CUBAN MEDICAL RESEARCH

Immunotherapy As An Innovative Treatment For Advanced Breast Cancer
Giselle Saurez, MD
Edmundo Rodríguez, MD
Mauricio Catalá, MD
Martha Osorio, MD
Adriana Carr, BS
Alain Díaz, BS
Zaima Mazorra, BS
María del C. Arango, MD
Elena Noris, MD
Rolando Camacho, MD
Luis E. Fernandez, PhD
Ana María Vázquez, PhD
Rolando Pérez, PhD
Agustín Lage, MD, PhD.

Abstract : Despite current standardized treatments for local and systemic control of breast cancer with high levels of objective anti-tumor response, many cases evolve towards recurrence or metastasis, and thus challenge clinical oncology to search for new therapeutic options to change the course of the disease. In Cuba, a specific active immunotherapy program with three therapeutic vaccines is used for advanced breast cancer. Two of these are based on gangliosides: NGcGM3/VSSP and NAcGM3/VSSP, while the third one is the anti-idiotype 1 E10. They have shown encouraging, while incipient, clinical and immunologic results.

Introduction

The natural history of breast cancer is characterized by long duration and patient heterogeneity. Current therapeutic treatment schemes permit survival of 100% of patients at five years, when they are diagnosed at stage 0 or in-situ. Less than 10% of patients show disseminated breast cancer at the time of diagnosis. Nevertheless, about 50% of women in stages I, II, III of breast cancer show distance dissemination at some point in their lives (1). About 85% of them develop dissemination within the first five years, though the risk of relapse is still present after 10 to 30 years.

Advances in screening, local-regional control through surgery and therapeutic radiations, and adjuvant therapy (cytotoxic chemotherapy and/or hormonal treatment) have achieved considerable improvement with anti-tumorous clinical response. Nevertheless, recurrence or evolution towards metastasis are unfortunately still unsolved problems for clinical oncology, mainly in certain groups of patients with parched tumors and high risk of relapse, where present day therapy has not proven significantly beneficial for survival (2-14).

These factors indicate the need to find new therapeutic forms that change this behavior in terms of survival and disease-free intervals in the most advanced stages of breast cancer (15).

Today, immunotherapy seems promising in this regard. This therapy, also called biological therapy, biological response modifier therapy, or biotherapy, activates the immunologic system through its specialized cells, antibodies and other involved organs, limiting or inhibiting the growth of tumorous cells that by various mechanisms have escaped immunologic surveillance.

Immunotherapy is based on the existence of tumor-associated antigens (ATA), antigens associated with the neoplastic cells. Since these antigens are not found or may be in a cryptic state in normal tissues, the immune system can recognize them (in tumorous tissues) and respond against them (16,17).

In breast cancer, experimental application of immune therapies has grown rapidly, with the use of interferons and interleukins (especially in combination schemes), monoclonal antibodies, anti-tumor vaccines and various immunotoxins(18). Thus, across nearly the whole spectrum of immunothesapy, agents have been studied with different mechanisms of action against the tumors.

Among the most successful agents for advanced breast cancer therapy is the humanized monoclonal antibody that is linked to receptor Her2-neu, Trastuzumab (Herceptin), indicated for metastatic breast cancer patients. This antibody was approved by the FDA in 1998, based on the evidence of its anti tumor efficacy as an agent in itself or in combination with cytotoxic treatment.

Monotherapy with Herceptin showed a response rate of 21% (19) in patients who have received previous treatment with chemotherapy and whose tumors overexpress Her2-neu. Other clinical studies concluded that the group of patients who received chemotherapy with Antraciclin and Herceptin were more successful than those who only received chemotherapy (25.4 months vs. 20.9 months, p = 0.05) with a positive receptor to Her-2. While clinical improvement in terms of survival has been associated with this treatment, it is not exempt from limitations in terms of toxicity, quite often enhanced when used in combination protocols with cytotoxic agents.

Another biological therapeutic modality is specific active immunotherapy, in the form of vaccines which direct the host’s immune response against malignant cells, more effective than results expected from passive immunotherapy with monoclonal antibodies (AcM) or other therapeutic forms. Unlike conventional vaccines used as prophylaxis against certain infectious diseases, therapeutic vaccines against cancer are applied once the presence of malignancy is established.

