1. No category

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1995 86: 4063-4075
Radioimmunotherapy of interleukin-2R alpha-expressing adult T-cell
leukemia with Yttrium-90-labeled anti-Tac [see comments]
TA Waldmann, JD White, JA Carrasquillo, JC Reynolds, CH Paik, OA Gansow, MW Brechbiel,
ES Jaffe, TA Fleisher and CK Goldman
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Radioimmunotherapy of Interleukin-2Ra-Expressing Adult T-cell
Leukemia With Y ttrium-90- Labeled Anti-Tac
By Thomas A. Waldmann, Jeffrey D. White, Jorge A. Carrasquillo, James C. Reynolds, Chang
H. Paik,
Otto A. Gansow, Martin W. Brechbiel, Elaine S. Jaffe, Thomas A. Fleisher, Carolyn K. Goldman, Lois E. Top,
Richard Bamford, Sara Zaknoen, Eric Roessler, Claude Kasten-Sportes, Richard England, Hariklia Litou,
John A. Johnson, Terri Jackson-White, Angela Manns, Barrie Hanchard, Richard P. Junghans, and David L. Nelson
Adult T-cell leukemia (ATL) is a malignancy of mature lymphocytescaused by the retrovirus humanT-celllymphotropic virus-l. It is an aggressive leukemia with a median
survival time of 9 months; no chemotherapy regimen appears successful in inducing long-term disease-free survival.
The scientific basis of the present study is that ATL cells
express high-affinityinterleukin-2receptorsidentifiedby the
anti-Tac monoclonal antibody, whereas
normal resting cells
do not. To exploit this difference, we administered anti-Tac
to 18 patients with ATL initially
armed with Yttrium-90 (9)
(first 9 patients) in a phaseI dose-escalationtrial and subsequently (second groupof 9 patients) in a phase II trial involving a uniform IO-mCi dose of S‘V-labeled anti-Tac. Patients
undergoing a remission were permitted to receive up to
eight additional doses.At the 5- to 15-mCi doses used, 9 of
16 evaluable patients responded to ’‘V anti-Tac with a partial
(7 patients) or complete (2 patients) remission.Theresponses observed represent improved efficacy in terms of
length of remission when compared with previous results
with unmodified anti-Tac. Clinically meaningful (rgrade 3)
toxicity was largely limited to the hematopoietic system.In
conclusion, radioimmunotherapy with 9 anti-Tac directed
toward the IL-2R expressed on ATL cellsmay provide auseful approach for treatment of this aggressive malignancy.
This is a US government work. There are no resfricfions on
ifs use.
T
receptor leads to an autocrine stimulation of the proliferation
of the HTLV-I-infected cells.”
The observation that IL-2Ra is not expressed by normal
resting cells but is expressed by ATL cells provided the
scientific basis for IL-2R-directed therapy of this neoplastic
disease. ATL is an aggressive malignancy of lymphocytes
that display a multilobulated nucleus and express a CD3+,
CD4+, CD8-, CD7-, and CD25+ (IL-2Ra, Tac+) phenotype.’.’6 Patients with ATL have serum antibodies to HTLVI and manifest monoclonal integration of this retrovirus in
their circulating malignant cells. Principal clinical features
include lymphadenopathy; hepatosplenomegaly; and skin,
central nervous system, and pulmonary inv~lvement.‘~
Patients with acute ATL manifest a striking degree of immunosuppression and develop opportunistic infections. Several
oncology groups, including the Lymphoma Study Group of
Japan, have reported a median survival time of 9 months in
the acute type of ATL and of 24 months in chronic ATL.”
Shimoyama and members of the Lymphoma Study Group’’
stated in 1991 that “the various combination chemotherapies
so far developed have not increased significantly the survival
of patients with ATL.”
In light of the disappointing results using conventional
HE DEVELOPMENT OF monoclonal antibody (MoAb)
technology by Kohler and Milstein’ rekindled interest
in the use of antibodies targeted to cell surface antigens to
treat cancer patients. However, MoAbs are just beginning to
fulfill the promise for immunotherapy inherent in their great
specificity for recognizing and selectively binding to abnormal cells. A number of factors underlie the low therapeutic
efficacy observed initially. Most of the mouse MoAbs used
were not cytocidal against neoplastic cells in humans. In
addition, in most cases, the antibodies used were not directed
against a vital cell surface structure such as a receptor for a
growth factor that is required for both tumor cell proliferation
and the prevention of apoptotic cell death induced by factor
deprivation.
We readdressed this issue using the interleukin-2 receptor
(IL-2R) as the target for immune interventi~n.~.~
The IL-2R
consists of at least three IL-2 binding subunits, ie, IL-2Ra,
IL-2Rp, and I L - ~ R Y .Identification
~-~
and characterization
of the IL-2Ra subunit was facilitated by our development
of an MoAb, anti-Tac, that binds to IL-2Ra and prevents its
interaction with L-2. Resting normal T cells, B cells, and
monocytes do not display IL-2Ra. In contrast to this lack
of IL-2Ra expression in
normal
resting mononuclear
cells, this receptor subunit is expressed by a proportion of
the abnormal cells in certain fonns of leukemia and lymphoma.’”*
We have focused our therapeutic studies of malignancy
on the distinct form of human T-cell lymphotropic virus
I (HTLV-I)-associated adult T-cell leukemia (ATL).3-’3,’4
Virtually all leukemic cells of patients with HTLV-I-associated ATL constitutively express 10,000 to 35,000 IL-2Ra
receptors per cell.’s316
An analysis of HTLV-I and its protein
products suggests a potential mechanism for the association
between HTLV-I and constitutive IL-2Ra expression on
ATL c e l l ~ .The
~ ~retrovirus
~’~
HTLV-I encodes a transactivating protein, tax, that plays an important role in the early
phases of HTLV-I-associated malignancy by indirectly inducing the expression of the cellular genes that encode IL2 and IL-2Ra. The induced expression of both IL-2 and its
Blood, Vol86, No 11 (December l), 1995: pp 9063.4075
From ?he Metabolism Branch, the Radiarion Oncology Branch,
the Laboratory of Pathology, and the Viral Epidemiology Branch,
National Cancer Institute, and the Clinical Pathology Department
and Nuclear Medicine Department of the Warren Grant Magnuson
Clinical Center, National institutes of Health, Bethesda, MD; and
the Parhology Department. University of the West Indies, Kingsfon,
Jamaica.
Submitted May 31, 1995; accepted July 21, 1995.
Address reprints to Thomas A. Waldmann, MD,Metabolism
Branch, National Cancer Institute, National Institutes of Health,
Bldg 10, Room 4N115, Bethesda, MD 20892.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely IO
indicate this fact.
This is a US government work. There are no restrictions on its use.
0006-4971/9.5/8611-0014$0.00/0
4063
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4064
WALDMANN ET AL
combination chemotherapy, new approaches to the treatment
of ATL are required. In our clinical trials, we have exploited
the observation that normal resting cells, including the normal T cells of patients withATL, do not display the a
subunit of the IL-2R (IL-2Ra), whereas malignant T cells
display 10,000 to 35,000 IL-2Ra per cell that are identified
by the anti-Tac MoAb.'s,'6 In our initial studies, we administered unmodified murine anti-Tac to patients with ATL in
an effort that was directed toward preventing the interaction
of IL-2 with its growth factor receptor, thereby inducing the
cytokine deprivation form of apoptotic cell death.3 Six of
the 19 treated patients developed a partial (4 patients) or
complete (2 patients) remission.3 Although the use of unmodified anti-Tac for the treatment of ATL wasencouraging,
13 of the 19 patients did not manifest even a partial remission. Furthermore, 5 of the 6 responding patients have relapsed. One possible explanation for failures of unmodified
anti-Tac therapy in certain patients with ATL is the observation that the leukemic cells of most patients in the aggressive
phase of ATL no longer produce IL-2 or require IL-2 for
their proliferation and surviva1.'8320
Nevertheless, the leukemic cells continue to express large numbers of IL-2a receptors. The limited efficacy of unmodified anti-Tac led to an
alternative approach, ie, the use of this agent as a carrier of
cytotoxic substances, including radionuclides. A number of
reviews concerning radionuclide selection and radioimmunotherapy of leukemiallymphoma with armed MoAbs have
been published
The major focus of our program
involving IL-2R-directed MoAbs armed with radionuclides
has been on the use of 90Y linked to anti-Tac.30the
In present
study, 18 patients with ATL have been treated with a total
of 55 doses of "Y-labeled murine anti-Tac, initially (first 9
patients) in a phase I dose-escalation trial and subsequently
(second group of 9 patients) in a phase I1 trial involving 10
mCi of goY-labeledanti-Tac per dose.
