Study of the humoral immune response induced with the CIMAvax-EGF vaccine of its relationship with the survival of patients with non-small cell lung cancer

July 9, 2024by CubaHeal Research0

Synthesis

The epidermal growth factor receptor (EGFR) system and its ligands are considered an attractive target for immunotherapy directed to tumors of epithelial origin.

Of non-small cell lung cancers (NSCLC), between 40 and 80% overexpress EGFR, which has been associated with poor prognosis and resistance to agents cytotoxic This tumor location has been chosen for the development of several clinical trials Phase I / II with the CIMAvax-EGF vaccine that are based on active immunization with the factor of epidermal growth (EGF), one of the main ligands of the EGFR. In this work, evaluate two different treatment schemes to demonstrate for the first time that the generation of anti-EGF neutralizing antibodies in patients with NSCLC, is able to inhibit the activation of EGFR in the presence of this ligand and correlations are found between neutralizing capacity and the magnitude of the response of specific antibodies generated with vaccination Additionally, a relationship was found between age and the clinical benefit of the vaccination, and data are offered on the relationship between the characteristics of the response of Anti-EGF antibodies generated and the clinical benefit of patients treated with CIMAvax-EGF. At the same time, the possibility of optimizing the vaccination scheme was demonstrated previously used in order to achieve a more powerful specific immune response, without toxicity related

INTRODUCTION

Therapeutic vaccination for the treatment of cancer depends on the induction of a specific immune response against antigens associated with the tumor and is currently considered as one of the most promising strategies for the therapy of this disease (Geldmacher and cols. 2011). The study of the mechanisms of action of vaccines in the control of Cell proliferation is essential for the development of these therapeutic strategies, and many Sometimes it requires the initial assessment of the capacity of different treatment schemes to induce specific responses against own molecules that are involved in regulation and cellular replication.

Since its inception, vaccinology has been based on immunization through the use of foreign antigens. This has greatly limited the knowledge about the reactivity of the immune system to own molecules, which has skewed the experiments of autoimmunization basically to the field of autoimmune diseases. However, there are experimental evidences that sustain the existence of a reactivity on the part of the system immune to own molecules. Among the evidences that sustain this theory are:

presence of natural auto-antibodies (Avrameas et al., 1981) and autoreactive T cells (Cohen 1986) in healthy individuals, the discovery of positive selection for generation of the T cell repertoire by own peptides at the level of the thymus (Nikolic-Zugic and Bevan 1990), and the existence of a functional immune system in animals maintained in a free environment pathogens (Pereira et al 1986).

These evidences support the theory of the existence of an immunological homunculus, which based on the belief that natural autoimmunity does not occur randomly, but rather that could be directed towards a particular sub-population of self-antigens (Cohen and Young 1991; Cohen 1992). Therefore, in its definition, the immunological homunculus is no more than term applied to the natural autoimmune repertoire directed against the dominant antigens, the which is composed of T and B cells capable of recognizing dominant epitopes of the own (Cohen and Young 1991). These autoimmune cells are regulated by cellular networks, which include idiotype / anti-idiotype interactions.

EGF is recognized by natural antibodies in healthy subjects, is expressed in the thymus and when is accompanied by secondary signals (adjuvants and transporter proteins) activates a more efficient immune response against it, which increases with the suppression of regulation of the immune system (González et al 2002). For all these characteristics, it can be considered this growth factor as a component of the immunological homunculus.

EGF is one of the major ligands of the epidermal growth factor receptor

(EGFR). The binding of EGF to the extracellular region of EGFR induces a dimerization of the receptor that results in its autophosphorylation and in the transmission of mitogenic signals (Prigent and Lemoine 1992; Prenzel et al. 2001). The over-activation and / or overexpression of the EGFR can induce the alteration of various biological functions, such as cell proliferation, migration, differentiation and apoptosis, which ultimately lead to the transformation of a normal cell in malignant. There are multiple evidences about the prognostic value of the EGFR expression in breast, lung, colon, stomach and prostate cancer (Pérez et al. 1984; Macias et al. 1987; Salomon et al. nineteen ninety five). In fact, several studies in gastric cancer who have examined the concurrent expression of EGFR and its ligands (Yonemura et al., 1992; Tokunaga et al. nineteen ninety five; Nicholson et al. 2001), reported great disadvantages in times of survivals or in tumor-free progression-free survival when there was co-expression of EGFR and its ligands EGF or transforming growth factor α (TGFα) “Transforming growth factor”).

The EGF / EGFR system is a very attractive target for the development of new

therapies aimed at the treatment of cancer (Arteaga 2003). Among the best-known strategies of EGF / EGFR system blocking are those of direct blocking of the receiver through monoclonal antibodies (mAbs) that specifically bind to the extracellular domain (DEC) of the molecule (Barker et al 2001, Norman 2001), as well as the use of low molecular molecular weight with receptor tyrosine kinase activity, competing for the binding site of the ATP (Crombet et al 2004).

Active immunotherapy against EGF is an emerging concept, in which it is proposed

manipulate the immune response of the individual, to generate specific antibodies against EGF, able to block the ligand / receptor binding and consequently the signaling through this latest. On the basis of this concept, the therapeutic vaccine CIMAvax-EGF was generated, which is formed by a chemical conjugate between the recombinant human EGF and the P64k protein of Neisseria menigitidis, mixed with the oil adjuvant Montanide ISA 51 (EGF / P64k / Montanide ISA 51). Both in patients with advanced cancer and in experimentation with animals, this vaccine has been administered intramuscularly and it has been possible to generate response of specific antibodies by EGF (González G et al., 1997). The neutralization of EGF mediated by antibodies generated with vaccination with CIMAvax-EGF, could prevent the activation of EGFR and stop the cell cycle of tumor cells that overexpress it.

In this way, other mechanisms can be triggered, such as apoptosis and inhibition of angiogenesis, which can lead to the destruction of the tumor or, simply, the stopping their growth. These mechanisms have not been previously studied.

Non-small cell lung cancer (NSCLC) is one of the most malignant diseases frequent throughout the world, with a very high mortality rate. Only 30% of Patients can be treated surgically. Traditional treatments have a modest efficacy in most patients (Bunn et al 2003). Between 40 and 80% of the NSCLC over-express EGFR (Salomon et al 1995), which has been associated with poor prognosis and resistance to cytotoxic agents (Ryan and Chabner 2000). For these reasons, patients with this type of tumor have been chosen for the development of several trials Phase I / II clinical trials with the CIMAvax-EGF vaccine. These clinical trials have shown that the vaccine is immunogenic and well tolerated. There has also been an increase in the survival of those patients who achieve a high antibody response specific against the EGF (González et al 2003). On the other hand, a publication of the year 2006 demonstrated that higher doses of EGF are well tolerated and that there is a relationship between doses, the antibody titer and the survival of the vaccinated patients. (Ramos et al 2006) However, the mechanism of action and the potentiation of the humoral immune response specific, as well as the relationship between the biological effect of the specific Antibodies generated by vaccination and the clinical benefit of the treated patients, have not been evaluated in depth in previous studies.

On the basis of the background described above, the following hypothesis was formulated job:

The specific antibodies generated by vaccination with CIMAvax-EGF in combination
with chemotherapy, in two different treatment schemes, reduce the concentration of EGF in the serum of patients with NSCLC and interfere with the activation of EGFR promoted by this ligand.

To demonstrate this hypothesis, the following objectives were set:

To evaluate the immunogenicity of the CIMAvax-EGF vaccine and its effect on EGF concentrations in the serum of NSCLC patients treated with different immunization schemes.
To evaluate the ability of anti-EGF antibodies to prevent the activation of EGFR.

Evaluate the relationship between the characteristics of the specific humoral immune response generated and the survival of patients treated with different schedules of treatment.

To achieve these objectives, we propose the following experimental tasks:
Determination of the titer of specific antibodies by recombinant human EGF

in the serum of patients in advanced stages of NSCLC (IIIb / IV) immunized or not with the CIMAvax-EGF vaccine in two treatment schemes: chemotherapy / vaccine / vaccine (QVV) and vaccine / chemotherapy / vaccine (VQV).

Determination of the response of specific antibodies by different regions of the EGF molecule.
Determination of EGF concentration in serum of patients under evaluation.
Determination of the percentage of inhibition of EGF binding to its receptor in vitro as a measure of the neutralizing capacity of the antibodies generated by the vaccination.
Determination of the inhibition percentage of phosphorylation of EGFR, induced in vitro by EGF, caused by patient sera.
Evaluation of the survival of the patients included in the study and of their correlation with the immunological variables studied.

The scientific novelty of this work is that, for the first time, it is demonstrated in NSCLC patients treated with the CIMAvax-EGF vaccine, the capacity of the anti-EGF antibodies generated to neutralize EGF and prevent phosphorylation of EGFR mediated by said ligand and the direct relationship of this neutralizing capacity with the magnitude of the specific antibody titers by the B loop of the EGF molecule. Additionally, it demonstrates that an immune response can be generated with the VQV treatment scheme specific anti-EGF more powerful than when using the QVV treatment scheme.

The contribution to knowledge consists in the demonstration that the humoral immune response. The specific vaccine generated by the CIMAvax-EGF vaccine can interfere with regulatory cell proliferation associated with the interaction between EGF and its receptor. In this way supports the antitumor activity of active immunotherapy based on EGF observed with the use of the two current treatment schemes.

The practical value of the results consists of supporting the use of the vaccine CIMAvax-EGF as a therapeutic alternative for the treatment of epithelial tumors dependent on EGF for its proliferation and to demonstrate the possibility of optimizing the vaccination scheme previously used in order to achieve a more powerful specific immune response.

The present results have been presented in eleven national and international events; between they, the International Workshops of Immunotherapy IT-2006 and IT-2008 (Havana) and the congresses EORTC 2006 (Prague, Czech Republic) and EACR20 2007 (Lyon, France); form part of three publications published in international journals with a high impact index (“Journal of Clinical Oncology”, “Clinical Cancer Research” and “Journal of Immunotherapy”) and are endorsed by the Prize of the Academy of Sciences of Cuba in the year 2008. They are part of a registry of the CIMAvax-EGF vaccine in Cuba and in Peru.

BIBLIOGRAPHICAL REVIEW

Non-small cell carcinoma: treatment and survival

The vast majority of lung cancers are carcinomas, that is, malignant tumors that are born from epithelial cells. There are two forms of lung carcinoma, categorized according to the size and appearance of the malignant cells seen histopathologically under a microscope: non-small cell tumors (80.4%) and small cell tumors (16.8%) (Travis 1995). This classification is based on histological criteria and has important implications for the treatment and prognosis of the disease (2008).

Non-small cell carcinoma (NSCLC) represents more than 75% of all neoplasms that originate in the lung, most of which presents in stage of disease locally advanced or metastatic at diagnosis (Jemal et al 2003; Jemal et al 2005).

