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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 activation of human T lymphocytes. The safety, pharmacokinetics, and immunosuppressive activity of humanized anti-Tac
were evaluated in 20 patients who developed acute graftversus-host disease (GVHD) after allogeneic marrow transplantation. Patients had developed acute GVHD at 5 t o 26
(median, 14) days after transplantation and had failed t o
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 sec11 t o 48
ond infusion of antibody was administered after
(median, 16) days in 8 patients who hadtransient improvement of GVHD after the first infusion. Acute side effects,
limited t o chills in 1patient anddiaphoresis in another, were
observed during or shortly
after the antibodyinfusion. Overall 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 improvementin
one organ but progression in another. Responses occurred
G
RAFT-VERSUS-HOST disease (GVHD) is a frequent
complication of allogeneic marrow transplantation
caused by donor T lymphocytes reactive to histocompatibility 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 interleukin-2 (IL-2).6 Binding of IL-2 to its receptor (IL-2R)
isan essential requirement for T-cell proliferation. Highaffinity IL-2Rs are complexes ofat least three proteins, a
p55 IL-2Ra,a p70 IL-2RP, and a p64 IL-2Ry.IL-2Ra
and IL-2RP, butnot IL-2Ry, are capable of binding IL-2
independently of the others, but with a much lower affinity
From the Clinical Research Division, Fred Hutchinson Cancer
Research Center, Department of Medicine, Division of Oncology,
University of Washington, Seattle WA; the Metabolism Branch, National 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 Disease.
Address reprint requests to Claudio Anasetti, MD,Fred Hutchinson Cancer Research Center, I124 Columbia St, E61 I , Seattle, WA
98104.
The publication costsof this article were defrayedin 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 caseswith skin disease, 3 of 15 with liver disease,
and 6 of 12 with gastrointestinal disease. Two patientssurvive at 529 and 645 days after antibody treatment. Two patients 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 theantibody was
44 t o 363 hours, with a harmonic mean of 79, 88, and 94
hours, respectively, for the three doses studied. Absolute
peripheral blood T-lymphocyte counts remainedunchanged
during the56 days after infusion of theantibody. A fraction
of circulatingT cells expressed the a! chain ofthe IL-2 receptor that, in some patients, was bound by antibody in vivo
up to 28 days after treatment. No patient developed a measurable antibody response t o humanized anti-Tac. Humanized anti-Tac has a long half-life after intravenous injection
in humans, superior t o any rodent monoclonal antibody specific for human T cells, and does not appear t o induce antibody formation in recipientsof marrow transplants. Improvement of steroid-refractory GVHD in 40% of patients
after only one or two antibody infusions indicates that humanized 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 Xlinked severe combined immunodeficiency disease in humans.' 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 inactivates IL-2Ra-bearing cells would specifically inhibit nascent or ongoing immune responses without suppressing naturalimmunity or the ability tomount a specific immune
response to an antigen encountered once the immunosuppressive therapy has been terminated.
In rodents, monoclonal antibodies (MoAbs) specific to IL2Ra 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 antibodies exert their immunosuppressive activity include
blocking IL-2 binding to its receptor and elimination of IL2Ra-bearing lymphoid cells. In humans, efficacy of the
murine monoclonal anti-IL-2Ra antibody anti-Tac was
demonstrated in a randomized clinical trial ofprevention
of cadaveric kidney allograft rejection.' Furthermore, three
distinct rodent MoAbs werefound to have some immunosuppressive 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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GVHDTREATMENT WITH HUMANIZED ANTI-TAC
the recipient to the heterotypic protein, and by the inefficient
interaction of the murine antibody with host effector mechanisms. Chimeric MoAbs consisting of human constant regions and mouse variable regions were developed to circumvent these problems. Techniques were developed to more
fully humanize mouse MoAbs by incorporating almost exclusively amino acids present in the complementary determining regions (CDRs) into human framework and Cregion sequences. The humanized anti-Tac is a selectively
modified human IgGl MoAb that binds specifically to IL2Ra.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 cellmediated 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 complement-dependent lysis in vitro.14 Humanized anti-Tac was