MATERIALS ANDMETHODS
Putienf population. Eighteen patients with histologically confirmed HTLV-I-associated ATL were studied (Table 1). Eachof
the patients manifested the following features: ( l ) a histologically
confirmed diagnosis of leukemia or lymphoma of mature T cells
with polymorphic indented or lobulated nuclei; (2) expression of the
Tac antigen (IL-2Ra) on at least 10% of their peripheral blood,
lymph node, or dermal T cells; (3) antibodies to HTLV-I demonstrable in the serum; and (4)no cytotoxic chemotherapy or radiation
therapy during the 4 weeks before entering into the trial. Patients
with or without previous chemotherapy were eligible for inclusion
in this study; 10 patients had received previous chemotherapy (Table
2). Patients with symptomatic central nervous system disease were
excluded; however, patients with malignant cells demonstrable in the
cerebrospinal fluid were included and received intrathecal cytosine
arabinoside andor methotrexate. Patients were required to have a
white blood cell (WBC) count of at least 3,00O/pL, a platelet count
of 75,OOO/pL, and a life expectancy of at least 1 month. In addition,
patients manifesting circulating human antimouse antibodies
(HAMA) were excluded. All patients fulfilling the entry criteria were
included inthe study. The patients rangedin age from 23 to 63
years (mean, 43 years). Five patients were men and 13 were women.
Sixteen were black, 1 was Hispanic, and 1 was of Japanese origin.
Five were from the United States, 5 from Jamaica, 2 from Guyana,
2 from Trinidad, and 1 each from Haiti, Grenada, St Vincent, and
Japan. Using the criteria of the Japanese Lymphoma Study Group,"
11 of the patients with ATL were in the acute stage, 2 manifested
ATL lymphoma, and 5 had chronic ATL.
Assay for antibodies to HTLV-I. The sera of ATL patients were
analyzed for antibodies to disrupted and inactivated HTLV-I using
an enzyme-linked immunosorbent assay (ELISA; Cellular Products,
Buffalo, NY).'
Production of the anti-Tuc MoAb. The anti-Tac MoAb, a mouse
The lots used
IgG2a MoAb, was produced as described previousl~.~
were greater than 99% pure IgG as assessed by high-performance
liquid chromatography (HPLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Chelation and radiolubeling of anti-Tuc. The anti-Tac preparation was conjugated to the 2-(4-isothiocyanatobenzyl)-6-methyl-diethylenetriamine penta-acetic acid (1B4M-DTPA) using the procedures of Brechbiel and Gansow3' and Mirzadeh et al."' Radiolabeling
was performed using *Y for therapy and "'In for imaging. In brief,
approximately 1 mg of conjugated anti-Tac was put into a propylene
vial that served as the reaction vessel and was allowed to react with
w'Y or "'In. Excess DTPAwas then added to complex unreacted
ionic metal isotope and the anti-Tac bound fraction was purified by
preparative size exclusion HPLC using a TSK G-3000 column (30
cm X 2 I .5 mm ID) using 0.05 m o m phosphate buffered saline, pH
6.2, at 5 mL/min. The radioactivity in the final product was more
than 98% protein bound as determined by instant thin-layer chromatography using silica gel impregnated glass fiber sheets (2:2: 1 10%
ammonium fomate in waterhethanoVcitric acid 0.2 mol/L) and by
paper chromatography using 5% HSA pretreated Whatman # l paper
(Whatman, Maidstone, UK). This radiochemical puritywas confirmed using analytical HPLC. All products passed sterility and pyrogen testing.
Therapeutic study plan. All patients were hospitalized andreceived the anti-Tac MoAb labeled with
intravenously over a 2hour period. Nine patients with ATL were initially treated in a phase
I dose-escalation trial. In this phase I trial, groups of 3 patients were
scheduled to receive escalating doses of
anti-Tac, which started
at 5 mCi and then in subsequent groups of patients increased by 5
mCi increments until a maximum tolerated dose not requiring support by bone marrow transplantation was determined. Three patients
each received 5, 10, and 15 mCi 9oY anti-Tac. Nine additional paanti-Tac in a phase I1 trial. The entry
tients received 10 mCi
criteria were identical for the phase I and phase I1 trials. Patients
that manifested a partial or complete remission were eligible to
receive up to eight additional cycles of treatment (with at least 6week intervals between cycles) provided that they did not develop
circulating HAMA. Retreatment was delayed until the blood counts
returned to the range thatwas originally required for entry. Retreatment with
anti-Tac was at the same dose as the initial
therapy for those patients that did not develop grade 2 3 hematopoetic toxicity after the previous dose, whereas it was reduced to 10
or 5 mCi (Table 2) for individuals who had developed grade 3 or
greater toxicity after therapy. Unlabeled unconjugated anti-Tac was
mixed with the radiolabeled anti-Tac to control the total quantity
(in milligrams) of antibody administered. The 9 patients in the initial
phase I study received 10 mg of anti-Tac per infusion. Based on in
vivo pharmacokinetic and bioavailability studies during the phase I
trial (see below), we (R.P.J., J.A.C., D.L.N., C.K.G., and T.A.W.,
unpublished observations) developed an algorithm to predict a dose
of total anti-Tac (sum in milligrams of unlabeled and labeled antibody) that was sufficient to overcome the effect of soluble antigen
levels (ie, sIL-2Ra) without excessively diluting antibody-specific
activity. Based on this algorithm, the 9 patients in the phase 11 trial
received a total quantity of anti-Tac in their initial treatment or
retreatment cycle that was determined by their soluble serum IL-2Ra
levels. Patients with a sIL-2Ra of less than 2,000 UlmL received a
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9@Y
4065
ANTI-TacTHERAPY OF AT1
Table 1. Demographic and Clinical Features of ATL Patients
Patient No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
slL-ZR
(U/mL)
AgeISeWRace
Type of ATL
Chronic
Acute
Acute
Acute
Acute
Chronic
Chronic
Chronic
Acute
Acute
Acute
Acute
Lymphoma
Chronic
Acute
Acute
Acute
Lymphoma
4WlB
32lFlH
24IFlB
55/M/B
34fflB
44/M/B
38ff /B
61lFlB
54FIB
48lFlB
23fflB
51/M/e
45/M/B
63/M/B
52fflA
37ffIB
34lWB
38IFlB
4,026
47,141
2,750
57,626
2,950
2,113
2,938
7,5962.30
2,097
57,065
34,825
81,585
102,266
3.241
20,755
40,927
54,629
4,727
LymphocytesIpL
Serum Ca'
ImmolJL)
14,600
23,800
6,400
20,100
11,200
7,100
8,400
1,100
11,990
5,255
2.32
2.35
4.50
3.45
2.55
6,900
32,600
37,200
6,500
13,900
35,600
14,700
11,100
8,900
36,200
39,300
112,800
5,800
2,105
27.87 5
32,100
1,640
780
14,800
2,590
370
4,820
32,280
32,900
89,720
670
2.60
2.43
IL-ZWlac-Expressing
Circulating
WBCJpL
2.40
2.03
3.51
2.48
2.80
2.29
2.29
2.32
4.30
2.54
Abbreviations: B, black; H, Hispanic; A, Asian.
Normal range for serum calcium is 2.05 to 2.5 rnmol/L.
total dose of 2 mg of anti-Tac, those with 2,OOO to 10,OOO U/mL
received 5 mg of anti-Tac, and those with more than 10,OOO UlmL
received 10 mg of anti-Tac.
Bioavailability, dosimetry, and imaging studies. Before the initiation ofthe 9 anti-Tac therapeutic trial, "'In-labeled anti-Tac
MoAb was administered to 5 patients with ATL on two occasions
in association with total doses of 1 and 50 mg of anti-Tac, respectively, to define the in vivo pharmacokinetics of radiolabeled antiTac and to determine the optimal quantity of anti-Tac to administer
to the ATL patients who manifest sIL-2Ra antigenemia to deliver
the highest fraction of the infused radiolabeled antibody to the tumor
cells. In addition, "lIn-labeled anti-Tac was infused on up to three
occasions per patient in association with 9oY anti-Tac administration
in the phase I and II therapeutic trials. Because soluble IL-2Ra is
present in the circulation, the bioavailability of '"In anti-Tac in ex
vivo plasma was determined before and at various times after infusion. In these studies, the radiolabeled "'In anti-Tac circulating in
the patient's serum was assessed immediately ex vivo for its capacity
to bind to the L-2R-expressing T-cell line HUT-102. In addition,
gamma camera scans were performed using the "'In anti-Tac at the
initiation of the therapeutic trials and during subsequent cycles.
These scans were interpreted by a single experienced reader and
were compared with prestudy physical examination and with other
appropriate radiographic studies.
Computer-assisted analysis of images obtained from patients receiving "'In MoAb, scintigraphic data, and serial pharmacokinetic
estimates were used in the dosimetry for ?-labeled anti-Tac. A
IO-mCi dose of 9 anti-Tac administered to a patient was calculated
to yield radiation of approximately 105 cGy to the marrow, 228 cGy
to the liver, and 336 cGy to the spleen. The calculated radiation
dose to the tumor represents a range of doses across a population
of patients who target 9 radiolabeled antibody to varying extents.
