Note: Descriptions are shown in the official language in which they were submitted.
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METHODS OF THERAPY FOR NON-HODGKIN' S LYMPHOMA
FIELD OF THE INVENTION
The present invention is directed to methods of therapy for non-Hodgkin's
lymphoma, more particularly to concurrent therapy with interleukin-2 and
monoclonal
antibodies targeting the CD20 B-cell surface antigen.
BACKGROUND OF THE INVENTION
The non-Hodgkin's lymphomas are a diverse group of malignancies that are
predominately of B-cell origin. In the Working Formulation classification
scheme,
these lymphomas been divided into low-, intermediate-, and high-grade
categories by
I S virtue of their natural histories (see "The Non-Hodgkin's Lymphoma
Pathologic
Classification Project," Cancer 49(1982):2112-2135). The low-grade lymphomas
are
indolent, with a median survival of 5 to 10 years (Horning and Rosenberg
(1984) N.
Ehgl. J. Med. 311:1471-1475). Although chemotherapy can induce remissions in
the
majority of indolent lymphomas, cures are rare and most patients eventually
relapse,
requiring further therapy. The intermediate- and high-grade lymphomas are more
aggressive tumors, but they have a greater chance for cure with chemotherapy.
However, a significant proportion of these patients will relapse and require
further
treatment.
Interleukin-2 (IL-2) is a potent stimulator of natural killer (NK) and T-cell
proliferation and function (Morgan et al. (1976) Science 193:1007-1011). This
naturally occurring lymphokine has been shown to have anti-tumor activity
against a
variety of malignancies either alone or when combined with lymphokine-
activated
killer (LAK) cells or tumor-infiltrating lymphocytes (TIL) (see, for example,
Rosenberg et al. (1987) N. Engl. J. Med. 316:889-897; Rosenberg (1988) Arcn.
Surg.
208:I21-135; Topalian et al, 1988) J. Clirc. ~~col. 6:839-853; Rosenberg et
al. (1988)
N. Engl. J. Med. 319:1676-1680; and Weber et al. (1992) .I, Clin. Orccol.
10:33-40).
The anti-tumor activity of IL-2 has best been described in patients with
metastatic
melanoma and renal cell carcinoma using Pxoleukin~ IL-2, a commercially
available
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IL-2 formulation. Other diseases, including lymphoma, also appear to respond
to
treatment with IL-2 (Gisselbrecht et al. (1994) Blood 83(8):2020-2022).
However,
high doses of IL-2 used to achieve positive therapeutic results with respect
to tumor
growth frequently cause severe side effects, including capillary leak,
hypotension, and
neurological changes (see, for example, Duggan et al. (1992) J. Inamunotherapy
I2:I 15-122; Gisselbrecht et al. (1994) Blood 83:2081-2085; and Sznol and
Parkinson
1994) Blood 83:2020-2022).
Cancer research turned to the use of monoclonal antibodies as therapeutic
agents. Produced in a similar fashion to diagnostic antibodies, therapeutic
antibodies
are designed to target tumor cells in order to facilitate their destruction.
The use of
therapeutic monoclonal antibodies has been hampered in the past primarily
because of
issues related to the antigenicity of the protein. Monoclonal antibodies have
traditionally been a mouse product, and therefore generate an anti-murine
response
when injected into humans. This so-called HAMA (human anti-mouse antibody)
I S response has imposed a great limitation on the use of monoclonal
antibodies, as
repeated dosing is nearly always precluded. In addition, serious
complications, such
as serum sickness, have been reported with the use of these agents. With the
advent
of chimeric and humanized antibodies, the therapeutic benefit of monoclonals
is being
realized. Using recombinant DNA technology, it is possible for a monoclonal
antibody to be constructed by joining the variable or antigen recognition site
of the
antibody to a human backbone. This construction greatly decreases the
incidence of
blocking or clearing of the foreign antibodies from the host. This development
allows
for multiple doses of antibody to be given, providing the opportunity for
reproducible
and sustained responses with this therapy.
Monoclonal antibodies have increasingly become a method of choice for the
treatment of lymphomas of the B-cell type. All B-cells express common cell
surface
markers, including CD20 and CD19. CD20 is a 33-37 kD phosphoprotein that is
expressed early in B-cell differentiation and normally disappears in mature
plasma
cells. CD I9 is closely associated with the B-cell antigen receptor and
functions to
send a signal when the cell engages antigen. CD20 and CD19 are expressed at
very
high levels on lymphoma cells. Approximately 90% of low-grade lymphomas
express CD20 while CD19 is nearly ubiquitously expressed from all B-cells
excluding
bone marrow progenitors and plasma cells.
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CD20 has become the premiere target for monoclonal therapy directed at B-
cell antigens. In vitro work has demonstrated that monoclonal antibodies
directed to
CD20 result in cell death by apoptosis (Shan et al. (1998) Blood 91:1644-
1652).
Other studies report that B-cell death is primarily mediated by antibody-
dependent
cytotoxicity (ADCC). ADCC is a cellular mechanism that depends on specific
effector cells carrying receptors for the monoclonal antibody bound to its
target.
These are in general receptors that are present on NK cells, neutrophils, and
cells with
monocyte/macrophage lineage. The NIA cells appear to be the relevant mediators
of
this phenomenon, and antibodies to CD20 mediate their cytotoxicity primarily
through ADCC.
Because of the possible immunological basis of the anti-tumor activity of anti-
CD20 antibodies, combinations with other cytokines that enhance NIA cell
function
have been examined. Cytokines such as IL-12, IL-15, TNP-alpha, TNF-beta, gamma-
IFN, and IL-2 have been tested for potentiation of ADCC, a distinct NIA
function. All
appear to be active in enhancing ADCC, although each agent is associated with
its
own specific toxicities.
The most compelling animal model is a nude mouse implanted with Daudi
cells. Daudi cells are cells from a cell line derived from a patient with
Burkitt's
lymphoma, a B-cell tumor that expresses CD20. In this model, IL-2 was tested
in
combination with unconjugated anti-CD20 antibody both as a prophylaxis and
after
tumors had been established (Hooijberg et al. (1995) cancer Research 55:2627-
2634). The Hooijberg study showed that IL-2, in combination with unconjugated
anti-CD20 antibody, is able to eliminate tumors completely in some animals.
The
combination was highly effective at affecting complete regression of tumors.
Other
cytokine combinations and the use of cytokines alone were much less effective
in
eliminating tumors. Hooijberg et al. also examined the combination in
preventing
tumor outgrowth and found that IL-2 and anti-CD20 were highly effective in
preventing tumor growth.
Thus, this animal model supports the notion that IL-2 in combination with
anti-CD20 is a potent mediator of B-cell tumor regression in prevention of
tumor
outgrowth. In this study, the IL-2 was given weekly and in a subcutaneous dose
of
200,000 units/mouse. The equivalent dose in humans is as high as 600 MILT,
which is
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greater than high-dose bolus used in treatment of renal cell carcinoma or
metastatic
melanoma.
Rituximab (IDEC-C2B8; IDEC Pharmaceuticals Corp., San Diego, California)
is a chimeric anti-CD20 monoclonal antibody containing human IgGl and kappa
constant regions with marine variable regions isolated from a marine anti-CD20
monoclonal antibody, IDEC-2B8 (Reff et al. (1994) Blood 83:435-445). The anti-
lymphoma effects of rituximab are in part due to complement mediated
cytotoxicity
(CMC) antibody-dependent cell mediated cytotoxicity (ADCC), inhibition of cell
proliferation, and finally direct induction of apoptosis. In early studies,
rituximab
induced a rapid depletion of CD20+ normal B-cells and lymphoma cells (Reff et
al.
(1994) Blood 83:435-445). Phase I trials of single doses up to 500 mg/m2 and
of 4
weekly doses of 375 mg/m2 demonstrated clinical responses with no dose-
limiting
toxicity in low-grade or follicular lymphoma patients (Maloney et al. (1994)
Blood
84:2457-2466. In a phase II trial, 4 weekly infusions of 375 mg/m2 induced
responses
in 17 of 34 evaluable low-grade or follicular lymphoma patients, with a median
time
to progression of 10.2 months (Maloney et al. (1997) Blood 90:2188-2195). Side
effects were in general associated with the first rituximab infusion and were
mild to
moderate. In a large pivotal phase II study, in 166 patients with low-grade or
follicular lymphoma, objective response was reported for 76 (50%) of 151
evaluable
patients and side effects were identical to those previously described
(McLaughlin et
al. (1998) ,l. Clip. Oncol. 16:2825-2833). Previous experience with rituximab
in
patients with the large cell histology is very limited, with fewer than 12
patients
having been included in the early phase I and phase II studies. Recent
studies,
however, show that rituximab has activity in diffuse large cell and mantle
cell
lymphoma patients and should be tested in combination with chemotherapy in
such
patients (Coiffier et al (1998) Blood 92:1927-1932).
However, the reality of all current antineoplastic therapies is that tumors
become resistant to therapy by a variety of mechanisms including tumor escape,
acquired drug resistance, and down-regulation of cell-surface target
molecules, among
others. In a recent study, it was shown that therapy of B-cell lymphoma with
anti-
CD20 antibodies can result in loss of the CD20 antigen expression (Davis et al
(1999)
Clin. Cahcer Res. 5:611-615). After two courses of therapy with rituximab, the
subject developed a transformed lymphoma that no longer expressed CD20.
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Thus, although IL-2 therapy alone and rituximab therapy alone have provided
a means for partial treatment of lymphoma, new therapies are needed that will
provide
prolonged treatment for this cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure lA-1F shows the time course for natural killer (NK) cell count
(CD16/CD56 cells) (lA), CD4 cell count (1B), and CD8 cell count (1C) in 11
patients
undergoing concurrent therapy with weekly rituximab therapy (375 mg/m2) and
thrice-weekly doses of Proleukin~ IL-2 for treatment of non-Hodgkin's
lymphoma.
Rituximab was administered by infusion over up to 6 hours on day 1 (D1), day 8
(D8), day 15 (D15), and day 22 (D22). Proleukin~ IL-2 was administered
subcutaneously three times per week for 4 weeks beginning on day 8. The doses
of
Proleukin~ IL-2 were 4.5 MIU (3 patients), 10.0 MIU (3 patients), 14.0 MIU (3
patients), and 18.0 MILD (data shown for 2 patients). The corresponding cell
counts
for the 9 patients that have both tumor evaluation and week-10 lymphocyte
subset
counts available are shown in 1D (NK cell count), lE (CD4 cell count) and 1F
(CD8
cell count). PD = progressive disease; SD = stable disease; CR/PR = complete
response or partial response.
Figure 2 shows median NK cell counts at baseline, at 4-weeks post-initiation,
and at 10-weeks post-initiation of concurrent therapy with Proleukin~ IL-2 and
rituximab for responders (complete response or partial response) versus non-
responders (stable disease or progressive disease) from the study described
for Figure
1. Statistical significance was calculated using the Wilcoxon Rank Sum Test.
Figure 3 shows the time course for natural killer (NK) cell activity using in
vitro assays for NK cell function, including NK-mediated cytolytic function
(NK),
and LAK- and ADCC-mediated function (LAK and ADCC, respectively) as
determined for a complete responder (CR). The CR patient participated in the
thrice-
weekly 18.0 MIU Proleukin~ IL-2/weekly rituximab dosing regimen described for
Figure 1. See Example 1 below for details regarding functional assays. The
data
demonstrate that NK activity is maintained in a CR patient.
SUMMARY OF THE INVENTION
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Methods for providing treatment to a human subject with lymphoma using a
combination of interleukin-2 (IL-2) or a variant thereof (hereinafter
collectively "IL-
2") and at least one anti-CD20 antibody or a fragment thereof (hereinafter
collectively
"anti-CD20 antibody") are provided. These two therapeutic agents are
administered
as separate pharmaceutical compositions, one containing IL-2, the other
containing at
least one anti-CD20 antibody, each according to a particular dosing regimen.
The
pharmaceutical composition comprising the anti-CD20 antibody is administered
according to a weekly dosing schedule. The pharmaceutical composition
comprising
IL-2 is administered according to a twice- or thrice-weekly constant IL-2
dosing
regimen, or is administered according to a two-level IL-2 dosing regimen. This
two-
level IL-2 dosing regimen comprises a first time period of IL-2 dosing,
wherein a
higher total weekly dose of IL-2 is administered to the subject, followed by a
second
time period of IL-2 dosing, wherein a lower total weekly dose of IL-2 is
administered
to the subject. The total weekly dose of IL-2 during the second time period of
IL-2
dosing is lower than the total weekly dose of IL-2 administered during the
first time
period of IL-2 dosing. The total weekly dose to be administered during the
first time
period and/or during the second time period of IL-2 dosing can be administered
as a
single dose. Alternatively, the total weekly dose administered during either
or both of
the first and second time periods of IL-2 dosing can be partitioned into a
series of
equivalent doses that are administered according to a two-, three-, four-,
five-, six- or
seven-times-a-week dosing schedule. In some embodiments, multiple maintenance
cycles of therapy with anti-CD20 antibody in combination with the two-level IL-
2
dosing are administered to a subject in need of treatment for non-Hodgkin's
lymphoma, wherein each maintenance cycle comprises administering the anti-CD20
antibody in combination with the two-level IL-2 dosing regimen. The need for
administering these multiple maintenance cycles is assessed by monitoring
natural-
killer (NIA) cell counts in subjects undergoing treatment with the methods of
the
invention. The methods also provide for an interruption in the two-level
dosing
regimen of IL-2, where the subject is given a time period off of IL-2
administration,
or a time period off of IL-2 and anti-CD20 antibody administration, between
the first
and second time periods of the two-level IL-2 dosing regimen.
Administering of these two agents together in the manner set forth herein
provides for greater therapeutic effectiveness than can be achieved using
either of
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these agents alone, resulting in a positive therapeutic response that is
improved with
respect to that observed with either agent alone. In addition, the beneficial
therapeutic
effects of these agents can be achieved using lower cumulative dosages of IL-
2,
thereby lessening the toxicity of prolonged IL-2 administration and the
potential for
tumor escape.
A method for predicting clinical response of a subject undergoing a time
period of concurrent therapy with anti-CD20 antibody and IL-2 is also
provided. The
method comprises monitoring natural killer (NK) cell expansion in said subject
at
about 1 week to about 14 weeks post-initiation of the time period of
concurrent
therapy. Threshold counts for NIA cell expansion that are predictive of
positive
therapeutic response in a subject undergoing concurrent therapy with these two
therapeutic agents are provided.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to methods of treating a human subject with
lymphoma, more particularly non-Hodgkin's B-cell lymphoma. The methods
comprise combination therapy with interleukin-2 or a variant thereof
(hereinafter
collectively "IL-2") and at least one anti-CD20 antibody or a fragment thereof
(hereinafter collectively "anti-CD20 antibody"). Combination therapy with
these two
therapeutic agents provides for anti-tumor activity. By "anti-tumor activity"
is
intended a reduction in the rate of cell proliferation, and hence a decline in
growth
rate of an existing tumor or in a tumor that arises during therapy, and/or
destruction of
existing neoplastic (tumor) cells or newly formed neoplastic cells, and hence
a
decrease in the overall size of a tumor during therapy. Subjects undergoing
therapy
with a combination of IL-2 and at least one anti-CD20 antibody in accordance
with
the methods of the present invention experience a physiological response that
is
beneficial with respect to treatment of B-cell lymphoma, more particularly non-
Hodgkin's B-cell lymphoma.
