Oncoimmunology: some fundamental problems of cancer immunotherapy

In this review we briefly discuss several central problems of modem oncoimmunology. We review the controversies surrounding the concept of immunological surveillance, as well as the problem of immunological tolerance to tumors. The discovery of tumor antigens has a great advance towards identification of possible therapeutic targets. However, antigen-specific vaccinations against cancer so far have a very limited use, mainly for prevention of virus-associated cancers, which is essentially based on anti-viral immune response. On the other hand, antibodies to cancer antigens are being widely used in cancer diagnostics, and there are remarkable examples of their therapeutic applications. The future opportunities in both theoretical and practical oncoimmunology will directly depend on further advances in basic science.     

Carbohydrate vaccines as immunotherapy for cancer

Carbohydrates have established themselves as the most clinically relevant antigens of those tested and subsequently developed for vaccines against infectious diseases. However, in cancer patients, many of the defined carbohydrate antigens are really altered 'self' antigens and for unclear reasons, the body does not react to them immunologically. Although these self antigens have been found to be potentially suitable targets for immune recognition and killing, the development of vaccines for cancer treatment is actually more challenging compared with those for infectious diseases mainly because of the difficulty associated with breaking the body's immunological tolerance to the antigen. These antigens lack the inherent immunogenicity associated with bacterial antigens and, therefore, methods to enhance immunological recognition and induction of immunity in vivo are under investigation. These include defining the appropriate tumour-associated antigen, successfully synthesizing the antigen to mimic the original molecule, inducing an immune response, and subsequently enhancing the immunological reactivity so that all components can work together. This has been successfully accomplished with several glycolipid and glycoprotein antigens using carriers such as keyhole limpet haemocyanin (KLH) together with a saponin adjuvant, QS-21. Not only can high titre IgM and IgG antibodies be induced, which are specific for the antigen used for immunization, but the antibodies can mediate complement lysis. The approaches for synthesis, conjugation, clinical administration and immunological potential are discussed.     

Therapeutic genes for cancer gene therapy

Cancer still represents a disease of high incidence and is therefore one major target for gene therapy approaches. Gene therapy for cancer implies that ideally selective tumor cell killing or inhibition of tumor cell growth can be achieved using nucleic acids (DNA and RNA) as the therapeutic agent. Therefore, the majority of cancer gene therapy strategies introduce foreign genes into tumor cells which aim at the immunological recognition and destruction, the direct killing of the target cells or the interference with tumor growth. To achieve this goal for gene therapy of cancer, a broad variety of therapeutic genes are currently under investigation in preclinical and in clinical studies. These genes are of very different origin and of different mechanisms of action, such as human cytokine genes, genes coding for immunstimulatory molecules/antigens, genes encoding bacterial or viral prodrug-activating enzymes (suicide genes), tumor suppressor genes, or multidrug resistance genes.     

Tumor-associated antigen arrays for the serological diagnosis of cancer

The recognition that human tumors stimulate the production of autoantibodies against autologous cellular proteins called tumor-associated antigens (TAAs) has opened the door to the possibility that autoantibodies could be exploited as serological tools for the early diagnosis and management of cancer. Cancer-associated autoantibodies are often driven by intracellular proteins that are mutated, modified, or aberrantly expressed in tumor cells and hence are regarded as immunological reporters that could help uncover molecular events underlying tumorigenesis. Emerging evidence suggests that each type of cancer might trigger unique autoantibody signatures that reflect the nature of the malignant process in the affected organ. The advent of novel genomic, proteomic, and high throughput approaches has accelerated interest in the serum autoantibody repertoire in human cancers for the discovery of candidate TAAs. The use of individual anti-TAA autoantibodies as diagnostic or prognostic tools has been tempered by their low frequency and heterogeneity in most human cancers. However, TAA arrays comprising several antigens significantly increase this frequency and hold great promise for the early detection of cancer, monitoring cancer progression, guiding individualized therapeutic interventions, and identification of novel therapeutic targets. Our recent studies suggest that the implementation of TAA arrays in screening programs for the diagnosis of prostate cancer and other cancers should be preceded by the optimization of their sensitivity and specificity through the careful selection of the most favorable combinations of TAAs.     

