1994 84: 1320-1327   

 Nash and R StorbC Anasetti, JA Hansen, TA Waldmann, FR Appelbaum, J Davis, HJ Deeg, K Doney, PJ Martin, R anti-Tac: an antibody that binds to the interleukin-2 receptorTreatment of acute graft-versus-host disease with humanized

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Treatment of Acute Graft-Versus-Host Disease With Humanized Anti-Tac: An Antibody That Binds to the Interleukin-2 Receptor

By Claudio Anasetti, John A. Hansen, Thomas A. Waldmann, Frederick R. Appelbaum, Jennifer Davis, H. Joachim Deeg, Kristine Doney, Paul J. Martin, Richard Nash, Rainer Storb, Keith M. Sullivan,

Robert P. Witherspoon, Mary-Helen Binger, Richard Chizzonite, John Hakimi, Diane Mould, Hiroko Satoh. and Susan E. Light

Humanized anti-Tac is a genetically engineered human lgGl monoclonal antibody specific for Tac. the (Y subunit of the interleukin-2 (IL-2) receptor, and blocks IL-2-dependent acti- vation of human T lymphocytes. The safety, pharmacokinet- ics, and immunosuppressive activity of humanized anti-Tac were evaluated in 20 patients who developed acute graft- versus-host disease (GVHD) after allogeneic marrow trans- plantation. Patients had developed acute GVHD at 5 t o 26 (median, 14) days after transplantation and had failed to respond t o primary therapy with glucocorticoids. Sequential groups of 4 patients each received a single l-hour infusion of antibody in escalating doses of 0.5, 1.0, or 1.5 mg/kg; 8 additional patients were then treated with 1.5 mg/kg. A sec- ond infusion of antibody was administered after 11 to 48 (median, 16) days in 8 patients who had transient improve- ment of GVHD after the first infusion. Acute side effects, limited t o chills in 1 patient and diaphoresis in another, were observed during or shortly after the antibody infusion. Over- all improvement of acute GVHD occurred in 8 patients, 6 of whom were treated with a single antibody infusion and 2 with two infusions. Four responses were complete and 4 were partial. Three additional patients had improvement in one organ but progression in another. Responses occurred

RAFT-VERSUS-HOST disease (GVHD) is a frequent complication of allogeneic marrow transplantation

caused by donor T lymphocytes reactive to histocompatibil- ity antigens of the host."' During early activation, T cells express a variety of surface molecules not found on resting cells including, among others, high-affinity receptors for in- terleukin-2 (IL-2).6 Binding of IL-2 to its receptor (IL-2R) is an essential requirement for T-cell proliferation. High- affinity IL-2Rs are complexes of at least three proteins, a p55 IL-2Ra, a p70 IL-2RP, and a p64 IL-2Ry. IL-2Ra and IL-2RP, but not IL-2Ry, are capable of binding IL-2 independently of the others, but with a much lower affinity

G

From the Clinical Research Division, Fred Hutchinson Cancer Research Center, Department of Medicine, Division of Oncology, University of Washington, Seattle WA; the Metabolism Branch, Na- tional Institutes of Health, Bethesda, MD; and Hoffmann-La Roche, Nutley, NJ.

Submitted December 16, 1993; accepted April 11, 1994. Supported in part by National Institutes of Health Grants No. CA

18029 and CA 18221 from the National Cancer Institute; Grant No. HL36444 from the National Heart, Lung and Blood Institute; and Grant No. AI33484 from the Institute of Allergy and Infectious Dis- ease.

Address reprint requests to Claudio Anasetti, MD, Fred Hutchin- son Cancer Research Center, I124 Columbia St, E61 I , Seattle, WA 98104.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1994 by The American Society of Hematology. 0006-4971/94/8404-0002$3.00/0

1320

in 9 of 16 cases with skin disease, 3 of 15 with liver disease, and 6 of 12 with gastrointestinal disease. Two patients sur- vive at 529 and 645 days after antibody treatment. Two pa- tients died after relapse of leukemia. Sixteen patients died of infection or organ failure between 5 and 211 (median, 55) days. The terminal elimination half-life of the antibody was 44 to 363 hours, with a harmonic mean of 79, 88, and 94 hours, respectively, for the three doses studied. Absolute peripheral blood T-lymphocyte counts remained unchanged during the 56 days after infusion of the antibody. A fraction of circulating T cells expressed the a! chain of the IL-2 recep- tor that, in some patients, was bound by antibody in vivo up to 28 days after treatment. No patient developed a mea- surable antibody response t o humanized anti-Tac. Human- ized anti-Tac has a long half-life after intravenous injection in humans, superior to any rodent monoclonal antibody spe- cific for human T cells, and does not appear t o induce anti- body formation in recipients of marrow transplants. Im- provement of steroid-refractory GVHD in 40% of patients after only one or two antibody infusions indicates that hu- manized anti-Tac is immunosuppressive. 0 1994 by The American Society of Hematology.

than that of the multimeric complex. IL-2RP and IL-2Ry are expressed constitutively on most lymphoid cells. IL-2RP plays a critical role in intracellular signaling induced by IL-2 binding. IL-2Ry is required for normal T- and B-cell development, because its genetic mutation results in X- linked severe combined immunodeficiency disease in hu- mans.' IL-2Ra is expressed on T, B, and natural killer (NK) cells only after these cells have become activated by antigen or IL-2. Hence, an antibody that selectively destroys or inac- tivates IL-2Ra-bearing cells would specifically inhibit na- scent or ongoing immune responses without suppressing nat- ural immunity or the ability to mount a specific immune response to an antigen encountered once the immunosup- pressive therapy has been terminated.

