Humanized anti-CD25 monoclonal antibody for prophylaxis of graft-vs-host disease (GVHD) in haploidentical bone marrow transplantation without ex vivo T-cell depletion

Experimental Hematology 31 (2003) 1019–1025

Humanized anti-CD25 monoclonal antibody for prophylaxis of graft-vs-host disease (GVHD) in haploidentical bone marrow transplantation without ex vivo T-cell depletion Hui-Ren Chena, Shu-Quan Jia, Heng-Xiang Wanga, Hong-Ming Yana, Ling Zhua, Jing Liua, Mei Xuea, and Chang-Qing Xunb a

Research Institution Of Air Force Hematology PLA, Beijing, China; bUniversity of Kentucky and VA Medical Center, Lexington, Ky., USA (Received 17 April 2003; revised 11 June 2003; accepted 19 June 2003)

Objective. To investigate the effects of a novel anti–IL-2 receptor (CD25) monoclonal antibody, basiliximab, on graft-vs-host disease (GVHD) and engraftment in haploidentical bone marrow transplantation (BMT). Materials and Methods. Thirteen consecutive high-risk leukemia patients (age 9–41) underwent haploidentical BMT with G-CSF–primed marrow as stem cells without ex vivo T-cell depletion. Basiliximab, along with a combination of cyclosporine (CSA), methotrexate (MTX), and mycophenolate mofetil (MMF), was used for GVHD prophylaxis. Immunophenotyping, limited-dilution assay, and colony-forming assays were used to measure the effect of basiliximab on the subsets of lymphocytes, cytotoxic T-lymphocyte precursors (CTLp), and hematopoietic cells. Results. All patients established successful trilineage engraftment with full donor chimerism. No patients developed grade II–IV acute GVHD. Patients who survived more than 12 months and were free of relapse showed limited chronic skin GVHD. Ten of 13 patients are currently alive with a Karnofsky performance score of 100% at median follow-up of 17 months (range 12– 24 months). Basiliximab significantly decreased alloreactive CTLp by 10-fold to 100-fold in limiting-dilution assays. It had no effect on hematopoietic stem and progenitor cells as determined by in vitro colony-forming assays. Conclusion. The addition of basiliximab to CSA, MMF, and MTX as GVHD prophylaxis effectively reduced severe lethal GVHD in haploidentical BMT. It is possible to selectively eliminate or reduce the number of alloreactive T cells with anti-CD25 antibody, which results in prevention of or a reduction in the severity of GVHD. 쑖 2003 International Society for Experimental Hematology. Published by Elsevier Inc.

The high incidence of severe graft-vs-host disease (GVHD) limits the application of haploidentical bone marrow transplantation (BMT), a curative procedure for leukemia patients without matched donors. It is known that donor T cells are strongly associated with GVHD and vigorous ex vivo T-cell depletion reduces GVHD. However, it also results in an increased incidence of graft failure and disease relapse [1–6]. Over the past decade, major attempts have been made to successfully ameliorate some of these problems to enable haploidentical BMT be performed with more promising long-term efficacy [7,8]. Using mega-doses of T cell–

Offprint requests to: Chang-Qing Xun, M.D., B412 Division of Hematology/Oncology, 1101 Veterans Drive, Lexington, KY 40402, USA; E-mail: [email protected]

depleted hematopoietic stem cells demonstrated that engraftment of three loci-disparate haploidentical transplants can be achieved when the standard T–depleted bone marrow preparation was supplemented with sevenfold to 10-fold more CD34 cells [9,10]. However, posttransplant infectious complications remain a major problem when using the T cell– depleted grafts. Studies have been done to induce T cell tolerance via coculture of donor and recipient cells in the presence of a T cell costimulator inhibitor, CTLA4-Ig, instead of removing T cells. In the cohort of patients treated with CTLA4-Ig, the incidence of GVHD was reduced but not completely eliminated. It is uncertain if such tolerance will be long-lasting in vivo after transplantation [11,12]. An alternative and equally promising approach for depletion of unwanted alloresponses is based on the elimination

