Marine Seagrass Extract of Thalassia testudinum Suppresses Colorectal Tumor Growth, Motility and Angiogenesis by Autophagic Stress and Immunogenic Cell Death Pathways.

Marine plants have become an inexhaustible reservoir of new phytopharmaceuticals for cancer treatment. We demonstrate in vitro/in vivo antitumor efficacy of a standardized polyphenol extract from the marine angiosperm Thalassia testudinum (TTE) in colon tumor cell lines (RKO, SW480, and CT26) and a syngeneic allograft murine colorectal cancer model. MTT assays revealed a dose-dependent decrease of cell viability of RKO, CT26, and SW480 cells upon TTE treatment with IC50 values of, respectively, 175, 115, and 60 µg/mL. Furthermore, TTE significantly prevented basal and bFGF-induced angiogenesis in the chicken chorioallantoic membrane angiogenesis assay. In addition, TTE suppressed bFGF-induced migration of endothelial cells in a wound closure assay. Finally, TTE treatment abrogated CT26 colorectal cancer growth and increased overall organism survival in a syngeneic murine allograft model. Corresponding transcriptome profiling and pathway analysis allowed for the identification of the mechanism of action for the antitumor effects of TTE. In line with our in vitro/in vivo results, TTE treatment triggers ATF4-P53-NFκB specific gene expression and autophagy stress pathways. This results in suppression of colon cancer cell growth, cell motility, and angiogenesis pathways in vitro and in addition promotes antitumor immunogenic cell death in vivo.

Nimotuzumab Site-Specifically Labeled with 89Zr and 225Ac Using SpyTag/SpyCatcher for PET Imaging and Alpha Particle Radioimmunotherapy of Epidermal Growth Factor Receptor Positive Cancers.

To develop imaging and therapeutic agents, antibodies are often conjugated randomly to a chelator/radioisotope or drug using a primary amine (NH2) of lysine or sulfhydryl (SH) of cysteine. Random conjugation to NH2 or SH groups can require extreme conditions and may affect target recognition/binding and must therefore be tested. In the present study, nimotuzumab was site-specifically labeled using ∆N-SpyCatcher/SpyTag with different chelators and radiometals. Nimotuzumab is a well-tolerated anti-EGFR antibody with low skin toxicities. First, ΔN-SpyCatcher was reduced using tris(2-carboxyethyl)phosphine (TCEP), which was followed by desferoxamine-maleimide (DFO-mal) conjugation to yield a reactive ΔN-SpyCatcher-DFO. The ΔN-SpyCatcher-DFO was reacted with nimotuzumab-SpyTag to obtain stable nimotuzumab-SpyTag-∆N-SpyCatcher-DFO. Radiolabeling was performed with 89Zr, and the conjugate was used for the in vivo microPET imaging of EGFR-positive MDA-MB-468 xenografts. Similarly, ∆N-SpyCatcher was conjugated to an eighteen-membered macrocyclic chelator macropa-maleimide and used to radiolabel nimotuzumab-SpyTag with actinium-225 (225Ac) for in vivo radiotherapy studies. All constructs were characterized using biolayer interferometry, flow cytometry, radioligand binding assays, HPLC, and bioanalyzer. MicroPET/CT imaging showed a good tumor uptake of 89Zr-nimotuzumab-SpyTag-∆N-SpyCatcher with 6.0 ± 0.6%IA/cc (n = 3) at 48 h post injection. The EC50 of 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher and 225Ac-control-IgG-SpyTag-∆N-SpyCatcher against an EGFR-positive cell-line (MDA-MB-468) was 3.7 ± 3.3 Bq/mL (0.04 ± 0.03 nM) and 18.5 ± 4.4 Bq/mL (0.2 ± 0.04 nM), respectively. In mice bearing MDA-MB-468 EGFR-positive xenografts, 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher significantly (p = 0.0017) prolonged the survival of mice (64 days) compared to 225Ac-control IgG (28.5 days), nimotuzumab (28.5 days), or PBS-treated mice (30 days). The results showed that the conjugation and labeling using SpyTag/∆N-SpyCatcher to nimotuzumab did not significantly (p > 0.05) alter the receptor binding of nimotuzumab compared with a non-specific conjugation approach. 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher was effective in vitro and in an EGFR-positive triple negative breast cancer xenograft model.

Aberrant expression of N-glycolyl GM3 ganglioside is associated with the aggressive biological behavior of human sarcomas.

