Scavenger receptor cysteine-rich (SRCR) domains are evolutionally conserved modules that display complex structures stabilized by key amino acids, while some other residues have evolved with a relative independence, thus allowing the functional diversity of these receptors. CD6, a highly glycosylated membrane protein predominantly expressed on lymphocytes, contains three SRCR domains. The lack of CD6 domain crystal structure has limited the characterization of the binding sites for the interacting molecules. The interaction between CD6 and its ligand, activated leukocyte-cell adhesion molecule (ALCAM)/CD166, through the membrane-proximal SRCR3 domain, has low affinity and involves conserved sites in both molecules mediating a cross-species binding. The CD6-ALCAM interaction has been involved in cell adhesion, maturation, regulation of activation, and survival processes, suggesting the potential relevance of this target for therapeutic interventions. Several anti-CD6 monoclonal antibodies (MAb) have been described but their affinity and epitope definition remain unclear. We found the murine and humanized T1 MAb versions have similar CD6 recognition profiles and affinity constants of about 6 x 10(8). These antibodies do not block the CD6-ALCAM interaction and recognize a conformational epitope independent of the CD6 N-glycosylation. This epitope was additionally found in the chimpanzee and contains an RXE/Q consensus motif located in the membrane-distal SRCR1. These results, together with the therapeutic evidence previously obtained with these MAbs, suggest a differential contribution of CD6 domains to lymphocyte biology. Potential mechanisms for T1 MAb therapeutic effect different from CD6-CD166 interaction blocking would be dissected.
"In addition, when lymphocytes are activated by PMA or by fetal calf serum, CD6 serine, threonine, and tyrosine (Y) residues are hyperphosphorylated and the MW of the related band increased from 105 to 130 kDa . Comparison of the CD6 SRCR domains between human and mouse shows 54% aa identity for SRCR-D1, 84% for SRCR-D2, and 81% for SRCR-D3 , and 69% for the transmembrane domain . The 244-aa cytoplasmic tail of CD6 harbors nine threonine and 32 serine residues. "
[Show abstract][Hide abstract] ABSTRACT: CD6 is a cell surface receptor expressed on the majority of T cells and a subset of B cells. When expressed, CD6 contributes to lymphocyte activation through its extracellular domain 1, while adhesion and cellular migration are related to the extracellular scavenger receptor cysteine-rich domain (SRCR-D)-3 of CD6. Itolizumab, clone T1h, is a newly developed humanized IgG1 monoclonal antibody that targets CD6 SRCR-D1 and blocks immune activation. Itolizumab has been proposed to be effective in autoimmune diseases such as rheumatoid arthritis, Sjögren's syndrome and multiple sclerosis. In Sjögren's syndrome, the utilization of itolizumab as therapeutic option is reinforced by our recent observation that ALCAM, the CD6 ligand, is overexpressed and that CD6-positive T and B cells are detected within salivary glands from Sjögren's syndrome patients. In this study, itolizumab-positive target cells were characterized within both peripheral blood and salivary glands in order to provide rational for anti-CD6 treatment in Sjögren's syndrome.
Immunologic Research 04/2013; 56(2-3). DOI:10.1007/s12026-013-8423-x · 3.10 Impact Factor
"In this regard, the clinical use of humanized mAbs has revealed a striking absence of adverse reactions. Based on these evidences, together with our previous finding that itolizumab exhibits the same CD6 recognition profile and a similar affinity constant, but was less immunogenic in monkeys than its murine or chimeric counterpart  , we expected itolizumab be less immunogenic and toxic than its predecessors, leading to additional benefits in RA patients. "
[Show abstract][Hide abstract] ABSTRACT: T cells are involved in the pathogenesis of rheumatoid arthritis (RA). CD6 is a co-stimulatory molecule, predominantly expressed on lymphocytes, that has been linked to autoreactive responses. The purpose of this study was to evaluate the safety, immunogenicity and preliminary efficacy of itolizumab, a humanized anti-CD6 monoclonal antibody, in patients with active rheumatoid arthritis. Fifteen patients were enrolled in a phase I, open-label, dose-finding study. Five cohorts of patients received a weekly antibody monotherapy with a dose-range from 0.1 to 0.8 mg/kg. Itolizumab showed a good safety profile, with no severe or serious adverse events reported so far. No signs or symptoms associated with immunosuppression were observed in the study. Objective clinical responses were achieved in more than 80% of patients after treatment completion, and these responses tend to be sustained afterwards. This clinical study constitutes the first evidence of the safety and positive clinical effect of a monotherapy using an anti-CD6 antibody in patients with rheumatoid arthritis.
Results in Immunology 12/2012; 2:204-211. DOI:10.1016/j.rinim.2012.11.001
"While 2H1 scFv binding was totally abolished by antigen reduction, Bevacizumab recognizes both reduced and native VEGF. Phage-mimotope identification in combination with computational tools has been successfully applied by other authors (Alonso et al., 2008; Enshell-Seijffers et al., 2003; Tarnovitski et al., 2006) to locate epitopes on antigens, but these predictions should be further confirmed. Additional studies, such as site-directed mutagenesis of the antigen, are currently undergoing in order to determine the precise nature of the 2H1 scFv epitope. "
[Show abstract][Hide abstract] ABSTRACT: Following the clinical success of Bevacizumab, a humanized monoclonal antibody that blocks the interaction between vascular endothelial growth factor (VEGF) and its receptors, the search for new neutralizing antibodies targeting this molecule has continued until now. We used a human VEGF variant containing three mutations in the region recognized by Bevacizumab to direct antibody selection towards recognition of other epitopes. A total of seven phage-displayed antibody fragments with diverse binding properties in terms of inter-species cross-reactivity and sensitivity to chemical modifications of the antigen were obtained from a human phage display library. All of them were able to recognize not only the selector mutated antigen, but also native VEGF. One of these phage-displayed antibody fragments, denominated 2H1, was shown to compete with the VEGF receptor 2 for VEGF binding. Purified soluble 2H1 inhibited in a dose dependent manner the ligand-receptor interaction and abolished VEGF-dependent proliferation of human umbilical vein endothelial cells. Our epitope disturbing strategy based on a triple mutant target antigen was successful to focus selection on epitopes different from a known one. Similar approaches could be used to direct phage isolation towards the desired specificity in other antigenic systems.
Journal of Biotechnology 01/2011; 151(2):166-74. DOI:10.1016/j.jbiotec.2010.12.007 · 2.87 Impact Factor
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