Oncol 16: 86. TABLE 1: Side effects reported in clinical trials with PROLEUKIN IL-2, involving a total of 525 patients with either renal carcinoma or metastatic melanoma System GRADE 3 GRADE 4 ALL EVENTS General Chills 70 (13%) 0 (0%) 271 (52%) Fever 29 (6%) 5 (1%) 154 (29%) Malaise Infection 43 (8%) 0 (0%) 143 (27%) 29 (6.0%) 7 (1%) 66 (13%) Cardiovascular System Hypotension 205 (39%) 15 (3%) 375 (71%) Cardiovascular Disorders 36 (7) 7 (1%) 59 (11%) Digestive System Diarrhea 113 (22%) 10 (2%) 350 (67%) Vomiting 111 (21%) 7 (1%) 261 (50%) Nausea 25 (5%) 0 (0%) 186 (35%) Nausea and Vomiting 25 (5%) 2 (0%) 101 (19%) Hematological Thrombocytopenia 59 (11%) 5 (1%) 192 (37%) Anemia 15 (3%) 1 (0%) 150 (29%) Metabolic and Nutritional Bilirubinemia 30 (6%) 13 (2%) 210 (40%) Creatinine Increase 14 (3%) 5 (1%) 174 (33%) SGOT Increase 17 (3%) 3 (1%) 122 (23%) Central Nervous System Confusion 57 (11%) 5 (1%) 177 (34%) Somnolence 19 (4%) 2 (0%) 115 (22%) Respiratory System Dyspnea 52 (10%) 5 (1%) 224 (43%) Respiratory Disorders 15 (3%) 14 (3%) 59 (11%) Urogenital System Oliguria 137 (26%) 33 (6%) 333 (63%) Anuria 13 (1%) 25 (5%) 36 (7%) Grade 3 and 4 Adverse Effects in > or equal to 3% of treated patients (n=525). Grade 4 Adverse Effects are those that compromise the lives of patients. The rate of drug-related death in 255 patients with metastatic renal carcinoma who received PROLEUKIN IL-2 was 4% (11/255). The rate of drug-related death in 270 patients with metastatic melanoma who received PROLEUKIN IL-2 was 2% (6/270). TABLE 2: General Characteristics of Patients Before the Initiation of Phase I/II Clinical Protocol Patient Age Gender Time of Evolution Before Starting Protocol Clinical Stage Primary Metastasis Previous Treatment CT001 34 F 36 months IV Thorax In transit and gland Surgery anterior mammary CT002 35 M ND IV Osseous (Vertebral) CT003 34 M 12 months IV Back Lung Surgery CT004 69 M 60 months IV Back Multiple Surgery, IL2 CT005 57 M 36 months IV EII Lung Surgery CT006 30 M 36 months IV Thorax Subcutaneous inguinal left Surgery CT007 53 F 6 months III EII Inguinal left lymph nodes Surgery CT008 37 F 36 months IV EII Pulmonary Surgery CT009 24 F 8 months III Back Axillary right lymph nodes Surgery CT010 39 M 60 months IV Malar Local dissemination Enucleation CT011 68 F 30 months IV Back Liver Surgery CT012 57 F 14 months IV Plant left In transit Surgery, IL2 CT013 62 M 36 months IV Scalp Lung Surgery Note: ND stands for "Not Determined." TABLE 3: Adverse Reactions and Clinical and Immunological Responses of Patients in Previous Phase I Clinical Protocol Patient No. DC/Dose Adverse Reactions Elispot DTH Vaccination Objective Regressions Post-Therapy Disease CT001 3-5 x 10^6 Negative + + 2nd cycle Regression of pulmonary nodules Progression of pulmonary nodules CT002 3-5 x 10^6 Negative Nd Nd - - Deceased CT003 3-5 x 10^6 Negative + + 3rd cycle - Deceased CT004 3-5 x 10^6 Negative - - 1st cycle - Stable disease CT005 3-5 x 10^6 Fever, arthralgias AAN and RF (-) - - 1st cycle Regression of cutaneous metastasis Progression of cutaneous metastasis CT006 3-5 x 10^6 Negative + + 2nd cycle - Progression CT007 3-5 x 10^6 Negative + - 1st cycle - Stable disease CT008 10 x 10^6 Negative + - Withdrawal (1st dose) - Deceased CT009 10 x 10^6 Negative + + 1st cycle - Stable disease CT010 10 x 10^6 Negative Nd - 1st cycle - Progression CT011 10 x 10^6 Negative Nd + 1st cycle - Stable disease CT012 10 x 10^6 Negative Nd - 2nd cycle - Progression CT013 10 x 10^6 Negative Nd - 1st cycle - Progression Note: Nd = not determined. Treatment of Malignant Melanoma with Dendritic Cells Pulsed with Tumor Extracts. Clinical Study Design PHASE I/II TRIAL IN STAGE IV MALIGNANT MELANOMA This protocol is ethically justified as advanced melanoma patients, once their tumor invasion has extended beyond the lymphatic lymph node barrier, are not amenable to satisfactory treatment with traditional methods such as surgery, radiotherapy, or conventional chemotherapy. Disseminated tumors are refractory to all standard treatments. 