0301-472X/03 $–see front matter. Copyright 쑖 2003 International Society for Experimental Hematology. Published by Elsevier Inc. doi: 1 0. 10 1 6 / S0 3 0 1- 4 7 2X ( 0 3) 0 0 22 8 - 5

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of alloreactive T cells, sparing T cells that have other functions. A preclinical study has demonstrated that allospecific Tcell depletion by using an immunotoxin directed against the α chain of interleukin-2 (IL-2) receptor was feasible and specific [13]. The IL-2 receptor comprises three transmembrane protein chains: α (CD25), β (CD122), and γ (CD132). The level of expression of CD25 is low on resting T lymphocytes but is induced to a high level after allogenic stimulation. CD25 does not transduce a signal but is responsible for the rapid association of IL-2 with the β and γ chains, which in turn trigger the antigen-activated T lymphocyte to enter mitosis and undergo clonal expansion. The expression of CD25 on activated T lymphocytes led to the identification of CD25 as a potential target with monoclonal antibodies (mAbs). T-cell depletion with anti-CD25 mAbs was effective in preventing both graft rejection and GVHD in a murine model with complete haplotype-mismatched hematopoietic stem cell transplants (HSCT) [14]. Basiliximab (trade name Simulect from Novartis, Basel, Switzerland) is a novel chimeric high-affinity monoclonal antibody, which is directed against the α chain of the IL-2 receptor, a critical signaling molecule in activation of the immune response. It competes effectively with IL-2 and inhibits IL-2-driven proliferative responses [15]. Basiliximab has a long mean terminal half-life (4–6 weeks), minimal immunogenicity, and lack of major toxicity that makes it potentially a valuable new agent for immunoprophylaxis [16]. In our current study, we investigated the effects of prophylactic basiliximab on the incidence of acute grade II–IV GVHD and engraftment in haploidentical BMT.

Materials and methods Clinical protocol Between November 2000 and November 2001, 13 consecutive leukemia patients, 6 with acute lymphocyte leukemia (ALL) (1 in complete remission [CR] 1, 5 in CR2), 2 with acute myelogenous leukemia (AML) (1 in CR1 and 1 in CR2), and 5 with chronic myeloid leukemia (CML) (2 in chronic phase [CP], 1 in accelerate phase [AP], 2 in blast phase [BP]) entered the study with the Institutional Review Board–approved protocol. Median age of patients was 17 years (range 9–41). Patients eligible for this trial had advanced disease or leukemia with other poor prognostic features. None of these patients had an HLA-matched sibling or singleantigen-mismatched related donor. Their need for transplantation was urgent. Patients and donors were HLA-typed, using serologic methods for HLA class I-A and B antigens and PCR-SSP (polymerase chain reaction amplification of sequence-specific primers) highresolution methods for HLA class II typing. All pairs of donors and recipients were identical for one HLA haplotype and incompatible at two or three loci (HLA-A, B, and DR) of the unshared haplotype. Of 13 donors, 9 were parents, 1 a son, and 3 siblings.

