Single-domain antibodies (sdAbs), which occur naturally in camelids, are endowed with many characteristics that make them attractive candidates as building blocks to create new antibody-related therapeutic molecules. In this study, we isolated from an immunized llama several high-affinity sdAbs directed against human carcinoembryonic antigen (CEA), a heavily glycosylated tumor-associated molecule expressed in a variety of cancers. These llama sdAbs bind a different epitope from those defined by current murine mAbs, as shown by binding competition experiments using immunofluorescence and surface plasmon resonance. Flow cytometry analysis shows that they bind strongly to CEA-positive tumor cells but show no cross-reaction toward nonspecific cross-reacting antigen, a highly CEArelated molecule expressed on human granulocytes. When injected into mice xenografted with a human CEA-positive tumor, up to 2% of the injected dose of one of these sdAbs was found in the tumor, despite rapid clearance of this 15 kDa protein, demonstrating its high potential as a targeting moiety. The single-domain nature of these new anti-CEA IgG fragments should facilitate the design of new molecules for immunotherapy or diagnosis of CEA-positive tumors. Abbreviations CDR, complementarity determining region; CEA, carcinoembryonic antigen; FITC-GAM, fluorescein isothiocyanate goat anti-mouse Ig; NCA, nonspecific cross-reacting antigen; sCEA, soluble carcinoembryonic antigen; sdAb, single-domain antibody; SPR, surface plasmon resonance.

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This Phase I/II study investigated myeloablative (131)I-rituximab radioimmunotherapy (RIT) and high-dose chemotherapy supported by one or two autologous stem cell transplantations in heavily pretreated patients with relapsed or refractory B cell non-Hodgkin lymphoma. Myeloablative RIT was safe and feasible when followed by autologous stem cell transplantation with low incidence of secondary late effects and could be a reasonable alternative regimen especially in elderly patients and in patients who have concerns about high-dose chemotherapy. Tandem myeloablative (131)I-rituximab RIT and high-dose chemotherapy supported by two autologous stem cell transplantations was also feasible. However, the toxicity was higher than after myeloablative RIT, therefore it might be recommended to restrict the tandem approach to lymphoma with poor prognosis.