Surgery is the therapeutic option with curative intent, but only one third of the cases at the time of diagnosis has a localized stage that allows said treatment (Esteban et al 2006). Despite the surgical effort many patients develop a local or distant recurrence due to non-detectable injuries at the time of surgery (Mountain 1997).

The encouraging results obtained in the treatment of small cell carcinoma of lung with chemotherapy in the mid-80s, he turned his attention to the treatment of non-small cell carcinoma based on this therapeutic modality. Starting then modest progress has been observed in terms of survival; Nevertheless, In general, the response rate is lower than that of the small cell.

A complete response is rarely observed and only 40% of patients can be achieved a partial response with current chemotherapies (Silvestri and Spiro 2006).

Cisplatin (CDDP), or its similar, carboplatin, are the chemotherapeutic agents that are they are used more frequently to treat NSCLC (Murray et al 2006). At the NSCLC in advanced state, the response rate obtained for the first chemotherapy regimens with platinum salts was generally low (10 to 15%), with only 6 weeks of extension of the median survival (Group 1995).

Later, in the decade of the 90s, new drugs were developed for the management of NSCLC including docetaxel, paclitaxel, vinorelbine and gemcitabine. This led to the completion of a large number of clinical trials evaluating these agents alone or in combination with cisplatin or carboplatin. These combination studies showed a significant improvement in the efficacy of NSCLC treatment (Kelly et al 2001, Scagliotti et al. cols. 2002). With the introduction of these new agents, it has been possible to extend the advantage in the survival in patients in a period of 3 to 4 months when compared to the palliative treatment alone. The results of treatment with at least 4 cycles of chemotherapy Based on current combinations, they offer a median survival of 7.8 to 8.1 months compared to 4 to 5 months when only supportive treatment is offered, with a survival a year from 31 to 36% compared to 18% obtained without chemotherapy (Schiller et al 2002).

Myelosuppression, that is, the reduction in the production of the granulocytic line of the white blood cells is the only adverse effect that limits the dosage of these medications (Katzung 2007). Currently, research continues in clinical studies the best way to use this combination of medications (Morera 2004). On the other hand, the main advantage that exhibits modern chemotherapy is related to a better toxicity profile than includes: lower incidence of nausea, vomiting, hair loss and sepsis due to neutropenia febrile.

Chemotherapy is currently considered as the standard treatment for cancer of lung followed by curative resection and the debate about the usefulness of the adjuvant treatment in the early stages (Ia and Ib); although there is clear evidence to favor its use in resectable stages II and IIIa (Esteban et al 2006). However, in the daily clinical practice there is little data available on the treatment of patients with Non-small cell lung carcinoma (Zietemann and Duell 2010).

2.1.3 Biological therapy in the treatment of lung cancer Given the modest success obtained from the application of standard therapies in the treatment of lung cancer, the scientific community has turned to the study of new schemes therapeutic for the management of this disease. Biological therapy or immunotherapy is one of the new strategies used for the treatment of lung cancer. These therapies biological agents use the immune system, either directly or indirectly, to fight cancer or to reduce the side effects that some treatments can cause (2010 c). The Immunotherapy can be indicated in conjunction with surgery, chemotherapy and radiotherapy.

Some of the most used compounds in this therapeutic modality at present include:

interferons, inhibitors of growth factors, monoclonal antibodies, therapeutic vaccines, gene therapy and non-specific immunomodulatory agents (2010 b).

The development of immunotherapy based on specific molecular targets present in the cancer cells is largely due to the progress made in recent years in the understanding of tumor biology and the oncogenetic mechanisms of lung cancer.

Some of these molecular targets include vascular growth factor and its receptors, as well as the epidermal growth factor receptor (Provencio et al 2010). The agents employees for this type of treatment appear to be, in general, more safe and effective in certain histological subtypes of lung cancer, particularly that of non-small cells and in its advanced stages (Rossi A 2010). The only disadvantage so far is that they require an adequate histological diagnosis of cancer.

EGF and its receptor as a target of antitumor therapies.

Growth factors:

Growth factors are a group of multifunctional polypeptides that are involved in a variety of physiological processes, including embryogenesis, cell differentiation and proliferation, angiogenesis and activation of the immune system (Klagsbrun 1990).

The different growth factors have been grouped into several families, based on their structure and its biological activity. These families are included by the family of the factor of epidermal growth (EGF), the family of fibroblast growth factor (FGF), the of platelet-derived growth factor (PDGF) and the growth factor family insulin-like (ILGF), among others.

The EGF family is formed in turn by several growth factors. The union of these growth factors to their respective receptors results in the activation of a tyrosine kinase associated with a cytoplasmic domain. This event triggers a number of events biological and enzymatic, which eventually lead to DNA synthesis and cell proliferation.

The EGF family:

This family of ligands groups structurally and functionally related proteins that come together to ErbB receptors and activate them. The basis of this structural relationship is the so-called domain type EGF, a sequence of 45 to 50 amino acids that contains 6 cysteines spaced from one specific and joined together by 3 intramolecular disulfide bridges, which determine a compact three-tiered tertiary structure. There is a lot of evidence that this structure is responsible for the binding of those ligands to their receptors and which discontinuous regions of theThey are involved in this recognition (Todaro et al., 1980).

Among the members of this group in addition to their natural ligand, the EGF, we find the factor of transforming growth alpha (TGF, from English, “transforming growth factor α”) (Todaro y cols. 1976), amphiregulin (Shoyab et al 1989), the growth factor binding heparin (HB-EGF, from the English, “heparine binding growth factor”) (Higashiyama et al., 1991), betacellulin (BTC) (Ciccodicola et al 1989) and epiregulin (Groenen et al 1994). Other Ligands of the EGFR family described below are neuregulins or heregulins, (Harari et al 1999) and tomorregulin (Kinugasa et al 2004).

Most ligands in this family are synthesized as membrane precursors that they are formed by the EGF type structure in the extracellular domain, an amino extension terminal, a transmembrane region and a cytoplasmic tail. The ligands are released as soluble factors, from the surface by a proteolytic cleavage in the extracellular domain; this event represents an important and efficient strategy to regulate the activity of a variety of membrane proteins (Dong and Wiley 2000).

The EGF

EGF was isolated in 1962 from mouse submaxillary glands and subsequently, in 1975. The human form of EGF was obtained from human urine (Cohen et al 1975; Starkey and cols. 1975). The discovery of this growth factor led to the search for identification of its cellular receptor (EGFR) and the detection and characterization of other members of the family.

EGF is a polypeptide of 53 amino acids with a molecular weight of 6045 Da, and 6 residues conserved of cysteine that form three covalent bonds by disulfide bridges, which suggests which is a structurally stable molecule. The EGF sequence also contains a relatively large number of glycine and tyrosine residues (Cohen et al 1975; Gregory 1975). It is a ubiquitous molecule, and like other growth factors and unlike the classic hormones, its production seems to be systemic and its action paracrine (Carpenter et al.1975).

The EGF precursor (pro-EGF) is distinguished by its large size (1200 amino acids) and its eight “EGF-like” sequences in the extracellular domain, where either of them can function as a ligand for the EGFR (Gray et al., 1983).

EGF is present in plasma at very low concentrations and is generally associated with platelets and other elements of blood coagulation (Savage et al 1986). In humans, have found high levels of EGF in prostatic, seminal and cerebrospinal fluid fluids, as well as in a wide range of epithelial cells such as lung, stomach, kidneys, duodenum, pancreas, skin, breast, salivary glands, thyroid, ovary, uterus and placenta. It is further stated that the liver can be a source of circulating EGF (Laborde et al 1988).

Although all the functions of EGF are not known exactly, a large variety of effects in mammals, both in experimental animals and in cell cultures coming from this species. Among these effects are: early opening of the eyelids and appearance of the teeth in newborn mice, in vitro stimulation of normal cells and tumors of ectodermal and mesodermal origin (Carpenter and Cohen 1990), as well as the development and maturation of other organs such as the lungs, the gastrointestinal tract, mammary gland, among others. In addition, it promotes angiogenesis, inhibits gastric secretion acid and has been shown to be effective in accelerating the healing of wounds (Schultz et al.cols. 1991).

All members of the EGF superfamily join the EGFR or another member of the EGF family of receptors with tyrosine kinase activity, but exhibit biological activities different

EGF receptor: the EGFR

The EGFR belongs to a family of receptors with tyrosine kinase activity that translate information of extracellular growth factors to signaling pathways intracellular These signals in turn modulate various biological functions, such as cell proliferation, migration, differentiation and apoptosis (Holbro and Hynes 2004). This family, also known as the ErbB family, consists of four recipients: the EGFR or Her1 / ErbB1 (which was the first to be cloned) (Ullrich et al 1984), Her2 / neu / ErbB2, Her3 / ErbB3 and Her4 / ErbB4 (Normanno et al., 2005).

The receivers of the ErbB family present a modular structure consisting of a domain extracellular ligand binding, a transmembrane hydrophobic domain and a domain intracellular. The latter has a very conserved cytoplasmic stem through evolution, with tyrosine kinase activity, with the exception of Her3, which has amino acid substitutions critical, lacking kinase activity. The extracellular domains are the least conserved in the family, which suggests that different receptors have different specificities in the binding to its ligands. The receptors are frequently co-expressed in the cells and in dependence of the activating ligand can form homodimers or heterodimers, which generates a complex network of signal transduction (Alroy and Yarden 1997, Riese and Stern 1998).

EGFR structure

The EGFR is a transmembrane glycoprotein with a molecular weight of 170 kDa, which polypeptide chain consists of 1186 amino acids, and contains 40 kDa of oligosaccharides linked to asparagine residues in 11 or 12 potential glycosylation sites (Ullrich et al. 1984; Stroop et al. 2000). The EGFR is formed by an extracellular domain of union to the ligand of 621 amino acids, a simple transmembrane domain of hydrophobic alpha helices of 23 amino acids and an intracellular domain of 542 amino acids (Ullrich and Schlessinger 1990).

The extracellular domain of EGFR has a molecular weight of 105 kDa, and its conformation in the space is independent of the rest of the molecule. It consists of four subdomains denominated I (L1), II (S1), III (L2) and IV (S2). Of these, subdomain II has the critical region for dimerization to occur between the receptors, and it has been called “arm of dimerization “(Garrett et al 2002, Ogiso et al 2002).

The intracellular domain comprises three subdomains: the juxtamembrane subdomain, which is required for protein kinase C mediated feedback; the carboxy-terminal tail, which it lacks catalytic activity and has six tyrosines that are transphosphorylation and binding sites of adapter and / or effector proteins such as Grb2 and phospholipase Cγ, respectively; and the Central subdomain with tyrosine kinase activity (src), which is a subdomain of homology 1 (SH1), which is responsible for the transphosphorylation of the six tyrosine residues in the region carboxy-terminal (Bazley and Gullick 2005). It has been found that the phosphorylated tyrosines in the EGFR ligand dependent activation has occurred (Sweeney and Carraway 2000) and the phosphorylation pattern determines the type of second messenger will be recruited (van der Geer and Pawson 1995).