Patients’ and donors’ characteristics are summarized in Table 1. Written informed consent was obtained from patients and/or guardians. Conditioning regimens Included were cytarabine (Cytosar, Pharmacia & Upjohn, Peapack, NJ, USA) 3.0 g/m2 twice a day for 3 consecutive days on days ⫺7, ⫺6, and ⫺5; cyclophosphamide (CY) 45 mg/kg/day for 2 consecutive days on days ⫺5 and ⫺4; total-body irradiation (TBI) with 1000 cGy by two fractions on days ⫺2 and ⫺1 using a highenergy linear accelerator with midline dose rate at 5–6 cGY/minute; and rabbit antithymocyte globulin (ATG) (Fresenius AG, Oberursel, Germany) 5 mg/kg on days ⫺4, ⫺3, ⫺2, and ⫺1. Donor priming and marrow collection Donors were primed with granulocyte colony-stimulating factor (GCSF; Lenograstim, Chugai Pharmaceutical, Tokyo, Japan) at 3–4 ug/ kg/day subcutaneously [17]. On the eighth day, bone marrow was harvested with a target volume of 18–20 mL/kg of recipient’s body weight. Fresh marrow was infused on day 0 without any ex vivo manipulation. GVHD prophylaxis Basiliximab (Simulect; Novartis) along with cyclosporine (CSA), methotrexate (MTX), and mycophenolate mofetil (MMF) were used for GVHD prophylaxis. Basiliximab was given in two doses of 20 mg each by intravenous infusion over 30 minutes on day 0 about 2 hours before transplantation and on day 4 after transplantation. CSA was given at 1.5 mg/kg/day intravenously on days ⫺7 to ⫺1, then 3 mg/kg/day as a 24-hour continuous intravenous infusion from day ⫺1 until the day of bowel function recovery, then switched to oral CSA. MTX was given at 15 mg/m2 on day ⫹1 and 10 mg/m2 on days ⫹3, ⫹6, and ⫹11. MMF 1.0 g/day was administered from days ⫹7 to ⫹100. Acute GVHD was graded and staged according to the consensus conference on GVHD grading [18]. Acute GVHD, grade I or more, was treated with methylprednisolone 1–2 mg/kg/day and tapered off quickly when symptoms resolved. Supportive care The patients were hospitalized in rooms with high-efficiency particulate air filters and received standard antibiotic prophylaxis consisting of oral trimethoprim-sulfamethoxazol (TMP-SMZ), fluconazole, acyclovir, and intravenous immunoglobulins. G-CSF (Lenograstim) 3–4 ug/kg/day was also administered to all recipients from the second day of transplant until absolute neutrophil counts (ANC) were greater than 0.5 × 109/L for three consecutive days. Patients were transfused if hemoglobin or platelets were below 8.0 g/dL or 20 × 109/L. All blood products were irradiated. Both donors and recipients were screened for serum cytomegalovirus (CMV) PP65 antigen prior to transplant. If positive, they were treated with standard course of ganciclovir until the PP65 antigen became negative and then transplanted. CMV PP65 antigen was monitored every 2 weeks after the transplant. If positive, prophylactic ganciclovir was given until the antigen became negative. The EBV-IgM and IgG antibodies were determined by enzyme-linked immunosorbent assay (ELISA). Studies of immune reconstitution Two-color immunophenotyping was used to measure T cells, B cells, and natural killer (NK) subsets at 1, 3, 6, 12, and 18 months after transplant and results were compared with normal donors. The

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Table 1. Patient characteristics and donor-recipient relationship UPN of Patients

Patients Age/Sex

Donors

Disease/Status

HLA Typing

Age

Relationship

Host

Donor

A24 B60 DR4 A11 B37 DR10 A33 B46 DR11 A33 B58 DR13 A2 B27 DR15(2) A11 B46 DRA2 B46 DR8 A24 B61 DRA2 B52 R15 A31 B7 DR15 A24 B61 DR1 A33 B58 DR17 A31 B61 DR11 A11 B35 DR13 A2 B35 DR15 A-B40 DR8 A24 B46 DR12 A2 B15 DR14 A11 B51 DR4 A1 B54 DR7 A2 B48 DR4 A- B13 DR12 A2 B40 DR9 A3 B52 DR14 A24 B44 DR4 A33 B75 DR7

A24 B60 DR4 A11 B46 DR9 A33 B46 DR11 A2 B37 DR9 A2 B27 DR15(2) A24 B71 DR12(5) A2 B46 DR8 A24 B51 DR9 A2 B52 R15 A11 B71 DR15 A24 B61 DR1 A11 B46 DR12 A31 B61 DR11 A2 B13 DR131 A2 B35 DR15 A33 B40 DR16 A24 B46 DR12 A26 B61 DR11 A11 B51 DR4 A24 B54 DR12 A2 B48 DR4 A1 B37 DR10 A2 B40 DR9 A30 B13 DR9 A24 B44 DR4 A3 B75 DR13

155

15/M

ALL/CR3

43

Mother

157

27/M

CML/CP

31

Sister

161

22/F

ALL/CR1

49

Mother

162

14/M

ALL/CR2

41

Mother

165

36/F

ALL/CR2

32

Sister

166

22/M

ALL/CR2

27

Brother

167

41/M

CML/AP

14

Son

168

22/M

ANLL/CR2

51

Father

169

17/M

ALL/CR2

41

Father

172

13/M

CML/CP

36

Mother

173

14/F

CML/BP

34

Father

174

12/M

CML/BP

35

Mother

176

9/M

37

Father

M4/CR2

CR, complete remission; CP, chronic phase; AP, accelerate phase; BP, blast phase; ALL, acute lymphocytic leukemia; CML, chronic myelocytic leukemia; ANLL, acute non-lymphocytic leukemia.