In rodents, monoclonal antibodies (MoAbs) specific to IL- 2Ra are immunosuppressive in vivo in several models of transplantation and autoimmune diseases.' Those antibodies can suppress delayed-type hypersensitivity responses, inhibit local or systemic graft-versus-host reactions, and prevent or reverse rejection of cardiac, skin, and pancreatic allografts. Possible mechanisms by which monoclonal anti-IL-2Ra an- tibodies exert their immunosuppressive activity include blocking IL-2 binding to its receptor and elimination of IL- 2Ra-bearing lymphoid cells. In humans, efficacy of the murine monoclonal anti-IL-2Ra antibody anti-Tac was demonstrated in a randomized clinical trial of prevention of cadaveric kidney allograft rejection.' Furthermore, three distinct rodent MoAbs were found to have some immunosup- pressive activity in patients with acute GVHD resistant to treatment with cyclosporine and glucocorticoids.'""'

The therapeutic efficacy of rodent MoAbs in humans is limited by their short half-life, by the immune response of

Blood, VOI 84, NO 4 (August 15), 1994: pp 1320-1327

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GVHD TREATMENT WITH HUMANIZED ANTI-TAC 1321

the recipient to the heterotypic protein, and by the inefficient interaction of the murine antibody with host effector mecha- nisms. Chimeric MoAbs consisting of human constant re- gions and mouse variable regions were developed to circum- vent these problems. Techniques were developed to more fully humanize mouse MoAbs by incorporating almost ex- clusively amino acids present in the complementary de- termining regions (CDRs) into human framework and C- region sequences. The humanized anti-Tac is a selectively modified human IgGl MoAb that binds specifically to IL- 2Ra.I3 Murine sequences comprise only about 10% of the engineered molecule. The affinity of the humanized antibody for IL-2Ra is approximately threefold lower than that of the parent mouse antibody. However, the humanized antibody has acquired the ability to mediate antibody-dependent cell- mediated cytotoxicity of IL-2Ra+ cells in vitro, in contrast to the native murine IgG2a antibody, which does not possess this a~tivity. '~ Humanized anti-Tac does not activate comple- ment-dependent lysis in vitro.14 Humanized anti-Tac was found to be less immunogenic, to have more favorable phar- macokinetics, and to be more immunosuppressive than na- tive murine antibody in cynomolgus monkeys transplanted with cardiac allografts.15."

We report here results of a phase 1-11 study of humanized anti-Tac used in one or two doses for treatment of acute GVHD in patients refractory to therapy with cyclosporine and glucocorticoids. We found that treatment with human- ized anti-Tac was not associated with appreciable side ef- fects, that the terminal elimination half-life was higher than those reported for any rodent MoAb in humans, and that there was no detectable antibody formation to the humanized anti-Tac antibody in the patients studied. Improvement of GVHD was seen in approximately 40% of the patients treated, indicating that humanized anti-Tac has immunosup- pressive activity in humans.

MATERIALS AND METHODS

Patients. Characteristics of the 20 patients enrolled in this study are summarized in Table 1. All patients received myeloablative treat- ment with cyclophosphamide plus total body irradiation or busulfan or both." Criteria for matching recipients and donors for human leukocyte antigens (HLA) have been GVHD prophy- lactic regimens included cyclosporine alone, cyclosporine and pred- nisone, or cyclosporine and methotrexate, as previously described.'.'' The clinical diagnosis of acute GVHD was confirmed by biopsy of skin, liver, or gastrointestinal tract in 16 of 20 patients. The onset of acute GVHD occurred at a median of 14 (range, 5 to 26) days after transplantation. Patients were treated with 6-methylprednisolone at a dose of 2 mgAcg/day intravenously (IV) for a median of 18 (range, 4 to 30) days. Two patients were enrolled on a randomized phase 111 primary GVHD therapy trial using 6-methylprednisolone in com- bination with either Xomazyme-CD5 plus or placebo administered at 0.1 mgkgld for 14 days." The study drug had been discontinued for at least 72 hours before treatment with humanized anti-Tac. One patient received humanized anti-Tac for overall grade I acute GVHD. In this patient, there was a relative contraindication to glucocorti- coids because of prednisone-induced psychosis. The GVHD grades at time of antibody therapy are shown in Table 1.

Treatment protocol and de$nition of clinical response. Patients gave informed consent for antibody administration according to treatment protocol no. 638 approved by the Institutional Review Board of the Fred Hutchinson Cancer Research Center. Hypersensi-

tivity to humanized anti-Tac was tested in all patients by IV adminis- tration of 100 pg of antibody without premedication. Patients receiv- ing prednisone or cyclosporine before humanized anti-Tac antibody continued treatment with these medications as tolerated by renal function. Antibody was administered as a l-hour IV infusion with vital signs monitored every 15 minutes. Three groups each consisting of 4 patients received a single infusion of humanized anti-Tac at doses of 0.5, 1.0, or 1 .S mgkg. The highest total single dose adminis- tered in any patient was 100 mg. Patients who showed transient improvement of GVHD were eligible for retreatment once at a subse- quent time with an identical dose of antibody. After the first 12 patients were treated, the protocol was amended to allow enrollment of an additional 8 patients to be treated at the 1.5 mgkg dose.