It is precisely in the field of biotechnology research in Cuba that therapeutic vaccines play a leading role in challenging breast cancer.

Since 1993, Havana’s Molecular Immunology Center has been developing a project of therapeutic vaccines mainly directed against breast carcinoma, which includes two vaccines based on ganglioside antigens (Nacetil GM3, Nglicolil GM3) and an anti-idiotype vaccine (1 E10), a mimicry of Ganglioside Nglicolil GM3

Why ganglioside vaccines?

Several types of vaccine compounds have been tried on breast cancer in the form of cellular, molecular, peptide and anti-idiotypical vaccines (20-23). Carbohydrate epitopes such as Sialyl-Tn (STn), (associated with MUC 1), and MUC 1 itself are used as targets for breast cancer immunotherapy (24-34).

The selection of ganglioside vaccines is based on the fact that gangliosides are components of the plasmatic membrane of all cells found in mammals. They are glicoesfingolipids with an exacerbated expression in tumorous cells that take part in a number of cellular processes such as: receptors for ligands in the adhesion cells, immuno-regulators of the prolific response of lymphocytes to antigens and mitogens, and as cell growth regulators, potentially involved in angiogenesis, in metastasis dissemination, and in tumor-induced immuno-depression (35).

The expression of gangliosides varies both in quality and quantity in the various stages of normal cellular differentiation, and during malignant transformation. In addition, there is a correlation between their aberrant expression and the tumor’s progression, and eventually with immunogenecity (36). This overexpression of gangliosides has been found in tumors of neuroectodermic origin like melanomas, astrocytomas, sarcomas, neuroblastomas, and small-cell lung cancer (37-38). Recently evidence has been found of their expression in ovarian, kidney and breast tumors (39). These findings make gangliosides attractive targets to treat such tumors with specific active immunotherapy.

Nacetil Ganglioside Vaccines

Experimental observations were made in the 1980’s, showing that the reactivity of certain antibodies specific to this molecule essentially depend on this glycolipid’s density as exposed in the cellular membrane. This density has a threshold value which is recognized by these antibodies either as all or nothing; and at the same time, it was noted that the amount of Nacetil GM3 present in tumorous tissue is higher than that found in normal tissue, explaining the cryptic or “hiding” phenomenon (40,41). That is, this molecule may be in a cryptic or hidden state in normal tissues, and exposed in tumors.

In 1992, Donald Morton’s group at the John Wayne Institute for Cancer Treatment and Research, Santa Monica, reported the characterization of human monoclonal antibody L612 (IgM), which had been obtained through the transformation of B Cells, of a regional lymphatic ganglion of a breast cancer patient. This antibody proved to be specific for Nacetil GM3 (41).

These passive immunotherapy elements led in 1994 to the first attempt to achieve specific active immunotherapy using ganglioside NacGM3 as an antitumorous target, with a chemical anti-idiotype monoclonal antibody, which behaved as an internal image of the ganglioside (42). This anti idiotype antibody was inoculated in patients with melanoma and it was possible to measure specific prolific response of lymphocytes in these patients. Another N-Acetil ganglioside used as a target for active immunotherapy with ganglioside GM2 in patients with melanoma (30,37). The Livingston group has used different forms of vaccines with antigen GM2 adsorbed in BCG in patients with melanoma, indicating that those patients with higher titres of anti-GM2 antibodies of isotypes IgM and IgG showed delay in their relapse (37). After his studies with melanoma cellular vaccines, Morton reported that the induction of a response of GM3, GD3, GM2, GD2 anti-ganglioside IgM antibodies is related to a beneficial impact on survival (43,44).

The present development of cancer vaccines using NAcGM3 as a target has moved from the tumor to self-immunity trying to direct an immune response against this molecule, which is abundant in normal non neural tissues. At the same time, it has moved towards the need to eliminate the relative immunologic ignorance of this ganglioside and to achieve a steady specific immune patient response.

The GM3/VSSP Vaccine, our experience:

We have found a group of immunologic and antitumorous evidence in animal models through new technology developed by the Center of Medical Research, and the group of molecular oncology of the University of Quilmes and the University of Buenos Aires, Argentina, with a vaccine preparation containing NAcGM3 ganglioside associated with the outer membrane protein of Neisseria meningitides (OMP, outer membrane protein) as a carrier.

