HST.175
11/21/00
Andrew H. Lichtman, M.D., Ph.D.

Tumor Immunology

Malignant tumor cells (cancer cells) may express antigens, called tumor antigens, which can be recognized by the host immune system. The concept of immune surveillance suggests tha a physiologic function of the immune system is to recognize and destroy clones of transformed cells before they grow into tumors, and to kill tumors after they are formed. Although the immune system does react against many tumors, the responses are often weak and ineffective. A major research goal in the field of tumor immunology is to identify tumor antigens and devise ways of enhancing host responses to them. In this lecture we will describe the types of antigens that are expressed by malignant tumors, how the immune system recognizes and responds to these antigens, and the application of immunological approaches to the therapy of cancer.

• Histopathologic studies
• Studies with transplantable carcinogen induced tumors in rodents

• Most tumors are weakly immunogenic: derived from self; no costimulators, produce inhibitors of immune responses.
• Rapid growth and spread of tumors may overwhelm the capacity of the immune system to eradicate tumor cells, and control of a tumor requires that all the malignant cells be eliminated.
• Tumors have specialized mechanisms for evading host immune responses.

A variety of tumor antigens that may be recognized by T and B lymphocytes have been identified in human and animal cancers. It is important to identify tumor antigens because they may be used as components of tumor vaccines, or antibodies and effector T cells generated against these antigens may be used for immunotherapy.

Classification of tumor antigens based on their patterns of expression.

• Tumor specific antigens (TSAs): antigens that are expressed on tumor cells but not on normal cells were called); some of these are unique to individual tumors.
• Tumor associated antigens (TAAs): antigens that are also expressed on normal cells; in most cases, normal cellular constituents whose expression is aberrant or dysregulated in tumors.

Classification of tumor antigens based on molecular nature of antigen.

Products of Mutated Oncogenes and Tumor Suppressor Genes

• Gene products that are required for malignant transformation or for maintenance of the malignant phenotype.
• Often produced by point mutations, deletions, chromosomal translocations, or viral gene insertions involving cellular proto-oncogenes or tumor suppressor genes to form oncogenes whose products have transforming activity.
• Synthesized in the cytoplasm of the tumor cells; may enter the class I antigen processing pathway..

Products of Other Mutated Genes

Tumor antigens may be produced by mutated genes whose products are not related to the transformed phenotype, and may have no known function.

The first examples were Tumor-specific transplantation antigens (TSTAs), in animal studioes, which are diverse mutants of host cellular proteins. Several human tumor antigens fit this description.

Aberrantly Expressed Normal Cellular Proteins

Tumor antigens may be normal cellular proteins that are over-expressed or aberrantly expressed in tumor cells. Some tumor antigens are normal proteins that are produced at low levels in normal cells and over-expressed in tumor cells. For example, tyrosinase, an enzyme involved in melanin biosynthesis which is expressed only in normal melanocytes and melanomas. Both class CD8⁺ CTL clones and CD4⁺ T cell clones from melanoma patients recognize peptides derived from tyrosinase.

Other tumor antigens may be derived from genes that are not expressed in normal tissues or are expressed only early during development, and are dysregulated as a consequence of malignant transformation of a cell. An example is the product of the Melanoma antigen (MAGE) genes, first isolated from human melanoma cells, encode cellular protein antigens recognized by melanoma-specific In normal tissues, MAGE expression is restricted to testis and placenta; it is postulated (? immunologically privileged sites where T cells do not respond effectively to antigens)

Tumor Antigens Encoded by Genomes of Oncogenic Viruses

The products of oncogenic viruses function as tumor antigens and elicit specific T cell reponses that may serve to eradicate the tumors. DNA viruses are implicated in the development of a variety of tumors in experimental animals and humans.

• Papovaviruses, including polyoma virus and simian virus (SV)40, and adenoviruses induce malignant tumors in neonatal or immunodeficient adult rodents.
• Epstein-Barr virus (EBV), which is associated with B cell lymphomas and nasopharyngeal carcinoma
• Human papilloma virus (HPV), which is associated with cervical carcinomas.

Virus-encoded protein antigens are found in the nucleus, cytoplasm, or plasma membrane of the tumor cells. These endogenously synthesized proteins can be processed, and complexes of processed viral peptides with class I MHC molecules may be expressed on the tumor cell surfaces. Because the viral peptides are foreign antigens, DNA virus-induced tumors are among the most immunogenic tumors known.

