Luis E. Fernández

A Mouse IgG1 Monoclonal Antibody Specific for N-Glycolyl GM3 Ganglioside Recognized Breast and Melanoma Tumors

14F7 murine monoclonal antibody (MAb) is an IgG1 immunoglobulin that is generated by immunizing Balb/c mice with GM3(NeuGc) ganglioside hydrophobically conjugated with human very-low-density lipoproteins and in the presence of Freunds adjuvants. 14F7 MAb binds specifically to GM3(NeuGc), whereas neither N-glycolyl or N-acetyl gangliosides, nor a sulfated glycolipid, are recognized as assessed by enzyme-linked immunosorbent assay or immunostaining on thin layer chromatograms. Immunohistochemical studies in fresh tumor tissues showed that 14F7 MAb strongly recognized in antigen expressed in human breast and melanoma tumors.

Enhancement of the immune response to poorly immunogenic gangliosides after incorporation into very small size proteoliposomes (VSSP)

Certain gangliosides are tumor-associated antigens that constitute potential targets for cancer immunotherapy. A major drawback in the design of ganglioside-based cancer vaccines, however, is the poor immunogenicity of these glycolipids. Here we report the immunological and physicochemical properties of very small size proteoliposomes (VSSP) obtained by using anionic detergents to incorporate gangliosides into the outer membrane protein complex (OMPC) of N. meningitidis. VSSP of three different gangliosides, GM3, NGcGM3 and GD3, were tested. These gangliosides differ in level of expression in normal tissues and in immunogenicity in different animal species. We show that the immunization with VSSP in an oil adjuvant consistently induced both IgM and IgG anti-ganglioside antibodies. In the mouse, the anti-ganglioside IgG fraction was not restricted to the typical T-independent isotype IgG3. Unexpectedly, significant levels of the T-dependent IgG1, IgG2a and particularly IgG2b were also found. VSSP-mediated enhancement of the immunogenicity was not restricted to the relatively immunogenic ganglioside GD3, satisfactory immune responses against highly tolerated GM3 and NGcGM3 were also obtained. Similar results were achieved in chickens and monkeys. No reactogenicity was observed even when self-gangliosides were used for immunization. VSSP overcame natural tolerance to gangliosides in an adjuvant dependent fashion.

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.

Bivalent binding by intermediate affinity of nimotuzumab: A contribution to explain antibody clinical profile

Nimotuzumab is an EGFR-targeting antibody that has demonstrated encouraging clinical results in the absence of severe side-effects observed with other approved anti-EGFR antibodies. We investigated whether different clinical behavior of nimotuzumab is related to its bivalent/monovalent binding profile. Binding properties of nimotuzumab and cetuximab, the most development of anti-EGFR antibodies, were studied in vitro using chip surfaces and cells with varying EGFR expression levels. Experimental observations demonstrated that in contrast to cetuximab, the intrinsic properties of nimotuzumab required bivalent binding for stable attachment to the cellular surface, leading to nimotuzumab selectively binding to cells that express moderate to high EGFR expression levels. At these conditions, both antibodies bound bivalently, and accumulated to similar degrees. When EGFR density is low, nimotuzumab monovalent interaction was transient, whereas cetuximab continued to interact strongly with the receptors. We compared the in vitro anti-tumor efficacy of nimotuzumab and cetuximab. Cetuximab decreased the cell viability and induced apoptosis for all the tested cell lines, effects which did not depend on EGFR expression level. In contrast, nimotuzumab also provoked significant anti-cellular effects, but its anti-tumor capacity decreased together with EGFR expression level. Cetuximab Fab fragment was able to impact tumor cell survival, whereas nimotuzumab fragment totally lost this effect. Tumor-xenograft experiments using cells with a high EGFR expression revealed similar tumor growth inhibiting effects for both antibodies. This study suggests an explanation for nimotuzumab clinical profile, whereby antitumor activity is obtained in absence of severe toxicities due to its properties of bivalent binding to EGFR. See commentary: Don’t jump to rash conclusions

