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Anti-epidermal growth factor receptor (EGFR) therapies have been proven clinically effective for a variety of epithelial tumours. Vaccination of mice with the extracellular domain (ECD) of autologous EGFR overcomes the tolerance to self-EGFR and has antimetastatic effect on EGFR+ tumour. Because EGF/EGFR-signalling plays an important role in the inflammation stage of wound healing, the main objective of this study was to explore the possible role of murine (m) EGFR-ECD vaccine in the croton-oil-induced ear oedema and wound healing process in mice as autologous experimental models, mimicking the possible post-surgical wound complication in patients treated with human EGFR-ECD/VSSP vaccine. Mice were intramuscularly immunised four times; biweekly with the mEGFR-ECD/VSSP/Mont. Seven days later, an 8 mm diameter, full-thickness skin wound was created on the back of each animal. Immunisation induced a strong specific humoral response against the mEGFR-ECD protein and a DTH dose-response curve but interestingly, animals treated with mEGFR-ECD/VSSP/Mont had similar inflammatory and healing speed responses compared to control ones. These data suggest that application of mEGFR-ECD/VSSP vaccine as a therapeutic approach in cancer patients could not elicit a poor healing process after surgery., (© 2012 The Authors. International Wound Journal © 2012 Medicalhelplines.com Inc and John Wiley & Sons Ltd.)

1E10 is a murine anti-idiotypic mAb specific for an idiotypic mAb that reacts with NeuGc-containing gangliosides, sulfatides, and Ags expressed in some human tumors. In melanoma, breast, and lung cancer patients, this anti-idiotypic Ab was able to induce a specific Ab response against N-glycosylated gangliosides, attractive targets for cancer immunotherapy as these glycolipids are not naturally expressed in humans. A clinical study with nonsmall cell lung cancer patients showed encouraging clinical benefits. Immunological studies performed in 20 of these patients suggested a correlation between the induction of Abs against NeuGcGM3 and longer survival times. The induced anti-NeuGcGM3 Abs recognized and directly killed tumor cells expressing the Ag, by a mechanism independent of complement activation. In the present work, we show that this cytotoxicity differs from apoptosis because it is temperature independent, no chromatin condensation or caspase 3 induction are detected, and the DNA fragmentation induced has a different pattern than the one characteristic for apoptosis. It is a very quick process and involves cytosqeleton reorganization. The Abs induce cellular swelling and the formation of big membrane lesions that allow the leakage of cytoplasm and the loss of the cell membrane integrity. All of these characteristics resemble a process of oncotic necrosis. To our knowledge, this is the first report of the active induction in cancer patients of NeuGcGM3-specific Abs able to induce complement independent oncotic necrosis to tumor cells. These results contribute to reinforcing the therapeutic potential of anti-idiotypic vaccines and the importance of NeuGcGM3 ganglioside as antitumor target.

1E10 mAb is an anti-Id murine mAb (Ab2 mAb) specific for an Ab1 mAb that reacts with NeuGc-containing gangliosides, sulfatides, and Ags expressed in some human tumors. In preclinical studies, this Ab2 Ab was able to mimic NeuGc-containing gangliosides only in animals lacking expression of these Ags in normal tissues. In this study, we report on the immune responses elicited in 20 non-small cell lung cancer patients treated with 1 mg of aluminum hydroxide-precipitated 1E10 mAb. In the hyperimmune sera from 16 of 20 patients, a strong specific Ab response of both IgM and IgG isotypes against NeuGcGM3 ganglioside was observed. Patient immune sera were able to induce complement-independent cell death of NeuGcGM3-expressing X63 murine myeloma target cells. Significant immunoreactivity to NeuGcGM3 was still detected after the complete abrogation of the reactivity against 1E10 mAb by the adsorption of patient sera with this Ab. We hypothesize that Id(-)Ag(+) Abs could reflect the activation of an autologous idiotypic cascade into the patients. Both Id(+)Ag(+) and Id(-)Ag(+) fractions were separated by affinity chromatography and characterized. Although IgG isotype Abs were found in both fractions, IgM isotype Abs were found only in the Id(-)Ag(+) fraction. Both Id(+)Ag(+) and Id(-)Ag(+) Abs were able to specifically recognize and induce cell death in NeuGcGM3-expressing X63 myeloma target cells. Patients that developed IgG and/or IgM Abs against NeuGcGM3 showed longer median survival times.

