Résumé
In an attempt to improve tumor targeting and tumor retention time of monoclonal antibodies (MAbs), we prepared biparatopic antibodies (BpAbs) having the capability of binding 2 different non‐overlapping epitopes on the same target antigen molecule, namely, the carcinoembryonic antigen (CEA). Six BpAbs were constructed by coupling 2 different Fab′ fragments from 4 different specific anti‐CEA MAbs recognizing 4 CEA epitopes (Gold 1–4). Demonstration of the double paratopic binding of these antibodies for CEA was confirmed in vitro by inhibition radioimmunoassay and cross‐inhibition analysis by surface plasmon resonance (SPR; BIACORE) technology. Using the latter technique, the affinity constants for CEA immobilized onto the sensor chip were found to range from 0.37 to 1.54 × 109 M−1 for the 4 parental F(ab′)2 fragments and from 1.88 to 10.14 × 109 M−1 for the BpAbs, demonstrating the advantage of biparatopic binding over conventional F(ab′)2 binding. The Ka improvement was particularly high for BpAb F6/35A7 and BpAb F6/B17 with a 9.5‐ and 8.1‐fold increase, respectively, as compared with the parental F(ab′)2. In vivo, the 6 BpAbs were compared with their 2 respective parental F(ab′)2 by injection of 131I‐BpAb/125I‐F(ab′)2 parental fragments into nude mice xenografted with the human colon carcinoma T380. Dissection 72 hr post‐injection demonstrated that BpAb B17/CE25 and BpAb F6/B17 gave higher tumor uptake than that of their parental F(ab′)2. This finding is particularly interesting for BpAb F6/B17, which compared favorably with the F6 F(ab′)2, one of the best parental F(ab′)2 fragments used in our study. Int. J. Cancer 81:285–291, 1999. © 1999 Wiley‐Liss, Inc.