomerase-specific cytotoxic T lymphocytes from tumor-bearing patients and healthy individuals.

Although high frequencies of T lymphocytes specific for certain tumor-associated antigens have been detected in some cancer patients, increasing evidence suggests that these T cells may be functionally defective in vivo and fail to induce meaningful clinical responses. One strategy to overcome this limitation is to target novel antigens that are ignored during the natural antitumor immune response but are nevertheless capable of triggering effector T-cell responses against tumors after optimal presentation by antigen-presenting cells. Here, we show that the telomerase catalytic subunit (hTERT)-a nearly universal tumor antigen identified by epitope deduction rather than from patient immune responses-is immunologically ignored by patients despite progressive tumor burden. Nevertheless, HLA-A2-restricted CTLs against hTERT are equivalently induced ex vivo from patients and healthy individuals and efficiently kill human tumor cell lines and primary tumors. Thus, telomerase-specific T cells from cancer patients are spared functional inactivation because of immunological ignorance. These findings support clinical efforts to target the hTERT as a tumor antigen with broad therapeutic potential.     

Delayed hypersensitivity in tumor-bearing mice. In vitro activation of "eclipsed" spleen cells.

At various stages during the progressive growth of a transplanted sarcoma in BALB/c mice, the delayed hypersensitivity response to tumor antigen was determined using the food-pad swelling test (FPS) and the leukocyte migration inhibition assay (LMI). A close correlation was observed between the in vivo and in vitro assays. "Early" recognition of tumor antigen was detected 24 h after tumor inoculation by both techniques and this positive response was maintained until day 15. As the tumor grew larger, the delayed hypersensitivity response in vivo vanished, while the delayed hypersensitivity response in vitro disappeared about 3 days later. This suppression or "eclipse" of the anti-tumor cellular immune response was specific for the type of tumor used, and could be reversed in vitro by means of a low pH treatment of lymphoid cells.     

Surface antigens on transplantable tumor cell lines producing mouse type C viruses.

A variety of transplantable mouse tumor lines were shown to contain murine type C viruses and virus-associated antigens. The type of virus isolated and antigens detected could not invariably be correlated with the original method of tumor induction, but testing of the majority of tumor lines for infectious virus at various levels of in vivo or in vitro passage yielded isolates that were consistent in tissue culture host range for each tumor. In contrast, during the course of in vivo transplantation, some of the lines underwent considerable change in the pattern of virus-associated cell-surface antigens. When the transplanted tumor lines were placed into culture, all showed some alteration in the detectable surface antigens. Upon retransplantation and passage of the cultured cells in mice, the surface antigens gradually returned to the original in vivo patterns and occasionally acquired additional type C virus-associated antigens not detected in the original tumor line. To test for association of antigens with infectious virus, appropriate tissue culture cell lines were infected with the viruses isolated from the tumors. In these infected indicator cells, some new virus-associated cell-surface and virion evelope antigens were detected, but the complete array of antigens found in the original tumor lines was not acquired. These findings indicated the presence of several different type C viruses in long transplanted cell lines and demonstrated that environmental and host cell factors may have major influences on expression of virus-associated antigens.     

Changes in tumor immunity during therapy determined by leukocyte adherence inhibition and dermal testing.

Leukocyte adherence inhibition (LAI) is an easily carried out in vitro test for tumor reactivity which is based on the prevention of adherence of leukocytes incubated with antigen to which the donor is immune. In this study a comparison is made with dermal response to tumor antigen. A total of 234 patients were tested, 74 of whom had melanoma, 111 of whom had squamous cell carcinoma of the head and neck, 18 of whom had neuroblastoma and 31 of whom had other tumors. Forty-two persons without tumors acted as controls for the LAI test. A high degree of correlation was found between LAI and dermal response. Furthermore, LAI exhibited marked tumor specificity and showed a ten-fold greater sensitivity than dermal response. LAI may be used to monitor serial changes in tumor reactivity in cancer patients.     

Immunology and immunotherapy of neuroblastoma

Purpose

This review demonstrates the importance of immunobiology and immunotherapy research for understanding and treating neuroblastoma.

Principal results

The first suggestions of immune system–neuroblastoma interactions came from in vitro experiments showing that lymphocytes from patients were cytotoxic for their own tumor cells and from evaluations of tumors from patients that showed infiltrations of immune system cells. With the development of monoclonal antibody (mAb) technology, a number of mAbs were generated against neuroblastoma cells lines and were used to define tumor associated antigens. Disialoganglioside (GD2) is one such antigen that is highly expressed by virtually all neuroblastoma cells and so is a useful target for both identification and treatment of tumor cells with mAbs. Preclinical research using in vitro and transplantable tumor models of neuroblastoma has demonstrated that cytotoxic T lymphocytes (CTLs) can specifically recognize and kill tumor cells as a result of vaccination or of genetic engineering that endows them with chimeric antigen receptors. However, CTL based clinical trials have not progressed beyond pilot and phase I studies. In contrast, anti-GD2 mAbs have been extensively studied and modified in pre-clinical experiments and have progressed from phase I through phase III clinical trials. Thus, the one proven beneficial immunotherapy for patients with high-risk neuroblastoma uses a chimeric anti-GD2 mAb combined with IL-2 and GM-CSF to treat patients after they have received intensive cyto-reductive chemotherapy, irradiation, and surgery. Ongoing pre-clinical and clinical research emphasizes vaccine, adoptive cell therapy, and mAb strategies. Recently it was shown that the neuroblastoma microenvironment is immunosuppressive and tumor growth promoting, and strategies to overcome this are being developed to enhance anti-tumor immunotherapy.

