Yuniel Fernández-Marrero

Crystal structure of an anti-ganglioside antibody, and modelling of the functional mimicry of its NeuGc-GM3 antigen by an anti-idiotypic antibody.

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.

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.

Antiatherosclerotic Effect of an Antibody That Binds to Extracellular Matrix Glycosaminoglycans

Objective—Subendothelial retention of proatherogenic lipoproteins by proteoglycans is critical in atherosclerosis. The aim of this study was to characterize the recognition and antiatherogenic properties of a chimeric monoclonal antibody (mAb) that reacts with sulfated molecules. Methods and Results—chP3R99 mAb recognized sulfated glycosaminoglycans, mainly chondroitin sulfate (CS), by ELISA. This mAb blocked ≈70% of low-density lipoprotein (LDL)–CS association and ≈80% of LDL oxidation in vitro, and when intravenously injected to Sprague-Dawley rats (n=6, 1 mg/animal), it inhibited LDL (4 mg/kg intraperitoneally, 1 hour later) retention and oxidation in the artery wall. Moreover, subcutaneous immunization of New Zealand White rabbits (n=19) with chP3R99 mAb (100 &mgr;g, 3 doses at weekly intervals) prevented Lipofundin-induced atherosclerosis (2 mL/kg, 8 days) with a 22-fold reduction in the intima-media ratio (P<0.01). Histopathologic and ultrastructural studies showed no intimal alterations or slight thickening, with preserved junctions between endothelial cells and scarce collagen fibers and glycosaminoglycans. In addition, immunization with chP3R99 mAb suppressed macrophage infiltration in aorta and preserved redox status. The atheroprotective effect was associated with the induction of anti-CS antibodies in chP3R99-immunized rabbits, capable of blocking CS-LDL binding and LDL oxidation. Conclusion—These results support the use of anti-sulfated glycosaminoglycan antibody–based immunotherapy as a potential tool to prevent atherosclerosis.

Is BOK required for apoptosis induced by endoplasmic reticulum stress

The B-cell lymphoma 2 (BCL-2)-related ovarian killer (BOK) shares sequence homology with the proapoptotic BCL-2 family members BAX and BAK. However, Bok −/− cells are not protected from classic apoptotic triggers and evidence for a proapoptotic role of BOK is derived mostly from overexpression studies (1). BOK localizes preferentially to the endoplasmic reticulum (ER) membrane, where it interacts with IP3-receptors (2, 3). Using cells from their newly generated Bok −/− mouse strain, Carpio et al. propose that BOK is a critical inducer of BAX/BAK-dependent apoptosis in response to ER stress (4). This proposal is in contrast to our earlier report, in which we showed that loss of BOK did not confer resistance toward ER stress in several cell types (2). Underlying reasons for this discrepancy may lie in the initial Sv129:C57BL/6 mixed genetic background of the strain used by Carpio et al. (4) [which may influence the phenotype despite backcrossing (5)] and their targeting strategy of the Bok locus. By targeting exons 2 (containing the START codon) and 3, alternative splicing of exon 1–4 is enabled and is indeed readily detectable based on the RT-PCR analysis in figure 1C of Carpio et al. (4). A resulting ∼1-kb transcript (exon1/4/5), which is not occurring naturally, contains several predicted ORFs and may influence the phenotype of these mice. In contrast, our Bok −/− strain was generated in a pure C57BL/6 genetic background, with no detectable transcript because of targeting of the exon 1 splice donor site along with exon 2 (1).

A shift from N-glycolyl- to N-acetyl-sialic acid in the GM3 ganglioside impairs tumor development in mouse lymphocytic leukemia cells.

Humans, in contrast to other mammals, do not synthesize N-glycolyl-neuraminic acid (Neu5Gc) due to a deletion in the gene (cmah) encoding the enzyme responsible for this conversion, the cytidine monophospho-N-acetyl-neuraminic acid hydroxylase (CMP-Neu5Ac hydroxylase). The detection of considerable amounts of Neu5Gc-sialoconjugates, in particular gangliosides, in human malignancies makes these antigens attractive targets for immunotherapy, in particular with monoclonal antibodies (mAbs). We have previously described a GM3(Neu5Gc) ganglioside-specific mAb, named 14F7, with the ability to kill tumor cells in a complement-independent manner. Silencing the cmah gene in GM3(Neu5Gc)-expressing L1210 mouse lymphocytic leukemia B cells caused the abrogation of this cytotoxic effect. We now show that cmah-silenced L1210 cells (cmah-kd) express a high level of GM3(Neu5Ac) and have an impaired ability for anchorage-independent cell growth and tumor development in vivo. No evidences of increased immunogenicity of the cmah-kd cell line were found. These results provide new evidences on the role of GM3(Neu5Gc), or Neu5Gc-sialoconjugates in general, in tumor biology. As an important tool in this study, we used the humanized version (here referred to as 7C1 mAb) of a recently described, rationally-designed mutant of 14F7 mAb that is able to bind to both GM3(Neu5Gc) and GM3(Neu5Ac). In contrast to its parental antibody, the humanized 14F7 (14F7hT) mAb, 7C1 mAb was able to kill not only GM3(Neu5Gc)-expressing L1210 wild type cells, but also GM3(Neu5Ac)-expressing cmah-kd cells, which endorses this antibody as a potential agent for cancer immunotherapy.

