We present a system for cancer targeting based on single-chain Fv (scFv) antibodies selected from combinatorial libraries, produced in bacteria and purified by using an engineered tag. Combinatorial libraries of scFv genes contain great diversity, and scFv antibodies with characteristics optimized for a particular task can be selected from them using filamentous bacteriophage. We illustrate the benefits of this system by imaging patients with carcinoembryonic antigen (CEA)-producing cancers using an iodine-123 labeled scFv anti-CEA selected for high affinity. All known tumor deposits were located, and advantages over current imaging technology are illustrated. ScFvs are produced in a cloned form and can be readily engineered to have localizing and therapeutic functions that will be applicable in cancer and other diseases.

No abstract

(Green et al., 1990). We have used this method to compare two antibody preparations in the clinical development of RIT. RIT had produced good response rates in radiosensitive tumours such as lymphoma when large amounts of radionuclide are given (Kaminski et al., 1993; Press et al., 1993). However, common epitheial tumours such as colorectal and breast carcinoma have not yet been treated so successfully because of their greater radioresistance, which diminishes the therpeutic ratio. In spite of this, responses to therapy have been reported , and it is likely that moderate increases in efficency in delivery of antibodymediated delivery of radiation could establish radioimmunotherapy as a useful form of therapy for metastatic colorectal carcinoma.Intact IgG antibodies with a molcular weight (MW) of 150 kilodaltons (kDa) may not penetrate well from blood through endothelium and extravascular tissues to the tumour (Yokota et al., 1992). It is proposed that F(ab% antibodies (MW 100 kDa) will achieve more effective penetration because of their smaler molcular size and that this will significantly improve the prospects for effective radioimmunotherapy of colorectal cancer.

MFE-23 is the first single-chain Fv antibody molecule to be used in patients and is used to target colorectal cancer through its high affinity for carcinoembryonic antigen (CEA), a cell-surface member of the immunoglobulin superfamily. MFE-23 contains an N-terminal variable heavy-chain domain joined by a (Gly(4)Ser)(3) linker to a variable light-chain (V(L)) domain (kappa chain) with an 11-residue C-terminal Myc-tag. Its crystal structure was determined at 2.4 A resolution by molecular replacement with an R(cryst) of 19.0%. Five of the six antigen-binding loops, L1, L2, L3, H1 and H2, conformed to known canonical structures. The sixth loop, H3, displayed a unique structure, with a beta-hairpin loop and a bifurcated apex characterized by a buried Thr residue. In the crystal lattice, two MFE-23 molecules were associated back-to-back in a manner not seen before. The antigen-binding site displayed a large acidic region located mainly within the H2 loop and a large hydrophobic region within the H3 loop. Even though this structure is unliganded within the crystal, there is an unusually large region of contact between the H1, H2 and H3 loops and the beta-sheet of the V(L) domain of an adjacent molecule (strands DEBA) as a result of intermolecular packing. These interactions exhibited remarkably high surface and electrostatic complementarity. Of seven MFE-23 residues predicted to make contact with antigen, five participated in these lattice contacts, and this model for antigen binding is consistent with previously reported site-specific mutagenesis of MFE-23 and its effect on CEA binding.