BACKGROUND: The aberrant expression of N-glycolyl GM3 ganglioside (NeuGcGM3) in patients with sarcomas was reevaluated by assessing the relation of this molecule with some clinicopathological features and overall survival (OS) of patients. METHODS: Fifty formalin-fixed and paraffin-embedded specimens from patients diagnosed with sarcomas were included. For the evaluation of NeuGcGM3, the 14F7 monoclonal antibody followed by a peroxidase avidin-biotin system was used. Clinicopathological features were obtained from patient records. Survival rates were estimated by the Kaplan-Meier method and compared with the log-rank test. For multivariate analyses, the Cox regression model was used to identify independent prognostic factors for OS. RESULTS: The majority of samples had high levels of NeuGcGM3 expression (66.0%) that showed statistical correlation with age (p = 0.014), TNM stage (p = 0.022), histological grade (p = 0.013) and proliferation rates (p = 0.012). In addition, a tendency for association with tumor depth (p = 0.070) was evidenced. In univariate survival analysis, TNM stage (p = 0.000), occurrence of metastasis (p = 0.000) and expression of NeuGcGM3 (p = 0.034) were significant prognostic factors for OS, while a tendency for association was evidenced for histological grade (p = 0.091). Among these variables, only the presence of metastasis (p = 0.001) was an independent prognostic factor on multivariate analysis. CONCLUSIONS: The present research suggests the evaluation of NeuGcGM3 expression as a complementary prognostic factor in sarcoma, although our results need to be validated in a larger series and prospective studies. Moreover, our results could support the use of this molecule as a target for immunotherapy.

An enzyme immunoassay for determining epidermal growth factor (EGF) in human serum samples using an ultramicroanalytical system.

Human epidermal growth factor is a small peptide consisting of 53 amino acid residues, which stimulates cell proliferation and is associated with several human carcinomas. A simple sandwich-type ultramicroELISA assay (UMELISA), based on the advantages of high affinity reaction between streptavidin and biotin has been developed for the measurement of EGF in human serum samples. Strips coated with a high affinity monoclonal antibody directed against EGF are used as solid phase, to ensure the specificity of the assay. The EGF assay was completed in 18 hr, with a measuring range of 39-2500 pg/mL. The intra- and inter-assay coefficients of variation were 4.4-7.3% and 0-5.1%, respectively, depending on the EGF concentrations evaluated. Percentage recovery ranged from 96-104%. Regression analysis showed a good correlation with the commercially available Human EGF Immunoassay Quantikine® ELISA kit (n = 130, r = 0.92, P < 0.01). The analytical performance characteristics of our UMELISA EGF endorse its use for the quantification of EGF in human serum samples.

Prognostic Significance of N-Glycolyl GM3 Ganglioside Expression in Non-Small Cell Lung Carcinoma Patients: New Evidences.

The prognostic role of N-glycolyl GM3 ganglioside (NeuGcGM3) expression in non-small cell lung carcinoma (NSCLC) still remains controversial. In this study, the NeuGcGM3 expression was reevaluated using an increased number of NSCLC cases and the 14F7 Mab (a highly specific IgG1 raised against NeuGcGM3). An immunohistochemical score integrating the percentage of 14F7-positive cells and the intensity of reaction was applied to reassess the relationship between NeuGcGM3 expression, some clinicopathological features, and the overall survival (OS) of NSCLC patients. The double and the triple expression of NeuGcGM3 with the epidermal growth factor receptor (EGFR) and/or its ligand, the epidermal growth factor (EGF), were also evaluated. NeuGcGM3 expression correlates with both S-Phase fraction (p = 0.006) and proliferation index (p = 0.000). Additionally, NeuGcGM3 expression was associated with a poor OS of patients in both univariate (p = 0.020) and multivariate (p = 0.010) analysis. Moreover, the double and/or the triple positivity of tumors to NeuGcGM3, EGFR, and/or EGF permitted us to identify phenotypes of NSCLC with a more aggressive biological behavior. Our results are in agreement with the negative prognostic significance of NeuGcGM3 expression in NSCLC patients. However, standardization of techniques to determine the expression of NeuGcGM3 in NSCLC as well as the implementation of a universal scoring system is recommended.

Prognostic Role of 14F7 Mab Immunoreactivity against N-Glycolyl GM3 Ganglioside in Colon Cancer.