50 uL of whole blood was incubated with appropriate amounts of undiluted anti-CD3, CD4, CD8, CD16, and CD56 (Coulter Immunology, Hialeah, FL, USA) mAb conjugated with either fluorescein isothiocyanate (FITC) or phycoerythrin (PE) respectively for 30 minutes at 4⬚C. The cells were washed and analyzed on an Epics profile flow cytometer (Coulter Immunology). Engraftment Neutrophil engraftment was defined as first of three consecutive days with ANCgreater than 0.5 × 109/L after neutrophils nadir. Platelet engraftment was noted when platelet count exceeded 20 × 109/L without platelet transfusions for at least 7 days. Chimerism was evaluated by cytogenetic and DNA analysis of one or more genetic differences between donor and recipient. Chimerism of peripheral blood and bone marrow cells was determined by PCR-SSP for detection of HLA class I and II antigens and monitored every 3 to 6 months after transplant. Isolation of bone marrow and blood mononuclear cells Mononuclear cells (MNC) were isolated from both 3–5 mL of normal donor bone marrow (BM) and recipient’s peripheral blood (PB) by Ficoll-Hypaque density-gradient centrifugation and washed twice in Hank’s balance salt solution. The MNCs were resuspended in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 ug/mL streptomycin for the following biological function assays.

Mixed lymphocyte reaction (MLR) One-way MLR was performed with and without the presence of basiliximab. In brief, 5 × 105/mL of MNCs from the donor (responder cells) were incubated with the irradiated peripheral blood MNCs from the recipient (stimulating cells) with and without basiliximab at the concentration of 0.025 mg/mL, 0.05 mg/mL, and 0.1 mg/mL in 20 mL of culture medium. The cells were incubated at 37⬚C, humidified with 5% CO2 for 48 hours, and then collected for flow cytometric analysis [19]. Flow cytometric analyses Lymphocyte subsets were analyzed by one- or two-color immunofluorescence on a FacScan analyzer (Becton-Dickinson, San Diego, CA, USA). Cells were washed with RPMI 1640 medium after the 48-hour incubation period described above and stained with anti-CD3, anti-CD4, anti-CD8, and anti-CD16 (all from Becton-Dickinson) for 30 minutes. The cells were washed and analyzed by FacScan and presented as the percentage of cells staining positive for each individual mAb. Cytotoxic T lymphocyte (CTL) assay CTL precursor frequency was determined in graft-vs-host direction with standard limiting-dilution assays (LDA) with and without the presence of basiliximab [20,21]. In brief, donor bone marrow mononuclear cells (BMMNC) (responder cells) were incubated with recipient’s irradiated peripheral blood mononuclear cells (PBMNC) (donor-recipient pair) at 37⬚C, 5% CO2 for 7 days to

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Table 2. Clinical outcome Grafts contents UPN of Patients 155 157 161 162 165 166 167 168 169 172 173 174 176

GVHD

TNC (× 108/kg)

CD3

CD34 (× 106/kg)

CFU-GM (× 109/kg)

Neutrophils ⬎0.5 × 109/L (days)

Platelets 20 × 109/L (days)

Acute Grade

Chronic

Follow-up (mo.)