The estimated dose to the tumor with the 10-mCi dose ranged from
140 to 243 cGy.
Evaluation of toxiciry. Toxicity was evaluated according to the
National Cancer Institute's Common Toxicity Criteria. Complete
blood cell and platelet counts were obtained before each infusion
and at 24 and 48 hours, at 6 to 10 days, and at 4 to 6 weeks
after each infusion. Hepatic enzyme studies, renal studies, electrolyte
studies, and urine analysis were performed at 24 hours and weekly
during the phase I study and at 4 to 6 weeks during phase 11.
The serum was assayed for soluble IL-2Ra using an ELISA technique described previously.' Furthermore, the serum was assayed
for human antimurine anti-Tac antibody using a two-arm capture
ELISA technique, as described p r e v i ~ u s l ySerum
. ~ ~ antimurine antiTac levels for a given patient were considered to be meaningfully
increased when the antibody level after therapy was on the linear
part of the curve and was greater than 250 ng/mL.
Tumor response. Tumor response was assessed by physical examination and CAT scan. In addition, pretreatment and posttreatment
"'In anti-Tac imaging studies were evaluated for follow-up of lymph
node, spleen, and skin involvement. Furthermore, the number of
circulating cells expressing leukemic cell phenotype was monitored
by direct and indirect immunocytofluoroscopy using a fluorescenceactivated cell sorter (FAC), as discussed previously.' Two antibodies
(anti-Tac and 7G7/86) that are directed toward different epitopes of
IL-2Ra were used to identify the expression of this receptor subunit.
Other MoAbs used included antibodies that react withhuman Tcell-associated antigens (CD2, CD3, C M , and CD8 [Ortho, Raritan,
NJ, and Becton Dickinson, Mountain View, CA]; CD7 (3A1) a gift
from Dr Barton Haynes [Duke University, Durham, NC]). Fluorescein isothiocyanate (FITC)-labeled goat antimouse IgGandIgM
reagent was obtained from Cal Tag (San Francisco, CA). The absolute number of cells in the circulation per cubic millimeter expressing
a particular antigen was determined from the product of ( l ) circulating WBC count per cubic millimeter, (2) the proportion of circulating
WBCs that were mononuclear cells as determined by routine hematologic analysis, and (3) the proportion of these mononuclear cells that
expresses the antigen under study as assessed by imrnunocytofluoroscopy.
In addition, molecular genetic analysis of T-cell antigen receptor
(Tcr) gene rearrangements and HTLV-I integration were performed
as described p r e v i o ~ s l y . These
~ ~ . ~ ~analyses were performed on pripheral blood mononuclear cells at the initiation of therapy and at
subsequent periods to monitor the efficacy of therapy in eliminating
the monoclonal T-cell receptor. The restriction enzymes BamH1,
EcoRI, and HindlII were used in the analysis of T-cell receptor gene
rearrangement, whereas EcoRI and Pst I (International Biotechnolo-
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4066
WALDMANN ET AL
Table 2. Effect of V-Anti-Tac Therapy
Patient
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
Total
Previous(per
Therapy
15
16
17
None
18
CHOP
14
Maximum Toxicity =Grade 31
First Cycle Manifested
BAM-M + RT; murine
45 (5,5, 5,5,5, 5, 5, None
anti-Tac
5)
5,
None
5) 5,20
5,(5.
neutropenia
and
3 Grade
thrombocytopenia cycle 4
ProMACE; DDIIAZTI
5 (5)
thrombocytopenia
3 Grade
suraminlsolumedroll
cycle 1
doxorubicin/
cyclophosphamide
murine anti-Tac
None
20 (10, 5, 5)
Grade 4 neutropenia cycle 1,
grade 3 thrombocytopenia
cycle 1. grade 3
heptotoxicity cycle 1,
grade 3 cardiac toxicity
cycle l*
CHOP; COP
45 (10, 10,10, IO, 5) Grade 3 thrombocytopenia
cycle 4
CHOP
66 (IO, 10, 10, 6. IO, Grade 3 neutropenia cycle 5
10, IO)
50 (15. 15, 10,
5, 5)
Grade 4 neutropenia and
None
thrombocytopenia cycle 3,
grade 3 hepatotoxicity
cycle 2
Grade 4 neutropenia and
CHOP
20 (15, 5)
grade 3 thrombocytopenia
cycle 1
Grade 3 neutropenia cycle 2
25 (15, 10)
ProMACE-CytaBOM
Grade 4 neutropenia cycle 1,
None
15 (IO, 5)
grade 3 hepatotoxicity
cycle 1, grade 3
thrombocytopenia cycle 2
None
ProMACE-CytaBOM
10 (IO)
None
ProMACE-CytaBOM;
10 (10)
cisplatinlcytarabinl
dexamethasone
CHOP, ProMACECytaBOM
None
None
None
cle
Doses of "Y-Anti-Tac
Administered
cycle; mCi)
Grade 4 thrombocytopenia
10 (IO)
1
10 (IO)
15 (10. 5)
20 (10, 10)
5, 5, 5, 5,(IO,35
10 (IO)
5)
None
Grade 4 neutropenia cycle 1
Grade 3 renal toxicity cycle
2
Grade 4 thrombocytopenia
cycle 6,grade 4
neutropenia cycle 6
None
PD
Development of
AntiMurine
Human
Anti-Tac Antibodies
(HAMA)
PD
Clinical
Response/
Duration (mo)
Freedom
From
Progressive
Disease (mol
-
+
CR 30
36
+
PR 12
13
-
NE
+
PR 10
13
PR 7
9
Unevaluable
CR
3,
PR
33+
disease
Stable
PR 6
PD
NE
-
+
+
~
PD
Died before
response
could be
determined
PD
45+
35+
5
6
1
1
1
1
PR 23
PR 1
PD (mixed
24
PR 13
13
2
1
response)
~
NE
1
Abbreviations: ProMACE-CytaBOM, prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide, cytosine arabinoside, bleomycin,
vincristine, leucovorin; BAM-M, bleomycin, adriamycin methotrexate and topical nitrogen mustard;
CHOP, cyclophosphamide, doxorubicin,
vincristine, prednisone; COP, cyclophosphamide, vincristine, prednisone; RT. radiotherapy; DDI, didanosine; AZT, zidovudine; CR. complete
remission; PR, partial response; SD, stable disease; PD, progressive disease; NE, not evaluated.
It
* T h e cardiac toxicity in this patient was orthopnea that occurred while he was receiving large fluid volume to control hypercalcemia.
quickly resolved with furosemide therapy and did not recur.
gies [New Haven, CT]andNewEnglandBiolabs[Beverly,
MA])
assessable disease lastingmore than 1 month; (2) partial response
were used in the analysis of HTLV-I integration to distinguish monowas defined as at least a 50% reduction of leukemic cell count, a
of this
retrovirus.
50% reduction in the
size
of all measurable
lesions,
and no
new
clonal
from
polyclonal
integration
for 1 month; (3) stable disease was defined as lessthana
The criteria for therapeutic response were as follows: (1) complete lesion
response with no new lesion or less thana 25% increase in
response was defined as thedisappearance of all measurableandpartial
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9ov ANTI-Tac THERAPY OF ATL
any measurable lesion; and (4) progressive disease was defined as
at least a 25% increase in leukemic cell count or an increase of 25%
or greater in any measurable lesion.
RESULTS
Patient characteristics. Eighteen patients with histologically confirmed HTLV-I-associated ATL were treated with
intravenously administered
anti-Tac (Tables 1 and 2).
Two patients had lymphoma-type ATL with normalnumbers
of peripheral lymphocytes. The peripheral blood WBC count
before therapy in the remaining 16 patients who had leukemia ranged from 6,400 to 112,800/pL (6.4 to 112.8 X lo9/
L; geometric mean, 18,977 X/+ 1.22//.~L).Patients with ATL
had pretherapy serum IL-2R (sIL-2Ra) levels of 2,097 to
102,266 U/mL (2.097 to 102.266 X 106U/L;geometric mean,
12,940 X / + 1.41 U/mL), whereas the upper limit of normal
levels is 502 U/mL (mean, 238 U/mL). T-cell leukemic populations were confirmed to be monoclonal by molecular genetic analysis of the genes encoding the T-cell receptor and
HTLV-I. Specifically, Southern blot analysis using a radiolabeled probe that hybridizes with the constant region of TcrP
chain showed a nongermline band indicating a clonal Tcr
gene rearrangement, a feature that is the hallmark of a clonally expanded population of T lymphocytes (Fig l). Furthermore, Southern blot analysis of HTLV-I proviral integration
in Psr I and EcoRI digests of DNA obtained from the circulating mononuclear cells of the patients demonstrated clonal
integration of HTLV-I provirus. Clinically, 10 patients rnanifested involvement of the skin. Eight were hypercalcemic,
with a serum calcium level in these cases ranging from 2.54
to 4.50 mmoVL (normal range, 2.05 to 2.50 mmoVL).