The therapeutic methods of the invention are directed to treatment of any non-
Hodgkin's B-cell lymphoma whose abnormal B-cell type expresses the CD20
surface
antigen. By "CD20 surface antigen" is intended a 33-37 kD integral membrane
phosphoprotein that is expressed during early pre-B cell development and
persists
through mature B-cells but which is lost at the plasma cell stage. Although
CD20 is
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expressed on normal B cells, this surface antigen is usually expressed at very
high
levels on neoplastic B cells. More than 90% of B-cell lymphomas and chronic
lymphocytic leukemias, and about 50% of pre-B-cell acute lymphoblastic
leukemias
express this surface antigen.
It is recognized that concurrent therapy with IL-2 and an anti-CD20 antibody
may be useful in the treatment of any type of cancer whose unabated
proliferating
cells express the CD20 surface antigen. Thus, for example, where a cancer is
associated with aberrant T-cell proliferation, and the aberrant T-cell
population
expresses the CD20 surface antigen, concurrent therapy in accordance with the
methods of the invention would provide a positive therapeutic response with
respect
to treatment of that cancer. A human T-cell population expressing the CD20
surface
antigen, though in reduced amounts relative to B-cells, has been identified
(see Hultin
et al. (1993) Cytomet~y 14:196-204).
It also is recognized that the methods of the invention are useful in the
therapeutic treatment of B-cell lymphomas that are classified according to the
Revised
European and American Lymphoma Classification (REAL) system. ~ Such B-cell
lymphomas include, but are not limited to, lymphomas classified as precursor B-
cell
neoplasms, such as B-lymphoblastic leukemia/lymphoma; peripheral B-cell
neoplasms, including B-cell chronic lymphocytic leukemia/small lymphocytic
lymphoma, lymphoplasmacytoid lymphoma/immunocytoma, mantle cell lymphoma
(MCL), follicle center lymphoma (follicular) (including diffuse small cell,
diffuse
mixed small and large cell, and diffuse large cell lymphomas), marginal zone B-
cell
lymphoma (including extranodal, nodal, and splenic types), hairy cell
leukemia,
plasmacytoma/ myeloma, diffuse large cell B-cell lymphoma of the subtype
primary
mediastinal (thymic), Burkitt's lymphoma, and Burkitt's like high grade B-cell
lymphoma; and unclassifiable low-grade or high-grade B-cell lymphomas.
By "non-Hodgkin's B-cell lymphoma" is intended any of the non-Hodgkin's
based lymphomas related to abnormal, uncontrollable B-cell proliferation. For
purposes of the present invention, such lymphomas are referred to according to
the
WorkirrgFormzslatio~ classification scheme (see "The Non-Hodgkin's Lymphoma
Pathologic Classification Project," Cancer 49(1982):2112-2135), that is those
B-cell
lymphomas categorized as low grade, intermediate grade, and high grade. Low-
grade
B-cell lymphomas include small lymphocytic, follicular small-cleaved cell, and
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follicular mixed small-cleaved cell lymphomas; intermediate-grade lymphomas
include follicular large cell, diffuse small cleaved cell, diffuse mixed small
and large
cell, and diffuse large cell lymphomas; and high-grade lymphomas include large
cell
immunoblastic, lymphoblastic, and small non-cleaved cell lymphomas of the
Burkitt's
and non-Burkitt's type.
While the methods of the invention are directed to treatment of an existing
non-Hodgkin's B-cell lymphoma, it is recognized that the methods may be useful
in
preventing further tumor outgrowths arising during therapy. The methods of the
invention are particularly useful in the treatment of subjects having low-
grade B-cell
lymphomas, particularly those subjects having relapses following standard
chemotherapy. Low-grade B-cell lymphomas are more indolent than the
intermediate- and high-grade B-cell lymphomas and are characterized by a
relapsing/remitting course. Thus, treatment of these lymphomas is improved
using
the methods of the invention, as relapse episodes are reduced in number and
severity.
In accordance with the methods of the present invention, IL-2 and at least one
anti-CD20 antibody as defined elsewhere below are used in combination to
promote a
positive therapeutic response with respect to non-Hodgkin's B-cell lymphoma.
By
"positive therapeutic response" is intended an improvement in the disease in
association with the combined therapeutic activity of these agents, and/or an
improvement in the symptoms associated with the disease. Thus, for example, an
improvement in the disease may be characterized as a complete response. By
"complete response" is intended an absence of clinically detectable disease
with
normalization of any previously abnormal radiographic studies, bone marrow,
and
cerebrospinal fluid (CSF). Such a response must persist fox at least one month
following treatment according to the methods of the invention. A complete
response
can be unconfirmed if no repeat evaluation of tumor status is done at least
one month
after the initial response is evaluated. Alternatively, an improvement in the
disease
may be categorized as being a partial response. By "partial response" is
intended at
least a 50% decrease in all measurable tumor burden (i.e., the number of tumor
cells
present in the subject) in the absence of new lesions and persisting for at
least one
month. Such a response is applicable to measurable tumors only. In addition to
these
positive therapeutic responses, the subject undergoing concurrent therapy with
these
two therapeutic agents may experience the beneficial effect of an improvement
in the
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symptoms associated with the disease. Thus the subject may experience a
decrease in
the so-called B symptoms, i.e., night sweats, fever, weight loss, and/or
urticaria.
Promotion of a positive therapeutic response with respect to a non-Hodgkin's
lymphoma in a human subject is achieved via concurrent therapy with both IL-2
and
at least one anti-CD20 antibody. By "concurrent therapy" is intended
presentation of
IL-2 and at least one anti-CD20 antibody to a human subject such that the
therapeutic
effect of the combination of both substances is caused in the subject
undergoing
therapy. Concurrent therapy may be achieved by administering at least one
therapeutically effective dose of a pharmaceutical composition comprising IL-2
and at
least one therapeutically effective dose of a pharmaceutical composition
comprising at
least one anti-CD20 antibody according to a particular dosing regimen. For
example,
in accordance with the methods of the present invention, concurrent therapy is
achieved by administering the recommended total weekly doses of a
pharmaceutical
composition comprising IL-2 in combination with the recommended
therapeutically
effective doses of a pharmaceutical composition comprising at least one anti-
CD20
antibody, each being administered according to a particular dosing regimen. By
"therapeutically effective dose or amount" is intended an amount of the
therapeutic
agent that, when administered with a therapeutically effective dose or amount
of the
other therapeutic agent, brings about a positive therapeutic response with
respect to
treatment of a B-cell lymphoma such as non-Hodgkin's lymphoma. Administration
of the separate pharmaceutical compositions can be at the same time (i.e.,
simultaneously) or at different times (i.e., sequentially, in either order, on
the same
day, or on different days), so long as the therapeutic effect of the
combination of both
substances is caused in the subject undergoing therapy.
The separate pharmaceutical compositions comprising these therapeutic agents
as therapeutically active components may be administered using any acceptable
method known in the art. Thus, for example, the pharmaceutical composition
comprising IL-2 can be administered by any form of injection, including
intravenous
(IV), intramuscular (IIV>], or subcutaneous (SC) injection. In some
embodiments of
the invention, the pharmaceutical composition comprising IL-2 is administered
by SC
injection. In other embodiments of the invention, the pharmaceutical
composition
comprising IL-2 is a sustained-release formulation, or a formulation that is
administered using a sustained-release device. Such devices are well known in
the
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art, and include, for example, transdermal patches, and miniature implantable
pumps
that can provide for drug delivery over time in a continuous, steady-state
fashion at a
variety of doses to achieve a sustained-release effect with a non-sustained-
release IL-
2 pharmaceutical composition. The pharmaceutical composition comprising the
anti-
s CD20 antibody is administered, for example, intravenously. When administered
intravenously, the pharmaceutical composition comprising the anti-CD20
antibody
can be administered by infusion over a period of about 1 to about 10 hours. In
some
embodiments, infusion of the antibody occurs over a period of about 2 to about
8
hours, over a period of about 3 to about 7 hours, over a period of about 4 to
about 6
hours, or over a period of about 6 hours, depending upon the anti-CD20
antibody
being administered.
Concurrent therapy with both of these therapeutic agents in the mamier set
forth herein provides for greater therapeutic effectiveness than can be
achieved using
either of these agents alone, resulting in a positive therapeutic response
that is
improved with respect to that observed with either agent alone. This positive
therapeutic response is achieved using lower cumulative doses of IL-2 than
would be
required to get similar therapeutic benefit using IL-2 as a single agent.
Thus, a dose
of IL-2 alone that is not normally therapeutically effective may be
therapeutically
effective when administered in combination with at least one anti-CD20
antibody in
accordance with the methods of the invention. The significance of this is two-
fold.
First, the potential therapeutic benef is of treatment of lymphoma with IL-2
can be
realized at IL-2 doses that minimize toxicity responses normally associated
with
prolonged IL-2 therapy or high-bolus IL-2 administration. Such toxicity
responses
include, but are not limited to, chronic fatigue, nausea, hypotension, fever,
chills,
weight gain, pruritis or rash, dysprea, azotemia, confusion, thrombocytopenia,
myocardial infarction, gastrointestinal toxicity, and vascular leak syndrome
(see, for
example, Allison et al. (1989) J. Clin. Oacol. 7(1):75-80). Secondly,
targeting of
specific molecules on a tumor cell surface by monoclonal antibodies can select
for
clones that axe not recognized by the antibody or are not affected by its
binding,
resulting in tumor escape, and loss of effective therapeutic treatment. Such
tumor
escape has been documented with repeated doses of an anti-CD20 antibody (Davis
et
al. (1999) Clih. Cancer Res. 5:611-615). The improved therapeutic benefit of
anti-
CD20 antibodies administered in combination with IL-2 may translate into less
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frequent administration of monoclonal antibodies, thereby lessening the
potential for
tumor escape.
The amount of at least one anti-CD20 antibody to be administered in
combination with an amount of IL-2, and the amount of either of these
therapeutic
agents needed to potentiate the effectiveness of the other therapeutic agent,
are readily
determined by one of ordinary skill in the art without undue experimentation
given
the disclosure set forth herein. Factors influencing the respective amount of
IL-2 to
be administered in combination with a given amount of at least one anti-CD20
antibody in accordance with the dosing regimens disclosed herein include, but
are not
limited to, the mode of administration, the particular lymphoma undergoing
therapy,
the severity of the disease, the history of the disease, and the age, height,
weight,
health, and physical condition of the individual undergoing therapy.
Similarly, these
factors will influence the necessity for repeated exposure to combination IL-
2lanti-
CD20 antibody therapy in the manner set forth herein. Generally, a higher
dosage of
the antibody agent is preferred with increasing weight of the subject
undergoing
therapy. '
In accordance with the methods of the present invention, the human subject
undergoing treatment with one or more weekly doses of anti-CD20 antibody as
defined herein below is also administered IL-2 as defined herein below
according to a
constant IL-2 dosing regimen or according to a two-level IL-2 dosing regimen.
The
first therapeutically effective dose administered to the subject can be the
anti-CD20
antibody or can be the IL-2, depending upon which IL-2 dosing regimen is used.
Generally, where the individual is to receive a constant IL-2 dosing regimen,
the
initial therapeutic agent to be administered is anti-CD20 antibody, while the
first dose
of IL-2 is administered subsequently, for example, within 10 days following
administration of the first therapeutically effective dose of the anti-CD20
antibody,
for example, within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some
embodiments, the
constant IL-2 dosing regimen is initiated by administering a first dose of IL-
2 within 7
days of administering the first therapeutically effective dose of anti-CD20
antibody,
such as within 1, 2, 3, 4, 5, 6, or 7 days. Where the individual is to receive
a two-
level IL-2 dosing regimen, either therapeutic agent can be administered first,
so long
as the subject has an overlapping period of time during which both therapeutic
agents
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are being administered to the subject, each according to the particular dosing
regimen
disclosed herein.
Thus, in one embodiment, the two-level IL-2 dosing regimen is initiated prior
to initiating weekly administration of therapeutically effective doses of anti-
CD20
antibody. In this manner, a first dose of IL-2 is administered up to one month
before
the first dose of anti-CD20 antibody is administered. By "up to one month" is
intended the first dose of IL-2 is administered at least one day before
initiating anti-
CD20 antibody administration, but not more than one month (i.e., 30 days)
before
initiating anti-CD20 antibody administration. Thus, IL-2 administration can
begin,
for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days (i.e., 1
week), 10
days, 14 days (i.e., two weeks), 17 days, 21 days (i.e., 3 weeks), 24 days, 28
days (4
weeks), or up to one month (i.e., 30 days) before administering the first
therapeutically effective dose of the anti-CD20 antibody.
In other embodiments, the two-level IL-2 dosing regimen and anti-CD20
antibody administration begin concurrently on the same day, either at the same
time
(i.e., simultaneous administration) or at different times (i.e., sequential
administration,
in either order). Thus, for example, in one embodiment where concurrent
therapy
with these two therapeutic agents begins on day 1 of a treatment period, a
first
therapeutically effective dose of anti-CD20 antibody and a first dose of IL-2
would
both be administered on day 1 of this treatment period.
In alternative embodiments, a first therapeutically effective dose of anti-
CD20
antibody is administered to the subject, for example, on day 1 of a treatment
period,
and the two-level IL-2 dosing regimen is initiated by administering a first
dose of IL-2
within 10 days of administering the first therapeutically effective dose of
anti-CD20
antibody. In such embodiments, preferably the two-level IL-2 dosing regimen is
initiated by administering a first dose of IL-2 within 7 days of administering
the first
therapeutically effective dose of anti-CD20 antibody, such as within l, 2, 3,
4, 5, 6, or
7 days. Depending upon the severity of the disease, the patient's health, and
prior
history of the patient's disease, repeated sessions of concurrent therapy with
IL-2 and
anti-CD20 antibody in accordance with the dosing regimens disclosed herein is
contemplated. Such repeated sessions are referred to herein as maintenance
cycles,
which are described in more detail below.
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In accordance with the methods of the present invention, a therapeutically
effective dose of anti-CD20 antibody is administered weekly in combination
with a
constant IL-2 dosing regimen or in combination with a two-level IL-2 dosing
regimen. The duration of weekly administration of a therapeutically effective
dose of
anti-CD20i antibody and the duration of either of the IL-2 dosing regimens
will
depend upon the subject's overall health, history of disease progression, and
tolerance
of the particular anti-CD20/IL-2 administration protocol. Generally, the
duration of
weekly anti-CD20 antibody administration is about 4 weeks to about 8 weeks,
including 4, 5, 6, 7, or 8 weeks. The duration of IL-2 administration is a
function of
the IL-2 dosing regimen used.
In some embodiments of the invention, the subject undergoing concurrent
therapy with these two therapeutic agents is administered a constant IL-2
dosing
regimen. By "constant IL-2 dosing regimen" is intended the subject undergoing
concurrent therapy with IL-2 and anti-CD20 antibody is administered a constant
total
weekly dose of IL-2. This total weekly dose of IL-2 is partitioned into a
series of
equivalent doses that are administered according to a two- or three-times-a-
week
dosing schedule. With respect to the constant IL-2 dosing regimen, a "two-
times-a-
week," "twice weekly," or "two times per week" dosing schedule is intended to
mean
that the total weekly dose of IL-2 is partitioned into two equivalent doses
that are
administered to the subject within a 7-day period, allowing for a minimum of
72
hours between doses and a maximum of 96 hours between doses. By "three-times-a-
week," "thrice weekly," or "three times per week" dosing schedule is intended
the
constant total weekly dose of IL-2 is partitioned into three equivalent doses
that are
administered to the subject within a 7-day period, allowing for a minimum of
25
hours between doses and a maximum of 72 hours between doses. The duration of
the
constant IL-2 dosing regimen is about 4 weeks to about 10 weeks, for example,
4, 5,
6, 7, 8, 9, or 10 weeks.