found to be less immunogenic, to have more favorable pharmacokinetics, and to be more immunosuppressive than native 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 humanized anti-Tac was not associated with appreciable side effects, 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 immunosuppressive 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 treatment with cyclophosphamide plus total body irradiation or busulfan
or both." Criteria for matching recipients and donors for human
leukocyte antigens (HLA) have been
GVHD prophylactic regimens included cyclosporine alone, cyclosporine and prednisone, 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
111primary GVHD therapy trial using 6-methylprednisolone in combination 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 glucocorticoids 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-
1321
tivity to humanized anti-Tac was tested in all patients by IV administration of 100 pg of antibody without premedication. Patients receiving 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 administered in any patient was 100 mg. Patients whoshowed transient
improvement of GVHD were eligible for retreatment once at a subsequent time with an identical dose of antibody. After thefirst 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 wererecordedweekly
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 wasdefined byan 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 definedby 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 response to humanized anti-Tac if the attending physician or the investigators believed that GVHD was the primary cause of organ dysfunction. Overall, complete response wasdefinedas resolution of
GVHD in all evaluable involved organs with no additional treatment
administered for acute GVHD. Partial response was defined as improvement in at least one evaluable organ without deterioration in
any other. Overall progression wasdefinedas 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 reported." 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 recognized by humanized anti-Tac.".'* Cells coated by humanized antiTac 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 evaluated 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-binding epitopes were also evaluated by comparing the fluorescent intensity 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
6467
6469
6498
6523
6535
6483
6568
6590
6606
6661
6696
6500
6789
6650
5477
6892
6930
6946
7056
6605
46lF
311F
47/M
35lM
49lF
47/F
34lM
361M
38/M
36lF
52lF
32lF
26lF
241M
45lM
32F
40lM
28/M
25lM
51lM
Primary
Disease/Status
Donor
Relation
HLA
Matching
GVHD
Prophylaxis
CMUCP
MDS/RAEB
NHURel
AMURel
CMUAP
NHURel
MDSIRAEBTR
CMUBC
CMUAP
CMUAP
NHURel
MDSIRAEB TA
CMUAP
Unrelated
Sibling
Sibling
Unrelated
Unrelated
Sibling
Sibling
Sibling
Unrelated
Unrelated
Sibling
Sibling
Unrelated
Unrelated
Unrelated
Unrelated
Unrelated
Unrelated
Unrelated
Unrelated
Identical
Identical
Identical
516loci
Identical
Identical
516loci
Identical
Identical
516loci
Identical
Identical
516loci
Identical
Identical
516loci
Identical
Identical
Identical
Identical
CsNMTX
CsA
CsA
CsNMTX
CsNMTX
CsNPred
CsNMTX
CsNMTX
CsNMTX
CsNMTX
CsNPred
CsNPred
CsNMTX
CsNMTX
CsNMTX
CsNMTX
CsNMTX
CsNMTX
CsNMTX
CsNMTX
RA
CMUCP
CMUCP
CMU3rd CP
CMUAP
ALURel
CMUAP
Day of
GVHD
Liver
Onset
GVHD Grade at Time of Antibody
Therapy
Skin
8
4
14
7
11
2
2
13
19
10
14
16
10
23
0
0
2
2
3
4
0
0
4
2
0
4
0
2
0
3
3
2
4
16
6
3
4
0
14
26
21
0
3
21
21
4
0
3
IV
111
I
II
3
0
0
3
3
3
111
IV
IV
IV
111
111
IV
IV
111
111
111
Ill
111
Ill
111
111
0
3
3
0
3
0
0
3
1
3
3
3
5
Overall
3
1
3
3
21
GI
0
0
3
0
0
3
2
1
Abbreviations: ALL, acute lymphocytic leukemia; AML, acute myeloid leukemia; AP, accelerated phase; BC, blast crisis; CML. chronic myelogenous 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 antigen; 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 enzymelinked 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 reagent. Plates were then incubated with serum containing soluble IL2Ra peptide and then washed. The detecting antibody was peroxidase-labeled humanized anti-Tac. The normal level of soluble
IL-2Ra in healthy volunteers is 200 to 500 U/mL, where 1 U equals
0.2 pg.
Human antibody against humanized anti-Tac. Formation of human 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 peroxidase-conjugated humanized anti-Tac as the detecting reagent. The
concentration of antibodies to humanized anti-Tac could be determined from a standard curve of purified goat antibodies specific to
humanized anti-Tac. The lower limit of detection was 7.8 p g L . The
second assay used was a dot blot assay. Complexes of specific
antibodies and humanized anti-Tac were captured by protein G Seph+
arose and then dissociated from protein G Sepharose and from each
other by treatment with pH 2.5 buffer. Dissociated Igs were immobilized 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-conjugated humanized anti-Tac. The concentration of each antibody was
determined from standard curves of mixtures of humanized antiTac and goat antibodies to humanized anti-Tac. The lower limit of
sensitivity of the dot blot assay for antibodies to humanized antiTac and for humanized anti-Tac were 3 12 and 80 p g L , respectively.
loo
l
Dose (mg/kg)
* 0.5
Cmax (mg/L)
T 1/2
AUC
0.Oll
0
10
20
(hours)
9.75
26.5
41.5
33.9
94
88
79
(rng/L*h) 778
30
* 1 .O
1886
40
I
431 0
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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GVHDTREATMENTWITH
1323
HUMANIZED ANTI-TAC
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 second 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 chemistries 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 patients with complete or partial overall responses later developed 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 antiTac. 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 survived 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 different dose levels and results of noncompartmental pharmacokinetic 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 intersubject 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 to1.251 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+ lymphocytes were low before treatment, but did not decrease significantly 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 treatment showed increasing counts of each lymphocyte subset
(Fig 3). Expression of IL-2Ra or P on the surface of circulating lymphocytes was not affected by treatment with humanized 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 antibody (Fig 5 ) . Lymphocyte-bound antibody wasno 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 humanized 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 glucocorticoids, cyclosporine, or polyclonal anti-thymocyte globulin,'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 humanized 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 patient survival was 76 days in this study, compared with 27
and 29 days in two preceding studies of glucocorticoid-refractory GVHD at our institution in which patients were
treated with murine monoclonal anti-T-cell antibodies specific 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 OKT3,2564.1,26or BC324;IL-2Raspecific antibodies 2A3" or BT563"; or anti-CD5 immunotoxin H65-RTA2'), 18 of 108 patients (17%) with glucocorticoid-resistant GVHD survived a minimum of 4 months.
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
ANASETTI ET AL
1324
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From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1325
GVHD TREATMENT WITH HUMANIZED ANTI-TAC
HAT epitope
7G7 epitope 0p75
loo/
Q
..
>
..e
30
ae
S 20
E
10
l
l
0.1
200
25
15
5
10
Days after Infusion
Better survival might be expected in patients treated earlier
with less advanced GVHD, as suggested by a study of IL2Ra-specific antibody BB-l0 in which 14 of 32 patients
(44%) were surviving at the time of the report with a followup 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. Another major factor that might have limited the therapeutic
efficacy of humanized anti-Tac is the advanced stage of
day on study
.
E
6
rl
1,000
0 m 7 0 2 8 m 5 6 0300
h
P
CD4
0
7
28
56
300
Day on study
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.
10,Ooo
n
"
CD3
NK
lymphocyte subset
Fig 3. Mean k SEM absolute counts of peripheral blood lymphocyte subsets bdore treatment In = 161in patients and after treatment
with humanized anti-Tacat 7 (n = 15),28 In = 81, 56 In = 61, and 300
days (n = 31.
Fig 4. Mean k SEM percentages of IL-2Ra' or IL-2RBf lymphocytes 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
(p751wasmeasured by staining cells with PE-conjugated Mik-p3
antibody. Representative datain 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 murine antibodie~.".'~In this study, there was no antibody formation 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 notformed
after the first infusion. The lack of immunogenicity of humanized anti-Tac in this study cannot be generalized with
confidence inviewof
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 decrease in the number of circulating T cells. In contrast, administration of either CD3-specific murine antibody OKT3,"
CD5-specific immunotoxin H65-RTA," or CD52-specific
humanized antibody Campath-lH3' is associated with profound and long-lasting lymphopenia. Circulating IL-2Ra+
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1326
ANASETTIETAL
DAYS AFTER INlTlATlONOF TREATMENT
CONTROL
ANTIBODY
0
9
28
56
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 antihuman IgG1. p55 bound by humanized anti-Tac in
vivo was measuredby staining cells using antiglobulin alone; free p55 was measured by staining cells
usingPE-conjugated humanized anti-Tac; and expression 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 antibody is long lasting in vivo. This observation does not exclude 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
or for human cytospecific for a tumor antigen (100
megalovirus (CMV)-specific Igs administered in the posttransplant period (5 to 10 day^),'^ but is lower than values
reported in patients receiving CMV-specific human MoAbs
(13 to 17 days)34or 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 betweendose groups.
The AUC o + of humanized anti-Tac increased in proportion 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 immunosuppression.
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 fluorescent reagents; and to Ursula Griffin for assistance in the preparation of this manuscript.
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From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1994 84: 1320-1327
Treatment of acute graft-versus-host disease with humanized
anti-Tac: an antibody that binds to the interleukin-2 receptor
C Anasetti, JA Hansen, TA Waldmann, FR Appelbaum, J Davis, HJ Deeg, K Doney, PJ Martin, R
Nash and R Storb
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