100% of patients who develop distant metastases will die due to their disease, either from complications or cachexia. Therefore, immunotherapy based on immunological stimulation with immunocompetent dendritic cells, combined with immunological reinforcement using IL-2, may, according to evidence from ongoing clinical protocols, lead to prolonged survival with improved quality of life and, in some cases, complete tumor regression. GENERAL AIM: To study the clinical and immunological response of patients treated with vaccines based on autologous dendritic cells loaded with tumor antigens derived from autologous or allogeneic melanoma extracts, supplemented with the intermittent use of low doses of recombinant human interleukin 2 (rhIL2) PROLEUKIN® (aldesleukin). MAIN SPECIFIC AIMS: • SAFETY: Safety in administering dendritic cell preparations; local and systemic toxicity estimation. Determination of adverse reactions such as fever, nausea, allergy, neurological and cardiovascular symptoms. Local toxicity at the administration site. • IMMUNE RESPONSE MEASUREMENT: Observe the immune response based on in vivo and in vitro parameters: • In vivo response: Measure the Delayed-Type Hypersensitivity response (DTH). It involves a cross-test comparing in vivo tissue response between dendritic cells sensitized with tumor extracts and their respective control dendritic cells without loading. • In vitro response: ELISPOT assays, measuring IFN-γ production in the peripheral blood of treated patients. Compare specific immune responses after each therapy cycle by measuring IFN-γ production by tumor-specific CTLs. Radioactive chromium releases cytotoxic assays to measure CTL and NK-mediated antitumor response. After each therapy cycle, ELISA assays for quantifying cytokines (IFN-γ, IL-10) in patient serum. • CLINICAL RESPONSE MEASUREMENT: To observe the objective response rate. The percentage of patients with complete response (CR) and patients with partial response (PR) will be reported. SECONDARY SPECIFIC AIMS: To evaluate: • Overall Survival. • Disease-Free Survival. • Quality of Life (assessed using EORTC global QLQ-C30 and EORTC disease-specific QLQ). • Performance status response: The percentage of patients with a 20% or more significant increase in activity measured by the Karnofsky index will be reported. MATERIAL AND METHODS Dendritic Cell Generation: Patient leukocytes will be obtained through leukapheresis at the Blood Bank of the Clinical Hospital of the UNIVERSITY OF CHILE, following the standard protocols used in that unit. Peripheral blood mononuclear cells (PBMCs) will be isolated from peripheral blood using a Ficoll-Hypaque gradient. PBMCs will be cultured for 2 hours at 37ºC and 5% CO2 in a suitable serum-free clinical research medium, AIM-V Clinical Research® (Life Technologies, USA). Non-adherent cells will be discarded, and adherent cells (monocytes) will be cultured in AIM-V supplemented with 500 U/ml of recombinant IL-4 (R&D Systems, INC., MN, USA) and 800 U/ml of GM-CSF (R&D Systems, INC., MN, USA). On the sixth day, monocytes differentiated into dendritic cells will be pulsed with melanoma extracts and then matured with TNF-α (R&D Systems, INC., MN, USA). Flow cytometry will confirm mature dendritic cells' CD14-, CD36low, and CD83high phenotype characteristics. Mature DCs will be washed twice and resuspended in 300 µl of sterile saline. All cell manipulation procedures for vaccine production will be performed under GMP conditions, as indicated. Melanoma Extracts: The melanoma cell lines used in this study were established by Dr. Flavio Salazar at The Institute of Biomedical Sciences, Universidad de Chile. These cell lines have been previously checked to ensure they are free from viruses or other contaminants. Cells are maintained under strict sterile conditions in the Immunology Laboratory of the Biomedical Sciences Institute at the University of Chile. When possible, tumor tissue obtained from the patient will be directly treated to obtain autologous tumor antigens. Dendritic cells will be loaded with lysates from three melanoma lines, 1x106 cell/per line. The lysate will be obtained by three freeze-thaw cycles, irradiated with a dose of 60 Gy, and sonicated for one 30-second cycle. The cell extract will be resuspended in 600 µl of sterile saline. Dendritic cells, approximately 6x106 per 6-well plate, will be pulsed with 100 µl of this extract per culture well for 6 hours at 37ºC and 5% CO2. Interleukin 2: PROLEUKIN (recombinant human Interleukin-2) will be administered in a dose of 2.4 x 106 IU/m2 (millions of international units per square meter of body surface), subcutaneously, reconstituted according to the manufacturer's instructions, for two days, after each vaccination for three consecutive days. The administration will be carried out by trained personnel in the appropriate facilities of the Clinical Hospital of the UNIVERSITY OF CHILE. Vaccination: The preparation of autologous dendritic cells loaded with tumor extract (approximately 10-20 