INSTITUTE OF BIOMEDICAL SCIENCES (ICBM) “PROF. DR. EDUARDO CRUZ-COKE LASSABE” DISCIPLINARY PROGRAM OF IMMUNOLOGY Clinical Hospital of UNIVERSIDAD DE CHILE PHASE II STUDY OF IMMUNIZATION WITH DENDRITIC CELLS AND LOW DOSE OF INTERLEUKIN-2, IN PATIENTS WITH ADVANCED MELANOMA. Flavio Salazar Carlos Ferrada Mercedes López Miguel Fodor Milton Larrondo Juan Honeyman Alejandro Escobar Santiago, Julio de 2001 Cancer continues to be one of the leading causes of death, especially among inhabitants of highly developed countries. Modern surgical and transplant techniques, new and more effective drugs, and improved radiation methods have yielded promising results in treating some of these conditions. However, some malignant cells often survive these therapies and spread throughout the body, necessitating the development of new treatments as an alternative or complement to current ones. One such approach would be manipulating the immune system to combat tumors, known as cancer immunotherapy. The use of modified immune system cells for oncological treatment, such as dendritic cells (DCs), is well-documented by international studies that have tested and are exploring various clinical protocols. Over the past five years, accumulated evidence has demonstrated that modified dendritic cell vaccines produce significant clinical and immunological responses in diseases such as malignant melanoma, non-Hodgkin's lymphoma, and other neoplasms in stages of the disease where no practical alternative therapy exists. On the other hand, experimental therapies based on Interleukin-2 (IL-2) have been used with relative success in some neoplasms since 1985. However, problems related to the toxicity of IL-2 are still under investigation. Although recombinant human IL-2 like PROLEUKIN has proven to be one of the most effective strategies in treating advanced renal carcinoma and melanoma, therapy based on high-dose intravenous IL-2 is not suitable for all patients. Our direct experience, based on clinical studies conducted at the Karolinska Institute in Sweden and international experience with PROLEUKIN, indicates that low doses (2.4x106 IU/m2) administered subcutaneously maintain the same beneficial effects for patients while almost wholly eliminating adverse effects. Therefore, we have decided to present to the respective committee a second protocol, leveraging the experience gained and the recent funding obtained from FONDEF, to develop an optimized protocol that alternates dendritic cell vaccines with the use of low doses of IL-2 as a complement to strengthen the immune system. For some time now, we have established a multidisciplinary group of professionals at the Faculty of Medicine of the University of Chile, involving the participation of preclinical and clinical researchers. We have recently completed a phase I clinical protocol for treating malignant melanoma using DCs loaded with tumor extracts. This study, the first of its kind in Chile and probably in Latin America, serves as the basis for the presentation of this project. The results can be summarized as follows: No adverse effects were observed from the subcutaneous application of dendritic cells, except for one patient who showed mild fever and arthralgia. Immunological effects were observed in vitro in 60% of the treated patients through an increase in specific T lymphocyte precursors against tumor cells in the patient's blood measured by ELISPOT, in addition to delayed-type hypersensitivity reactions (DTH) in 50% of the treated patients when tumor extract was injected subcutaneously after vaccinations. Finally, partial regression or mixed response was observed in two patients. One showed regression of lung metastases, and the other showed regression of cutaneous metastases. In most cases, disease stability was observed, although it cannot be concluded that a significant relationship exists between vaccination and the survival of treated patients. These results motivate further exploration of the methodology, primarily intending to provide therapeutic alternatives to patients with no other options. Theoretical Formulation of the Project Melanoma is a malignant tumor of neuroectodermal origin whose incidence has progressively increased in the last century. The only way to cure a malignant melanoma is early detection and appropriate surgical treatment, as once it reaches an advanced state, it is highly resistant to conventional treatments such as surgery, radiotherapy, and chemotherapy (1-3). Globally, melanoma is the fastest-growing and most deadly type of skin cancer in Europe and the United States among various cutaneous cancer types. This situation is also observed in Chile; only in Santiago, between 1992 and 