Using flow cytometric phenotypic analysis of circulating
mononuclear cells, we showed that, in the 16 cases with
leukemia, the predominant mononuclear cell population expressed the CD3', CD4+, CD8-, CD25+ phenotype. Circulating mononuclear cells of the patients expressed the Tac
antigen (CD25) on a relatively homogeneous cell population
manifesting high fluorescence intensity. In15 of the 16
cases, the abnormal cell population did not react with the
CD7 MoAb that reacts with normal T-cell precursors and
with at least 70% of normal mature T lymphocytes.
Studies with "'In-labeled anti-Tac to monitor impact of
sIL-2Ra on delivery of radiolabeled anti-Tac to tumor cells.
Before the initiation of the therapeutic O
' Y anti-Tac trial,
Ill
In-labeled anti-Tac MoAb was administered to 5 patients
to define the pharmacokinetics of radiolabeled anti-Tac and
to monitor the impact of circulating sIL-2Ra on our ability
to deliver radionuclide-labeled anti-Tac to tumor cell targets.
In the initial studies, "'In-labeled anti-Tac was administered
to 5 patients in association with a total anti-Tac antibody
dose of 1 mg (radiolabeled and unlabeled). One week later,
the same dose of radioindium-labeled anti-Tac was administered to the same patients in association with a total dose of
50 mg of the anti-Tac MoAb. When a low total quantity (1
mg of total antibody) of radiolabeled anti-Tac was administered, high levels of circulating antigen (sIL-2Ra) were
shown to interfere with tumor cell targeting by binding to
the administered antibody, thus reducing antibody access to
cellular targets. Soluble IL-2Ra expression had an effect on
4067
the bioavailability of infused anti-Tac. For example, when
1 mg of radiolabeled antibody was administered to patients
with high sIL-2Ra levels (eg, 230,370 U/mL), virtually no
radioindium bound to circulating leukemic cells, there was
only minimal anti-Tac binding to these circulating Tac-expressing cells demonstrable by flow cytometry. To define
the bindability of circulating radioindium-labeled anti-Tac,
we used a quantitative assay to determine the bioactivity ex
vivo of infused antibodies as a function of circulating sIL2Ra levels. In these studies, the radiolabeled "'In anti-Tac
circulating in the patient's serum was assessed ex vivo for
its capacity to bind to the JL-2R-expressing T-cell line HUT
102 over the 30 minutes of incubation at 4°C. When a small
quantity such as the 1 mgpatient dose of high specific activity
anti-Tac was administered to patients with
high
sIL-2Ra levels, only a small fraction of the anti-Tac in the
circulation remained unblocked by bound sIL-2Ra and was
therefore able tobind to the HUT 102 cells exvivo. The
bioavailable fraction of the indium-labeled anti-Tac increased markedly whenthe quantity of anti-Tac administered
in association with the radiolabeled antibody was increased
from 1 mg to 50 mg per patient. We extended these observations by administering "'In anti-Tac to 16 of the patients
receiving anti-Tac as part of the Yttrium-90 therapeutic protocols and showed that a linear relationship exists between
sIL-2Ra concentration and the amount of antibody required
to achieve different fractions of circulating bioavailable antibody. In the phase I trial discussed below, we administered
a total dose of 10 mg of anti-Tac to all patients. However,
based on the bioavailability data obtained during this initial
phase of the study, in the phase I1 trial we administered a
quantity of anti-Tac (2, 5, or 10 mg) at each infusion that
was chosen on the basis of the patient's soluble slL-2R
concentration.
Toxicity. Eighteen patients with ATL were treated with
goY-labeledanti-Tac. Three patients each were studied at 5 ,
10, and 15 mCi doses. The remaining 9 patients were studied
subsequently in a phase I1 trial involving an initial dose of
10 mCi of "Y-labeled anti-Tac per dose. Patients undergoing
a partial or complete remission were permitted to receive up
to eight additional doses of "Y-labeled anti-Tac. The mean
number of dose cycles was three (range, 1 to 9). The 18
patients received a total of 55 distinct cycles of therapy with
an aggregate dose for individual patients ranging from 5
to 66 mCi over the total treatment course (mean, 24 mCi/
patient).
The predominant toxicity observed in the ATL patients
after 90Yanti-Tac administration was hematologic depression (Table 2). However, grade 3 transient hepatic toxicity
occurred in 3 of the 54 evaluable treatment cycles and 4
patients developed transient proteinuria. One patient died of
unexplained cardiac asystole 23 days after the administration
of
anti-Tac. This patient, who did not manifest hematologic toxicity, was an individual with end-stage glaucoma,
hypertension, diabetes, and hypercalcemia before therapy.
One patient died of progressive disease 10 days after therapy.
The most common pattern of toxicity observed in the patients
was thrombocytopenia and granulocytopenia appearing initially at weeks 4 to 5 after
anti-Tac therapy, withnadir
From www.bloodjournal.org by guest on November 10, 2014. For personal use only.
WALDMANN ET AL
4068
Patient
Patient
Germline
Germline
Disease in
Relapse
714 Days
Therapy
Post
Patient
Disease in
Relapse
Patient
714 Days
Post Therapy
-~
4-
4Kb
+a
-
-"
-"
EcoRl
- ,
i
'-
4-
- Y-
Hind 111
Fig 1. Analysis of Tcrp gene rearrangements to monitor
anti-Tac MoAb treatment of patient no. 7 with ATL using a Tcrp constant
region probe (Cpl. The Tcrp constant region genes are on 4- and 11-kb EcoRl fragments and on 3.5-, 6.5-, and 8.0-kb Hindlll fragments in
germline DNA as indicated (-4. The digest of patient peripheral blood DNA during an active phase of the disease before the initiation of
therapy yielded a diminished 11-kb EcoRl band as well as one nongermline band I-) that identified a monoclonal pattern of Tcrp gene
rearrangement. Furthermore, there was a diminution of the8.0-kb Hindlll germline band and the appearance of one nongermline bandI-) in
the Hindlll digest that reflects a monoclonal Tcrp pattern ofgene rearrangement as well. This Southern blot patternindicates that one Tcrp
allele in the leukemic clone rearranged t o C p l .whereas the otherallele rearranged t o Cp2. Digests of patient DNA obtained in remission after
"Y anti-Tac therapy did not show thetwo nongermline bands, thus confirming the elimination of the circulating monoclonalleukemic cell
population. In theschematic diagram of the germline
arrangement of the Tcrp gene, we indicate the locationsof the EcoRl (E) and Hindlll (H)
restriction endonuclease sites as well as the Cp regions recognized by the cDNA probe used.
values usually occurring during weeks S to 7 after treatment
(Table 2). A single patient manifested a late toxic event that
wasthe development of a myelodysplastic syndrome and
Sweet's syndrome that progressed to myelogenous leukemia
and death approximately 3 years after the initial induction
of a complete remission. The patient had received 3 months
of BAM-M (bleomycin adriamycin. methotrexate. and topical nitrogen mustard) chemotherapy. 30 Gy of external beam
irradiation to the lumbar spine. and courses of unmodified
murine anti-Tac before entry into the "'Y anti-Tac trial.
T~tmorresponse. Sixteen of the 18 patients with histologically confirmedATLhad measurable disease and survived for at least 3 weeks after therapy and were thus evaluable for their therapeutic response (Table 2 ) . Seven of these
16 patients had either a transient response (< I month). had
a mixed response with a 50% to 95% reduction in the number
of circulating leukemic cells butan increase in the size of
lymph nodes. or developed progressive disease after their
initial "'Y anti-Tac therapy. Four of these patients who did
not respond were treated with "'Y anti-Tac at a period when
they were experiencing progressive disease despite the fact
that they had been receiving multiagent chemotherapy up to
I to 2 months before beginning the "'Y anti-Tac protocol.
The remaining 9 patients had a more favorable response to
therapy. Seven patients ( I with chronic ATLand 6 with
acute ATL) developed a partial remission. The duration of
thesepartial remissions rangedfrom 1.6 to 22.4 months
(mean, 9.2 months). Six patients of this group manifesting
partial remissions received subsequent courses of '"'YantiTac until they developed HAMA,persistenthematologic
toxicity. or progressive disease.
Two additional patients developed a complete remission.