Purpose. To assess the prognostic role of 14F7 Mab immunoreactivity, against N-Glycolyl GM3 ganglioside, in patients with colon cancer (CC) and to evaluate the relationship between its expression and clinicopathological features. Methods. Paraffin-embedded specimens were retrospectively collected from 50 patients with CC operated between 2004 and 2008. 14F7 Mab staining was determined by immunohistochemistry technique and its relation with survival and clinicopathologic features was evaluated. Results. The reactivity of 14F7 Mab was detected in all cases. Most cases had high level of immunostaining (70%) that showed statistical correlation with TNM stage (P = 0.025). In univariate survival analysis, level of 14F7 Mab immunoreactivity (P = 0.0078), TNM Stage (P = 0.0007) and lymphovascular invasion (0.027) were significant prognostic factors for overall survival. Among these variables, level of 14F7 Mab immunoreactivity (HR = 0.268; 95% CI 0.078-0.920; P = 0.036) and TNM stage (HR = 0.249; 95% CI 0.066-0.932; P = 0.039) were independent prognostic factors on multivariate analysis. Conclusions. This study is the first approach on the prognostic significance of 14F7 Mab immunoreactivity in patients with colon adenocarcinoma and this assessment might be used in the prognostic estimate of CC, although further studies will be required to validate these findings.

NGcGM3/VSSP vaccine as treatment for melanoma patients.

Gangliosides are glycosphingolipids that are present in the plasma membranes of vertebrates and are involved in multiple cellular processes. In the Center of Molecular Immunology an NGcGM3 ganglioside based vaccine has been developed and is conceptualized as a targeted therapy in cancer. NGcGM3/VSSP vaccine had been used as treatment of metastatic melanoma patients and had showed to be safe and immunogenic. The treatment improved antitumoral response or maintain the response obtained with previous onco-specific treatment as chemotherapy. The results indicate that the vaccine improved overall survival of metastatic melanoma patients after first line-chemotherapy. The clinical trial ongoing currently will allow corroborating these results.

Tissue Reactivity of the 14F7 Mab Raised against N-Glycolyl GM3 Ganglioside in Tumors of Neuroectodermal, Mesodermal, and Epithelial Origin.

The expression of N-glycolylneuraminic acid forming the structure of gangliosides and/or other glycoconjugates (Hanganutziu-Deicher antigen) in human has been considered as a tumor-associated antigen. Specifically, some reports of 14F7 Mab (a highly specific Mab raised against N-glycolyl GM3 ganglioside) reactivity in human tumors have been recently published. Nevertheless, tumors of epithelial origin have been mostly evaluated. The goal of the present paper was to evaluate the immunohistochemical recognition of 14F7 Mab in different human tumors of neuroectodermal, mesodermal, and epithelial origins using an immunoperoxidase staining method. Samples of fetal, normal, and reactive astrocytosis of the brain were also included in the study. In general, nontumoral tissues, as well as, low-grade brain tumors showed no or a limited immunoreaction with 14F7 Mab. Nevertheless, high-grade astrocytomas (III-IV) and neuroblastomas, as well as, sarcomas and thyroid carcinomas were mostly reactive with 14F7. No reaction was evidenced in medulloblastomas and ependymoblastomas. Our data suggest that the expression of N-glycolyl GM3 ganglioside could be related to the aggressive behavior of malignant cells, without depending on the tumor origin. Our data could also support the possible use of N-glycolyl GM3 as a target for both active and passive immunotherapies of malignancies expressing this molecule.

Immunoreactivity of the 14F7 Mab Raised against N-Glycolyl GM3 Ganglioside in Primary Lymphoid Tumors and Lymph Node Metastasis.

The reactivity of the 14F7 Mab, a highly specific IgG1 against N-glycolyl GM3 ganglioside (NeuGcGM3) in normal tissues, lymphomas, lymph node metastasis, and other metastatic sites was assessed by immunohistochemistry. In addition, the effect of chemical fixation on the 14F7 Mab staining using monolayers of P3X63Ag.653 cells was also evaluated. Moreover, the ability of 14F7 to bind NeuGcGM3 ganglioside inducing complement-independent cytotoxicity by a flow cytometry-based assay was measured. The 14F7 Mab was reactive in unfixed, 4% paraformaldehyde, 4% formaldehyde, and acetone fixed cells. Postfixation with acetone did not alter the localization of NeuGcGM3, while the staining with 14F7 Mab was significantly eliminated in both cells fixed and postfixed with methanol but only partially reduced with ethanol. The staining with 14F7 Mab was evidenced in the 89.2%, 89.4%, and 88.9% of lymphomas, lymph node metastasis, and other metastatic sites, respectively, but not in normal tissues. The treatment with 14F7 Mab affected both morphology and membrane integrity of P3X63Ag.653 cells. This cytotoxic activity was dose-dependent and ranged from 24.0 to 84.7% (10-1000 µ g/mL) as compared to the negative control. Our data could support the possible use of NeuGcGM3 as target for both active and passive immunotherapy against malignancies expressing this molecule.