This protein is able to behave simultaneously as a multiepitopic antigen for serotype B in N. Meningitides and as a carrier and adjuvant for the polysaccharide antigen of serotype C of the VAMENGOC BC bacteria in the vaccine. This proved an alternative and powerful way of ganglioside presentation into the immune system (45).

Before the clinical application, an evaluation was made of the vaccine’s immunogenecity with mice, monkeys and chickens, with a plan of repeated immunization doses, using Montanide ISA 51 as an adjuvant. Seroconversion to specific IgM and IgG against antigen NAcGM3 was achieved with the three animal models, without any signs of toxicity with the doses used.

C57BL/6 mice that were first immunized subcutaneously with the B16 Melanoma model and were then confronted with the tumor, showed increased global survival, delay in tumor appearance, tumor size reduction, as well as cytotoxic activity mediated by antibodies, thus indicating powerful antitumorous protective activity (46,47).

Clinical Trials with the GM3/VSSP/Montanide ISA 51 vaccine

We started a pilot clinical trial in 1998, with the approval of the Cuban Regulatory Agency (CECMED), in two institutions in Havana: The National Institute of Oncology and Radiology (INOR) and the Hermanos Ameijeiras Hospital (HHA) in 21 patients with histological diagnoses of breast cancer at stages IIIa, IIIb, IV or with an evolutionary metastatic disease, in order to assess immunogenecity and safety in the vaccine.

The criteria for patient selection were the following:

1. Patients who had received their latest onco-specific therapy at least four weeks before their inclusion in the study, and were not susceptible to any other treatment, except for Tamoxifen, which could be concurrent.

2. A signed consent waiver

3. Patients whose physical condition is 0-2 according to WHO parameters.

4. Aged between 18 and 80..

5. A life expectancy of at least 6 months.

6. Clinical Laboratory Parameters within the established protocol normal values.

Treatment:

The patients received 7 immunizations of 120 μ g of NacGM3, which were administered intramuscularly every 14 days, starting on day 0 of the trial until day 56. The remaining two immunizations were given every 28 days until the completion of the 7 doses.

They were all clinically and immunologically assessed at the start of treatment and were followed up for one year. These assessments included: complete physical examination, US, Thorax XR, CT tests and studies of immune response by means of antibody values (Acs.) IgG. IgM specifically against GM3.

Results:

The assessment made at the moment of inclusion found that 8 patients were at stage III, 2 were at stage IV, and the remaining 11 were in the middle of an evolutionary metastasic stage (EMS). 20 out of 21 patients received several therapies previous to vaccination, and may or may not have concurrent therapy with Tamoxifen, with an average age of 49.8 years. Fourteen patients concluded the seven dose treatment plan, three completed the first 5 doses (one quit and 2 progressed), and the remaining ones did not complete the first five doses (1 abandoned and 3 progressed with considerable deterioration of their physical condition). Those patients who received at least the first 5 doses of the vaccine preparation (induction phase) were considered to be immunologically assessable, though in all cases the Acs response against NAcGM3 (Table 1) was determined.

The results of immunogenecity showed that titres of specific IgM pre-immune Acs against ganglioside N-Acetil GM3 were found in 14 patients. In 12 out of 17 assessable patients (those who received at least 5 doses), vaccination succeeded in the induction of titres of antibodies IgM or IgG, or both. An increase of IgM was found in 11 patients, more than doubling seroconversion values in comparison with the initial titre. As regards IgG antibody response, only 6 showed seroconversion. No humoral response was reported at any time in the assessment of three patients, in spite of the fact that they had received the complete vaccination program. Antibody response ranged between 1:80 and 1:2560 for IgM, and 1:80 to 1:10240 for IgG (Tables 3 and 4).

The immunoglobulin isotype with the highest impact in anti-tumorous response is currently under discussion. Ravindranath et al. advocate the value of IgM in isolating the circulating Ags to restore immunocompetence (48). Morton, on the other hand, studied melanoma cellular vaccines and reported that the induction of an antibody response with IgM anti-gangliosides GM3, GD3, GM2, GD2 is related to a beneficial impact on survival (43). Livingston also supports the value of IgG for its effective characteristics and great affinity (49). IgM together with cell surface carbohydrates, like gangliosides ,better activates the complement in the intravascular space, while IgG1 and IgG3 do the same in the extravascular space.