Patients were monitored for blood cell and platelet counts, serum chemistry, and renal function tests daily; for liver function every other day; and with a chest x-ray once weekly for 56 days. GVHD severity was graded as de~cribed.'~ Measurements of involved skin surface area, serum bilirubin, stool volume (3-day average), visible blood in stool, and abdominal cramping were recorded weekly through day 56 after the administration of humanized anti-Tac or until death.

Responses were evaluated on day 29 after humanized anti-Tac antibody therapy, at initiation of alternative GVHD therapy, or at death, whichever occurred first. Response of skin disease was defined as resolution or decrease of rash involving the skin surface by 225%. Progressive skin disease was defined by an increase in involved surface area by 225%. Response of liver disease was defined by a decrease in serum bilirubin to less than 2 mg/dL for patients with baseline values of 2 to 4 mg/dL, a decrease of 2 2 mg/dL for patients with baseline values of 4 to 8 mg/dL, and a decrease of 225% for patients with baseline values greater than 8 mg/dL. Progressive liver disease was defined as an increase of serum bilirubin by 2 2 mg/dL for patients with baseline values less than 8 mg/dL or as an increase by 2 2 5 % in serum bilirubin for patients with baseline values 2 8 mg/dL. Response of gut disease was defined by resolution of diarrhea or decrease in the 3-day average stool volume by 2500 mL/d with clearing of cramps and bleeding if present. Progressive gut disease was defined by an increase in the 3-day average stool volume by 2500 mL or development of new cramps or bleeding. Liver or gut complications other than GVHD were ignored while scoring re- sponse to humanized anti-Tac if the attending physician or the inves- tigators believed that GVHD was the primary cause of organ dys- function. Overall, complete response was defined as resolution of GVHD in all evaluable involved organs with no additional treatment administered for acute GVHD. Partial response was defined as im- provement in at least one evaluable organ without deterioration in any other. Overall progression was defined as deterioration in at least one evaluable organ. No change was defined by the absence of any difference sufficient to meet minimal criteria for response or progression in any evaluable organ after treatment.

Lymphocyte studies. Blood samples were obtained just before treatment, and 18 hours, 7, 28, 56, and 360 days after each antibody infusion. Cell surface phenotype was evaluated as previously re- ported." IL-2Ra+ cells were monitored by staining with fluorescein isothiocyanate (FITC)-conjugated antibody 7G7-B6, specific for the CD25 antigen but binding to an epitope different from the one recog- nized by humanized anti-Tac.".'* Cells coated by humanized anti- Tac were identified by staining with a phycoerythrin (PE)-labeled murine IgG2b antibody specific for human IgGl (clone HP-6000; obtained from American Type Culture Collection, Rockville, MD). Saturation of humanized anti-Tac binding epitopes in vivo was eval- uated by comparing the fluorescent intensity of cells incubated with PE-conjugated HP-6000 antiglobulin to that of cells incubated with excess exogenous humanized anti-Tac plus HP-6000. Free Tac-bind- ing epitopes were also evaluated by comparing the fluorescent inten- sity of cells incubated with PE-conjugated humanized anti-Tac or

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1322 ANASETTI ET AL

Table 1. Characteristics of Patients Treated With Humanized Anti-Tac

Disease/Status Primary

Relation Donor HLA

Matching GVHD

Prophylaxis

Day of GVHD Onset

GVHD Grade at Time of Antibody Therapy

Skin Liver GI Overall

6467 46lF 6469 311F 6498 47/M 6523 35lM 6535 49lF 6483 47/F 6568 34lM 6590 361M 6606 38/M 6661 36lF 6696 52lF 6500 32lF 6789 26lF 6650 241M 5477 45lM 6892 32F 6930 40lM 6946 28/M 7056 25lM 6605 51lM

CMUCP MDS/RAEB NHURel AMURel CMUAP NHURel MDSIRAEB TR CMUBC CMUAP CMUAP NHURel MDSIRAEB TA CMUAP RA CMUCP CMUCP CMU3rd CP CMUAP ALURel CMUAP

Unrelated Sibling Sibling Unrelated Unrelated Sibling Sibling Sibling Unrelated Unrelated Sibling Sibling Unrelated Unrelated Unrelated Unrelated Unrelated Unrelated Unrelated Unrelated

Identical Identical Identical 516 loci Identical Identical 516 loci Identical Identical 516 loci Identical Identical 516 loci Identical Identical 516 loci Identical Identical Identical Identical

CsNMTX CsA CsA CsNMTX CsNMTX CsNPred CsNMTX CsNMTX CsNMTX CsNMTX CsNPred CsNPred CsNMTX CsNMTX CsNMTX CsNMTX CsNMTX CsNMTX CsNMTX CsNMTX

8 14 7

11 13 19 10 14 16 10 23 21 16 6

14 26 21 5

21 21

4 2 2 3 3 2 4 0 3 3 2 4 3 4 0 3 0 3 4 0

3 1 0 0 2 3 0 4 2 0 4 0 2 0 3 1 3 3 2 1

3 3 0 0 3 3 3 0 3 3 0 3 0 0 0 0 3 0 0 3

IV 111 I II

111 IV IV IV 1 1 1 111 IV IV 111 111 111 Ill 111 Ill 111 111

Abbreviations: ALL, acute lymphocytic leukemia; AML, acute myeloid leukemia; AP, accelerated phase; BC, blast crisis; CML. chronic myeloge- nous leukemia; CP. chronic phase; CsA, cyclosporine; GI, gastrointestinal tract; loci, HLA A, B, D loci; MDS, myelodysplastic syndrome; MTX, methotrexate; NHL, non-Hodgkin's lymphoma; Pred, 6-methylprednisolone; RA, refractory anemia; RAEB(TR), refractory anemia with excess blasts (in transformation); Rel, relapse.