One of these patients developed a myelodysplastic picture
progressing to myelogenous leukemia that terminated in her
death 36 months after initiation of therapy. Althoughthe
cause of death in this case was the secondary myelogenous
leukemia. cells with ATL morphology were detected in the
skin at autopsy examination. The remaining patient continues in complete remission more than 3 years after therapy
initiation. The observations thatsupportthese conclusions
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ANTI-Tac THERAPY OF ATL
4069
BeforeTreatment
After 2 Doses
-Y- CY -Tac
Fig 2. CAT scan of thorax of patient no. 4 before treatment (top)
and after t w o cycles of
anti-Tac therapy (bottom). There was a
marked reduction in the size of the axillary lymph nodes in the scan
obtained during the period when the patient was in an wY anti-Tac
therapy-induced partial remission.
concerning thefavorabletherapeutic responses includethe
demonstration of a reduction in size of all measurable lesions
as assessed by physical examination. CAT scan (Fig 2). and
gamma camera imaging studies after intravenous coadministrationof "'In anti-Tac (Fig 3). Furthermore.theclinical
responses in all patients with leukemia were associated with
a reduction in the number of peripheral blood leukemic cells
enumerated by FACS analysis (Fig 4A). by a decline in the
slL-2Ra concentration (Fig 4B). and by a normalization of
the Southern blot patterns of Tcrp gene arrangement and
HTLV-I integration (Fig l ) . Normal T cells can be distinguished from leukemic cells in the patients by FACS analysis
in that the normal cells express the CD7 antigen. whereas
the leukemic cells of most patients do not. In patients manior complete remission.theCD2S'CD7
festingapartial
leukemic cells werereduced
in number or wereabsent.
whereas the CD7' expressing CD25 nonexpressing normal
T cells persisted at near pretherapy levels (Fig 4A). indicating good Tac-expressing tumor cell specificity of the therapeutic response.
Among the 9 patients with a partial or complete remission
after '"'Y anti-Tac therapy. 3 had received previous chcmotherapy. 3 manifested hypercalcemia. and 3 hod Iiwr function abnormalities before ""Y ;anti-Tac therapy.During the
period of partial or complete remission. there w a s :a normalization o f the serum calcium level in each case. Furthermore.
liver function tests normalized or improved after therapy i n
each of the 3 patients that manifested liver function abnormalities before therapy.
The routine hematologic and immunofluorescence nnolyses discussed above usually yield valid conclusions conccrning the efticacy of MoAb therapy. However. care nlust be
takenwheninterpretingimlnunoRuorescence
analyses because an MoAb could theoretically cause modulation o f its
target antigen from the cell surface without leading to cell
death. Furthermore.antibodytherapy
might select for the
expansion of a variant leukemic cell subpopulation that does
not express the antigen targeted by the antibody. To address
this concern. theobservedclinicalremissionswere
confirmed by molecular genetic analysis of the arrangement of
the geneencoding thechain
of the Tcr. In particular.the
two complete clinic:d remissions were conlirmecl by r~lolecular analysis of the Tcrp gene arrangement by showing that
the novel nongermline band on Southern analysis of peripheralblood mononuclearcells that was characteristic of ;I
monoclonalexpansion of T cells observedbeforetherapy
was no longer demonstrable after therapy (Fig 1 ). Furthermore, the complete remissions in these 2 patients were confirmed by molecular genetic analysis of integrated HTLV-I
provirus in the circulating mononuclear cells. Before thcrapy. the patients manifested a monoclonal HTLV-I
integrntion pattern on EcoRI and Pst l digests of their mononuclear
cell DNA. The band(s) on Southern analysis that had establishedthemonoclonalHTLV-Iintegrationpretherapy
was
decreased in intensity in each of the S patients undergoing
:a partial remission who were re-evaluated during this period
and was no longer demonstrable in the cells of the 2 patients
who were evaluated when they werein a complete remission.
In~nlrrrlologicc o ~ n p e t e ~ trrlrl
l c ~ prodlrctiorl of m 1 HAMA
response to the infirserl MOA/?. One of themajorclinical
features associated with ATL is aprofoundimmunodeticiency state affecting both cellular and humoral immunity."
Before therapy. only 4 of the 18 patients manifested a positiveskin test response to one or moreof the seven recall
skin test antigens assessed by the Merieux multitest skin test
procedure. Furthermore, only I of thepatientsmanifested
HAMA within the 8 weeks after initiation of anti-Tnc therapy. Noneofthe 7 patientsfailing to respond to anti-Tac
therapy developed apositiveskin
test response to recall
antigens after therapy or made an HAMA response to the
infused mouse MoAb. Eight patients who were anergic before therapy manifested a partial or complete remission after
anti-Tac therapy. Five of these eight initially anergic patients
developed a delayedhypersensitivityresponse
to one or
more of the seven recall skin test antigens during remission.
Furthermore. S of the 9 patients who underwent a partial or
complete clinicalremission.as
well ;as 1 patient without
evaluable disease, initially developed HAMA to the infused
anti-Tac after the first. second. third ( 2 cases). seventh. or
ninth course of "'Y murineanti-Tac administration. This
From www.bloodjournal.org by guest on November 10, 2014. For personal use only.
WALDMANN ET AL
4070
Fig 3. '"In anti-Tac imaging
studies of patient no. 1 before
treatment and at the timeof the
fourth treatment with 9oY antiTac, when the patient was in a
complete remission. Beforetherapy, "'In anti-Tacwas deposited
in sites of malignant T-cell infiltration of the skin of the hands,
whereas no such depositionwas
evident at the timeof the fourth
study, confirming the complete
remission.
development o f HAMA precluded further administration of
'"'Y anti-Tac. Thus. effective IL-?R-directed therapy of patients with ATL is associated in some cases with a return of'
cellular and humoral immune functions.
DISCUSSION
Adult T-cell leukemia is ;I malignancy of T lymphocytes
with a median survival time of 9 months in the acute and
24 months in the chronic form of the disease.".'" Various
combination chemotherapieshave not significantly increased
the survival of patients with ATL. In light of the disappointing resultsusing conventional combination chemotherapy.
IL-?R-directed therapy was developed to exploit the observation that normal resting cells, includingtheunaffected
normal T cells of patients with ATL. do not display IL-2Ra.
whereas the leukemic cells express this interleukin receptor
subunit.'".''
The present therapeutic trial using "'Y anti-Tac was initiated t o exploit the observation that theleukemic cells of
patients in the IL-2-independent aggressive phase of their
disease continued t o express large numbers of the IL-2Ra
receptor. When the S- to 15-mCi doses were used. 9 of the
I6 evaluahle patients responded to '"'Yanti-Tac with a partial
o r complete remission. The patientstreated with "'Y antiTnc were not studied simultaneously with the group of patientstreated with unmodifiedanti-Tac. In addition.there
W;IS a difference in the total quantity of anti-Tac administered
between the two studies with an initial course of 200 mg of
anti-Tac i n divided doses used in the study with unmodified
anti-Tac ;IS compared with the 2 to I O mg administered in
the present study in association with "'Y anti-Tac. Therefore.
differences between the results obtained with the two groups
may reflect factors other than the form of anti-Tnc administered. Nevertheless. the I X patients in the present study were
quite comparahle t o the previously studied I O patients who
weretreated with unmodified anti-Tac.' In particular. the
two groups were comparable in terms of age (mean age. 43
1.41 years): ATL type (identical with the exception that there
were 2 additional patients with lymphoma type ATL and I
less with chronicATL in the unmodified anti-Tacstudy):
mean sIL-2Ra levels; number of circulating Tac-expressing
lymphocytes:andincidenceofhypercalcemia.abnormal
liverfunction tests. andimmunodeficiency.' The response
to therapy with either unmodified or '"'Y-labeled anti-Tac
correlated with the disease classificationandresponse
to
previous chemotherapy. In particular. 7 of the 9 patients in
the twostudies with chronicATLdeveloped a partial or
complete remission. Eight of the 22 patients with acute- or
lymphoma-type ATL who were not failing to respond t o an
ongoingcourse of chemotherapy manifested ;I remission.
whereas no remissions were observed in the h patients who
were studied within I to 2 months o f completion of an ineffective course of aggressivechemotherapy.
A KaplnnMeier"' plot of event-free survival (surviving patients without progressive disease) in patients treated using unmodified
anti-Tac and in those receiving '"'Y-labeled.anti-Tac is presented in Fig SA and R. '"'Y-labeled anti-Tac therapy suggests improved efficacy when compared with treatment with
unmodifiedmurineanti-Tac.However.
it should be noted
that this comparison must be viewed with caution becnuse
the patients were not studied sinlultaneously in :I randomized
controlled trial. One of the patients included in the present
From www.bloodjournal.org by guest on November 10, 2014. For personal use only.
9oV
407 1
ANTI-Tac THERAPY OF ATL
CD7 +calls
/
A I
Fig 4. (A) Effect of 9 anti-Tac therapy on the
absolutenumber of Tac-expressingATLleukemic
and normal T cells permicroliter of patient no. 7 . 9
anti-Tac MoAb waa administered intravenously to
the patient at the doses and on the days indicated
by the arrows 1 4 . The patient initially had 27,875
circulating Tac-expressing malignant cellslpL 10).
