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The heavy chain of anti-N-glycolyl-ganglioside P3 mAb plays the main role in its binding properties. At least one hybrid idiotype consisting on the P3 VH and an unrelated VL domain retains antigen recognition. Moreover, the unusual immunogenic properties of P3 idiotype could be modified by single mutations of H-CDR residues. Here, we show that DNA gene gun immunization with the P3 VH combined with an unrelated VL domain or with itself (VH dimer, VHD) is enough for inducing anti-idiotypic antibodies, independently of antigen recognition by the resulting molecule. The scFv fragment of P3 mAb was also able to induce an anti-idiotypic response. For both the P3 and the P3 anti-idiotypic 1E10 mAbs, heavy chains dominate the induction of antibodies against the respective idiotypes.

This report is focused on the molecular basis for the interaction of a monoclonal antibody (mAb) and its anti-idiotypic mAb. P3 mAb (Ab1) recognizes N-glycolyl-gangliosides, and 1E10 mAb is one of its anti-idiotypic mAbs (Ab2). Chimeric versions of both antibodies retained their specificity. Charged residues in their H-CDRs, particularly H-CDR3, were considered to play a major role in their binding and immunogenic properties. P3 mAb has the unusual property of generating a strong antibody response in syngeneic mice, even when it is administered in saline. We selected phagotopes from a 12mer peptide library displayed on filamentous phage to characterize amino acid motifs recognized by these antibodies. The peptides were enriched in charged amino acids similar to those present in P3 and 1E10 mAb H-CDR3. We also report the construction of four mutants of the P3 antibody, where arginine residues in the heavy chain CDRs were substituted by serine residues, and the characterization of their interaction with 1E10 mAb and GM3(NeuGc) ganglioside, as well as their immunogenic properties in Balb/c mice. H-CDR1 R31 residue appears to have a central role in P3 mAb reactivity and antigenicity. H-CDR3 R100a residue seems to be more involved in the immunogenicity of the P3 idiotype.

P3 monoclonal antibody (MAb) is a murine IgM that specifically recognizes N-glycolyl (NeuGc)-gangliosides and sulfatides. It also reacts with antigens expressed in human breast tumors and melanoma. In syngeneic model, P3 MAb is able to elicit a strong anti-idiotypic (Ab2) antibody response, even in the absence of adjuvants or carrier proteins. 1E10 MAb is an anti-idiotypic antibody specific for P3 MAb that has demonstrated anti-tumoral effects in syngeneic and allogeneic animals. Here we report the construction of the human IgG(1) chimeric P3 and 1E10 antibodies, and the evaluation of the maintenance of the main properties of the murine MAbs. Chimeric P3 antibody specifically reacted with GM3(NeuGc) and GM2(NeuGc) gangliosides, and not with their acetylated variants. Also, it strongly recognized the anti-idiotypic 1E10 MAb. Chimeric 1E10 antibody specifically reacted with P3 MAb. Upon immunization of Balb/c mice with both chimeric antibodies, we were able to demonstrate the immunodominance of their variable regions. The anti-idiotypic response induced by both antibodies was strong and in most of the mice was even significantly higher than the anti-isotypic response, despite the fact that 70% of the chimeric molecule is xenogenic with respect to the animal model.

Abstract: 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 &y& Elsevier]

Abstract: 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 E99 →R mutant antibody did not exclusively depend on ganglioside expression on tumor cells. [Copyright &y& Elsevier]

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-glycolilated 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 cytosqueleton 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. [ABSTRACT FROM AUTHOR]