x 106 cells/dose) will be administered subcutaneously to selected patients in a volume of 300 µl of sterile saline solution, in a plastic container, labeled with the patient identification number, date, number of dendritic cells inoculated and dose. Patients will receive their first dose seven days after undergoing the leukapheresis procedure, which will be considered day 0, their second dose on day 10, followed by IL2 at the indicated doses on days 12, 13, and 14. Subsequently, their third dose will be injected on day 30, followed by IL2 on days 32, 33, 34, and its fourth dose on day 50, followed by IL2 on days 52, 53, and 54 (see scheme). IL2 IL2 IL2 12 13 14 32 33 34 52 53 54 -7 0 10 30 50 Leukapheresis 1st DCV 2nd DCV 3th DCV 4th DCV DCV= Dendritic Cell Vaccination Preclinical Trials: CELL LINES AND MEDIA: The melanoma cell lines Mel-1, Mel-2 and Mel-3 were established at the Institute of Biomedical Sciences, Faculty of Medicine, University of Chile and screened to ensure viruses' absence. The T2 cell line, a TAP-deficient cell line derived from a T-cell/ B-cell leukemia hybrid and C1R-A2, a cell line deficient in MHC class I expression and transfected with HLA-A2, will be maintained in RPMI 1640 medium (GIBCO BRL.) supplemented with 10% Fetal Bovine Serum (FBS) (GIBCO BRL.), 1% L-glutamine (GIBCO BRL.), and 1% Penicillin-Streptomycin (GIBCO BRL.). ISOLATION OF MONONUCLEAR CELLS FROM PERIPHERAL BLOOD: Leukocyte concentrates from patients (leukapheresis) will be mixed in equal parts with sterile PBS. Each sample will be separated into 50 ml conical tubes containing 10 ml of Limphoprep® (NYCOMED PHARMA, Oslo, Norway.) and then centrifuged for 20 minutes at 1200 rpm without brake at 18°C using a refrigerated IEC MP4R centrifuge (MA, USA). The mononuclear cell layer will then be transferred with a pipette to conical tubes and washed three times in 20 ml of PBS for 5 minutes at 1200 rpm, resuspended in a final volume of 20 ml for quantification and viability determination by Trypan Blue exclusion. 1.8 x 108 PBMCs per 6 well plate will generate DCs; the rest will be cryopreserved for future use. ELISPOT ASSAY FOR IFNγ RELEASE BY ANTIGEN-SPECIFIC CTLs: In brief, 96-well plates (Multiscreen; Millipore, MAIP N45) will be sensitized with 2 µg/ml of primary anti-IFNγ monoclonal antibody (acMo) (Mabtech 1-DIK) overnight at 4°C, washed, and blocked with 1% PBS/BSA for 2 hours at 37°C. Effector and target cells will be added to the wells for 4 hours at 40°C, followed by washing before incubation for 20 hours at 4°C with 75 µl of secondary antibody (0.75 µg/ml biotin-conjugated anti-IFNγ acMo; Mabtech, 7-B6-biotin). Streptavidin-ALP will be added for 1 hour at room temperature in the dark. The wells will then be washed and incubated for 5 minutes with NBT/BCIP for complex visualization. Spots will be counted using a microscope. The response will be considered positive if there are >10 spot-forming cells (SFC) per 2x105 respective controls. TYPING OF CD8, CD4, AND NK CELL POPULATIONS IN PATIENT BLOOD: To detect cellular markers in PBMCs from melanoma patients, the cells to be analyzed will be washed three times with ice-cold PBS and then incubated with 2 µg/ml of either FITC- or PE-conjugated anti-CD3, anti-CD4, anti-CD8, and anti-CD56 monoclonal antibodies (Pharmigen). The tests will be analyzed using the FAScan instrument (Beckton Dickinson, CA, USA) at the Disciplinary Program of Immunology at the ICBM, University of Chile. ELISA ASSAY FOR THE DETERMINATION OF IL-10 AND IFNγ IN PATIENT SERUM: In brief, plates sensitized with primary anti-IFN-γ monoclonal antibody (acMo) (OptEIA, Pharmigen) and primary anti-IL-10 monoclonal antibody (acMo) will be blocked with 1% PBS/BSA for 1 hour at room temperature. Then, 100 µl of each patient's serum will be added to each well and incubated for 2 hours at room temperature. The wells will then be washed and incubated for one hour at room temperature with 100 µl of secondary antibody (0.75 µg/ml biotin-conjugated anti-IFN-γ acMo Avidin-HRP (Pharmigen)). Substrate (TMB and hydrogen peroxide) will be added for 30 minutes at room temperature in the dark. The wells will then be washed, and the STOP solution will be added. Absorbance at 450 nm will be read using an ELISA reader (BIORAD, model 3550) at the Disciplinary Program of Immunology at the ICBM, University of Chile. Dose and Dosing Rationale: Selected patients will be treated with four cycles of therapy over 60 days. In each dose, 10 to 20 x 106 DCs loaded with extracts from 3x106 allogeneic