EGFR activation and signal transduction

It is postulated that the binding of a TGF- or EGF monomer to the extracellular domain of EGFR stabilizes a conformation that allows homodimerization or heterodimerization with others members of the EGFR family. The binding of the ligand to subdomains I and III cause spatial rearrangements that expose the subdomain II or “dimerization arm” of the molecule. The dimerization allows cross-phosphorylation in tyrosine residues that serve as sites of recruitment and anchoring of proteins with Src type 2 homologous domains (SH2, from “Src homology 2”) (Koch et al., 1991) or phosphotyrosine binding domains (PTB). “Phospho-tyrosine-binding”) (Garrett et al., 2003) that initiate multiple signaling pathways intracellular.

The network of signals transmitted through the EGFR is extremely complex. The diversity of ligands, together with the possibility of forming combinations of homodimers and heterodimers in the ErbB family, in turn coupled to various intracellular signaling pathways, determine this high degree of complexity in the so-called EGF / EGFR system. There are three ways fundamental signaling through the EGF / EGFR system. The first of these ways of signaling is the so-called pathway of protein kinases activated by mitogens (MAPKs, del English, “mitogen-activated protein kinases”). This way is possibly the way to more studied intracellular signaling associated with the EGF / EGFR system. The biological effects observed after the activation by ligands of the EGFR, could be associated to the Activation of the Ras / Raf / ERK path. When the activation is mediated by the EGF, these effects they are fundamentally associated with cell proliferation (Di Fiore PP 1987).

Another intracellular signaling pathway of activated EGFR is associated with phosphatidyl inositol 3-kinase (PI3K, from English, “phosphotidylinositol-3 kinase”), which stimulates anti-apoptotic signals mediated by the nuclear factor of transcription  (NF-B, from English, “nuclear factor-B”) and signs of cell division. Therefore, the activation of the PI3K pathway mediated by the EGFR is directly related to the survival of tumor cells.

A third signaling pathway occurs through c-Src, a cytoplasmic tyrosine kinase involved in numerous cellular processes, including mitogenic signals (Prenzel et al 2001). The transcription factor and transducer family is activated by this protein of signals 3 (STAT3, from English, “signal transducer and activator of transcription 3”), which has particular importance in the proliferation and survival of tumor cells (Rubin Grandis et al. 1998). A member of this family, STAT3, regulates the production of the factor of vascular endothelial growth (VEGF), the vascular endothelial growth factor which increases cell proliferation and is closely linked to angiogenesis phenomena in tumors (Niu et al 2002).

All intracellular signaling pathways activated through ErbB receptors converge in the nucleus where the cell cycle regulators and the transcription factors control the biological response to the activation of said receptors.

EGFR and cancer

The oncogenic effects related to the alteration of the EGFR’s physiology have turned

EGF / EGFR system in a very attractive and promising target for intervention

immunotherapeutic based on specific antagonist agents of EGFR constitutes a protein widely expressed in the tissues of the organism, it is considered an antigen associated with tumors (TAA, from English, “tumor associated antigen”).

The first line of thought suggests that EGFR is involved in the pathogenesis of human carcinomas is derived from a large number of studies that have shown over-expression of this protein in the majority of solid tumors of epithelial origin. He EGFR has an increased expression in lung tumors (Hendler and Ozanne 1984), breast (Pérez et al 1984), head and neck (Dassonville et al 1993), colon (Lockhart and Berlin 2005), esophagus, prostate (Liu et al 1993, Zhau et al 1996), bladder (Neal et al 1985), pancreas (Tan et al. 2004), ovary (Gullick et al 1986) and brain tumors (Salomon et al 1995). The Frequency of expression of EGFR in tumors of epithelial origin is generally high. By example, in the case of carcinomas of the head and neck, the EGFR is expressed in 100% of the cases studied, and in non-small cell lung carcinoma this frequency is approximately 80% (Normanno et al., 2003). The over-expression of the EGFR by Tumor cells are due to various mechanisms, which include gene amplification, alteration of transcriptional events, as well as deletions or mutations that generate constitutively active receptors (Nicholson et al., 2001).

The oncogenic effects related to the alteration of the EGFR’s physiology have turned EGF / EGFR system in a very attractive and promising target for intervention immunotherapeutics based on specific antagonist agents of EGFR constitutes a protein widely expressed in the tissues of the organism, it is considered an antigen associated with tumors (TAA, from English, “tumor associated antigen”).

Moreover, over-expression of EGFR is associated with poor prognosis in tumors of the head, neck and lung (Brabender et al 2001; Khademi et al 2002), with a high risk of recurrence of the disease (Chow et al 1997, Turkeri et al 1998) and with decreased survival in patients with ovarian, colon, bladder, thyroid and head cancer and neck (Akslen et al 1993; Fischer-Colbrie et al 1997; Grandis et al 1998). Further, Numerous studies show that the presence of elevated levels of EGFR directly correlates with resistance to conventional therapies (Holbro et al 2003).

Tumors possessing EGFR constitutive activation tend to be more aggressive in regarding the formation of metastasis. There is evidence in the literature that over expression EGFR leads to an increase in the metastatic ability of tumor cells some models, and that this is due to an increase in the capacity of intravasation of themselves (Xue et al., 2006). In addition, it has been found that many types of tumor cells migrate autocrine activation stimulated by EGFR (Obeid et al. 1997).

Growth factors also play a crucial role in the cell cycle. Some of they are part of the competent factors, which lead the cells of the phase G0 to the phase earliest of G1. The EGF in particular, is a factor of cell cycle progression, since it is in charge of stimulating the molecular events necessary for the occurrence of transition through the restriction point (point R) located in phase G1. Once this point, the cells can progress to the G2 phase, and from here continue to the next phases of the cycle even in the absence of growth factors. For that reason, the presence of excessive stimuli, causes a continuous progression of the cycle and the consequent cell division, leading to an uncontrolled proliferation of cells. This is the case in cells tumors, which also become independent of these stimuli through the Increased expression of signal receptor molecules, such as EGFR (Lui and Grandis 2002).

However, the correlation between expression levels and transformation of EGFR

Neoplastic in vivo is still controversial (Gullick and Srinivasan 1998). Robertson and collaborators showed that from 40 to 60% of the neoplasms examined show normal levels of EGFR expression (Robertson 1996 KW). In those cases the changes in the ligands of the receptor could have more influence on the antitumor activity than the changes related to the levels of expression of the EGFR. However, at present it is known that the levels of EGFR expression are very high in certain neoplasias and constitute a marker of bad prognosis For all the above exposed the system of EGFR and its ligands are in currently an excellent target for antitumor therapy.

Immunotherapy of cancer

Most cancer patients are treated with a combination of surgery, chemotherapy and radiotherapy. Surgery alone is not enough for eradication full of cancer cells. On the other hand, radiation and chemotherapy affect both malignant cells as to normal cells, causing severe adverse effects. For the In general, the immune system of a patient can not supplant these antitumor treatments, instead, it eliminates antigens that it recognizes as not own, while it generally ignores those that he considers his own. Tumors are made up of own cells that are malignant and that is the reason why they can elude the surveillance of the immune system (Cappuccio et al. 2007).

In recent years, interest in cancer immunotherapy has grown as a way to increase the response of the immune system against the tumor. There are two strategies essential for the evaluation of new oncological drugs: passive immunotherapy and active immunotherapy.

Passive immunotherapy and EGFR

Passive immunotherapy is based on the use of specific monoclonal antibodies by the extracellular domain of the epidermal growth factor receptor (EGFR). The accessibility which possesses the extracellular domain of the EGFR for antibodies and immunotoxins, the expression differential of the receptor on the surface of the cells (high in tumor cells and low in tissues normal) epithelial, and the biology of the EGFR / ligand system, has become a target molecule important for the development of passive immunotherapy of cancer of origin epithelial. In fact, they have been produced and characterized monoclonal antibodies against EGFR human, which bind to the receptor with an affinity similar to that of natural ligands, compete with them and inhibit the activation induced by the ligand (Kawamoto et al 1983; Sato et al. 1983).

The effects derived from the use of anti-tumor therapies with monoclonal antibodies against the EGFR are attributed to interference with ligand-receptor interactions that result in elimination of the proliferative stimulus. These antibodies could play a key role in the recruitment and activation of cytotoxic effector cells intervening in a mechanism such as antibody-mediated cellular cytotoxicity (ADCC)dependent cell cytotoxicity “) (Bier et al 1998).

One of these antibodies with therapeutic activity is the chimeric monoclonal antibody of isotype IgG1, ICM-C225 / Cetuximab / Erbitux. Cetuximab binds specifically to Subdomain III of the extracellular domain of EGFR, competing with the EGF ligand. So This antibody blocks receptor activation, affects the dimerization process, and induces efficient internalization and degradation of the EGFR (Shiqing et al., 2005). Also I know has shown that it can induce ADCC through the activation of mononuclear cells from peripheral blood of humans, suggesting another potential mechanism of anti-tumor action of said drug (Naramura et al 1993). Cetuximab is the first blocking antibody of the EGFR approved by the regulatory entity of drugs and foods in the USA (FDA “US Food and Drug Administration”) for use in patients. Initially he received the approval for use as monotherapy or in combination with irinotecan in patients with metastatic colorectal carcinoma with detectable expression of EGFR (Systems-ImClone 2004) and subsequently, for use in patients with squamous cell carcinomas of the head and neck in combination with radiotherapy. In addition, there are ongoing phase II and III clinical studies where they are including patients with advanced stage pancreatic carcinoma (Xiong and cols. 2004). For non-small cell lung carcinoma (NSCLC) (Lynch TJ et al. 2004; Rosell et al. 2004) have already completed phase III trials and this product has been registered in Europe. Although therapy with Cetuximab is tolerated, adverse effects often appear associated with the use of the drug such as allergic reactions, hypomagnesemia, fatigue and toxicity on the skin in the form of eruptions (Lacouture 2006). Sometimes these adverse events can be severe and lead to a reduction in the dose or early suspension of treatment (Tejpar et al 2007, Boland and Bebb 2010). Another antibody with encouraging clinical results is the humanized monoclonal antibody h-R3 / TheraCIM / Nimotuzumab. The Nimotuzumab, generated in the laboratories of the Center of Molecular Immunology was obtained from its murine predecessor, the ior egf / r3 developed by previous immunization of mice with placenta human enriched with EGFR (Fernández A et al., 1992).

Nimotuzumab, like Cetuximab, is a specific antibody against the extracellular domain of EGFR. Nimotuzumab was registered in Cuba by the Center for State Control of the Quality of Medicines (CECMED) in 2004 for use in patients with advanced head and neck tumors in combination with radiotherapy (Crombet et al. 2004). Subsequently, this registry was extended to other tumor sites such as tumors brain and esophagus. They are also in advanced stages of clinical studies that support its possible use in patients with tumors of the breast, prostate and lung, in the which have obtained encouraging results (Crombet et al., 2006).

