Tumor antigens can be divided into tumor-specific antigens and tumor-associated antigens. Tumor-specific antigens are not found on normal tissue, and they permit the host to recognize a tumor as foreign. Tumor-specific antigens have been shown to exist in oncogenesis models utilizing chemical, physical, and viral carcinogens but appear to be less common in models of spontaneous tumor development.
The identification of tumor-specific antigens led to the theory of immune surveillance, which suggests that the immune system is continuously trolling for foreign (tumor-specific) antigens. This theory is supported by the observation that at least some cancers are more common in immune-suppressed patients such as transplant patients or human immunodeficiency virus–infected individuals. However, many cancers are not overrepresented in these patient populations. Furthermore, spontaneous tumor models, which more closely resemble human carcinogenesis, appear to have a less extensive repertoire of tumor-specific antigens but instead have been found to express many tumor-associated antigens.
Tumor-associated antigens are found in normal cells but either become less prevalent in normal tissue after embryogenesis (eg, alpha-fetoprotein [AFP]) or remain present in normal tissue but are overexpressed in cancer cells (eg, prostate-specific antigen [PSA]). In either case, the more ubiquitous nature of these antigens appears to cause reduced immune reactivity (also known as tolerance) to the specific antigen. The mechanisms of tolerance are complex and may be due in part to the absence of other required costimulatory molecules (such as B7, a molecule required for T-cell stimulation). Recent evidence has also implicated a number of immune checkpoints that result in downregulation of the cellular immune response. In particular, two molecules, CTLA-4 and PD-1, have been identified on activated lymphocytes that dampen the immune response, and therefore have been exploited as potential therapeutic targets.
The development of monoclonal technology has allowed the development of many antibodies against many tumor-associated antigens and has provided insight into the regulation and expression of these antigens. The reexpression or upregulation of these tumor-associated antigens during carcinogenesis may lead to immune response (or loss of tolerance). Many novel therapeutic approaches have sought to break this tolerance, and approaches to enhance a patient's immune response will be discussed.
A large number of monoclonal antibodies have been developed against a variety of tumor-associated antigens. Oncofetal antigens such as AFP and beta-human chorionic gonadotropin (β-hCG) are important markers in germ cell tumors. β-hCG is also expressed in a small percentage of patients with bladder carcinoma. Antibodies directed against specific targets such as vascular endothelial growth factor (VEGF) have been developed and have been tested for the treatment of advanced prostate cancer, renal cell carcinoma (RCC), and transitional cell carcinoma (TCC).
Antibodies in Cancer Diagnosis and Detection
Immunoassays are used to test both body fluids and tissues for the presence of tumor-associated antigens. In the urologic cancers, the most obvious example is the development of monoclonal antibodies against PSA. The utility and limitations of PSA are described elsewhere in this volume. Other antigens that have been tested in prostate cancer include prostatic acid phosphatase, which has largely been replaced by PSA in screening programs and in patients with low tumor burden. Prostatic acid phosphatase may be of some use in detecting or following bone metastases and as a predictive marker of response to therapy for metastatic disease. Antibodies to prostate-specific membrane antigen (PSMA) have been used, primarily for immunohistochemistry.
Unfortunately, there are as yet no well-established antigens (or antibodies) that can be used to reliably evaluate and monitor RCC, although a variety of target antigens are being evaluated.
Two oncofetal antigens, β-hCG and carcinoembryonic antigen, are expressed by a minority (≤20%) of TCCs. These markers are not routinely used, but in diagnostic dilemmas, measurement of serum levels of β-hCG or staining of tissue for this antigen may be useful.
As described in Chapter 23, antibodies to hCG and AFP are routinely used to detect shed antigens from germ cell tumors in the bloodstream. These antigens can also be detected in tissue samples. While the use of serum markers in germ cell tumors is reviewed elsewhere, it is worth noting that the presence of the oncofetoprotein AFP, either in serum or in tissue specimens, is pathognomonic for a nonseminomatous germ cell tumor, regardless of results of routine pathologic evaluation. In addition to their diagnostic utility, AFP and hCG can be used as markers of response to therapy and as predictive factors of outcome. For example, the international germ cell tumor risk classification schema for patients with metastatic disease relies heavily on AFP and hCG levels as well as levels of a nonspecific marker, lactate dehydrogenase, to assign patients with nonseminomatous germ cell tumors to one of three risk levels (see Chapter 23).
