Cancer Imrnunol Immunother (1988) 26:237-242 ancer mmunology mmunotherapy © Springer-Verlfig 1988 Scintigraphic detection in mice of inflammatory lesions and tumours by an indium-labelled monoclonal antibody directed against Mac-I antigen* Brigitte Collet 1, Pascal Pellen 1, Anne Martin 1, Annick Moisan 1, Dominique Bourel 2, and Louis Toujas 1 Centre R~gional de Lutte contre le Cancer Pontchaillou, F-35033 Rennes-Cedex, France 2 Centre R~gional de Transfusion Sanguine de Rennes, F-35033 Rennes, France Summary. The monoclonal antibody, 3A33, directed against Mac-1 antigen which is expressed essentially on macrophages and polymorphonuclear cells, was injected i.v. into mice, as part of an attempt to visualize inflamma- tory lesions and tumours by external scintigraphy. The monoclonal antibody, a rat IgG2a, was conjugated with a bifunctional chelating agent, diethylenetriaminepentaac- etic acid at a 1:1 molecular ratio and complexed with 1U-indium, a procedure which apparently did not alter its binding to peritoneal macrophages and provided relatively stable cell labelling. An unrelated rat IgG2a of unknown specificity radiolabelled in the same manner as 3A33 served as a control. The uptake of i.v. injected 3A33 by peritoneal macrophages was up to 50 times that of unrelat- ed IgG2a. After i.v. inoculation, the antibody accumulated in the liver, spleen, lung, in foot-pad inflammatory reac- tions induced by injection of Freund's adjuvant and in experimentally grafted tumours. The 3A33: non-specific IgG2a uptake ratio in inflammatory lesions and tumours, however, was much lower than for peritoneal macro- phages and was generally close to 2. This was sufficient to obtain scintigraphic images of inflammations and tu- mours. The images obtained after injection of 3A33 were clearly of better quality than those given by the non-specif- ic immunoglobulin. They could be improved by subtrac- tion of the vascular images obtained after injection of 99m-technetium serum albumin. The labelling of Mac-l- positive blood mononuclear cells by in vitro incubation with radioactive 3A33 was not intense enough to allow scinti- graphic imaging after in vivo re-infusion but seemed more selective than the injection of whole antibody in detecting inflammatory reactions. These results seem interesting in view of the potential human application to the detection inflammatory lesions and the appreciation of tumour in- flammatory components. Possible improvements in the technique are discussed. Introduction Radiolabelled monoclonal antibodies (MAb) directed against tumour cells and injected i.v. have been found to * This work was supported by the F6d6ration Nationale des Cen- tres de Lutte contre le Cancer and the Association pour la Re- cherche contre le Cancer Offprint requests to: B. Collet accumulate predominantly in cancer lesions and to allow the visualization of tumours by external scintigraphy [8]. Cancerous tissues are often infiltrated with inflammatory cells, especially macrophages [1, 9]. The purpose of this work was to determine whether a MAb directed against cells present in the inflammatory reaction and labelled with a gamma emitter was suitable for the scintigraphic de- tection of tumours. The MAb used, 3A33, was a rat IgG2a prepared by im- munizing a rat against mouse macrophages and shown previously to react with Mac-1 antigen [11]. This antigen, associated with a 95-170 × 103 dalton protein complex which corresponds to the C3bi receptor, is known to be ex- pressed on macrophages, polymorphonuclear and natural killer cells [2]. After in vivo inoculation, MAb 3A33 was shown to bind macrophages and to be internalized into cytoplasmic vacuoles. However, markers such as fluores- cein or 12Liodine attached to the antibody, were rapidly re- leased from the cells [5]. In the present work, MAb 3A33 was conjugated with the bifunctional chelate diethylenetri- aminepentaacetic acid (DTPA) and combined with ULin- dium. This radiomarker has been reported to bind to cells more firmly than iodine [14]. The ability of ill-indium- labelled 3A33 injected i.v. to accumulate in macrophages of the peritoneal cavity and in s. c. inflammations was first in- vestigated, then the imaging of grafted experimental tu- mours was attempted. Materials and methods Radioactivity labelling of MAb 3A33 and control IgG2a. MAb 3A33, a rat IgG2a, was obtained by immunization of a rat against mouse macrophages and by inter-species cell fusion between immune lymphocytes and mouse plasma- cytoma cells. The hybridoma was grown as ascites follow- ing i.p. implantation into nude mice as described previ- ously [11]. Ascitic fluid centrifuged at 10,000 g for 30 min was layered on an anion exchanging column (Mono-Q, FPLC system, Pharmacia, Uppsala, Sweden). Elution was performed with an NaC1 gradient in 0.025 M Tris buffer at pH 7.7, the antibody eluting at a NaC1 concentration of 0.3 M. It was then concentrated to 3-5 mg/ml in 0.15 M NaC1 and conjugated with DTPA [8]. The DTPA anhy- dride, kindly provided by J-C Saccavini (Commissariat/~ l'6nergie atomique, Saclay, France), was dissolved in dimethylsulphoxide and added in the proportion of 5 mol- ecules per molecule of antibody. After 20 min incubation