Current status

8.5 9.2 7.6 4.2 9.9 7.5 6.3 10.5 4.5 10.9 7.7 8.5 11.5

55 46 59 45 49 52 52 50 48 58 50 53 67

6.3 7.0 5.6 6.2 8.2 6.1 5.8 8.1 3.9 9.1 5.1 7.9 10.8

69 72 55 59 50 62 45 56 39 76 68 62 70

16 20 18 20 18 20 19 19 24 19 19 18 16

18 20 21 29 20 22 23 23 29 22 22 21 19

I I I I I I I I I I I I I

L L L L L L L L L L L

24 21 20 14 19 18 3 16 2 14 13 12 12

Alive in CCR Alive in CCR Alive in CCR Died from relapse Alive in CCR Alive in CCR Died from CMV Alive in CCR Died from TTP Alive in CCR Alive in CCR Alive in CCR Alive in CCR

CCR, Continued complete remission; TTP, Thrombotic thrombocytopenia purpura; L, Limited chronic GVHD; CMV, Cytomegalovirus.

generate human CTL as effector cells. Negative control effector cells were the cells incubated with irradiated cells from the same donor. Target cells were allogeneic PBMNC stimulated with PHA (PHA blasts) and labeled with 51-Cr at 37⬚C for 1 hour. The 51Cr-labeled PHA blast target cells were then incubated with different concentrations (limiting dilution) of effector cells for 4 hours in 96-well U-bottomed plates in triplicate. The 51-Cr cells released to the supernatant were measured by gamma counter and percentage of specific lytic activity was calculated as previously described [22]. Progenitor cell assays CFU-GM and BFU-E were grown in complete methylcellulose medium (StemCell Technologies, Toronto, ON, Canada) according to manufacturer’s instructions. CFU-GM colonies having more than 40 cells per cluster were counted on day 7 after plating. BFU-E colonies were scored on day 14 of culture [23]. CFU-Meg cultures were performed utilizing a plasma clot assay. Briefly, BMMNC were grown in IMDM medium (Gibco, Baltimore, MD, USA) containing 250 ug/mL calcium chloride (Sigma, St. Louis, MO, USA), 15% preselected human AB-plasma, and 15% bovine plasma (Sigma). Clotting was induced by adding thrombin at 2.5 u/mL (Sigma). The clots were cultured in quadruplicate at 37⬚C with 5% CO2 in 4well plates (Nalge NUNC, Rochester, NY, USA). After 12 days of culture, clots were air dried, methanol-fixed, washed with phosphate-buffered saline (PBS), and stored at 4⬚C before being stained for CD61 expression. Immunofluorescence staining was performed utilizing conjugated CD61-FITC antibodies with appropriate isotype control. CFU-Meg were identified microscopically as groups having more than three CD61⫹ cells. Statistical analysis Analyses for clinical data were performed with all patients on November 15, 2002 after a median follow-up of 17 months, minimum 12 months (range 12–24 months). Event-free survival was estimated using the Kaplan-Meier method. The experimental results are expressed as the mean ⫾ standard deviation (SD). Statistical analysis was performed by means of the nonparametric paired Wilcoxon rank-sum test. Frequency of proliferating cells was based on the Poisson distribution of nonresponder culture cells. For comparison, the independent t-test was used. A p value of less than 0.05 was considered significant.

Results Transplantation outcomes All patients achieved successful trilineage engraftment (Table 2). The median time to neutrophil recovery greater than 0.5 × 109/L was 19 days (range 16–24 days) and to platelet recovery greater than 20 × 109/L was 22 days (range 18–29 days). Sustained engraftment was achieved in all patients; no late rejection was observed. Chimerism confirmed by sex chromosome or PCR-SSP for HLA typing showed all recipients had greater than 95 donor cells in the marrow one month posttransplant and throughout the entire follow-up period. All patients developed grade I acute skin GVHD and responded very quickly to a short course (about 2 weeks) of methylprednisolone. No patients developed grade II–IV acute GVHD (aGVHD). Eleven patients who survived through day 100 were evaluated for chronic GVHD. After 12 months follow-up, all 11 patients had somewhat limited chronic GVHD with dry mouth and skin, but no extensive chronic GVHD. Five patients developed CMV PP65 antigemia after transplants (38.5%) and received standard course of ganciclovir treatment. One patient (UPN 168) died of CMV disease. One patient (UPN 162) became EBV-positive in serology testing but did not have any clinical symptoms. No patients developed lymphoproliferative disease. Ten of 13 patients are currently alive with a median follow-up of 17 months (range 12–24 months). All surviving patients have a Karnofsky clinical performance status of 100%. Three patients died; one died of leukemic relapse, one from a CMV infection, and one from thrombotic thrombocytopenia purpura (Table 2). Immune reconstitution Fifteen donors and recipients were compared for CD3, CD4, CD8, CD20, and CD56 cells in the blood before and after transplant. CD3⫹, CD8⫹, CD20⫹, and CD56⫹ cells in the

