M.D., Metabolism Branch, National Cancer Institute,
National Institutes of Health, Bethesda, MD 20892 USA
Patients with human T -cell Iymphotrophic virus I (HTL V -1)-associated
leukemia/lymphoma were treated with different forms of IL-2 receptor
(IL-2R)-directed therapy that exploit the difference in IL-2R expression
between normal and malignant cells. Using unmodified anti- Tac monoclonal
antibody, one-third of the patients with adult T -cell leukemia
(A TL) treated have undergone a remission, in two cases complete.
There was little toxicity observed; however, unmodified monoclonal
antibodies are limited by their immunogenicity and their poor effector
functions. To address these issues, "humanized" anti- Tac was produced
that contains the complementaritydetermining regions from the mouse
with the remainder of the molecule derived from human IgG 1k This
antibody is dramatically less immunogenic than the murine version,
has improved pharmacokinetics, and, in contrast to the parent antibody,
manifests antibodydependent cellular cytotoxicity (ADCC). To enhance
its effector function, anti- Tac was armed with toxins and alfa-
and ß-emitting radionuclides. In a clinical trial of 90y -anti-
Tac in A TL patients, at the doses used (5, 10, and 15 mCi 90y -anti-
Tac per patient), 10 of the 15 patients with A TL treated to date
underwent sustained partial or complete remission. Thus, the clinical
application of IL-2R-directed therapy represents anew perspective
for the prevention of allograft rejection and for the treatment
of graft-versus-host disease, select autoimmune disorders, and leukemia/lymphoma.
The development of the hybridoma technique by Köhler and Milstein
(1) rekindled interest in the use of antibodies to treat cancer
patients. However, monoclonal antibodies are just beginning to fulfill
the promise for immunotherapy inherent in their great specificity
for recognizing and binding to abnormal cells. That is, despite
wide-ranging interest, the "magic bullet" of antibody therapy that
has been a dream since the time of Paul Ehrlich (2) has proven elusive.
One of the factors in this low therapeutic efficacy is that the
murine monoclonal antibodies are immunogenic when administered to
humans. Another critical factor is that most of the monoclonal antibodies
employed are not effective cytocidal agents against human neoplastic
cells. Furthermore, in most cases the antibodies were not directed
against a vital structure present on the surface of malignant cells,
such as a growth factor receptor required for tumor cell proliferation
and survival. We readdressed this issue by (a) using genetic engineering
to create less immunogenic and more effective monoclonal antibodies,
(b) arming antibodies with toxins or radionuclides to enhance their
effector functions, and ( c ) selecting the IL-2R on abnormal cells
as a target for effective monoclonal antibody action. The scientific
basis for the use of this target is that resting cells do not express
-IL-2R alfa, whereas this receptor subunit is expressed by a proportion
of the abnormal cells in certain forms of lymphoid neoplasia (e.g
., adult T -cellleukemia/lymphoma [A TL], cutaneous T -cell lymphoma,
hairy cell leukemia, and Hodgkin's disease), in select autoimmune
diseases (e .g ., rheumatoid arthritis, sarcoidosis, scleroderma,
and tropical spastic paraparesis [TSP]), in individuals undergoing
graft-versus-host disease, and by the T cells involved in allograft
rejection (3- 7).
RESULTS AND DISCUSSION
STRUCTURE AND FUNCTION OF THE MULTISUBUNIT IL-2R
Successful T -cell-mediated immune responses require that these
cells change from a resting to an activated state. Activation requires
two sets of signals from cell surface receptors to the nucleus.
The first signal is initiated when an appropriately processed and
presented foreign antigen interacts with a heterodimeric T -cell
receptor (Tcr) for that specific antigen. Following this encounter
with antigen, T cells enter a program of cellular activation leading
to de novo synthesis and secretion of IL-2 (3-5). Resting T cells
do not express high-affinity IL-2R's, but receptors are rapidly
expressed after T -cell activation with antigen or mitogen (3, 6-8).
