The Use of CD45 Monoclonal Antibodies
There is a very extensive literature concerning the actions of CD45 mAbs on T cells, and earlier publications have been reviewed elsewhere (Alexander 1997). It should first be noted that there is an important difference in the potential mechanism of action of CD45 mAbs, depending whether or not secondary cross-linking antibody is utilised in the experimental system. During the late 1980s and early 1990s many publications appeared claiming that the dominant action of CD45 was negative with respect to TCR signal transduction coupling (reviewed in Alexander 1997). These results were based on experiments in which CD3 mAb against the relatively rare TCR was cross-linked with the very abundant CD45 molecule. Since in many of the cell systems utilised CD3 ligation was required to induce intracellular signals, extensive cross-linking with abundant CD45 thereby produced a 'dilution effect' whereby CD3 ligation became less effective and, not surprisingly, the intracellular signals were inhibited. However, it was later shown using chemically engineered divalent antibodies consisting of F(ab') fragments against both CD3 and CD45, that when this dilution effect is prevented by fixing the CD3/CD45 ratio, then there is no inhibition (Alexander et al. 1992; Shivnan et al. 1992). In fact, under these conditions the enforced juxtaposition of CD45 with the TCR amplifies T cell activation responses, consistent with the dominantly positive effect of CD45 on TCR signal transduction coupling. Therefore results based on cross-linking the abundant CD45 molecule with other receptors using secondary antibody should be treated with caution, particularly in the absence of relevant controls.
By contrast, there are many instances in which specific CD45 mAbs have striking effects on T cells under conditions in which cross-linking is not an issue. In animal models, one of the main uses of CD45 mAbs has been to induce tolerance to tissue transplants. The most extensive studies have been carried out using a CD45RB mAb called MB23G2 that has been shown to induce long-term engraftment of islets tissue into MHC-disparate diabetic mice (Auersvald et al. 1997; Basadonna et al. 1998), to prevent renal allograft rejection (Lazarovits et al. 1996; Zhong and Lazarovits 1998) and to be useful in the treatment of preclinical models of autoimmunity (Zhong and Lazarovits 1998). Tolerance could be adoptively transferred by transfusion of tolerant mouse CD4+ splenic lymphocytes into naive allografted animals (Gao et al. 1999). The induction of tolerance was associated with a partial depletion of peripheral blood lymphocytes and with a shift in CD45 isoform expression on T cells from higher molecular weight isoforms to a proportionally greater increase in CD45R0 (Basadonna et al. 1998). In further studies to investigate the mechanism of action of MB23G2 in tolerance induction, it was found that mAb treatment of mice was followed by a rapid up-regulation on T cells of CTLA-4, a receptor that mediates negative signals, inhibiting T cell activation (Fecteau et al. 2001). Administration of a blocking CTLA-4 mAb at the time of transplantation prevented the anti-CD45RB therapy from prolonging the survival of islet allografts, suggesting that CTLA-4 up-regulation was playing a role in tolerance induction. In addition, treatment with cyclosporin A blocked the MB23G2-induced CTLA-4 expression and promoted acute rejection. How the CD45RB mAb treatment causes up-regulation of CTLA-4 on T cells remains an intriguing question.
Rat models have also been developed to investigate the actions of CD45 mAbs in vivo. The rat anti-rat RT7 mAb has been extensively used. In the rat, two allomorphic forms of the RT7 antigen exist, known as RT7a and RT7b (previously known as ART-1 and Ly-1) (Wonigeit 1979a,b). In animals expressing the RT7a allotype, the RT7a mAb binds to all CD45+ cells. Treatment of rats with the RT7a mAb causes a massive depletion of peripheral leucocytes as well as bone-marrow precursor cells (Dahlke et al. 2002), and this is associated with tolerance induction to fully MHC-mismatched grafts (Ko et al. 2001). Interestingly, mature B cells, although well coated with the mAb, are protected from depletion. Leucocyte depletion showed an identical pattern in complement-deficient as in normal rats, suggesting that complement lysis is probably not important for the depleting effect (Dahlke et al. 2002). However, RT7a mAbs (IgG2b) of other isotypes were much less effective at depletion, indicating that Fc-receptor-mediated interactions are important. Besides the actions of such mAbs in tolerance induction, depletion of stem cells has potential in the therapy of haematopoietic malignancies and also in the conditioning of bone-marrow for other indications.
The mechanism of action of the RT7a mAb in depleting leucocytes remains unknown, but CD45 ligation using certain mAbs has been shown to directly induce apoptosis in either murine or human T and B cells (Klaus et al. 1996; Lesage et al. 1997). The ligation of CD45 is associated with its localisation to the detergent-insoluble cell fraction. Results based on expression of CD45 mutants in the CD45-deficient BW5147 thymoma T cell line have suggested that CD45 PTPase activity and in fact most of the cytoplasmic tail are not required for the apoptosis induced by CD45 ligation (Fortin et al. 2002). The role of CD45 in regulating apoptosis has also been suggested by the finding that in CD45_/_ CD4+CD8+ thymocytes there is a marked increase in basal apoptosis (Byth et al. 1996) that is reversed upon expression of the active lckY505F transgene (Baker et al. 2000), implicating CD45-regulat-ed p56lck in the induction of survival signals. It is, therefore, possible that perturbation of CD45 using specific mAbs might interfere with the ability of CD45-regulated p56lck to maintain survival signals. However, this does not appear to be the mechanism in the BW5147 experimental system, since the CD45 PTPase activity appears to be dispensable (Fortin et al. 2002).
CD45 mAbs have also been widely used with some success in pre-clinical radiotherapy animal model systems to target CD45+ cancer cells with various isotopes (Matthews et al. 1992, 1999; Ruffner et al. 2001; Nemecek and Matthews 2002; Sandmaier et al. 2002; Vallera et al. 2003). Isotopes used in these studies include 125I, 131I, 213Bi and 90Y. The potential for CD45 mAbs in therapy for haematological malignancies has also been highlighted by animal studies in which the CD45 mAb inhibited the proliferation of cancer cells (Nemecek and Matthews 2002). For example, a CD45 mAb was effective in inhibiting growth of systemically disseminated human non-Hodgkin's lymphoma B cells in SCID mice (Dekroon et al. 1996).
Increasing understanding of the differential molecular actions of the different CD45 isoforms will influence future strategies used for developing CD45 mAbs as therapeutic reagents. For example, if the heterodimerisation model of CD45 isoform actions developed using cell line model systems (Dornan et al. 2002) proves to be relevant to primary T cells, then potentially mAbs could be developed that blocked, for example, the proposed interaction between CD4 and CD45R0, thereby down-regulating T cell activation.
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