Natural antibodies against ganglioside GM3 were found in our patients, but these findings were not related to a better patient prognosis, the sample used in this study does not allow us to affirm it..

The administration of the vaccine preparation caused slight toxicity of degrees I and II according to the WHO classification criteria. Toxicity consisted mainly in a local reaction in the injection site: local erythema with underlying induration, moderate pain in the injection site and degrees I-II fever (Table 2).

As for the clinical evaluation, the median global survival was calculated, resulting in 7.97 months with a 95% reliability interval between 3.21 and 12.72 months (2.43 standard error) (Table 2, Figure 1).

These preliminary results led to the evaluation of several dose levels in this very location in Stage 1 clinical trials, with the purpose of identifying the optimum biological dosage and security profile of this vaccine. A Stage II therapeutic evaluation study was also initiated in several parts of the country. These trials are now underway.

Ganglioside NGcGM3 Vaccine

The use of therapeutic vaccines with N-glycolil gangliosides could be efficient in treating those tumors that express them, as their absence in normal human tissues gives them a certain lack of identity, which makes them more immunogenic. Two important experimental results related with NGcGM3 uphold the hypothesis underlying the use of this ganglioside in breast cancer patients. First, the biochemical characterization of the expression of ganglioside NGcGM3, combining the gas chromatography – mass spectrometry technique with the use of AcMs against N-glycolil gangliosides (39), where ganglioside levels amounted to values between 5 and 12% of the total of sialic acid and lipids, which represents some 10 7 molecules of ganglioside per cell. These cuantities are 10 times higher if we refer to the receptors in the epidermic growth factor per cell (50). In the second place, the intense and homogenious recognition of breast tumors and their metastasis by AcM 14F7 as a highly specific antibody for NGcGM3 (51). Immunohistochemichal studies with this antibody indicated intense marking on the membrane and the cytoplasm in more than 90% of these structures, in cuts of human breast cancer tissues.

The vaccine department of the Medical Research Center has another vaccine preparation containing N glycolilate, associated with the protein in the outer membrane of Neisseria Meningitidis , as a platform technology applied to our vaccines in the form of very small size proteoliposomes (VSSP).

Preclinical studies with chickens, which are animals with no cellular expression of this antigen either, different vaccine form variants with NGcGM3 were evaluated. An increase in the levels of specific antibodies of isotype IgC against this ganglioside was observed, without any evidence of toxicity in these animals.

Clinical Trial with the NGcGM3/VSSP Montanide ISA 51 vaccine

A pilot clinical trial was conducted in 1997 in the Mastopathy Service and the Experimental Chemotherapy Ward at the National Institute of Oncology and Radiology, in order to evaluate this product’s immunogenecity and safety. 21 patients with histological diagnosis of advanced breast cancer participated in the study, following selection criteria that were similar to those used in the previously described trial. Only one dose level (200 μ g) of the vaccine preparation was used (52, 53), (Table 1).

Treatment Plan:

The administration plan of the NGcGM3/VSSP Montanide ISA 51 vaccine formula consisted in 51 doses administered intramuscularly. The first 5 doses were injected every 14 days, while the remaining 10 doses were administered every 28 days.

Results:

Most patients had received some therapy before they were vaccinated. 10 of them were at stage III when they were included in the trial, while the remaining 11 were at stage IV or the disease was disseminated. 8 of the 21 patients in the trial finished the one-year treatment plan (15 doses). 6 were at stage III, and 2 with disseminated disease. Two patients left treatment on their own will, and another 11 showed considerable physical deterioration.

Patients who received at least the first five doses were considered to be immunologically assessable. Four patients in this group did not complete the induction phase. (Table 1).