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.

The membrane activity of BOK involves formation of large, stable toroidal pores and is promoted by cBID

The BCL‐2 family members are key regulators of the intrinsic apoptotic pathway, which is defined by permeabilization of the mitochondrial outer membrane by members of the BAX‐like subfamily. BOK is classified as a BAX‐like protein; however, its (patho‐)physiological role remains largely unclear. We therefore assessed the membrane permeabilization potential of C‐terminally truncated recombinant BOK, BOK∆C. We show that BOK∆C can permeabilize liposomes mimicking the composition of mitochondrial outer membrane, but not of endoplasmic reticulum, forming large and stable pores over time. Importantly, pore formation was enhanced by the presence of cBID and refractory to the addition of antiapoptotic BCL‐XL. However, isolated mitochondria from Bax−/−Bak−/− cells were resistant to BOK‐induced cytochrome c release, even in the presence of cBID. Taken together, we show that BOK∆C can permeabilize liposomes, and cooperate with cBID, but its role in directly mediating mitochondrial permeabilization is unclear and may underlie a yet to be determined negative regulation.

Brain regeneration in Drosophila involves comparison of neuronal fitness.

Summary Darwinian-like cell selection has been studied during development and cancer [1–11]. Cell selection is often mediated by direct intercellular comparison of cell fitness, using “fitness fingerprints” [12–14]. In Drosophila, cells compare their fitness via several isoforms of the transmembrane protein Flower [12, 13]. Here, we studied the role of intercellular fitness comparisons during regeneration. Regeneration-competent organisms are traditionally injured by amputation [15, 16], whereas in clinically relevant injuries such as local ischemia or traumatic injury, damaged tissue remains within the organ [17–19]. We reasoned that “Darwinian” interactions between old and newly formed tissues may be important in the elimination of damaged cells. We used a model of adult brain regeneration in Drosophila in which mechanical puncture activates regenerative neurogenesis based on damage-responsive stem cells [20]. We found that apoptosis after brain injury occurs in damage-exposed tissue located adjacent to zones of de novo neurogenesis. Injury-affected neurons start to express isoforms of the Flower cell fitness indicator protein not found on intact neurons. We show that this change in the neuronal fitness fingerprint is required to recognize and eliminate such neurons. Moreover, apoptosis is inhibited if all neurons express “low-fitness” markers, showing that the availability of new and healthy cells drives tissue replacement. In summary, we found that elimination of impaired tissue during brain regeneration requires comparison of neuronal fitness and that tissue replacement after brain damage is coordinated by injury-modulated fitness fingerprints. Intercellular fitness comparisons between old and newly formed tissues could be a general mechanism of regenerative tissue replacement.

Survival control of malignant lymphocytes by anti-apoptotic MCL-1

Programmed apoptotic cell death is critical to maintain tissue homeostasis and cellular integrity in the lymphatic system. Accordingly, the evasion of apoptosis is a critical milestone for the transformation of lymphocytes on their way to becoming overt lymphomas. The anti-apoptotic BCL-2 family proteins are pivotal regulators of the mitochondrial apoptotic pathway and genetic aberrations in these genes are associated with lymphomagenesis and chemotherapeutic resistance. Pharmacological targeting of BCL-2 is highly effective in certain indolent B-cell lymphomas; however, recent evidence highlights a critical role for the BCL-2 family member MCL-1 in several lymphoma subtypes. MCL-1 is recurrently highly expressed in various kinds of cancer including non-Hodgkin’s lymphoma of B- and T-cell origin. Moreover, both indolent and aggressive forms of lymphoma require MCL-1 for lymphomagenesis and for their continued survival. This review summarizes the role of MCL-1 in B- and T-cell lymphoma and discusses its potential as a therapeutic target.

Lonely killers: Effector cell- and complement-independent non-proapoptotic cytotoxic antibodies inducing membrane lesions

The majority of the most effective monoclonal antibodies (mAbs) currently in the clinics bind to cancer or immune cells. Classic mechanisms of cell killing by therapeutic mAbs include antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity and induction of apoptosis by engagement of specific cell ligands. A few reports have described mAbs whose cytotoxic activity is Fc-independent and that do not induce the morphological and biochemical changes associated with the apoptosis-type of cell death. Even fewer works describe mAbs able to directly induce membrane lesions. Here, we discuss the available data on those molecules and their cell killing activity, with particular attention to the case of a mAb specific for the tumor-associated N-glycolyl (Neu5Gc)-GM3 ganglioside (GM3(Neu5Gc)). Some similarities are found in the cell death pathways triggered by these mAbs, but data are not abundant. We conclude that the usefulness of mAbs with a direct cytotoxic activity for immunotherapeutic strategies deserves deeper research.