1996, 318 malignant melanoma cases were detected, representing 10.6% of all types of skin cancer (1). While it is true that malignant melanoma is almost entirely curable (>95%) when detected early, the survival probability in patients with metastatic melanoma decreases significantly (<15%). Although surgery, chemotherapy, and radiotherapy are the primary conventional methods for treating this disease, implementing new therapies is essential, especially in patients with stage III and IV melanoma, where chemotherapy and radiotherapy are ineffective. Cancer remains one of the leading causes of death, especially among residents of highly developed countries. Modern surgical techniques and transplants, new and more effective drugs, and improved irradiation methods have allowed promising results in treating some diseases. A clear example of this progress is reflected in the treatment of childhood leukemia with a new generation of anti-cancer drugs (2). Often, some malignant cells survive these therapies and spread throughout the body, necessitating the development of new treatments as alternatives or complements to current ones. One such approach is manipulating the immune system to combat tumors, known as cancer immunotherapy. Since the 1980s, when cytotoxic T lymphocytes (CTL) with antitumor activity were isolated from peripheral blood, lymph nodes, and tumor tissue of melanoma patients, there has been growing interest in using the immune system in cancer treatment. Cytokines such as Interleukin-2 (IL-2) and Interferon gamma (IFN-γ) are commonly used as alternative therapies (4,5) in some neoplasms, and adoptive therapies, using in vitro stimulated CTL, have been tested in some clinical protocols with varying responses (6). Over the last thirty years, significant progress has been made in understanding the functioning of the immune system, especially regarding antigen recognition (3,7). On the other hand, the significant advances in molecular biology techniques that facilitate gene cloning and the production of recombinant proteins have allowed for identifying tumor-associated antigens (AAT) and their subsequent characterization. Cytotoxic T Lymphocytes and Antitumor Immune Response Historically, pioneering experiments demonstrated that the growth of syngeneic tumors in mice could be prevented through prior immunization with the same tumor (8,9). Guided by these studies in animal models, it has been observed that human T lymphocytes have also shown the ability to lyse autologous tumors in vitro (10,11) specifically. Furthermore, T cells are capable of proliferating in response to stimulation with autologous tumor cells, secreting cytokines such as IL-2, interferon-gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-α) (12). Antitumor T cells can be expanded in large quantities in vitro and adoptively transferred to treat significant tumor burdens in mice and humans (6). These observations have enabled the identification of tumor antigens recognized by human autologous T cells using molecular cloning techniques (13,14). These results provide undeniable evidence of a T cell-mediated response against autologous tumors. Cytotoxic T lymphocytes (CTLs, CD8+) can recognize tumors through 8-10 amino acid peptide fragments derived from cytoplasmic or nuclear proteins associated with major histocompatibility complex (MHC) class I (15). Melanoma-specific CTLs, either adoptively transferred or activated in vivo by melanoma-associated antigen epitopes, have therapeutic potential, capable of inducing the regression of tumors and their micrometastases (16,17). Melanoma-Associated Antigens Over the past five years, several melanoma-associated antigens (MAAs) recognized by CTLs have been identified, and their peptide epitopes are characterized using genetic (13,14) and biochemical methods (18). The cloning of genes encoding human MAAs has allowed their characterization and classification into different types. The first category consists of so-called tumor-specific antigens, among which the MAGE gene family stands out (19-21). These antigens are derived from proteins whose genes are actively transcribed in various types of tumors but not in normal adult tissue cells and are related to the primary stages of embryonic development (19-21). Another tumor-specific antigen in melanoma consists of those originating from mutated genes. A few examples of such antigens have been described in melanoma, with one of them being derived from cyclin-dependent kinase 4 (CDK4) (22). The primary group of antigens, whose CTL response has been most frequently described in melanoma patients, comprises differentiation antigens, also known as tissue-associated antigens. This category includes