NeuGcGM3 ganglioside is especially attractive because it is expressed on melanoma cells but it is minimally or not expressed at all on most normal human tissues. A Phase Ib/IIa clinical trial was carried out in patients with advanced cutaneous and ocular malignant melanomas, to evaluate immunogenicity and toxicity of an intramuscularly administered cancer vaccine and composed by NeuGcGM3 in a proteoliposome of Neisseria meningitides with Montanide ISA 51 as adjuvant. Twenty two patients were included, twelve at dose level of 200 microg and 10 at 400 microg. The first five doses were administered every other week and then monthly until 9 doses. 12 patients received additional immunizations. Vaccination induced specific anti-NeuGcGM3 IgM, IgG and IgA antibodies responses. Titers of IgM were greater for the highest vaccine doses. Vaccination also elicited DTH response in 45.5% of patients in the lower doses and 77.8% in the higher doses. Toxicities were mostly grade 1 or 2, according CTC-NCI criteria. Interestingly, 3 patients developed vitiligo at the lower dose (none in the highest dose) although the nominal antigen NeuGcGM3 is not present in melanocytes. Survival analysis was not the goal of this Phase I trial; nevertheless, the fact that seven patients are alive for more than 2 years after inclusion is noteworthy. Safety and immunogenicity with NeuGcGM3 vaccine treatment in advanced melanoma patients were established. The prognostic value of autoimmunity and the possibilities of dissociating anti-tumor immunity from autoimmunity deserve further research.

A phase I clinical trial was conducted in patients with stage III/IV breast cancer who were treated with the anti-idiotype mAb 1E10 specific to an Ab1 mAb able to react specifically with N-glycolyl-containing gangliosides and with antigens expressed on human melanoma and breast carcinoma cells. Patients were treated with 1 or 2 mg of aluminum hydroxide-precipitated 1E10 mAb every other week for six injections. Two patients at each dose were reimmunized 7-9 months after completing the induction phase. In hyperimmune sera from eight of the nine patients who received at least four doses of anti-Id vaccine preparations, strong specific responses were observed both against 1E10 mAb and NeuGc-GM3 ganglioside (Ab3 Id+Ag+). Nonclassical Ab1' antibodies (Id-Ag+) were also elicited by 1E10 mAb vaccine treatment. There were no differences between the two levels of dose tested in relation to toxicity and immunogenicity. No evidence of serious or unexpected effects was observed.

Purpose: A heterophilic ganglioside cancer vaccine was developed by combining NeuGcGM3 with the outer membrane protein complex of Neisseria meningitidis to form very small size proteoliposomes (VSSP). A phase I clinical trial was performed to determine safety and immunogenicity of this vaccine., Patients and Methods: Stage III to IV breast cancer patients received up to 15 (200 micro g) doses of the vaccine by intramuscular injection. The first five doses (induction phase) were given at 2-week intervals, with the remaining treatment (maintenance) administered on a monthly basis., Results: Twenty-one patients, 11 of whom had metastatic disease, were included. Main toxicities included erythema and induration at the injection site, sometimes associated with mild pain, and low-grade fever (World Health Organization grades 1 and 2). All treated patients who completed the induction phase developed anti-NeuGcGM3 antibody titers between 1:1,280 and 1:164,000 immunoglobulin G (IgG), and 1:640 and 1:164,000 IgM. Noteworthy specific IgA antibodies were induced by vaccination in all stage III patients and in three stage IV patients. Serum antibody levels were higher in the stage III patients, with the larger increases observed after week 32. The antiganglioside IgG subclasses were mainly IgG1 and IgG3. Hyperimmune sera increased complement-mediated cytotoxicity versus P3X63 myeloma cells and a marked IgG differential reactivity against human mammary ductal carcinoma samples., Conclusion: NeuGcGM3/VSSP/Montanide ISA 51 is an unusual immunogenic ganglioside vaccine and also seems to be safe in this small trial. Immunologic surrogates of activity indicate that this reagent warrants further investigation.