Thus, in one such embodiment, concurrent therapy with these two therapeutic
agents comprises weekly administration of a therapeutically effective dose of
at least
one anti-CD20 antibody for a period of 4 weeks in combination with
administration of
a 4-week to 8-week constant IL-2 dosing regimen, wherein each recommended
total
weekly dose of IL,-2 is partitioned into three equivalent doses that are
administered to
the subject within a 7-day period according to a three-times-a-week dosing
schedule
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(i.e., allowing for a minimum of 25 hours between doses and a maximum of 72
hours
between doses). In one embodiment, the constant IL-2 dosing regimen has a
duration
of 4 weeks; in another embodiment, the constant IL-2 dosing regimen has a
duration
of 8 weeks. For example, a therapeutically effective dose of at least one anti-
CD20
antibody is administered on days 1, 8, 15, and 22 of a treatment period, and a
4-week
or 8-week constant TL-2 dosing regimen is initiated on day 3, 4, 5, 6, 7, 8,
9, or 10 of
the same treatment period. In one such embodiment, the 4-week or 8-week
constant
IL-2 dosing regimen begins on day 8 of the same treatment period, with each
recommended total weekly dose of IL-2 being partitioned into three equivalent
doses
that are administered according to a three-times-a-week dosing schedule (i.e.,
4 total
weekly doses of IL-2, which are administered as a total of 12 equivalent
doses; or 8
total weekly doses of IL-2, which are administered as a total of 24 equivalent
doses).
Thus, for example, where a 4-week constant IL-2 dosing regimen is
contemplated, therapeutically effective doses of anti-CD20 antibody are
administered
on days 1, 8, 15, and 22 of a treatment period, while the 12 equivalent doses
of IL-2
are administered on days 8, 10, 12, 15, 17, 19, 22, 24, 26, 29, 31, and 33 of
the same
treatment period. As the three equivalent doses of IL-2 that are to be
administered
each week are staggered to allow for a minimum of 25 hours between IL-2 doses
and
a maximum of 72 hours between IL-2 doses, the three equivalent doses within
any
given week can be administered on, for example, days 1, 2, and 5 of any given
week
of IL-2 administration; on days 1, 3, and 5 of any given week; on days 1, 3,
and 6 of
any given week; on days 1, 4, and 5 of any given week; on days 1, 4, and 6 of
any
given week; or on days 1, 4, and 7 of any given week; so long as the time
period
between IL-2 doses is a minimum of 25 hours and a maximum of 72 hours, and the
entire constant total weekly dose of IL-2 is administered.
In another embodiment of the invention, a similar dosing regimen is used, with
the exception of administering the weekly therapeutically effective doses of
at least
one anti-CD20 antibody for a total of 8 weeks in combination with a constant
IL-2
dosing regimen that has a duration of about 4 weeks to about 10 weeks,
including 4,
5, 6, 7, 8, 9, or 10 weeks. In this embodiment, concurrent therapy with these
two
therapeutic agents comprises administration of a therapeutically effective
dose of at
least one anti-CD20 antibody on days 1, 8, 15, 22, 29, 36, 43, and 50 of a
treatment
period, for a total of 8 therapeutically effective doses of anti-CD20
antibody, and
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initiation of the 4-week to 10-week constant IL-2 dosing regimen beginning on
day 3,
4, 5, 6, 7, 8, 9, or 10 of the same treatment period, where each recommended
total
weekly dose of IL-2 is partitioned into three equivalent doses that are
administered
according to a three-times-a-week dosing schedule as described above. In one
embodiment, the constant IL-2 dosing regimen begins on day 8 (i.e., at the
start of
week 2) of the treatment period, and continues over 8 consecutive weeks (i.e.,
the
recommended constant total weekly dose is administered for weeks 2-9) of the
same
treatment period.
In each of the foregoing embodiments, the recommended total weekly dose of
IL-2 that is to be administered over the 4-week to 10-week constant IL-2
dosing
regimen can alternately be partitioned into two equivalent doses that are
administered
according to a two-times-a-week dosing schedule. In this manner, during each
week
of the constant IL-2 dosing regimen, the two equivalent doses are administered
each
week, beginning on day 1 of the first week of IL-2 administration, with a
minimum of
72 hours between doses and a maximum of 96 hours between doses. Thus, for
example, if the 4-week to 10-week constant IL-2 dosing regimen begins on day 8
of a
treatment period (i.e., 8 days after the first therapeutically effective dose
of anti-CD20
antibody is administered), the second dose for that week can be administered
on day
11 or day 12 of the treatment period, with the next therapeutically effective
dose of
IL-2 being administered on day 15 of this same treatment period.
Thus, in one embodiment of the invention, concurrent therapy with these rivo
therapeutic agents comprises administration of a therapeutically effective
dose of at
least one anti-CD20 antibody on days 1, 8, 15, 22, 29, 36, 43, and 50 of a
treatment
period, for a total of 8 therapeutically effective doses of anti-CD20
antibody, with
initiation of the 4-week to 10-week constant IL-2 dosing regimen beginning on
day 3,
4, 5, 6, 7, 8, 9, or 10 of the same treatment period, where each of the
recommended
total weekly doses of IL-2 are partitioned into two equivalent doses that are
administered according to a two-times-a-week dosing schedule. In one such
embodiment, the constant IL-2 dosing regimen begins on day 8 (i.e., at the
start of
week 2) of the treatment period, and has a duration of 4 weeks (i.e., 4 total
weekly
doses of IL-2, which are administered as a total of 8 equivalent doses) or 8
weeks
(i.e., 8 total weekly doses of IL-2, which are administered as a total of 16
equivalent
doses). Thus, for example, where IL,-2 administration occurs over an 8-week
period
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and begins 1 week following administration of the first therapeutically
effective dose
of anti-CD20 antibody, the complete treatment period occurs over 9 weeks. In
an
alternative embodiment, this 4-week or 8-week constant IL-2 dosing regimen is
followed (i.e., recommend total weekly dose of IL-2 administered during weeks
2-5
or weeks 2-9, respectively, of a treatment period), while a therapeutically
effective
dose of the anti-CD20 antibody is administered once a week over the first 4
weeks of
the treatment period, i.e., on days 1, 8, 15, and 22, for a total of 4
therapeutically
effective doses of the antibody anti-tumor agent.
In other embodiments of the invention, concurrent therapy with these two
therapeutic agents comprises a "two-level IL-2 dosing regimen: ' By "two-level
IL-2
dosing regimen" is intended the subject undergoing concurrent therapy with IL-
2 and
anti-CD20 antibody is administered IL-2 during two time periods of IL-2
dosing,
which have a combined duration of about 4 weeks to about 16 weeks, including,
4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In one embodiment, the two-
level IL-2
dosing regimen has a combined duration of about 4 weeks to about 12 weeks; in
other
embodiments, the two-level IL-2 dosing regimen has a combined duration of
about 4
weeks to about 8 weeks, including about 4, 5, 6, 7, or 8 weeks. The total
weekly dose
of IL-2 that is to be administered during the first and second time periods of
the two-
level IL-2 dosing regimen is chosen such that a higher total weekly dose of IL-
2 is
given during the first time period and a lower total weekly dose of IL-2 is
given
during the second time period. The duration of the individual first and second
time
periods of the two-level IL-2 dosing regimen can vary, depending upon the
health of
the individual and history of disease progression. Generally, the subject is
administered higher total weekly doses of IL-2 for at least 1 week out of the
4-week to
16-week two-level IL-2 dosing regimen. In one embodiment, higher total weekly
doses of IL-2 are administered during the first half of the two-level IL-2
dosing
regimen, with lower total weekly doses being administered during the second
half of
the two-level IL-2 dosing regimen. Thus for example, where the two-level IL-2
dosing regimen has a combined duration of 8 weeks, the higher total weekly
doses of
IL-2 would be administered for the first 4 weeks of IL-2 dosing, and the lower
total
weekly doses of IL-2 would be administered for the second 4 weeks of IL-2
dosing.
Though specific dosing regimens are disclosed herein below, it is recognized
that the invention encompasses any administration protocol that provides for
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concurrent therapy with an anti-CD20 antibody and a two-level IL-2 dosing
regimen
that provides for initial exposure to higher total weekly doses of IL-2, and
subsequent
exposure to lower total weekly doses of IL-2. While not being bound by theory,
it is
believed that administering a higher dose of IL-2 during the initial stages of
IL-2
dosing provides for an initial stimulation of NIA cell activity that can be
maintained by
a lower dose during the subsequent weeks of IL-2 dosing. As IL-2 side effects
are
dose-related, the lowered dose will increase tolerability during the extended
treatment
period.
Thus, the methods of the invention contemplate treatment regimens where a
therapeutically effective dose of at least one anti-CD20 antibody is
administered once
a week for one or more weeks, for example, for 4 weeks or 8 weeks, in
combination
with a two-level IL-2 dosing regimen having a combined duration of about 4
weeks to
about 16 weeks, including 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16
weeks. Either
agent could be administered first, as explained above for this two-level IL-2
dosing
regimen. For example, in one embodiment, a therapeutically effective dose of
anti-
CD20 antibody is administered first, for example, on day 1 of a treatment
period,
followed by initiation of the two-level IL-2 dosing regimen within 10 days,
preferably
within 7 days of the first administration of the anti-CD20 antibody, for
example,
within 1, 2, 3, 4, 5, 6, or 7 days. During the two-level IL-2 dosing regimen,
a higher
total weekly dose of IL-2 is administered in the first time period of the two-
level IL-2
dosing regimen, for example, over the first 1-4 weeks of IL-2 administration,
and
lower total weekly doses of IL-2 are administered during the second time
period of
the two-level IL-2 dosing regimen (i.e., over the remaining course of the two-
level IL-
2 dosing regimen).
In one embodiment, the methods of the invention provide for weekly
administration of a therapeutically effective dose of a pharmaceutical
composition
comprising at least one anti-CD20 antibody over a period of 4 weeks in
combination
with a two-level IL-2 dosing regimen having a combined duration of 4 weeks to
8
weeks, including 4, 5, 6, 7, or 8 weeks. In this manner, a therapeutically
effective
dose of at least one anti-CD20 antibody is administered on days l, 8, 15, and
22 of a
treatment period, and the 4-week to 8-week two-level IL-2 dosing regimen is
initiated
beginning on day 3, 4, 5, 6, 7, 8, 9, or 10 of the same treatment period.
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In one such embodiment, therapeutically effective doses of the pharmaceutical
composition comprising the anti-CD20 antibody are administered weekly for 4
weeks
beginning on day 1 of a treatment period and the two-level IL-2 dosing regimen
begins on day 8 of the same treatment period and continues for 8 weeks (i.e.,
during
weeks 2-9 of the treatment period). In an alternative embodiment, this 8-week
two-
level IL-2 dosing regimen is followed (i.e., IL-2 administration occurring
during
weeks 2-9 of a treatment period), while a therapeutically effective dose of
the
pharmaceutical composition comprising the anti-CD20 antibody is administered
once
a week over the first 8 weeks of the treatment period (i.e., on day l, 8, 1 S,
22, 29, 36,
43, and 50 of the treatment period).
For human subjects undergoing concurrent therapy with weekly administration
of a therapeutically effective dose of anti-CD20 antibody in combination with
a two-
level IL-2 dosing regimen, the total weekly dose of IL-2 during the first and
second
time periods of this two-level IL-2 dosing regimen can be administered as a
single
1 ~ dose, or can be partitioned into a series of equivalent doses that are
administered
according to a two- three-, four-, five-, six-, or seven-times-a-week dosing
schedule.
Thus, for example, the higher total weekly dose during the first time period
can be
administered as a single dose, or can be partitioned into a series of
equivalent doses
that are administered according to a two- three-, four-, five-, six-, or seven-
times-a-
week dosing schedule. Similarly, the lower total weekly dose during the second
time
period can be administered as a single dose, or can be partitioned into a
series of
equivalent doses that are administered according to a two-, three-, four-,
five-, six-, or
seven-times-a-week dosing schedule. For purposes of the two-level IL-2 dosing
regimen, a "two-, three-, four-, five-, six-, or seven-times-a-week dosing
schedule" is
intended to mean that the total weekly dose is partitioned into two, three,
four, five,
six, or seven equivalent doses, respectively, which are administered to the
subject
over the course of a 7-day period, with no more than one equivalent dose being
administered per 24-hour period. The series of equivalent doses can be
administered
on sequential days, or can be administered such that one or more days occur
between
any two consecutive doses, depending upon the total number of equivalent doses
administered per week.
Thus, for example, where a series of two equivalent doses of IL-2 are
administered per week (i.e., over a 7-day period) and the first equivalent
dose of that
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week is administered on day 1, the second equivalent dose of IL-2 can be
administered on day 2, 3, 4, 5, 6, or 7 of that week. In one embodiment, the
total
weekly dose of IL-2 is partitioned into two equivalent doses that are
administered to
the subject within a 7-day period, allowing for a minimum of 72 hours between
doses
and a maximum of 96 hours between doses.
Similarly, where a series of three equivalent doses of IL-2 are administered
per week and the first equivalent dose of that week is administered on day 1,
the
second equivalent dose can be administered on day 2, 3, 4, 5, or 6 of that
week, and
the third equivalent dose can be administered on day 3, 4, 5, 6, or 7 of that
week, so
long as about 24 hours occur between administration of the second and third
equivalent doses. In one embodiment, the total weekly dose of IL-2 is
partitioned into
three equivalent doses that are administered to the subject within a 7-day
period,
allowing for a minimum of 25 hours between doses and a maximum of 72 hours
between doses.
I S Where a series of four equivalent doses of IL-2 are administered per week
and
the first equivalent dose of that week is administered on day 1, the second
equivalent
dose can be administered on day 2, 3, 4, or 5 of that week, the third
equivalent dose
can be administered on day 3, 4, 5, or 6 of that week, and the fourth
equivalent dose
can be administered on day 4, 5, 6, or 7 of that week, so long as about 24
hours occur
between administration of any two consecutive doses (i.e., between the first
and
second equivalent doses, between the second and third equivalent doses, and
between
the third and fourth equivalent doses).
Where a series of five equivalent doses are administered per week and the
first
equivalent dose of that week is administered on day l, the second equivalent
dose can
be administered on day 2, 3, or 4 of that week, the third equivalent dose can
be
administered on day 3, 4, or 5 of that week, the fourth equivalent dose can be
administered on day 4, 5, or 6 of that week, and the fifth equivalent dose can
be
administered on day 5, 6, or 7 of that week, so long as about 24 hours occur
between
administration of any two consecutive doses (i.e., between the first and
second
equivalent doses, between the second and third equivalent doses, between the
third
and fourth equivalent doses, and between the fourth and fifth equivalent
doses).