melanosomal antigens MART-1/Melan A, gp100, and Tyrosinase (23-28). Genes encoding these antigens are expressed not only in melanomas but also in normal melanocytes and the pigmented epithelium of the retina, making them autoantigens (23-28). CTLs specific for these antigens can be isolated from lymph nodes, tumor-infiltrating lymphocytes (TILs), or peripheral blood lymphocytes (PBLs) of melanoma patients (26-28). This T-cell recognition has been interpreted as a breakdown of immunological tolerance against self-antigens, perhaps due to the overexpression of these proteins in tumor cells. Dendritic Cells: Key Professional Antigen-Presenting Cells A T cell-mediated response induction requires antigen presentation in a specialized cellular context provided by antigen-presenting cells (APCs) (14). These specialized cells trigger a primary immune response and include macrophages, B cells, and dendritic cells (DCs). DCs play a fundamental role in generating an antitumor T cell-mediated response, as they can capture tumor antigens and present them in association with MHC molecules (15–17). Additionally, they express many co-stimulatory molecules, which provide crucial signals ensuring the effectiveness of the T cell-mediated response (18). DCs, also called professional APCs, are strategically located at antigen concentration sites, efficiently internalizing, processing, and presenting soluble antigens in the context of MHC class I and II, being the most efficient in inducing a primary T cell response (19–21). DCs were first visualized in the skin as Langerhans cells in 1868, but their characterization began only 25 years ago. DCs are also potent stimulators of B cells and mediate the isotype switching of immunoglobulins. The main obstacles to the characterization and utilization of DCs (low relative frequency and the lack of specific cellular markers) have recently been overcome. Indeed, relatively high quantities of DCs can be obtained in vitro from peripheral blood monocytes stimulated with GM-CSF and interleukin-4 (IL-4) (29). The cells generated in this way have the same characteristics as immature primary DCs isolated in vivo. They have low expression of MHC and other co-stimulatory molecules like CD86 and CD40 but are highly well-equipped to capture antigens (29) through mechanisms that include endocytosis, pinocytosis, and membrane fusion (30). Interleukin-2 Interleukin-2 (IL-2) is a 15 kDa glycoprotein primarily synthesized and secreted by helper T lymphocytes (Th) as a result of activation mediated by mitogens or the interaction between the TCR/CD3 complex and the MHC complex on the surface of antigen-presenting cells (31-35). The response of T lymphocytes to activation is the induction of IL-2 production and the expression of its receptor (IL-2R) on the cell membrane, leading to the clonal expansion of T lymphocytes specific to the antigen. IL-2 can act as an autocrine factor, stimulating the expansion of the producing cells themselves, or as a paracrine factor, stimulating the activation of other immune system cells, such as CD8+ T lymphocytes, B cells (36,37), and natural killer (NK) cells (38-46). Furthermore, so-called lymphocyte-activated killer cells (LAK) (47,48), used in adoptive therapies, are believed to derive from NK cells and CD8+ lymphocytes activated by IL-2 in vitro. Other cellular subtypes influenced by IL-2 include neutrophils (49,50), monocytes (51), and gamma-delta T lymphocytes (52), which have shown increased activation, enhanced effector functions, and improved cell survival. IL-2 was first biologically characterized in JURKAT cells, a human leukemia cell line. In its natural state, IL-2 is differentially glycosylated, and these changes in glycosylation levels explain the variations in its molecular weight, ranging from 15 to 18 kDa (53). The IL-2 gene was later cloned and expressed in eukaryotic cells (53) and E. coli (54). The recombinant analog of IL-2 is known as PROLEUKIN® (aldesleukin). Given the pivotal role of IL-2's interaction with IL-2R in mediating the immune response, it is evident that research and manipulation of this system for therapeutic or diagnostic purposes are crucial. IL-2 has shown promising effects as an anti-cancer drug due to its ability to activate and proliferate LAK cells and tumor-infiltrating lymphocytes (TILs) (55-58). IL-2-mediated effects observed in renal carcinoma and melanoma since 1985 have earned recent approval by the United States Food and Drug Administration (FDA) for treating these neoplasms (59-63). However, issues related to IL-2 toxicity are still under investigation (64). Although PROLEUKIN IL-2 has proven to be one of the most effective strategies in treating advanced