Divisione di Oncologia Sperimentale E, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milano, Italy We describe the cloning and expression of a new cDNA from the filamentous fungus Aspergillus clavatcls I F 0 8605. This cDNA contains an open reading frame (ORF) that predicts a putative ribonuclease precursor with high similarity to the a-sarcin family of ribosome-inactivating proteins (RIPS). The cDNA encoding the mature protein was expressed in Escherichiu coli, and the recombinant protein, a 17-kDa polypeptide designated clavin was purified and characterized. Clavin shows typical type-1 RIP properties : specific cleavage of ribosomal and synthetic RNA and inhibition of protein synthesis in cellfree and cellular systems. When selectively targeted to a tumour cell antigen by coupling to a monoclonal antibody (mAb) clavin was able to inhibit protein synthesis at nanomolar concentration. Pharmacokinetics analysis in mice indicated an elimination half-life (tl,ZP) of 7.4 h with no particular accumulation in major organs. Liver toxicity was very limited and transient while no alteration of kidney function was observed. Clavin induced a late and very low antibody response in mice. The iii vitro and in vivo biological characteristics of clavin, together with its availability in large amounts, suggest the usefulness of this toxin in the production of toxic chemical conjugates.

Summary The present paper describes two immunoconjugates consisting of an anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MAb), named Mint5, covalently linked to the type 1 ribosome-inactivating proteins (RlPs) ocymoidine (Ocy) and pyramidatine (Pyra) from Saponaria ocymoides and Vaccaria pyramidata respectively. Both antibody and toxins are shown to retain their respective biological properties upon chemical conjugation. The immunoconjugates exert specific inhibition of EGFR expressing target cell proliferation and protein synthesis in in vitro assays and also inhibit the growth of grafted human tumour cells in nude mice.Keywords: immunoconjugate; anti-epidermal growth factor receptor; ocymoidine; pyramidatineThe clinical use of immunotoxins for the treatment of cancer is currently under evaluation worldwide. The therapeutic potentiality of immunotoxins and preclinical and clinical results over the last 20 years have been reviewed recently (Thrush et al, 1996), indicating that, although immunotoxins seem promising for systemic therapy of haematological malignancies, a number of different problems still need to be solved, especially for the treatment of solid tumours. In particular, the refining of dose regimen and administration route, the combination with chemotherapy and the reduction of immunogenicity are the major goals of future research. In this paper, we describe the preparation of two new immunoconjugates made of an anti-epidermal growth factor receptor monoclonal antibody, named MintS, chemically linked to either ocymoidine or pyramidatine, toxins from Saponaria ocymoides and Vaccaria pyramidata respectively.The epidermal growth factor (EGF) and its receptor play a critical role in the growth and regulation of many normal and malignant cell types. EGFR overexpression is a common feature in most carcinomas and correlates with poor prognosis (Fox et al, 1994).The potential value of EGFR as a target for the diagnosis and therapy of human tumours has been recognized for several years, and the use of anti-EGFR monoclonal antibodies may provide therapeutic tools in the treatment of tumours overexpressing the receptor (Ennis et al, 1991).Moreover, immunoconjugates of EGFR-specific monoclonal antibodies to either gelonin (Ozawa et al, 1989) or ricin A chain (Masui et al, 1989) were shown to reduce the growth of human tumour cells transplanted into athymic mice.

The goal of the present study was to establish the condition to obtain preparative amounts of the recombinant cytotoxin alpha-sarcin to be used for immunoconjugate production. alpha-Sarcin cDNA was isolated from Aspergillus giganteus strain MDH 18,894 and its expression in Escherichia coli was attempted by the use of both two-cistron and fusion protein-expression systems. Whereas the former resulted in low intracellular expression level of recombinant alpha-sarcin (r-Sar), the latter allowed high-level expression of the fusion protein in the culture supernatant. A variant form of alpha-sarcin with an additional threonine residue in position 1 (Thr-Sar) was obtained by proteolytic processing of the fusion protein with a final yield after purification of 40 mg/L of culture. Both recombinant proteins r-Sar and Thr-Sar were identical to native a-sarcin with respect to the biochemical properties and to the in vitro biological activity.

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