Conclusions

Our understanding of the immunobiology of neuroblastoma has increased immensely over the past 40 years, and clinical translation has shown that mAb based immunotherapy can contribute to improving treatment for high-risk patients. Continued immunobiology and pre-clinical therapeutic research will be translated into even more effective immunotherapeutic strategies that will be integrated with new cytotoxic drug and irradiation therapies to improve survival and quality of life for patients with high-risk neuroblastoma.     

Vaccination for malignant melanoma: recent developments

The identification of tumor-associated antigens recognized by cellular or humoral effectors of the immune system has opened new perspectives for cancer immunotherapy. Different categories of cancer-associated antigens have been described as targets for CD8+ T cells in vitro and in vivo: (1) 'cancer-testis' (CT) antigens expressed in different tumors and normal testis; (2) melanocyte differentiation antigens; (3) point mutations of normal genes; (4) antigens that are overexpressed in malignant tissues, and (5) viral antigens. Clinical trials with antigenic peptides have been initiated to induce specific immunological responses in vivo. Immunological and clinical parameters for the assessment of peptide-specific reactions have been defined: DTH, CD8+ T cell, autoimmune and tumor regression responses. Preliminary results show that tumor-associated peptides alone elicit specific DTH and CD8+ T cell responses associated with tumor regression after intradermal vaccination. Granulocyte macrophage colony-stimulating factor has been shown to enhance peptide-specific immune reactions by amplification of dermal antigen-presenting dendritic cells. Complete tumor regressions have been observed after the induction of CD8+ T cell responses by peptide immunization. Based on these results, active immunotherapy with tumor-associated antigens may be a promising approach for patients in adjuvant treatment situations, who are at high risk for tumor recurrence. Recently, a strategy utilizing spontaneous antibody responses to tumor-associated antigens (SEREX) has led to the identification of a new CT antigen, NY-ESO-1. NY-ESO-1-specific spontaneous humoral and cellular immune responses were found in approximately 50% of patients with NY-ESO-1-positive tumors. Clinical studies have been initiated to evaluate the immunological effects of immunization with NY-ESO-1 peptides in cancer patients with detectable or absent immunity against NY-ESO-1.     

Identification of beta-tubulin isoforms as tumor antigens in neuroblastoma.

There is currently substantial interest in the identification of human tumor antigens for diagnosis and immunotherapy of cancer. We have implemented a proteomic approach for the identification of tumor proteins that elicit a humoral response in cancer patients, which we have applied to neuroblastoma. Proteins from neuroblastoma tumors and cell lines were separated by two-dimensional PAGE and transferred to poly(vinylidene difluoride) membranes. Sera from 23 newly diagnosed patients with neuroblastoma, from 12 newly diagnosed children with other solid tumors, and from 13 normal individuals were screened for IgG and IgM autoantibodies against neuroblastoma proteins by means of Western blot analysis. Sera from 11 patients with neuroblastoma and from 1 patient with a primitive neuroectodermal tumor, but none of the other controls exhibited IgG-based reactivity against a protein constellation with an estimated Mr 50,000. NH2-terminal sequence and mass spectrometric analysis identified the major constituents of this constellation as beta-tubulin isoforms I and III. The IgG antibodies were additionally characterized to be of the subclass IgG1. Neuroblastoma patient sera that contained anti-beta-tubulin IgG antibodies also contained IgM antibodies specific against the full-length beta-tubulin molecule and against COOH-terminal beta-tubulin cleavage products. Neuroblastoma patient sera that reacted with beta-tubulin I and III isoforms in neuroblastoma tissues did not react with beta-tubulin I and III isoforms found in normal brain tissue. Our findings indicate the occurrence of beta-tubulin peptides in neuroblastoma, which are immunogenic. The occurrence of immunogenic peptides in neuroblastoma may have utility in diagnosis and in immunotherapy of this aggressive childhood tumor.     

Growth inhibition of murine colonic adenocarcinoma by tumor immune but not by IL-2-activated or alloactivated lymphocytes

The antigenic profile of C-26 and C-51 BALB/c colonic adenocarcinomas was examined by in vivo and in vitro assays. Mice immunized with irradiated C-26 or C-51 tumor cells from freshly excised tumor nodules or from in vitro-growing cell lines were able to reject a challenge of both tumors. Spleen lymphocytes of immune but not of normal mice were effective in cross-inhibiting tumor growth in vivo in a Winn assay. Tissue-associated antigens common to C-26 and C-51 and to their metastases but not to other syngeneic neoplasms were detected in vitro by cytotoxic T lymphocytes obtained after 5 days of a secondary culture of immune lymphocytes and irradiated tumor cells. Activated lymphocytes were obtained by exposure of spleen cells to interleukin 2 or by allostimulation. Such lymphocytes, although cytotoxic in vitro on C-26 and C-51 carcinomas, were unable to significantly reduce in vivo tumor growth in the Winn assay.