1756 DJ Flavell et al immunity, which for the purposes of this paper are termed NOD/SCID mice. Moreover, the profound B- and T-cell defi- ciency acquired through the scid mutation means that NOD/SCID mice do not develop IDDM but they do have a high incidence for developing spontaneous thymomas (Prochazka et al, 1992) giving them a life expectancy of 12 months or less. The profound immun- odeficient state of NOD/SCID mice has resulted in better and more robust engraftment levels with normal and malignant haema- tological material (Wang et al, 1995) that has allowed for a quanti- tative evaluation of leukaemic stem cell populations in primary patient derived samples (Dick, 1996) and the establishment of intact human haematopoietic systems (Bhatia et al, 1997). SCID mice have in the past provided a useful tool for the preclinical evaluation of novel antibody-based therapies for human acute leukaemias and lymphomas. Nearly all of these previous studies have been based upon cell line models, which may not accurately recapitulate the disease process and behaviour in humans (Flavell, 1996). A major advance in this area would be to develop the capability of engrafting a sufficiently large enough cohort of animals with human leukaemia cells from one indi- vidual patient in order to undertake therapy studies with sufficient statistical power. In this context several workers have recently reported the several-fold expansion of human T-cell leukaemia (Yu et al, 1999) and myeloma cells (Pilarski et al, 2000) in preconditioned NOD/SCID. As a preliminary undertaking to progressing the idea of developing in vivo therapy models for individual patients for the preclinical evaluation of antibody- based therapeutics we wanted to ensure that such therapy studies were feasible in NOD/SCID mice utilizing an existing cell line model of human T-ALL, which has been well defined in the SCID mouse. Our strategy has been to compare the therapeutic efficacy of the murine anti-CD7 IgG1 antibody HB2 and the immunotoxin HB2-SAPORIN in SCID and NOD/SCID mice engrafted with the human CD7+ T-ALL cell line HSB-2. We clearly demonstrate that the therapeutic effectiveness of HB2 antibody is significant in SCID mice but is non-existent in NOD/SCID mice and also show that HB2-SAPORIN IT treatment is significantly reduced in NOD/SCID when compared with the outcome obtained in SCID mice. These current experimental findings taken together with our previous observations (Flavell et al, 1998) lead us to propose that the NOD/SCID mouse model of HSB-2 human leukaemia more accurately reflects the intrinsic in vivo therapeutic effectiveness of HB2-SAPORIN treatment which unlike the SCID mouse model is not influenced by the presence of NK cells which we have previously shown can have significant effects on the in vivo therapeutic potency of IT treatment. MATERIALS AND METHODS SCID and NOD/SCID mice Pathogen free CB.17 scid / scid (SCID) mice of both sexes were produced from our own breeding colony. NOD/LtSz-scid / scid mice (NOD/SCID) were also provided from our own breeding colony with breeding pairs kindly supplied by Dr Finbarr Cotter (Institute of Child Health, London, UK). All animals were main- tained in filter top micro isolator cages and provided with sterile water and food ad libitum under conditions complying with British Government Home Office regulations. All manipulations of experi- mental animals were conducted in a laminar flow hood using strictly controlled procedures adhering to the UKCCCG Guidelines British Journal of Cancer (2000) 83(12), 1755–1761 for the Welfare of Animals in Experimental Neoplasia to minimize stress or suffering (Workman et al, 1998). HSB-2 human T-ALL cell line and YAC-1 murine lymphoma cell line The CD7+ human cell line HSB-2 (Adams et al, 1970) and the murine lymphoma cell line YAC-1 (Cikes et al, 1973) were main- tained in the logarithmic phase of growth in RPMI 1640 medium containing 10% fetal calf serum and supplements of 2 mM of sodium pyruvate and glutamine (R10 medium) at 37˚C under a humidified atmosphere of 5% CO2. Antibodies The rat anti-mouse antibodies AT37 (anti-CD2), 6D5 (anti-CD19) and F4/80 (anti-macrophage) and the mouse anti-mouse NK cell antibody NK1-1 directly conjugated to phycoerythrin were obtained from Serotec Ltd (Kidlington, UK). The rat antibody 2.4G2 directed against mouse FcγRII/III (anti-CD16/32) was obtained from PharMingen (San Diego, CA). The murine hybridoma cell line HB2 producing an IgG1 antibody directed against the human CD7 mole- cule was obtained from the American Tissue Culture Collection (Bethesda, MD). Bulk HB2 antibody was produced in vitro from this hybridoma on a Cellex Accusyst R hollow fibre bioreactor system (Cellex, Minneapolis, MN, USA). F(ab )2 fragments of HB2 antibody were produced by pepsin digestion of native HB2 antibody employing an ImmunoPure F(ab )2 kit from Pierce, (Rockford, IL, USA) following the manufacturers instructions. F(ab )2 HB2 antibody produced in this way gave a single band of 110 kD molecular weight on SDS-PAGE analysis under non- reducing conditions and was wholly free of detectable contami- nating Fc. Immunotoxin construction The immunotoxins HB2-SAPORIN and HB2-F(ab )2-SAPORIN were constructed by conjugating molar equivalent amounts of HB2 antibody or its F(ab )2 fragment to saporin using the heterobifunc- tional cross linking reagent N-succinimidyl 3-(2-pyridyldithio)propi- onate (SPDP) as described previously (Thorpe et al, 1985). Only IT containing 2 saporin moieties per IT molecule were used in these studies because of their well defined characteristics and potency as described by us previously (Flavell et al, 1997). The purity of immunotoxins was confirmed by SDS-PAGE and were then dial- ysed into PBS pH7.2, sterilized by passage through a 0.2 µ m filter and stored deep frozen in 100 µ g aliquots at 80˚C. Chromium release assay Details of chromium release assays used to determine the lytic capability of SCID and NOD/SCID splenocytes for HSB-2 and YAC-1 cells both constitutively and as effectors in antibody- dependent cellular cytotoxicity (ADCC) have been fully described previously (Flavell et al, 1998). Flow cytometry Expression of the various antigen markers on SCID and NOD/ SCID splenocytes was determined on a Coulter Epics XL flow © 2000 Cancer Research Campaign