Where a series of six equivalent doses of IL-2 are administered per week and
the first equivalent dose of that week is administered on day l, the second
equivalent
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dose can be administered on day 2 or 3 of that week, the third equivalent dose
can be
administered on day 3 or 4 of that week, the fourth equivalent dose can be
administered on day 4 or S of that week, the fifth equivalent dose can be
administered
on day 5 or 6 of that week, and the sixth equivalent dose can be administered
on day 6
or 7 of that week, so long as about 24 hours occur between administration of
any two
consecutive doses (i.e., between the first and second equivalent doses,
between the
second and third equivalent doses, between the third and fourth equivalent
doses,
between the fourth and fifth equivalent doses, and between the fifth and sixth
equivalent doses).
In one embodiment, the total weekly dose of IL-2 is partitioned into seven
equivalent doses, which are administered daily over the 7-day period, with
about 24
hours occurring between each consecutive dose.
It is not necessary that the same dosing schedule be followed for the first
and
second periods of the two-level IL-2 dosing regimen. Thus, the dosing schedule
can
be adjusted to accommodate an individual's tolerance of prolonged IL-2 therapy
in
combination with anti-CD20 antibody therapy, and to reflect the individual's
responsiveness to concurrent therapy with these two therapeutic agents. The
preferred
dosing schedule during these two time periods is readily determined by the
managing
physician given the patient's medical history and the guidance provided
herein.
Thus, the present invention provides methods for treating a human subject
with non-Hodgkin's lymphoma using concurrent therapy with weekly
administration
of a therapeutically effective dose of anti-CD20 antibody in combination with
either a
constant IL-2 dosing regimen or a two-level IL-2 dosing regimen. For purposes
of the
present invention, the therapeutically effective dose of at least one anti-
CD20
antibody to be administered weekly is in the range from about 100 mg/m2 to
about
550 mg/m2, about 125 mg/m2 to about 500 mglm2, about 225 mg/m2to about 400
mg/m2, or about 375 mg/m2. The pharmaceutical composition comprising the anti-
CD20 antibody is administered, for example, intravenously, as noted herein
above.
The IL-2 is administered, for example, by IV, IM, or SC injection, in
combination
with the anti-CD20 antibody therapy so as to provide the recommended total
weekly
doses of IL-2 during the constant IL-2 dosing regimen or during the two-level
IL-2
dosing regimen as described more fully below. The following embodiments
provide
guidance as to suitable dosing regimens, though any number of different dosing
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regimens can be contemplated by one of skill in the art apprised of the
disclosure set
forth herein.
For purposes of the following discussion of total weekly doses of IL-2 to be
administered during the constant or two-level IL-2 dosing regimen, the
multimeric IL-
2 pharmaceutical composition commercially available under the tradename
Proleukin~ IL-2 (Chiron Corporation, Emeryville, California) is used as the
reference
IL-2 standard. By "reference IL-2 standard" is intended the formulation of IL-
2 that
serves as the basis for determination of the total weekly IL-2 doses to be
administered
to a human subject with lymphoma in accordance with the desired constant or
two-
level IL-2 dosing regimen in combination with at least one anti-CD20 antibody
to
achieve the desired positive effect, i.e., a positive therapeutic response
that is
improved with respect to that observed with either of these therapeutic agents
alone.
Where Proleukin~ IL-2 is to be administered according to a constant IL-2
dosing regimen, the total weekly dose is about 30.0 MIU to about 54.0 MILT,
I S depending upon the duration of the treatment period and whether the IL-2
is dosed on
a twice-a-week or thrice-a-week dosing schedule, while the therapeutically
effective
dose of anti-CD20 antibody to be administered weekly is in the range from
about 100
mg/m2 to about 550 mg/m2, about 125 mg/m2 to about 500 mg/m2, about 225 mg/m2
to about 400 mg/m2, or about 375 mg/m2. Thus, for example, in some
embodiments,
the total amount of Proleukin~ IL-2 that is to be administered per week as
part of a
constant IL-2 dosing regimen is about 30.0 MILT, 32.0 MIU, 35.0 MIU, 37.0 MIU,
40.0 MIU, 42.0 MIU, 45.0 MIU, 47.0 MILT, 50.0 MIU, 52.0 MILT, or 54.0 MIU, and
the total amount of anti-CD20 antibody is about 225, 250, 275, 300, 325, 350,
375, or
400 mg/m2/weekly dose. When the total weekly dose of Proleukin~ IL-2 is about
30.0 MIU to about 42.0 MIU, the total amount of anti-CD20 antibody is about
325,
350, 375, or 400 mg/mz/weekly dose. In one embodiment, the total weekly dose
of
Proleukin~ IL-2 is about 42.0 MIU, and the total amount of anti-CD20 antibody
is
about 375 mg/m2/weekly dose. As previously noted, the total weekly dose of IL-
2
during a constant IL-2 dosing regimen is partitioned into two or three
equivalent
doses that are administered according to a two- or three-times-a-week dosing
schedule, respectively. Thus, for example, where the total weekly dose of
Proleukin~
IL-2 is 30.0 MIU, the three equivalent doses of this reference IL-2 standard
to be
administered during each week would be 10.0 MIU, and the two equivalent doses
of
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this reference IL-2 standard to be administered during each week would be 15.0
MIU.
Similarly, where the total weekly dose of Proleukin~ IL-2 is 54.0 MIU, the
three
equivalent doses of this reference IL-2 standard to be administered during
each week
would be 18.0 MIU, and the two equivalent doses of this reference IL-2
standard to be
administered during each week would be 27.0 MIU.
Where Proleukin~ IL-2 is to be administered according to a two-level IL-2
dosing regimen, the higher total weekly dose that is administered during the
first time
period of this dosing regimen is about 30.0 MIU to about 54.0 MIU, and the
lower
total weekly dose that is administered during the second time period of this
dosing
regimen is about 18.0 MIU to about 39.0 MIU. As previously noted, the total
weekly
dose administered during the first time period of the two-level IL-2 dosing
regimen,
for example, during the first half of this dosing regimen, is always higher
than the
total weekly dose administered during the second time period of the two-level
IL-2
dosing regimen, for example, during the second half of this dosing regimen.
Thus, in some embodiments, the higher total weekly dose of Proleukin~ IL-2
that is administered during the first time period of the two-level IL-2 dosing
regimen
is about 30.0 MIU to about 54.0 MIU, including about 30.0 MIU, 32.0 MIU, 35.0
MIU, 37.0 MIU, 40.0 MIU, 42.0 MIU, 45.0 MIU, 47.0 MIU, 50.0 MIU, 52.0 MIU, or
54.0 MIU, and other such values falling within this higher dosing range; and
the lower
total weekly dose of Proleukin~ IL-2 is about 18.0 MIU to about 39.0 MIU,
including
18.0 MIU, 20.0 MIU, 23.0 MILT, 25.0 MIU, 27.0 MIU, 30.0 MIU, 32 MIU, 35.0 MIU,
37.0 MIU, and 39.0 MIU, and other such values falling within this lower dosing
range. In one embodiment, the two-level IL-2 dosing regimen has a combined
duration of 4 weeks to 8 weeks, where the higher total weekly dose of
Proleukin~ IL-
2 that is administered during the first time period of the two-level IL-2
dosing
regimen is about 30.0 MIi1 to about 42.0 MIU, such as 30.0 MIU, 32.0 M1U, 34.0
MIU, 36.0 MIU, 38.0 MIU. 40.0 MIU, and 42.0 MIU, and the lower total weekly
dose
of Proleukin~ IL-2 that is administered during the second time period of the
two-level
IL-2 dosing regimen is about 18.0 MICT to about 30.0 MIU, such as 18.0, 20.0,
22.0,
24.0, 26.0, 28.0, and 30.0 MILD. In one such embodiment, the higher total
weekly
dose of Proleukin~ IL-2 that is administered during the first time period is
42.0 MILT
and the lower total weekly dose of Proleukin~ IL-2 that is administered during
the
second time period is 30.0 MIIJ. As previously noted, the total weekly dose of
IL-2
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during the first and second time periods of a two-level IL-2 dosing regimen is
administered as a single dose, or is partitioned into a series of equivalent
doses that
are administered according to a two-, three-, four-, f ve-, six-, or seven-
times-a-week
dosing schedule. Thus, for example, where the total weekly dose of Proleukin~
IL-2
during the first period of the two-level IL-2 dosing regimen is 42.0 MIU, the
three
equivalent doses of this reference IL-2 standard to be administered during
each week
would be 14.0 MIU, and the two equivalent doses of this reference IL-2
standard to be
administered during each week would be 21.0 MIU. Similarly, where the total
weekly dose of Proleukin~ IL-2 during the second period of the two-level IL-2
dosing
regimen is is 30.0 MIU, the three equivalent doses of this reference IL-2
standard to
be administered during each week would be 10.0 MIU, and the two equivalent
doses
of this reference IL-2 standard to be administered during each week would be
15.0
MIU.
In accordance with the methods of the present invention, the subject is
administered this two-level IL-2 dosing regimen in combination with weekly
administration of a therapeutically effective dose of anti-CD20 antibody. The
therapeutically effective dose of anti-CD20 antibody to be administered weekly
is in
the range from about 100 mg/m2 to about 550 mg/m2, about 125 mg/m2 to about
500
mg/m2, about 225 mg/m2to about 400 mg/m2, or about 375 mg/m2. Thus, for
example, in some embodiments, the total amount of anti-CD20 antibody is about
225,
250, 275, 300, 325, 350, 375, or 400 mg/m2/weekly dose. In other embodiments,
the
total amount of anti-CD20 antibody is about 325, 350, 375, or 400 mg/m2/weekly
dose.
In a preferred embodiment, the therapeutically effective dose of anti-CD20
antibody is administered once a week for 4 weeks or 8 weeks beginning on day 1
of a
treatment period, and the two-level IL-2 dosing regimen is initiated on day 8
of this
treatment period and has a combined duration of 8 weeks. In this embodiment,
the
higher total weekly dose of IL-2 administered during weeks 2-5 of the
treatment
period is about 30.0 MIU to about 54.0 MILT, preferably about 30.0 MIU to
about 42.0
MIU, and the lower total weekly dose of IL-2 administered during weeks 6-9 is
about
18.0 MIU to about 39.0 MIU, preferably about 18.0 MILT to about 30.0 MILT. The
a
higher and lower total weekly doses of IL-2 are administered as a single dose,
or are
partitioned into equivalent doses that are administered according to a two-,
three-,
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four-, five-, six-, or seven-times-a-week dosing schedule. In one such
embodiment,
the higher total weekly dose of IL-2 during weeks 2-S of the treatment period
is about
30.0 MIU to about 42.0 MIU, for example, 42.0 MICT, and the lower total weekly
dose
or IL-2 is about 18.0 MIU to about 30.0 MIU, for example, 30.0 MIU. In this
embodiment, each of the higher and lower total weekly doses of IL-2 are
partitioned
into two equivalent doses that are administered according to a two-times-a-
week
dosing schedule, where the two equivalent doses are administered to the
subject
within a 7-day period, allowing for a minimum of 72 hours between doses and a
maximum of 96 hours between doses. In an alternative embodiment, each of the
higher and lower total weekly doses of IL-2 are partitioned into three
equivalent doses
that are administered according to a three-times-a-week dosing schedule, where
the
three equivalent doses are administered to the subject within a 7-day period,
allowing
for a minimum of 25 hours between doses and a maximum of 72 hours between
doses.
The foregoing therapeutically effective doses of the reference IL-2 standard
Proleukin~ IL-2 are expressed in terms of MIU, which represent total amounts
or
absolute doses that are to be administered to a human subject on a weekly
basis. The
corresponding relative total weekly dose of Proleukin~ IL-2 to be administered
to a
person to can readily be calculated. The average person is approximately 1.7
m2.
Thus, where the absolute total weekly dose of Proleukin~ IL-2 to be
administered is
about 30.0 MICT to about 54.0 MIU, the corresponding relative total weekly
dose of
Proleukin~ IL-2 is about 17.6 MIU/m2 to about 31.8 MIUlm2. Similarly, when the
absolute total weekly dose is 30.0 MIU, 32.0 MIU, 35.0 MIU, 37.0 MIU, 40.0
MIU,
42.0 MIU, 45.0 MIU, 47.0 MIU, 50.0 MIU, 52.0 MIU, or 54.0 MIU, the
corresponding relative total weekly dose is 17.6 MIU/m2, 18.8 MIU/m2, 20.6
MIU/m2, 21.8 MIU/m2, 23.5 MIU/m2, 24.7 MIU/m2, 26.5 MIU/m2, 29.4 MIU/m2,
30.6 MILT/m2, and 31.8 MIII/m2, respectively. These relative total weekly
doses of
IL-2 represent those doses that are to be administered in accordance with the
constant
IL-2 dosing regimen, and also represent the range of relative total weekly
doses of IL-
2 that are to be administered during the f rst time period of the two-level IL-
2 dosing
regimen. Those absolute total weekly doses that are to be administered during
the
second time period of the two-level IL-2 dosing regimen (i.e., within the
range of
about 18.0 MIU to about 39.0 MIU, including, for example, 18.0 MIU, 20.0 MIIJ,
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23.0 MIU, 25.0 MIU, 27.0 MIU, 30.0 MIU, 32 MIU, 35.0 MIU, 37.0 MIU, and 39.0
MIU) have corresponding relative total weekly doses of about 10.6 MIU/m2 to
about
22.9 MIU/m2, including 10.6 MIU/m2, 11.8 MIU/m2, I3.5 MIU/m2, 14.7 MIU/m2,
15.9 MIU/m2, 17.6 MIU/mz, 18.8 MIU/m2, 20.6 MIU/m2, 21.8 MILT/m2, and 22.9
MIU/m2, respectively.
MIU represents an international unit for a protein's biological activity. The
international unit for IL-2 biological activity was established in 1988 by the
World
Health Organization (WHO) International Laboratory for Biological Standards.
The
IL-2 biological reference materials provided by the National Institute for
Biological
Standards and Control (NIBSC), which belongs to WHO, has 100 international
units
per ampoule of native human, Jurkat-derived IL-2. Activity of an IL-2 product
can be
measured against this international standard in an i~ vitro potency assay by
HT-2 cell
proliferation. Thus, for example, Proleukin~ IL-2 has a biological activity of
about
MIU per mg of this IL-2 product as determined by an HT-2 cell proliferation
assay
15 (see, for example, Gearing and Thorpe (1988) J. Inamu~ological Methods
114:3-9;
Nakanishi et al. (1984) J. Exp. Med. 160(6):1605-1621). The active moiety used
in
this product is the recombinant human IL-2 mutein aldesleukin (referred to as
des-
alanyl-1, serine-125 human interleukin-2; see U.S. Patent No. 4,931,543).
Using this
information, one can calculate the recommended absolute total weekly dose of
Proleukin~ IL-2 in micrograms. Hence, where the absolute total weekly dose of
Proleukin~ IL-2 is about 30.0 MIU to about 54.0 MIU, the corresponding
absolute
total weekly dose of Proleukin~ IL-2 in micrograms is about 2000 ~,g to about
3600
pg of this product. Similarly, where the absolute total weekly dose in MIU is
about
I8.0 MIU to about 39.0 MIU, the corresponding absolute total weekly dose in
~,g is
about 1200 pg to about 2600 pg. Thus, given an absolute total weekly dose of
Proleukin~ IL-2 expressed in MILT, one of skill in the art can readily compute
the
corresponding relative total weekly dose expressed in MIU/m2, or the absolute
total
weekly dose expressed in ~g of this IL-2 product. See also Example 7 below.
