Pharmacokinetics of CAMPATH-1H in BMT patients

Cytotherapy (2001) Vol. 3, No. 4, 261–267

Pharmacokinetics of CAMPATH-1H in BMT patients P Rebello1, K Cwynarski 2, M Varughese2, A Eades2, JF Apperley2 and G Hale1 1

2

Sir William Dunn School of Pathology, University of Oxford, UK Department of Haematology, Imperial College School of Medicine, Hammersmith Hospital, London, UK

Background

CAMPATH-1 (CD52) Abs are used in stem-cell transplantation for prevention of GvHD and rejection. The humanized Ab CAMPATH1H has recently replaced the rat Ab CAMPATH-1G. There was a concern whether it might have a longer half-life in vivo and, possibly,

plant were well above the level necessary for opsonization of lymphocytes. The peak Ab concentration was 6.1 µg/mL in Group A and 2.5 µg/mL in Group B. CAMPATH-1H could be detected in Group A for

cause prolonged immunosuppression post-transplant.

23 days post-transplant, significantly longer than in Group B (11 days). The terminal half-life in the two groups was similar (range 15–21 days) and contrasts with the half-life of < 1 day previously esti-

Methods

mated for CAMPATH-1G. There were no cases of graft failure and the incidence of GvHD was similar in the two groups.

Serum samples were collected pre- and post-transplant from patients receiving CAMPATH-1H at 10 mg/day according to two protocols:

Discussion

(A) from Day 25 to Day +4 (total dose, 100 mg), (B) from Day 210 to Day 26 (total dose, 50 mg). The Ab concentrations were measured using an immunofluorescence assay.

The humanized Ab CAMPATH-1H appears to persist in the circulation for longer than the original rat Ab CAMPATH-1G. This might

Results

contribute to delayed lymphocyte recovery and prohibit the use of early donor-lymphocyte infusions. A short course of treatment given early pre-transplant is likely to be preferable to the extended course given both

Lymphocytes were substantially depleted by the second day of treatment

pre- and post-transplant.

and were below 0.1 3 109/L by the day of transplant and for at least 1 month post-transplant. By Day 90 there was a greater recovery in Group B, to a median of 0.32 3 109/L compared with 0.25 3 109/L

Keywords

in Group A. By Day 180, both groups had recovered to approx 0.52 3 109/L. Serum concentrations of CAMPATH-1H on the day of trans-

Introduction CAMPATH-1H is a humanized IgG1 MAb directed against the CD52 Ag of human lymphocytes, which has frequently been used as an immunosuppressive agent in stem-cell transplantation for prevention of rejection and GvHD [1–3]. Current studies are based on previous experience with the homologous rat IgG2b Ab CAMPATH-1G [4,5]. The lympholytic activity of the two Abs appears to be generally similar [6]. However, clearance rates in humans may be different. If so, this might have important implications for their use in stem-cell transplants and the design of

CAMPATH, CD52, stem cell, bone marrow, T-cell depletion, pharmacokinetics, half-life.

the appropriate dose regimen. For example, if the humanized Ab persists in the patient for a significant time after the transplant, it might interfere with immune reconstitution, or the anti-tumor activity of donor lymphocytes. In a previous small study with CAMPATH-1G, Ab levels were measured in 12 patients who had been treated with 10 mg/day from Day 25 to Day 21 before receiving allogeneic BM on Day 0 [5]. The concentration of Ab directly before stem-cell infusion was 0.9 6 0.6 µg/mL in 10 patients, but < 0.13 µg/mL in the other two. By just 1 h after the stem-cell infusion, levels were < 0.13 µg/mL in

Correspondence to: Geoff Hale, Therapeutic Antibody Centre, Old Road, Headington, Oxford, OX3 7JT, UK © 2001 ISHAGE

