Structural Basis of GD2 Ganglioside and Mimetic Peptide Recognition by 14G2a Antibody

Monoclonal antibodies targeting GD2 ganglioside (GD2) have recently been approved for the treatment of high risk neuroblastoma and are extensively evaluated in clinics in other indications. This study illustrates how a therapeutic antibody distinguishes between different types of gangliosides present on normal and cancer cells and informs how synthetic peptides can imitate ganglioside in its binding to the antibody. Using high resolution crystal structures we demonstrate that the ganglioside recognition by a model antibody (14G2a) is based primarily on an extended network of direct and water molecule mediated hydrogen bonds. Comparison of the GD2-Fab structure with that of a ligand free antibody reveals an induced fit mechanism of ligand binding. These conclusions are validated by directed mutagenesis and allowed structure guided generation of antibody variant with improved affinity toward GD2. Contrary to the carbohydrate, both evaluated mimetic peptides utilize a “key and lock” interaction mechanism complementing the surface of the antibody binding groove exactly as found in the empty structure. The interaction of both peptides with the Fab relies considerably on hydrophobic contacts however, the detailed connections differ significantly between the peptides. As such, the evaluated peptide carbohydrate mimicry is defined primarily in a functional and not in structural manner.Malignant transformation is universally accompanied by changes in cell surface glycosylation. A glycolipid, GD2 ganglioside (GD2)¹, is one of the most prominent tumor-associated antigens, ranking in the 12th position of the NCI prioritized list of cancer vaccine targets (1). GD2 is embedded in the outer plasma membrane with its ceramide tail (fatty acid coupled sphingosine). The sugar moiety is exposed to the extracellular milieu and is composed of glucose (Glc; linked to ceramide), galactose (Gal) and N-acetylgalactosamine (GalNAc). Two additional sialic acid residues (N-acetylneuraminic acid, NeuAc) branch form Gal and provide GD2 with a negative charge (Fig. 1). Overexpression of GD2 is well documented in neuroblastoma, melanoma, certain osteosarcomas, small cell lung cancers, and soft tissue sarcomas (2–4).Open in a separate windowFig. 1.Recognition of GD2 ganglioside by monoclonal antibody 14G2a at the cell surface. (top panel) Antigen combining region of 14G2a antibody recognizes the sugar moiety of GD2 ganglioside (yellow), which is exposed to the extracellular milieu. The lipid part of the ganglioside is buried inside the cell membrane. GD2 bound Fab structure determined in this study is shown in color. Fc fragment (PDB ID: 1igt) and membrane model derived from published data are shown in corresponding scale and colored gray. (bottom panel) Chemical structure of GD2 ganglioside and sugar ring nomenclature used throughout the study.The concept of therapeutic targeting of GD2 is currently most advanced in neuroblastoma, the most common extracranial tumor of childhood. Neuroblastoma is a heterogenous and complex disease. Spontaneous remissions are sometimes observed, but more than a half of the patients are diagnosed with a high-risk neuroblastoma of poor prognosis. This highlights the demand for treatment modalities that would offer major clinical benefits for this group of patients (5). High and stable presence of GD2 on cancer cells in neuroblastoma and limited expression on relevant normal tissues (i.e. neurons, peripheral nerve fibers and skin melanocytes) allows diagnosis, detection of metastases, treatment monitoring and, most importantly, targeting of the tumor itself.GD2-specific monoclonal antibodies have been extensively tested in clinics. This includes a mouse 14G2a antibody (IgG2a; derived from a mouse 14.18 antibody of IgG3 subclass), and improved modifications thereof including a chimeric antibody ch14.18, and recently a humanized antibody hu14.18K322A. Moreover, mouse 3F8 antibody (IgG3) and recently its humanized derivative hu3F8 were also evaluated. The antibodies were demonstrated to engage antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against neuroblastoma (5). Additionally, direct cytotoxic effects were observed in neuroblastoma models (6). The results of a randomized clinical trial published in 2010, evaluating ch14.18, interleukin-2 and granulocyte and macrophage-colony stimulating factor combined with a standard maintenance agent 13-cis retinoic acid demonstrated significant improvement of outcome in high-risk neuroblastoma patients (7). Based on these and further findings, the Food and Drug Administration (FDA) has just recently approved Unituxin (dinutuximab; ch14.18) combination therapy for high risk neuroblastoma (8). Therefore, the standard care treatment protocols may now be extended with monoclonal antibodies targeting GD2 for a better expected outcome.Antibodies against gangliosides other than GD2 are considered as potential therapeutic agents in different types of cancer. Ganglioside-specific antibodies are moreover involved in various types of autoimmune diseases (9). Nevertheless, the molecular mechanism of ganglioside recognition remains unknown because not a single crystal structure of antibody–ganglioside complex has been determined to date. In particular, it is not known how the specificity against GD2 is achieved in antibodies evaluated in clinics. Although crystal structures of empty ME36.1 antibody specific for GD2 and GD3 (10) and empty 3F8 antibody specific for GD2 (11) were determined, the conclusions concerning GD2 binding have to be treated with caution because of general limitations in reliable prediction of binding modes of complex, flexible ligands in dynamic pockets.The success of GD2-specific antibodies in treatment of neuroblastoma fuels investigation on active immunization strategies. To overcome poor antigenicity of GD2, glycolipid surrogates including peptide mimetics are being developed. The idea of a peptide vaccine eliciting anticarbohydrate response has been precedented in the case of Group B Streptococcus polysaccharide (12). Multiple peptides mimicking GD2 in its binding to specific antibodies were selected using phage display (13, 14) and some have been demonstrated to elicit protective, GD2 directed response in preclinical studies. However, the structural basis of peptide-ganglioside mimicry and its relation to the potential of particular peptides to induce GD2 directed immune response remain unknown.Here, we analyze the interactions guiding ganglioside recognition by an antibody and the structural basis of peptide-ganglioside mimicry. The crystal structure of Fab fragment of 14G2a antibody in a complex with the sugar moiety of GD2 ganglioside is provided and the binding mode is discussed in detail. Structure of an empty 14G2a antibody is reported for reference. The major conclusions are verified by directed mutagenesis and antibody variant with increased affinity toward GD2 is developed using structure guided approach. The binding modes of two largely divergent peptide mimics of GD2 (15) at the antigen-binding site of 14G2a antibody are reported and compared with that of the carbohydrate. Mouse 14G2a antibody was chosen for this study because it contains the same antigen binding region as the ch14.18 chimeric antibody recently approved by FDA (8).