Monoclonal antibodies to a specific antigen can be radiolabeled, and the preferential binding of the monoclonal antibody to tumor cells can be exploited. Theoretically, such an approach could be used for the presurgical evaluation of disease, postsurgical evaluation for minimal residual disease, confirmation of cancer identified by other imaging modalities, and detection of recurrent disease. There are several potential impediments to successful tumor radioimmunodetection. These include dilution of antibody in the bloodstream; metabolism of the antibody; nonspecific binding in liver, reticuloendothelial system, bone marrow, and elsewhere; binding of antibody by circulating or shed antigen; and the development of neutralizing human antimouse antibodies.
The only radioimmunodetection system for urologic cancers at this time is 111In-capromab pendetide (Prostascint), a murine monoclonal antibody to PSMA. Its use has been hampered by a fairly laborious administration process, operator dependence in interpretation of scans, and a less than satisfactory positive predictive value. The use of 111In-capromab pendetide is described in Chapter 10.
Immunotherapy with Monoclonal Antibodies
Immunotherapy with monoclonal antibodies alone (“naked antibodies”) has been fairly extensively evaluated. The use of monoclonal antibodies against tumor-associated antigens has met with only limited success in patients with solid tumors including prostate and kidney cancer. In lymphoproliferative disorders such as leukemia and lymphoma, some antibodies to tumor-associated surface antigens appear to result in tumor cell death. The mechanism for these effects is certainly multifactorial but may, in part, be mediated by resultant complement fixation.
Direct antiproliferative effects of antibodies on cancer cells can be achieved by antibodies against functionally important antigens. Thus, the inhibition of growth factors and growth factor receptors and the activation or inhibition of signal transducing molecules are attractive therapeutic targets. In urologic cancers, while no approved monoclonal antibody therapy exists, trials of antibodies against growth factors, VEGF (an angiogenic molecule), and signal transduction molecules are being undertaken. Kidney cancer is highly dependent on angiogenesis, and bevacizumab (an antibody agent against VEGF) has been shown to prolong time to progression (TTP) in metastatic disease. Two independent randomized phase 3 trials have demonstrated the superiority of alpha-interferon plus bevacizumab over alpha-interferon alone, with an improvement in TTP, leading to approval by the U.S. Food and Drug Administration (FDA) of bevacizumab for this indication. By contrast, in prostate cancer patients with progressive disease despite androgen deprivation therapy, the addition of bevacizumab to conventional chemotherapy with docetaxel failed to demonstrate a prolongation in overall survival, (although TTP was improved).
An alternative approach to naked antibodies is to conjugate any of a variety of cytotoxic agents to an antibody. The advantage of this approach is a “bystander effect,” making it unnecessary to use an antibody that binds each and every cell. This can be achieved in a variety of ways. The most straightforward is to use the monoclonal antibody as a means of providing some targeting specificity for the cytotoxic agent used. Cytotoxic agents used include radioisotopes, chemotherapy, and toxins such as ricin. Other means of providing some specificity is to bind a prodrug (with an antibody) to the tumor site and then to activate the bound prodrug. Finally, targeting with bispecific antibodies (eg, to antigen and to an effector T cell, or to antigen and toxin) has been undertaken. These approaches have all been tested in prostate cancer, but all remain investigational at this point.
There is considerable evidence, both clinical and preclinical, that tumor-associated antigens can elicit a cell-mediated immune response. In some models, when carcinogen-induced tumors in mice are resected and the mouse is reinoculated with tumor cells, the tumor fails to regrow, suggesting the development of immunity to specific antigens. Specific antigens that are rejected in immunized hosts are termed transplantation antigens. The specificity of tumor rejection has since been demonstrated to reside in T lymphocytes (at a minimum). Lymphocytes of cancer patients can sometimes be stimulated in vitro to recognize specific tumor-associated antigens and consequently demonstrate properties of cytolytic T lymphocytes. Unfortunately, the phenomenon of tumor rejection is by no means universal, either in the laboratory or clinically, and it is unusual to detect cytolytic T-lymphocyte activity against many tumor-associated antigens.
Nevertheless, there are several clinical scenarios that suggest that cell-mediated antitumor responses exist. These observations have promoted a broad search for the means of enhancing patients' immune responses to tumor-associated antigens. RCC is in many ways the prototypical immune-mediated tumor and, along with melanoma, has until recently been the primary target of immune manipulations.