renal carcinoma and melanoma, high-dose intravenous IL-2 therapy is unsuitable for all patients. Its use is restricted to adult patients in good overall health. The basic biology of IL-2-based strategies for treating infectious diseases, transplants, and AIDS has been reviewed recently (65,66). Since 1985, PROLEUKIN IL-2 has been administered to thousands of patients. As experience with PROLEUKIN IL-2 has grown over the years, treating physicians have become familiar with most of the side effects that have arisen and have learned to manage them appropriately. In most patients, these effects occur during treatment and are controlled within seventy-two hours after treatment completion. Therefore, it is recommended that intravenous IL-2 administration be carried out with the patient hospitalized. Most of the side effects of IL-2 use described in the literature are based on experiences with high doses (18 million units per square meter of body surface area) and intravenous administration, as reviewed in Table 1. Our direct experience, based on clinical studies conducted at the Karolinska Institute in Sweden (67), as well as international experience (68-70), indicates that low doses (2.4 million units per square meter of body surface area) and subcutaneous administration maintain the same beneficial effects for patients while nearly eliminating adverse effects (67-70). Significance of these Studies Several preclinical studies have documented the ability of certain genetically modified tumors to induce protective immunity against a challenge of native tumors (71-73). Based on this data and shared antigens recognized by CTLs, several clinical studies have been developed using vaccines with modified allogeneic tumor lines expressing cytokines such as IL-2 or co-stimulatory molecules like B7-1 (74). There is ample evidence and substantial preclinical and clinical data indicating the importance of T cell-mediated responses in immune-dependent tumor regression (6,9-12). These results have stimulated research, leading to the development of vaccines against defined antigens. Several vectors containing defined antigens, such as MAGE1, MAGE3, gp100, and Tyrosinase, have been produced for clinical studies. Due to the lack of knowledge about the best immunization methods and the absence of appropriate animal models, various immunization protocols have been clinically tested. Recently, therapies based on immunization with peptides derived from the gp100 antigen modified to increase their HLA affinity (75) or dendritic cell vaccines pulsed with peptides (76) or melanoma extracts (77) have been shown to induce specific CTL responses in most treated melanoma patients, along with objective regressions of their tumor masses (76-79). This type of therapy has also been used in other types of neoplasms. In the case of Follicular Lymphoma, the positive effects of chemotherapy have been combined with subsequent immunotherapy based on dendritic cells pulsed with anti-idiotypic antigens. The results in treating minimal residual disease have been more than satisfactory, with 8 out of 11 treated patients eliminating residual malignant cells after vaccination (80). Vaccination of Melanoma Patients Using DCs Loaded with Allogeneic Melanoma Extracts In Chile, we have completed the first phase I clinical trial for treating advanced malignant melanoma using dendritic cell vaccines loaded with allogeneic tumor extracts. Since chemotherapy and radiotherapy only offer palliative effects in advanced melanoma, resulting in an inferior prognosis for these patients, we intended to develop a protocol that would adapt to the reality of our country, allowing for a combination of high technology and low costs. In summary, the results obtained in the Phase I clinical trial, which were published in (Escobar et al. Clin. Exp. Immunol. 2005. doi:10.1111/j.1365-2249.2005.02948.x), can be summarized as follows: 1- No adverse effects to the subcutaneous application of dendritic cells were observed, except for one patient who showed mild fever and arthralgia, probably not associated with the vaccine, as shown in Table 2- Immunological effects were observed in vitro in 60% of the treated patients, through an increase in precursor T lymphocytes specific to tumor cells in the patient's blood, as measured by ELISPOT. 3- Delayed-type hypersensitivity reactions (DTH) were observed in 50% of the treated patients when tumor extract was injected subcutaneously after vaccinations. 