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Table 3. Immune reconstitutions of lymphocyte subsets (Absolute cell numbers/uL immunophenotyping) Recipients after the transplants Subsets CD3⫹ CD3⫹CD4⫹ CD3⫹CD8⫹ CD19⫹ CD(16⫹56)⫹

Normal donors n ⫽ 15 1600 (970–2100) 980 (826–1090) 610 (370–1180) 350 (150–540) 350 (86–620)

1 month n ⫽ 13 98 31 60 22 36

3 months n ⫽ 11

(59–166)∗ (19–52)∗ (35–130)∗ (8–30)∗ (14–60)∗

310 110 210 90 185

(170–505)∗ (70–205)∗ (125–421)∗ (50–146)∗ (152–295)

6 months n ⫽ 11 450 201 370 170 210

(380–870)∗ (110–470)∗ (315–700) (72–227)∗ (162–320)

12 months n ⫽ 11 948 425 716 252 325

18 months n ⫽ 5

(830–1520) (260–878)∗ (425–1118) (125–340) (218–570)

1498 832 920 366 412

(910–1790) (418–1380) (488–1120) (240–570) (284–710)

∗Significantly different compared to normal donors at p ⬍ 0.05.

blood were recovered within 6 to 12 months. The CD4⫹ cells were recovered relatively slower, but reached above a 200/uL level in 6 months and became normal within 18 months after transplant (Table 3). Effects of basiliximab on T cell subsets and CTLp production in vitro Incubation of BMMNC with basiliximab for 2 days in MLR did not change CD3, CD4, CD8, and CD16 counts compared with controls (Table 4). However, basiliximab significantly reduced the number and frequency of alloreactive cytolytic T lymphocytes (CTLp) in recipient cells using limitingdilution assays (Table 5). Frequency of specific alloreactive CTLp was 1 CTLp per median of 30,000 cells (range 10,000– 60,000) for the control group and 1 CTLp per a median of 1,200,000 cells (range 600,000–1,800,000) for the basiliximab-treated group ( p ⫽ 0.02), a 10-fold to 100-fold reduction. Effects of basiliximab on progenitor cells in the marrow Colony-forming unit assays were performed using donor BM in the presence or absence of basiliximab. No significant difference in CFU-GM, BFU-E, and CFU-Meg units was noted between control and basiliximab-treated marrow cells (Table 6).

Discussion Our study was performed with 13 high-risk leukemia patients who underwent haploidentical bone marrow transplantation using G-CSF–primed donor BM as the stem cell source with addition of basiliximab to baseline immunosuppression for GVHD prophylaxis without ex vivo T-cell depletion. All Table 4. The effect of basiliximab on lymphocyte subsets (mean X ⫾ SD) in one-way mixed lymphocyte reactions (n ⫽ 13) Experiment

CD3 (%)

CD4 (%)

CD8 (%)

Basiliximab 0.025 mg/mL 0.05 mg/mL 0.1 mg/mL Control

36 ⫾ 11 33 ⫾ 10 32 ⫾ 10 36 ⫾ 11

20 ⫾ 5 18 ⫾ 4 17 ⫾ 4 22 ⫾ 6

12 ⫾ 4 12 ⫾ 3 13 ⫾ 4 13 ⫾ 4

CD16 (%) 19 ⫾ 7 20 ⫾ 7 18 ⫾ 6 20 ⫾ 6

p ⬎ 0.05 at all comparisons between the control and basiliximab-treated marrow cells