Immunotherapy Involving Cell-Mediated Immunity
Evidence of cell-mediated immunity playing a role in tumor rejection lies in the results of a variety of immunotherapeutic interventions. Immunotherapy can be broadly classified as active or passive. This classification refers to the role the host's immune system plays. Thus, the passive transfer of preformed antibodies is contrasted to a vaccination program in which the host's immune system must be capable of mounting an immune response. Adoptive therapy refers to a middle ground in which efforts are made to reconstitute, modify, or bolster one of the effector cells involved ex vivo, followed by reinfusion into the patient, where the rest of the immune cascade must then be recruited.
Active Immunotherapy: Vaccination
Autologous vaccination programs (the vaccination of patients with their own tumor cells) have been extensively explored. The advantage of autologous vaccination is that the vaccine bears the antigens of the patient's tumor, although the distinct disadvantage is that not every patient has a tumor available for vaccine preparation, and the preparation of each vaccine is tremendously labor intensive. By contrast, allogeneic vaccines (the use of a generic vaccine or “off-the-shelf” antigen) have the benefit of mass production and ease of use, and the identification of specific tumor rejection antigens allows specific antigenic targeting. However, this approach runs the risk of a more narrow shared antigenic spectrum with the patient's tumor. Both autologous and allogeneic vaccination strategies have been evaluated in RCC and prostate cancer.
Several means exist to undertake vaccination. The simplest is to use intact but inactivated tumor cells. Inactivation can be achieved with ultraviolet radiation, external beam (photon) radiation, or freeze-thawing. Crude extracts of cells can also be used. The advantage of using cell extracts is that inactivation is not necessary and small particles and proteins that might be more easily phagocytosed are available. One can also enhance the immunogenicity of inoculated cells by growing the cells in cytokines, coinjecting with cytokines (nonspecific active immunotherapy, described later), or transfecting these cells with the genes for immune stimulatory cytokines or the costimulatory molecule B7. Clinical trials using prostate cancer cell lines transfected with the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene (GVAX; Cell Genesys, South San Francisco, CA) for vaccination in patients with metastatic hormone refractory prostate cancer failed to show a therapeutic benefit. Purified protein or peptides represent a second potential vaccination schema. A trial of PSA in a vaccina and fowlpox (ProstaVax) vector demonstrated clinical activity, and confirmatory trials are underway. A third way of undertaking specific vaccination is to attempt to bypass the antigen-presenting function of the immune system and to directly stimulate professional antigen-presenting cells, such as dendritic cells, ex vivo. These cells can be stimulated by pulsing them with protein or peptides of interest or by transfecting them with a gene encoding the antigenic peptide of interest before reinfusion. Sipuleucel-T is an autologous dendritic cell product that has been shown to prolong life, and it is appropriate for patients with castration-resistant metastatic prostate cancer who do not have cancer-associated pain, visceral metastases, rapidly progressive disease, or the need for systemic steroids.
Nonspecific Active Immunotherapy: Cytokines and Biologic Response Modifiers
Bacillus Calmette-Guérin (BCG) is a live attenuated form of tubercle bacillus that appears to have local activity against some tumors but has been largely disappointing as systemic therapy. The utility of BCG in the treatment of superficial bladder cancer is well described and is beyond the scope of this chapter. The mechanism by which BCG can elicit a local immune response in the uroepithelium and thereby exhibit impressive anticancer activity is not well delineated. However, possible mechanisms of action include macrophage activation, lymphocyte activation, recruitment of dendritic cells, and natural killer cells. It is intriguing that this is strictly a local phenomenon and that BCG has no role in the treatment of muscle-invasive or metastatic disease.
