No abstract

Summary A humanised IgGl/k version of A33 (hA33) has been constructed and expressed with yields up to 700 mg 1' in mouse myeloma NS0 cells in suspension culture. The equilibrium dissociation constant of hA33 (KD= 1.3 al., 1990, 1994). A phase I/II study has been conducted (Welt et al., 1994) with this murine antibody, in which some tumour responses were observed at the maximum tolerated dose (75 mCi m-2). The major limiting toxicity was haematological, as observed in almost all therapy studies with radioimmunoconjugates. All patients treated developed a human anti-mouse antibody (HAMA) response after one administration, and this led to very rapid clearance of the conjugate upon retreatment, consistent with all previous results with rodent antibodies. These data suggest that A33 is a promising antibody for successful radioimmunotherapy of colon cancer, and the purpose of this study has been to design and develop a second generation reagent based upon it. The key to the development of successful radioimmunotherapy will be the identification of reagents capable of delivering a killing dose to tumour cells without unacceptable toxicity to normal tissues. To this end we are evaluating several alternative radioimmunotherapeutic strategies including the use of isotopes which require internalisation into the cell for cytotoxicity, such as 125I (which are less toxic to normal tissues), and engineering the antibody for the optimal delivery of highly cytotoxic agents such as 90Y.The radioisotope 9'Y has been used in several radioimmunotherapy studies and is an attractive isotope for this purpose owing to its appropriate physical properties. As a pure high-energy P-emitter 9Y has advantages over the more

Immunization with dendritic cells (DCs) transfected with genes encoding tumor-associated antigens (TAAs) is a highly promising approach to cancer immunotherapy. We have developed a system, using complexes of plasmid DNA expression constructs with the cationic peptide CL22, that transfects human monocyte-derived DCs much more efficiently than alternative nonviral agents. After CL22 transfection, DCs expressing antigens stimulated autologous T cells in vitro and elicited primary immune responses in syngeneic mice, in an antigen-specific manner. Injection of CL22-transfected DCs expressing a TAA, but not DCs pulsed with a TAA-derived peptide, protected mice from lethal challenge with tumor cells in an aggressive model of melanoma. The CL22 system is a fast and efficient alternative to viral vectors for engineering DCs for use in immunotherapy and research.

Summary The monoclonal anti-CEA antibody, A5B7, has previously been administered to patients for radioimmunotherapy (RIT). Long circulation time and the formation of an immune response have limited therapeutic success in the clinic. Antibody fragments can be used to reduce the in vivo circulation time, but the best combination of fragment and radioisotope to use for therapy is far from clear. In this study we have compared the biodistribution of A5B7 IgG and F(ab')2 with chemically cross-linked divalent (DFM) and trivalent (TFM) A5B7 Fab' fragments in nude mice bearing human colorectal tumour xenografts. The crosslinkers were designed to allow site-specific labelling using yttrium 90 (90Y), a high-energy f-emitter. We have also compared the above antibody forms conjugated to both 13'I and 9'Y. Both DFM and TFM were fully immunoreactive and remained intact after radiolabelling and incubation in serum at 37°C for 24 h. Biodistribution results showed similar tumour uptake levels and an identical blood clearance pattern for F(ab')2 and DFM with high tumour-blood ratios generated in each case. However, unacceptably high kidney accumulation for both F(ab')2 and DFM and elevated splenic uptake of DFM labelled with 9'Y was observed.Kinetic analysis of antigen binding revealed that DFM had the fastest association rate (kass = 1.6 x 105 Ms-1) of the antibody forms, perhaps owing to increased flexibility of the cross-linker. This advantage implies that DFM may be more suitable than F(ab')2 radiolabelled with 13'I for RIT. TFM cleared from the blood significantly faster than A5B7 IgG when labelled with both 13'I and 9'Y, producing an improved therapeutic tumour-blood ratio. Kidney accumulation was not observed for [90Y]TFM, but a slightly higher splenic uptake was observed that may indicate reticuloendothelial system (RES) uptake. Overall, tumour uptake was higher for 90Y-labelled antibodies than for '3'I-labelled antibodies. Because of the faster clearance, it should be possible to administer a higher total dose of 90Y-labelled TFM than IgG, which is attractive for RIT. Both A5B7 DFM and TFM, therefore, show favourable properties compared with their parent antibody forms.

Attachment of poly(ethylene glycol) (PEG) to proteins can greatly alter their pharmacological properties, including extending the plasma half-life and reducing immunogenicity, both of which are potentially beneficial to tumour targeting. IgG, F(ab')2 and Fab' fragments of the anti-CEA antibody A5B7 were chemically modified with PEG (M(r) 5,000), labelled with 125I and their pharmacokinetics compared with the unmodified forms in the LS174T colonic xenograft in nude mice. PEG modification of the intact antibody had little effect on biodistribution, although tumour localisation was slightly reduced. In contrast, similar modification of F(ab')2 and Fab'A5B7 significantly prolonged plasma half-life and increased radioantibody accumulation in the tumour and to a lesser extent in normal tissues, but reduced tissue to blood ratios. Prior to modification, Fab' A5B7 (M(r) 50,000) cleared more rapidly from the circulation than F(ab')2 (M(r) 100,000), but after PEG attachment their biodistributions converged, while the tumour to blood ratios were reduced and resembled that of the intact antibody. The enhanced tumour accumulation, reduced normal tissue to blood ratios and potentially reduced immunogenicity of fragments after PEG attachment may therefore prove superior to either unmodified fragments or intact antibody for antibody-targeted therapy, although the increased plasma half-life may necessitate the use of a clearance mechanism.