The patient received 50 mCi of 9 anti-Tac during
the first 14 months of therapy in divided doses. By
10 months after the initiation of therapy, the patient
had undergonea complete remission that has been
maintainedfor the more than 35 months of obsenration. There was an initial modest reduction in the
number of normal Tcells (0: normal T cells are
CD7+CD25-). However, the number of thesenormal
T cells subsequentlyreturned to pretreatment levels
during the remaining period when the patient was
in a sustained complete remission. (B) Effect of 9
anti-Tac therapy on the serum concentration of slL2Rrr of the same patient. The serum slL-2Rrr level
of the patient before therapy was 2,938 UlmL. The
concentration sIL-2Ra returned to normal or below
normal levels after therapy, confirming the complete
remission.
9
a
I
12
4
1
I
15
18
*
Months
B
--\
study (patient no. 1) had been included in the previous trial
involving unmodified murine anti-Tac. This patient developed an initial partial remission after therapy with unmodified anti-Tac but her disease ultimately relapsed and was no
longer responsive to therapy with this agent. Although this
patient was not responsive to unmodified anti-Tac, she sustained a complete remission when treated with "Y-labeled
anti-Tac, supporting the view that the therapeutic efficacy
of unmodified antibody may be augmented by arming it with
a cytotoxic radionuclide.
In the 40 years since Korngold and Pressman37used I3'Ilabeled antibodies to localize tumors in rodents, numerous
clinical trials have been performed using radiolabeled
MoAbs for the therapy of cancer. Many of the therapeutic
trials have been performed in patients with solid tumors.
The results have been discouraging and generally have not
resulted in clinically significant responses. Therapy of leukemia has generally been more encouraging, with several studies reporting clinical
The results using 90Y
anti-Tac in the treatment of ATL reported in the present
study also are encouraging. Thus, it may be of value to
consider the various elements contributing to the remissions.
Months
Such an analysis may be useful in designing new strategies
involving receptor-directed radioimmunotherapy in diverse
clinical circumstances.
Factors that appear critical in developing an effective radioimmunotherapeutic regimen include (1) the choice of radionuclide, (2) the selection of the chelate used to link the
radionuclide to the MoAb, and (3) the choice of the MoAb.
An appropriate choice of radionuclide would be one that has
a short distance of action (eg, one with a p or a emission)
that will thereby maintain the antigen specificity of the
MoAb and kill antigen-expressing tumor cells and a few
adjacent antigen nonexpressing cells but will spare distant normal cells. @emitting radionuclides, such as I3'I,
90Y,lX6Re,Ig8Re,and 67Cu,have been useful in immunotherapy.21-25
13'1 is by far the most frequently used radionuclide
in radioimmunotherapy. Effective radioimmunotherapy of
B-cell lymphoma was achieved with I3'I-labeled anti-CD20
and anti-CD37 antibodie~:'.~' However, in most cases, 13'1labeled MoAbs have been relatively ineffective due to limitations of this radionuclide as a therapeutic agent. After uptake
of a radioiodinated MoAb into a tumor cell, there is degradation of the antibody with release of radioiodine from the
From www.bloodjournal.org by guest on November 10, 2014. For personal use only.
4072
WALDMANN ETAL
Loop:
radiotherapy of patients with enlarged malignant lymph
nodes.
A second pivotal issue in designing an optimal radioimmunotherapeutic reagent is the choice of the method used
to link the radionuclide to the MoAb. In the case of metals,
it is critical that the chelate retain the radiometal tightly and
not permit its release from the MoAb chelate complex in
vivo. This is especially important for wY because it is a bone
seeker, ie, any 90Yreleased into the circulation wouldbe
rapidly
cleared into boneandwould produce undesirable
C"""
I"
irradiation of the bone marrow, the critical organ. We chose
f1-b60+
I
IB4M-DTPA as our chelate based on our previous study in
OO
3
6
9
12
20
which
we compared the in vivo stability, in mice, of seven
Months
radioimmunoconjugates thatused different polyaminocarboxylate chelating agents with that of complex radioyttrium
to anti-Tac.3" A number of the chelating agents evaluated,
including those that have been used in other immunotherapeutic trials (eg, ethylenediaminetetraacetic acid) provided
unstable coupling of the radioyttrium to the antibodies.'" In
contrast, the 1B4M-DTPA chelating agent chosen for use in
I
the present study emerged as a promising immunotherapeutic
reagent because it behaved essentially identically to coadministered radioiodine-labeled antibody and was associated
with only a modest accumulation into bone of the injected
radioyttrium.
A third critical component to consider is the selection of
OO
3
6
9
12
20
the MoAb that serves to carry the radionuclide to the tumor
Months
target. An MoAb is selected in part based on the distribution
Fig 5. A Kaplan-Meier plot" of event-free survival (surviving paof its antigenic targetandonthespecificityandbinding
tients without progressive disease)in (A) patients treated with unaffinity of the antibody to its target. In the present study, we
modified anti-Tac (---I and 18) those receiving 9 anti-Tac 1-1.
selected anti-Tac because of its ability to bind to the IL-2a
subunit, a subunit that is not expressed on resting normal
cells but is expressed on the surface of a number of leukemic
tumor cell into the body fluids, resulting in loss of action on
cells, including the HTLV-I-associated ATL. An additional
the tumor cell and delivery of irradiation to normal tissues
with consequent development of severe myel~suppression.~' feature that may be critical in developing an effective agent
for radiolabeled MoAb treatment is to choose an antibody
In light of the limitations of radioiodine, metallic radionuthat, in its unmodified state, has an antitumor effect, especlides that can be securely linked to antibodies may provide
cially one that involves induction of apoptosis in sensitive
a better choice. Most of the metallic radionuclide remains
tumor cells either by depriving the cells of a required growth
within the tumor cell on uptake of the radiolabeled antibody.
factor or by acting as an agonist of a negative signaling
Our own studies have focused on Yttrium-90. Yttrium-90
p a t h ~ a y . ~An
' , ~antibody
~
that induces apoptosis may work
had been used previously linked to an anti-idiotype MoAb
synergistically with protracted low-dose irradiation tokill
in the treatment of B-cell lymphoma42antifenitin in the treattumor
Low-dose
irradiation induces apoptosis of
ment of end-stage Hodgkin's d i ~ e a s e ~and
' . ~ ~to an antiovarmalignant
cells
provided
that
they express the normal prodian antibody in the intraperitoneal radioimmunotherapy of
uctofthe
p53 tumor-suppressor gene, which maybean
ovarian cancer."-46 Yttrium-90 has a high &energy emission
essential element of the apoptotic pathway initiated by radia(maximum, 2.29 MeV; average, 0.94 MeV) and has a desirtion
Hematopoietic colony-stimulating factors
able @-hour half-life. Yttrium-90 is attractive for therapy
as
well
as
IL-2
inhibit
apoptosis of growth factor-dependent
of lymphoma because it decays with high-energy p but no
leukemic cells.51~52
Withdrawal of such growth factors or the
y emissions. The energy released per unit of activity is apintroduction of antibodies that prevent the interaction of the
proximately five times greater than that of I3lI and would
growth factor with its receptor may result in programmed
yield a significantly higher radiation dose delivered to the
cell death (apoptosis). There is considerable precedence for
tumor." The high-energy p emission of '9
may be of spethe therapeutic synergy between an MoAb that is directed
cial value for large tumors because this emission manifests
against a growth factor receptor and a therapeutic agent that
greater tissue penetration than the low-energy p emission
induces a p o p t o s i ~ .For
~ ~ example,
~~~
MoAbs to the c-erb
of I3'I. Therefore, "Y-labeled MoAbs can kill nontargeted
B-2 protein and to the epidermal growth factor receptor
antigen-nonexpressing tumor cells through a cross fire effect
complement the cytotoxic action of cis-diammine-dichlorofrom neighboring antigen-expressing cells that have been
platium on appropriate tumor cells.s3"s Furthermore, the eftargeted by the radiolabeled MoAb. In the present study, this
ficacies of
anti-CD20 and I3'I anti-CD37 maybe due in
cross fire effect may have been especially important in the
- - - - Anti-Tac
(unmodified)
-
"
"
"
:
From www.bloodjournal.org by guest on November 10, 2014. For personal use only.