Other monoclonal antibodies specific for the EGFR have obtained approval for Use in patients are ABX-EGF / Panitumumab (Tiseo et al 2004, Amato 2005, Tyagi 2005), and the EMD 72 000 / Zalutumumab (Graeven et al., 2006).

Another strategy currently used that is based on the blocking of signaling through the EGFR is the inhibition of tyrosine kinase activity using low molecular weight drugs:

the tyrosine kinase inhibitors (TKI, from English, “tyrosine kinase inhibitors”). The results more promising in clinical phase with this type of compounds have been obtained with the analogues of the ATP, from the family of pyridopyrimidine and quinazoline, which act by blocking the site of ATP binding in the kinase domain of the receptor, inhibiting its activation. The initial studies conducted with the quinazoline ZD1839 / IRESSA / Gefinitib showed that this drug possesses anti-tumor activity in vitro and in vivo, both as monotherapy, and in combination with conventional therapies (Ciardiello et al 2000; Sirotnak et al 2000).

Other strategies employed to inhibit EGFR activity associated with the use of small interferon ribonucleic acid (RNA) molecules (siRNA, from English, “small interfering ribonucleic acid “) (Zhang et al 2004; Kang et al 2006); toxin-ligand fusion proteins against EGFR, through the conjugation of EGF or TGF-α to the truncated forms of the Pseudomonas exotoxin A and other less immunogenic cellular toxins (Fitzgerald 1996;

Shao et al. 1998; Uckun et al. 1998; Noonberg and Benz 2000).

Active immunotherapy

Bases of specific active immunotherapy

Active specific immunotherapy has re-emerged vigorously since the end of the decade

However, more than 100 years ago, William Coley described remission for the first time of tumors in cancer patients treated with bacteria or their respective extracts (Wiemann and Starnes 1994). Some of these bacterial products were powerful activators of the system immune and consequently the tumors were destroyed (Krieg and Wagner 2000). From this discovery has accumulated a lot of evidence about the recognition of

the tumors by the immune system. Many antigens have been discovered and characterized they are overexpressed in malignant cells compared to their expression levels in normal cells. In addition, the hypothesis of the immunological surveillance of the cancer, in which the antigen-presenting cells (APC) cells “) recognize transformed and mutagenic cells, and this allows them to activate other components of the immune system to eliminate them (Sahin et al 1995, Kaplan et al 1998). From In fact, the boom in the use of specific active immunotherapy has been motivated by Multiple scientific evidences demonstrating the existence of tumor immunosurveillance (Burnet 1970), the possibility of defining and characterizing antigens associated with the tumor (Ko et al.

2003), and a better understanding of the molecular mechanisms of processing and presentation of antigens and induction of the immune response. Currently more than 50

vaccines are being evaluated in clinical studies, and more than 400 trials have been conducted clinical trials (Lage et al., 2005). Cancer vaccines are an application of these

knowledge and are based on the principle of the induction of an effective immune response to eliminate the tumor.

Because cancer vaccines were initially evaluated in patients with tumors advanced and therefore could not prevent the disease, they have been called vaccines therapeutic In the field of active immunotherapy, it is considered that there are two generations of cancer vaccines, which currently coexist in different clinical trials: a first generation based on cellular preparations, known as cellular vaccines and other based on molecular entities, called molecular vaccines.

The vaccines of the first generation, have used autologous tumor cells irradiated or

extracts thereof as a source of tumor antigens, in combination with adjuvants (Mitchell et al 1988, Morton et al 2002). Also, vaccines with cells have been developed autologous or heterologous tumors modified chemically, enzymatically or genetically (Mach and Dranoff 2000; Berd et al. 2001; Salgia et al. 2003).

The development of vaccines based on antigens associated with the tumor, which constitute a own molecules accounts for which mostly operate the deletion mechanisms

central thymic, finds its theoretical framework in the theory of damage, postulated by Polly Matzinger (Matzinger 1994). According to this theory, the main function of the immune system lies in the need to distinguish between harmful and harmless, instead of distinguishing between what is proper and what is not own. In this way, vaccination against one’s own could be successful if it occurs in a context of “danger signals”, for which the design of the adjuvants.

Therefore, the vaccination strategies used in the treatment of cancer will depend on

large measure of the quality of the selected antigen (Berzofsky et al., 2004) and of the adjuvant used for the activation of the immune response, leading to an efficient activation of the APC and subsequent activation of CD4 + T cells and CD8 + T cells to exercise

functions.

In the field of vaccination against tumors there are also recent reports that point

that the diversification of antigenic recognition that occurs with some strategies

vaccines may be beneficial for the antitumor response. This phenomenon very well described in models of autoimmune diseases, it starts with the response against a simple epitope antigenic This epitope is presented in the MHC II of the presenter cells, which activates Th cells and effector T cells that cause chronic inflammation and cell death

White. The cellular waste, which contains other antigens, is then captured by the cells

presenters and presented cross-wise by the MHC I, with the consequent activation of

new CD8 + T cells specific for other epitopes in the same antigen (diversification

intramolecular) or in other molecules (intermolecular diversification). There are some evidences experimental that sustain this phenomenon. El-Shami and collaborators published that in mice vaccinated with an OVA peptide (ovoalbumin) and challenged with EG7OVA (thymoma line EL-4 transfected with OVA) are able to generate a CTL response against other antigens. This response showed protection to the subsequent challenge with EL-4 that does not express OVA (el-Shami et al 1999). In the case of human studies, it has been published that patients who over-express the epidermal growth factor receptor type 2 (HER-2 / neu, from English “human epidermal growth factor receptor type 2”) in breast and ovarian tumors and that have been vaccinated with peptides of this molecule combined with the colony-stimulating factor of granulocytes and macrophages (GM-CSF, from English “granulocyte-macrophage colony stimulating

factor “), show an immune response against peptides not included in the vaccine.

This type of response also correlates with the antitumor response observed in

vaccinated patients (Brossart et al 2000).

Due to the low immunogenicity of the tumor cells and the molecular definition of the

tumor antigens, in the 80s the investigations diverted their attention to the use of

molecular entities in active therapy against cancer (Lage et al., 2005). One of the

strategies used in this type of vaccines has been the insertion of the genes coding for these tumor antigens in recombinant viral vectors (Liu 1998) and the use of the corresponding naked nucleic acid (Polo and Dubensky 1998). Another type of molecular vaccine is composed of peptides derived from various tumor antigens. Among these, the most used are those derived from melanoma antigens such as MART-1, gp-100, tyrosinase and antigens of the MAGE family. Vaccination with these peptides has been successful in induction of specific CD8 + T cells, but the clinical response has been variable and ineffective (Slingluff et al 2003; Akiyama et al 2005). Other strategies with molecular vaccines it comprises the use of synthetic or purified proteins from the tumor (Naftzger et al 1996). These vaccines include those that contain heat stress proteins (HSP,

English “heat shock protein”); mutated oncoproteins such as K-ras and p53; glycoproteins of family of mucins such as MUC-1; the carcinoembryonic antigen (CEA)

“carcinoembryonic antigen”), expressed in gastrointestinal tumors; the prostate antigen

specific (PSA, prostate-specific antigen), overexpressed in prostate cancer, among other. In this sense, an intense effort has been made to design vaccines based on autologous HSPs derived from tumors (Belli et al 2002). These endogenous proteins, highly conserved, they form complexes with tumor peptides that induce a response of

specific T cells when presented in the APC (Rivoltini et al 2003).

Another strategy, which has been widely used, is vaccination with dendritic cells (DCs)

(Nestle et al 2001). These presenter cells capture, process and present peptides to

virgin T cells in the secondary lymphoid organs. This process is the first sign

that together with the interaction of costimulatory molecules between the DCs and the T lymphocytes leads to the activation of the latter and the subsequent elimination of the tumor cells that they express the antigen. DCs can be stimulated with autologous or allogeneic tumor cells (Celluzzi and Falo 1998), tumor extract (Ashley et al 1997, Nair et al 1997) and bodies apoptotic (Chang et al 2002). In addition, they can be fused by electroporation with irradiated tumor cells (Siders et al., 2003). It is also possible the incorporation of acid deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) exogenous in these cells by recombinant viruses, plasmids or electroporation (Akiyama et al 2000; Esslinger et al. 2002). Most of these preparations have been immunogenic and have caused rejection of tumor in animal models. In the clinical scenario, the most advanced DC vaccine is Provenge, also known as Sipuleucel-T. This vaccine, used in prostate cancer advanced, relies on DCs loaded ex vivo with a recombinant fusion protein that It contains prostatic acid phosphatase and GM-CSF. The results of the phase III trial

showed a significant increase in the survival of vaccinated patients with respect to

control that only received placebo, as well as an appreciable decrease in serum levels of

PSA (Basler and Groettrup 2007). This therapy constitutes the first approved therapeutic vaccine by the FDA for cancer (Kantoff et al 2010; Madan and Gulley 2011).

The main benefit of active specific immunotherapy in the treatment of cancer is the

possibility of directing the attack of the immune system towards the tumor cells of the own individual. This specificity makes them more effective and probably less toxic than others therapeutic agents used so far.

On the other hand, despite the experience accumulated to date in the field of immunotherapy specific active, it is very common to observe the lack of association between response induction specific immune system with a favorable clinical response in patients. Among other causes, this could be due to a low sensitivity in the techniques used to measure the responses generated, by the inability to measure the response that takes place at the site of the tumor and / or in secondary lymphoid organs or by the use of suboptimal vaccination schemes for generation of a specific response really effective.

Optimization of vaccine strategies

Although there is no solid evidence in the scientific literature about optimal schemes

of treatment with cancer vaccines, the studies coming from the field of diseases

Infectious diseases suggest that in some scenarios, the application of high doses of vaccines composed of recombinant proteins is associated with a robust specific response by the antigen and an increase in the frequency of responding individuals. This is the case of a test carried out on animals, which were immunized with different doses of a vaccine

against the herpes virus, obtaining greater responses of specific T lymphocytes in those animals immunized with the highest dose (Martina et al 2003). In another trial conducted in healthy donors, the group that received the highest dose of a vaccine based on Recombinant proteins of human papillomavirus (HPV) Virus “) generated the highest frequency of responders among the vaccinated subjects (de Jong and cols. 2002). On the other hand, the administration of high doses of vaccine is associated to the development

of a memory response after vaccination, as to the generation of a response of high avidity antibodies by the antigen (Kundig et al 1996).

