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SUMMARY Three monoclonal antibodies MT1, L60 , and DF-Tl, were reported independently as recognising human T cells in routinely processed, paraffin wax embedded tissue. The present study was performed to compare these three reagents in terms of their immunocytochemical reactions and target molecule(s). On Western blotting of white cell extracts the three antibodies reacted with antigens of the same molecular weight (range 110-160 kilodaltons). Furthermore, their immunocytochemical reactivity with normal human cells, as analysed by two-colour flow cytometry, was essentially identical (labelling ofmonocytes, most T lymphocytes, and weak reactions with some B cells), and the antibodies gave closely similar reactions on 54 white cell derived neoplasms. To identify the target antigen for these three reagents, antibodies from the Third International Workshop on Leucocyte Antigens were reviewed and it was shown that the Western blotting and immunocytochemical reactions of MTl, L60 (Leu-22), and DF-T1 were identical with those of the reagents which defined the CD43 antigen (also known as leucosialin or sialophorin). Furthermore, all these antibodies reacted with cells transfected with a cDNA clone encoding CD43. It is concluded that antibodies MTl, L60 (Leu-22), and DF-T1 all recognise the heavily glycosylated myeloid/lymphoid associated CD43 antigen.There has been much recent interest in monoclonal antibodies which detect fixation resistant antigens and which can be used to distinguish between B and T cell lymphomas in routinely processed paraffin wax sections.' One widely used reagent of this sort is MTI2 which recognises human T cells and myeloid cells. Antibody L60

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Summary Immunotoxins that carry two toxin molecules to the target cell should in theory have a greater anti-tumour effect than those that carry just one. We have investigated the therapeutic efficacy of two anti-CD7-saporin immunotoxins constructed with one saporin (HB2-Sap 1 -mer) or two saporin molecules (HB2-Sap 2-mer) per immunotoxin molecule. In vitro, the 2-mer immunotoxin was 5.6 times more effective than the 1 -mer immunotoxin at inhibiting protein synthesis in the CD7+ human T-cell acute lymphoblastic leukaemia (T-ALL) cell line HSB-2 and was also more effective at inhibiting HSB-2 cell proliferation. Flow cytometry revealed that the 2-mer immunotoxin had a reduced binding capacity to HSB-2 cells compared with the 1-mer immunotoxin or native HB2 antibody. In therapy studies in SCID mice with disseminated HSB-2 human leukaemia, the 2-mer immunotoxin performed marginally better than the 1-mer immunotoxin, but log-rank analysis did not reveal any significant differences between the two therapy groups. We therefore conclude that, although the 2-mer immunotoxin performed better than the 1 -mer immunotoxin against target HSB-2 cells in vitro, this improved performance was not reflected as an improved in vivo therapeutic outcome in the SCID mouse model. Keywords: immunotoxin; CD7; saporin; T-cell acute lymphoblastic leukaemiaImmunotoxins (ITs) have promising potential as therapeutic agents for cancer, particularly in the treatment of leukaemias and lymphomas, in which they have been shown to have marked activities Falini et al, 1992;Amlot et al, 1993; Grossbard et al, 1993). Comprising a monoclonal antibody component linked to a toxin or ribosome-inactivating protein (rip), such as ricin A chain or saporin, immunotoxins selectively deliver the toxin moiety only to cell populations expressing the target antigen recognized by the antibody. There are, however, a multitude of factors that can effect the potency of a given immunotoxin for its target cell, which can severely limit its therapeutic value. At the mechanistic level, such factors include the isotype and affinity (van-Oosterhout et al, 1994) of the antibody component, the proximity of the target antigen epitope to the target cell membrane (Press et al, 1988, the expression by the target cell of antigens unrelated to the target antigen and that probably assist in the internalization of immunotoxin by receptor-mediated endocytosis (van-Oosterhout et al, 1994) and, of pivotal importance, the fundamental nature of the target antigen and the ease with which it internalizes to the appropriate intracellular compartment once antibody has bound to its target ligand , Preijers 1988, Wargalla and Reisfeld 1989. Ultimately, the key determinants governing the therapeutic potency of any given immunotoxin revolve around two key issues: firstly, the in vivo accessibility of immunotoxin to the tumour and, secondly, the Correspondence to: D J Flavell efficiency of the internalization and trafficking process which delivers immunotoxin to the appropriate intracellular compa...

Summary We have investigated the effectiveness of three different F(ab'y)2 bispecific antibodies (BsAb) for delivering the ribosome inactivating protein (RIP) saporin via the CD7 or CD38 cell surface molecules to the human T-ALL cell lines HSB-2 and HPB-ALL. Inhibition of 3H-leucine uptake by target cells was used as the parameter of cellular cytotoxicity. Used singly against HSB-2 cells in the presence of varied concentrations of saporin, an anti-CD7 BsAb, (HB2 x DB7-18) and an anti-CD38 BsAb (OKT1O x RabSap), gave 435-and 286-fold increases in saporin toxicity, respectively. For HPB-ALL cells the anti-CD7 BsAb performed poorly giving only an eight-fold increase in toxicity whilst on the same cell line the anti-CD38 BsAb was highly potent giving an 80,000-fold increase in saporin toxicity.A combination of both BsAb used together against HSB-2 cells was ten times more effective, than the best single BsAb HB2 x DB7-18 used alone. Kinetic studies conducted with HSB-2 cells revealed that the BsAb combination also gave an increased rate of protein synthesis inactivation in comparison to either BsAb used alone. These investigations clearly demonstrate a synergistic action when both BsAb are used in combination to target saporin against CD7 and CD38 expressed on the surface of the HSB-2 cell line.