Evidence that the adaptive immune system can prevent the growth of DNA virus-induced tumors

• EBV-associated lymphomas and HPV-associated skin cancers arise more frequently in immunosuppressed individuals, such as allograft recipients receiving immunosuppressive therapy and acquired immunodeficiency syndrome (AIDS) patients, than in normal individuals.
• Tumor transplantation/immunization experiments show virus-specific protection.

Thus, a competent immune system may play a role in surveillance against virus-induced tumors because of its ability to recognize and kill virus-infected cells.

Oncofetal Antigens

Oncofetal antigens are proteins that are expressed at high levels on cancer cells and in normal developing (fetal) but not adult tissues. The genes encoding these proteins are silenced during development, and are derepressed upon malignant transformation. Oncofetal antigens were identified with antibodies, and their main importance is that they provide markers that aid in tumor diagnosis. But, their expression in adults is not limited to tumors. The proteins are increased in tissues and in the circulation in various inflammatory conditions, and are found in small quantities even in normal tissues. There is no evidence that oncofetal antigens are important inducers or targets of anti-tumor immunity. The two most thoroughly characterized oncofetal antigens are carcinoembryonic antigen (CEA, CD66) and alpha-fetoprotein (AFP).

Carcinoembryonic antigen (CEA, CD66) is a highly glycosylated integral membrane protein that is a member of the Ig superfamily. CEA expression is increased in many carcinomas of the colon, pancreas, stomach, and breast, resulting in a rise in serum levels. The level of serum CEA is used to monitor the persistence or recurrence of metastatic carcinoma after treatment.

Alpha-fetoprotein (AFP) is a circulating glycoprotein normally synthesized and secreted in fetal life by the yolk sac and liver. Serum levels of AFP can be significantly elevated in patients with hepatocellular carcinoma, germ cell tumors, and, occasionally, gastric and pancreatic cancers. An elevated serum AFP level is a useful indicator of advanced liver or germ cell tumors, or of recurrence of these tumors after treatment. Furthermore, the detection of AFP in tissue sections by immunohistochemical techniques can help in the pathologic identification of tumor cells.

Altered Glycolipid and Glycoprotein Antigens

Most human and experimental tumors express higher than normal levels of and/or abnormal forms of surface glycoproteins or glycolipids, which may be diagnostic markers and targets for therapy. These altered molecules include gangliosides, blood group antigens, and mucins. This class of tumor-associated antigens is a target for cancer therapy with specific antibodies.

Glycolipids: GM2, GD2 and GD3 are expressed at high levels in melanomas are the gangliosides. Clinical trials of anti-GM2 or anti-GD3 antibodies, and immunization with vaccines containing GM2, are underway in melanoma patients.

Mucins: In tumors there is often dysregulated expression of the enzymes which synthesize the carbohydrate side chains of mucins, leading to the appearance of tumor-specific epitopes. Mucins that have been the focus of diagnostic and therapeutic studies, include CA-125 and CA-19-9, expressed on ovarian carcinomas, and MUC-1, expressed on breast carcinomas.

Tissue-Specific Differentiation Antigens

Tumors express molecules that are normally present on the cells of origin. These antigens are called differentiation antigens because they are specific for particular lineages or differentiation stages of various cell types. Their importance is as potential targets for immunotherapy, and for identifying the tissue of origin of tumors. Examples include CD10 (CALLA), and CD20 on B cell tumors, prostate specific antigen on prostatic carcinomas.

The effector mechanisms of both cell-mediated immunity and humoral immunity have been shown to kill tumor cells in vitro. It is not clear which of these mechanisms may contribute to protective immune responses against tumors. Tumor immunologists are trying to enhance these effector mechanisms in ways that are tumor specific.

The principal mechanism of tumor immunity is killing of tumor cells by CD8⁺ CTLs., Tumor-specific CTLs can be isolated from animals and humans with established tumors, such as melanomas. Furthermore, mononuclear cells derived from the inflammatory infiltrate in human solid tumors, called tumor-infiltrating lymphocytes (TILs), also include CTLs with the capacity to lyse the tumor from which they were derived. The role of CD4⁺ helper T cells in tumor immunity is less clear. CD4⁺ cells may play a role in anti-tumor immune responses by providing cytokines for effective CTL development. In addition, helper T cells specific for tumor antigens may secrete cytokines, such as TNF and IFN-g, which can increase tumor cell class I MHC expression and sensitivity to lysis by CTLs. IFN-g may also activate macrophages to kill tumor cells.