The correct identification of effective adjuvants is also a critical aspect in the

development of cancer vaccines. The requirements of an adjuvant used for the

development of therapeutic vaccines for the treatment of cancer patients, differ from those requirements of conventional adjuvants. First, the patients who will receive the

immunotherapy are immunocompromised patients, who have damaged mechanisms of

processing and presentation of antigens, and the inhibition of the reactivity to antigens themselves controlled by regulatory T cells is reinforced (O’Garra and Vieira 2004). In second Instead, the tumor antigens are usually their own and therefore relatively little

immunogenic Finally, tumors develop mechanisms to evade the immune system,

as for example, the mechanisms of tumor editing, the low presence or absence of expression of MHC-I molecules, as well as the secretion of cytokines or suppressor molecules (Ko and cols. 2003). Therefore, adjuvants of vaccines for the treatment of cancer should not only stimulate responses to poorly immunogenic antigens, but also reverse immunosuppression induced by tumors. These adjuvants then have to face various challenges in the different stages of the generation of antitumor immunity during vaccination. In fact, Its main functions within vaccinal preparations are: serve as a reservoir to slowly release the antigen to the area of inflammation in which DCs are recruited; to mature the DCs; mediate the cross-presentation of antigens coupled to them, modulate and polarize the immune response, as well as activate the cytotoxic T lymphocytes (CTL, from English: “cytotoxic T lymphocytes “).

Among exogenous “danger signals” that have demonstrated excellent adjuvant capacity

Bacillus Calmette-Guérin (BCG) and unmethylated bacterial DNA with sequences are found CpG (CpG). BCG has been used with some success as an antitumor agent in the

treatment of superficial bladder cancer and in melanomas (Alexandroff et al 1999; Soiffer et al. cols. 2003). For its part, CpG has shown the ability to stimulate powerful responses

specific against several types of tumors in preclinical trials (Brunner et al 2000). These

preclinical investigations were the precedent for the performance of numerous trials

clinical trials that are currently underway using the CpG or BCG sequences

as adjuvants of therapeutic vaccines against cancer (Speiser et al., 2005). Another derivative bacterial to which important immunopotentiating properties are attributed and an effect antitumor in preclinical trials is the OmpA protein of the outer membrane of the bacterium Gram-negative Klebsiella pneumoniae (Jeannin et al 2000). The capacity of all these derivatives of activating the DCs has been demonstrated in various scenarios. For example, both BCG as CpG activates human and mouse DCs, promoting the increase in

expression of major histocompatibility complex molecules (MHC) “major hiscompatibility complex”) type I and II, of the CD80 and CD86 molecules and of the secretion of IL-12 (Jakob et al 1998, Goldmann et al 2002). There are also experiments

carried out with the recombinantly expressed OmpA protein of N. meningitidis, which

show their ability to induce complete maturation of DCs through receptors Toll type (TLR) (Jeannin et al 2000).

The administration routes used for vaccination may also be important capital in obtaining an optimal effect with a specific vaccine preparation. In the cancer vaccination schemes have been used mostly the routes of subcutaneous or intradermal immunization. The results derived from numerous trials carried out on animals, as well as some clinical studies, suggest that the pathways intratumoral or intraganglionar administration could be the most effective. Kudo-Saito and collaborators for example, demonstrated that the sequential use of a vaccine subcutaneously followed by an intratumoral vaccination strategy is more effective in terms of effect antitumor, than the use of a single immunization route (Kudo-Saito et al., 2005b).

Finally, a strategy that has recently begun to be applied is the application of combined therapies aimed at obtaining effective antitumor responses. To achieve this objective should be considered therapies that combine the elimination of suppressive elements such as regulatory T cells and suppressive cytokines, the addition of active components of the immune system as the adoptive transfer of effector T cells, the stimulatory cytokines and the use of DCs as presenters. In addition, molecules that act must be added directly on the malignant cells, such as those that inhibit angiogenesis and the growth of the tumor.

Vaccines in the treatment of non-small cell lung cancer

Historically, the first attempt to treat NSCLC by active immunotherapy has been the use of autologous or allogeneic cell vaccines, which are made more antigenic by means of genetic manipulations to express immunostimulatory molecules such as GM-CSF.

This therapeutic approach is attractive because it does not require prior knowledge of a antigen as target to treat. The use of these vaccines has had little success in other locations such as melanoma and prostate cancer, however one of them (Lucanix) has reached stage III of development for the treatment of NSCLC (Mellstedt et al., 2011).

With the identification of tumor antigens expressed by the NSCLC they have been developed better characterized products for active immunotherapy in the NSCLC, among which find Gvax, LBLP25 or Stimuvax, Belagenpumatucel or Lucanix, Mage-A3 vaccine, EP2101 (IDM 2101) and dendritic cell vaccine (O’Mahony et al 2005; Nemunaitis and Murray 2006; Nemunaitis 2007; Barve et al. 2008; Dy and Adjei 2009; Gerard and Debruyne 2009; Hirschowitz and Yannelli 2009).

Gvax is a vaccine of autologous cells transfected with an adenovirus that has the gene of GM-CSF. This vaccine is specific to each patient and requires a tissue sample individual tumor for its development. Genetic manipulation induces the secretion of cytokines and immunosuppressive proteins at the injection site. It has been investigated in a Phase study I / II of 43 patients with NSCLC (33 with advanced disease). The toxicity profile was satisfactory with local reaction grade 1-2, while the secretion of GM-CSF correlated with the evolution of patients. A problem associated with the use of this vaccine is that it is prepared with own tissue of patients in advanced stage of the disease which can produce a unfavorable delay in the beginning of this therapy.

Belagenpumatucel (Lucanix) is composed of multiple NSCLC cell lines transfected with the growth factor transforming gene β2 (TGF β2) that inhibits cellular expression of TGF β2 making the tumor cells more immunogenic. Lucanix has been studied in open phase II trial in patients with NSCLC stage II, III and IV after the first line of treatment with chemotherapy. In this trial the authors demonstrated that no significant adverse events were presented. Patients in advanced stages who received the higher dose had a higher response rate and advantages in the survival at one and two years when compared with those who received lower doses of treatment. These results allowed the start of a Phase III trial (Nemunaitis et al. 2006).

The Liposomal vaccine MUC 1 (STIMUVAX) (L-BLP25) targets the nucleus of the antigen tumor-associated MUC1.The BLP25 is a lipopeptide of 25 amino acids that gives it the specificity of MUC1 (Mucin 1). MUC1 is a transmembrane protein, which is expressed in secretory epithelial cells and contains large amounts of sugars bound by bonds of O-glycosidic type. It is overexpressed in many types of cancer. The MUC1 associated with tumors is antigenically distinct from that of normal cells (Nemunaitis 2007).

Its expression is associated with reduced apoptosis, immunosuppression, resistance to chemotherapy and limited survival. Stimuvax is a liposomal vaccine that incorporates a lipid adjuvant and three lipids to increase the distribution of the vaccine to cells presenters In a phase II trial (Butts et al., 2005) in patients with stage IIIB / IV NSCLC, after completing the first line of treatment and having submitted a response objective or stabilization of the disease, LBLP25 was administered weekly for weeks at 4 injection sites and with a low dose of cyclophosphamide 3 days before the vaccination. The treatment was tolerable, the most common adverse events were: symptoms flu and reactions at the injection site. The median survival was 17.4 months for the vaccinated group compared to 13 months for the control group. The patients in Stage IIIB were those with the highest survival. These results led to the realization of a Phase III study currently underway.

EP2101 (IDM 2101) is a vaccine generated to induce cytotoxic T lymphocyte response against multiple epitopes and tumor associated antigens. It was designed to induce that response against five antigens frequently overexpressed in NSCLC: carcinoembryonic antigen (CEA), p53 protein (p53), HER-2 / neu and melanoma antigens MAGE 2 and MAGE 3, which have have been studied in trials with previous vaccines (Marshall et al 2000, Brabender et al 2001; Fong et al. 2001). Two previous phase I trials demonstrated the safety and immunogenicity of This vaccine, which is why Phase IIIB / IV patients were included in a Phase II trial.

NSCLC that resulted positive HLA (Barve et al., 2008); no adverse events were found significant, only erythema and mild pain at the injection site. The survival per year was 60% and the median survival was 17 months.

The MAGE A3 vaccine was designed to stimulate the immune system to respond to antigen MAGE A3 that was isolated first in melanoma cells. Phase I trials in melanoma and NSCLC (Marchand et al 2003) gave evidence that this protein is little immunogenic by itself, so it requires an adjuvant system to induce a robust and sustained immune response (Atanackovic et al 2004). It was evaluated in a study Phase II placebo-controlled in 182 patients, stages Ib and II of NSCLC after having been resected and in tumors that express MAGE A3. After 28 months of follow-up, a 27% increase in the disease-free interval when compared with the control group.

The adverse events described in this trial were slight (Vansteenkiste et al., 2007). With These results were designed as a multinational Phase III trial that is still in the recruitment phase of patients.

Other therapies tested in patients with NSCLC are dendritic cell-based vaccination. The dendritic cells have all the necessary elements to start and enhance a specific antigen immune response. For these therapies, the cell precursor dendritic cells are obtained by leukapheresis, are cultured and delivered in vaccine form to the patient. In a phase I study, CD14 + precursor dendritic cells were generated.were pulsed with apoptotic bodies of a NSCLC cell line that over-expressedHer2 / neu, CEA, MAGE-2. Of the 16 patients in the vaccinated group, none experienced serious adverse events, demonstrating that the vaccine is safe and presented biological activity in11 of the patients evaluated (Hirschowitz and Yannelli 2009). However, subsequent trials they have not shown evidence of effectiveness in this type of tumor.

On the other hand, the only clinical study related to active therapy based on the EGFR and its ligands for the treatment of NSCLC, it is vaccination EGF ligand. Previous results of our group have shown the possibility of making a active immunotherapy of cancer based immunization with this ligand of EGFR (Rodriguez et al., 2010).

Combined immunotherapy treatments in cancer

Cancer therapy has evolved towards the strategic integration of different modalities of treatment with a view to optimizing healing opportunities. Surgery and radiation therapy have been used to achieve locoregional control of the disease, while the therapies systemic (chemotherapy, endocrine therapy, molecular-targeted therapies) have been used in the control of diffuse disease (in hematological neoplasms) or of the has expanded beyond the primary site (in solid tumors) (Emens 2010). In addition, multiple drugs with complementary mechanisms of action are often combined to obtain additive or synergistic antitumor efficiencies.

Cancer chemotherapy typically disrupts fundamental, essential regulatory pathways for the survival and growth of tumor cells but are limited by mechanisms of resistance to the drug and by toxic collateral damage on normal tissues.

Recently it has been described as the main determinant of both the clinical course and the clinical response in malignant tumors, the interaction between the tumor and the host (Zitvogel et al. 2008; Formenti 2010; Pages et al. 2010). Among the elements of the response of the host that are critical for tumor progression and growth include:

stromal fibroblasts, host endothelial cells, macrophages associated with the tumor, NK cells and tumor-specific lymphocytes. The mechanism of action of multiple drug is based on the manipulation of the interaction between the tumor and the host to favor tumor regression Bevazucimab, Sunitinib and Sorafenib act on the biology of the endothelial cell so that they affect the vasculature associated with the tumor (Heath and Bicknell 2009).

Some monoclonal antibodies bind specifically to their target molecule in the cell and cause the recruitment of effector cells of innate immunity to mediate in cytotoxicity on tumor cells (Weiner et al., 2009).