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five patients and reduced in the others. Although further samples were not collected for analysis, it was estimated that the effective half-life of the rat Ab was approximately 13 h. In the present study, patients with CML received CAMPATH-1H during their preparative regimen for unrelated stem-cell transplant. Two different Ab regimens were used: (A) CAMPATH-1H, 10 mg/day for 10 days from Day 25 to Day +4, (B) CAMPATH-1H, 10 mg/day for 5 days from Day 210 to Day 26. We have previously reported increased transplant-related mortality from infectious complications in CMV seropositive patients [7,8] and the less intensive protocol (B) was devised in order to reduce the duration of immunosuppression consequent on CAMPATH-1H administration. This provided an opportunity to measure and compare the levels of therapeutic Ab at various times pre- and post-transplant. Measurement of a humanized MAb in human serum is challenging, because the antibody, typically < 20 µg/mL, is so similar to normal human immunoglobulin, which has a concentration of approximately 10 000 µg/mL. The assay should measure intact Ab and not break-down fragments. It needs to exploit the unique property of the Ab, viz its antigenic specificity, to discriminate it from normal IgG. Many different methods have been used to measure the binding of CAMPATH-1H to the CD52 Ag [9,10], but for this application the limiting factors are the relatively low affinity of the Ab (which limits the ultimate sensitivity) and the need to avoid non-specific detection of normal human IgG. After evaluation of different methods, we concluded that the most suitable was indirect immunofluorescence. Target cells are incubated with the test samples, followed by a fluorescent detection reagent specific for the Fc domain of human IgG. Fluorescence is measured by flow cytometry to indicate the amount of bound Ab, and the concentration is calculated by interpolation from a standard curve. The results have important implications for the optimization of CAMPATH-1H therapy in stem-cell transplantation.

Patients and methods Treatment regimens Fifteen patients (10 male, 5 female) with CML in the first chronic phase and one patient (female) with pre-B acute leukemia in first complete remission received stem cells from volunteer unrelated donors (15) or an HLA-

mismatched relative (one). The median age of the patients was 32 years (range 21–51 years). The median patient weight was 78 kg (range 50–112 kg). All patients received a standard conditioning regimen consisting of 120 mg/kg cyclophosphamide and six fractions of TBI to a total dose of 1440 cGy. CAMPATH-1H [11] was administered at 10 mg/day, either from Days 25 to +4 (Regimen A, total 100 mg) or from Days 210 to 26 (Regimen B, total 50 mg). Unmanipulated stem cells were infused on Day 0. Prophylaxis for GvHD was cyclosporin and methotrexate, as previously described [12]. At the onset of this study CMV negative and positive patients were treated according to regimens A and B respectively. During the course of the study, the incidence and severity of GvHD was found to be similar in both groups. Thereafter, the final seven patients were all treated according to Regimen B, irrespective of CMV status.

Assay for CAMPATH-1H in patient serum Samples for pharmacokinetic analysis were collected immediately before each dose of CAMPATH-1H and daily following the last dose for 1–10 days and then every 2–7 days for up to 30 days. The schedule was adjusted to fit in with routine patient follow-up. Sera were stored frozen until analysis. Before analysis, the test samples were incubated at 56°C for 30 min to inactivate complement. This procedure does not affect the CAMPATH-1H activity. Samples were analyzed by indirect immunofluorescence as follows. Target cells (HUT-78, ECACC Accession no. 88041901) from a working cell-bank were cultured for 1–10 days, then harvested by centrifugation and resuspended in wash buffer (PBS containing 0.1% BSA and 0.05% sodium azide) to give approximately 1.25 6 3 10 cells/mL. Test samples of sera were diluted with wash buffer as required (normally two-fold) and 50 µL of the diluted sample were mixed with 50 µL of cell suspension in a 96-well microtiter plate. The mixture was incubated on ice for 30 min. Wash buffer was added to a final volume of 200 µL, the cells were pelleted by centrifugation at 200 g for 1 min and the supernatants were removed. This was repeated four times. The cells were resuspended in 50 µL of detection reagent; FITClabelled polyclonal anti-human IgG Fc domain (Sigma cat. no. F-9512) at a final dilution of 1:50 in wash buffer. They were incubated on ice for 30 min, washed three times as before, and finally resuspended in 100 µL wash