FITC-conjugated antibody 2A3 (murine IgGl specific for Tac anti- gen; distributed by Becton Dickinson, San Jose, CA) before and after treatment. Surface expression of IL-2Rb was identified by antibody Mikp3.23

Pharmacokinetics. Serum concentrations of humanized anti-Tac were measured before and at various intervals through 56 days after administration. Antibody was measured by competitive enzyme- linked immunosorbent assay (ELISA) using purified soluble IL-2Ra protein as the capture reagent, with the test serum competing with peroxidase-labeled humanized anti-Tac incubated simultaneously. The lower limit of detection for this assay was 25 p g L Model independent pharmacokinetic analysis was performed. Values for terminal elimination half-life (t1/2P), volume of distribution (VdP). systemic clearance (CLs), and area under the curve (AUC o + x )

were determined. Pharmacokinetic parameters are listed in the table accompanying Fig 1.

Circulating soluble IL-2Ra. Unbound soluble IL-2Ra protein circulating in serum was measured by a sandwich ELISA assay. Solid-phase-bound 7G7/B6 antibody was used as the capture re- agent. Plates were then incubated with serum containing soluble IL- 2Ra peptide and then washed. The detecting antibody was peroxi- dase-labeled humanized anti-Tac. The normal level of soluble IL-2Ra in healthy volunteers is 200 to 500 U/mL, where 1 U equals

Human antibody against humanized anti-Tac. Formation of hu- man antibodies specific for humanized anti-Tac was evaluated by two assays. The first assay was a sandwich ELISA using humanized anti-Tac bound to a microtiter plate as the capture reagent and perox- idase-conjugated humanized anti-Tac as the detecting reagent. The concentration of antibodies to humanized anti-Tac could be deter- mined from a standard curve of purified goat antibodies specific to humanized anti-Tac. The lower limit of detection was 7.8 pgL. The second assay used was a dot blot assay. Complexes of specific antibodies and humanized anti-Tac were captured by protein G Seph-

0.2 pg.

arose and then dissociated from protein G Sepharose and from each other by treatment with pH 2.5 buffer. Dissociated Igs were immobi- lized on a polyvinyldiene fluoride (PVDF) membrane. Humanized anti-Tac was detected by peroxidase-conjugated IL-2Ra. Antibodies specific for humanized anti-Tac were detected by peroxidase-conju- gated humanized anti-Tac. The concentration of each antibody was determined from standard curves of mixtures of humanized anti- Tac and goat antibodies to humanized anti-Tac. The lower limit of sensitivity of the dot blot assay for antibodies to humanized anti- Tac and for humanized anti-Tac were 3 12 and 80 pgL, respectively.

loo l Dose (mg/kg) * 0.5 * 1 .O

Cmax (mg/L) 9.75 26.5

T 1/2 (hours) 79 88

AUC (rng/L*h) 778 1886

4 1 . 5

33.9

94

431 0

0.Oll I 0 10 20 30 40 50

Days after Infusion

Fig 1. Mean f SEM serum concentrations of humanized anti-Tac in patients treated at doses of 0.5 (n = 4). 1.0 (n = 4). or 1.5 mglkg (n = 12).

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GVHD TREATMENT WITH HUMANIZED ANTI-TAC 1323

RESULTS

Clinical safety and eficacy. Treatment with humanized anti-Tac was administered at a median of 32 (range, 16 to 50) days after transplantation (Table 2). Humanized anti-Tac was administered IV as a single infusion over 1 hour at 0.5 mgkg in 4 patients, at 1 .0 mgkg in 4 patients, and at 1.5 mgkg in 12 patients. Eight patients who manifested transient improvement after the first antibody infusion received a sec- ond infusion at the same dose, a median of 16 (range, 11 to 48) days after the first infusion. Two of 28 antibody infusions (7%) were followed by side effects possibly related to the infusion of the antibody: chills in 1 patient and diaphoresis in another. There were no consistent changes in serum chem- istries after treatment with humanized anti-Tac. There was no evidence of other toxicities.

Four patients had complete resolution of acute GVHD, 4 had a partial overall improvement, 3 had improvement in one organ but deterioration in another, 3 had no changes, and 6 had progression. Six responses were achieved after one infusion of humanized anti-Tac, and 2 responses were achieved after the second infusion. Nine of 16 patients (56%) had response to treatment of skin GVHD; complete response in 5 and partial response in 4. Three of 15 patients (20%) responded to treatment of liver GVHD; complete response in 1 and partial response in 2. Six of 12 patients (50%) responded to treatment of gastrointestinal GVHD; complete response in 4 and partial response in 2. One patient with a complete overall response did not develop chronic GVHD and is alive on day 645, off immunosuppression. Seven pa- tients with complete or partial overall responses later devel- oped chronic GVHD. One is alive on day 529, 2 died after relapse of leukemia, and 4 died with pneumonia on days 100, 112, 170, and 21 1 after treatment with humanized anti- Tac. Twelve patients who failed to respond to humanized anti-Tac died of organ failure, infection, or bleeding at a median of 40 (range, 5 to 98) days after treatment with humanized anti-Tac. Median patient survival was 76 days after treatment with humanized anti-Tac. Causes of death are listed in Table 2. Among the 4 patients who did not have a biopsy performed to confirm the clinical diagnosis of acute GVHD, 2 patients achieved a complete response and sur- vived at least l year, and the other 2 patients had progression of GVHD and died.