On the other hand, cancer vaccines can actively induce immune responses specific on tumors of both humoral and cell types (Finn 2008). The advantages of this type of immunotherapy with respect to traditional therapies, are due to the exquisite specificity for the tumor cells, with a minimum of adverse effects and its potential to produce long-lasting effects without requiring continuous active therapy as a consequence of the immunological memory that is created.

At the same time, the ability of certain chemotherapies to increase efficacy of tumor cell vaccines and of promoting tumor T cell activity specifically transferred adoptively (Baxevanis et al., 2010). Among the mechanisms proposed for the enhancement of the immune response induced by chemotherapy is finds the induction of cell death, increasing the cross-presentation of antigens tumor in vivo. Additionally, myelosuppression induced by chemotherapy can induce the production of cytokines that favor homeostatic proliferation and / or ablation of immunosuppression mechanisms.

So, the combination of the effect of the therapies established for the treatment of cancer with the effects achieved by immunotherapy has become a strategy therapeutic that promises to be more effective for the treatment of cancer.

At present, several clinical trials have addressed these combinatorial strategies. As examples of these strategies is the use of antibodies against anticytotoxic antigen expressed in T cells (CTLA-4, from the English “cytotoxic T lymphocyte antigen”) with which blocks the negative regulatory signal given by this molecule. In animal models, the combination of this antibody with whole-cell vaccines or peptide vaccines had as a result a significant increase in the antitumor activity of said immunogens (Davila et al 2003, Hurwitz et al 2003). Similarly, the combination of antibodies against CD4 + CD25 + regulatory T cells with DCs vaccines is another of the new combined therapy strategies that include vaccination, aimed at raising the effectiveness of immune response against cancer (Kudo-Saito et al 2005a, Prasad et al 2005).

In phase II clinical studies in recurrent advanced NSCLC, an increase in the time of disease progression and overall survival when combining the Bevacizumab antibody (Avastin) with platinum regimens (Adjei et al., 2010).

Severe adverse events including pulmonary hemorrhage have been described in patients with cancer lung squamous histology. Based on these results, the Phase III study 4599 ECOG (from English: “Eastern Cooperative Oncology Group”) combined Bevacizumab in the same type of patients but not scaly histology.

On the other hand, a phase II trial in the first line of combined treatment with chemotherapy in Advanced NSCLC demonstrated an increase in the response rate in patients treated with the combination Figitumumab / Chemotherapy compared with chemotherapy alone (54% vs 42%) (Karp et al., 2009). Figitumimab (CP-751871) is a potent monoclonal antibody Humanized that binds selectively to the insulin growth factor receptor (IGF1R) and prevents the binding of insulin-like growth factor type I to its receptor and as Consequently, it inhibits tumor growth (Cohen et al., 2005).

The Oncophage vaccine, composed of the HSP 96 to which peptides are complexed tumors, is an example of the use of this type of therapy, specifically in patients with stage IV renal carcinoma. The results of a phase III clinical trial already concluded, showed an increase in the disease-free interval in the group of patients treated with the combination of surgery, vaccine and IL-2 compared to the group treated with surgery and IL-2 (Belli et al 2002).

Immunotherapy based on EGFR has also been the target of therapeutic combinations. In the case of Cetuximab, it has been proven that it increases the sensitivity to chemotherapy both in vitro and in humans (Kim et al., 2009). Cetuximab has been studied in several clinical trials in combination with chemotherapy in the first and second line. Results of the Multicenter Phase I / II study of Cetuximab in combination with paclitaxel and carboplatin in Patients with stage IV NSCLC demonstrated that the treatment was safe and well tolerated and they found response rate, time to progression and median survival slightly higher than historical controls treated only with carboplatin and paclitaxel (Thienelt and cols. 2005). Subsequently, the authors of the Phase III study called FLEX indicated that the addition of Cetuximab to the chemotherapy regimen with cisplatin / vinorelbine in the first line, caused a modest increase in overall survival with an increase in the response in patients with advanced NSCLC who express in EGFR (Pirker et al., 2009; Stinchcombe and Socinski 2009). The main adverse event that this antibody produces is grade 2-3 rash.

With all this knowledge, the potential to maximize biological activity and benefit the clinical profile of immunotherapies has never been greater. The strategic integration of the immunotherapy with chemotherapies to transform the tumor microenvironment and reduce different mechanisms of suppression and immune tolerance, could support an immune response Sustained and vigorous antitumour. Choosing carefully the doses and times of administration of drugs in the interaction with immunotherapy, using models of laboratories relevant to the clinic and in pilot clinical trials, it will be possible to accelerate the development of clinical trials in the higher phases to convert chemo-immunotherapy into a clinically significant reality (Emens 2010).

CIMAvax-EGF vaccine

The induction of an EGF deficiency as a consequence of active immunotherapy has been an emerging concept developed by Cuban scientists at the Center for Immunology Molecular Studies since 1992. This concept involves the manipulation of the individual’s immune system to produce antibodies against the EGF molecule with the aim of preventing tumor progression (Pérez et al 1984; Macias et al 1987; González et al 1996; Arteaga 2003; Lage et al. 2003; González and Lage 2007).

CIMAvax-EGF is a therapeutic cancer vaccine based on the binding of human EGF recombinant conjugate to a tansportadora protein (P64k of Neisseria meningitidis) that has been completely developed in Cuba. Previous results of our group have shown the possibility of performing an active cancer immunotherapy with the EGF-based vaccine. In fact, both preclinical and clinical evidence has been obtained about its immunogenicity and low toxicity (González et al 1996).

Pre-clinical studies

Preclinical studies for the development of the most effective formulation in the induction of immune response against the autologous EGF began in the early 90s Initially The possibility of the recognition of own EGF in mice and monkeys was evaluated (González et al 1996). The mice produced antibodies against the mouse EGF when they were immunized with EGF bound to the carrier protein. The antibodies produced showed immunological memory of IgG isotype. In the monkeys, the production of anti-EGF antibodies as were vaccinated with human EGF conjugated the transporter protein It was also shown that immunization with EGF modifies the biodistribution of EGF injected into mice (González et al 1996).

Additionally, 7 studies were conducted on animals to improve the immunogenicity of the recombinant human EGF vaccine with different carrier proteins and determine the better formulation. The transporter proteins studied were: Tetanus Toxoid, the P64k of Neisseria meningitidis, T3 (murine monoclonal antibody that recognizes CD3 human) and B7 (murine monoclonal antibody that recognizes human B cells). The adjuvants studied were: incomplete and complete Freund’s adjuvant (AIF / ACF), alumina and Montanide ISA 51 (oil adjuvant produced by SEPPIC, France). They also studied different routes of administration, different schemes and treatments combined with chemotherapy and / or immunosuppression (González G et al 1997; González et al. 2002).

All the experiments showed that immunization with EGF bound to a protein transporter in an appropriate adjuvant, generates an anti-EGF antibody response that it has an antitumor effect in experimental animals. In addition, no damage was observed histological in any of the treated animals (González et al 1996).

Clinical trials

The clinical development of CIMAvax-EGF began in 1995 with a phase I / II trial (Pilot I) performed at the CIMEQ hospital in Havana. 10 trials were included in this trial patients with primary malignancies in different locations, who had received previously the first line of chemotherapy. The main objective of this study was the evaluation of the immunogenicity of the incipient vaccine formulation (González et al., 1998). The result of this trial was the first published scientific evidence on the possibility of inducing an immune response against autologous EGF in patients with tumors in advanced stage. Additionally, the use of the P64k protein was confirmed as the immunopotentiator suitable for the conjugation and formulation of the vaccine CIMAvax-EGF.

A key factor in the development of the final formulation of the vaccine was the selection of the appropriate tumor location for the introduction of this novel vaccination strategy. NSCLC was selected because it is a very frequent pathology and due to the high expression of EGFR in tumor tissues during the development and progression of lung neoplasms (Lage et al. cols. 2003; González and Lage 2007). The magnitude of the expression of the EGFR has been reported in Literature as a predictive factor of response to biological therapies in patients with NSCLC (Toffoli et al., 2007).

The following published clinical trials coincided in time (pilot II, 1997-1999 and pilot III, 1998-2001) and were also Phase I / II trials developed in hospitals of the Havana city. The Pilot II trial included 20 patients with advanced stage NSCLC (IIIb and IV). Ten of the included patients received the vaccine in the adjuvant Alumina and the another 10 received the vaccine in the adjuvant Montanide ISA 51 (González et al 2003). The Pilot III trial also included 20 patients with the same characteristics as the Pilot trial II, but this time both treatment groups received cyclophosphamide (200 mg / m2 of body surface) 72 hours before starting the vaccination (González et al 2003). The Cyclophosphamide has been widely studied in the treatment of cancer. Their effects Immunomodulators have different repercussions according to dose and scheme therapeutic used (Man et al 2002; Ghiringhelli et al 2004). This treatment prior to vaccination was introduced with the aim of interfering immunological tolerance to EGF and enhance the induction of immunogenicity against this own molecule from the first dose (González et al 2003).

Both clinical trials confirmed the induction of a specific humoral immune response against EGF and made it possible to classify immunized patients into two subgroups: bad respondents (MR) and good respondents (BR) according to the magnitude of the response of anti-EGF antibodies generated and to the seroconversion of the initial response.

In both studies, survival was greater for the BR group (mean: 12.41 months; median: 9.1 months), than for the MR group (mean: 5.47 months, median: 4.5 months). The difference in survival between these two subgroups of patients was statistically significant (Log rank; p <0.05). Additionally, in these studies a greater immunogenicity when the adjuvant Montanide ISA 51 was used in the vaccine formulation as well as the benefits of treatment with low doses of cyclophosphamide 72 hours before to initiate vaccination (González et al 2003, González et al 2007).

Finally, in the period 2000-2003 a fourth clinical trial was also developed, phase I / II (pilot IV) with the aim of evaluating two different dose levels and higher than those used in the previous tests. Forty-three patients with advanced stage NSCLC (IIIb and IV) randomized into two groups to receive 71 μg or 142 μg of EGF. The lower dose was administered in one of the deltoid regions and the upper one was distributed between the two regions deltoides (Ramos et al 2006). The treated patients showed superior survivals (average: 9.83 months, median: 8 months) to those of a historical control of patients with equals demographic characteristics (mean: 6.2 months, median: 4.1 months).

Immuno-pharmacological variables in the optimization of the vaccination scheme

The immunopharmacology of cancer vaccines is not yet fully understood and there are very limited data on the immunopharmacological determinants that influence vaccines Therapeutics for the treatment of cancer (Starzl and Zinkernagel 1998; Zinkernagel and Hengartner 2001; Couch et al. 2003; Berd et al. 2004; Lage et al. 2005; Aucouturier et al. 2006; Gardiner et al. 2006). Variables such as dose, route of administration, interval of administration of doses and optimal combinations with established therapies should be evaluated in greater depth. This process is even more complex when it comes to optimize a scheme based on the generation of antibodies against a molecule of its own, with the intention to eliminate said circulating molecule and prevent the interaction thereof with its receptor in tumor cells (Rodriguez et al., 2010).