The interaction of IL-2 with the induced cellular receptor for this
lymphokine then triggers proliferation of these cells, culminating
in the emergence of effector T lymphocytes mediating helper, suppressor,
or cytotoxic functions. Thus, the expression of the genes encoding
IL- 2 and its receptor and the subsequent interaction of this ligand
with its receptor are critical events in T -cell activation and
are required for full expression of the human immune response. There
are three forms of cellular receptor for IL-2 based on their affinity
for ligand, including one with a very high IL2 binding affinity
(10 high-11 M), one with an intermediate affinity (10 high-9 M),
and one with a lower affinity (10 high-8 M) (810). We have used
monoclonal antibodies, affinity crosslinking with radiolabeled IL-
2, coprecipitation analysis, flow cytometric resonance energy transfer
measurement, and recovery following fluorescence photobleaching
to identify multiple subunits of this receptor (6, 11, 12). Initially,
a monoclonal antibody (antiTac) that we prepared was used to identify
a 55-kDa IL- 2R protein (now termed IL-2R alfa) (6). The receptor
protein identified by anti- Tac was characterized as a densely glycosylated
and sulfated structure with an apparent molecular mass of 55 kDa
that is composed of 251 amino acids as well as an NH2 terminal 21-amino-acid
signal peptide cleaved in vivo (13). Using crosslinking methodology
with radiolabeled IL-2, we defined a second 70/75-kDa IL-2 binding
protein, now termed IL-2Rß (11). We proposed a multi subunit model
for the high-affinity IL2R. In this model, an independently existing
IL-2R alfa or IL-2Rß subunit would generate low- and intermediateaffinity
receptors, respectively, whereas high-affinity receptors would be
formed when both receptors are expressed and noncovalently associated
in a receptor complex (11). An additional 64-kDa molecule (termed
IL2Ry) has been identified that plays a critical role in IL-2 binding,
internalization, and signaling mediated by the IL2R (14).
IL-2R EXPRESSION IN MALIGNANCY
We have used the IL-2R as a target for immune intervention. The
scientific basis for the approach utilizing the IL-2R alfa subunit
as a target for immunotherapy is that resting normal cells do not
express the Tac peptide of the IL-2R. In contrast, we and others
have shown that this receptor is expressed by a proportion of the
abnormal cells in certain forms of lymphoid neoplasia, select autoimmune
diseases, and in individuals rejecting allografts (3, 15, 16). That
is, a proportion of the abnormal cells in these diseases express
the Tac antigen on their surface. Furthermore, Nelson and coworkers
(17) have shown that the serum concentration of the soluble form
of IL- 2R alfa is elevated in patients with these disorders. In
terms of neoplasia, certain T -cell, B-cell, monocytic, and even
granulocytic leukemias express the Tac antigen. Specifically, the
malignant T cells of patients with human T -celllymphotrophic virus
I (HTLV-I)-induced ATL constitutively express IL-2R alfa (18). Furthermore,
the malignant T cells of the skin and lymph nodes of patients with
cutaneous T -cell lymphoma (mycosis fungoides and the sezary syndrome)
express the Tac antigen (19). In addition, virtually all of the
malignant cells of patients with hairy cell B-cellleukemia express
the Tac antigen, and a proportion of other B-celllymphomas are also
Tac-positive (20). Finally, true histiocytic leukemia cells and
the Reed-Sternberg cell of Hodgkin's disease also manifest IL-2R
alfa (21). In addition to these Tac-expressing leukemias and lymphomas,
we have shown that there are certain leukemias (e.g., acute lymphoblastic
leukemia and large granular lymphocytic leukemia) that do not express
IL-2R alfa, yet express the IL-2Rß subunit of the IL-2R (22). Disorders
of IL-2R Expression in HTLV-I-Associated ATL. We focused our initial
IL-2R immunotherapeutic studies on A TL, a distinct form of aggressive
T -cell leukemia (23). HTLV-I is the primary etiologic agent in
ATL (24). ATL is a malignant proliferation of CD3/CD4-expressing
T cells that typically infiltrates the skin, lungs, and liver. All
populations of leukemic cells we have examined from patients with
HTL V -I -associated A TL constitutivel y express high- and low-affinity
IL- 2R, including very large numbers of the IL-2R alfa defined by
the anti- Tac monoclonal antibody (18). An analysis of HTLV-I and
its protein products suggests a potential mechanism for the association
between HTLV-I and constitutive IL-2R alfa expression (25). The
retrovirus HTL V -I encodes a 42-kDa protein (now termed "tax")
that plays an important role in the early phases of HTL V -I -induced
malignancy by deregulating the expression of the cellular genes
that encode IL-2 and a 55-kDa Tac IL-2R peptide that controls T
-cell proliferation (25, 26).