Pharmacokinetics of CAMPATH-1H in BMT patients

buffer. Fifty microliters of 3% formaldehyde in PBS were added to fix the cells. Plates were stored at 4°C until analysis by flow cytometry on a FACScan, equipped with a FACSmate robotic sampler for microtitre plates. All fluorescent-labelled samples were protected from undue exposure to light. List mode data were collected from the FACScan, including forward and sideways scatter and green fluorescence. Data were analyzed using the computer package WinMDI (Joseph Trotter, Scripps Institute, Version 2.8). A region was set on the scatter cytogram to select cells that were viable at the time of staining. The median fluorescence intensity (MFI) of all cells in this region was plotted versus Ab concentration for standard samples ranging from 0.025 to 100 µg/mL. The curve of best fit was computed using the equation: plateau MFI = ——————————–—— + background (titer/concentration) slope + 1 Plateau is the MFI at infinite Ab concentration. Titer is the Ab concentration at which the MFI is half of the plateau. Slope is a constant, characteristic of a particular assay, generally in the range 0.5–2.5. Background is a constant, representing the MFI of cells not exposed to CAMPATH-1H (i.e. negative control). These parameters were calculated by non-linear least squares minimization using the SciApps software package (Version 1.50, R Ferguson, 1992) on an Acorn RiscPC. The assay for serum CAMPATH-1H was extensively validated (P. Rebello and G. Hale, unpublished work). The following validation parameters were determined: limit of detection 0.15 µg/mL (0.3 µg/mL allowing for serum dilution), limit of quantitation 0.25 µg/mL (0.5 µg/mL allowing for serum dilution), analytical range 0.25–10.0 µg/mL, linearity 0.999, overall precision 13%, bias +9%. There was no interference by a range of normal and patient control sera, and no reactivity with F(ab’)2 fragments of CAMPATH-1H. The assay was robust with respect to variations in cell-culture time, cell concentration, detection of reagent concentration, blood-clotting conditions, and sample storage conditions and working limits were set for each of these factors.

Lymphocyte reconstitution Post-transplant absolute lymphocyte counts were available from an additional 55 patients who received

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stem-cell transplants from volunteer unrelated donors at the Hammersmith Hospital between December 1994 and October 2000. Thirty-six patients received CAMPATH1H according to Regimen A and were evaluable on Day 90. Results were also available from 33 of these patients on Day 180. Data from Days 90 and 180 were available from 19 and 16 patients respectively, treated on Regimen B. Absolute lymphocyte counts were determined using a Sysmex SE 9000 Automated Haematology Analyser. Some of these patients had received corticosteroids for the management of GvHD. Systemic steroids were commenced at a dose of i.v. methylprednisolone of 1 mg/kg/day (skin and upper gastrointestinal tract GvHD Grades II–IV) or 2 mg/kg/day (lower gastrointestinal tract and liver GvHD Grades II–IV). The steroid dosages were subsequently modified according to clinical response. On Day 90, 19 of 29 evaluable patients from Regimen A and 10 of 19 patients from Regimen B were receiving steroids. By Day 180, 19 of 28 evaluable patients from Regimen A and 10 of 16 from Regimen B remained on steroids.

Results Depletion of blood lymphocytes The median lymphocyte count in the study group directly before CAMPATH-1H treatment was 1.72 3 109/L (range 0.54–3.96 3 109/L) and was not significantly different between the two groups (Group A 1.74 3 109/L, Group B 1.70 3 109/L). The kinetics of lymphocyte depletion were similar in both groups (Figure 1), and the lymphocyte count was < 0.3 3 109/L in all 12/13 patients where it was measured after the first two doses of CAMPATH-1H. There appeared to be a small rebound of lymphocyte counts on about Days 3–5 of the Ab treatment in Group B. In every case, the lymphocyte count was < 0.1 3 109/L by the day of the transplant and remained so for at least 1 month post-transplant. Lymphocyte subsets were not analyzed in this study owing to the very small numbers of cells found in the majority of samples.

Lymphocyte recovery Recovery of lymphocytes was retrospectively analyzed in a population of 55 patients who had been treated with the same regimens. The median absolute lymphocyte count on Day 90 was lower in patients who received Regimen A (median 0.25 3 109/L, range 0.02–0.96 3 109/L, n = 36)

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The mean Ab levels for each group are plotted in Figure 2. The trough level rose steadily each day during Ab administration and the final concentration reached after 10 days in Group A was close to double the level reached after 5 days in Group B. After reaching a peak, the Ab level declined in a biphasic fashion, with an initial rapid clearance from plasma over 3–5 days and a second, slower clearance with an apparent half-time of approximately 15 days for Group A and 21 days for Group B (not significantly different) (Figure 3).