Clinical evaluation of systemic and local immune responses in cancer: time for integration

The immune system has a dual role in cancer development and progression. On the one hand, it can eradicate emerging malignant cells, but on the other hand, it can actively promote growth of malignant cells, their invasive capacities and their ability to metastasize. Immune cells with predominantly anti-tumor functionality include cells of the innate immune system, such as natural killer cells, and cells of adaptive immunity, such as conventional dendritic cells and cytotoxic T lymphocytes. Immune cells with predominantly pro-tumor functionality include a broad spectrum of cells of the innate and adaptive immune system, such as type 2 neutrophils and macrophages, plasmacytoid DC, myeloid-derived suppressor cells and regulatory T lymphocytes. The presence of immune cells with tumor-suppressive and tumor-promoting activity in the cancer microenvironment and in peripheral blood is usually associated with good clinical outcomes and poor clinical outcomes, respectively. Significant advances in experimental and clinical oncoimmunology achieved in the last decade open an opportunity for the use of modern morphologic, flow cytometric and functional tests in clinical practice. In this review, we describe an integrated approach to clinical evaluation of the immune status of cancer patients for diagnostic purposes, prognostic/predictive purposes (evaluation of patient prognosis and response to treatment) and for therapeutic purposes.     

Dissecting tumor responsiveness to immunotherapy: the experience of peptide-based melanoma vaccines

Recent years have witnessed important breakthroughs in our understanding of tumor immunology. A variety of immunotherapeutic strategies has shown that immune manipulation can induce the regression of established cancer in humans. The identification of the genes encoding tumor-associated antigens (TAA) and the development of means for immunizing against these antigens have opened new avenues for the development of an effective anticancer immunotherapy. However, an efficient immune response against tumor requires an intricate cross-talk between cancer and immune system cells, which is still poorly understood. Only when the molecular basis underlying tumor susceptibility to an immune response is deciphered could new therapeutic strategies be designed to fit biologically defined mechanisms of cancer immune rejection. In this article, we address some of the critical issues that have been identified in cancer immunotherapy, in part from our own studies on immune therapies in melanoma patients treated with peptide-based vaccination regimens. This is not meant to be a comprehensive overview of the immunological phenomena accompanying cancer patient vaccination but rather emphasizes some emergent findings, puzzling controversies and unanswered questions that characterize this complex field of oncology. In addition to reviewing the main immunological concepts underlying peptide-based vaccination, we also review the available data regarding naturally occurring and therapeutically induced anticancer immune response, both at the peripheral and intratumoral level. The hypothesized role of innate immunity in predetermining tumor responsiveness to immunotherapeutic manipulation is also discussed.     

Exogenous DNA transcription in cells with their native DNA inhibited. 4. Demonstration of specific antigens codified by exogenous DNA

It is possible to obtain antisera to cancer-specific antigens of mouse Ehrlich ascites cancer (EAC) cells when chimera rabbits previously made unresponsive to immunological stimulation by normal mouse cells antigens are immunized with mouse cancer cells. Employing this specifically anti-cancerous immunoserum this work shows that EAC cells and TC-SV40 cells contain cross-reacting cancer-specific antigens. Exogenous DNA from EAC cells is demonstrated as being able to display its coded information when it is incorporated to endogenous TC-SV40 cell DNA whose activity has been previously inhibited by 5'-bromodeoxyuridine.     

Therapeutic targets and new directions for antibodies developed for ovarian cancer

Antibody therapeutics against different target antigens are widely used in the treatment of different malignancies including ovarian carcinomas, but this disease still requires more effective agents. Improved understanding of the biological features, signaling pathways, and immunological escape mechanisms involved in ovarian cancer has emerged in the past few years. These advances, including an appreciation of the cross-talk between cancer cells and the patient's immune system, have led to the identification of new targets. In turn, potential antibody treatments with various mechanisms of action, including immune activation or toxin-delivery, that are directed at these targets have been developed. Here, we identify established as well as novel targets for antibodies in ovarian cancer, and discuss how they may provide fresh opportunities to identify interventions with enhanced therapeutic potential.     

Pilot study of a novel combination of two therapeutic vaccines in advanced non-small-cell lung cancer patients

Cancer vaccines contain tumor antigens in a pro-inflammatory context with the purpose to generate potent antitumor immune responses. However, tumor cells develop different immunosuppressive mechanisms that limit the effectiveness of an anticancer immune response. Therefore, therapeutic vaccine treatment alone is usually not sufficient to generate tumor regression or survival improvement, especially in the advanced disease scenario in which most clinical studies have been conducted. Combining cancer vaccines with different anticancer therapies such as chemotherapy, radiotherapy and other immunotherapeutic agents has had different levels of success. However, the combination of cancer vaccines with different mechanisms of action has not been explored in clinical trials. To address this issue, the current review summarizes the main clinical and immunological results obtained with two different therapeutic vaccines used in advanced non-small-cell lung cancer patients, inducing an immune response against epidermal growth factor (CIMAvax-EGF) and NGcGM3 ganglioside (racotumomab). We also discuss preliminary findings obtained in a trial of combination of these two vaccines and future challenges with these therapies.     