Challenges facing adjuvants for cancer immunotherapy

An adjuvant is defined as a product that increases or modulates the immune response against an antigen (Ag). Based on this general definition many authors have postulated that the ideal adjuvant should increase the potency of the immune response, while being non‐toxic and safe. Although dozens of different adjuvants have been shown to be effective in preclinical and clinical studies, only aluminium‐based salts (Alum) and squalene–oil–water emulsion (MF59) have been approved for human use. However, for the development of therapeutic vaccines to treat cancer patients, the prerequisites for an ideal cancer adjuvant differ from conventional adjuvants for many reasons. First, the patients that will receive the vaccines are immuno‐compromised because of, for example, impaired mechanisms of antigen presentation, non‐responsiveness of activated T cells and enhanced inhibition of self‐reactivity by regulatory T cells. Second, the tumour Ag are usually self‐derived and are, therefore, poorly immunogenic. Third, tumours develop escape mechanisms to avoid the immune system, such as tumour editing, low or non‐expression of MHC class I molecules or secretion of suppressive cytokines. Thus, adjuvants for cancer vaccines need to be more potent than for prophylactic vaccines and consequently may be more toxic and may even induce autoimmune reactions. In summary, the ideal cancer adjuvant should rescue and increase the immune response against tumours in immuno‐compromised patients, with acceptable profiles of toxicity and safety. The present review discusses the role of cancer adjuvants at the different phases of the generation of antitumour immunity following vaccination.

Induction of Immunogenic Apoptosis by Blockade of Epidermal Growth Factor Receptor Activation with a Specific Antibody

Despite promising results in the use of anti-epidermal growth factor receptor (EGFR) Abs for cancer therapy, several issues remain to be addressed. An increasing emphasis is being placed on immune effector mechanisms. It has become clear for other Abs directed to tumor targets that their effects involve the adaptive immunity, mainly by the contribution of Fc region-mediated mechanisms. Given the relevance of EGFR signaling for tumor biology, we wonder whether the oncogene inhibition could contribute to Ab-induced vaccine effect. In a mouse model in which 7A7 (an anti-murine EGFR Ab) and AG1478 (an EGFR-tyrosine kinase inhibitor) displayed potent antimetastatic activities, depletion experiments revealed that only in the case of the Ab, the effect was dependent on CD4+ and CD8+ T cells. Correspondingly, 7A7 administration elicited a remarkable tumor-specific CTL response in hosts. Importantly, experiments using 7A7 F(ab′)2 suggested that in vivo Ab-mediated EGFR blockade may play an important role in the linkage with adaptive immunity. Addressing the possible mechanism involved in this effect, we found quantitative and qualitative differences between 7A7 and AG1478-induced apoptosis. EGFR blocking by 7A7 not only prompted a higher proapoptotic effect on tumor metastases compared with AG1478, but also was able to induce apoptosis with immunogenic potential in an Fc-independent manner. As expected, 7A7 but not AG1478 stimulated exposure of danger signals on tumor cells. Subcutaneous injection of 7A7-treated tumor cells induced an antitumor immune response. This is the first report, to our knowledge, of a tumor-specific CTL response generated by Ab-mediated EGFR inhibition, suggesting an important contribution of immunogenic apoptosis to this effect.

Isolation and characterization of an antitumor active agar-type polysaccharide of Gracilaria dominguensis

Cold water extraction of the red alga Gracilaria dominguensis, followed by cetyltrimethylammonium bromide fractionation, gave a highly sulfated, agar-type polysaccharide which inhibited the transplantation of Ehrlich ascites carcinoma in mice. The structure of the polysaccharide has been investigated by methylation analysis, and 1H- and 13C-n.m.r. spectroscopy, and was shown to be mainly composed of alternating (1----3)-linked beta-D-galactopyranosyl 6-sulfate and (1----4)-linked 3,6-anhydro-alpha-L-galactopyranosyl residues.

