Cristina Mateo de Acosta

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. [Mol Cancer Ther 2008;7(7):2033–41]

Detection and Characterization of N-Glycolyated Gangliosides in Wilms Tumor by Immunohistochemistry

Gangliosides are glycolipids present on the cell surface. The N-glycolylated ganglioside NeuGc-GM3 has been described in some neoplasms, such as breast carcinoma and melanoma, but is usually not detected in normal human cells. Our aim was to evaluate the presence of NeuGc-GM3 in Wilms tumor by immunohistochemistry. Postchemotherapy tumors were grouped into different histologic subtypes considering the main preserved component. Formalin-fixed, paraffin-embedded tumor samples were cut into 5-μm sections. The monoclonal antibody 14F7, a mouse IgG1 that specifically recognizes NeuGc-GM3, and a peroxidase-labeled polymer conjugated to secondary antibodies were used. Sections from breast carcinoma were employed as positive controls. Presence of NeuGc-GM3 was evident in 22 of 25 (88%) cases. The staining was stronger in the epithelial component, with a membrane pattern and cytoplasmic diffusion. The stromal component expressed cytoplasmic NeuGc-GM3 in cells with rhabdomyoblastic differentiation. Tubules of adjacent renal tissue were also positive, but no expression of NeuGc-GM3 was detected in nontumoral fetal kidney. Until now, the expression of N-glycolylated gangliosides in pediatric solid tumors has not been investigated. The present study evidenced the expression of NeuGc-GM3 in a high proportion of Wilms tumors, suggesting its potential utility as a specific target of immunotherapy.

Chimeric anti-N-glycolyl-ganglioside and its anti-idiotypic MAbs: Immunodominance of their variable regions

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.

Switching on cytotoxicity by a single mutation at the heavy chain variable region of an anti-ganglioside antibody.

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.

A cytotoxic humanized anti-ganglioside antibody produced in a murine cell line defective of N-glycolylated-glycoconjugates

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.

Expression and biological characterization of an anti-CD20 biosimilar candidate antibody: a case study.

The CD20 molecule is a non-glycosylated protein expressed mainly on the surface of B lymphocytes. In some pathogenic B cells, it shows an increased expression, thus becoming an attractive target for diagnosis and therapy. Rituximab is a chimeric antibody that specifically recognizes the human CD20 molecule. This antibody is indicated for the treatment of non-Hodgkin lymphomas and autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. In this work, we describe the stable expression and biological evaluation of an anti-CD20 biosimilar antibody. While rituximab is produced in fed-batch culture of recombinant Chinese hamster ovary (CHO) cells, our biosimilar antibody expression process consists of continuous culture of recombinant murine NS0 myeloma cells. The ability of the purified biosimilar antibody to recognize the CD20 molecule on human tumor cell lines, as well as on peripheral blood mononuclear cells from humans and primates, was demonstrated by flow cytometry. The biosimilar antibody induced complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and apoptosis on human cell lines with high expression of CD20. In addition, this antibody depleted CD20-positive B lymphocytes from peripheral blood in monkeys. These results indicate that the biological properties of the biosimilar antibody compare favorably with those of the innovator product, and that it should be evaluated in future clinical trials.

Construction of a Recombinant Non-Mitogenic Anti-Human CD3 Antibody

IOR-T3, a mouse monoclonal antibody specific for human CD3, has been successfully used in the treatment of acute transplant rejection due to its potential as T-cell immunosuppressant. In the present work we report the construction of a human IgG1 chimeric variant of IOR-T3, named T3q. In order to reduce the T-cell activating capacity of the newly obtained chimeric molecule, the two leucine residues at positions 234 and 235 of the CH2 region were replaced by alanines, obtaining the T3q(Ala/Ala) molecule. In vitro evaluation of T3q and T3q(Ala/Ala) showed that there were no differences in the recognition of human CD3 in comparison with murine IOR-T3. However, the Fc-mutated version T3q(Ala/Ala) displayed a much weaker FcgammaR binding capacity than the unmutated chimeric molecule T3q, as well as a reduced ability to induce T-cell proliferation, proinflammatory cytokine release (TNFalpha and IL-6), and early activation surface marker expression (CD25 and CD69). We also found that, unlike treatment with T3q, the reduction in T-cell proliferation was less marked on CD8(+) cells compared to the CD4(+) cells after treatment with T3q(Ala/Ala). These properties make T3q(Ala/Ala) an attractive clinical alternative as an immunoregulatory agent endowed with reduced toxicity.

Immunopotentiating properties of a multispecific α-anti-idiotype antibody

Multispecificity is not a well-understood property of some antibodies. Different functions have been attributed to multispecific natural antibodies, commonly associated with the neutralization and clearance of antigens. Much less is known about the role of antibodies like these, based on their idiotypic connectivity. B7Y33 is a chimeric IgG1 version of a polyreactive α anti-idiotype antibody that is able to interact with different immunoglobulin and non-immunoglobulin antigens. Here we report the capacity of this antibody to enhance the immunogenicity of several autologous IgMs in adjuvant-free conditions. Our results suggest that the formation of immune complexes seems to be necessary, but not sufficient, to this activity. The potential involvement of the interaction of B7Y33 with the FcγRIIb is discussed.