How do tumors stimulate CD8⁺ T cell responses specific for tumor antigens? Most tumor cells are not professional APCs, and cannot stimulate naïve T cells on their own. A likely possibility is cross priming: tumor cells or their antigens are picked up by host APCs, the tumor antigens are then processed inside the APCs, and peptides derived from these antigens are displayed bound to class I MHC molecules for recognition by CD8⁺ T cells. The professional APCs express costimulators that may provide the signals needed for the differentiation of the CD8⁺ T cells into anti-tumor CTLs, and the APCs express class II MHC molecules that may activate CD4⁺ helper T cells as well (Fig. 17-3).

Tumor-bearing hosts may produce antibodies against various tumor antigens. For example, patients with EBV-associated lymphomas have serum antibodies against EBV-encoded antigens expressed on the surface of the lymphoma cells. Antibodies may kill tumor cells by activating complement, or by antibody-dependent cell-mediated cytotoxicity in which Fc receptor-bearing macrophages or NK cells mediate the killing. However, there is little evidence for effective humoral immunity against tumors.

Natural killer (NK) cells kill many types of tumor cells, especially cells that have reduced class I MHC expression and can escape lysis by CTLs. In vitro, NK cells can lyse virally infected cells and certain tumor cell lines, especially hematopoietic tumors. NK cells respond to the absence of class I MHC molecules, and, some tumors lose expression of class I molecules, presumably as an adaptation to escape CTL lysis. This makes the tumors particularly good targets for NK cells. In addition, NK cells can be targeted to IgG antibody-coated cells by Fc receptors. The tumoricidal capacity of NK cells is increased by cytokines, including interferons, interleukin-2 (IL-2), and interleukin-12, and the anti-tumor effects of these cytokines are partly attributable to stimulation of NK cell activity. IL-2-activated NK cells, called lymphokine-activated killer (LAK) cells, are derived by culturing peripheral blood cells or TILs from tumor patients with high doses of IL-2. The use of LAK cells in adoptive immunotherapy of tumors will be discussed later.

The role of NK cells in tumor immunity in vivo is unclear. It has been suggested that the reason why T cell-deficient mice do not have a high incidence of spontaneous tumors is because they have normal numbers of NK cells that serve an immune surveillance function. A few patients have been described with deficiencies of NK cells and increased incidence of EBV-associated lymphomas.

In vitro, activated macrophages can lyse many tumor cells more efficiently than normal cells. How macrophages are activated by tumors is not known. The mechanisms of macrophage killing of tumor target cells are probably the same as the mechanisms of macrophage killing of infectious organisms. TNF may kill tumors by direct toxic effects and indirectly by effects on tumor vasculature. Direct toxicity is mediated by binding of TNF to receptors on tumor cells, which activates a signaling pathway that results in tumor cell apoptosis. The relative importance of these mechanisms of TNF-induced tumor killing may vary in different tumors.

Malignant tumors have mechanisms that enable them to evade or resist host immune responses. The process of evasion, often called tumor escape, may be a result of several mechanisms.

• Class I MHC expression may be down-regulated on tumor cells so that they cannot be recognized by CTLs..
• Tumors lose expression of antigens that elicit immune responses.
• Tumors may fail to induce CTLs because most tumor cells do not express costimulators or class II MHC molecules.
• The products of tumor cells may suppress anti-tumor immune responses. An example of an immunosuppressive tumor product is transforming growth factor-b
• Tumor antigens may induce specific immunologic tolerance.
• The cell surface antigens of tumors may be hidden from the immune system by glycocalyx molecules, such as sialic acid-containing mucopolysaccharides.

Immunotherapy has the potential of being the most tumor-specific treatment that can be devised to treat cancer patients. Immunotherapy for tumors is aimed at augmenting the weak host immune response to the tumors (active immunity), or at administering tumor-specific antibodies or T cells, a form of passive immunity. In this section, we describe some of the modes of tumor immunotherapy that have been tried in the past or are currently being investigated.

Vaccination with Tumor Cells and Tumor Antigens

Immunization of tumor-bearing individuals with killed tumor cells or tumor antigens may result in enhanced immune responses against the tumor. The identification of peptides recognized by tumor specific CTLs and the cloning of genes that encode tumor-specific antigens recognized by CTLs have provided many candidates for tumor vaccines.