“Y ANTI-Tac THERAPY OF ATL
4073
3. Waldmann TA, White JD,
Goldman CK, Top L, Grant A,
part to the fact that the CD20 and CD37 antibodies in
Bamford
R,
Roessler
E,
Horak
ID,
Zaknoen S, Kasten-Sportes C,
their unmodified form can induce cell cycle arrest or
England R, Horak E, Mishra B, Dipre M, Hale P, Fleisher TA,
apptosis~40.41.48.56 The efficacy observed in the present study
Junghans RP, Jaffe ES, Nelson DL: The interleukin-2 receptor:
with wY anti-Tac may be due in part to the fact that antiA target for monoclonalantibodytreatment
of human T-cell
Tac inhibits the interaction of the growth factor IL-2 with
lymphotrophicvirusI-induced
adult T-cell leukemia. Blood
the high-affinity IL-2R expressed on ATL cells. Such with82:1701,1993
drawal of IL-2 action has been shown to activate an apoptotic
4. Robb RI, Munck A, Smith KA: T-cell growth factor receptors.
suicide program in IL-2-dependent T cells.52This may be
J Exp Med 154:1455, 1981
the critical element in the therapeutic action of unmodified
5. Uchiyama T, Broder S, Waldmann TA: A monoclonal antibody
(anti-Tac) reactive with activated and functionally mature human T
anti-Tac in the subset of patients that have leukemic cells
cells. I. Production of anti-Tac monoclonal antibody and distribution
that still produce and respond to IL-2. In the present trial
of Tac’ cells. J Immunol 126:1393, 1981
involving 9’% anti-Tac treatment, it is possible that several
6. Tsudo M, Kozak RW, Goldman CK, Waldmann TA: Demondifferent antitumor mechanisms may be working in concert,
stration of a non-Tac peptide that binds interleukin-2: A potential
including low-dose irradiation coupled with antibody-inparticipant in a multichain interleukin-2 receptor complex. Proc Natl
duced apoptosis.
Acad Sci USA 83:9694, 1986
Although murine MoAbs including murine anti-Tac are of
7. Takeshita T, Asao H, Ohtani K, Ishii N, Kumaki S, Tanaka
value in the therapy of certain diseases, their effectiveness is
N, Munakata H, Nakamura M, Sugamura K: Cloning of the y chain
limited because rodent MoAbs induce an immune response
of the human IL-2 receptor. Science 257:379, 1992
that neutralizes their therapeutic effect. In the present study,
8. Waldmann TA: The interleukin-2 receptor. J Biol Chem
266:2681, 1991
6 patients developed HAMAafterone or moretreatment
9. Rubin LA, Nelson DL: The soluble interleukin-2 receptor: Bicycles, which precluded further therapy despite objective
eviology, function and clinical application. Ann Intern Med 113:619,
dence of excellent responses in 5 cases. To circumvent this
1990
impediment totreatment,geneticallyengineeredantibody
10. Motoi T, Uchiyama T, Hori T, Itoh K, Uchino H, Ueda R:
variants of anti-Tac were produced that combined the rodent
Elevated serum-soluble interleukin-2 receptor (Tac antigen) levels
Ig hypervariable regions with human constant and framework
in chronic myelogenous leukemia patients with blastic crisis. Blood
region^.^'^^^ This humanized versionof anti-Tac is less immu- 74:1052, 1989
nogenic than the murine version, has improved pharmacoki11. Korsmeyer SJ, Greene WC, Cossman J, Hsu SM, Jensen JP,
netics, and, in contrast to the parent MoAb, manifests antiNeckers LM, Marshall SL, Bakhshi A, Depper JM, Leonard WJ,
body-dependent cell-mediated cytotoxicity
with
human
Jaffe ES, Waldmann TA: Rearrangement and expression of immunomononuclear cells.3’~JR The predicted reduction
in immunogeglobulin genes and expression of Tac antigen in hairy cell leukemia.
Proc Natl Acad Sci USA 80:4522, 1983
nicity of humanized anti-Tac was observed in clinical trials
12. Schwarting R, Gerdes J, Stein H: Expression of interleukinperformed in cynomolgus monkeys and in patients receiving
this agent for the treatment of graft-versus-host di~ease.’’~~~2 receptor on Hodgkin’s and non-Hodgkin’s lymphomas and macrophages. J Clin Path01 38:1196, 1985
In light of its low immunogenicity, we have initiated clinical
13. Uchiyama T, Yodoi J, Sagawa K, Takatsuki K, Uchino H:
trials involving the repeated administrationof goy-labeled huAdult T-cell leukemia: Clinical and hematologic features of 16 cases.
manized anti-Tac to patients with
Tac-expressingATL as well Blood 50:481, 1977
as to patients with other L-2Ra-expressing mature T-cell
14. Poiesz BJ, Ruscetti F W , Gazdar AF, BunnPA, Minna JD,
leukemias and lymphomas, including those malignancies that Gallo RC: Detection and isolation of type C retrovirus particles from
are not associated with immunodeficiency.
fresh and cultured lymphocytes of a patient with cutaneous T-cell
In conclusion, the emerging understanding of the L-2/ILlymphoma. Proc Natl Acad Sci USA 77:7415, 1980
15. Waldmann TA, Greene WC, Sarin PS, Saxinger C , Blayney
2R system opens the possibility for more specific immune
DW, Blattner WA, Goldman CK, Bongiovanni K, Sharrow S, Depintervention. This understanding, taken in conjunction with
per JM, Leonard W, Uchiyama T, Gallo RC: Functional and phenothe ability to produce humanized antibodies to the L-2R by
typic comparison of human T cell leukemiallymphoma virus positive
genetic engineering and to arm these antibodies with a- or
adult T cell leukemia with human T cell leukemia/lymphoma virus
&emitting radionuclides, may provide a rational therapeutic
negative SCzary leukemia, and their distinction using anti-Tac monostrategy for the treatment of IL-2R-expressing leukemias and
clonal antibody identifying thehuman receptor for T cell growth
lymphomas, including HTLV-I-associated ATL.
factor. J Clin Invest 73:1711, 1984
ACKNOWLEDGMENT
We thank Patricia Perentesis for assistance with patient studies;
Mark Rotman, Hnat Le, and R.K. Leedham for formulation of the
radiolabeled product; and the National Institutes of Health nuclear
medicine technologists, particularly Millie Whatley for her assistance in imaging the patients.
REFERENCES
1. Kohler G, Milstein C: Continuous cultures of fused cells secreting antibody of pre-defined specificity. Nature 256:495, 1975
2. Waldmann TA: Multichain interleukin-2 receptor: A target for
immunotherapy in lymphoma. J Natl Cancer Inst 81:914, 1989
16. Uchiyama T, Hori T, Tsudo M, Wan0 Y, Umadome H: Interleukin-2 receptor (Tac antigen) expressed on adult T-cell leukemia
cells. J Clin Invest 76:446, 1985
17. Seiki M, Hattori S, Hirayama Y, Yoshida M: Human adult
T-cell leukemia virus: Complete nucleotide sequence of the provirus
genome integrated in leukemia cell DNA. Proc Natl Acad Sci USA
80:3618, 1983
18. Maeda M, Arima N, Daitoku Y, Kashihara M, Okamoto H,
Uchiyama T, Shirono K, Matsuoka M, Hattori T, Takatsuki K, Ikuta
K, Shimizu A, Honjo T, Yodoi J: Evidence for the interleukin-2
dependent expansion of leukemic cells in adult T cell leukemia.
Blood 70: 1407, 1987
19. Shimoyama M, and members of the Lymphoma Study Group
From www.bloodjournal.org by guest on November 10, 2014. For personal use only.
4074
(1984-1987): Diagnostic criteria and classification of clinical subtypes
of adult T-cell leukaemia-lymphoma.Br J Haematol 79428, 1991
20. Tendler CL, Greenberg SJ, Burton JD, Danielpour D, Kim
S-J, Blattner WA, Manns A, Waldmann TA: Cytokine induction in
HTLV-I associated myelopathy and adult T-cell leukemia: Alternate
molecular mechanisms underlying retroviral pathogenesis. J Cell
Biochem 46:302, 1991
21. Wessels BW, Rogus RD: Radionuclide selection and model
absorbed dose calculations for radiolabeled tumor associated antibodies. Med Phys 1 1 :638, 1984
22. Yorke ED, Beaumier PL, Wessels BW, Fritzberg AR, Morgan
AC: Optimal antibody-radionuclide combinations for clinical radioimmunotherapy: A predictive model based on mouse pharmacokinetics. Nucl Med Biol 18:827, 1991
23. Macklis RM, Lin JY, Beresford B, Atcher RW, Hines JJ,
Humm L:Cellular kinetics, dosimetry, and radiobiology of a particle radioimmunotherapy: Induction of apoptosis. Radiat Res
130:220, 1992
24. Carrasquillo JC: Radioimmunoscintigraphy with polyclonal
or monoclonal antibodies, in Zalutsky M (ed): Antibodies in Radiodiagnosis and Therapy. Boca Raton, FL, CRC, 1989, p 169
25. Vriesendorp HP, Quadri SM, Stinson RL, Onyekwere OC,Shao
Y, Klein JL, Leichner PK, Williams
R:Selectionof reagents for human
radioimmunotherapy. Int J Radiat Oncol Biol Phys 22:37, 1991
26. Larson SM, Sgouros G, Cheung N-KV: Radioisotope conjugates, in DeVita VT Jr, Hellman S, Rosenberg SA (eds): Biologic
Therapy of Cancer: Principals and Practice (ed 2). Philadelphia, PA,
Lippincott, 1994, p 534
27. Jurcic JG, Scheinberg DA: Recent developments in the radioimmunotherapy of cancer. Curr Opin Immunol 6:715, 1994
28. Goldenberg DM: Cancer Therapy with Radiolabeled Antibodies. Boca Raton, FL, CRC, 1995
29. Carrasquillo JA: Radioimmunotherapy of leukemia and
lymphoma, in Wagner H (ed): Principals of Nuclear Medicine. Philadelphia, PA, Saunders (in press)
30. Kozak RW, Raubitschek A, Mirzadeh S, Brechbiel MW, Junghans R, Gansow OA, Waldmann TA: Nature of the bifunctional
chelating agent used for radioimmunotherapy with yttrium-90 monoclonal antibodies: Critical factors in determining in vivo survival
and organ toxicity. Cancer Res 49:2639, 1989
31.Brechbiel MW, Gansow OA: Backbone-substituted DTF’A ligands for 9-radioimmunotherapy. Bioconjugate Chem 2:187, 1991
32. Mirzadeh S, Brechbiel MW, Atcher B, Gansow OA: Radiometal labeling of immunoproteins: Covalent linkage of 2-(4isothiocyanatobenzy1)diethylene-triaminepentaaceticacid ligands to
immunoglobulin. Bioconjugate Chem 159, 1990
33. Brown PS Jr, Parenteau GL, Dirbas FM, Garsia RI, Goldman
CK, Bukowski MA, Junghans RP, Queen C, Hakimi J, Benjamin
W, Clark RE, Waldmann TA: Anti-Tac-H, a humanized antibody to
the interleukin-2 receptor prolongs primate cardiac allograft survival.
