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Gangliosides containing the N-glycolyl (NGc) form of sialic acid are tumor-associated antigens and promising candidates for cancer therapy. We previously generated the murine 14F7 monoclonal antibody (mAb), specific for the N-glycolyl-GM3 ganglioside (NGcGM3), which induced an oncosis-like type of cell death on malignant cell lines expressing this antigen and recognized breast carcinoma by immunoscintigraphy in cancer patients. As humanization is expected to enhance its use for human cancer therapy, herein we describe the design and generation of two humanized versions of the 14F7 mAb by disrupting potential human T cell epitopes on its variable region. No differences in antigen reactivity or cytotoxic properties were detected among the variants tested and with respect to the chimeric counterpart. Humanized 14F7 genes were transfected into the NGcGM3-expressing NS0 cell line. Therefore, in the industrial scaling-up of the transfectoma in serum-free medium, cell viability was lost due to the cytotoxic effect of the secreted antibody. This shortcoming was solved by knocking down the CMP-N-acetylneuraminic acid hydroxylase enzyme, thus impairing the synthesis of NGc-glycoconjugates. Humanized 14F7 mAb is of potential value for the therapy of NGcGM3-expressing tumors., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Gangliosides are sialic acid-containing glycosphingolipids present in the plasma membrane of most mammalian cells. In humans, the expression of the N-glycolylated (Neu5Gc) variant of the sialic acid has been associated with malignant transformation, constituting therefore an attractive target for cancer immunotherapy. P3 monoclonal antibody (mAb) recognizes Neu5Gc-containing gangliosides, as well as sulfatides. Heavy chain CDR3 (H-CDR3) arginine residues have been shown to be crucial for ganglioside recognition, but less important for anti-idiotypic antibody binding. Here, we describe the effect on antibody reactivity of different mutations involving a single H-CDR3 acid residue. Substitution of glutamate 99 (Kabat numbering) by arginine, aspartate or serine residues resulted in no differences in anti-idiotype binding. However, the first mutation caused increased reactivity with the antigen, including a cytotoxic effect of the antibody on ganglioside-expressing cells previously unseen for the wild type antibody. Another antibody that recognizes N-glycolyl-GM3 ganglioside (GM3(Neu5Gc)), but not other glycolipids, named 14F7, exhibits also an arginine-enriched H-CDR3 and a complement-independent cell death activity. Unlike 14F7 mAb, the cytotoxicity of the P3 E(99)→R mutant antibody did not exclusively depend on ganglioside expression on tumor cells., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.

N-Glycolylated (NeuGc) gangliosides are tumor-specific antigens and as such represent attractive targets for cancer immunotherapy. The chimeric antibody chP3 selectively recognizes a broad variety of NeuGc gangliosides, showing no cross-reactivity to the highly similar N-acetylated (NeuAc) gangliosides that are common cellular antigens in humans. Here, we report the crystal structure of the chP3 Fab and its computer-docking model with the trisaccharide NeuGcalpha3Galbeta4Glcbeta, which represents the carbohydrate moiety of the tumor-antigen NeuGc-GM3. The interaction involves only the heavy chain of the chP3 antibody. The modelled complex is consistent with all available experimental data and shows good surface complementarity. The negatively charged sialic acid residue NeuGc is buried in a pocket flanked by two arginine residues, VH Arg31 and VH Arg100A. We have further investigated the interaction of chP3 with its anti-idiotypic antibody, 1E10 (also known as Racotumomab), currently in clinical trials as a cancer vaccine. While many of the chP3 residues predicted to interact with the NeuGc ganglioside also feature prominently in the modelled complex of chP3 and 1E10, we do not observe structural mimicry. Rather, we suspect that the anti-idiotype 1E10 may serve as an imprint of the structural characteristics of the chP3 idiotype and, consequently, give rise to antibodies with P3-like properties upon immunization.

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.

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.

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.

P3 mAb is an IgM monoclonal antibody specific for N-glycolyl-containing gangliosides. The immunogenicity of the P3 idiotype has been previously described by immunizing syngeneic BALB/c mice with the purified murine IgM or the mouse-human chimeric IgG antibody. In the present work we study the antibody response against the idiotype of P3 mAb through immunization with DNA. We used small immune proteins (SIP) consisting on the idiotype in the scFv format, covalently linked to gamma1CH3, the self-dimerizing domain of murine IgG1. SIPs were previously shown to be appropriate to induce specific anti-idiotypic responses. By gene gun immunization, a polyspecific response was occasionally generated, particularly with the P3 idiotype. A single shot of DNA was sufficient to induce a strong and long-lasting anti-P3 idiotype response. In addition, by delivery of the same DNA construct with a recombinant adeno-associated virus the unique immunogenicity of the P3 idiotype was demonstrated. The requirement of T cells in the anti-P3 idiotype response was indicated by the lack of P3-specific anti-idiotypic antibodies following immunization of both, allogeneic C57BL/6 and athymic BALB/c mice.