IL-2R alfa AS A TARGET FOR THERAPY IN PATIENTS WITH HTLV-I-ASSOCIATED
Unmodified Anti-Tac Monoclonal Antibody. No conventional treatment
program is successful in inducing long-term disease-free survival
in A TL patients, with an overall mortality of approximately 50%
within 5 months. However, the HTLV-I-induced ATL cells constitutively
express the IL-2R alfa chain identified by the anti- Tac monoclonal
antibody, whereas normal resting cells do not. This observation
provided the scientific basis for IL-2Rdirected immunotherapy with
this monoclonal antibody. IL-2R-directed immunotherapeutic agents
could theoreticall y eliminate IL- 2R alfa-expressing leukemic cells
or abnormally activated T cells involved in other disease states
while retaining the Tac-nonexpressing normal T cells and their precursors
that express the antigen receptors for T -cell-mediated immune responses.
In our initial studies, we administered unmodified murine anti-
Tac to patients with ATL (15, 16). The leukemic cells of each patient
with A TL reacted with anti- Tac. Our goal was to inhibit the interaction
of IL-2 with its growth factor receptor expressed on the malignant
cells. The 20 patients treated in this study did not have untoward
reactions related to the immunotherapy. Only patients undergoing
a remission produced antibodies to the monoclonal antibody. Seven
of the 20 treated patients had transient mixed (1 patient), partial
( 4 patients), or complete (2 patients) remission, lasting from
1 to more than 30 months after anti- Tac therapy. This was assessed
by elimination of measurable skin and lymph nodal disease, normalization
of serum calcium levels, and routine hematologic and phenotypic
tests of circulating cells. Further, elimination of clonal malignant
cells was shown by molecular genetic analysis of HTL V -I proviral
integration and by definition of monoclonal T -cell antigen receptor
gene rearrangements. Although this approach to the treatment of
A TL was encouraging, 13 of the 20 patients did not manifest at
least a partial remission. Furthermore, six of the seven responding
patients ultimately relapsed. This relapse was not associated with
the loss of IL- 2R alfa expression by the A TL cells. However, it
did reflect the fact that certain A TL cells no longer produce nor
require IL- 2 for their proliferation. Since anti- Tac functions,
in part, by preventing the interaction of IL-2 with IL-2R alfa,
it is no longer effective at this stage of the patient's disease.
Nevertheless, IL-2R alfa is still expressed on the leukemIc cells,
but not on the normal cells of the patients, thus providing a target
for immunotherapy with anti- Tac armed with toxins or radionuclides.
Humanized Antibodies to IL-2R. There are two problems with murine
monoclonal antibodies in general: their immunogenicity and their
ineffectiveness at recruiting hosteffector functions. We have addressed
these issues by producing "humanized" antibodies. These humanized
antiTac molecules, produced in conjunction with Cary Queen, retain
the complementarity-detem1ining region from the mouse, but virtually
all the remainder of the molecule is derived from human IgG 1 K.