For purposes of describing this invention, the doses of IL-2 have been
presented using Proleukin~ IL-2 as the reference IL-2 standard. One of skill
in the
art can readily determine what the corresponding doses would be for any IL-2
product
comprising any form of IL-2 using a conversion factor based on comparative
pharmacokinetic (PK) data and the serum concentration-time curve (AUC) for PK
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data collected during a 24-hour period for Proleukin~ IL-2. Using PIE data,
the IL-2
exposure in human subjects that were administered a single dose of the
reference IL-2
standard was determined. These subjects were selected such that they had not
previously received exogenous IL-2 therapy (i.e., these subjects were naive to
IL-2
therapy). By "exogenous IL-2 therapy" is intended any intervention whereby a
subject has been exposed to an exogenous source of IL-2, as opposed to
exposure that
occurs with the body's production of naturally occurring IL-2. Some of these
subjects
had received a single dose of 4.5 MILD of the reference IL-2 standard, while
others had
received a single dose of 7.5 or 18.0 MIU of the reference IL-2 standard. See
Example 8 herein below.
Following administration of the single dose of the reference IL-2 standard,
the
IL-2 exposure in the blood serum was monitored over the first 10 to 12 hours
post-
injection, then extrapolated to 24 hours, and the resulting area under the
serum
concentration-time curve (AUC) for data collected during that 24-hour period
was
calculated. This area under the serum concentration-time curve is referred to
herein
as the AUCo_24. Methods for measuring IL-2 exposure in this manner are well
known
in the art. See, for example, Gustavson (1998) J. Biol. Response Modifiers
1998:440-
449; Thompson et al. (1987) Cazzcer Research 47:4202-4207; Kirchner et al.
(1998)
Br. J. Clin. Pharmacol. 46:5-10; Piscitelli et al. (1996) Pharznacotherapy
16(5):754-
759; and Example 8 below. Thus, for those subjects receiving a dose of 4.5 MIU
(300
~cg) of Proleukin~ IL-2, the AUCo_Z4 value was 56 IU*hr/ml (SD = 15); for
those
subjects receiving a dose of 7.5 MIU (500 p.g) of Proleukin~ IL-2, the AUCo_Za
value
was 86 IU*hr/ml (SD = 31.5); and for those subjects receiving the 18.0 MIU
dose of
Proleukin~ IL-2, the AUCo_24 value was 375 IU*hr/ml (SD = 139). When such
AUCo_
24 data is determined for the reference IL-2 standard, Proleukin~ IL-2, the
therapeutically effective doses described herein result in an IL-2 exposure
within a
range from about 22 IU*hour/ml serum to about 653 IU*hourlml serum (see
Example
8 below).
The sum of individual AUCo_24 from individual doses will comprise the total
weekly AUCo_24 in partitioned individual doses. For example, if a dose of 18
MICT is
administered three-times-a-week, the individual AUCo_z4 is estimated at 375
ILT*hr/ml, and the total weekly AUCo_z4 will be 1125 IU*hr/ml based on linear
assumption of increased AUCn_24 with dose as shown in the Table 1 below.
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Table 1: AUCo_24 values obtained after administration of Proleukin~ IL-2.
Proleukin~ IL-2 AUCo_z4
Dose
(MIU/~,g) (IU*hr/ml)
18/I200 375
30/2000 625
42/2800 875
54/3600 1125
The same total weekly AUCo_z4 of 1125 IU*hr/ml could also be obtained by
dosing
two-times-a-week at 27 MIU or dosing five-times-a-week at about 1 I MIU.
For any other source of IL-2 (i.e., any other IL-2 formulation or any form of
IL-2, including native or biologically active variant thereof such as
muteins), a
comparable recommended dose for use in the methods of the invention can be
determined based on this AUCn_z4 data for Proleukin~ IL-2. In this manner, a
single
dose of the IL-2 source of interest is administered to a human subject, and
the level of
IL-2 in the serum following this initial IL-2 exposure is determined by
collecting PK
data and generating an AUCo_za for the IL-2 source of interest. By "initial IL-
2
exposure" is intended the subject used to measure IL-2 exposure has not
previously
undergone therapy with an exogenous source of IL-2 as noted above. This
ALTCp_24 iS
then compared to the AUCo_zø for Proleukin~ IL-2 to determine a conversion
factor
that can be used to calculate a dose of the IL-2 source that is comparable to
the
recommended dose for Proleukin~ IL-2. See, for example, the calculations for a
representative monomeric IL-2 formulation, L2-7001, that are shown in Example
8
below. Thus, for any IL-2 source used in the methods of the present invention,
the
total weekly dose of IL-2 to be administered during a constant IL-2 dosing
regimen,
or during a two-level IL-2 dosing regimen, is in an amount equivalent to the
recommended total weekly dose of the reference IL-2 standard, i.e., Proleukin~
IL-2,
as determined by the area under the serum concentration-time curve from human
PK
data.
The methods of the present invention also contemplate embodiments where a
subject undergoing concurrent therapy with weekly administration of
therapeutically
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effective doses of anti-CD20 antibody and administration of a two-level IL-2
dosing
regimen is given a "drug holiday" or a time period off from IL-2 dosing, or
from the
IL-2 dosing and the anti-CD20 antibody dosing, between the conclusion of the
frst
time period of the two-level IL-2 dosing regimen and the initiation of the
second time
period of the two-level IL-2 dosing regimen. In these embodiments, the two-
level IL-
2 dosing regimen is interrupted such that IL-2 dosing is withheld for a period
of about
1 week to about 4 weeks following conclusion of the first time period of the
two-level
IL-2 dosing regimen during which the higher total weekly dose has been
administered. During this time period off of IL-2 dosing, the subject can
continue to
receive weekly administration of a therapeutically effective dose of anti-CD20
antibody, or alternatively, the anti-CD20 antibody administration can also be
stopped.
The length of this interruption will depend upon the health of the subject,
history of
disease progression, and responsiveness of the subject to the initial IL-
2/antibody
therapy received during the first time period of the two-level IL-2 dosing
regimen.
During this drug holiday (i.e., time period off of IL-2 administration, or
time
period off of IL-2 and anti-CD20 antibody administration), natural-killer (NK)
cell
counts are monitored to determine when the two-level IL-2 dosing regimen, or
the
two-level IL-2 dosing regimen and weekly administration of anti-CD20 antibody,
are
to be resumed. In this manner, NK cell counts are measured bi-weekly or
monthly
during the two-dose IL-2 dosing regimen, and at the conclusion of the first
time
period of the two-level IL-2 dosing regimen before the drug holiday is
initiated.
Where NK cell count exceeds an acceptable threshold level, the two-level IL-2
dosing
regimen can be interrupted. By "acceptable threshold level" is intended the
subject
undergoing treatment has an NIA cell count that is about 150 cells/~l or
greater,
preferably 200 cells/~l or greater. Following discontinuance of the IL-2
dosing,
which may or may not include discontinuance of anti-CD20 antibody
administration,
NIA cell counts are then measured once per week or twice per week thereafter,
preferably once per week. An NK cell count falling below the acceptable
threshold
level of about 150 cells/~1, for example, an NIA cell count of less than 150
cells/~,1, is
indicative of the necessity to resume the two-level IL-2 dosing regimen, or
the two-
level IL-2 dosing regimen and the anti-CD20 antibody dosing regimen where the
drug
holiday also includes time off of anti-CD20 antibody administration.
Preferably the
two-level IL-2 dosing regimen is resumed when NK cell count falls below a
threshold
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WO 03/049694 PCT/US02/39253
level of about 200 cells/~l, i.e., an NIA cell count of less than 200
cells/~1. At this
time, the subject is administered the second time period of the two-level IL-2
dosing
regimen, where lower total weekly doses of IL-2 are administered in
combination
with the weekly administration of therapeutically effective doses of the anti-
CD20
antibody.
Where a subject undergoing therapy in accordance with the previously
mentioned dosing regimens exhibits a partial response, or a relapse following
a
prolonged period of remission, subsequent courses of concurrent therapy may be
needed to achieve complete remission of the disease. Thus, subsequent to a
period of
time off from a first treatment period, which may have comprised a constant IL-
2
dosing regimen or a two-level IL-2 dosing regimen, a subject may receive one
or
more additional treatment periods comprising either constant or two-level IL
dosing
regimens in combination with anti-CD20 antibody administration. Such a period
of
time off between treatment periods is referred to herein as a time period of
I S discontinuance. It is recognized that the length of the time period of
discontinuance is
dependent upon the degree of tumor response (i.e., complete versus partial)
achieved
with any prior treatment periods of concurrent therapy with these two
therapeutic
agents.
Thus, for example, where a subject is undergoing concurrent therapy with
weekly doses of anti-CD20 antibody and a two-level IL-2 dosing regimen, which
may
or may not include a drug holiday between the first and second time periods of
the
two-level IL-2 dosing regimen, their treatment regimen may include multiple
treatment sessions, each of which comprises concurrent therapy with weekly
doses of
anti-CD20 antibody and a two-level IL-2 dosing regimen. These multiple
treatment
sessions are referred to herein as maintenance cycles, where each maintenance
cycle
comprises anti-CD20 antibody administration in combination with a completed
two-
level IL-2 dosing regimen. By "completed two-level IL-2 dosing regimen" is
intended the subject has been administered both the first period of higher
total weekly
dosing and the second period of lower total weekly dosing. The necessity for
multiple
maintenance cycles can be assessed by monitoring NK cell count in a manner
similar
to that used to determine when a drug holiday is warranted, and when such a
drug
holiday must be concluded. Thus, upon completion of the two-level IL-2 dosing
regimen in any given maintenance cycle, the treating physician obtains a
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measurement of NK cell count. This indicator is then measured at monthly
intervals
(i.e., once a month) following completion of any given two-level IL-2 dosing
regimen. As with drug holidays, an NK cell count falling below an acceptable
threshold level (i.e., below about 150 cells/~1, preferably below about 200
cells/~,1) is
indicative of the need for administering another maintenance cycle to the
subject. The
duration between maintenance cycles can be about 1 month to about 6 months,
including 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 3.5 months, 4
months, 4.5 months, 5 months, 5.5 months, 6 months, or other such time periods
falling within the range of about 1 month to about 6 months.
Thus, the administration methods of the present invention provide an
improved means for managing non-Hodgkin's B-cell lymphomas in a human patient.
Constant IL-2 dosing according to a twice-weekly or thrice-weekly dosing
schedule
provides an intermittent dosing schedule that allows for less frequent
administration
of the IL-2 during anti-CD20 antibody therapy, and better tolerability of long-
term IL-
2 therapy. The two-level IL-2 dosing regimen offers the opportunity to provide
a
patient with higher total weekly doses of IL-2, which provide for expansion of
NK
cell numbers that can be maintained by a lower dose during the subsequent
weeks of
IL-2 dosing. As IL-2 side effects are dose-related, the lowered dose will
increase
tolerability during the extended treatment period. This administration
protocol has the
additional attraction of providing drug holidays between the higher and lower
total
weekly dosing schedules, again contributing to increased tolerability of
concurrent
therapy with anti-CD20 antibody and IL-2.
The term "IL-2" as used herein refers to a lymphokine that is produced by
normal peripheral blood lymphocytes and is present in the body at low
concentrations.
IL-2 was first described by Morgan et al. (1976) Science 193:1007-1008 and
originally called T cell growth factor because of its ability to induce
proliferation of
stimulated T lymphocytes. It is a protein with a reported molecular weight in
the
range of 13,000 to 17,000 (Gillis and Watson (1980) J. Exp. Med. 159:1709) and
has
an isoelectric point in the range of 6-8.5. For purposes of the present
invention, the
term "IL-2" is intended to encompass any source of IL-2, including mammalian
sources such as, e.g., mouse, rat, rabbit, primate, pig, and human, and may be
native
or obtained by recombinant techniques. The IL-2 may be the native polypeptide
sequence, or can be a variant of the native IL-2 polypeptide as described
herein
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below, so long as the variant IL-2 polypeptide retains the IL-2 biological
activity of
interest as defined herein. Preferably the IL-2 polypeptide or variant thereof
is
derived from a human source, and includes human IL-2 that is recombinantly
produced, such as recombinant human IL-2 polypeptides produced by microbial
hosts,
and variants thereof that retain the IL-2 biological activity of interest. Any
pharmaceutical composition comprising IL-2 as a therapeutically active
component
can be used to practice the present invention.
The IL-2 molecule useful in the methods of the invention may be a
biologically active variant of native IL-2. Such variant IL-2 polypeptides
should retain
the desired biological activity of the native polypeptide such that the
pharmaceutical
composition comprising the variant polypeptide has the same therapeutic effect
as the
pharmaceutical composition comprising the native polypeptide when administered
to
a subject. That is, the variant polypeptide will serve as a therapeutically
active
component in the pharmaceutical composition in a manner similar to that
observed for
the native polypeptide. Methods are available in the art for determining
whether a
variant polypeptide retains the desired biological activity, and hence serves
as a
therapeutically active component in the pharmaceutical composition. Biological
activity can be measured using assays specifically designed for measuring
activity of
the native polypeptide or protein, including assays described in the present
invention.
Additionally, antibodies raised against a biologically active native
polypeptide can be
tested for their ability to bind to the variant polypeptide, where effective
binding is
indicative of a polypeptide having a conformation similar to that of the
native
polypeptide.
For purposes of the present invention, the IL-2 biological activity of
interest is
the ability of IL-2 to activate and/or expand natural killer (NIA) cells to
mediate
lymphokine activated killer (LAIC) activity and antibody-dependent cellular
cytotoxicity (ADCC). Thus, an IL-2 variant (for example, a mutein of human IL-
2)
for use in the methods of the present invention will activate and/or expand
natural
killer (NIA) cells to mediate lymphokine activated killer (LAK) activity and
antibody-
dependent cellular cytotoxicity (ADCC). NK cells mediate spontaneous or
natural
cytotoxicity against certain cell targets referred to as "NK-cell sensitive"
targets, such
as the human erythroleukemia I~562 cell line. Following activation by IL-2, NK
cells
acquire LAK activity. Such LAIC activity can be assayed by the ability of IL-2
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activated NK cells to kill a broad variety of tumor cells and other "NK-
insensitive"
targets, such as the Daudi B-cell lymphoma line, that are normally resistant
to lysis by
resting (nonactivated) NK cells. Similarly, ADCC activity can be assayed by
the
ability of IL-2 activated NK cells to lyse "NIA-insensitive" target cells,
such as Daudi
B-cell lymphoma line, or other target cells not readily lysed by resting NK
cells in the
presence of optimal concentrations of relevant tumor cell specific antibodies.
Methods for generating and measuring cytotoxic activity of NK/LAI~ cells and
ADCC are known in the art. See for example, Current Protocols in Immunology:
ImnZUnologic Studies irz Humans, Supplement 17, Unit 7.7, 7.18, and 7.27 (John
Wiley & Sons, Inc., 1996). For purposes of the present invention, NIA cells
activated
by an IL-2 variant for use in the methods of the present invention demonstrate
a
specific lysing activity of NIA-insensitive cells in the presence (ADCC
activity) or
absence (LAIC activity) of antibody, more particularly NIA-insensitive Daudi
cells in
the presence of B-cell specific antibodies including,rituximab, that is at
least about
IS 20% greater, or at least about 25%, or 30%, or 35%, or 40% greater than
baseline
lysing activity of resting NIA cells (i..e., nonactivated) as measured using
effector to
target ratios between 12.5 to 50:1 in a standard 4-hr SICr-release
cytotoxicity assay
(see Current Protocols ire Immunology: Immuhologic Studies ih Hunaaus, Unit
7.7,
Supplement 17, Section 17.18.1 (John Wiley & Sons, Inc., 1996). In some
embodiments, the specific lysing activity of these IL-2 variant-activated NIA
cells is at
least about 45% greater, at least about 50% greater, at least about 55%
greater, or at
least about 60% greater than baseline lysing activity of resting NIA cells
when
measured as noted above.
