Phase I/II Trial In Stage III/IV Malignant Melanoma Using Autologous Dendritic Cells Loaded With Tumor Cell Lysates And Low Doses Of Proleukin
Phase: PHASE1
Conditions: Stage III Malignant Melanoma of Skin AJCC V6, Stage IV Malignant Melanoma of Skin
Sponsor: University of Chile

 
 
 
 
 
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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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.