Proc Natl Acad Sci USA 88:2663, 1991
34. Korsmeyer SJ, Greene WC, Cossman J, Hsu SM, Jensen JP,
Neckers LM, Marshall SL, Bakhshi A, Depper JM, Leonard WJ,
Jaffe ES, Waldmann T A Immunoglobulin gene rearrangement and
cell surface antigen expression of acute lymphocyte leukemias of
T-cell and B-cell precursor origins. J Clin Invest 71:301, 1983
35. Waldmann TA: The arrangement of immunogiobulin and Tcell receptor genes in human lymphoproliferative diseases. Adv Immunol 40:247, 1987
36. Kaplan EL, Meier P Non-parametric estimation from incomplete observations. J Am Stat Assoc 53:457, 1958
37. Komgold L, Pressman D: The localization of antilymphosarcoma antibodies in the Murphy lymphosarcoma of the rat. Cancer
Res 14:96, 1954
38. Vriesendorp HM, Herpst JM, Germack MA, Klein JL,
WALDMANN ET AL
Leichner PK, Loudenslager DM, Order SE: Phase 1-11 studies of
yttrium-labeled antifemtin treatment for end-stage Hodgkin’s disease, including radiation therapy oncology group 87-01.J Clin Oncol
9:918, 1991
39. Goldenberg DM, Horowitz JA, Sharkey RM, Hall TC, MurR, Siege1 JA, Izon DO,
thy S , Goldenberg H, LeeRE,Stein
Burger K, Swayne LC, Belisle E, Hansen HJ, Pinksy CM: Targeting dosimetry and radioimmunotherapy of B-cell lymphomas
with iodine-131-labeled LL-2 monoclonal antibody. J Clin Oncol
9548, 1991
40. Kaminski MS, Zasadny KR, Francis R,Milik AW, Ross CW,
Moon SD, Crawford SM, Burgess JM, Petry NA, Butchko GM, Glenn
SD, Wahl RL: Radioimmunotherapy of B-cell lymphoma, with [‘3’1]
anti-Bl (anti-CD20) antibody. N f i g 1 J Med 329:459, 1993
41. Press OW, Eary JF, Appelbaum FR, MartinPJ,BadgerCC,
DC,
Nelp W B , Glenn S, Butchko G,Fisher D, Porter B, Matthews
FisherLD,Bemstein
ID: Radiolabeled-antibodytherapy of B-cell
lymphomawithautologousbonemarrowsupport.
N Engl J Med
329:1219, 1993
42. Parker BA, Vassos AB, Halpem SE, Miller RA, HupfH,
Amox DG, Simoni JL, Starr W,Green MR, Royston I: Radioimmunotherapy of human B-cell lymphoma with Y-90 conjugated antiidiotype monoclonal antibody. Cancer Res 50:1022s, 1990
43. Bierman PJ, Vose JM, Leichner PK, Quadri SM, Armitage
3 0 , Klein JL, Abrams RA, Dicke KA, Vriesendorp HM: Yttrium
90-labeled antifenitin followed by high-dose chemotherapy and autologous bone marrow transplantation for poor-prognosis Hodgkin’s
disease. J Clin Oncol 11:698, 1993
44. Hnatowich DJ, Chino1 M, Siebecker DA, Gionet M, Griffin
T, Doherty PW, Hunter R, Kase KR: Patient biodisuibution of intraperitoneally administered yttrium-90-labeled antibody. J Nucl Med
29:1428, 1988
45. Hird V, Maraveyes A, Snook D, Dhokia B, Soutter WP,
Meares C, Stewart JSW, Mason P, Lambert HE, Epenetos AA:
Adjuvant therapy of ovarian cancer with radioactive monoclonal
antibody. Br J Cancer 68:403, 1993
46. Maraveyas A, Snook D,Hird V, KosmasC, Meares CF, Lambert
HE, Epenetos AA: Pharmacokineticsandtoxicity ofan ymium-90CITC-DTPA-HMFGl radioimmunwnjugate for intraperitoneal radioimmunotherapy of ovarian cancer. Cancer 73:1067, 1994
47. Macklis RM, Beresford BA, Palayoor S, Sweeney S, Humm
JL: Cell cycle alterations apoptosis and response to low-dose-rate
radioimmunotherapy in lymphoma cells. Int J Radiat OncolBiol
Phys 27643, 1993
48. Vitetta ES, Uhr JW: Monoclonal antibodies as agonists: An
expanded role for their use in cancer therapy. Cancer Res 545301,
I 994
49. Sellins KS, Cohen JJ: Gene induction by y-irradiation leads
to DNA fragmentation in lymphocytes. J Immunol 139:3199, 1987
50. Lowe SW, Schmitt EM, Smith SW, Osborne BA, Jacks T:
p53 is required for radiation-induced apoptosis in mouse thymocytes.
Nature 362:847, 1993
5 I. Sachs L, Lotem J: Control of programmed cell death in normal and leukemic cells: New implications for therapy. Blood 82: 15,
1993
52. Duke RC, Cohen JJ: IL-2 addiction: Withdrawal of growth
factor activates a suicide program in dependent T cells. Lymphokine
Res 5:289, 1986
53. Fan Z, Baselga J, Masui H, Mendelsohn J: Antitumor effect
of anti-epidermal growth factor receptor monoclonal antibodies plus
cis-Diamminedichloroplatium on well established A43 1 cell xenografts. Cancer Res 53:4637, 1993
54. Hancock MC, Langton BC, Chan T, Toy P, Monahan JJ, Mischak RP, Shawver LK A monoclonal antibody against c-erbB-2 protein
From www.bloodjournal.org by guest on November 10, 2014. For personal use only.
'9 ANTI-Tac THERAPY OF ATL
enhances the cytotoxicity of cis-Diamminedichloroplatiumagainst human breast and ovarian tumor cell lines. Cancer Res 51:4575, 1991
55. Arteaga CL, Winnier AR, Poirer MC, Lopez-Larraza DM,
Shawver LK, Hurd SD, Stewart SJ: p185E~e*B'2
signaling enhances
cisplatin-induced cytotoxicity in human breast carcinoma cells: Association between an oncogenic receptor tyrosine kinase and druginduced DNA repair. Cancer Res 54:3758, 1994
56. Ghetie M-A, Picker W, Richardson JA, Tucker K, Uhr JW,
Vitetta ES: Anti-CD19 inhibits the growth of human B-cell tumor
lines in vitro and of Daudi cells in SCID mice by inducing cell cycle
arrest. Blood 83:1329, 1994
57. Queen C, Schneider WP, Selick HE, Payne PW, Landolfi NF,
Duncan JF, Avdalovic NM, Levitt M, Junghans RP, Waldmann TA:
4075
A humanized antibody that binds to the interleukin 2 receptor. Proc
Natl Acad Sci USA 86:10029, 1989
58. 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 malignant
and
immune
disorders.
Cancer Res
50:1495, 1990
59. Anasetti C, Hansen JA, Waldmann TA, Appelbaum FR, Davis
J, Deeg HJ, Doney K, Martin PJ, Nash R, Storb R, Sullivan KM,
Witherspoon RP, Binger M-H, Chizzonite R, Hakimi 3, Mould D,
Satoh H, Light SE: Treatment of acute graft-versus-host disease with
humanized anti-Tac: An antibody that bindstothe
interleukin-2
receptor. Blood 84: 1320, 1994