Pharmacokinetics of humanized anti-Tac. Mean serum concentrationhime curves for patient groups treated at differ- ent dose levels and results of noncompartmental pharmacoki- netic analysis are shown in Fig 1. Observed maximum serum concentration (Cmax) values were predictable for each dose of humanized anti-Tac administered. AUC o + m values showed a dose-proportional increase with increasing doses. Terminal elimination half-life values showed high intersub- ject variability (44 to 360 hours), with the overall harmonic mean half-life being 88 hours. The mean VdP was 83 (range, 50 to 167) mLkg, greater than the plasma volume, indicating tissue penetration. Systemic serum clearance values were variable among patients, and the mean values were low (CL, 0.6 [range, 0.21 to 1.251 ml/kg/h). Values obtained from patients after a first or a second infusion of antibody had similar clearance values (Fig 2).

Human antibody formation against humanized anti-Tac. No human antibody responses against humanized anti-Tac were detected by either ELISA or dot blot assays in any of the 15 patients who survived at least 28 days after therapy and were tested weekly through day 56 after infusion of the antibody.

Circulating soluble IL-2Ra. Levels of soluble IL-2Ra antigen circulating free in the serum varied widely patient to patient, from 170 to 23,328 (median, 3,116) U/mL, and did not correlate with severity of acute GVHD at the time of treatment with humanized anti-Tac (data not shown). By day 28, soluble IL-2Ra levels decreased in 4 of 8 patients who showed improvement of GVHD and increased 6 of 7 patients who did not show improvement of GVHD.

Circulating peripheral blood lymphocytes. Absolute counts of CD3+, CD4+, CD8+, and CD16+/CD56+ lympho- cytes were low before treatment, but did not decrease sig- nificantly after the infusion of humanized anti-Tac. Cell counts remained low for at least 56 days after treatment. Three patients studied between 300 and 360 days after treat- ment showed increasing counts of each lymphocyte subset (Fig 3). Expression of IL-2Ra or P on the surface of circulat- ing lymphocytes was not affected by treatment with human- ized anti-Tac (Fig 4). The IL-2Ra epitope recognized by humanized anti-Tac was saturated on the cells of all patients who were studied on day 7 after treatment and on the cells of most patients studied on day 28 (Fig 4, D). Staining of freshly isolated lymphocytes with an antihuman globulin showed that cells from patients isolated 7 through 28 days after treatment were coated with humanized anti-Tac anti- body (Fig 5) . Lymphocyte-bound antibody was no longer detectable in most patients studied on day 56 after treatment with humanized anti-Tac.

DISCUSSION

Clinical improvement of GVHD was documented in 40% of the patients treated with one or two infusions of human- ized anti-Tac. This rate of response is similar to the rate of 41% previously reported by our group in 420 patients after second-line therapy of GVHD using multiple doses of gluco- corticoids, cyclosporine, or polyclonal anti-thymocyte glob- ulin,' and appears to be better than the 9% response rate observed in 11 patients treated with seven infusions of an IL-2Ra-specific murine antibody. I ' Administration of hu- manized anti-Tac appeared to be safe, and adverse patient outcome was caused by limited efficacy and progression of GVHD rather than to therapy-related toxicity. Median pa- tient survival was 76 days in this study, compared with 27 and 29 days in two preceding studies of glucocorticoid-re- fractory GVHD at our institution in which patients were treated with murine monoclonal anti-T-cell antibodies spe- cific for IL-2Ra or C D ~ E . " , ~ ~ Four of 20 patients (20%) in the current study recovered completely from acute GVHD, 2 are alive after more than 1 year, and 2 died of leukemia relapse. In published reports of phase I/II studies of various anti-T-cell murine MoAbs tested for anti-GVHD activity (ie, anti-CD3 antibodies OKT3,25 64.1,26 or BC324; IL-2Ra- specific antibodies 2A3" or BT563"; or anti-CD5 immuno- toxin H65-RTA2'), 18 of 108 patients (17%) with glucocor- ticoid-resistant GVHD survived a minimum of 4 months.

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1324 ANASETTI ET AL

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GVHD TREATMENT WITH HUMANIZED ANTI-TAC

loo/

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0 5 10 15 20 25 30

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Fig 2. Mean k SEM wrum concentrations of humanized anti-Tac in unique patient nos. (UPN) 6789,6660,5477,6946,7056, and 6605 after the (W) first or (AI second infusion.

Better survival might be expected in patients treated earlier with less advanced GVHD, as suggested by a study of IL- 2Ra-specific antibody BB-l0 in which 14 of 32 patients (44%) were surviving at the time of the report with a follow- up between 2 and 14 months after treatment."

Incomplete therapeutic success of humanized anti-Tac in this first trial might be related to the study design that limited the use of the antibody to one or two infusions to carefully evaluate acute safety and terminal half-life of the antibody. Because neutralizing antibodies were not developed by any patient, there is a rationale to believe that administration of multiple doses may achieve a better therapeutic effect. An- other major factor that might have limited the therapeutic efficacy of humanized anti-Tac is the advanced stage of

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Fig 4. Mean k SEM percentages of IL-2Ra' or IL-2RBf lympho- cytes isolated from peripheral blood from all patients alive a t the time indicated. Total lL-2Ra expression (767 epitope) was measured by staining cells with fluorascin-conjugated antibody 767, which binds to an IL-2Ra epitope distinct from the epitope recognized by humanized anti-Tac. Free IL-2Ra antigen recognized by humanized anti-Tac (HAT epitope), but not bound in vivo, was measured by incubating cells with fluoresceinconjugated antibody 2A3. IL-2w (p751 was measured by staining cells with PE-conjugated Mik-p3 antibody. Representative data in UPN 6661 is shown in Fig 5.