Immunologically speaking, most of the patients who received the vaccine showed good antibody response against NGlycolil GM3 (NGcGM3) of both isotype IgM and IgG (Tables 5 and 6). Antibody response ranged between 1:640 and 1:160 000 for IgM, and 1:280 to 1:164 000 for IgG. IgG response was mainly of isotype IgG1 and IgG3, generating a Th1 response pattern in correspondence with the IgG subclasses found, in relation with cytotoxic activity. A tendency for titre increase was observed in stage III patients as compared with stage IV or EMS patients. Specific IgA response was also present against ganglioside NGcGM3, this being the first time IgA is reported against gangliosides (52, 53).

The anti-GM3 (NeuGc) response of isotype IgM is easily inducible in all patients, and remains for a long period of time. Though specific IgG response requires a larger number of doses to generate, it has high titres and durability through time (Tables 2, 5 and 6).

This generation of important IgG and IgM titres against the ganglioside in the patients who were treated with the GM3(NeuGc)/ VSSP Montanide ISA 51 vaccine demonstrates a powerful immunogenic capacity of this product. IgG and IgM generation is much higher than found in other molecular vaccines with GM2 (54), Fuc GM1(55) gangliosides, or sialilated carbohydrates as Theratope (33, 56, 57) where the number of titres ranges between 1/40 and 1/10240. The highest number of titres in our patients, however, fluctuates between 1/640 and 1/164 000. Although the results of our research do not indicate direct correlation between this Acs response and the vaccine’s antitumorous properties, two patients with EMS remained sTable for more than 20 and 32 months respectively from the beginning of the trial, one of them was still alive after 40 months. The global survival analysis indicated an average of 23.47 months, with a 95% reliability interval between 9.21 and 37.72 months (standard error 7.27), (Table 2, Figure 1).

As regards toxicity, the patients showed local reaction in the injection site, mainly local pain, induration, and erythema for 24 to 48 hours. Some patients showed degree I-II fever (following the WHO classification criteria). Headache, chills, muscle pain, nausea and vomiting, in order of frequency, are other adverse events related with the product (Table 2).

Other studies presently underway in this very area, and with patients suffering from metastatic melanoma have evaluated several dose levels (100 μ g, 200 μ g and 400 μ g) to determine the optimum biological dose, maintaining the immunogenic property of the vaccine, as well as its low toxicity it terms of the previously described adverse reactions. The Toxicity Common Criteria classification was used in these clinical trials (58) (Table 2).

We have also initiated a controlled and randomized Phase II clinical trial, on patients with metastatic breast cancer who have shown antitumorous response to oncological treatment for this stage (chemotherapy and/or hormone therapy) previous to their inclusion. The 200 μ g dose is evaluated in this trial.

Anti-Idiotypic 1 E10 Vaccine.

New perspectives opened up after 1974 for enriching the therapeutic arsenal for antitumorous immunotherapy, when Niels Jerne outlined the theory of the Idiotypical Network, in which he presented the immune system as a complex network of interacting idiotypes which are in balance until the arrival of the antigen (59).

Two main approaches have developed along with this theory for the design of vaccines against many antigens, including those associated with tumors. The first approach is based on the existence of “inner image” or Ab2 b antibodies in the idiotypical repertoire, which appear as stereochemical copies of the antigens able to overcome the species barrier by mimicking the three-dimensional structure of the antigenic epitope. At the same time, it is presented in a different molecular environment, which adds a significant advantage, mainly for non-protein antigens, along with the possibility of obtaining large quantities by means of monoclonal antibody production technologies (AcMs). Their application as vaccines has induced protective responses against viruses, bacteria and parasites (60, 61). Anti-idiotypical antibodies have also been used to generate immune response against tumor-associated antigens. Positive results have been achieved in preclinical studies with different animal models and clinical trials with humans (62-65). Nevertheless, the inner image b character of anti-idiotype antibodies has not proved sufficient to predict its protective character (66).

The other approach for the use of anti-idiotype antibodies in vaccine design is based on the manipulation of the tumor-associated idiotypical network by means of regulatory idiotopes. This concept, initially proposed by Paul and Bona in 1982, suggests the existence of a special class of idiotypes with unique regulatory functions before antigenic stimulation. Evidence has been accumulated that these anti-idiotype antibodies are also able to produce protective immunity by means of mechanisms still not understood (67, 68).