Interleukin-2 (IL-2) is a naturally occurring cytokine that has multiple immunoregulatory properties. The observation that exogenously administered IL-2 could result in tumor regression in patients with RCC and melanoma was the first unequivocal indication that cancer regression could be mediated by immune manipulations. IL-2 stimulates lymphocyte proliferation, enhances cytolytic T-cell activity, induces natural killer cell activity, and induces gamma-interferon and tumor necrosis factor production. IL-2 has no direct cytotoxicity, but when administered exogenously will activate effector cells of the host immune system, including lymphocytes, natural killer cells, lymphokine-activated killer cells, and tumor-infiltrating lymphocytes. IL-2 has been administered in RCC in several different schemas, including high-dose intravenous bolus (IL-2 is FDA approved with this schedule), continuous intravenous infusion, and at lower doses subcutaneously. The high-dose regimens must be administered on an inpatient basis and are characterized by significant, albeit manageable, toxicities, including fever; malaise; vascular leak syndrome; hypotension; and cardiac, renal, and hepatic dysfunction. Subcutaneous IL-2 is self-administered by patients in the outpatient setting, and while clearly less toxic, still has associated malaise and constitutional symptoms. The optimal dosing regimen is not well established, and overall response proportions rarely exceed 20%. Durable complete responses of 5–8% have been reported with some of the high-dose regimens. IL-2 has also been combined with other active agents such as alpha-interferon and chemotherapy, although it is not clear if these combinations provide additional benefit. High-dose IL-2 is, to date, the only therapy of inducing durable complete remissions and potential cures. However, its toxicity profile makes this approach realistic for very carefully selected young patients with an excellent performance status, limited extent of metastases, and no significant comorbidities.
Alpha-interferon is a naturally occurring cytokine that has direct cytotoxic and possibly antiproliferative properties, but it also has immunoregulatory properties. It enhances major histocompatibility complex expression, thereby potentially increasing the efficiency of antigen processing and recognition. Alpha-interferon has anticancer activity in both RCC and superficial bladder cancer. Its primary toxicity is fever, malaise, and constitutional symptoms, although at higher doses, it can result in bone marrow toxicity, central nervous system toxicity, and hepatic toxicity. In RCC, as a single agent, alpha-interferon can result in clinical responses in up to 20% of patients. In contradistinction to IL-2 as a single agent, durable complete responses are quite rare. Nevertheless, in randomized trials, alpha-interferon appears to confer a modest survival advantage over other agents now known to be largely inactive. As noted earlier, the addition of bevacizumab to alpha-interferon is superior to alpha-interferon alone. Alpha-interferon is also used as an intravesical treatment in superficial bladder cancer, where it has established activity, and is not infrequently used as second-line therapy after BCG. A recent trial of BCG plus interferon failed to demonstrate superiority over BCG alone in patients with superficial bladder cancer.
GM-CSF is perhaps the most important cytokine in eliciting cellular immune responses. Administered systemically as a subcutaneous injection, GM-CSF has been shown to reduce PSA in patients with both hormone-sensitive and hormone-resistant prostate cancer. However, the use of GM-CSF is neither proven to be of clinical benefit nor approved for this indication and must be considered investigational.
A myriad of immunosuppressive factors exist within cancer patients that may serve to dampen antitumor immune responses. Some of these molecules represent natural pathways to inhibit autoimmunity, while some molecules may have been usurped by the tumor to evade immune recognition. Novel approaches are now being developed to target these pathways. For example, CTLA-4 is an inhibitory molecule that blocks activation of T cells. By preventing the action of CTLA-4, an anti-CTLA-4 antibody (ipilimumab) can augment and prolong T-cell immune responses. In animal models, ipilimumab can induce tumor rejection in immunogenic tumors and, in combination with antitumor vaccination, can induce rejection of minimally immunogenic tumors, including in the transgenic adenocarcinoma of mouse/prostate (TRAMP) prostate cancer model. Ipilimumab has been demonstrated to have modest anticancer activity as monotherapy in patients with metastatic CRPC, although it is not yet approved. The combination of CTLA-4 blockade with vaccination is of interest and is under investigation. The potential benefits of CTLA-4 blockade need to be balanced against not insignificant autoimmune toxicity, which can, rarely, result in colitis, dermatitis, hepatitis, and hypophysitis (panhypopituitarism).
Adoptive immunotherapy is the transfer of cellular products (effector cells) to the host or patient in an effort to develop an immune response. The use of adoptive immunotherapy was prompted by the observation that T cells derived from patients with melanoma or RCC had the ability to recognize antigens on the primary tumor. Thus, it was hoped that these cells could be harvested, activated ex vivo, and then reinfused into patients. Lymphokine-activated killer cells and tumor-infiltrating lymphocytes have been used to treat patients with metastatic RCC in the investigational setting, frequently along with IL-2. However, randomized trials comparing IL-2 alone with IL-2 plus cellular products have failed to demonstrate an improvement in response proportions or survival. Chapter 22 gives specific details of immunotherapy in RCC.