GVHD in most patients. IL-2Ra-specific antibodies can efficiently block generation of alloreactive cytotoxic T cells, the putative effectors in GVHD.28-30 However, once GVHD is established, one would expect that mature effector T cells lose expression of IL-2Ra and become less susceptible to the effect of IL-2Ra-specific agents. Humanized anti-Tac may be more effectively used at onset of GVHD, and perhaps even better if used for prevention rather than treatment of established GVHD.

Experiments in nonhuman primates have suggested that humanized anti-Tac might be more immunosuppressive than murine antibodies directed towards the same ~pecificity.'~ Humanized anti-Tac has a half-life of 94 hours at the highest dose studied, as opposed to approximately 40 hours for mu- rine antibodie~.".'~ In this study, there was no antibody for- mation against humanized anti-Tac, in contrast to 4 of 8 patients treated with murine anti-IL-2Ra antibody 2A3.I' The observation that the rates of antibody elimination were similar for the first and the second dose is consistent with the concept that neutralizing antibodies were not formed after the first infusion. The lack of immunogenicity of hu- manized anti-Tac in this study cannot be generalized with confidence in view of the profound immunodeficiency of patients early after allogeneic marrow transplantation.

Whether humanized anti-Tac destroys IL-2Ra+ cells in vivo could not be determined by our study. Administration of humanized anti-Tac induced a small, insignificant de- crease in the number of circulating T cells. In contrast, ad- ministration of either CD3-specific murine antibody OKT3," CD5-specific immunotoxin H65-RTA," or CD52-specific humanized antibody Campath-lH3' is associated with pro- found and long-lasting lymphopenia. Circulating IL-2Ra+

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1326

DAYS AFTER INlTlATlON OF TREATMENT CONTROL ANTIBODY 0 9 28 56

ANASETTI ET AL

Fig 5. Flow cytometric analysis of peripheral blood lymphocytes of UPN 6661 before, during, and after treatment with 1.5 mglkg humanized anti-Tac. Total expression of p55 llL-2Ra) was measured by staining cells wiM FlTCconjugated 7G7/B6 antibody and by humanized anti-Tac plus PE-conjugated anti- human IgG1. p55 bound by humanized anti-Tac in vivo was measured by staining cells using antiglobu- lin alone; free p55 was measured by staining cells using PE-conjugated humanized anti-Tac; and ex- pression of IL2Rf.3 (p751 antigen was measured by staining with PE-conjugated Mik-p3 antibody.

cells are bound by humanized anti-Tac up to 4 weeks after a single infusion, indicating that binding activity of the anti- body is long lasting in vivo. This observation does not ex- clude the possibility that humanized anti-Tac might induce killing of IL-2Ra+ cells, because newly activated T cells may be generated and enter circulation at the same rate that others are desuoyed.

Pharmacokinetic studies demonstrated that the terminal elimination half-life of humanized anti-Tac had a harmonic mean value of approximately 4 days. This value appears similar to those reported for another humanized antibody specific for a tumor antigen (100 or for human cyto- megalovirus (CMV)-specific Igs administered in the post- transplant period (5 to 10 day^),'^ but is lower than values reported in patients receiving CMV-specific human MoAbs (13 to 17 days)34 or other human Igs (20 days).” Antibody clearance is determined by two distinct processes: binding to the specific antigen and antigen-independent breakdown of the antibody molecule. High intersubject variability makes comparison of half-life values difficult between dose groups. The AUC o + of humanized anti-Tac increased in propor- tion to the dose administered, and it is possible that not all antigen binding sites were saturated even at the highest dose tested, and that administration of a higher dose of antibody may be required to achieve saturation. However, it remains to be tested whether therapeutic efficacy requires a saturating dose of antibody.

The data presented here provide the basis for initiating clinical trials investigating the effectiveness of multiple doses of humanized anti-Tac for prevention or treatment of GVHD and for exploring the activity of this agent in other disorders that might benefit from selective T-cell immuno- suppression.

ACKNOWLEDGMENT

The authors are grateful to Diane Doles, RN, Ann Klosterman, RN, and Michele Tesler, RN, for the care of the patients and the

collection of clinical data; to Lars Lindhardt for data management; to Ping Lin and Robert Pilson for preparing and characterizing fluo- rescent reagents; and to Ursula Griffin for assistance in the prepara- tion of this manuscript.

