Analysis of human and primate CD2 molecules by protein sequence and epitope mapping with anti-human CD2 antibodies.
A panel of anti-human CD2 monoclonal antibodies (mAb) and soluble human CD58 (LFA-3) were tested for binding to human peripheral blood mononuclear cells (PBMCs), recombinant human CD2 and mononuclear cells from Cynomolgus, Rhesus and African green monkey, Stump-tail, Pig-tail and Assamese macaque, Chimpanzee and Baboon. This analysis revealed that whilst some antibodies recognized all species, there were differential binding profiles with others. Three antibodies, MEDI-507, 6F10.3 and 4B2, recognized CD2 from human and Chimpanzee but not that from the other primates. We have cloned eight of the previously unknown primate CD2 molecules and report here their sequences for the first time. This analysis revealed that 12 amino acids formed a common set of residues in the extra cellular domain of human and Chimpanzee CD2. Using a "knock-in" mutagenesis approach starting with Baboon CD2, which does not bind MEDI-507, 6F10.3 and 4B2, we have identified three residues in the adhesion domain of human CD2 which are critical for its binding to these mAbs. These residues, N18, K55 and T59 define a region located outside of the previously described binding regions on CD2. Affinity measurements of the mutants revealed a variety of degrees of binding restoration for MEDI-507, 6F10.3 and 4B2, indicating that there are fine differences within a given epitope. Furthermore, the analysis of the competition of several of the anti-human CD2 antibodies with each other and CD58 demonstrated the existence of a continuum of overlapping epitopes on human CD2, which is in contrast to the commonly held belief that epitopes on human CD2 are clearly segregated.
Protein Engineering Department, MedImmune Inc., One MedImmune Way, Gaithersburg, MD 20878, USA.
Melissa M
MM
Damschroder
Alexander A
AA
Kozhich
Robert M
RM
Woods
Li
L
Cheng
Brian A
BA
Mullikin
Susan D
SD
Wilson
Nancy D
ND
Ulbrandt
Christine M
CM
Bachy
Herren
H
Wu
JoAnn A
JA
Suzich
Peter A
PA
Kiener
William F
WF
Dall'Acqua
Wendy I
WI
White
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D018801:CD2 Antigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D018968:CD58 Antigens / Q000276:immunology; D018604:Epitope Mapping; D000882:Haplorhini / Q000235:genetics / Q000276:immunology; D006801:Humans; D002679:Pan troglodytes / Q000235:genetics / Q000276:immunology; D020539:Sequence Analysis, Protein; D013601:T-Lymphocytes / Q000276:immunology
D000906:Antibodies; D018801:CD2 Antigens; D018968:CD58 Antigens