patients achieved rapid engraftment and full donor type chimerism. Incidence of grades II–IV acute GVHD was 0% with no GVHD-related deaths. Eleven of 13 patients developed limited chronic GVHD within the median follow-up of 17 months (range 12–24 months). Ten of 13 patients with highrisk leukemia are alive and disease-free with a Karnofsky performance score of 100%. Haploidentical BMT has been associated with a very high incidence of severe acute and chronic GVHD. Although the pathogenesis of GVHD is complicated, it is widely accepted that alloreactive T cells in the donor grafts recognize antigenic differences in the recipients. The cells become activated and undergo clonal expansion along with stimulation of IL-2. Activated and expanded T cells mediate their effects by direct cytolytic destruction of host tissue or indirectly via the release of inflammatory cytokines, e.g., tumor necrosis factor-α (TNF-α), IL-1 that either are cytotoxic or activate other immune mechanisms [24–26]. Basiliximab inhibits the alpha chain of IL-2 receptors, the initial step in T-cell activation and expansion. Therefore, we added basiliximab for the purpose of prophylaxis rather than treatment of GVHD. In our previous study of haploidentical BMT for 15 Table 5. Basiliximab on CTLp frequency in limiting-dilution assays∗ 1/Frequency

UPN of Patients

Non-basiliximab

Basiliximab

155 157 161 162 165 166 167 168 169 172 173 174 176

25,000 ⫾ 8700 30,000 ⫾ 17,000 20,000 ⫾ 10,000 45,000 ⫾ 7200 12,000 ⫾ 6000 40,000 ⫾ 20,000 60,000 ⫾ 20,000 10,000 ⫾ 8600 15,000 ⫾ 8700 35,000 ⫾ 21,000 25,000 ⫾ 8600 40,000 ⫾ 17,000 30,000 ⫾ 26,000

1,000,000 ⫾ 860,000 1,800,000 ⫾ 600,000 1,400,000 ⫾ 340,000 900,000 ⫾ 300,000 1,200,000 ⫾ 520,000 1,000,000 ⫾ 860,000 600,000 ⫾ 340,000 800,000 ⫾ 340,000 1,200,000 ⫾ 600,000 1,000,000 ⫾ 500,00 1,400,000 ⫾ 340,000 1,200,000 ⫾ 400,000 1,500,000 ⫾ 750,000

∗Limiting-dilution assays were set up as described in the methods. The frequency of CTLp was calculated based on the Poisson distribution of nonresponding cultures. Data are presented as 1/frequency of cytolytic cells rounded to the nearest thousand. Values are the mean of three replicate experiments ⫾ SD.

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Table 6. Effect of basiliximab on colony-forming progenitor cells (n ⫽ 13) CFU-GM

CFU-MK

(Colony/2 × 105 MNC)

Experiment Basiliximab 0.025 mg/mL 0.05 mg/mL 0.1 mg/mL Control

BFU-E

49 ⫾ 8.5 47 ⫾ 5.5 47 ⫾ 8.2 50 ⫾ 10.1

121 ⫾ 18.8 122 ⫾ 19.9 120 ⫾ 18.5 124 ⫾ 10.8

53 ⫾ 10.0 50 ⫾ 9.8 50 ⫾ 8.7 55 ⫾ 9.1

p ⬎ 0.05 at all comparisons between the control and basiliximab-treated marrow cells.