To make immunization more efficient with the CIMAvax-EGF vaccine, the researchers returned to preclinical experimentation with the hypothesis of generating an immune response more powerful, able to stay for a long period of time after successive immunizations. To this end they immunized animals of different genetic backgrounds (mice BALB / c and C57BL / 6) with the vaccine CIMAvax-EGF (EGF / P64k / Montanide ISA 51) and tested different doses, number of immunizations and dose intervals, both during the phase of induction as during the re-immunization phase. In this way they managed to induce a Anti-EGF antibody response, early, robust and long-lasting (Rodriguez et al. 2008). For the activation period, fractionation of the dose and intramuscular administration in 4 anatomically different sites increased the titre values antibodies and extended the duration of the response. It was also shown that repeated reinmunizations they converted MRs into BR. The results of these studies lead to the conclusion that vaccination should be administered in high fractional doses at sites anatomically different to bring the vaccine to the regional lymph nodes and achieve synergy of the immune response (Rodriguez et al., 2008).

Among other vaccines designed for the treatment of lung cancer, the vaccine CIMAvax-EGF is the only vaccine in the world that induces anti-EGF antibody titers that neutralize to endogenous EGF, removing this important growth factor from the tumor (Hirschowitz and Yannelli 2009).

The safety and immunogenicity results obtained with the use of the EGF vaccine in cancer, are a stimulus for future research and for the development of new therapeutic schemes in cancer immunotherapy.

MATERIALS AND METHODS

Components of the vaccine CIMAvax-EGF

The recombinant human epidermal growth factor (EGF) was produced in the Center for Genetic Engineering and Biotechnology (CIGB, Havana, Cuba) by technology of DNA. It is expressed in Sacaromise sereviciae and is composed of a mixture of EGF of 51 and 52 amino acids. This non-glycosylated molecule has shown an activity biological equivalent to that of the complete 53 amino acid molecule (Calnan et al. 2000).
The P64k protein from Neisseria meningitidis was also produced at the CIGB, La Havana Cuba. It is a recombinant protein that is expressed in Escherichia coli, contains 599 amino acid residues including 7 cysteine residues (Raúl Gómez 1999).
The Montanide ISA 51 (Seppic, France) was the adjuvant used in the preparation vaccination It is an adjuvant for vaccines ready to be used and must be mixed 50/50 (v / v) with the aqueous antigenic phase.

Preparation

The vaccine is composed of recombinant human epidermal growth factor (EGF) conjugated to the recombinant protein P64k of Neisseria meningitidis as a protein transporter for the conjugation, 0.05% glutaraldehyde was added to the protein mixture and the reaction was maintained for one hour, under constant stirring and at room temperature (25 ° C). Then the conjugate was dialyzed and filtered under sterile conditions. The final conjugate contained two molecules of EGF per molecule of P64k (González G et al., 1997; et al. 1998).

All the procedures were carried out in compliance with Good Manufacturing Practices. Montanide ISA51 was used as an adjuvant, it was mixed until the emulsification with equal volume of the EGF vaccine, immediately before the injection.

Patients and Methodology

Selection of patients

Phase II trial

Patients were included, with diagnosis confirmed by cyto-histological techniques, cancer of non-small cell lung in stages IIIb-IV with measurable lesions according to the criteria for the evaluation of the response to treatment in solid tumors (RECIST) “Response Evaluation Criteria in Solid Tumors”). All patients received the last cycle of chemotherapy at least 4 weeks before vaccination and had a life expectancy more than 3 months. The remaining inclusion criteria are listed below:

Patients with measurable lesions, defined as those that may be exactly measures in at least one dimension (it refers to the largest diameter) and possess a diameter equal to or greater than 20 mm using conventional techniques (emission tomography of positrons (PET), computerized axial tomography (CAT), nuclear magnetic resonance (NMR) or X-ray) or a diameter greater than or equal to 10 mm using spiral CT
Age > 18 years
General condition according to ECOG < 2 (Karnofsky > 60%)
Patients who have normal functioning of organs and defined bone marrow by the following parameters:
Hemoglobin > 9 g / L
Leukocytes > 3000 / uL
Absolute neutrophil count > 1500 / uL
Platelet count > 100000 / uL
Total bilirubin: Within normal limits
Glutamic pyruvic transaminase (TGP) and glutamic oxalacetic transaminase. (TGO) <5 times the upper normal institutional limit
Creatinine: Within normal limits for each institution
Female patients of childbearing age must have a pregnancy test negative and employing contraceptive methods such as intrauterine devices, hormonal contraceptives, barrier methods or tubal ligation
Patients who have signed the informed consent

Outcomes of the clinical trial

In both trials the following criteria were met for the definitive exit of the protocol of study:

RESULTS

Evaluation of the antibody response generated after vaccination with CIMAvax-EGF:

The humoral immune response induced by immunization with the CIMAvax-EGF vaccine evaluated in the two treatment schemes under test (QVV and VQV). In general, observed the existence of anti-EGF antibody titers before immunizing with the vaccine CIMAvax-EGF in 100% of the patients evaluated, with values equal to or greater than 1: 500 in 32% of them. During the first month of treatment there was an increase in the titers of the specific antibodies generated in both with both treatment schemes. The particularities of the immune response generated in each test are described to continuation.

Humoral immunity induced by the CIMAvax-EGF vaccine in the QVV assay:

In this trial, vaccination was administered after chemotherapy. The evaluation of

Immunogenicity was performed by determining the specific antibody response

anti-EGF in 42 patients (26 of the vaccinated group and 16 of the control group).

As indicated above, a certain level of title was found in all patients of antibodies before immunization. Of them, in 31% (n = 13) of patients evaluated (9 in the vaccinated group and 4 in the control group) the total antibody titer Specified by EGF showed values greater than 1: 100 (between 1: 500 and 1: 2000). Average geometry of the inverse of the title of specific antibodies against the EGF (anti-EGF) observed before immunization was 244 for the vaccinated group and 158 for the control group. The difference was not statistically significant (Mann Whitney U test, p> 0.05).

As a result of the immunization, the antibody titers increased. In the case of the group vaccinated, a significant increase was observed after the first month of treatment, titre of specific total antibodies with respect to the values detected before the start of immunization (Figure 1), according to the signed Wilcoxon rank test (p <0.05). The difference in the title of specific antibodies was maintained throughout the time of evaluation of the response, and maximum values of up to 1: 128000 were reached in patients treated for more than one year. More than 70% of the vaccinated patients (n = 19) were classified as good responders (BR), taking into account the magnitude of the anti-EGF (≥ 1: 4000) and seroconversion achieved. None of the patients in the control group met this criterion. The difference in antibody titers detected after the immunization, between the vaccinated group and the control group was statistically significant, according to the Mann Whitney U test (p <0.01).

Figure 1. Anti-EGF humoral immune response in patients with NSCLC included in the trial QVV. Patients treated with the vaccine received (1-month post-chemotherapy) immunizations weekly with 50 μg of EGF during the first month of treatment and then monthly during the phase of maintenance. The patients in the control group received only palliative treatment after the chemotherapy. Blood samples were taken on days 0, 14, 28 and monthly during the time follow-up of each patient. The anti-EGF antibody titers in the serum were measured by ELISA. The plates were coated with recombinant human EGF and the reactivity of the sera was detected with a anti-human IgG goat antiserum conjugated to alkaline phosphatase. It is represented in scale logarithmic geometric mean and 95% confidence interval (95% CI) of antibody titres anti-EGF assayed, in vaccinated patients (n = 26) and controls (n = 16) at different times. The asterisks (**) indicate the significant differences between both groups, according to the Mann U test Whitney, p0.01. The arrow indicates the time when the significant increase in titles begins against the EGF in the vaccinated group with respect to the zero day, according to the test of the ranges with sign of Wilcoxon, p <0.05.

Immunization with the CIMAvax-EGF vaccine could induce a cross-reactivity against

the human transforming growth factor (TGFα), which is another of the molecules belonging to the ligand family of the REGF. This possibility was evaluated in both the serum pre-immune (day 0) as in the post-immune (PI: between 3 and 6 months of treatment) of the vaccinated patients. Likewise, the existence of anti-TGFα antibodies was determined before immunizing.

As for EGF, the presence of specific natural antibodies was detected against the TGFα (geometric mean of the inverse of the titer, 202) with antibody titers between 1: 500 and

1: 2000 in 35% of the patients evaluated.

Immunization with EGF, which increased the antibody titers against this ligand did not

induced response against TGFα. After immunization, anti-viral antibody titers

EGF (geometric mean of the inverse of the title, 8724) were significantly higher than the

anti-TGFα titers (geometric mean of the inverse of the title, 208), according to the Mann U test Whitney (p <0.0001). No significant differences were found between the response levels against TGFα obtained before vaccination and those measured in the post-immunization phase (Figure 2).

As a result of these evaluations we can summarize that, using the scheme of QVV treatment, the specific humoral immune response against EGF was increased. East

increase resulted in 70% of patients developing a good response from

anti-EGF antibodies. Also, immunization with EGF did not produce an increase in

response against TGFα.

As a result of these evaluations we can summarize that, using the scheme of QVV treatment, the specific humoral immune response against EGF was increased.

increase resulted in 70% of patients developing a good response from anti-EGF antibodies. Also, immunization with EGF did not produce an increase in response against TGFα.

Figure 2. Anti-TGFα and anti-EGF humoral immune response in patients with NSCLC included in the QVV test. The patients were treated and evaluated, as described in Figure 1. It is represented in logarithmic scale the geometric mean and 95% CI of the antibody titer against each ligand of the REGF, in pre-immune and post-immune sera (with 3 or 6 months of vaccination) of patients treated with CIMAvax-EGF. The asterisks (***) indicate the significant differences between the groups, according to the Mann Whitney U test with p0,0001 for the comparison between post-immune times (PI) and according to the test of the Wilcoxon signed ranges, for the comparisons between the Day 0 and the Post-I in each case.

Humoral immunity induced by the CIMAvax-EGF vaccine in the VQV assay

In this second treatment scheme, patients received four times the dose of the previous scheme and in addition the first two immunizations were administered before the chemotherapy. The doses were distributed in four injection sites and were administered with intervals of every 14 days prior to chemotherapy and monthly, after the chemotherapy.

In the 20 patients in this trial, in terms of the natural response of anti-EGF antibodies,

They obtained results very similar to those found in the previous trial. Average geometric inverse of the titles was 242.

After the second week of treatment, a significant increase in the mean of the inverse of the specific total antibody titer with respect to the detected values before the start of immunization (Figure 3), according to the signed Wilcoxon rank test, p <0.01. This difference was maintained throughout the evaluation time of the response and reached maximum values of titles between 1: 128000 and 1: 1024000 in several patients (n = 6) with less than a year of treatment.

One of the most interesting observations in this treatment scheme was the fact that during the period of chemotherapy did not significantly decrease the titles of anti-EGF antibodies (p> 0.05, Kruskal Wallis test), despite not receiving the vaccination (Figure 3). After the completion of chemotherapy and prior to the restoration of monthly immunizations, the geometric mean of the inverse of the antibody titers specific against the EGF was 10642.