Suitable biologically active variants of native or naturally occurring IL-2
can
be fragments, analogues, and derivatives of that polypeptide. By "fragment" is
intended a polypeptide consisting of only a part of the intact polypeptide
sequence
and structure, and can be a C-terminal deletion or N-terminal deletion of the
native
polypeptide. By "analogue" is intended an analogue of either the native
polypeptide
or of a fragment of the native polypeptide, where the analogue comprises a
native
polypeptide sequence and structure having one or more amino acid
substitutions,
insertions, or deletions. "Muteins", such as those described herein, and
peptides
having one or more peptoids (peptide mimics) are also encompassed by the term
analogue (see International Publication No. WO 91/04282). By "derivative" is
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intended any suitable modification ofthe native polypeptide of interest, of a
fragment
of the native polypeptide, or of their respective analogues, such as
glycosylation,
phosphorylation, polymer conjugation (such as with polyethylene glycol), or
other
addition of foreign moieties, so long as the desired biological activity of
the native
polypeptide is retained. Methods for making polypeptide fragments, analogues,
and
derivatives are generally available in the art.
For example, amino acid sequence variants of the polypeptide can be prepared
by mutations in the cloned DNA sequence encoding the native polypeptide of
interest.
Methods for mutagenesis and nucleotide sequence alterations are well known in
the
art. See, for example, Walker and Gaastra, eds. (1983) Techniques in Molecular
Biology (MacMillan Publishing Company, New York); I~unkel (1985) Proc. Natl.
Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods E~zyfnol. 154:367-382;
Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring
Harbor, New York); U.S. Patent No. 4,873,192; and the references cited
therein.
Guidance as to appropriate amino acid substitutions that do not affect
biological
activity of the polypeptide of interest may be found in the model of Dayhoff
et al.
(1978) in Atlas of Protein Sequence aid Structure (Natl. Biomed. Res. Found.,
Washington, D.C.). Conservative substitutions, such as exchanging one amino
acid
with another having similar properties, may be preferred. Examples of
conservative
substitutions include, but are not limited to, Gly~Ala, Val~Ile~Leu, Aspe~Glu,
Lys~Arg, Asn~Gln, and Phe~Trp~Tyr.
In constructing variants of the IL-2 polypeptide of interest, modifications
are
made such that variants continue to possess the desired activity. Obviously,
any
mutations made in the DNA encoding the variant polypeptide must not place the
sequence out of reading frame and preferably will not create complementary
regions
that could produce secondary mRNA structure. See EP Patent Application
Publication
No. 75,444.
Biologically active variants of IL-2 will generally have at least 70%,
preferably at least 80%, more preferably about 90% to 95% or more, and most
preferably about 98% or more amino acid sequence identity to the amino acid
sequence of the reference polypeptide molecule, which serves as the basis for
comparison. Thus, where the IL-2 reference molecule is human IL-2, a
biologically
active variant thereof will have at least 70%, preferably at least 80%, more
preferably
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WO 03/049694 PCT/US02/39253
about 90% to 95% or more, and most preferably about 98% or more sequence
identity
to the amino acid sequence for human IL-2. A biologically active variant of a
native
polypeptide of interest may differ from the native polypeptide by as few as 1-
15
amino acids, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or
even 1
amino acid residue. By "sequence identity" is intended the same amino acid
residues
are found within the variant polypeptide and the polypeptide molecule that
serves as a
reference when a specified, contiguous segment of the amino acid sequence of
the
variants is aligned and compared to the amino acid sequence of the reference
molecule. The percentage sequence identity between two amino acid sequences is
calculated by determining the number of positions at which the identical amino
acid
residue occurs in both sequences to yield the number of matched positions,
dividing
the number of matched positions by the total number of positions in the
segment
undergoing comparison to the reference molecule, and multiplying the result by
100
to yield the percentage of sequence identity.
For purposes of optimal alignment of the two sequences, the contiguous
segment of the amino acid sequence of the variants may have additional amino
acid
residues or deleted amino acid residues with respect to the amino acid
sequence of the
reference molecule. The contiguous segment used for comparison to the
reference
amino acid sequence will comprise at least twenty (20) contiguous amino acid
residues, and may be 30, 40, 50, 100, or more residues. Corrections for
increased
sequence identity associated with inclusion of gaps in the variants' amino
acid
sequence can be made by assigning gap penalties. Methods of sequence alignment
are
well known in the art for both amino acid sequences and fox the nucleotide
sequences
encoding amino acid sequences.
Thus, the determination of percent identity between any two sequences can be
accomplished using a mathematical algorithm. One preferred, non-limiting
example
of a mathematical algorithm utilized for the comparison of sequences is the
algorithm
of Myers and Miller (1988) CABIOS 4:11-17. Such an algorithm is utilized in
the
ALIGN program (version 2.0), which is part of the GCG sequence alignment
software
package. A PAM120 weight residue table, a gap length penalty of 12, and a gap
penalty of 4 can be used with the ALIGN program when comparing amino acid
sequences. Another preferred, nonlimiting example of a mathematical algorithm
for
use in comparing two sequences is the algorithm of Marlin and Altschul (1990)
Proc.
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Natl. Acad. Sci. USA 87:2264, modified as in Marlin and Altschul (1993) Proc.
Natl.
Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST
and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403. BLAST
nucleotide searches can be performed with the NBLAST program, score =100,
wordlength =12, to obtain nucleotide sequences homologous to a nucleotide
sequence
encoding the polypeptide of interest. BLAST protein searches can be performed
with
the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences
homologous to the polypeptide of interest. To obtain gapped alignments for
comparison purposes, Gapped BLAST can be utilized as described in Altschul et
al.
(1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to
perform
an iterated search that detects distant relationships between molecules. See
Altschul
et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast
programs, the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Also see the ALIGN
program (Dayhoff (1978) in Atlas ofProtei~ Sequehce avcd Structure S:Suppl. 3
(National Biomedical Research Foundation, Washington, D.C.) and programs in
the
Wisconsin Sequence Analysis Package, Version 8 (available from Genetics
Computer
Group, Madison, Wisconsin), for example, the GAP program, where default
parameters of the programs are utilized.
When considering percentage of amino acid sequence identity, some amino
acid residue positions may differ as a result of conservative amino acid
substitutions,
which do not affect properties of protein function. In these instances,
percent
sequence identity may be adjusted upwards to account for the similarity in
conservatively substituted amino acids. Such adjustments are well known in the
art.
See, for example, Myers and Miller (1988) ComputerApplic. Biol. Sci. 4:11-17.
The precise chemical structure of a polypeptide having IL-2 activity depends
on a number of factors. As ionizable amino and carboxyl groups are present in
the
molecule, a particular polypeptide may be obtained as an acidic or basic salt,
or in
neutral form. All such preparations that retain their biological activity when
placed in
suitable environmental conditions are included in the definition of
polypeptides
having IL-2 activity as used herein. Further, the primary amino acid sequence
of the
polypeptide may be augmented by derivatization using sugar moieties
(glycosylation)
or by other supplementary molecules such as lipids, phosphate, acetyl groups
and the
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like. It may also be augmented by conjugation with saccharides. Certain
aspects of
such augmentation are accomplished through post-translational processing
systems of
the producing host; other such modifications may be introduced in vitro. In
any event,
such modifications are included in the definition of an IL-2 polypeptide used
herein
so long as the IL-2 activity of the polypeptide is not destroyed. It is
expected that such
modifications may quantitatively or qualitatively affect the activity, either
by
enhancing or diminishing the activity of the polypeptide, in the various
assays.
Further, individual amino acid residues in the chain may be modified by
oxidation,
reduction, or other derivatization, and the polypeptide may be cleaved to
obtain
fragments that retain activity. Such alterations that do not destroy activity
do not
remove the polypeptide sequence from the definition of IL-2 polypeptides of
interest
as used herein.
The art provides substantial guidance regarding the preparation and use of
polypeptide variants. In preparing the IL-2 variants, one of skill in the art
can readily
I S determine which modifications to the native protein nucleotide or amino
acid
sequence will result in a variant that is suitable for use as a
therapeutically active
component of a pharmaceutical composition used in the methods of the present
invention.
The IL-2 for use in the methods of the present invention may be from any
source, but preferably is recombinant IL-2. By "recombinant IL-2" is intended
interleukin-2 that has comparable biological activity to native-sequence IL-2
and that
has been prepared by recombinant DNA techniques as described, for example, by
Taniguchi et al. (1983) Nature 302:305-310 and Devos (1983) Nucleic Acids
Research 1 I :4307-4323 or mutationally altered IL-2 as described by Wang et
al.
(1984) Science 224:1431-1433. In general, the gene coding for IL-2 is cloned
and
then expressed in transformed organisms, preferably a microorganism, for
example E.
coli, as described herein. The host organism expresses the foreign gene to
produce IL-
2 under expression conditions. Synthetic recombinant IL-2 can also be made in
eukaryotes, such as yeast or human cells. Processes for growing, harvesting,
disrupting, or extracting the IL-2 from cells are known in the art as
evidenced by, for
example, U.S. Patent Nos. 4,604,377; 4,738,927; 4,656,132; 4,569,790;
4,748,234;
4,530,787; 4,572,798; 4,748,234; and 4,931,543.
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For examples of variant IL-2 proteins, see European Patent (EP) Publication
No. EP 136,489 (which discloses one or more of the following alterations in
the
amino acid sequence of naturally occurring IL-2: Asn26 to G1n26; Trpl21 to
Phe121;
Cys58 to Ser58 or A1a58, Cys105 to Ser105 or A1a105; Cys125 to Ser125 or
A1a125;
deletion of all residues following Arg 120; and the Met-1 forms thereof); and
the
recombinant IL-2 muteins described in European Patent Application No.
83306221.9,
filed October 13, 1983 (published May 30, 1984 under Publication No. EP
109,748),
which is the equivalent to Belgian Patent No. 893,016, and commonly owned U.S.
Patent No. 4,518,584 (which disclose recombinant human IL-2 mutein wherein the
cysteine at position 125, numbered in accordance with native human IL-2, is
deleted
or replaced by a neutral amino acid; alanyl-ser125-IL-2; and des-alanayl-
ser125-IL-
2). See also U.S. Patent No. 4,752,585 (which discloses the following variant
IL-2
proteins: a1a104 ser125 IL-2, a1a104 IL-2, a1a104 a1a125 IL-2, va1104 ser125
IL-2,
va1104 IL-2, va1104 a1a125 IL-2, des-alal a1a104 ser125 IL-2, des-alal a1a104
IL-2,
des-alal a1a104 a1a125 IL-2, des-alal va1104 ser125 IL-2, des-alal va1104 IL-
2, des-
alal va1104 a1a125 IL-2, des-alal des-pro2 a1a104 ser125 IL-2, des-alal des-
pro2
a1a104 IL-2, des-alai des-pro2 a1a104 a1a125 IL-2, des-alal des-pro2 va1104
ser125
IL-2, des-alal des-pro2 va1104 IL-2, des-alal des-pro2 va1104 a1a125 IL-2, des-
alal
des-pro2 des-thr3 a1a104 ser125 IL-2, des-alai des-pro2 des-thr3 a1a104 IL-2,
des-
alal des-pro2 des-thr3 a1a104 a1a125 IL-2, des-alal des-pro2 des-thr3 va1104
ser125
IL-2, des-alal des-pro2 des-thr3 va1104 IL-2, des-alal des-pro2 des-thr3
va1104
a1a125 IL-2, des-alal des-pro2 des-thr3 des-ser4 a1a104 ser125 IL-2, des-alal
des-
pro2 des-thr3 des-ser4 a1a104 IL-2, des-alal des-pro2 des-thr3 des-ser4 a1a104
a1a125
IL-2, des-alal des-pro2 des-thr3 des-ser4 va1104 ser125 IL-2, des-alal des-
pro2 des-
thr3 des-ser4 va1104 IL-2, des-alal des-pro2 des-thr3 des-ser4 va1104 a1a125
IL-2,
des-alal des-pro2 des-thr3 des-ser4 des-ser5 a1a104 ser125 IL-2, des-alal des-
pro2
des-thr3 des-ser4 des-ser5 a1a104 IL-2, des-alal des-pro2 des-thr3 des-ser4
des-ser5
a1a104 a1a125 IL-2, des-alal des-pro2 des-thr3 des-ser4 des-ser5 va1104 ser125
IL-2,
des-alal des-pro2 des-thr3 des-ser4 des-ser5 val 104 IL-2, des-alal des-pro2
des-thr3
des-ser4 des-ser5 va1104 a1a125 IL-2, des-alai des-pro2 des-thr3 des-ser4 des-
ser5
des-ser6 a1a104 a1a125 IL-2, des-alal des-pro2 des-thr3 des-ser4 des-ser5 des-
ser6
a1a104 IL-2, des-alal des-pro2 des-thr3 des-ser4 des-ser5 des-ser6 a1a104
ser125 IL-
2, des-alal des-pro2 des-thr3 des-ser4 des-ser5 des-ser6 va1104 ser125 IL-2,
des-alai
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WO 03/049694 PCT/US02/39253
des-pro2 des-thr3 des-ser4 des-ser5 des-ser6 va1104 IL-2, and des-alal des-
pro2 des
thr3 des-ser4 des-ser5 des-ser6 va1104 a1a125 IL-2 ) and U.S. Patent No.
4,931,543
(which discloses the IL-2 mutein des-alanyl-1, serine-125 human IL-2 used in
the
examples herein, as well as the other IL-2 muteins).
Also see European Patent Publication No. EP 200,280 (published December
10, 1986), which discloses recombinant IL-2 muteins wherein the methionine at
position 104 has been replaced by a conservative amino acid. Examples include
the
following muteins: ser4 des-ser5 a1a104 IL-2; des-alai des-pro2 des-thr3 des-
ser4 des-
ser5 a1a104 a1a125 IL-2; des-alal des-pro2 des-thr3 des-ser4 des-ser5 g1u104
ser125
IL-2; des-alai des-pro2 des-thr3 des-ser4 des-ser5 g1u104 IL-2; des-alal des-
pro2 des-
thr3 des-ser4 des-ser5 g1u104 a1a125 IL-2; des-alai des-pro2 des-thr3 des-ser4
des-
ser5 des-ser6 a1a104 a1a125 IL-2; des-alal des-pro2 des-thr3 des-ser4 des-ser5
des-
ser6 a1a104 IL-2; des-alai des-pro2 des-thr3 des-ser4 des-ser5 des-ser6 a1a104
ser125
IL-2; des-alai des-pro2 des-thr3 des-ser4 des-ser5 des-ser6 g1u104 ser125 IL-
2; des-
alai des-pro2 des-thr3 des-ser4 des-ser5 des-ser6 g1u104 IL-2; and des-alal
des-pro2
des-thr3 des-ser4 des-ser5 des-ser6 g1u104 a1a125 IL-2. See also European
Patent
Publication No. EP 118,617 and U.S. Patent No. 5,700,913, which disclose
unglycosylated human IL-2 variants bearing alanine instead of native IL-2's
methionine as the N-terminal amino acid; an unglycosylated human IL-2 with the
initial rnethionine deleted such that proline is the N-terminal amino acid;
and an
unglycosylated human IL-2 with an alanine inserted between the N-terminal
methionine and proline amino acids.