The questionable immunogenicity of certain neoplasms

Summary The possibilities of immunological intervention in neoplastic diseases are discussed in light of the accumulating findings that many spontaneously arising tumors of animals are not immunogenic in the host of origin or are not susceptible to immunological attack, and of the doubts as to the existence of tumor-associated antigens for many human neoplasms. Although some spontanous tumors do in fact display immunogenic properties and are subject to cytotoxic immune reactions, there are persuasive arguments for anticipating that a large proportion of naturally appearing malignant growths will be found to lack both immunogenicity and sensitivity to immunological effector mechanisms in the course of the host-tumor association. Even in the case of such tumors, however, significant opportunities for therapy and prophylaxis by extrinsic immunological manipulation remain cogent, as do eventualities of immunodiagnosis. These possibilities are discussed. It is concluded that whereas the simplistic assumptions which for some years set expectations in the field of tumor immunology must now be abandoned, reassessment of the nature of immunological parameters of host-tumor relationships and of the scope of immunological intervention on the basis of the information now available suggests that the immunological approach to the control of cancer continues to be a profitable area of investigation.     

Toll-like receptor expression and function in human dendritic cell subsets: implications for dendritic cell-based anti-cancer immunotherapy

Dendritic cells (DCs) are central players of the immune response. To date, DC-based immunotherapy is explored worldwide in clinical vaccination trials with cancer patients, predominantly with ex vivo-cultured monocyte-derived DCs (moDCs). However, the extensive culture period and compounds required to differentiate them into DCs may negatively affect their immunological potential. Therefore, it is attractive to consider alternative DC sources, such as blood DCs. Two major types of naturally occurring DCs circulate in peripheral blood, myeloid DCs (mDCs) and plasmacytoid (pDCs). These DC subsets express different surface molecules and are suggested to have distinct functions. Besides scavenging pathogens and presenting antigens, DCs secrete cytokines, all of which is vital for both the acquired and the innate immune system. These immunological functions relate to Toll-like receptors (TLRs) expressed by DCs. TLRs recognize pathogen-derived products and subsequently provoke DC maturation, antigen presentation and cytokine secretion. However, not every TLR is expressed on each DC subset nor causes the same effects when activated. Considering the large amount of clinical trials using DC-based immunotherapy for cancer patients and the decisive role of TLRs in DC maturation, this review summarizes TLR expression in different DC subsets in relation to their function. Emphasis will be given to the therapeutic potential of TLR-matured DC subsets for DC-based immunotherapy.     

Liposome-Based Cancer Vaccines

Abstract

Specific active immunotherapy employing crude or purified tumour cell extracts containing tumour-associated antigens (TAA) is of interest as a therapeutic modality for cancer treatment. Weak immunogenicity of TAA preparations and difficulties associated with the extraction of TAA from tumour cells have been major problems, but may be overcome by various strategies. In this context, we studied the potential of well-characterized reconstituted tumour cell membranes to induce protective tumour immunity. The weakly immunogenic SL2 lymphosarcoma syngeneic to DBA/2 mice was used as a tumour model. It was found that immunization with reconstituted membranes induces specific and systemic tumour immunity. Presentation of TAA on a membrane structure was essential. The tumour rejection potency of the reconstituted membranes was dramatically enhanced by the addition of muramyl tripeptide phosphatidyl-ethanolamine (MTP-PE) and interleukin-2. In addition, the effect of liposome encapsulation of IL-2 on its immunomodulating effect was monitored. Finally, insight was obtained into the immunological basis of the protective tumour immunity. On the basis of these findings, suggestions are made to improve liposome-based cancer vaccines.     

Modulatory efficacy of green tea polyphenols on glycoconjugates and immunological markers in 4-Nitroquinoline 1-oxide-induced oral carcinogenesis-A therapeutic approach

Green tea polyphenols (GTP) has been used as a chemopreventive agent world wide against chemically induced cancer. The present study is aimed to understand the therapeutic action of GTP on glycoconjugates and immunological markers in 4-Nitroquinoline 1-oxide (4-NQO)-induced oral cancer over a period of 30 days at 200mg/kg, p.o., Oral cancer was induced by painting 4-NQO for 8 weeks followed by administration of GTP after 22 weeks, for 30 days. Glycoconjugates such as hexose, hexosamine, sialicacid, fucose and mucoprotein were analysed. Expression of glycoconjugates was examined through histology and SDS-PAGE. Immunological markers such as circulating immune complex and mast cell density were studied. Oral cancer-induced animals showed a significant increase in levels of glycoconjugates and its expression, similar to that observed for immunological markers. Treatment with GTP altered the expression of glycoconjugates as well as immunological markers. The results suggest that GTP modulates both the expression of glycoconjugates and immunological markers resulting in regression of oral cancer.     