Abstract: Detailed information on the immunological relevance of α-type anti-idiotypic antibodies is lacking after more than 30 years since Jerne postulated his Idiotypic Network Theory. The B7Y33 mutant is a mouse-human chimeric version of the B7 MAb, a polyreactive α-type anti-idiotypic antibody, generated against an anti-GM2 ganglioside IgM Ab1 antibody. It retained the unusual self-binding activity and multispecificity of the parental murine antibody, being able to recognize several anti-ganglioside IgM antibodies as well as non-immunoglobulin antigens. Previous work with the murine B7 MAb suggested that this antibody might have immunoregulatory properties, and therefore we investigated the possible interaction of B7Y33 with immune cells. We found that B7Y33 binds to human and murine B lymphocytes. Inhibition assays using flow cytometry indicated that this antibody is capable of binding the Fc γ receptor II (FcγRII). The recognition of FcγRII-expressing K562, Raji and Daudi human cell lines, together with the capability of inhibiting the binding of an anti-human FcγRII antibody to these cells, suggest that B7Y33 interacts with both the FcγRIIa and FcγRIIb isoforms. We evaluated the contribution to the binding of different surface-exposed residues at the top of the heavy chain variable region (VH) CDR loops through the construction of mutants with substitutions in the three conventional VH CDRs (HCDRs) and the “HCDR4”, located in the framework 3 (HFR3). In addition, we assessed the involvement of the Fc region by performing key mutations in the CH2 domain. Furthermore, chimeric hybrid molecules were obtained by combining the B7Y33 heavy chain with unrelated light chains. Our results indicate that the multispecificity and self-binding properties of B7Y33 are not linked to its recognition of B lineage cells, and that this phenomenon occurs in a non-classical way with the participation of both the variable and constant regions of the antibody. Two possible models for this interaction are proposed, with B7Y33 binding to two FcγRIIb molecules through the Fc and Fv regions, or simultaneously to FcγRIIb and another unknown antigen on B cells. The FcγRIIb has recently received great attention as an attractive target for therapies directed to B lymphocytes. The recognition of peripheral B lymphocytes from B cell chronic lymphocytic leukemia (B-CLL) patients by B7Y33 suggests its potential application for the treatment of B cell malignancies. [Copyright &y& Elsevier]

Abstract: 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 NeuGcα3Galβ4Glcβ, 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

P3 mAb is a murine IgM that specifically recognizes N-glycolyl (NeuGc)-gangliosides and sulfatides. It also reacts with antigens expressed in human breast tumors and melanoma. In syngeneic model, P3 mAb is able to elicit a strong anti-idiotypic (Ab2) antibody response, even in absence of adjuvants or carrier proteins. Here we reported the construction of the human IgG1 chimeric P3 antibody, and the evaluation of the maintenance of the main properties of the murine P3 mAb. Chimeric P3 antibody specifically reacted with GM3(NeuGc) and GM2(NeuGc) gangliosides, and not with their acetylated variants. Also, it recognized the anti-idiotypic 1E10 mAb. Upon immunization of Balb/c mice with chimeric P3 antibody, we were able to demonstrate the immunodominance of its variable region. The anti-idiotypic response induced by this antibody was very strong and in three out of four mice was even significantly higher than the anti-isotypic response, despite the fact that 70% of the chimeric molecule is xenogenic with respect to the animal model. In chickens, the immunogenicity of chimeric P3 antibody idiotype was also evaluated. [ABSTRACT FROM AUTHOR]

We developed a phase I clinical trial for patients with stage III/IV breast carcinoma treated with the anti-idiotype mAb 1E10 (Ab2) specific to an Ab1 mAb (P3) able to react specifically with N-glycolyl-containing gangliosides and with antigens expressed on human melanoma and breast carcinoma cells. In animal models this Ab2 mAb induced Ab3 antibody responses sharing immunochemically-defined idiotopes with the Ab1, but unable to recognize N-glycolyl-containing gangliosides. The treatment of tumor-bearing mice with 1E10 mAb induced a strong antitumor activity. In a previous clinical trial carried out with anti-id mAb 1E10 in patie nts with advanced malignant melanoma, the vaccine proved to be safe and immunologically effective, with most patients being able to generate a specific immune response against 1E10 and NeuGc-GM3 ganglioside. The purpose of the present study was to evaluate the toxicity, the immune response to 1E10 anti- id vaccine and to test two levels of doses of the vaccine preparation. Ten patients of breast carcinoma, distributed in two groups of five, received six doses of 1 and 2 mg respectively, of aluminum hydroxide precipitated 1E10 anti-id mAb, intradermally into multiple sites at two weeks intervals. In eight of the nine patients who received at least four doses of the anti- id vaccine strong specific responses were observed both against 1E10 mAb and the NeuGc-GM3 ganglioside (Ab3Id+Ag+). Non-classical Ab1' antibodies (Id-Ag+) were also elicited by 1E10 mAb vaccine treatment. The comparative analyses of the responses indicated that there were not differences between the two levels of dose tested in relation to toxic ity and immunogenicity. No evidences of serious or unexpected effects were observed. The results of this clinical trial showed that 1E10 anti-id vaccine was well tolerated and immunologically active, capable of inducing a specific immune response against 1E10 and NeuGc-GM3 ganglioside in most breast cancer patients. [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]