Multiple B Cell Clones Producing Antibodies Directed to the Spacer and Disintegrin/TSR1 Domains of ADAMTS13 in a Patient with Acquired TTP.

Thrombotic thrombocytopenic purpura (TTP) is associated with a severe deficiency of the von Willebrand factor-cleaving protease ADAMTS13. In plasma of the majority of patients with acquired TTP, antibodies that inhibit ADAMTS13 activity have been detected. In this study we have used phage display to isolate anti-ADAMTS13 antibodies from the total immunoglobulin repertoire of a patient with acquired TTP. The immunoglobulin G variable heavy chain (V_H) gene repertoire was amplified from CD19 positive B cells and combined with a variable light chain (V_L) gene repertoire. The resulting library consisted of 3.4 x 10⁸ individual clones. Combined V_H and V_L segments, expressed as single-chain Fv fragments (scFv) on the surface of filamentous phage, were selected for binding to an ADAMTS13 fragment consisting of the disintegrin/ first thrombospondin type 1 repeat (TSR1)/ cysteine-rich/ spacer domains. After three rounds of selection, six different scFv antibody clones were identified that were assigned to four groups based on the use of VH germline gene segments. ScFv 9, 10, and 27, all use the V_H1–69 germline gene segments, possess the same CDR3, and show a similar pattern of somatic hypermutation. These results suggest that scFv 9, 10 and 27 are derived from a common B cell precursor. ScFv 26 also belongs to the V_H1 family, but uses germline gene segment V_H1–02 instead. Clones 16 and 41 are both part of the V_H3 family and are derived from germline gene segments V_H3–07 and V_H3–09 respectively. The affinity of the scFv for the ADAMTS13 disintegrin/ TSR1/ cysteine-rich/ spacer fragment was determined by surface plasmon resonance analysis. Purified disintegrin/ TSR1/ cysteine-rich/ spacer fragment was immobilized on an activated CM5-sensor chip and subsequently different concentrations of purified scFv were added. Binding and dissociation of scFv was followed for 2 minutes and the affinity of the different scFv for the immobilized ADAMTS13 fragment was calculated from the obtained binding curves. ScFv 9 binds with high affinity to the disintegrin/ TSR1/ cysteine-rich/ spacer fragment (K_d 3.6 ± 0.8 nM); whereas scFv 10, 16, 26, 27 and 41 displayed a somewhat lower affinity (K_d ranging from 20–200 nM). Epitope mapping using several smaller ADAMTS13 fragments in the disintegrin/ TSR1/ cysteine-rich/ spacer region revealed that scFv 9, 10, and 41, bind specifically to the ADAMTS13 spacer domain. The epitope of scFv 16 however, resides in the disintegrin/ TSR1 domains. Our results indicate that multiple B cell clones that produce antibodies recognizing epitopes in the spacer and disintegrin/ TSR1 domains of ADAMTS13 are present in the immunoglobulin repertoire of a patient with acquired TTP.