On the basis of computer modeling of the structure of this antibody,
murine elements close to the complementarity-determining regions
were identified, and those that were believed to be important to
maintain the appropriate conformation of this antibody were retained
One primary goal in these studies is to maintain the affinity and
functional capacity of the mouse monoclonal antibody. The parent
anti- Tac molecule had an affinity of 9 x 10 high 9/M, whereas the
hyperchimeric "humanized" version has an affinity of 3 x 10 high
9/M, still very high (27). The parent monoclonal and the humanized
version showed comparable inhibition of T -cell proliferation in
response to tetanus antigen (28). The humanized version of anti-
Tac had improved pharmacokinetics when compared to the murine version,
with an in vivo survival that is 2.5- fold longer (terminal t112'
103 hr vs. 38 hr). In addition, humanized anti- Tac was less immunogenic
than murine anti- Tac when administered to cynomolgus monkeys undergoing
heterotopic cardiac allografting (29). A final goal of this project
is to make an antibody that is abetter effector of cell killing
than is murine anti- Tac. Therefore, we were greatly encouraged
by the observation that although the parent mouse anti- Tac could
not function in antibody-dependent cellular cytotoxicity (ADCC)
with human mononuclear cells, the hyperchimeric IgG 1 anti -Tac
manifests ADCC with human mononuclear cells (28). Clinical trials
will be initiated using humanized anti- Tac in the treatment of
patients with leukemia/lymphoma or with graft-versus-host disease.
M onoclonal Antibody-Cytotoxic Agent Conjugates: IL-2R Directed
Immunotoxins. To continue to take advantage of the difference in
IL- 2R expression between normal and leukemic cells and to improve
the effectiveness of IL-2Rdirected therapy, different approaches
were initiated to modify the antibody for clinical purposes. In
one approach, anti- Tac or IL-2 was used to deliver modified toxins
to IL-2R-expressing cells without incurring severe systemic toxicity.
Hwang and coworkers (30) generated. a genetically modified form
of Pseudomonas exotoxm (PE40), from which the first domain responsible
for ubiquitous cell binding was deleted. To generate a homogeneous
product, a single-chain antibody fusion protein [e.g., anti- Tac
(Fv)-PE40] in which the variable regions of anti- Tac are joined
in protein linkages wIth PE40 was constructed and expressed in E.
coli. Anti- Tac (Fv)-PE40 is very cytotoxic to IL-2R-bearing human
cell lines as well as fresh cells derived from patients with IL-2R
alfa-expressing A TL, but is not cytotoxic to receptor negative
cells (31). IL-2-PE40, a chimeric protein composed of human IL-2
genetically linked to the amino terminus of PE40, was constructed
to provide an alternative (lymphokine-mediated) method of delivering
the truncated toxin to the surface of IL- 2R -expressing cells (32).
Monoclonal Antibodies Armed with alfa- and ß-Emitting Radionuclides.
The action of toxins conjugated to monoclonal antibodies depends
on their ability to be internalized by the cell and translocated
to the cytoplasm. In fact, the toxin conjugates do not pass easily
from the endosome to the cytosol. Furthermore, large protein toxins
are immunogenic and thus provide only a short therapeutic window
prior to the development of host antibodies directed toward the
toxin. In the future these problems will probably be resolved. However,
to circumvent these limitations, radiolabeled monoclonal antibodies
were developed as alternative immunoconjugates to deliver a cytotoxic
agent to target cells. There are a number of advantages over other
approaches in the use of radiolabeled monoclonal antibody conjugates
for therapy. One is that with the appropriate choice of radionuclide,
radiolabeled monoclonal antibodies kill cells at distances of several
cell diameters; therefore, a radiolabeled monoclonal antibody binding
to an antigen-expressing cell may kill adjacent antigen-nonexpressing
cells, thereby overcoming the tumor cell antigenic heterogeneity
that presents a problem for most other monoclonal-antibody-mediated
approaches. Furthermore, the radiolabeled antibody need not be internalized
to kill the tumor cell. Nuclear chemistry has provided a selection
of radioisotopes that could be linked . to Immunoprotems. In studies
performed in collaboration with Otto Gansow, we turned to alfa-
and ß-emitting radionuclides as alternative cytotoxic agents that
could be conjugated to anti- Tac. In initial studies, a series of
chelating agents was developed that did not compromise antibody
specificity and did not permit premature release of the radionuclide
in viva. Our choice of isotopes is based on the desire to have agents
with a short distance of action that will act on the cell in question
and on a small number of bystander cells without unwanted toxicity.