We generated the 1E10 gamma-type anti-idiotype mAb (Ab2) specific to an Ab1 mAb able to react specifically with N-glycolyl-containing gangliosides and with Ags expressed on human melanoma and breast carcinoma cells. This Ab2 mAb induced an Ab response in animal models sharing immunochemically defined idiotopes with the Ab1. The treatment of tumor-bearing mice with 1E10 mAb induced a strong antitumor activity. A clinical trial was conducted in 20 patients with advanced malignant melanoma. Patients were treated with six intradermal injections of aluminum hydroxide-precipitated 1E10 anti-Id mAb given at 2-wk intervals. Sixteen of the 17 patients who received at least four doses of the anti-Id vaccine develop Ab3 Abs capable of inhibiting Ab2 binding to Ab1 (Ab3Id+). In contrast to the incapacity of 1E10 mAb to generate Ab3 Abs with the same antigenic specificity as the Ab1 mAb in mice, a very specific and strong Ab3 response against N-glycolyl-containing gangliosides was induced in 16 patients (Ab3Ag+). No evidence of serious or unexpected adverse effects has been observed in this clinical trial. 1E10 anti-Id vaccine was safe, well tolerated, and immunologically effective, with most patients being able to generate a specific immune response against 1E10 and Neu-glycolyl-GM(3) ganglioside.

Transforming growth factor alpha (TGFα) is a potent ligand of the epidermal growth factor receptor (EGFR). EGFR is frequently over-expressed in epithelial tumors and endogenous ligands, mostly TGFα, are frequently co-expressed with EGFR, potentially resulting in autocrine stimulation of tumor cell growth. Therefore, different therapeutic approaches aim for the inactivation of TGFα/EGF/EGFR signaling system, but no approach is based on TGFα as a target. The principal goal of this work was to assess the potential of an active specific immunotherapy approach to block the TGFα/EGFR autocrine loop. For the proof of the concept, a fusion protein between human TGFα (hTGFα) and P64k protein from Neisseria meningitidis was generated, and its immunogenicity characterized in a mouse model using different adjuvants. All immunogens were effective for the generation of specific humoral responses against hTGFα. The inmunodominant epitope of hTGFα when immunizing mice with the fusion protein involved the C-loop/C-terminal region. This region includes key residues for hTGFα binding to EGFR. The anti-hTGFα immune mice sera recognized the natural hTGFα precursor in A431 cells and hTGFα-transfected 3T3 fibroblasts as revealed by flow cytometry analysis and immunoblotting. They inhibited the binding of 125I-TGFα to the EGFR, EGFR-autophosphorylation, and downstream activation of MAP kinases as well as proliferation of two EGFR-expressing human carcinoma cell lines. These data suggest that EGFR signaling activation by the hTGFα autocrine loop may be inhibited in vivo by induction of specifically blocking antibodies. The fusion protein reported in this paper could be a potential immunogen for the development of a new cancer vaccine. [ABSTRACT FROM AUTHOR]

The Epidermal Growth Factor Receptor (EGFR) plays an essential role in regulating neoplastic processes. EGFR over-expression in many human epithelial tumors has been correlated with disease progression and bad prognosis. Passive EGFR-directed immunotherapy has been introduced in medical oncology practice, but no active specific approaches have been used. We designed a vaccine candidate based on the extracellular domain (ECD) of human EGFR (HER1), and the homologous vaccine for mice based on murine EGFR. This vaccine candidate uses the Very Small-Sized Proteoliposomes from Neisseria meningitidis (VSSP) and Montanide ISA 51 -VG as adjuvants. The autologous vaccine circumvents the tolerance to self EGFR by inducing a specific immune response with an anti-metastatic effect on EGFR+ tumors. The vaccine candidate based on HER1 -ECD induced anti-EGFR polyclonal antibodies (PAb) that bind EGFR+ human tumor cell lines from different tissues. These anti-EGFR PAb abrogate ligand-dependent EGFR phosphorylation, provoking the inhibition of tumor cell growth and apoptosis. Preclinical results further encouraged the evaluation of the HER1 vaccine candidate in phase I clinical trials. [ABSTRACT FROM AUTHOR]