Effect of vaccination with N-glycolyl GM3/VSSP vaccine by subcutaneous injection in patients with advanced cutaneous melanoma.

NeuGc-containing gangliosides have been described in melanoma cells and are an attractive target for cancer immunotherapy because they are minimally or not expressed in normal human tissues. Melanoma patients treated with a vaccine based on N-glycolyl gangliosides have shown benefit in progression free survival and overall survival. We conducted a multicenter Phase I/II clinical trial in patients with metastatic cutaneous melanoma treated with the N-gycolyl GM3/very-small-size proteoliposomes vaccine by the subcutaneous route. Selecting the optimal biological dose of the vaccine was the principal objective based on immunogenicity, efficacy, and safety results. Six dose levels were studied and the treatment schedule consisted of five doses administered every 2 weeks and then monthly until 15 doses had been given. Dose levels evaluated were 150, 300, 600, 900, 1200, and 1500 µg with five patients included in each dose level except the 900 µg dose (n = 10). Immunogenicity was determined by antibody titers generated in patients after vaccination. Antitumor effect was measured by response criteria of evaluation in solid tumors and safety was evaluated by common toxicity criteria of adverse events. The vaccine was safe and immunogenic at all doses levels. The most frequent adverse events related to vaccination were mild to moderate injection site reactions and flu-like symptoms. Vaccination induced specific anti-NeuGcGM3 immunoglobulin M and immunoglobulin G antibody responses in all patients. Disease control (objective response or stable disease) was obtained in 38.46% of patients. Global median overall survival was 20.20 months. Two patients achieved overall survival duration of about 4 and 5 years, respectively. The 900 µg dose resulted in overall survival duration of 19.40 months and was selected as the biological optimal dose.

Immunoreactivity of the 14F7 Mab (Raised against N-Glycolyl GM3 Ganglioside) as a Positive Prognostic Factor in Non-Small-Cell Lung Cancer.

Lung carcinoma is the leading cause of cancer-related mortality worldwide. Therefore, numerous studies are focusing on the assessment of other biological and molecular prognostic factors in these tumors. We evaluated the relationship between 14F7 Mab reactivity, pathological features, DNA-content and S-phase fraction (SPF), and their impact in the survival of NSCLC patients. Hematoxylin and eosin staining and immunohistochemistry optical microscopy assays as well as DNA content and SPF measuring using flow cytometry were performed. The 14F7 reactivity was widely observed in NSCLC sections, no depending of the clinicopathological characteristics. We also obtained differences in the intensity of reaction with 14F7 as well as in the SPF between diploid and aneuploid carcinomas. Patients with diploid tumors showing higher SPF and 14F7 reaction joint to a low mitotic index displayed higher survival rates. Our results are in agreement with the assumption of the possible positive prognostic value of 14F7 staining in NSCLC.

Immunoreactivity of the 14F7 Mab Raised against N-Glycolyl GM3 Ganglioside in Epithelial Malignant Tumors from Digestive System.

The limited expression of N-Glycolyl GM3 (NeuGcGM3) ganglioside in human normal tissues, as well as its presence in melanoma and breast carcinoma using 14F7 Mab (anti-NeuGcGM3), has been previously reported. In this work we evaluated for the first time the 14F7 Mab immunorecognition in some digestive system tumors. Immunohistochemical assays were made with 14F7, followed by anti-mouse biotinylated antibody and ABC/HRP system in normal and pathological human tissues were made. No immunoreaction was evidenced in normal tissues. The reactivity of 14F7 was detected in all adenocarcinomas of the stomach (12/12), colon (12/12), and pancreas (11/11). A finely granular immunorecognition in esophageal tumors (5/15), epidermoid carcinoma of the rectum (5/7), and basaloid carcinoma (4/5) of the latter as well as in hepatocellular carcinoma (13/14) was also observed. Our results are in agreement with the assumption that NeuGcGM3 ganglioside may be considered as target for passive and active immunotherapy in digestive system malignancies expressing this molecule.

Immunohistochemical Reactivity of the 14F7 Monoclonal Antibody Raised against N-Glycolyl GM3 Ganglioside in Some Benign and Malignant Skin Neoplasms.