In one case, we bound the ß-emitting 90y to anti- Tac using chelates
that did not permit elution of radiolabeled yttrium from the monoclonal
antibody (33). Monkeys that received xenografts or allografts of
cynomolgus hearts showed a marked prolongation of graft survival
following administration of 90y -labeled anti- Tac (29). Following
preclinical efficacy and toxicity studies, we initiated a dose escalation
trial of 90y anti- Tac for the treatment of patients with HTL V
-I-associated Tac-expressing A TL. At the doses utilized (5, 10,
and 15 mCi per patient) 10 of the 15 patients underwent partial
(8 patients) or complete (2 patients) remission. Thus, antiTac armed
with a radionuclide provides meaningful therapy for a form of leukemia
that was previously universally fatal.
Future development of isotopic monoclonal antibody chelates may
focus on alfa-emitting radionuclides, which may be the most effective
agents at killing tumor targets without damaging distant normal
tissues. Radionuclides emitting alfa particles release high-energy
emissions (6-9 MeV, 10 times as great as ß or yemitters) over a
short distance ( 40--80 µm) and are efficient at killing individual
target cells, such as those found in leukemia, without significantly
penetrating normal tissues. U nder hypoxic conditions that permit
little cellular repair , alfa irradiation is efficient at killing
cells. Suitable alfa-emitting nuclides available for immunotherapy
that are under our investigation include 212Bi, 212pb, and 211 At.
A major future direction for our IL-2R-directed therapeutic program
will be to arm the humanized version of the antiTac monoclonal antibody
with alfa-emitting radionuclides. Two chelates have been developed
(DOT A and CHX) that permit the linkage of 212Bi to humanized anti-
Tac. We have shown that 212Bi conjugated to anti- Tac by use of
these bifunctional chelates is well suited to effectively kill Tac-expressing
A TL cells at doses that have only a modest effect on IL-2R alfa-nonexpressing
lines. Activity levels of 0.5 µCi targeted by 212Bi-Iabeled
anti- Tac eliminated more than 98% of the proliferative capacity
of the IL-2Rexpressing HTL V -I -associated A TL line, HUT -102-
B 2, with only a modest effect on an IL-2R-nonexpressing line (34
). This specific therapeutic effect was blocked by an excess of
unlabeled anti- Tac but not human IgG. On the basis of in vitra
binding studies and in viva pharmacokinetic analyses in mice, the
two chelating agents fulfilled the criteria for a suitable radioimmunotherapeutic
agent. Thus, one of the most promising directions for the future
development of armed monoclonal antibodies for the treatment of
human cancer involves the linkage of alfa-emitting radionuclides
such as 212Bi to humanized monoclonal antibodies. Such conjugates
may prove to be relatively nonimmunogenic agents that are effective
in eliminating malignant cells when used alone or as part of multimodality
treatment with conventional chemotherapy.
In summary, our present understanding of the IL-2/IL-2R system
has opened the possibility for more specific immune intervention
strategies. The IL-2R is proving to be an extraordinarily useful
therapeutic target. The clinical applications of anti-IL-2R-directed
therapy, especially the use of humanized antibodies armed with radionuclides,
represents anew perspective for the prevention of allograft rejection
and for the treatment of graft-versus-host disease, select autoimmune
disorders, and leukemia/lymphoma.
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