Immunogenicity of synthetic TF-KLH (keyhole limpet hemocyanin) and sTn-KLH conjugates in colorectal carcinoma patients

Mucins of colorectal carcinomas overexpress the cancer-associated disaccharides Thomsen-Friedenreich antigen (TF) and sialyl-Tn antigen (sTn), making these antigens suitable for active specific immunotherapy. Patients at high risk for recurrent colon cancer, but free from disease after surgical resection, were immunized with synthetic TF and sTn covalently attached by a two-carbon crotyl linker to keyhole limpet hemocyanin (KLH). Four groups of patients were treated with TF-KLH without adjuvant, TF-KLH plus the immunological adjuvant Detox, sTn-KLH plus Detox, or sTn-KLH plus the immunological adjuvant QS-21, and the serological response was monitored. Enzyme-linked immunosorbent assay (ELISA), do-blot immunostains, and inhibition assays were used to identify antibody responses against synthetic TF and sTn epitopes and against natural antigens, including asialoglycophorin expressing TF antigen, and ovine submaxillary mucin and the human colon cancer line LS-C expressing sTn antigen. Our results demonstrate that vaccines containing TF or sTn-KLH conjugates plus immunological adjuvants Detox and especially QS-21 induced high IgM and IgG antibody titers against the respective synthetic disaccharide epitopes. However, when tested against natural antigens expressing these disaccharide epitopes, IgM antibodies showed weak to moderate reactivity, while IgG antibodies were almost totally unreactive. On the basis of these results we are continuing to test modifications of synthetic TF and sTn epitopes to identify those that induce IgM and IgG antibodies that are more reactive with these antigens as they are expressed on tumor mucins.This work was supported by grants from the National Institutes of Health (CA33049, CA52491, CA08748) and the Chemotherapy Foundation     

Lack of ADAM2, CALR3 and SAGE1 Cancer/Testis Antigen Expression in Lung and Breast Cancer

Immunotherapy is emerging as a supplement to conventional cancer treatment, and identifying antigen targets for specific types of cancer is critical to optimizing therapeutic efficacy. Cancer/testis antigens are highly promising targets for immunotherapy due to their cancer-specific expression and antigenic properties, but the expression patterns of most of the more than 200 identified cancer/testis antigens in various cancers remain largely uncharacterized. In this study, we investigated the expression of the cancer/testis antigens ADAM2, CALR3 and SAGE1 in lung and breast cancer, the two most frequent human cancers, with the purpose of providing novel therapeutic targets for these diseases. We used a set of previously uncharacterized antibodies against the cancer/testis antigens ADAM2, CALR3 and SAGE1 to investigate their expression in a large panel of normal tissues as well as breast and lung cancers. Staining for the well-characterized MAGE-A proteins was included for comparison. Immunohistochemical staining confirmed previous mRNA analysis demonstrating that ADAM2, CALR3 and SAGE1 proteins are confined to testis in normal individuals. Negative tissues included plancenta, which express many other CT antigens, such as MAGE-A proteins. Surprisingly, we detected no ADAM2, CALR3 and SAGE1 in the 67 lung cancers (mainly non-small lung cancer) and 189 breast cancers, while MAGE-A proteins were present in 15% and 7–16% of these tumor types, respectively. Treatment with DNA methyltransferase inhibitors has been proposed as an attractive strategy to increase the expression of cancer/testis antigens in tumors before immunotargeting; however, neither ADAM2, CALR3 nor SAGE1 could be significantly induced in lung and breast cancer cell lines using this strategy. Our results suggest that ADAM2, CALR3 and SAGE1 cancer/testis antigens are not promising targets for immunotherapy of breast and lung cancer.     

