Construction and characterization of a recombinant murine monoclonal antibody directed against human fibrin fragment‐D dimer

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Abstract
CDNA libraries in λ phage were generated from the murine hybridoma secreting mAb‐15C5, a monoclonal antibody directed against fragment‐D dimer of crosslinked human fibrin [Holvoet et al. (1989) Thromb. Haemo‐stasis 61, 307–313], and clones encoding fragments of the heavy (γ1) and the light (k) chain were isolated. The κ‐chain cDNA was reconstructed from two overlapping clones encoding 20 amino acids of signal sequence and the 214 amino acids of the mature protein chain. The γ1‐chain cDNA was reconstructed from the mAb‐15C5 κ‐chain signal sequence, the mAb‐15C5 γ1 variable‐domain coding sequence and murine γ1‐gene and γ1‐chain cDNA fragments encoding the constant domains. These cDNAs were expressed in Chinese hamster ovary cells, selected cell lines were scaled up in roller bottle culture, and recombinant mAb‐15C5 was purified from the conditoned medium by chromatography on Zn‐chelate–Sepharose, protein‐A–Sepharose and insolubilized fragment‐D dimer, with a yield of 50 μg/1 and a recovery of 20%. SDS‐gel electrophoresis without reduction revealed a homogeneous band, and after reduction a light‐chain band with identical and a heavy‐chain band with a somewhat slower mobility than that of the natural mAb‐15C5. Competitive binding revealed a comparable affinity of natural and recombinant mAb‐15C5, for fibrin fragment‐D dimer. Thus recombinant mAb‐15C5, obtained by co‐expression of the reconstructed cDNAs of the k and γ1 chain in Chinese hamster ovary cells, has very similar properties to natural mAb‐15C5. These recombinant mAb‐15C5 cDNAs may be useful for the construction of a humanized monoclonal antibody for thrombus imaging, and for targeting of thrombolytic agents to fibrin.