melanoma cells will be injected. The extracts will be prepared from irradiated melanoma cells lysed through continuous freezing, thawing, and sonication. This dose is estimated to be sufficient to induce an antitumor immune response, as preclinical and clinical trials have demonstrated clinical and Laboratory immune responses, supporting the method's safety. To support the patient's immune system stimulation, interleukin-2 (IL-2) will be administered subcutaneously at 2.4 x 106 IU/m2 two days after the second vaccine dose for three consecutive days. This regimen will be repeated after each vaccination. Currently, standard protocols administer between 18 x 106 and 9 x 106 IU/m2 of IL-2 per dose (recommended by distributor CHIRON BV Holland). The low doses used in this study are based on the project director's experience, supported by studies published in international journals and recent literature (67-70). The intention is to avoid IL-2-mediated side effects while preserving its beneficial effects. Treatment: a) Treatment Regimen: For administering each dendritic cell dose, the treating investigator will observe and treat patients on an outpatient basis in an adequately equipped outpatient clinic. The 300 µl dose in a sterile saline solution will be injected subcutaneously, preferably near the primary tumor or near unaffected lymph nodes. b) Immediately after treatment, the patient must be monitored. Vital signs should be taken 15 minutes before and after each treatment, and the patient should be observed in the clinic for at least one hour. After the observation period, the patient will be sent home. c) Interleukin-2 will be administered subcutaneously, on an outpatient basis, in a properly equipped outpatient clinic for three consecutive days. Vital signs will be monitored, and the patient will be observed in the clinic for 30 minutes. d) Patients will be followed to detect and evaluate potential toxic reactions to treatment. The next dose can be repeated the following week if: - There was no grade IV toxicity with the previous treatment, - There was no obvious disease progression - The patient does not meet the exclusion criteria for the protocol. Inclusion Criteria: • Patients with histologically confirmed melanoma of the skin: Breslow, Clark, histological type. Location of the primary, clinical, or pathological regional status. • Complete staging demonstrating the presence of distant metastases, either visceral or in soft tissues or bone tissue: Brain, Lung, Abdomen, and Pelvis CT and bone scintigraphy. • Objectively measurable disease by clinical or radiological means • Karnofsky's Performance Status is more significant than 70% • Life expectancy greater than three months • Patients over 18 years old • Informed consent from the patient to participate in the protocol Exclusion Criteria: Patients meeting any of the following criteria will be removed from the study: • Age greater than or equal to 65 years • Ongoing active infections, including viral immunodeficiency • Previous chemotherapy within less than two months • Concomitant malignant tumors (e.g., Chronic Lymphocytic Leukemia, etc.) • Uncontrolled concomitant diseases (Hypertension, unstable Diabetes mellitus, renal diseases requiring dialysis) • Situations or conditions requiring urgent surgical intervention, such as intestinal obstruction due to metastasis • Pregnancy or lactation • Concurrent participation in other therapeutic research protocols • Any condition that compromises the objectives of this study. Removal Criteria from the Study: Patients will be removed from this study if any of the following conditions occur: • By the patient's decision to discontinue the study. • Intolerable adverse reactions, according to WHO Criteria Grading Toxicities • Intercurrent illness that may compromise the patient's life or interfere with the treatment study evaluation • Requirements for concomitant medication that may interfere with study results. • Failure to complete the study entirely • Failure to follow up on the patient. Sample Size Recruitment will be conducted to terminate the study after a specific number of patients have been enrolled if a minimum number of responses have not been observed. The lowest significant response rate will be 20% (alpha = 0.10; beta 0.10). If at least the minimum number of objective responses are observed to complete recruitment, 30 evaluable patients will be enrolled. For example, if there is no response in any of the first 12 treated patients or only 1 out of the first twenty, the