Cancer treatment: the combination of vaccination with other therapies

Harnessing of the immune system by the development of ‘therapeutic’ vaccines, for the battle against cancer has been the focus of tremendous research efforts over the past two decades. As an illustration of the impressive amounts of data gathered over the past years, numerous antigens expressed on the surface of cancer cells, have been characterized. To this end, recent years research has focussed on characterization of antigens that play an important role for the growth and survival of cancer cells. Anti-apoptotic molecules like survivin that enhance the survival of cancer cells and facilitate their escape from cytotoxic therapies represent prime vaccination candidates. The characterization of a high number of tumor antigens allow the concurrent or serial immunological targeting of different proteins associated with such cancer traits. Moreover, while vaccination in itself is a promising new approach to fight cancer, the combination with additional therapy could create a number of synergistic effects. Herein we discuss the possibilities and prospects of vaccination when combined with other treatments. In this regard, cell death upon drug exposure may be immunogenic or non-immunogenic depending on the specific chemotherapeutics. Also, chemotherapy represents one of several options available for clearance of CD4⁺ Foxp3⁺ regulatory T cells. Moreover, therapies based on monoclonal antibodies may have synergistic potential in combination with vaccination, both when used for targeting of tumor cells and endothelial cells. The efficacy of therapeutic vaccination against cancer will over the next few years be studied in settings taking advantage of strategies in which vaccination is combined with other treatment modalities. These combinations should be based on current knowledge not only regarding the biology of the cancer cell per se, but also considering how treatment may influence the malignant cell population as well as the immune system. This article is a symposium paper from the conference “Progress in Vaccination against Cancer 2007 (PIVAC 7)”, held in Stockholm, Sweden, on 10–11 September 2007.     

Immune based therapies in cancer

Immunotherapy of cancer has become a more promising approach in the past decade. Developments in both basic immunology and tumor biology have increased our knowledge of the interactions between the tumor cells and the immune system. The molecular identification of tumor-associated antigens and understanding of immunological pathways have cleared the way for development of different strategies for anti-tumor vaccines. The success of any cancer vaccine relies on the induction of an effective tumor-specific immune response to break tolerance and to elicit a long lasting anti-tumor immunity. It is also increasingly clear that the interactions of host-tumor are quite complicated leading to tumor escape mechanisms, which add another level of difficulty to this interaction. This review will summarize the recent developments in tumor immunotherapy as well as the clinical trials addressing novel immunotherapeutic approaches to cancer.     

An overview of cancer immunotherapy

The survival of patients with cancer has improved steadily but incrementally over the last century, with the advent of effective anticancer treatments such as chemotherapy and radiotherapy. However, the majority of patients with metastatic disease will not be cured by these measures and will eventually die of their disease. New and more effective methods of treating these patients are required urgently. The immune system is a potent force for rejecting transplanted organs or microbial pathogens, but effective spontaneous immunologically induced cancer remissions are very rare. In recent years, much has been discovered about the mechanisms by which the immune system recognizes and responds to cancers. The specific antigens involved have now been defined in many cases. Improved adjuvants are available. Means by which cancer cells overcome immunological attack can be exploited and overcome. Most importantly, the immunological control mechanisms responsible for initiating and maintaining an effective immune response are now much better understood. It is now possible to manipulate immunological effector cells or antigen-presenting cells ex vivo in order to induce an effective antitumour response. At the same time, it is possible to recruit other aspects of the immune system, both specific (e.g. antibody responses) and innate (natural killer cells and granulocytes).     

Bicistronic DNA Vaccines Simultaneously Encoding HIV,HSV and HPV Antigens Promote CD8+ T Cell Responses and Protective Immunity

Millions of people worldwide are currently infected with human papillomavirus (HPV), herpes simplex virus (HSV) or human immunodeficiency virus (HIV). For this enormous contingent of people, the search for preventive and therapeutic immunological approaches represents a hope for the eradication of latent infection and/or virus-associated cancer. To date, attempts to develop vaccines against these viruses have been mainly based on a monovalent concept, in which one or more antigens of a virus are incorporated into a vaccine formulation. In the present report, we designed and tested an immunization strategy based on DNA vaccines that simultaneously encode antigens for HIV, HSV and HPV. With this purpose in mind, we tested two bicistronic DNA vaccines (pIRES I and pIRES II) that encode the HPV-16 oncoprotein E7 and the HIV protein p24 both genetically fused to the HSV-1 gD envelope protein. Mice i.m. immunized with the DNA vaccines mounted antigen-specific CD8⁺ T cell responses, including in vivo cytotoxic responses, against the three antigens. Under experimental conditions, the vaccines conferred protective immunity against challenges with a vaccinia virus expressing the HIV-derived protein Gag, an HSV-1 virus strain and implantation of tumor cells expressing the HPV-16 oncoproteins. Altogether, our results show that the concept of a trivalent HIV, HSV, and HPV vaccine capable to induce CD8⁺ T cell-dependent responses is feasible and may aid in the development of preventive and/or therapeutic approaches for the control of diseases associated with these viruses.