Other IL-2 muteins include the those disclosed in WO 99/60128 (substitutions
of the aspartate at position 20 with histidine or isoleucine, the asparagine
at position
88 with arginine, glycine, or isoleucine, or the glutamine at position126 with
leucine
or gulatamic acid), which reportedly have selective activity for high affinity
IL-2
receptors expressed by cells expressing T cell receptors in preference to NK
cells and
reduced IL-2 toxicity; the muteins disclosed in U.S Patent No. 5,229,109
(substitutions of arginine at position 38 with alanine, or substitutions of
phenylalanine
at position 42 with lysine), which exhibit reduced binding to the high
affinity IL-2
receptor when compared to native IL-2 while maintaining the ability to
stimulate
LAIC cells; the muteins disclosed in International Publication No. WO 00/58456
(altering or deleting a naturally occurring (x)D(y) sequence in native IL-2
where D is
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WO 03/049694 PCT/US02/39253
aspartic acid, (x) is leucine, isoleucine, glycine, or valine, and (y) is
valine, leucine or
serine), which are claimed to reduce vascular leak syndrome; the IL-2 pl-30
peptide
disclosed in International Publication No. WO 00/04048 (corresponding to the
first 30
amino acids of IL-2, which contains the entire a-helix A of IL-2 and interacts
with the
b chain of the IL-2 receptor), which reportedly stimulates NK cells and
induction of
LAK cells; and a mutant form ofthe IL-2 pl-30 peptide also disclosed in WO
00/04048 (substitution of aspartic acid at position 20 with lysine), which
reportedly is
unable to induce vascular bleeds but remains capable of generating LAK cells.
Additionally, IL-2 can be modified with polyethylene glycol to provide
enhanced
solubility and an altered pharmokinetic profile (see U.S. Patent No.
4,766,106).
The term IL-2 as used herein is also intended to include IL-2 fusions or
conjugates comprising IL-2 fused to a second protein or covalently conjugated
to
polyproline or a water-soluble polymer to reduce dosing frequencies or to
improve IL-
2 tolerability. For example, the IL-2 (or a variant thereof as defined herein)
can be
fused to human albumin or an albumin fragment using methods known in the art
(see
WO 01/79258). Alternatively, the IL-2 can be covalently conjugated to
polyproline
or polyethylene glycol homopolymers and polyoxyethylated polyols, wherein the
homopolymer is unsubstituted or substituted at one end with an alkyl group and
the
poplyol is unsubstituted, using methods known in the art (see, for example,
U.S.
Patent Nos. 4,766,106, 5,206,344, and 4,894,226).
Any pharmaceutical composition comprising IL-2 as the therapeutically active
component can be used in the methods of the invention. Such pharmaceutical
compositions are known in the art and include, but are not limited to, those
disclosed
in U.S. Patent Nos. 4,745,180; 4,766,106; 4,816,440; 4,894,226; 4,931,544; and
5,078,997. Thus liquid, lyophilized, or spray-dried compositions comprising IL-
2 that
are known in the art may be prepared as an aqueous or nonaqueous solution or
suspension for subsequent administration to a subject in accordance with the
methods
of the invention. Each of these compositions will comprise IL-2 as a
therapeutically or
prophylactically active component. By "therapeutically or prophylactically
active
component" is intended the IL-2 is specifically incorporated into the
composition to
bring about a desired therapeutic or prophylactic response with regard to
treatment,
prevention, or diagnosis of a disease or condition within a subject when the
pharmaceutical composition is administered to that subject. Preferably the
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pharmaceutical compositions comprise appropriate stabilizing agents, bulking
agents,
or both to minimize problems associated with loss of protein stability and
biological
activity during preparation and storage.
In preferred embodiments of the invention, the IL-2 containing pharmaceutical
compositions useful in the methods of the invention are compositions
comprising
stabilized monomeric IL-2, compositions comprising multimeric IL-2, and
compositions comprising stabilized lyophilized or spray-dried IL-2.
Pharmaceutical compositions comprising stabilized monomeric IL-2 are
disclosed in International Publication No. WO 01/24814, entitled "Stabilized
Liquid
Polypeptide-Containing Pharmaceutical Compositions." By "monomeric" IL-2 is
intended the protein molecules are present substantially in their monomer
form, not in
an aggregated form, in the pharmaceutical compositions described herein. Hence
covalent or hydrophobic oligomers or aggregates of IL-2 are not present.
Briefly, the
IL-2 in these liquid compositions is formulated with an amount of an amino
acid base
sufficient to decrease aggregate formation of IL-2 during storage. The amino
acid
base is an amino acid or a combination of amino acids, where any given amino
acid is
present either in its free base form or in its salt form. Preferred amino
acids are
selected from the group consisting of arginine, lysine, aspartic acid, and
glutamic
acid. These compositions further comprise a buffering agent to maintain pH of
the
liquid compositions within an acceptable range for stability of IL-2, where
the
buffering agent is an acid substantially free of its salt form, an acid in its
salt form, or
a mixture of an acid and its salt form. Preferably the acid is selected from
the group
consisting of succinic acid, citric acid, phosphoric acid, and glutamic acid.
Such
compositions are referred to herein as stabilized monomeric IL-2
pharmaceutical
compositions.
The amino acid base in these compositions serves to stabilize the IL-2 against
aggregate formation during storage of the liquid pharmaceutical composition,
while
use of an acid substantially free of its salt form, an acid in its salt form,
or a mixture
of an acid and its salt form as the buffering agent results in a liquid
composition
having an osmolarity that is nearly isotonic. The liquid pharmaceutical
composition
may additionally incorporate other stabilizing agents, more particularly
methionine, a
nonionic surfactant such as polysorbate 80, and EDTA, to further increase
stability of
the polypeptide. Such liquid pharmaceutical compositions are said to be
stabilized, as
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WO 03/049694 PCT/US02/39253
addition of amino acid base in combination with an acid substantially free of
its salt
form, an acid in its salt form, or a mixture of an acid and its salt form,
results in the
compositions having increased storage stability relative to liquid
pharmaceutical
compositions formulated in the absence of the combination of these two
components.
These liquid pharmaceutical compositions comprising stabilized monomeric
IL-2 may either be used in an aqueous liquid form, or stored for later use in
a frozen
state, or in a dried form for later reconstitution into a liquid form or other
form
suitable for administration to a subject in accordance with the methods of
present
invention. By "dried form" is intended the liquid pharmaceutical composition
or
formulation is dried either by freeze drying (i.e., lyophilization; see, for
example,
Williams and Polli (1984) J. Parenteral Sci. Technol. 38:48-59), spray drying
(see
Masters (1991) in Spray-Drying Handbook (5th ed; Longman Scientific and
Technical, Essez, IJ.I~.), pp. 491-676; Broadhead et al. (1992) Drug Devel.
Ind.
Plaar»a. 18:1169-1206; and Mumenthaler et al. (1994) Phar»z. Res. 11:12-20),
or air
drying (Carpenter and Crowe (1988) Cryobiology 25:459-470; and Roser (1991)
Biophartn. 4:47-53).
Other examples of IL-2 formulations that comprise IL-2 in its nonaggregated
monomeric state include those described in Whittington and Faulds (1993) Drugs
46(3):446-514. These formulations include the recombinant IL-2 product in
which
the recombinant IL-2 mutein Teceleukin (unglycosylated human IL-2 with a
methionine residue added at the amino-terminal) is formulated with 0.25% human
serum albumin in a lyophilized powder that is reconstituted in isotonic
saline, and the
recombinant IL-2 mutein Bioleukin (human IL-2 with a methionine residue added
at
the amino-terminal, and a substitution of the cysteine residue at position 125
of the
human IL-2 sequence with alanine) formulated such that 0.1 to 1.0 mg/ml IL-2
mutein
is combined with acid, wherein the formulation has a pH of 3.0 to 4.0,
advantageously
no buffer, and a conductivity of less than 1000 mmhos/cm (advantageously less
than
500 mmhos/cm). See EP 373,679; Xhang et al. (1996) Pharrnaceut. Res. 13(4):643-
644; and Prestrelski et al. (1995) Pharmaceut. Res. 12(9):1250-1258.
Examples of pharmaceutical compositions comprising multimeric II,-2 are
disclosed in commonly owned U.S. Patent No. 4,604,377. By "multimeric" is
intended the protein molecules are present in the pharmaceutical composition
in a
microaggregated form having an average molecular association of 10-50
molecules.
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These multimers are present as loosely bound, physically associated IL-2
molecules.
A lyophilized form of these compositions is available commercially under the
tradename Proleukin~ IL-2 (Chiron Corporation). The lyophilized formulations
disclosed in this reference comprise selectively oxidized, microbially
produced
recombinant IL-2 in which the recombinant IL-2 is admixed with a water soluble
carrier such as mannitol that provides bulk, and a sufficient amount of sodium
dodecyl sulfate to ensure the solubility of the recombinant IL-2 in water.
These
compositions are suitable for reconstitution in aqueous injections for
parenteral
administration and are stable and well tolerated in human patients. When
reconstituted, the IL-2 retains its multimeric state. Such lyophilized or
liquid
compositions comprising multimeric IL-2 are encompassed by the methods of the
present invention. Such compositions are referred to herein as multimeric IL-2
pharmaceutical compositions.
The methods of the present invention may also use stabilized lyophilized or
spray-dried pharmaceutical compositions comprising IL-2, which may be
reconstituted into a liquid or other suitable form for administration in
accordance with
methods of the invention. Such pharmaceutical compositions are disclosed in
International Publication No. WO O 1 /24814, entitled "Methods for Pulmonary
Delivery ofl~terleukin-2." These compositions may further comprise at least
one
bulking agent, at least one agent in an amount sufficient to stabilize the
protein during
the drying process, or both. By "stabilized" is intended the IL-2 protein or
variants
thereof retains its monomeric or multimeric form as well as its other key
properties of
quality, purity, and potency following lyophilization or spray-drying to
obtain the
solid or dry powder form of the composition. In these compositions, preferred
carrier
materials for use as a bulking agent include glycine, mannitol, alanine,
valine, or any
combination thereof, most preferably glycine. The bulking agent is present in
the
formulation in the range of 0% to about 10% (w/v), depending upon the agent
used.
Preferred carrier materials for use as a stabilizing agent include any sugar
or sugar
alcohol or any amino acid. Preferred sugars include sucrose, trehalose,
raffinose,
stachyose, sorbitol, glucose, lactose, dextrose or any combination thereof,
preferably
sucrose. When the stabilizing agent is a sugar, it is present in the range of
about 0%
to about 9.0% (w/v), preferably about 0.5% to about 5.0%, more preferably
about
1.0% to about 3.0%, most preferably about 1.0%. When the stabilizing agent is
an
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WO 03/049694 PCT/US02/39253
amino acid, it is present in the range of about 0% to about 1.0% (w/v),
preferably
about 0.3% to about 0.7%, most preferably about 0.5%. These stabilized
lyophilized
or spray-dried compositions may optionally comprise methionine,
ethylenediaminetetracetic acid (EDTA) or one of its salts such as disodium
EDTA or
other chelating agent, which protect the IL-2 against methionine oxidation.
Use of
these agents in this manner is described in International Publication No. WO
01/24814. The stabilized lyophilized or spray-dried compositions may be
formulated
using a buffering agent, which maintains the pH of the pharmaceutical
composition
within an acceptable range, preferably between about pH 4.0 to about pH 8.5,
when in
' a liquid phase, such as during the formulation process or following
reconstitution of
the dried form of the composition. Buffers are chosen such that they are
compatible
with the drying process and do not affect the quality, purity, potency, and
stability of
the protein during processing and upon storage.
The previously described stabilized monomeric, multimeric, and stabilized
I S lyophilized or spray-dried IL-2 pharmaceutical compositions represent
suitable
compositions for use in the methods of the invention. However, any
pharmaceutical
composition comprising IL-2 as a therapeutically active component is
encompassed
by the methods of the invention.
As used herein, the term "anti-CD20 antibody" encompasses any antibody that
specifically recognizes the CD20 B-cell surface antigen, including polyclonal
anti-
CD20 antibodies, monoclonal anti-CD20 antibodies, human anti-CD20 antibodies,
humanized anti-CD20 antibodies, chimeric anti-CD20 antibodies, xenogeneic anti-
CD20 antibodies, and fragments of these anti-CD20 antibodies that specifically
recognize the CD20 B-cell surface antigen. Preferably the antibody is
monoclonal in
nature. By "monoclonal antibody" is intended an antibody obtained from a
population of substantially homogeneous antibodies, i.e., the individual
antibodies
comprising the population are identical except for possible naturally
occurring
mutations that may be present in minor amounts. Monoclonal antibodies are
highly
specific, being directed against a single antigenic site, i.e., the CD20 B-
cell surface
antigen in the present invention. Furthermore, in contrast to conventional
(polyclonal) antibody preparations that typically include different antibodies
directed
against different determinants (epitopes), each monoclonal antibody is
directed
against a single determinant on the antigen. The modifier "monoclonal"
indicates the
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character of the antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring production
of the
antibody by any particular method. For example, the monoclonal antibodies to
be
used in accordance with the present invention may be made by the hybridoma
method
first described by Kohler et al. (1975) Nature 256:495, or may be made by
recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). The
"monoclonal
antibodies" may also be isolated from phage antibody libraries using the
techniques
described in Clackson et al. (1991) Nature 352:624-628 and Marks et al. (1991)
J.
Mol. Biol. 222:581-597, for example.
Anti-CD20 antibodies of murine origin are suitable for use in the methods of
the present invention. Examples of such murine anti-CD20 antibodies include,
but are
not limited to, the B1 antibody (described in U.S. Patent No. 6,OlS,S42); the
1FS
antibody (see Press et al. (1989) J. Clin. O~col. 7:1027); NKI-B20 and BCA-B20
anti-CD20 antibodies (described in Hooijberg et al. (1995) Cancer Research
55:840-
846); and IDEC-2B8 (available commercially from IDEC Pharmaceuticals Corp.,
San
Diego, California); the 2H7 antibody (described in Clark et al. (1985) Froc.
Natl.
Acad. Sci. USA 82:1766-1770; and others described in Clark et al. (1985) supra
and
Stashenko et al. (1980) J. Immu~ol. 125:1678-1685.