study should be terminated. The confidence interval for the response rate is >20%. Evaluable Patient: An evaluable patient meets the inclusion criteria, does not meet any of the removal criteria, receives all four vaccination cycles, and has been clinically and radiologically followed until the end of this trial. Study Conduct • Signing of informed consent • Complete medical history • Complete physical examination • Karnofsky Performance Status measurement • Estimation of survival quality using the EORTC scale • If applicable, photographs of the tumor and metastases • Hematological studies, including albumin and total proteins. • Electrocardiogram • CD3, CD4, CD8 cell counts, and total lymphocyte count • Blood sample collection for in vitro immunological studies. • Staging radiological studies: Computed tomography of the brain, lungs, abdomen, and pelvis, and bone scintigraphy • Estimation of metastasis size through physical and radiological examination • Delayed hypersensitivity test (DTH) Enrollment After the procedures mentioned above, each eligible patient will be enrolled. Each patient will be given an identification number. Data will be maintained in a computerized file with restricted access for the research team for each patient. Patient Monitoring Patients will be clinically monitored once a week through the following parameters: • Weight • Karnofsky Index • Vital signs • EORTC Quality of Life Index • Comprehensive physical examination • Monitoring for toxicity symptoms according to WHO Criteria Grading Toxicities and reporting adverse effects Additionally, they will be evaluated through ELISPOT and ELISA assays, with blood samples taken before each vaccination, to assess the response of specific CTLs to DC stimulation and the presence of cytokines in the patient's serum. Measurement at the End of Treatment • Clinical evaluation of the patient • Estimation of clinical or radiological tumor response: determining tumor volume • Comprehensive physical examination • Karnofsky Index • Photography • Quality of life and pain assessments • Delayed hypersensitivity test (DTH): for in vivo response measurement • Hematological and chemical tests: Assays for measuring in vitro immune response. Long-Term Follow-Up Once the patient has completed the full four-cycle treatment, they must be followed up every four weeks for one year. Every two months during the second year. The date of death must be determined in applicable cases. During each visit: • Directed physical examination • Karnofsky Index • Estimation of clinical and radiological tumor size • Photography • EORTC Quality of Life Assessment • Reporting of adverse effects Clinical Response Assessment TUMOR RESPONSE CRITERIA CR: Complete disappearance of the tumor PR: Tumor regression > 50% MR: Tumor regression > 25% - <50% SD: Decrease or growth < 25% PD: Increase in tumor size during treatment Statistical Analysis The primary objective of this study is to estimate the objective response and toxicity associated with treatment using dendritic cells loaded when administered to patients with disseminated melanoma. A stratified recruitment design will be used in this study. In the first stage, 12 patients will be enrolled and treated with loaded dendritic cells. If none of the patients responds to the treatment, enrollment for this study will be terminated. If at least one patient responds to the treatment, the treatment will continue. If, after the enrollment of patient number 20, at least two patients respond to the treatment, the study should continue until a minimum of 30 evaluable patients with 3 out of 4 completed cycles. Assuming that the study will be completed with thirty patients as planned, the 90% confidence interval for the estimated response rate will be 12%, assuming a 20% response rate. If sufficient evidence indicates that the valid response rate is greater than 20%, it would be given if the lower limit of the confidence interval is more significant than 0.20 (alpha = 0.05, 1-tailed -test). Primary Efficacy Analysis Because this uncontrolled study enrolls a small number of patients, formal hypothesis tests are not planned. However, comparing different subgroups may generate hypotheses that can be studied in subsequent clinical trials. Infrastructure and Equipment Provided by the Sponsor Academic Units for the Project This project will primarily utilize the Tumor Immunology Laboratory's infrastructure and the Immunology Program facilities at the Institute of Biomedical Sciences, Faculty of Medicine, University of Chile. This Laboratory is equipped for the culture and expansion of cell lines in a Good Manufacturing Practice (GMP) compliant culture room. This system ensures that our vaccines are produced and continuously monitored according to international standards, verifying sterility and quality control (World Health Organization TRS 908, Annex 4). Additionally, there are suitable conditions for molecular and biochemical studies. The Laboratory is equipped for working with low-activity radioisotopes such as 3H and 51Cr. Furthermore, the group of researchers involved has the necessary experience, as reflected in their publications. 