British zyxwvutsrqponm Journal of Huemutology, 1995. 90. 572-577 zyxwvutsrq Differential sensitivity of CD30+ neoplastic cells to gelonin delivered by anti-CD3O/anti-gelonin bispecific antibodies SABRINA SFORZINI,’ ANDREA BOLOGNESI,2 RAFPAECLA MEAZZA,1*3 SABRINA MARCIANO,’ PATRIZIA CASALINI, HCJRST DijRKOP,4 PIER LIJIGI TAZZARI,2 HARALD STEIN,4 FIORENZO sTIRPE2 AND SILVANO FERRINll ‘lstituto Nazionale per la Ricerca zyxwvutsrq sul Cancro, Genova, Italy, ’Dipartimento di Patologia Sperimentale, Universith di Bologna, Italy, ‘lstituto di Oncologia dell’Universita di Genova, Italy, 41nstitute of Pathology. Klinikum Steglitz, Free University of Berlin, Germany, and ’lstituto Nazionale Tumori, Milano, Italy Received 21 December 1994; acceptedfor publication 31 March 1995 Summary. Lymphocyte activation antigens, such as CD30, rcpresent suitable target molecules for antibody-driven drug delivery in haemopoietic malignancies. A ribosome- inactivating protein (RIP) type 1 of potential interest for mAb targeting is gelonin, which displays a lower toxicity, as compared to other RIPs. In this study, two anti-CD30/anti- gelonin bispecific monoclonal antibodies (bimAbs), secreted by hybrid hybridomas, were used to deliver this RIP to CD30+ tumour cells. The two bimAbs, termed D4 and A18, were produced using the same anti-CD30 mAb and two anti-gelonin mAbs, directed to unrelated epitopes of the gelonin molecule. These bimAbs enhanced gelonin toxicity (ICs0 5 zyxwvutsrqp x zyxwvutsrq M, in the absence of mAbs) against the CD30+ L540 Hodgkii’s lymphoma cell line in a protein synthesis inhibition assay. Thus, in the presence of M D4 bimAb, protein synthesis was inhibited with an ICso of 5 x 10-l’~ as gelonin, whereas with A18 bimAb the ICs0 was 8 zyxwvu x lo-” M. More interestingly, the combined use of the two bimAbs had a synergistic effect, since the lCso of gelonin reached 6 x 10-I’~. Among CD30 tumour cell lines, the Hodgkin’s lymphoma L428 was also sensitive to gelonin delivered by bimAbs (IC50 zyxw 6 x lo-“ M), whereas the COLE Hodgkin’s cell line and the T-ALI. Jurkat were completely resistant to the toxic effect of gelonin and bimAbs. COLE and Jurkat cells were also resistant to a gelonin/anti-CD30 conventional immunotoxin, whereas they were sensitive to a saporin/anti-CD30 immunotoxin. This suggests that the resistance to gelonin is not related to a lack of internalization through the CD30 molecule but is associated with some property of the RIP. Keywords: CD30, gelonin, bispecific monoclonal antibodies, cytotoxicity, Hodgkin’s lymphoma. The use of monoclonal antibodies (mAbs) directed to surface tumour-associated antigens (TAA) to target tumour cells selectively with cytotoxic agents has been extensively studied (Vitetta et al, 1993). One of the possible approaches is represented by the chemical linkage of a toxic moiety to an antibody to product an ‘immunotoxin’. Ricin A chain has been widely used to prepare immunotoxins, but several single-chain ribosome-inactivating proteins (type 1 RIPS) have been isolated (Barbieri et al, 1987, 1993). These RIPs are easier and safer to purify, display a lower toxicity, and can be used to prepare powerful immunotoxins (Barbieri et al. 1993; Vitetta et al, 1993). Several lymphocyte activation antigens have been regarded as suitable target molecules for antibody-driven Correspondence: Dr Silvano Perrini. Istituto Nazionale per la Ricerca sul Cancro, V. le Benedetto XV 10, 161 32 Genova, Italy. drug delivery in haemopoietic malignancies, because they are not expressed on normal resting lymphocytes or on haemopoietic cells. For example, anti-CD2 5 or anti-CD30 mAbs may be utilized for the targeting against Hodgkin’s lymphomas. anaplastic large cell lymphomas (ALC) and acute T-cell leukaemias. Immunotoxins prepared with anti- CD30 mAbs have been reported to be extremely potent in vitro (Tazzari zyxwv et al, 1992; Engert et al. 1990) and to induce anti-tumour effects in untreatable Hodgkin’s disease (Falini et al, 1992). Bispecific monoclonal antibodies (bimAbs). reacting with a cytotoxic agent and with a TAA, may represent a suitable approach, alternative to immunotoxins, to deliver toxic compounds to the tumour. A few anti-toxin/anti-TAA bimAbs have been produced for the selective delivery of type 1 RIPS (Glennie et al. 1988; Flavell et al, 1992). Good zy in vitro results have been reported with anti-CD22/anti-saporin 5 72 zyxwvuts 0 1995 Blackwell Science Ltd