• Immunization with purified tumor antigens with adjuvants.
• Immunization with professional APCs, such as dendritic cells, that are purified from the patients and either incubated with tumor antigens or transfected with genes encoding these antigens
• DNA vaccines expressing tunor antigens.

The cell-based and DNA vaccines may be the best ways to induce CTL responses, because the cells and DNA are taken up by APCs and the encoded antigens are synthesized in the cytoplasm and enter the class I MHC pathway of antigen presentation.

The development of virally induced tumors can be blocked by preventive vaccination with viral antigens or attenuated live viruses.This approach is successful in reducing the incidence of feline leukemia virus-induced hematologic malignancies in cats and in preventing the herpesvirus-induced lymphoma called Marek's disease in chickens. In humans, the ongoing vaccination program against the hepatitis B virus (HBV) may reduce the incidence of hepatocellular carcinoma, a liver cancer associated with HBV infection.

Augmentation of Host Immunity to Tumors With Cytokines and Costimulators

Cell-mediated immunity to tumors may be enhanced by expressing costimulators and cytokines in tumor cells, and by treating tumor-bearing individuals with cytokines that stimulate the proliferation and differentiation of T lymphocytes and NK cells.

• Animal experiments in which tumor cells are transfected with genes that encode B7 costimulatory molecules.
• Therapeutic trials in which a sample of a patient's tumor is propagated in vitro, transfected with costimulator genes, irradiated, and reintroduced into the patient..
• Tumor cells may be transfected with cytokine genes in order to localize the cytokine effects to where they are needed. When rodent tumors transfected with IL-2, IL-4, IFN-g, or GM-CSF genes are injected into animals, the tumors are rejected or begin to grow and then regress. The injection of cytokine-secreting tumors induces specific, T cell-mediated immunity to subsequent challenges by unmodified tumor cells. Thus, the local production of cytokines may augment T cell responses to tumor antigens, and cytokine-expressing tumors may act as effective tumor vaccines. Several clinical trials with cytokine gene-transfected tumors are underway in patients with advanced cancer.

Cytokines may also be administered systemically for the treatment of various human tumors. The largest clinical experience is with IL-2 administered in high doses alone or in conjunction with adoptive cellular immunotherapy (discussed later). After administration of IL-2, there are increased numbers of blood T and B lymphocytes and NK cells, an increase in NK cell activity, and increases in serum TNF, IL-1, and IFN-g. The limitation of this treatment is that it can be highly toxic, causing fever, pulmonary edema, and vascular shock. There is great interest in the potential of IL-12 to enhance anti-tumor T cell- and NK cell-mediated immune responses, and early trials in advanced cancer patients are now under way. Hematopoietic growth factors, including GM-CSF, G-CSF and IL-11, are used in cancer treatment protocols to shorten periods of neutropenia and thrombocytopenia after chemotherapy or autologous bone marrow transplantation.

Nonspecific Stimulation of the Immune System

Immune responses to tumors may be stimulated by local administration of inflammatory substances or by systemic treatment with agents that function as polyclonal activators of lymphocytes.

• Bacille Calmette-Guerin (BCG) mycobacterium, as an adjuvant at the sites of tumor growth.
• Activating anti-CD3 antibodies, in mice, results in polyclonal activation of T cells and, concomitantly, prevention of tumor growth. Early clinical trials with anti-CD3 antibody in patients with disseminated tumors are ongoing.

Passive immunotherapy involves the transfer of immune effectors, including tumor-specific T cells and antibodies, into patients. Passive immunization against tumors is rapid, but does not lead to long-lived immunity. Several approaches for passive immunotherapy are being tried, with variable success.

