TARGETING BCL 1 LYMPHOMA WITH ANTI-IDIOTYPE ANTIBODIES: BIODISTRIBUTION KINETICS OF DIRECTLY LABELED ANTIBODIES AND BISPECIFIC ANTIBODY-TARGETED BIVALENT HAPTENS Corine MANETTI 1 *, Eric ROUVIER 1 , Emmanuel GAUTHEROT 1 , Eric LOUCIF 1 , Jacques BARBET 1 and Jean Marc LE DOUSSAL 2 1 Imaging and Therapeutics Department, Immunotech SA, Marseille, France 2 University of Lausanne Institute of Biochemistry, Epalinges, Switzerland The mouse BCL 1 lymphoma model has been used for evaluating immunotherapy with anti-idiotype (anti-Id) anti- bodies, including Id immunisation, IgG therapy and bispecific (Bs) antibody-targeted cytotoxicity. H ere, we provide quanti- tative data on the targeting of small (25 6 12 mg) intrasplenic BCL 1 tumours, using anti-Id IgG, F(ab8) 2 and anti-Id 3 anti- hapten BsF(ab8) 2 covalently labelled with 125 iodine, as well as noncovalent complexes of BsF(ab8) 2 and 125 I-labelled bivalent hapten. The results are the following: 1) up to 115% of the injected dose per gram (%ID/g) of spleen can be localised in the first hour, corresponding to approximately 600% ID/g of tumour; 2) localisation is specific for cell-surface Id; 3) opti- mal doses can overcome circulating Id; 4) circulating Id markedly increases the catabolism of IgG, thus impairing tumour localisation; 5) bivalent reagents are internalised by the target cells; 6) iodine covalently bound to bivalent antibod- ies [IgG, F(ab8) 2 ] is rapidly (T 1/2 : 6–9 hr) released from the tumour; in contrast, the bivalent hapten is retained for a longer time (T 1/2 : 25 hr); and 7) in the absence of bivalent hapten, the monovalent BsF(ab8) 2 is not rapidly internalised and dissociates from tumour cell-surface Id. Our results suggest that monovalent anti-Id, lacking Fc, can efficiently be targeted to the BCL 1 tumour surface. For radioimmuno- therapy, the intracellular targeting of catabolism-resistant 125 I-labelled bivalent hapten provides optimal tissue selectiv- ity. Int. J. Cancer, 71:1000–1009, 1997. r 1997 Wiley-Liss, Inc. The syngeneic BALB/c B lymphoma BCL 1 , described by Slavin and Strober (1978), causes large splenomegaly followed by leukae- mia. The disease is thus similar to human prolymphocytic chronic leukaemia. BCL 1 cells express on their surface monoclonal lambda IgM and IgD, which may be used as specific tumour markers (Vitetta et al., 1979). The therapy of this lymphoma has met with a certain success using bispecific monoclonal antibodies (BsMAbs) (Brissinck et al., 1991) and immunotoxins (Krolick et al., 1982; Fulton et al., 1988) targeting surface idiotype (sId). Although lymphomas are considered very favourable targets for radioimmu- notherapy, owing to the large array of available MAbs and to their high radiosensitivity, radioimmunoconjugates have not yet been used in the BCL 1 model. In this model, the idiotype (Id) immunoglobulin is expected to be secreted and internalised when used as a target antigen. With anti-Id Abs, tumour localisation may then be impeded by the presence of circulating Id and decreased via catabolism by tumour cells. In human B-cell tumours, impressive clinical response rates have been achieved (Press et al., 1993; Kaminski et al., 1993) with iodine-labelled Abs targeted to noninternalised antigens (CD20 and CD37) (Press, 1995). In contrast, when the target antigen is internalised (CD5, CD19, CD22, CD33 or IgM), the iodine- labelled Abs are catabolized and radioactive peptides are released (Press et al., 1989; Naruki et al., 1990), less favourable clinical results have been obtained (Zimmer et al., 1988; Appelbaum et al., 1992; Juweid et al., 1995). It is thus interesting to study the biodistribution of anti-Id Abs according to their valency and structure. We first studied BCL 1 Id secretions in relation with lymphoma growth, and we evaluated the biodistribution of iodine-labelled bivalent anti-Id Abs bearing or not bearing an Fc portion [IgG and F(ab8) 2 ], and of a monovalent anti-Id reagent, BsF(ab8) 2 . We also tested the biodistribution of a bivalent hapten specifically targeted to BCL 1 cells by anti-Id 3 anti-hapten BsF(ab8) 2 (Affinity Enhance- ment System, AES). The AES has been successfully used in the clinic to image carcinoembryonic antigen-expressing tumours (Le Doussal et al., 1993; Peltier et al., 1993; Chetanneau et al., 1994). The AES has also been shown to increase the retention of radioiodine inside human B lymphoma cells targeted through membrane IgM (Manetti et al., 1995). The results are discussed with respect to the influence of competition by circulating Id and catabolism by normal organs and target tumour cells. MATERIAL AND METHODS Mice Female BALB/c mice, 6–8-week-old, were obtained from IFFA- CREDO (Lyon, France). Tumour cell lines The BALB/c-derived B-cell tumour BCL 1 was kindly provided by Dr. K. Thielemans (Brussels, Belgium). A primary tumour cell bank was constituted with spleen cells from female BALB/c mice injected with BCL 1 cells and harbouring marked splenomegaly. For each experiment, 10 7 cells from this bank were injected i.v. to 3 BALB/c mice. After 2–3 weeks, these mice were sacrificed and their spleen served as a source of fresh tumour cells. For in vivo experiments, BALB/c mice were injected i.v. with 10 6 BCL 1 cells in RPMI-1640 (Axcell, L’Argentie `re, France). For in vitro tests, spleen were teased and red blood cells were lysed in 5 mM EGTA-0.06% saponin (Sigma, St. Louis, MO) for 2 min at 20–25°C. BCL 1 cells were washed and resuspended in 10 mM phosphate buffer, 150 mM NaCl, pH 7.2 (PBS), supplemented with 0.1% BSA and 0.02% NaN 3 . Monoclonal antibodies Clone B1, secreting the mouse IgG 1 anti-Id MAb, and an IgM-secreting BCL 1 hybridoma (Brissinck et al., 1991) were kindly provided by Dr. K. Thielemans. The anti-Id MAb was purified from ascites fluids by affinity chromatography on protein A-sepharose (Pharmacia, Orsay, France). The pentameric BCL 1 IgM was purified from ascites fluid by 50% ammonium sulphate precipitation and size exclusion chromatography on a Superdex 200 (16 3 600 mm) column (Pharmacia). The anti-histamine- succinyl-glycine (anti-HSG) MAb (Morel et al., 1990), a mouse IgG 1 , was supplied as the purified IgG by Immunotech (Marseille, France). F(ab8) 2 fragments were prepared by pepsin (Sigma) digestion (5 for 2 hr at 37°C). Contract grant sponsor: French Ministry of Research and Technology, ‘‘Saut Technologique’’92C 0429. *Correspondence to: Imaging and Therapeutics Department, Immuno- tech SA, 130 Avenue de Lattre de Tassigny, BP 177, 13276 Marseille Cedex 9, France. Fax: 00 33 4 91 17 27 62. Received 5 September 1996; revised 12 December 1996 Int. J. Cancer: 71, 1000–1009 (1997) r 1997 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer