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Keywords:

  • basiliximab;
  • CD25;
  • cyclosporin A;
  • daclizumab;
  • graft-versus-host disease

SUMMARY

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Humanized or chimeric monoclonal antibodies (MoAbs) directed against the interleukin-2 (IL-2) receptor α-chain, CD25, are promising immunosuppressive agents due to improved pharmacokinetic profiles and less toxicity. These MoAbs have been used effectively in preventing and/or treating rejection in solid organ transplantation and are currently under investigation for prevention/treatment of graft-versus-host disease (GvHD) in stem cell transplantation. We analysed the in vitro activities of the chimeric anti-CD25 MoAb basiliximab and the humanized anti-CD25 MoAb daclizumab in various test systems for alloimmune response and T cell activation in comparison to cyclosporin A (CsA) and prednisolone. Anti-CD3- and alloantigen-induced T cell proliferation were decreased significantly by the anti-CD25 MoAbs in a dose-dependent fashion. At a concentration of 10 ng/ml daclizumab and CsA synergistically decreased T cell proliferation of mixed lymphocyte cultures, whereas basiliximab showed only subadditive activity. Simultaneous addition of the anti-CD25 MoAbs and prednisolone did not result in combined activity. Addition of exogenous IL-2 completely overcame the inhibitory effect on T cell proliferation of both anti-CD25 MoAbs, but not that of CsA and prednisolone. Anti-CD25 MoAbs inhibited the generation of antigen-specific cytotoxic T lymphocytes in a limiting dilution assay, whereas they showed no effect on the cytolytic activity of established antigen-specific T cell clones. This in vitro study demonstrates strong immunosuppressive activity by both chimeric and humanized MoAbs against CD25. The combined activity with CsA justifies their early use for prevention rather than treatment of GvHD.


INTRODUCTION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Acute graft-versus-host disease (GvHD) remains a major complication following allogeneic stem cell transplantation (SCT) despite immunosuppression with cyclosporin A (CsA), methotrexate ± prednisolone [1–3]. Activated T lymphocytes expressing the high-affinity interleukin-2 (IL-2) receptor play a crucial role in mediating GvHD so that monoclonal antibodies (MoAbs) against the α-chain of the IL-2 receptor (CD25) should provide a route for targeting only those T cells actively involved [4,5].

The use of murine anti-CD25 MoAbs was limited by the early development of neutralizing antimouse antibodies although effective T cell modulation was demonstrated in vitro[6–8]. Chimeric or humanized MoAbs with murine and human variable and human constant regions produced by recombinant DNA technology retain the binding affinity of the original murine anti-CD25 MoAbs while being less immunogeneic [9]. Both chimeric (basiliximab) and humanized anti-CD25 MoAbs (daclizumab) have been evaluated and proven effective in the prophylaxis of acute rejection after solid organ transplantation [10–14]. Preliminary data in patients with steroid-refractory acute GvHD demonstrate activity in a substantial proportion of patients [15–18].

To have a better rationale for earlier, i.e. preventive, rather than therapeutic use we investigated the in vitro immunomodulatory potential of basiliximab and daclizumab in more detail.

MATERIALS AND METHODS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Compounds

Stock solutions (5 mg/ml) of basiliximab (Simulect®; Novartis, Vienna, Austria), daclizumab (Zenapax®; Hoffmann-La Roche, Grenzach-Wyhlen, Germany), prednisolone (Solu-Dacortin®; Merck, Vienna, Austria) and CsA (Sandimmun®; Novartis) were prepared.