The evaluation of 14F7 Mab (anti-N-glycolyl GM3 ganglioside) immunorecognition in normal skin, cutaneous malignant melanoma (CMM), and in lymph node metastases (LNM) has been previously reported. In this work we extended the study to benign (BMN) and dysplastic (DMN) melanocytic nevi, basal (BCC), and squamous cell carcinoma (SCC). Immunohistochemical assays with 14F7 followed by a biotinylated link universal and streptavidin-AP in normal and pathological tissues were made. No reaction of 14F7 in normal skin (0/10) as well as a low reactivity in BMN (2/11) and DMN (1/7) was detected. A limited staining in BCC (2/13) and in SCC (4/8) was also evidenced, while 14F7 Mab were mostly reactive in CMM (28/28) and in LNM (6/7). These results suggest that 14F7 reactivity could be closely related with the more aggressive biological behavior of CMM and also support the use of NeuGcGM3 as target for both passive and active melanoma immunotherapy.

Immunogenicity and safety of a NeuGcGM3 based cancer vaccine: Results from a controlled study in metastatic breast cancer patients.

Increased levels of NeuGc-containing gangliosides have been described in human breast cancer. A controlled Phase II clinical trial was conducted in patients with metastatic breast cancer to evaluate immunogenicity, safety and to identify evidences of biological activity of a cancer vaccine composed by NeuGcGM3 in a proteoliposome of Neisseria meningitidis together with Montanide ISA 51 as adjuvant. After first line chemotherapy, 79 women were randomized 1:1 to receive the vaccine candidate or best supportive care. All patients achieved at least stable disease to the first line therapy for the metastatic condition. Treatment consisted on 5 vaccine doses every 2 weeks and then, monthly re-immunization to complete 15 doses. Vaccination with the NeuGcGM3 based vaccine was safe and the most frequent adverse events consisted on injection site reactions, fever, arthralgia and chills. The vaccine was immunogenic and a sustained increase of both IgG and IgM antibody titters against NGcGM3 was observed after the second vaccination month. Antibodies were able to recognize the NeuGcGM3(+) murine tumor cell line L1210 and the myeloma cell line P3X63. Humoral response was specific since vaccination did not result in Neu-Acetyl GM3 or GM2-antibody response. Hyperimmune sera from vaccinated patients were able to prevent the NeuGcGM3 mediated CD4 down-modulation on T lymphocytes. In the intent to treat analysis, there was a trend toward a survival advantage for the vaccine group and this effect was significant for women bearing non-visceral metastasis. Two phase III clinical studies with this vaccine candidate are ongoing.

Anti-ganglioside antibody-induced tumor cell death by loss of membrane integrity.

Gangliosides have been involved in multiple cellular processes such as growth, differentiation and adhesion, and more recently as regulators of cell death signaling pathways. Some of these molecules can be considered as tumor-associated antigens, in particular, N-glycolyl sialic acid-containing gangliosides, which are promising candidates for cancer-targeted therapy because of their low expression in normal human tissues. In this study, we provided the molecular and cellular characterization of a novel cell death mechanism induced by the anti-NGcGM3 14F7 monoclonal antibody (mAb) in L1210 murine tumor cell line but not in mouse normal cells (B and CD4(+) T lymphocytes) that expressed the antigen. Impairment of ganglioside synthesis in tumor cells abrogated the 14F7 mAb cytotoxic effect; however, exogenous reincorporation of the ganglioside did not restore tumor cell sensitivity to 14F7 mAb-induced cytotoxicity. 14F7 F(ab')(2) but not Fab fragments retained the cytotoxic capacity of the whole mAb. By contrary, other mAb, which recognizes N-glycolylated gangliosides, did not show any cytotoxic effect. These mAbs showed quite different capacities to bind NGcGM3-positive cell lines measured by binding inhibition experiments. Interestingly, this complement-independent cell death mechanism did not resemble apoptosis, because no DNA fragmentation, caspase activation, or Fas mediation were observed. However, NGcGM3 ganglioside-mediated 14F7 mAb-induced cell death was accompanied by cellular swelling, membrane lesion formation, and cytoskeleton activation, suggesting an oncosis-like phenomenon. This novel mechanism of cell death lets us to support further therapeutic approaches using NGcGM3 as a molecular target for antibody-based cancer immunotherapy.