The antibody heavy chain is generally more important than the light chain for the interaction with the antigen, although many reports demonstrate the influence of the light chain in the antibody binding properties. The heavy chains of anti-N-glycolyl-ganglioside P3 mAb and anti-idiotypic 1E10 mAb display complementary charged residues in their H-CDRs, particularly in H-CDR3. A basic residue in P3 mAb H-CDR1 was shown to be crucial for the interaction with the antigen and 1E10 mAb. The immunogenetic features of three other P3 mAb anti-idiotypic mAbs are now analyzed. One of them bears the same heavy chain as 1E10 mAb and a different light chain, but differs in its binding to P3 mAb mutants where H-CDR basic residues were replaced and in the binding to 1E10-specific phagotopes. Chimeric hybrid antibodies with P3 and 1E10 mAb heavy chains and unrelated light chains were obtained to further determine the importance of heavy chains in P3 and 1E10 mAb binding properties. One of the P3 heavy chain hybrid antibodies retained the specificity of P3 mAb with slight affinity differences. The heavy chains appear to play the main role in these mAb interactions, with the light chains modulating the affinity to their ligands.

The heavy-chain variable regions (VH) from 14F7 MAb, an IgG1 antibody specific for GM3(NeuGc) ganglioside, and its anti-idiotype, the 4G9 MAb, were cloned and sequenced. Comparison with previously reported sequences showed that VH 14F7 belongs to the J558(VHI) gene family and that it is highly mutated. VH 4G9 belongs to the Q52(VHII) gene family. The HCDR3 14F7 sequence contains three basic residues that could be involved in the binding to 4G9 MAb, which bears acidic residues in its HCDR3. Studies performed in the syngeneic model showed that 14F7 MAb requires both coupling to KLH and the use of Freund's adjuvant to induce an effective anti-idiotypic IgG (Ab2) response. In contrast, P3 MAb, a germline gene-encoded Ab1 that also recognizes the GM3(NeuGc) ganglioside through a basic motif in its H-CDRs, has been reported to be immunogenic in syngeneic mice, even when injected in saline. In addition, when Leghorn chickens were immunized with 14F7 or P3 MAbs emulsified in Freund's adjuvant, only P3-immunized animals were able to develop antibodies that recognized NeuGc-containing gangliosides, antigens which are not present in the normal tissues of this animal species. This phenomenon could be due to the lack of idiotypic connectivity of 14F7MAb.

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.

We have previously generated a murine anti-idiotype (Ab2) monoclonal antibody (mAb) to a murine Ab1 mAb, named P3, which selectively binds Neu-glycolyl (NeuGc)-sialic acid on several monosialo- and disialogangliosides, and also reacts with sulfatides and antigens expressed in human melanoma and breast tumors. This Ab2 mAb, designated as 1E10, induced anti-anti-idiotype antibodies (Ab3) in mice and cancer patients. These Ab3 generated by 1E10 mAb were characterized by bearing P3 mAb idiotopes (Ab3, Id +). But when the specificity of these Ab3 antibodies was tested, no specific humoral response against NeuGc-containing gangliosides was detected in sera from immunized mice. However, hyperimmune sera from melanoma and breast cancer patients vaccinated with this Ab2 mAb were able to react specifically with these gangliosides. The different expression of NeuGc-containing gangliosides in the normal tissues of mice and humans could explain these results. In order to demonstrate these findings in other animal species with a different NeuGc-sialic acid expression, we performed similar studies in monkeys and chickens. In monkeys, as in most mammals, NeuGc-containing gangliosides are self-antigens. In contrast, chickens, like humans, lack the expression of these antigens in normal tissues. Here we report that the antibody response against NeuGc-containing gangliosides induced by immunization with 1E10 mAb was completely different in both species. No specific antibody response against these gangliosides was detected in hyperimmune monkey sera. In contrast, a strong and specific Ab3 response against GM3(NeuGc) and GM2(NeuGc) gangliosides (Ab3, Ag+) was generated in chickens due to the administration of 1E10 mAb.

The 14F7 monoclonal antibody (MAb), IgG1 isotype, which reacts specifically to GM3(NeuGc) ganglioside induced a specific IgG anti-idiotypic antibody (Ab2) response in syngeneic mice when it was administered coupled with KLH and in the presence of Freund's adjuvant. Spleen cells from these mice were used in somatic-cell hybridization experiments using the murine myeloma cell line P3-X63-Ag8 653 as fusion partner. An IgG1 Ab2 MAb was selected. This Ab2 MAb, called 4G9, was able to block the binding of 14F7 MAb to GM3(NeuGc) ganglioside and developed a strong IgG anti-anti-idiotypic antibody (Ab3) response, when injected into syngeneic mice. These Ab3 antibodies were characterized to bear 14F7 MAb idiotopes, but did not have the same specificity as 14F7 MAb. In the other hand, a very specific anti-NeuGc-containing ganglioside response was generated in chickens immunized with this Ab2 MAb, thus behaving, in this species as an "internal image" antibody.

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.