Differential influence of the tumour-specific non-human sialic acid containing GM3 ganglioside on CD4+CD25− effector and naturally occurring CD4+CD25+ regulatory T cells function

Increasing evidences suggest that the aberrant expression of certain gangliosides on malignant cells could affect hosts anti-tumour-specific immune responses. We have recently documented the relevance of the N-glycolylated variant of GM3 ganglioside (NGcGM3), a tumour-specific non-human sialic acid containing ganglioside, for tumour progression. However, evidences about the implication of hosts immunity in NGcGM3-promoted cancer progression had not been obtained previously. In this work, we compared tumour growth of X63 myeloma cells pre-treated or not with an inhibitor of the glucosylceramide synthase enzyme, in wild or CD4+ T cell-depleted BALB/c mice. Results clearly showed a relationship between the agonistic effect of NGcGM3 in tumour growth and the presence of CD4+ T lymphocytes. For the first time, a description of a ganglioside-differential effect over purified CD4+CD25- and naturally occurring regulatory CD4+CD25+ T cells is provided. While NGcGM3 similarly down-modulated the CD4 expression in both cell populations, the inhibitory capacity of the CD4+CD25+ lymphocytes and their proliferation, induced by an anti-CD3 mAb and IL2, were not modified. In a different fashion, a reduction in proliferative capacity and a noteworthy secretion of anti-inflammatory cytokines were detected when CD4+CD25- T cells were cultured in the presence of NGcGM3. Considering the relevance of dendritic cells (DC) on primary activation of T cells, the effect of NGcGM3 over DC differentiation and TLR4-mediated maturation was also assessed. Our results indicate that NGcGM3 contributes to cancer progression mainly by influencing DC and CD4+CD25- T lymphocyte functions, rather than increasing the inhibitory capacity of naturally occurring regulatory T cells.

Inhibition of Tumor-Induced Myeloid-Derived Suppressor Cell Function by a Nanoparticulated Adjuvant

The interaction between cancer vaccine adjuvants and myeloid-derived suppressor cells (MDSCs) is currently poorly understood. Very small size proteoliposomes (VSSP) are a nanoparticulated adjuvant under investigation in clinical trials in patients with renal carcinoma, breast cancer, prostate cancer, and cervical intraepithelial neoplasia grade III. We found that VSSP adjuvant induced a significant splenomegaly due to accumulation of CD11b+Gr-1+ cells. However, VSSP-derived MDSCs showed a reduced capacity to suppress both allogeneic and Ag-specific CTL response compared with that of tumor-induced MDSCs. Moreover, splenic MDSCs isolated from tumor-bearing mice treated with VSSP were phenotypically more similar to those isolated from VSSP-treated tumor-free mice and much less suppressive than tumor-induced MDSCs, both in vitro and in vivo. Furthermore, different from dendritic cell vaccination, inoculation of VSSP-based vaccine in EG.7-OVA tumor-bearing mice was sufficient to avoid tumor-induced tolerance and stimulate an immune response against OVA Ag, similar to that observed in tumor-free mice. This effect correlated with an accelerated differentiation of MDSCs into mature APCs that was promoted by VSSP. VSSP used as a cancer vaccine adjuvant might thus improve antitumor efficacy not only by stimulating a potent immune response against tumor Ags but also by reducing tumor-induced immunosuppression.

Ganglioside-based vaccines and anti-idiotype antibodies for active immunotherapy against cancer

This review shall present an update in anticancer ganglioside-based immunotherapies, with particular emphasis on molecular vaccines and anti-idiotype mAbs produced by the Center of Molecular Immunology (Havana, Cuba). The project comprises vaccines of N-acetyl or N-glycolylneuraminic acid GM3 ganglioside incorporated into very small proteoliposomes and anti-idiotype antibodies to glycolylated gangliosides. Development of these vaccine preparations from preclinical models of melanoma, breast and lung cancer to human investigation is summarized. A brief discussion on the progress and limitations of present-day clinical trials and future prospects is also included.