2.0

Lymphocyte count (310^9 /L)

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4

6

0.2 0

5

10

15

20

25

30

35

40

Time since first dose of antibody (days)

Figure 1. Depletion of lymphocytes with CAMPATH-1H. The mean blood lymphocyte counts are plotted starting from immediately before the first Ab dose. l Group A, 10 mg for 10 days, s Group B, 10 mg for 5 days. than in those treated according to Regimen B (median 0.32 3 109/L, range 0.13–1.3 3 109/L, n = 19, P = 0.04). There was no significant difference in the lymphocyte counts recorded on Day 180 (Regimen A, median 0.5 3 109/L, range 0.1–1.2 3 109/L, Regimen B, median 0.54 3 109/L, range 0.16–2.0 3 109/L; P = 0.2). No significant differences were observed between patients who did or did not receive steroids at either time-point in either regimen.

Antibody concentration (mg/ml)

0.0

5 4 3 2 1 0 210

25

0

5

10

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35

Time from transplant (days)

Figure 2. Blood levels of CAMPATH-1H. The mean serum-Ab levels are plotted with respect to the day of transplant (Day 0). l Group A, 10 mg for 10 days, s Group B, 10 mg for 5 days. 10

Sera from all 16 patients were tested in duplicate at a final dilution of 1:2. The pre-treatment serum of one patient (Group A) gave a weak positive result for CAMPATH1H (0.61 µg/mL). It was assumed that this was due to natural human Abs (eg anti-HLA), which recognized the HUT-78 target cells and this level was subtracted from all subsequent measurements. Another patient in Group A gave apparently high levels of CAMPATH-1H, even up to 3 months post-treatment. Unfortunately, a pretreatment sample was not available, so an extra sample was collected at 9 months post-treatment. This gave a positive result of 1.65 µg/mL in the assay. It was assumed that this was also a baseline level, representing natural Abs and it was subtracted from all the previous results. The corrected results for both patients have been included in the subsequent analysis, although omission would not make a significant difference to the conclusions.

Antibody concentration (mg/ml)

Blood levels of CAMPATH-1H

1

0.1 0

5

10

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30

35

Time since last dose (days)

Figure 3. Pharmacokinetics of CAMPATH-1H. The mean blood Ab levels are plotted starting from the peak level at the end of the last dose. The terminal half-life (t 12 ) is calculated from the data collected between Days 5 and 35. l Group A, 10 mg for 10 days, t 12 = 15 days; s Group B, 10 mg for 5 days, t 12 = 21 days.

Pharmacokinetics of CAMPATH-1H in BMT patients

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Table 1. CAMPATH-1H concentration in patient serum Regimen Number of patients CAMPATH-1H (first day) CAMPATH-1H (days) CAMPATH-1H (total dose mg) Antibody concentrations Peak concentration (µg/mL 6 SD) Concentration on day of stem cell infusion (µg/mL 6 SD) Day of last sample above 0.5 µg/mL (LOQ)b

Group A

Group B

5 25 10 100

11 210 5 50

6.1 6 2.3 2.3 6 0.5 23 6 5

2.5 6 0.9 1.0 6 0.5a 11 6 6

a

Serum was not collected on the day of stem-cell transfusion from four patients in Group B and the level of CAMPATH-1H was calculated by interpolation from the levels measured on the days before and after. The difference in mean concentrations on Day 0 is significant whether or not these four samples are included P<0.01, students t-test). b This is a minimum estimate, since samples were not taken daily.

There was a significant difference between Groups A and B in the plasma levels of CAMPATH-1H on the planned day of the transplant (Table 1). However, even the lowest levels (1.7 µg/mL in Group A and 0.52 µg/mL in Group B) were still well above the optimum required for Ab-dependent cell-mediated killing of lymphocytes, which is estimated to be approx 0.1 µg/mL [6]. CAMPATH-1H persisted above the limit of quantitation (0.5 µg/mL) for significantly longer in Group A (at least 23 days after the last dose, equivalent to 28 days after the transplant), than in Group B (at least 11 days after the last dose, equivalent to 6 days after the transplant).