Proliferative response to phytohaemagglutinin (PHA) and anti-CD3 MoAb

Peripheral blood mononuclear cells (PBMC) separated from heparinized PB of healthy volunteers by density gradient centrifugation on Ficoll-Isopaque (Lymphoprep; Axis-Shield PoC AS, Oslo, Norway; 5 × 104) were incubated with 1% PHA (Difco, Detroit, MI, USA) or 100 ng/ml anti-CD3 MoAb OKT3 (Orthoclone®, Janssen-Cilag, Buckinghamshire, UK) in a U-bottomed microtiter plate (Greiner Bio-One, Frickenhausen, Germany) at 37°C in a humidified air atmosphere having a CO2 content of 5% for 72–96 h, respectively. All experiments were performed in six replicates. Appropriate concentrations of the compounds (0·1–10 µg/ml) were added to the cultures either from the beginning or after 24, 48, 72 and 96 h. For the last 12–16 h of culture, each well was exposed to 2 µCi of [3H]thymidine (40–60 Ci/mmol, Amersham, Arlington Heights, IL, USA). Cells were harvested using a semiautomated device, and [3H]thymidine uptake expressed in counts per minute (cpm) was measured in a liquid scintillation counter (Beckmann LS 1801, Galway, Ireland).

Mixed lymphocyte reaction (MLR)

Proliferative responses of PBMC to allogeneic stimulation were determined in a one-way MLR as described [19]. Briefly, 5 × 104 responder PBMC was cocultured in six replicates with 5 × 104 mitomycin C-treated (Sigma, St Louis, MO, USA) allogeneic PBMC in U-bottomed microtiter plates at 37°C in a humidified air atmosphere having a CO2 content of 5%. The compounds were added simultaneously with recombinant (r) IL-2 (100 U/ml) at the onset of culture. On day 6 [3H]thymidine uptake was measured as described above.

Analysis of cytolytic activity

Cytolytic activity was determined in a classical chromium release assay [20]. Briefly, target cells were labelled with Cr51 (Na2Cr51O4, specific activity 300–500 Ci/g chromate; NEN, Dreieich, Germany) for 1 h, washed three times and incubated with responder cells for 4 h at 37°C in U-bottomed microtiter plates. The supernatant was harvested and measured in a liquid scintillation counter (Clinigamma; Pharmacia LKB, Bromma, Sweden). Results are expressed as specific lysis defined as percentage of maximal lysis according to the formula: specific release = (experimental release – spontaneous release)/(maximum release – spontaneous release) × 100. Experimental release represents cpm released from target cells in the presence of effector cells, maximum release cpm from target cells incubated with Triton X-100 (Sigma) and spontaneous release cpm from target cells cultivated with plain tissue culture medium.

Cytotoxic limiting dilution analysis

Limiting dilution analyses were performed as described [21]. Briefly, graded numbers (25000–3125 cells) of PBMC were co-cultured with 50000 irradiated (30 Gy) allogeneic PBMC. The compounds were added at culture onset at a final concentration of 1 µg/ml (basiliximab, daclizumab, prednisolone) and 0·1 µg/ml (CsA). From every dilution step 24 replicates were assessed in U-bottomed microtiter plates. On days 3 and 6 a final concentration of 5 U/ml human rIL-2 was added. After 10 days of culture at 37°C in 5% CO2/95% air atmosphere each microculture was tested individually for cytolytic activity against PHA-stimulated allogeneic PBMC generated on day 0.

T cell clone

The generation and specificity of the minor histocompatibility antigen (mHag)-specific T cell clone were described recently [22]. The T cell clone was thawed and specifically restimulated twice. After a total culture period of 14 days cytolytic activity was determined against the specific stimulator cells. For the last 24, 48 or 72 h of culture the T cell clone was incubated with final concentrations of 1 µg/ml basiliximab or daclizumab and 0·1 µg/ml CsA.

Statistical evaluation

Differences between runs of experiments with variable concentrations of the compounds were computed using Student's t-test.