patients with high-risk leukemia using the similar protocol without basiliximab, 5 of 15 patients developed grade II– IV acute GVHD and 3 died of acute GVHD [27]. In our current study using basiliximab for GVHD prophylaxis, the grade II–IV acute GVHD was essentially controlled. Andre-Schmuts et al. reported pediatric patients who received 1–8 × 105/kg of allodepleted donor T cells with antiCD25 immunotoxin ex vivo depletion on days 15 and 47 after the haploidentical hematopoietic stem cell transplant and no other treatment for GVHD. No patients developed grade II–IV GVHD [28]. Results of anti-CD25 mAb for GVHD treatment were somewhat controversial. The Cahn group compared the efficacy of using a combination of in vivo anti-CD25α (BT563), cyclosporine, and steroid vs placebo and cyclosporine-steroid for the treatment of grade II–III acute GVHD in 69 patients. Response rates were not statistically different, 63% for the placebo and 70% for the BT563-treated groups [29]. Other groups reported that antiCD25α had substantial activity for the treatment of acute GVHD and improvement of steroid-refractory GVHD with a response rate of 29 to 40% [30,31]. The Przepiorka group reported that intensified treatment at the beginning rather than later had a better response. Complete response rates were 29% with the regimen given on days 1, 8, 15, 22, and 29 vs 47% with the regimen given on days 1, 4, 8, 15, and 22 [30]. The data suggest that prophylaxis was a superior treatment and earlier rather than later treatment was better. Although vigorous ex vivo T-cell depletion of the graft reduces the GVHD, it leads in turn to an increased graft rejection and delayed immune reconstitution resulting in concordant increased occurrences of opportunistic infections and leukemic relapse. Aversa et al. reported successful engraftment with megadosing of CD34⫹ cells and vigorous ex vivo T-cell depletion for GVHD prophylaxis in haploidentical HSCT. However, immune reconstitution after transplant with vigorous T-cell depletion was slower with CD4 cells reaching 100/uL in 10 months and 200/uL in 16 months along with increased risk of infection and mortality [32]. Ruggeri et al. found that alloreactivity of NK cells in the graft could eliminate leukemia and graft rejection while protecting patients against GVHD, and immune reconstitution was significantly faster without ex vivo T-cell depletion [33].

Rao et al. demonstrated the major reactions of both GVHD and host-vs-graft reaction (HVGR) that occur during initial phase of transplant [34]. Pathogenesis of acute and chronic GVHD is a dynamic course and each stage has its major pathogenetic players in the development of disease. Each major player at different stages is also connected to form a dynamic cascade that leads to further development of disease. Our strategy has been to focus on the dynamic course of GVHD development using different agents to block the major players at different stages to prevent the GVHD cascade. We used CSA and MTX as our baseline and added different immunosuppressants at different stages of the transplant course with an emphasis up front to prevent both GVHD and HVGR. This allowed us to perform the haploidentical BMT without ex vivo T-cell depletion. Our study showed basiliximab significantly reduced the number of alloreactive CTLp which are associated with GVHD by limiting-dilution analyses. Interestingly, basiliximab had no effect on the vast majority of other T cells as shown by FACS (fluorescein-activated cell sorting) analysis. Immune reconstitution after haplo-BMT was relatively good. CD3⫹, CD8⫹, CD19⫹, and NK cells were within normal range by 6 to 12 months. It is possible that basiliximab eliminates these alloantigen-activated T cells without affecting the majority of other T cells present in the graft. Andre-Schmutz performed 16 selective donor T-cell depletions with anti-CD25α chain and noticed less than 1% residual anti-host alloreactivity in 12 of 16 depletions [28]. Without any GVHD treatment, no patients developed grade II–IV GVHD after the donor lymphocyte infusions on days 15 and 47 of mismatched related HSCT. Other immune responses were preserved with evidence of early T-cell expansion in three patients who had continuous viral infection. Strong cytolytic activities specific for antiviral responses were noted [28,35]. It is possible to prevent severe GVHD without compromising immune function. Many studies have shown that ex vivo T-cell depletion increased the risk of graft rejection, especially in mismatched stem cell transplant settings [1–6]. Our study showed that basiliximab did not affect the hematopoietic colony-forming cells in vitro and all transplanted patients achieved trilineage engraftment with basiliximab added to GVHD prophylaxis. Similar findings were noticed by other groups [36]. All studies have shown that anti-CD25 mAb was well tolerated [28–31]. In conclusion, experiments have demonstrated that it is possible to selectively eliminate or reduce the alloreactive T cells in the bone marrow grafts and significantly reduce the severity of GVHD. The application of different immunosuppressive agents at different stages of BMT, particularly the addition of basiliximab, allowed us to cross the major histocompatibility complex (MHC) barriers without teetering on the brink of severe lethal GVHD in haploidentical BMT. A randomized study with more patients and longer follow-up is warranted.

H. Chen et al. / Experimental Hematology 31 (2003) 1019–1025

Acknowledgment Dr. Xun is supported by grant number NIH/NCI CA 1795.

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