1. Tumor Immunology Laboratory: Analytical and precision balances, spectrophotometer, refrigerators, -20°C and -70°C freezers, immunodot and immunoelectrotransfer multi-analysis system, gradient former, Eppendorf centrifuge, refrigerated centrifuge, magnetic stirrers, vortex, thermostatic bath with agitation, power supplies, PCR thermocycler, microscope, digital camera, equipment for immunohistochemistry and immunofluorescence, chemical hood. Cell Culture Room: GMP-compliant culture room, temperature-regulated filtered air, laminar flow hoods, regulated CO2 incubators, inverted microscope, thermostatic bath, refrigerator, and liquid nitrogen tanks. The project includes a wide range of human cell lines, cytokines such as IL-2, IL-4, IL-10, GM-CSF, TNF, IFN-γ, IL-12, and IL-15, hybridomas, monoclonal antibodies specific for MHC class I and different haplotypes (HLA-A2, HLA-B7, among others), gene expression plasmids for receptors (MC1R, HER2/neu), human and murine IL-2, IL-4, and IL-10, as well as primers for T cell receptor repertoire detection with PCR and synthetic peptides of various melanoma-associated antigens. 2. High-Efficiency and Conventional Chromatography Laboratory: FPLC/HPLC system and columns, cold chamber, conventional columns, peristaltic pump, UV flow detector, fraction collectors, recorder. 3. Laboratory for Biological Probes Radiolabeled with High and Low-Energy Isotopes: Negative flow safety hood and volatile radioisotope absorption system with activated carbon, orbital shaker, visible light microscope, and safety cabinet for storing and decaying radioactive waste. 4. Flow Cytometry Room: Flow cytometer (FACSort), analysis computers. 5. Darkroom for Radioautographic Processing will be available, including a Polaroid camera, developing reagents, and radioautography cassettes. 6. Washing, Sterilization, Distilled Water, and Ice Room: Material drying oven, floor autoclave, flake ice maker, water distillers, water deionizer, ultra-pure water production system. 7. A Central Laboratory containing pH meters, a spectrophotometer, an ELISA reader, and a 10-head gamma counter will be available. 8. Immunology Library includes ten journal subscriptions, a database for bibliographic reference search, and over 100 immunology and related books. 9. Others: Word and graphics processing computer systems, fax, direct phone line, email, and internet access. 10. Central Services: In addition to the infrastructure within the Immunology Program, the Faculty of Medicine has other general-use equipment that may be relevant to this project, such as an ultra-centrifuge, scintillation counter, confocal microscope, peptide, and oligonucleotide synthesis and sequencing, among others. There is also a central animal facility designed for maintaining pure strains of mice, infected and immunized mice, and rabbits. 11. Clinical Hospital of the University of Chile: The infrastructure of the blood bank at the Clinical Hospital of the University of Chile is available, featuring equipment for leukapheresis sample collection and the Cryopreservation Laboratory equipped with a modern automated biological sample freezing system. 12. Associated Companies in the Project: Oncobiomed Ltda., Oncomed Ltda., and Tecnofarma S.A. have provided their clinics for the selection and treatment of some patients and their administrative teams for technological transfer, respectively. Funding: This study will be financially supported through contributions from the FONDEF Project No. DO2I1088: "Research and Development of Biological Vaccines for Oncological Immunotherapy," for 310 million Chilean pesos, for a duration of three years (2003-2005). Additionally, the project's associated companies, ONCOMBIOMED Ltda. (85 million Chilean pesos) Furthermore, Tecnofarma S.A. (35 million Chilean pesos) has committed to providing funds through personnel financing and cytokine grants such as IL-2.