Adoptive Cellular Therapy

Adoptive cellular immunotherapy is the transfer of cultured immune cells that have anti-tumor reactivity into a tumor-bearing host. The cells to be transferred are expanded from the lymphocytes of patients with the tumor. One way of doing this is to generate lymphokine activated killer (LAK) cells by removing peripheral blood leukocytes from tumor patients, culturing the cells in high concentrations of IL-2, and injecting the LAK cells back into the patients. LAK cells are derived mainly from NK cells. Adoptive therapy with autologous LAK cells, in conjunction with in vivo administration of IL-2 or chemotherapeutic drugs, has yielded impressive results in mice, with regression of solid tumors. Human LAK cell therapy trials have so far been largely restricted to advanced cases of metastatic tumors, and the efficacy of this approach appears to be variable from patient to patient. A variation of this approach is to isolate tumor infiltrating lymphocytes (TILs) from the inflammatory infiltrate present in and around solid tumors, obtained from surgical resection specimens, and expand the TILs by culture in IL-2. The rationale for this approach is that TILs may be enriched for tumor-specific CTLs and for activated NK cells. Human trials with TIL therapy are ongoing.

Therapy with Anti-Tumor Antibodies

Tumor-specific monoclonal antibodies may be useful for specific immunotherapy of tumors. The potential of using antibodies as "magic bullets" has been alluring to investigators for many years and is still a very active area of research. Anti-tumor antibodies eradicate tumors by the same effector mechanisms that are used to eliminate microbes, including opsonization and phagocytosis, and activation of the complement system. A monoclonal antibody specific for the oncogene Her-2/Neu, which is expressed at high levels in some tumors, has shown success in breast cancer patients, and is now approved for clinical use. The use of "humanized" antibodies helps avoid anti-antibody responses by the patient. Failures often attributed to outgrowth of antigen loss variants of the tumor cells that no longer express the antigens that the antibodies recognize.

Tumor-specific antibodies may be coupled to toxic molecules, radioisotopes, and anti-tumor drugs, to promote delivery of these cytotoxic agents specifically to the tumor. Toxins such as ricin or diphtheria toxin are potent inhibitors of protein synthesis and can be effective at extremely low doses if they are carried to tumors attached to anti-tumor antibodies; such conjugates are called immunotoxins. Because of toxicity, interaction with host Fc receptors, antibody responses to reagents, etc., clinical trials of immunotoxins have been of variable and modest success.

Anti-idiotypic antibodies have been used to treat B cell lymphomas that express surface Ig with particular idiotypes. The approach has not proved generally successful, largely because of the selective outgrowth of tumor cells with altered idiotypes that do not bind the anti-idiotypic antibody.

Anti-tumor antibodies are also used to remove cancer cells from the bone marrow prior to autologous marrow transplantation. In this protocol, some of the patient's bone marrow is removed, and the patient is given lethal doses of radiation and chemotherapy, which destroy tumor cells as well as the remaining normal marrow cells in the patient. The bone marrow cells removed from the patient are treated with antibodies or immunotoxins specific for tumor antigens, to kill the tumor cells. The treated marrow, having been purged of tumor cells, is transplanted back into the patient to reconstitute the hematopoietic system destroyed by irradiation and chemotherapy.

• Tumors express antigens that are recognized by the immune system, but most tumors are weakly immunogenic, and immune responses often fail to prevent the growth of tumors. The immune system can be stimulated to effectively kill tumors.
• Tumor antigens recognized by CTLs are the principal inducers of and targets for anti-tumor immunity. These antigens include mutants of oncogenes and other cellular proteins, normal proteins whose expression is dysregulated or increased in tumors, and products of oncogenic viruses.
• Antibodies specific for tumor cells recognize antigens that are used for diagnosis and are potential targets for antibody therapy. These include oncofetal antigens, which are expressed normally during fetal life and whose expression is deregulated in some tumors, and molecules that are normally expressed on the cells from which the tumors arise, and are thus differentiation antigens for particular cell types.
• Immune responses that are capable of killing tumor cells consist of CTLs, NK cells, and activated macrophages. The role of these immune effector mechanisms in protecting individuals from tumors is not well defined.
• Tumors evade immune responses by several mechanisms, including down-regulating the expression of MHC molecules, selecting cells that do not express tumor antigens, producing immunosuppressive substances, and inducing tolerance to tumor antigens.
• Immunotherapy of tumors is designed to augment active immune responses against these tumors, or to administer tumor-specific immune effectors to patients. Immune responses may be actively enhanced by vaccination with tumor cells or antigens, administration of tumors modified to express high levels of costimulators or cytokines that stimulate T cell proliferation and differentiation, and systemic administration of cytokines. Approaches for passive immunotherapy include the administration of anti-tumor antibodies, antibodies conjugated with toxic drugs (immunotoxins), and tumor-reactive T cells and NK cells isolated from patients and expanded by culture with growth factors.