The AcM 1E10 of isotype IgG1 was obtained from immunization of Balb/c mice with the P3 monoclonal antibody, which recognizes gangliosides that carry N-glycolilated sialic acid (69-70), and was also obtained in melanoma and breast carcinoma tumors. AcM 1E10 is highly specific against AcM P3 and inhibits the union of the latter with the GM3(NeuGc) ganglioside (70).

Syngeneic animal immunization with AcM 1E10 induced a strong anti-anti-idiotypical (Ab3) antibody response. These antibodies shared idiotopes with AcM P3, but differed from them in their antigenic specificity (Id+Ag-) (71). In xenogenic models, specifically in monkeys, AcM 1E10 was able to induce Ab3 Id+Ag- antibody response, while the immunodominance of this AcM idiotypical determinant was demonstrated (72). The antitumorous effect of 1E10 was demonstrated in syngeneic and alogenic mice, according to experiments whose results indicate that this AcM, which does not behave as an internal image in the animal models that were studied, may activate more than one antitumorous response mechanism against tumorous breast and melanoma cells (68).

The results of the experiments with animals using AcM 1E10 encouraged the evaluation of its effects in human beings. It was first tried on patients with advanced melanoma, where the vaccine’s low toxicity and immunogenic property were demonstrated as it generated humoral response against AcM 1E10 and N glycolated gangliosides (73). A phase 1 trial with scaled doses was later initiated on patients with advanced breast cancer, which we will now describe.

Clinical Trial with the anti-Idiotypical 1 E10 vaccine in Alumina

Characteristics of the study:

10 patients who were diagnosed with breast cancer at stages III, IV or with disseminated disease were chosen for the study in the year 2000. Other inclusion criteria similar to those followed in previously described studies in this article were taken into account for this clinical trial.

Prior to their inclusion in the study, all the patients received a complete physical examination, haematological and hemo chemical studies, plus initial and periodical evaluation through X-Rays, Ultra Sound, Gamma rays and CT.

Treatment Plan:

The patients were placed in two groups of five patients, and were treated with 1 and 2 mg respectively of AcM 1 E10 precipitated in alumina. All patients received 6 intradermal injections every 14 days (induction phase). Four patients, two from each dose level, were re- immunized 7 to 9 months after the induction phase and continued to be vaccinated in three-month intervals until the progression of their disease.

Results:

The average age was 58.5 years, ranging from 38 to 72. The organs involved in the nine patients with disseminated disease included metastasis in bones, lungs, lymphatic ganglia, and subcutaneous cellular tissue (SCT). All patients had received previous therapy, including surgery, radiotherapy, chemotherapy, and hormone therapy. Seven patients received 6 doses of the 1 E10 antibody vaccine preparation, and four of them were re-immunized receiving a total of 8 to 10 injections. Two patients were out of the trial after 5 doses due to the progression of their disease. One patient abandoned the study after two doses of the vaccine (Table 1).

The disease progression time median was 7 months in the group of patients who received up to 6 immunization doses. Conversely, progression time for the four patients who received more than 6 doses reached 15 months, and two of them remained sTable for 27 and 25 months respectively. As regards global survival in all the assessable patients, the median was 33.13 months (standard error 32.41) ( Table 2, Figure 1 ).

In comparing the group of patients who were not re-immunized (up to 6 doses) with those who did receive additional vaccines (re-immunized), a median of 7 months was obtained, in an interval of 2.71-11.29 months (95% reliability interval) for the first group, while it reached 24 months (95% reliability interval for 20.61-27.39 months) for the group of patients who continued to be re-immunized with more than 6 doses. (Diaz, A. Clinical Immunology).

The toxicity of the 1E10-Alumina vaccine consisted mainly in local reaction at the site of injection with erythema and induration, occasionally associated with some pain, which disappeared in few days (24-72 hours). Some patients had I-II degree fever (according to the WHO classification), itching, arthralgia, slight headache, and higher blood pressure in the three patients who suffered from hypertension (Table 2).

Immune Response:

A strong Ab3 antibody response IgG type developed in those patients who received at least four doses of the vaccine. This response was detectable after they had received between 2 and 5 doses, and it increased with the number of doses, thus demonstrating the validity of the vaccine and the treatment plan (Figure 3).