The combination effects of the anti-CD25 MoAbs with CsA and prednisolone were evaluated according to the following formulas: synergistic drug interaction: surviving fraction (SF) of drugs A and B  < SF (A) × SF (B); additive interaction: SF (A + B) = SF (A) × SF (B); subadditive interaction: SF (A + B) > SF (A) × SF (B) and < SF (B) if SF (A) > SF (B)

RESULTS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Effect of anti-CD25 MoAbs on anti-CD3- and PHA-induced T cell proliferation

Whereas PHA-induced T cell proliferation was not affected by addition of anti-CD25 MoAbs (data not shown), anti-CD3-induced T cell proliferation was significantly reduced in a dose-dependent fashion by all compounds (Fig. 1). Interestingly, at lower concentrations the chimeric MoAb basiliximab was slightly more effective than the humanized anti-CD25 MoAb daclizumab (reduction from 100% to 41 ± 8%versus 69 ± 12% with 0·1 µg/ml of each MoAb, P= n.s.; Fig. 1). Delayed addition of the compounds beyond 48 h of culture had no significant effect on anti-CD3-driven T cell proliferation (data not shown).

image

Figure 1. Dose-dependent effect of anti-CD25 MoAbs on anti-CD3-induced T cell proliferation compared to CsA and prednisolone. The mean proliferation ± s.e. of 13 experiments is shown. Proliferation in the absence of the compounds was set at 100%.

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Cell viability (determined by eosin dye staining) was not affected by either compound in any of the experiments (data not shown).

Effect of anti-CD25 MoAbs and exogenous IL-2 on alloantigen-induced proliferation

Alloantigen-induced T cell proliferation in the MLR was reduced significantly in the presence of all compounds (Fig. 2). Addition of exogenous rIL-2 at a concentration of 100 U/ml reversed almost completely the inhibitory effects of both anti-CD25 MoAbs but not that of CsA or prednisolone (Fig. 2).

image

Figure 2. Effect of addition of 100 U/ml IL-2 on T cell proliferation reduced by 1 µg/ml of basiliximab, daclizumab and prednisolone and 0·1 µg/ml CsA. The results (mean ± s.e.) of four experiments are shown.

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Combination effects of anti-CD25 MoAbs and CsA

To evaluate whether anti-CD25 MoAbs and CsA have a synergistic or only additive inhibitory effect on proliferation in the MLR, suboptimal concentrations (1 or 10 ng/ml) of both compounds were added to the cultures. All combinations of basiliximab and CsA resulted in a subadditive inhibitory effect on T cell proliferation. Also, daclizumab and CsA at 1 ng/ml acted subadditively in decreasing T cell proliferation (data not shown). However, daclizumab and CsA, both at 10 ng/ml, reduced T cell proliferation synergistically in MLR (Fig. 3).

image

Figure 3. Combination effect of anti-CD25 MoAbs with CsA. The mean proliferation ± s.e. from four experiments is shown. Proliferation without compounds was set at 100%.

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The addition of suboptimal concentrations of both anti-CD25 MoAbs and prednisolone did not result in an additive or synergistic effect on the inhibition of proliferation of MLR (data not shown).

Effect of anti-CD25 MoAbs on the generation of cytotoxic T cells and their cytolytic activity

Limiting dilution analyses were performed to test whether anti-CD25 MoAbs had an effect on the generation of alloantigen-specific cytotoxic T cell precursors. As shown in Fig. 4a, the frequencies of alloantigen-specific cytotoxic T cell precursors were reduced three-fold when generated in the presence of both anti-CD25 MoAbs (P = n.s.).

imageimage

Figure 4. (a) Effect of the anti-CD25 MoAbs on the generation of cytotoxic T lymphocyte precursor cells in a limiting dilution assay. The mean frequency ± s.e. of reacting T cells from four different experiments is shown. (b) Anti-CD25 MoAbs have no effect on the cytolytic activity of established mHag-specific T cell clones. The mean specific lysis ± s.e. of three experiments at effector : target (E : T) ratios of 25 : 1 and 5 : 1 is shown. Specific lysis without compounds at an E : T ratio of 25 : 1 was set at 100%.

Only the addition of 0·1 µg/ml CsA achieved borderline significance by reducing the frequency of alloreactive cytotoxic T cells (P = 0·0622).