4- Partial regression or mixed response were observed in 2 patients. One showed regression of lung metastases, and the other showed regression of cutaneous metastases (Table 3). In most cases, disease stability was observed, although it cannot be concluded that a significant relationship exists between vaccination and the survival of treated patients. The idea of this Phase I/II clinical trial will be an adaptation of the protocol by Nestle and colleagues. After obtaining approval from the human research ethics committee, we recruited 80 patients (Table 2) who were duly informed about the scope of these protocols and the realistic prospects of success. They then underwent a comprehensive health check to assess their overall health. The selected patients underwent leukapheresis at the blood bank of the Clinical Hospital of the University of Chile. Peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll-Hypaque gradient in the laboratory of Dr. Flavio Salazar at the Institute of Biomedical Sciences of the University of Chile. Some of the cells were cryopreserved in liquid nitrogen for future use. According to the protocol, approximately 1.8x108 PBMCs were placed in 6-well culture plates and incubated for two hours at 37°C and 7% CO2. The suspended cells, peripheral blood lymphocytes (PBLs), were removed and frozen for later use. The monocytes adhering to plastic were incubated for seven days in serum-free culture medium (AIM-V therapeutic, GIBCO, BRL) in the presence of 500 U/ml of IL-4 and 800 U/ml of GM-CSF, as described in detail in the Materials and Methods section. On the sixth day, the DCs were pulsed with tumor extracts from allogeneic melanoma cell lines, previously checked for sterility, and incubated for 6 hours at 37°C and 7% CO2, followed by maturation with TNF-alpha for 12 to 18 hours. Mature DCs were characterized by flow cytometry. Approximately 5 to 15 x 106 pulsed DCs were subcutaneously injected. This routine was repeated weekly for at least four times and a maximum of 6 times. After the fourth dose, the patient's general condition, possible clinical regressions, adverse side effects, and the effects of the treatment on the immune system were evaluated. This evaluation was done through in vivo delayed-type hypersensitivity (DTH) assays using melanoma extracts and their respective controls. Planned in vitro assays included the detection of anti-melanoma CTL precursors by ELISPOT (77), cytokine production, proliferation assays, and quantifying the CD4/CD8 ratio by flow cytometry. Several obstacles must be overcome to make this type of treatment effective in patients. First, immunization is hindered in patients due to the state of immunosuppression that many experience due to tumor burden, chemotherapy (81,82), or other as-yet-undetermined factors. The heterogeneity of antigenic expression in tumors must also be considered, so vaccination with tumor extracts guarantees immunization with multiple antigens. Another difficulty to be considered is the ability of tumors to evade immune system recognition (83). These escape strategies range from inhibiting antigen presentation to producing immunosuppressive substances such as TGF-beta and IL-10 (84). The relative importance of these potential mechanisms in reducing the potential of vaccine-based immunotherapy has yet to be established. Nevertheless, it is crucial to investigate these obstacles and develop appropriate clinical protocols that harness the full potential of cellular vaccine-based immunotherapy, which has been convincingly demonstrated in murine models. Therefore, we have decided to present a second study to the respective committee, building on the experience gained and the recently obtained funding from FONDEF, to develop an optimized protocol that alternates dendritic cell vaccines with the use of low doses of IL- 2 as a complement to strengthen the immune system. This optimization is based on studies by Steve Rosenberg of the NIH, which showed improved responses by combining dendritic cell vaccines loaded with modified antigenic peptides in the presence of IL-2 (75). Additionally, as previously described, the use of high-dose IL-2 has side effects that, while manageable, are severe and temporarily affect patients' quality of life. Therefore, based on our own experience (67) and that of others (68-70), we have decided to use low doses of subcutaneously administered IL-2, which do not produce marked adverse effects (67,70). Few studies worldwide have been conducted in this area, one recent in renal carcinoma (70). Overall, most studies demonstrate that IL-2 enhances the ability of dendritic cells to induce an immune response (68-70). Finally, the received funding will allow us to conduct these studies in GMP (good manufacturing procedure) laboratories with high biological safety, complying with all international standards for clinical studies. 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