The epidermal growth factor receptor (EGFR) plays a central role in the development and progression of many tumors and its overexpression is associated with decreased survival, being an attractive target for cancer treatment. We introduce here a new therapeutic cancer vaccine based in the extracellular domain of human EGFR (EGFR-ECD). Vaccination of mice with EGFR-ECD induced both specific IgG secreting B cells and CD4+ T cells. Besides, sera from immunized mice reacted with antigen-positive cell lines and inhibited the EGF/EGFR binding. Furthermore immunization of mice with murine EGFR ECD (mEGFR-ECD) in appropriated adjuvants, produced specific high titer antibody response with a TH1 isotype profile. No toxicity associated to vaccine administration was detected. [ABSTRACT FROM AUTHOR]

Transforming Growth Factor alpha (TGFα) is an endogenous growth factor that is associated with the loss of growth regulation of the transformed phenotype and its action is by the interaction with the epidermal growth factor (EGF) receptor. Also this molecule is over-expressed by carcinomas as a tumor-associated antigen. In this regard the principal goal of this work is to development an active immunoteraphy capable to block the autocrine loop TGFα-EGFR and/or to elicit a specific cellular response against tumor that over-expressed this growth factor. To our knowledge TGFα has not been used in active specific cancer immunotherapy. A new vaccine containing a fusion protein between human TGFα (hTGFα) and P64k from Neisseria meningitidis, was generated and characterized in a mouse model using different adjuvants. Interestingly a good correlation between anti-hTGFα serum antibody titers, mainly IgG, and antibody secreting B cells in the lymph nodes of immunized mice was seen. The isotype profile of hTGFα-specific antibody responses was similar when Freund adjuvant complete or incomplete was used showing increase levels of IgG2a and IgG2b immunoglobulins respect to aluminum hydroxide. Noteworthy, those anti-hTGFα antibodies recognize the hTGFα precursor in cell line expressing this growth factor. For 4 peptide represent the different loops of hTGFα tested, serum from immunized mice mainly recognize the C-loop and C-terminal region. That region is important in the TGFα/EGF-R binding. Not crossreactivity against human or murine epidermal growth factor (EGF) was found. [ABSTRACT FROM AUTHOR]

An heterophilic ganglioside cancer vaccine, obtained by combining NeuGcGM3 with the outer membrane protein complex of Neisseria meningitidis to form very small size proteoliposomes (VSSP), and incomplete Freund's adjuvant (Montanide ISA 51) was developed. A phase I clinical trial was performed to determine safety and immunogenicity of this vaccine. Stage III- IV breast cancer patients received up to fifteen (200 (g) doses of the vaccine by intramuscular injection. The first 5 doses (induction phase) were given at two weeks intervals, while in the remaining of the treatment (maintenance) monthly administrations were given. Patients (21), 11 of whom had metastatic disease, were included. Main toxicity's included erythema and induration at the injection site, sometimes associated with mild pain, and low-grade fever (WHO grades I, II). All treated patients, who completed the induction phase, developed anti-NeuGcGM3 antibody titers between 1: 1,280 to 1:164,000 (IgG) and 1:640 to 1:160,000 (IgM). Noteworthy specific IgA antibodies were induced by vaccination in all stage III patients and in 3 stage IV patients. Serum antibody levels were higher in the stage III patients, with the larger increases being observed after week 32. Hyper-immune sera marked IgG differential reactivity against human mammary ductal carcinoma samples. NeuGcGM3/VSSP/Montanide ISA 51 is the highly immunogenic ganglioside vaccine and is also safe. Post-immune serum specific IgA, and IgG reactivity vs human mammary ductal carcinoma, warrants further exploration. [ABSTRACT FROM AUTHOR]