Anti-ganglioside antibody-induced tumor cell death by loss of membrane integrity

Gangliosides have been involved in multiple cellular processes such as growth, differentiation and adhesion, and more recently as regulators of cell death signaling pathways. Some of these molecules can be considered as tumor-associated antigens, in particular, N-glycolyl sialic acid¨Ccontaining gangliosides, which are promising candidates for cancer-targeted therapy because of their low expression in normal human tissues. In this study, we provided the molecular and cellular characterization of a novel cell death mechanism induced by the anti-NGcGM3 14F7 monoclonal antibody (mAb) in L1210 murine tumor cell line but not in mouse normal cells (B and CD4+ T lymphocytes) that expressed the antigen. Impairment of ganglioside synthesis in tumor cells abrogated the 14F7 mAb cytotoxic effect; however, exogenous reincorporation of the ganglioside did not restore tumor cell sensitivity to 14F7 mAb-induced cytotoxicity. 14F7 F(ab¡ä)2 but not Fab fragments retained the cytotoxic capacity of the whole mAb. By contrary, other mAb, which recognizes N-glycolylated gangliosides, did not show any cytotoxic effect. These mAbs showed quite different capacities to bind NGcGM3-positive cell lines measured by binding inhibition experiments. Interestingly, this complement-independent cell death mechanism did not resemble apoptosis, because no DNA fragmentation, caspase activation, or Fas mediation were observed. However, NGcGM3 ganglioside-mediated 14F7 mAb-induced cell death was accompanied by cellular swelling, membrane lesion formation, and cytoskeleton activation, suggesting an oncosis-like phenomenon. This novel mechanism of cell death lets us to support further therapeutic approaches using NGcGM3 as a molecular target for antibody-based cancer immunotherapy(AU)

Heterophilic NeuGcGM3 ganglioside cancer vaccine in advanced melanoma patients: results of a Phase Ib/IIa study.

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.

Clinical evidences of GM3 (NeuGc) ganglioside expression in human breast cancer using the 14F7 monoclonal antibody labelled with (99m)Tc.

The relevance of certain gangliosides in tumour growth and metastatic dissemination has been well documented, reasons for considering these molecules as potential targets for cancer immunotherapy and diagnosis. GM3(NeuGc) ganglioside is particularly interesting due to its restrictive expression in normal human tissues according to immunohistochemical studies, using either polyclonal or monoclonal antibodies. But both immunohistochemical and biochemical methods have strongly suggested its over-expression in human breast tumours. Nevertheless, the lack of a direct evidence of this antigenic display in human breast cancer has kept the subject controversial. For the first time, we described herein the "in vivo" detection of GM3(NeuGc) ganglioside in human breast primary tumours using a radioimmunoscintigraphic technique with 14F7, a highly specific anti-GM3(NeuGc) ganglioside monoclonal antibody, labelled with (99m)Tc. In an open, prospective Phase I/II clinical trial, including women diagnosed in stage II breast cancer, the 14F7 monoclonal antibody accumulation in tumours at doses of 0.3 (n=5), 1 (n=5) and 3 mg (n=4) was evaluated. Noteworthy, the immunoscintigraphic study showed antibody accumulation in 100% of patients' tumours for the 1 mg dose group. In turn, the radioimmunoconjugate injected at doses of 0.3 mg or 3 mg of the antibody, was uptaken by 60 and 33.3% of breast tumours, respectively. "In vivo" immune recognition of GM3(NeuGc) in breast tumours reinforces the value of this peculiar target for cancer immunotherapy.

Immunotherapy of advanced breast cancer with a heterophilic ganglioside (NeuGcGM3) cancer vaccine.

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.

In vivo and in vitro anti-tumor effect of 14F7 monoclonal antibody.

The 14F7 monoclonal antibody (MAb) is an IgG(1) antibody that reacts specifically with GM3 (NeuGc) and with tissue sections of human tumors. We demonstrated here that this MAb is agglutinin that specifically agglutinated horse erythrocytes. Additionally, the capacity of 14F7 MAb to mediate cytotoxicity against GM3 (NeuGc)-positive murine myeloma cells, in vitro and in vivo, was evaluated. High concentrations of 14F7 MAb were needed to induce complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) against the murine myeloma cells. The most relevant finding was the ability of this MAb to directly kill the target cells without participation of complement. This cytotoxicity was dependent on the temperature and MAb concentration and the number of the target cells. In vivo, the passive treatment with 14F7 MAb produced a strong anti-tumor activity, similar to the anti-tumoral response obtained with standard chemotherapy treatment.