Characteristics and performance of a bispecific F (ab'γ)2 antibody for delivering saporin to a CD7+ human acute T-cell leukaemia cell line

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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.

Section: Discussionmentioning

confidence: 64%

Effectiveness of combinations of bispecific antibodies for delivering saporin to human acute T-cell lymphoblastic leukaemia cell lines via CD7 and CD38 as cellular target molecules

Flavell¹,

Cooper²,

Morland³

et al. 1992

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“…In previous studies in our laboratory utilising a bispecific antibody, one Fab arm of which was constructed with the same anti-CD7 antibody HB2 and the other Fab arm with an anti-saporin antibody (HB2 x DB7-18), the delivery of saporin to HSB-2 cells was less efficient with an achieved IC50 of 0.23 nM (expressed as the concentration of free saporin in the system) (Flavell et al, 1991). The improved effectiveness of the HB2-Sap immunotoxin over the bispecific antibody HB2 x DB7-18 is probably because the bispecific antibody attaches to the cell surface univalently and therefore has a lower binding avidity and also does not cross-link adjacent CD7 molecules on the cell surface.…”

Section: Discussionmentioning

confidence: 99%

“…In a previous study (Flavell et al, 1991) we showed that the ribosome-inactivating protein saporin has potent in vitro cytotoxicity when targeted with a bispecific antibody to the CD7 surface antigen of HSB-2 cells. In the present study we have clearly demonstrated the effective and selective in vitro, cytotoxicity of an immunotoxin constructed with the anti-CD7 monoclonal antibody HB2 and saporin.…”

Section: Discussionmentioning

confidence: 99%

“…In vitro cytotoxicity of the HB2-Sap immunotoxin was assessed in a [3H]leucine incorporation assay that we have described previously (Flavell et al, 1991). Triplicate cultures of HSB-2 cells at a density of 1 x 105 cells per well in 96-well microculture plates were exposed for 48 h at 37°C to immunotoxin or equimolar concentrations of saporin and HB2 antibody at each experimental concentration.…”

Section: Cytotoxicity Of Immunotoxin In Vitromentioning

confidence: 99%

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Effectiveness of HB2 (anti-CD7) – saporin immunotoxin in an in vivo model of human T-cell leukaemia developed in severe combined immunodeficient mice

Morland

Barley²,

Boehm³

et al. 1994

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Summary The transplantation of the human T-cell acute lymphoblastic leukaemia (T-ALL) cell line HSB-2 into severe combined immunodeficient (SCID) mice was found to produce a disseminated pattern of leukaemia similar to that seen in man. The intravenous injection of 107 HSB-2 cells was associated with a universally fatal leukaemia. Histopathological examination of animals revealed the spread of leukaemia initially from bone marrow to involve all major organs including the meninges. An immunotoxin (HB2-Sap) was constructed by conjugating the anti-CD7 MAb HB2 to the ribosome-inactivating protein saporin. An in vitro protein synthesis inhibition assay revealed specific delivery of HB2-Sap immmunotoxin (IT) to CD7+ HSB-2 target cells with an IC50 of 4.5 pM. When SCID mice were injected with 106 HSB-2 cells and then treated 8 days later with a single intravenous dose of 10 Lg of immunotoxin there was a significant therapeutic effect evidenced by the numbers of animals surviving in the therapy group compared with untreated controls (X2 = 5.348, P = 0.021). These results demonstrate the useful application of human leukaemia xenografts in SCID mice and the potential therapeutic effect of an anti-CD7 immunotoxin in human T-ALL.

“…The ability of HB2-Sap 1-mer or 2-mer immunotoxins to inhibit protein synthesis in the HSB-2 target cell line was evaluated using a [3H]leucine uptake assay described by us previously (Flavell et al, 1991). Briefly, triplicate cultures of 1 x 105 target HSB-2 cells were exposed to individual equimolar concentrations of each IT or saporin in RIO medium and incubated at 370C for 48 h in a humidified atmosphere of 5% carbon dioxide 95% air.…”

Section: Protein Synthesis Inhibition Assaymentioning

confidence: 99%

Comparison of the potency and therapeutic efficacy of the anti-CD7 immunotoxin HB2-saporin constructed with one or two saporin moieties per immunotoxin molecule

Flavell

Boehm²,

Noss³

et al. 1997

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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...