REFERENCES

1. Storb R, Deeg HJ, Whitehead J, Appelbaum F, Beatty P, Ben- singer W, Buckner CD, Clift R, Doney K, Farewell V, Hansen J, Hill R, Lum L, Martin P, McGuffin R, Sanders J, Stewart P, Sullivan K, Witherspoon RP, Yee G, Thomas E: Methotrexate and cyclosporine compared with cyclosporine alone for prophylaxis of acute graft versus host disease after marrow transplantation for leu- kemia. N Eng J Med 314:729, 1986

2. Nash RA, Pepe MS, Storb R, Longton G, Pettinger M, Anasetti C, Appelbaum FR, Bowden RA, Deeg HJ, Doney K, Martin PJ, Sullivan KM, Sanders JE, Witherspoon RP: Acute graft-versus-host disease: Analysis of risk factors after allogeneic marrow transplanta- tion and prophylaxis with cyclosporine and methotrexate. Blood 80: 1838, 1992

3. Anasetti C, Beatty PG, Storb R, Martin PJ, Mori M, Sanders JE, Thomas ED, Hansen JA: Effect of HLA incompatibility on graft- versus-host disease, relapse and survival after marrow transplanta- tion for patients with leukemia or lymphoma. Human Immunol 29:79, 1990

4. Martin PJ, Schoch G, Fisher L, Byers V, Anasetti C, Appel- baum FR, Beatty PF, Doney K, McDonald GB, Sanders JE, Sullivan KM, Storb R, Thomas ED, Witherspoon RP, Lomen P, Hannigan J, Hansen JA: A retrospective analysis of therapy for acute graft-ver- sus-host-disease: Initial treatment. Blood 76: 1464, 1990

5. Martin PJ, Schoch G, Fisher L, Byers V, Appelbaum FR, Mc- Donald GB, Storb R, Hansen JA: A retrospective analysis of therapy for acute graft-versus-host disease: Secondary treatment. Blood 77:1821, 1991

6. Minami Y, Kono T, Miyazaki T, Taniguchi T: The IL-2 recep- tor complex: Its structure, function, and target genes. Annu Rev Immunol 1 I :245, 1993

7. Noguchi M, Yi H, Rosenblatt HM, Filipovich AH, Adelstein S, Modi SW, McBride OW, Leonard WJ: Interleukin-:! receptor gamma chain mutation results in X-linked severe combined immuno- deficiency in humans. Cell 73:147, 1993

For personal use only. by guest on July 13, 2011. bloodjournal.hematologylibrary.orgFrom

GVHD TREATMENT WITH HUMANIZED ANTI-TAC 1327

8. Strom TB, Kelley VR, Murphy JR, Nichols J, Woodworth TG: Interleukin-2 receptor-directed therapies: Antibody or cytokine- based targeting molecules. Annu Rev Med 44:343, 1993

9. Kirkman RL, Shapiro ME, Carpenter CB, McKay DB, Milford EL, Ramos EL, Tihey NL, Waldmann TA, Zimmerman CE, Storm TB: A randomized prospective trial of anti-Tac monoclonal antibody in human renal transplantation. Transplantation 51:107, 1991

10. Herve P, Wijdens J, Bergerat JP, Bordigoni P, Milpied N. Cahn JY, Clement C, Beliard R, Morel-Founier B, Racadot E, Troussard X, Benz-Lemoine E, Gaud C, Legros M, Attal M, Kloft M, Peters A: Treatment of corticosteroid resistant acute graft-versus- host disease by in vivo administration of anti-interleukin-2 receptor monoclonal antibody (B-B10). Blood 75:1017, 1990

11. Anasetti C, Martin PJ, Hansen JA, Appelbaum FR, Beatty PG, Doney K, Harkonen S, Jackson A, Reichert T, Stewart P, Storb R, Sullivan KM, Thomas ED, Warner N, Witherspoon RP: A phase 1-11 study evaluating the murine anti-IL-2 receptor antibody 2A3 for treatment of acute graft-versus-host disease. Transplantation 50:49, 1990

12. Blaise D, Olive D, Him M, Vines P, Lafage M, Attal M, Stoppa AM, Gabert J, Gastaut JA, Camerlo J, Mannoni P, Mawas C, Maraninchi D: Prevention of acute GVHD by in vivo use of anti- interleukin-2 receptor monoclonal antibody (33B3.1): A feasibility trial in 15 patients. Bone Marrow Transplant 8:105, 1991

13. Queen C, Schneider WP, Selick HE, Payne PW, Landolfi NF, Duncan JF, Avdalovic NM, Levitt M, Junghans RP, Waldmann TA: A humanized antibody that binds to the interleukin 2 receptor. Proc Natl Acad Sci USA 86:10029, 1989

14. Junghans RP, Waldmann TA, Landolfi NF, Avdalovic NM, Schneider WP, Queen C: Anti-Tac-H, a humanized antibody to the interleukin 2 receptor with new features for immunotherapy in malig- nant and immune disorders. Cancer Res 50:1495, 1990

15. Brown PS Jr. Parenteau GL, Dirbas FM, Garsia RJ, Goldman CK, Bukowski MA, Junghans RP, Queen C, Hakimi J, Benjamin WR, Clark RE, Waldman TA: Anti-Tac-H, a humanized antibody to the interleukin 2 receptor, prolongs primate cardiac allograft sur- vival. Proc Natl Acad Sci USA 88:2663, 1991

16. Hakimi J, Chizzonite R, Luke D, Familletti PC, Bailon P, Kondas JA, Pilson RS, Lin P, Weber DV, Spence C, Mondini LJ, Tsien WH, Levin JL, Gallati VH, Korn L, Waldman TA, Queen C, Benjamin W: Reduced immunogenicity and improved pharmacoki- netics of humanized anti-Tac in cynomolgus monkeys. J Immunol 147:1352, 1991

17. Buckner CD, Clift RA, Appelbaum FR, Storb R, Fefer A, Petersen FB, Sanders JE, Sullivan K, Thomas ED, Hansen JA: Ef- fects of treatment regimens on post marrow transplant relapse. Semin Hematol 28:32, 1991 (suppl 4)