The analysis of the specificity of this response found that the serum of all patients showed more recognition of the F(ab´)2 fragments of AcM 1E10 than of the rest of the same isotype AcMs used as control, which suggests the induction of a specific response against the AcM 1E10 idiotype. This was corroborated with the detection of a strong remanent reactivity against AcM 1E10 in the serum of the patients previously adsorbed with an irrelevant monoclonal antibody.

The degree of response against the AcM 1E10 idiotype was significantly higher that the anti-isiotypical response in all the patients (Diaz, A. Clinical Immunology). The medium titre of the anti-anti-idiotypical response was 1:15000, ranging from 1:10000 to more than 1:100000.

An important result of this study was the patients’ serum inhibited the union of AcM 1E10 (Ab2) with AcM P3 (Ab1). This suggests the presence of antibodies that share idiotopes with AcM P3 in the serum of the patients who were immunized with the 1E10-Alumina vaccine. These antibodies may be found in the site of recognition of AcM P3 by AcM 1E10, or in a location that is closely related with the union site.

Ab3 antibodies were detected in the immunized patients with the same specificity as AcM P3. They are able to selectively recognize N glycolilated gangliosides by ELISA and HPTLC immunotincture. These Ab1’ antibodies were able to inhibit the union of AcM P3 with the NGcGM3 ganglioside. Titres of up to 1:12800 and 1:6400 were obtained of anti-gangliosides antibodies of IgM and IgG isotypes respectively.

Anti-ganglioside antibodies were detected in the patients 7 to 9 months after the conclusion of the treatment induction stage, indicating the generation of a long-term response. Subsequent re-immunization with this vaccine produced a sTable increase of anti-NGcGM3 titres up to 1:1600 (Diaz, A. Clinical Immunology).

On the other hand, a considerable difference has been found in isotype patterns and the subclasses of immunoglobulin produced by the patients against AcM 1E10, in comparison with N glycolilated gangliosides. This suggests the activation of two different populations of B cells which are able to recognize these gangliosides.

Remanent reactivity against GM3(NeuGc) was detected in experiments in which complete inhibition was achieved in serum recognition by AcM 1E10 due to absorption with this AcM. This indicates that a high percentage of the positive antigen response induced by the 1E10-Alumina vaccine is not idiotype positive. This suggests some regulation of a parallel set of cells that produce N-glycolilated anti-ganglioside antibodies, which are present in a natural form in breast carcinoma patients. (Diaz, A. Clinical Immunology), (Figure 5).

All these studies show the therapeutic potential in research and development that have been achieved in almost a decade. Though our research does not favor a particular product yet, their results are encouraging, given that we have only carried out Phase 1 studies.

Unlike internationally developed vaccines, our products have rendered evidence of direct antitumorous response in cases of sTable disseminated disease for more than 20 months after treatment start-up. This is an important scientific achievement in the field of immunotherapy, especially in breast cancer.

Each of the vaccines has its own immunologic advantages. We do not seem to have found direct links with antitumorous response, but they do show immune restoration capacity in cases of long evolution (more than five years after diagnosis), or with multiple onco-specific treatments: surgery, radiotherapy, hormone therapy, chemotherapy, that affect these patients’ immune system. This has been demonstrated by the seroconversion of specific antibody titres for the antigens expressed by each vaccine.

No statistical significance has been found from the start of treatment in trying to relate immunized patient survival with any of the GM3 (NGcGM3, NAcGM3) ganglioside or 1 E10 anti-idiotypical vaccines (Figure 1). Variability in patient characteristics, based on the stage in which they are included in the trial, previously administered oncological treatment, and time of diagnosis of metastasis before inclusion in the study, influence this result.

In addition, these are vaccine products with a wide safety profile which reinforces the strategy of using this new therapeutic alternative against advanced breast cancer with potential low-risk benefits.

No vaccine that is still under development has been registered for breast cancer. It is our opinion that the strategy to modify the evolution of this disease at its advanced stages cannot be solely achieved with immunologic rescuing or an increase in patient survival. It is important to make a change in multidisciplinary behavior, where prevention of modifiable risks and immunotherapy can really prevent the appearance of the disease; and in those cases in which the disease is present, early action can be taken to prevent recurrence and achieve better life quality.

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