Towards the Definition of a Chimpanzee and Human Conserved CD6 Domain 1 Epitope Recognized by T1 Monoclonal Antibody

Abstract

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.

The CD6 Multiple Sclerosis Susceptibility Allele Is Associated with Alterations in CD4+ T Cell Proliferation

“…It has been previously shown that the CD6 ligand, ALCAM, is induced on CD4 + T cells after activation, and blocking this interaction with inhibitory anti-CD6 mAb inhibits sustained entry into cell cycle (12,(25)(26)(27)(28)(29)(30). Thus, we activated highly purified CD4 + T cells from donors with the rs17824933 CC , rs17824933 CG , or rs17824933…”

Immunological and histological evaluation of clinical samples from psoriasis patients treated with anti-CD6 itolizumab

“…Although a full pharmacokinetic study was not performed, circulating itolizumab was detected by ELISA using as antigen CD6 SRCR1, the domain recognized by this antibody. 26 Results are shown in Figure S2. In spite of the higher dose administered during the maintenance phase in group A, the amount of itolizumab in sera from most of the patients appeared to be roughly the same at AI and AM weeks, with only slightly increased values at the latter point, at least as determined by the absorbance measurements.…”

“…26 Itolizumab did not inhibit soluble ALCAM binding to CD6-expressing HEK293 cells 26 and did not cause T cell depletion in rheumatoid arthritistreated patients. 27 In experiments in vitro with peripheral blood mononuclear cells (PBMC) from healthy donors, itolizumab inhibited proliferation in the presence of soluble ALCAM and IL-2; downregulated the phosphorylation of intracellular proteins involved in the CD6-mediated activation pathway; and reduced IFN-γ, IL-6 and TNF production.…”

Immunological evaluation of rheumatoid arthritis patients treated with itolizumab

“…This antibody recognizes the membrane-distal domain 1 of CD6 and was not able to inhibit soluble ALCAM binding to CD6-expressing HEK-293 cells. 32 Moreover, itolizumab did not cause T cell depletion when used as monotherapy in RA patients. 33 In experiments in vitro with peripheral blood mononuclear cells (PBMC) from healthy donors, itolizumab reduced IFN-g, IL-6 and TNF production.…”