Figure 3. Anti-EGF humoral immune response measured in sera from patients with NSCLC included in the QVV and VQV tests. Patients treated according to the VQV scheme received immunizations every 14 days, with 200 μg of EGF (distributed in 4 different injection sites) during the first month, then received the corresponding chemotherapy cycles (4 cycles, 1 every 21 days) and 29 days after the end of chemotherapy the immunizations were resumed but this time, monthly. Blood samples were taken on days 0, 14, 28 and monthly during the time follow-up of each patient, and the anti-EGF antibody titers in the serum were measured by ELISA The plates were coated with recombinant human EGF and the reactivity of the sera was detected with a total human anti-IgG goat antiserum conjugated to alkaline phosphatase. The scheme of

QVV treatment was described in figure 1. The geometric mean and the logarithmic scale are plotted 95% CI, of the anti-EGF antibody titers tested in vaccinated patients (n = 26) and controls (n = 16) included in the study of the QVV scheme and those vaccinated according to the VQV scheme (n = 20) in the Different times. The arrow indicates the time when the significant increase in the titles begins of anti-EGF antibodies with the VQV scheme, according to the Wilcoxon signed rank test, p <0.01.

Applying the VQV treatment scheme, where multiple changes were introduced with relation to the QVV scheme: changes in the dose, the frequency of vaccination in the phase of induction, four administration sites and combination with chemotherapy, 95% of the Patients were classified as good responders (GR). This classification was reached in 80% of patients with only one month of treatment (Figure 4), while using the QVV scheme requires two months to achieve this classification in the same percentage of patients When we apply a classification criterion of the magnitude of the response of more rigorous antibodies (antibody titres equal to or greater than 1: 64,000), 55% of the Patients treated with the present scheme were classified as super-responders (SBR),while with the previous scheme only 15.3% of patients adjusted to this classification.

These results indicate that the treatment scheme can be optimized in order to achieve

Antibodies responses of greater magnitude, in a larger number of patients and in less

treatment time. Likewise, the results obtained show that when vaccinated prior to chemotherapy, anti-EGF titers do not decrease markedly during this period. Additionally, it is important to note that the increase in immunogenicity achieved When the VQV treatment scheme is used, it is not associated with an increase in toxicity (data not shown).

Figure 4. Percentages of good responder patients (BR) in the first four months of

immunization with the vaccine CIMAvax-EGF in both treatment schemes, VQV and QVV. calculation, monthly, the percentage of patients that has achieved the BR classification during the 4 first months of treatment, for both treatment schemes. BR: if they reached titles of antibodies greater than or equal to 1: 4000 (serum dilution) and at least four times higher than the value of the title measured prior to the first immunization.

Survival and toxicity associated with vaccination with CIMAvax-EGF

Survival of patients treated with the QVV scheme. Influence of age

For the total number of patients evaluated clinically (n = 74) in the QVV trial, the survival of those vaccinated were 6.47 months and 5.33 months in unvaccinated patients. The Survival reached statistical significance within the subgroup of patients under 60 years of age (11.57 months vs. 5.33 months in controls). In the patients of the group control, age was not a predictor of survival (Neninger Vinageras et al., 2008). In the sample of 42 patients chosen for this study (Table 4), the tendency to increase in survival as a result of vaccination, and the differences between the responses of the group under 60 years old and the group over 60 years old.

Table 4. Demographic characteristics of the 42 patients selected for the

study of the QVV scheme.

In congruence with the results observed for the total sample of patients evaluated clinically in the QVV trial, survival (median, 13 months) of patients vaccinated under 60 years (n = 17) was higher (median: 7.03 months) than that of patients controls (n = 12) of the same age group (figure 20a) in the group of 42 selected patients for studies of the specific immune response. In the same way, it occurred within the group of vaccinated patients (Figure 20b), where the subgroup of patients under 60 years of age of age, had a significantly higher survival, than that of the treated patients older than 60 years (n = 9, median: 7.43 months). So, we can say that, although with somewhat higher values in the survivorship medians, the patients selected for the Immunological study make up a representative sample of what happens in the total sample of patients included in the QVV trial, in terms of overall survival.

Figure 20. Survival functions in patients chosen for the immunological study of the

QVV assay. a) Kaplan Meier curves showing the survival of vaccinates and controls in the group of patients under 60 years old. b) The survival of the vaccinated group is shown according to the age groups (log rank, p <0.05).

This influence of age on the response to vaccination was independent of the levels of response of anti-EGF antibodies, given that the antibody titers obtained by the vaccination were not significantly different between the two age groups and the proportion of good and bad responders was similar in both age groups. In fact, the influence of age in the survival of patients treated with the QVV treatment scheme,

it was evident even within the group of good responders (BR) to vaccination (Table 5). The patients with good anti-EGF antibody response, product of vaccination with

CIMAvax-EGF, and under 61 years of age, showed better survival time than Patients older than 60 years.

Age did not correlate with the biological effect of the antibodies in their function of neutralizing the EGF and prevent binding with your REGF receptor.

Table 5. Relationship of the immune response and age with patient survival

vaccinated according to the QVV scheme.

However, an inverse correlation was found between age and concentration values of

TGFα, measured in the immune serum of vaccinated patients. Patients 60 years old or younger who received the vaccination, had an increase in the concentrations of TGFα in the serum immune (Figure 21). It remains to be proven whether this is an age-dependent or vaccination, since the control group evaluated did not have enough sample to address this type of analysis.

In summary, vaccination with CIMAvax-EGF produced a survival advantage within

patients under 60 years of age of the QVV trial. In this subgroup of patients, after vaccination TGFα levels higher than those found in the over 60 years old.

Survival of patients treated with the VQV scheme.

In the VQV trial, 20 patients with advanced NSCLC were evaluated. At the moment of diagnosis, 50% of the patients were in stage IV and the other 50% in stage IIIB of

the disease (Table 6). All patients received two doses (200μg) of the vaccine CIMAvax-EGF prior to the first line of chemotherapy and at least one dose after having finished the chemotherapy.

The analysis of the response to treatment of the 20 patients included in this trial resulted in 85% of the control of the disease, since two complete answers were obtained (RC), five partial responses (RP) and stabilization of the disease in 10 patients (EE). When studied survival, the median for the 20 patients was 12.8 months. In the subgroup of 17 patients where some objective response was achieved (RC or RP) or at least EE, a median survival of 16.2 months. When survival was estimated from the first month after the end of the first line of chemotherapy (time that corresponds to the moment in which survival is measured when the QVV treatment scheme was used),

a median of 9.3 months of survival was obtained. This value was superior to survival

achieved for patients vaccinated according to the QVV scheme (median: 6.47 months), and when was compared with the control group of that trial (median: 5.33 months), it was significantly higher (Log rank, p <0.05) (data not shown).

In both treatment schemes the vaccine was safe and well tolerated.

The most common adverse effects observed were: chills, fever, nausea, vomiting and fatigue; all classified as grade 1 or 2 (mild or moderate) according to the Common Toxicity Criteria of the National Cancer Institute No adverse effect of grade 3 or 4 was observed that was related to the treatment. In the case of the QVV assay, no differences were observed between the hematological and blood chemical parameters of the control group and the vaccinated (data not shown).

Relationship of the specific immune response with survival in patients of CPCNP immunized with CIMAvax-EGF

Relationship between the characteristics of the specific immune response and the

Survival of NSCLC patients treated with the QVV scheme:

Within the group of patients vaccinated in the QVV trial, patients with good response

of Antibodies (BR; n = 19) post-treatment, showed significant survival greater (median: 11.7 months) than the group of poor responders (MR, n = 7) to the treatment

(Figure 22a), a previously described finding for this type of vaccination (González et al.

2003).

Survival was also associated with the reduction produced by the vaccine in serum EGF concentrations. 85% of the patients classified as BR, managed to reduce EGF concentrations lower than 168 pg / mL. In those patients (n = 17) in which the EGF concentration was reduced to values lower than 168 pg / mL, the times of Survival were significantly greater (median: 13 months) than in patients (n = 9) where a reduction was not achieved at levels lower than this value (median: 5.6 months; Log rank test, p = 0.0023) (Figure 22b).

With the measurement of the Transforming Growth Factor (TGFα) a result was obtained different. When survival was studied associated with post-immunization TGFα levels (Figure 22c), significantly longer survival times were found in those

patients (n = 6) who showed higher concentrations of TGFα (median: 33.5 vs. 8.47

months).

Figure 22. Survival curves related to the characteristics of the immune response

humoral test against human EGF in patients with NSCLC treated according to the

QVV scheme).

a) Survival as a function of the magnitude of the response of anti-EGF antibodies (Log rank, p = 0.001).
b) Survival of vaccinated patients that reduce EGF concentrations to lower values than 168 pg / mL vs. those that do not reduce up to those levels with the treatment (Log rank p = 0.0023).
c) shows the survival of vaccinated patients according to the concentration of TGFα in sera immune (Log rank; p = 0.0073).
d) The survival of vaccinated patients according to the ability of immune sera to inhibit EGF / REGF binding (Log rank; p = 0.0001). e) Survival of patients vaccinated according to the recognition of the immunodominant epitope (B loop) of the EGF molecule (Log rank, p = 0.012).

Patients (n = 10) whose immune serum was able to inhibit the binding between EGF and its receptor, showed longer survival times than those patients in whom observed a post-immunization inhibition (median: 11.7 vs. 5.63 months, Log rank test, p= 0.0001; Figure 22d). In the case of the immunodominance of B-loop of the EGF molecule, the patients (n = 6) in the which the generated antibody response was preferentially against the central loop (B loop) of the EGF molecule, reached longer survival times (median: 33.5 months), whereas in those in which an immunodominance of the B-loop of the

molecule, the median survival was 7.43 months (Log rank test, p = 0.012;

22e). All these associations occurred in the vaccinated patients but not in the patients of the control group.

Relationship between the characteristics of the specific immune response and the

Survival of NSCLC patients treated with the VQV scheme.

In the optimized VQV scheme it was also verified that there is a greater survival for the patients with better antibody response. In correspondence with the results of the

clinical trials conducted previously, when the influence of the magnitude of the antibody response in the survival of the treated patients, were observed survival times in patients who showed very high anti-EGF titers (SBR). The SBR patients (n = 14) showed a significantly higher survival (Figure 23) (median: 19.3 months) than the BR (median: 8.4 months) according to the Log rank test (p = 0.0036).

For the rest of the immunological parameters evaluated, no relationship was found with the survival, since all the patients chosen for these determinations were SBR and for both had similar characteristics in terms of the specific immune response

developed post-vaccination and survival times. Additionally, in this trial it was observed that patients who had a high concentration of serum EGF before starting the vaccination, they later had a better survival, and this difference was statistically significant according to the Log rank test (p = 0.015). The same trend was observed in the patients of the QVV scheme, but in this case significance was not reached statistics.

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