18. Beatty PG, Anasetti C, Hansen JA, Longton GM, Sanders JE, Martin PJ, Mickelson EM, Choo SY, Petersdorf EW, Pepe MS, Appelbaum FR, Bearman SI, Buckner CD, Clift RA, Petersen FB, Singer J, Stewart PS, Storb RS, Sullivan KM, Tesler MC, With- erspoon RP, Thomas ED: Marrow transplantation from unrelated donors for treatment of hematologic malignancies: Effect of mis- matching for one HLA locus. Blood 81:249, 1993

19. Santos GW, Tutschka PJ, Brookmeyer R, Sara1 R, Beschorner WE, Bias WB, Braine HG, Bums WH, Farmer ER, Hes AD, Kaizer H, Mellitis D, Sensenbrenner LL, Stuart R, Yeager AM: Cyclosporine plus methylprednisolone versus cyclosphosphamide plus methylprednisolone as prophylaxis for graft-versus-host dis- ease: A randomized double-blind study in patients undergoing allo- geneic marrow transplantation. Clin Transplant 1:2l, 1987

20. Byers VS, Henslee PJ, Kernan NA, Blazar BR, Gingrich R, Phillips GL, LeMaistre CF, Gilliland G, Antin JH, Martin P, Tutscha PJ, Trown P, Ackerman SK, O’Reilly RJ, Scannon PJ: Use of anti- pan T-lymphocyte ricin a chain immunotoxin in steroid-resistant acute graft-versus-host disease. Blood 75: 1426, I990

21. Rubin LA, Kurman CC, Biddison WE, Goldman ND, Nelson DL: A monoclonal antibody 7G7/B6, binds to an epitope on the human interleukin-2 (IL-2) receptor that is distinct from that recog- nized by IL-2 or anti-tac. Hybridoma 4:91, 1985

22. Hakimi J, Seals C, Anderson LE, Podlaski FJ, Lin P, Danho W, Jenson JC, Perkins A, Donadio PE, Familletti PC, Pan Y-CE, Tsien WH, Chizzonite RA, Casabo L, Nelson DL, Cullen BR: Bio- chemical and functional analysis of soluble human interleukin-2 receptor produced in rodent cells. J Biol Chem 262:17336, 1987

23. Tsudo M, Kitamura F, Miyasaka M: Characterization of the interleukin 2 receptor p chain using three distinct monoclonal anti- bodies. Proc Natl Acad Sci USA 86:1982, 1989

24. Anasetti C, Martin PJ, Storb R, Appelbaum FR, Beatty PG, Davis J, Doney K, Hill HF, Stewart P, Sullivan KM, Witherspoon RP, Thomas ED, Hansen JA: Treatment of acute graft-versus-host disease with a nonmitogenic anti-CD3 monoclonal antibody. Trans- plantation 54:844, I992

25. Gratama JW, Jansen J, Lipovich RA, Tanke HJ, Goldsetin G, Zwaan FE: Treatment of acute graft-versus-host disease with anti-CD3 monoclonal antibodies. Am J Kidney Dis 1 1 : 149, 1988

26. Martin PJ, Hansen JA, Anasetti C, Zutter M, Dumam D, Storb R, Thomas ED: Treatment of acute GVHD with anti-CD3 monoclonal antibodies. Am J Kidney Dis 11:149, 1988

27. Cuthbert RJG, Phillips GL, Barnett MJ, Nantel SH, Reece DE, Shepherd JD, Klingemann HG: Anti-interleukin-2 receptor monoclonal antibody (BT 563) in the treatment of severe acute GVHD refractory to systemic corticosteroid therapy. Bone Marrow Transplant 10:45 I , 1992

28. Uchiyama T, Nelson DL, Fleisher TA, Waldmann TA: A monoclonal antibody (anti-Tac) reactive with activated and function- ally mature human T cells. J Immunol 126:1398, 1981

29. Depper J, Leonard W, Waldmann TA, Greene W: Blockade of interleukin-2 receptor by anti-Tac antibody: Inhibition of human lymphocyte activation. J Immunol 131:690, 1983

30. Tan P, Anasetti C, Martin PJ, Hansen JA: Alloantigen-specific T suppressor-inducer and T suppressor-effector cells can be activated despite blocking the IL-2 receptor. J Immunol 145:485, 1990

3 1. Isaacs JD, Watts RA, Hazleman BL, Hale G, Keogan MT, Cobbold SP, Waldmann H: Humanized monoclonal antibody therapy for rheumatoid arthritis. Lancet 340:748, 1992

32. LoBuglio AF, Wheeler RH, Trang J, Haynes A, Rogers K, Harvey EB, Sun L, Ghrayeb J, Khazaeli MB: Mousehuman chimeric monoclonal antibody in man: Kinetics and immune response. Proc Natl Acad Sci USA 86:4220, 1989

33. Rand KH, Houck H, Ganju A, Babington RG, Elfenbein GJ: Pharmacokinetics of cytomegalovirus specific IgG antibody follow- ing intravenous immunoglobulin in bone marrow transplant patients. Bone Marrow Transplant 4:679, 1989

34. Drobyski WR, Gottlieb M, Carringan D, Ostberg L, Grebenau M, Schran H, Magid P, Ehrlich P, Nadler PI, Ash RC: Phase I study of safety and pharmacokinetics of a human anticytomegalovirus monoclonal antibody in allogeneic bone marrow transplant recipi- ents. Transplantation 5 1 : 1 190, 1991

35. Morel1 A, Terry WD, Waldmann TA: Metabolic properties of IgC subclasses in man. J Clin Invest 49:673, 1970

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