Multiple Mapping of Peptide Sequences Recognized by Various Monoclonal Anti-ADAMTS13 Antibodies with Functional or Nonfunctional Effects On the Catalytic Activity.

Abstract 3182

Poster Board III-119

Fourteen mouse anti-ADAMTS13 monoclonal antibodies (MoAb#1∼#13, A10) were individually analyzed for their precise epitope peptide sequences in each domain using lambda phage surface display system. A phage library expressing random peptide fragments of ADAMTS13 on its surface was constructed, thereby selecting phage clones bound to each MoAbs immobilized on microtiter plates. Binding epitope sequences for eleven MoAbs were defined, although MoAb#3, #4 and #5 were not clarified. Among 11 epitope-determined MoAbs, epitopes were relatively short (6 to 23 amino acids) in MoAb#1, #2, #8, #11, #12 and #13, recognizing metallopretease, disintegrin-like, TSP1-4, TSP1-8, CUB1 and C-terminus domains, respectively. On the other hand, epitopes were relatively long (49 to 72 amino acids) in A10, MoAb#6, #7, #9 and #10, recognizing disintegrin-like, TSP1-2, TSP1-3, TSP1-5 and TSP1-7 domains, respectively. MoAb#1, #2 and A10 demonstrated inhibitory effects on the cleavage activity of ADAMTS13 evaluated by FRETS-VWF73 assay. MoAb#1 recognized Gln159 to Asp166 in the metalloprotease domain, and MoAb#2 and A10 recognized Asn308 to Glu327, Tyr305 to Glu376 in the disintegrin-like domain, respectively. From findings using C-terminal truncated mutants of ADAMTS13, MoAb#3 and #5 were supposed to recognize TSP1-1 and spacer domain, respectively, although only C-terminal tail peptide sequences were selected from both of the screening, suggesting the possibility of intramolecular association between the C-terminal region and TSP1-1/spacer domains. MoAb#4 was supposed to recognize disintegrin-like domain, although we could not obtain any significant ADAMTS13 peptide sequence from the screening. We speculate that these 3 epitope-undetermined MoAbs may recognize complex conformational structure of ADAMTS13. Alternatively, intact peptide structure of ADAMTS13 might not be expressed properly on the phage surface. In conclusion, we defined precise epitope sequences of 11 monoclonal anti-ADAMTS13 antibodies. Three of them, recognizing metalloprotease or disintegrin-like domains inhibited the cleavage activity of ADAMTS13. Analysis of the epitope sequences may elucidate the correlation between the molecular conformation and the catalytic activity.