To investigate further the immunosuppressive potential of anti-CD25 MoAbs basiliximab and daclizumab were added at an appropriate concentration (1 µg/ml) to a mHag-specific T cell clone 24–72 h prior to the final cytotoxicity assays as described in Material and methods (Fig. 4b). Specific lysis of host-specific Epstein–Barr virus-transformed B-lymphoid cell lines was decreased twofold when basiliximab was added to the cultures (P = n.s.). Only marginal effects were observed in the presence of daclizumab or CsA.

DISCUSSION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

The present in vitro study demonstrates clearly that both the chimeric as well as the humanized anti-CD25 MoAbs (basiliximab, daclizumab) effectively suppress alloantigen- and anti-CD3-induced T cell proliferation when used at concentrations achievable in vivo[23] and effective in saturating the IL-2 receptor [24,25]. When these results were compared with the published data for the murine counterpart which inhibits PHA-induced T cell proliferation in only suboptimal lectin doses [6], none of the antibodies were seen to have a significant effect on 1% PHA-stimulated T cell proliferation. Furthermore, both antibodies were less active in preventing the development of cytotoxic T lymphocyte precursors in allogeneic co-cultures compared to their murine counterpart, which has been shown to be very potential (>95%) in abrogating the generation of cytotoxic T cells [6], suggesting some loss of activity through the humanization procedure. The effect on the generation of antigen-specific cytotoxic T cells was about five times less than that observed with CsA at a concentration of 100 ng/ml. As seen already with the murine anti-Tac antibody, both anti-CD25 MoAbs had no effect on the cytolytic activity of an already established mHag-specific, HLA-A2-restricted T cell clone, suggesting that anti-CD25 MoAbs will be most effective clinically when used in the early phase rather than in reversion or treatment of already established GvHD or allograft rejection. Surprisingly, regarding human allogeneic SCT, only data on treatment of steroid-refractory GvHD are available so far for daclizumab and basiliximab, although encouraging response rates of 29% to 71% have been reported in a limited number of patients with an otherwise poor prognosis [16–18].

It is of note that both the humanized as well as the chimeric MoAb were equally effective, at least in vitro, although there was a tendency to a more potent suppression of anti-CD3-induced T cell proliferation and cytolytic activity in favour of the chimeric MoAb basiliximab. This may be explained by a 10-fold lower affinity of daclizumab to its target compared to basiliximab. However, whereas basiliximab inhibited only T cell proliferation subadditively with CsA, the humanized MoAb daclizumab showed synergistic activity with CsA in vitro. Synergistic activity in inhibiting activated T lymphocytes has also been observed with mouse MoAbs against rat IL-2 receptor, resulting in significant prolongation of survival of renal allograft recipients [26] and reduced graft-versus-host reaction [27]. These findings are in line with the clinical observations of an approximately 30–40% reduction in the incidence of allograft rejection when using either MoAb in combination with standard immunosuppression [10,11].

Simultaneous addition of IL-2 overcomes completely the inhibitory effect of both anti-CD25 MoAbs on alloantigen-induced T cell proliferation. This important observation suggests that while CsA- and steroid-induced inhibition of T cell activation by alloantigens seems to be complete, even in the presence of 100 U/ml IL-2, inhibition of allorecognition by anti-CD25 MoAbs is weaker and reversible by exogenous IL-2. This probably reflects that CsA inhibits transcription of IL-2 and other ‘early’ T cell activation genes which are obviously not affected by the anti-CD25 MoAbs.

In conclusion, the in vitro immunosuppressive potential of the tested humanized and chimeric monoclonal anti-CD25 antibodies argues strongly in favour of a prophylactic use of these compounds in allogeneic haematopoietic SCT. The low rate of infusion-related side effects, the obvious lack of an increased rate of infectious complications when used for prophylaxis and, of immense importance in SCT, namely the reduced relapse rate seen with anti-T cell antibodies with narrow compared to broad specificity or even with rat anti-CD25 MoAbs [28–31], should prompt clinicians to test both MoAbs in randomized trials for their efficacy in preventing rather than treating acute and/or chronic GvHD.

REFERENCES

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES
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