Engraftment and GvHD One patient (Group B) died on Day +12 of acute renal failure and was not evaluable for engraftment or GvHD. All the remaining patients engrafted with a median time to reach 0.5 3 109 neutrophils/L slightly longer for Group A (26 days) than for Group B (22 days). This did not reach statistical significance (P > 0.5, Mann–Whitney U test), but the difference was significant when all of the patients treated according to these protocols were considered [7]. The incidence and severity of GvHD was comparatively low in both groups. In Group A, three of five evaluable patients suffered Grade II acute GvHD, and no patients suffered chronic GvHD. In Group B, five of 10 evaluable patients suffered Grade II and one suffered Grade III acute GvHD, whereas four of eight evaluable patients suffered limited chronic GvHD. There were no cases of severe chronic GvHD.

Discussion Measurement of the humanized Ab CAMPATH-1H in human serum is not straightforward owing to its rather low affinity. The most reliable method is immunofluorescence using a human lymphocyte T cell-line (HUT-78) as the target cell, although the limit of quantitation is only 0.5 µg/mL. Validation of the assay using many samples of serum from normal donors and patients with CLL did not give any significant false-positive results (PR and GH, unpublished data). However, two of the 16 patients in this study gave a positive reaction, either with pre-treatment serum, or with post-treatment serum collected 9 months after the Ab therapy. It is possible that various human allo-antibodies might be found in patients who have received blood transfusions, and these could give positive binding to the human target cells in our assay. Even though the method is imperfect, and not as sensitive as we would like, it was useful to evaluate the data in order to guide future studies of CAMPATH-1H in stem-cell transplants. Both regimens gave adequate blood levels of CAMPATH-1H on the day of stem-cell infusion for potential clearance of donor T cells by ADCC. This may explain the relatively low incidence of GvHD seen in this patient population. Furthermore, the administration of CAMPATH-1H resulted in rapid depletion of host lymphocytes, which reached a nadir after just 2–3 days of treatment, similar to previous reports where the rat Ab CAMPATH1G was used [13]. This would be expected to reduce the risk of graft rejection. Though this small study did not

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have adequate power to demonstrate differences in graft rejection, larger studies using essentially the same protocols have shown that the use of CAMPATH-1H in vivo in unrelated donor transplants is associated with a low incidence of graft rejection of approximately 2–6% [3]. The terminal half-life of CAMPATH-1H was determined in this study to be approximately 2–3 weeks. The precise value is uncertain, because the calculation depends on measurements of samples that are close to the limit of quantitation, and there was considerable variability between individual patients. A more accurate estimate of the half-life will be available from other studies in progress, where CAMPATH-1H has been used to treat patients with CLL. Nevertheless, this half-life is comparable with that of normal human IgG [14] and similar to other chimeric MAbs [15,16]. CAMPATH-1H persisted at levels sufficient to give optimal cell-mediated killing of lymphocytes for up to 30 days post-transplant in some patients. In contrast, a study on patients who were treated with CAMPATH-1G at 10 mg per day from Day 25 to Day 21, showed that the Ab levels were already below the limit of quantitation in many patients by the day after the transplant [5]. We therefore have to consider the possibility that CAMPATH-1H may interfere with immune reconstitution to a greater extent than the rat Ab CAMPATH-1G, even though they appear to have similar cytolytic effects. At the present time it is difficult to determine the quantitative effects on the immune system, although the issue of rapid and frequent CMV reactivation, which was the original motivation for the change of protocol in this study, has also been raised by other investigators [17]. Furthermore, if it is planned to infuse donor lymphocytes post-transplant, either for promotion of anti-tumor effects, or for restoration of immunity, then the potential effects of residual CAMPATH-1H need to be considered. CAMPATH-1H seems to be just as effective at reducing the risk of GvHD when given in lower doses pretransplant (i.e. Regimen B), and so we believe that such a protocol is to be preferred, especially considering that lymphocyte recovery appeared to be somewhat more rapid. Since lymphocyte depletion and effective immunosuppression can be achieved in solid organ transplants with as little as 2 days treatment [18], there may be further scope for reducing the dose and mitigating any undesirable effects on immune reconstitution.

Acknowledgements We thank the team at the Therapeutic Antibody Centre, who provided Abs for this study, Prof Herman Waldmann for his constant guidance and encouragement, and gratefully acknowledge the support of: UK Medical Research Council, Millennium Pharmaceuticals Inc., and the EP Abraham’s Trust.

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