The term "anti-CD20 antibody" as used herein encompasses chimeric anti-
CD20 antibodies. By "chimeric antibodies" is intended antibodies that are most
preferably derived using recombinant deoxyribonucleic acid techniques and
which
comprise both human (including immunologically "related" species, e.g.,
chimpanzee) and non-human components. Thus, the constant region of the
chimeric
antibody is most preferably substantially identical to the constant region of
a natural
2S human antibody; the variable region of the chimeric antibody is most
preferably
derived from a non-human source and has the desired antigenic specificity to
the
CD20 cell surface antigen. The non-human source can be any vertebrate source
that
can be used to generate antibodies to a human CD20 cell surface antigen or
material
comprising a human CD20 cell surface antigen. Such non-human sources include,
but
are not limited to, rodents (e.g., rabbit, rat, mouse, etc.; see, for example,
U.S. Patent
No. 4,816,567) and non-human primates (e.g., Old World Monkey, Ape, etc.; see,
for
example, U.S. Patent Nos. 5,750,105 and 5,756,096). Most preferably, the non-
human
component (variable region) is derived from a murine source. As used herein,
the
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phrase "immunologically active" when used in reference to chimeric anti-CD20
antibodies means a chimeric antibody that binds human Clq, mediates complement
dependent Iysis ("CDC") of human B lymphoid cell lines, and lyses human target
cells through antibody dependent cellular cytotoxicity ("ADCC"). Examples of
chimeric anti-CD20 antibodies include, but are not limited to, IDEC-C2B8,
available
commercially under the name rituximab (IDEC Pharmaceuticals Corp., San Diego,
California) and described in U.S. PatentNos. 5,736,137, 5,776,456, and
5,843,439;
the chimeric antibodies described in U.S. Patent No. 5,750,105; those
described in
U.S. Patent Nos. 5,500,362; 5,677, I80; 5,721,108; and 5,843,685.
Humanized anti-CD20 antibodies are also encompassed by the term anti-
CD20 antibody as used herein. By "humanized" is intended forms of anti-CD20
antibodies that contain minimal sequence derived from non-human immunoglobulin
sequences. For the most part, humanized antibodies are human imrnunoglobulins
(recipient antibody) in which residues from a hypervariable region of the
recipient are
replaced by residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the desired
specificity, affinity, and capacity. See, for example, U.S. Patent Nos.
5,225,539;
5,585,089; 5,693,761; 5,693,762; 5,859,205. In some instances, framework
residues
of the human immunoglobulin are replaced by corresponding non-human residues
(see, for example, U.S. Patents 5,585,089; 5,693,761; 5,693,762). Furthermore,
humanized antibodies may comprise residues that are not found in the recipient
antibody or in the donor antibody. These modifications are made to further
refine
antibody performance (e.g., to obtain desired affinity). In general, the
humanized
antibody will comprise substantially all of at least one, and typically two,
variable
domains, in which all or substantially all of the hypervariable regions
correspond to
those of a non-human immunoglobulin and all or substantially all of the
framework
regions are those of a human immunoglobulin sequence. The humanized antibody
optionally also will comprise at least a portion of an immunoglobulin constant
region
(Fc), typically that of a human immunoglobulin. For further details see Jones
et al.
(1986) Nature 331:522-525; Riechmann et al. (1988) Nature 332:323-329; and
Presta
(1992) Curr. Op. Struct. Biol. 2:593-596.
Also encompassed by the term anti-CD20 antibodies are xenogeneic or
modifzed anti-CD20 antibodies produced in a non-human mammalian host, more
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particularly a transgenic mouse, characterized by inactivated endogenous
immunoglobulin (Ig) loci. In such transgenic animals, competent endogenous
genes
for the expression of light and heavy subunits of host inununoglobulins are
rendered
non-functional and substituted with the analogous human immunoglobulin loci.
These transgenic animals produce human antibodies in the substantial absence
of light
or heavy host immunoglobulin subunits. See, for example, U.S. Patent No.
5,939,598.
Fragments of the anti-CD20 antibodies are suitable for use in the methods of
the invention so long as they retain the desired affinity of the full-length
antibody.
Thus, a fragment of an anti-CD20 antibody will retain the ability to bind to
the CD20
B-cell surface antigen. Fragments of an antibody comprise a portion of a full-
length
antibody, generally the antigen binding or variable region thereof. Examples
of
antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv
fragments and single-chain antibody molecules. By "single-chain Fv" or "sFv"
antibody fragments is intended fragments comprising the VH and VL domains of
an
antibody, wherein these domains are present in a single polypeptide chain.
See, for
example, U.S. Patent Nos. 4,946,778; 5,260,203; 5,455,030; 5,856,456.
Generally, the
Fv polypeptide further comprises a polypeptide linker between the VH and VL
domains that enables the sFv to form the desired structure for antigen
binding. For a
review of sFv see Pluckthun (1994) in The Pharmacology of Monoclonal
A~rtibodies,
Vol. 113, ed. Rosenburg and Moore (Springer-Verlag, New York), pp. 269-315.
Antibodies or antibody fragments can be isolated from antibody phage
libraries generated using the techniques described in McCafferty et al. (1990)
Nature
348:552-554 (1990). Clackson et al. (1991) Nature 352:624-628 and Marks et al.
(1991) J. Mol. Biol. 222:581-597 describe the isolation of murine and human
antibodies, respectively, using phage libraries. Subsequent publications
describe the
production of high affinity (nM range) human antibodies by chain shuffling
(Marks et
al. (1992) BiolTechnology 10:779-783), as well as combinatorial infection and
in vivo
recombination as a strategy for constructing very large phage libraries
(Waterhouse et
al. (1993) Nucleic. Acids Res. 21:2265-2266). Thus, these techniques are
viable
alternatives to traditional monoclonal antibody hybridoma techniques for
isolation of
monoclonal antibodies.
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A humanized antibody has one or more amino acid residues introduced into it
from a source that is non-human. These non-human amino acid residues are often
referred to as "donor" residues, which are typically taken from a "donor"
variable
domain. Humanization can be essentially performed following the method of
Winter
and co-workers (Jones et al. (1986) Nature 321:522-525; Riechmann et al.
(1988)
Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536), by
substituting rodent CDRs or CDR sequences for the corresponding sequences of a
human antibody. See, for example, U.S. Patent Nos. 5,225,539; 5,585,089;
5,693,761; 5,693,762; 5,859,205. Accordingly, such "humanized" antibodies may
include antibodies wherein substantially less than an intact human variable
domain
has been substituted by the corresponding sequence from a non-human species.
In
practice, humanized antibodies are typically human antibodies in which some
CDR
residues and possibly some framework residues are substituted by residues from
analogous sites in rodent antibodies. See, for example, U.S. Patent Nos.
5,225,539;
5,585,089; 5,693,761; 5,693,762; 5,859,205. See also U.S. Patent No.
6,180,370, and
International Publication No. WO 01/27160, where humanized antibodies and
techniques for producing humanized antibodies having improved affinity for a
predetermined antigen are disclosed.
Various techniques have been developed for the production of antibody
fragments. Traditionally, these fragments were derived via proteolytic
digestion of
intact antibodies (see, e.g., Morimoto et al. (1992).Iou~aal ofBiochemical and
Biophysical Methods 24:107-117 (1992) and Brennan et al. (1985) Science
229:81).
However, these fragments can now be produced directly by recombinant host
cells.
For example, the antibody fragments can be isolated from the antibody phage
libraries
discussed above. Alternatively, Fab'-SH fragments can be directly recovered
from E.
coli and chemically coupled to form F(ab')Z fragments (Carter et al. (1992)
BiolTechhology 10:163-167). According to another approach, F(ab')2 fragments
can
be isolated directly from recombinant host cell culture. Other techniques for
the
production of antibody fragments will be apparent to the skilled practitioner.
Further, any of the previously described anti-CD20 antibodies may be
conjugated prior to use in the methods of the present invention. Such
conjugated
antibodies are available in the art. Thus, the anti-CD20 antibody may be
labeled
using an indirect labeling or indirect labeling approach. By "indirect
labeling" or
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"indirect labeling approach" is intended that a chelating agent is covalently
attached
to an antibody and at least one radionuclide is inserted into the chelating
agent. See,
for example, the chelating agents and radionuclides described in Srivagtava
and
Mease (1991) Nuel. Med. Bio. 18: 589-603. Alternatively, the anti-CD20
antibody
may be labeled using "direct labeling" or a "direct labeling approach", where
a
radionuclide is covalently attached directly to an antibody (typically via an
amino acid
residue). Preferred radionuclides are provided in Srivagtava and Mease (1991)
supra.
The indirect labeling approach is particularly preferred. See also, for
example, labeled
forms of anti-CD20 antibodies described in U.S. Patent No. 6,015,542.
The anti-CD20 antibodies are typically provided by standard technique within
a pharmaceutically acceptable buffer, for example, sterile saline, sterile
buffered
water, propylene glycol, combinations of the foregoing, etc. Methods for
preparing
parentally administerable agents are described in RenZington.'s
Pharrrxaceutical
Seiences (18~' ed.; Mack Pub. Co.: Eaton, Pennsylvania, 1990). See also, for
example, International Publication No. WO 98/56418, which describes stabilized
antibody pharmaceutical formulations suitable for use in the methods of the
present
invention.
The present invention further provides a method for predicting clinical
response of a subject undergoing a time period of concurrent therapy with anti-
CD20
antibody and IL-2 in accordance with the dosing regimens disclosed herein. The
method comprises monitoring natural killer (NIA) cell expansion in said
subject at
about 1 week to about 7 weeks post-initiation, preferably at about 1 to about
14 weeks
post-initiation of the time period of concurrent therapy with these two
therapeutic
agents. Preferably NK cell counts are determined prior to the start of
concurrent
therapy, and are monitored throughout the time period of concurrent therapy so
that
the time course of NK cell expansion can be followed. In this manner, NK cell
counts
are determined weekly in a patient over the course of concurrent therapy with
anti-
CD20 antibody and IL-2 and for a period of 4-6 weeks following the final IL-2
administration. Methods for determining NK cell counts are known in the art.
See,
for example, methods disclosed in Suzuki et al. ((1983) J. Immureol. 130:981-
987;
Herberman (1987) Prog. Clin. Biol. Res. 244:267-274; and Meropol et al. (1998)
Cancer Imrnunol. Irnmunother. 46:318-326. When undergoing combination therapy
with IL-2, and anti-CD20 antibody as outlined herein with twice-weekly, thrice-
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weekly, or daily IL-2 dosing for 4 weeks, those patients having expansion of
NK cell
counts to greater than about 200 cells/~l at 10 weeks post-initiation of
therapy are
predicted at a week-14 evaluation to be non-progressors, i.e., to be complete
responders, partial responders, or will be characterized by stable disease. In
contrast,
those patients having expansion of NK cell counts to less than about 200
cellsl~.1 at 10
weeks post-initiation of therapy are predicted to be progressors, i.e., to
have relapse or
progressive disease, at the week-14 evaluation. Thus monitoring of NK cell
expansion
in patients undergoing combination therapy with rituximab and IL-2 can serve
as an
important diagnostic tool for a patient's prognosis with this therapy.
Thus, the present invention provides a method for treating lymphoma, more
particularly non-Hodgkin's B-cell lymphoma in a human subject, comprising
administering to the subject at least one therapeutically effective dose of an
anti-CD20
antibody and providing a means for maintaining natural-killer (NK) cell count
in this
subject at or above an acceptable threshold level. This acceptable threshold
level is
I S an NK cell count of about 150 cells/~1, preferably an NK cell count of
about 175
cells/~1. In some embodiments, the methods effectively maintain an NK cell
count in
the subject of about 200 cells/~1 or above. The means by which NK cell count
is
maintained includes any protocol by which IL-2 is administered to the subject
such
that at least one therapeutically effective dose of IL-2 in an amount that
results in an
initial IL-2 exposure within a range from about 22 IU*hour/ml serum to about
653
IU*hour/ml serum is administered to the subject, wherein said IL-2 exposure is
measured as the area under the serum concentration-time curve (AUC) as
determined
by human pharmacokinetic (PK) data.
One means for maintaining NK cell count above the acceptable threshold level
comprises administering IL-2 according to the constant IL-2 dosing regimen
disclosed
herein. Thus, the subject is administered at least one therapeutically
effective dose of
IL-2 in an amount necessary to achieve the same initial IL-2 exposure as a
dose of a
reference IL-2 standard (i.e., Proleukin~ IL-2) in a range from about 666.67
~g to
about 1200 ~,g as determined by the area under the serum concentration-time
curve
from human PK data. In such an embodiment, the subject is also administered a
therapeutically effective dose of anti-CD20 antibody in the range from about
125
mg/m2 to about 500 mg/m2, which is administered according to a weekly dosing
schedule as noted herein above. Where the means for maintaining NK cell count
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above the acceptable threshold level comprises a constant IL-2 dosing regimen
disclosed herein, the therapeutically effective dose of IL-2 can be
administered
according to a two-times-a-week or three-times-a-week dosing schedule, such
that a
total weekly dose of IL-2 in an amount equivalent to a total weekly dose of
the
reference IL-2 standard in a range from 2000 ~g to 3600 ~,g, for example, in a
range
from 2800 ~g to 3600 p.g, as determined by the area under the serum
concentration-
time curve from human PK data is administered to the subject. The duration of
dosing of the anti-CD20 antibody can be about 4 weeks to about 8 weeks, and
the
duration of the constant IL-2 dosing regimen can be about 4 weeks to about 10
weeks,
as noted herein above. Further guidance as to particular dosing regimens for
the anti
CD20 antibody in combination with constant IL-2 dosing are provided herein
above.
Another means for maintaining the NK cell count above the acceptable
threshold level comprises administering a two-level dosing regimen of IL-2,
where
the two-level dosing regimen of IL-2 comprises a first time period, wherein a
higher
total weekly dose of IL-2 is administered to said subject, followed by a
second time
period, wherein a lower total weekly dose of IL-2 is administered to said
subject, as
disclosed herein above. In this embodiment, the higher total weekly dose of IL-
2 is in
an amount equivalent to a total weekly dose of the reference IL-2 standard in
a range
from 2000 p.g to 3600 p.g as determined by the area under the serum
concentration
time curve from human pharmacokinetic (PK) data, and the lower total weekly
dose
of IL-2 is in an amount equivalent to a total weekly dose of a reference IL-2
standard
in a range from 1200 p,g to about 2600 p,g as determined by the area under the
serum
concentration-time curve from human PK data. As previously noted above, the
lower
total weekly dose of IL-2 is lower than the higher total weekly dose of IL-2.
In such
embodiments, the therapeutically effective dose of anti-CD20 antibody is in
the range
from about 125 mg/m2 to about 500 mg/m2.
Where the means for maintaining NK cell count above the acceptable
threshold level is a two-dose regimen of IL-2, a first dose of IL-2 can be
administered
to the subject prior to administering a first dose of anti-CD20 antibody, for
example,
about 1 week to about 30 days prior to administering the first dose of anti-
CD20
antibody. Alternatively, a first dose of IL-2 can be administered to the
subject
concurrently with (i.e., on the same day, either simultaneously or
sequentially, in
either order) a first dose of anti-CD20 antibody. In yet another embodiment, a
first
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dose of IL-2 is administered to the subject after a first dose of anti-CD20
antibody is
administered to the subject, for example, within 10 days, preferably within 7
days, of
administering the antibody to the subject. As noted herein above, the higher
total
weekly dose of IL-2 can be administered as a single dose or can be partitioned
into a
first series of equivalent doses that are administered according to a two-,
three-, four-,
five-, six- or seven-times-a-week dosing schedule, and the lower total weekly
dose of
IL-2 can be administered as a single dose or can be partitioned into a second
series of
equivalent doses that are administered according to a two-, three-, four-,
five-, six- or
seven-times-a-week dosing schedule. The duration of dosing of the anti-CD20
antibody can be about 4 weeks to about ~ weeks, and the duration of the two-
level IL-
2 dosing regimen can be about 4 weeks to about 16 weeks, as noted above. This
means of maintaining NK cell count may further comprise giving the subject a
drug
holiday between the first period of the two-level IL-2 dosing regimen (i.e.,
where
higher total weekly doses of IL-2 are administered) and the second period of
the two-
level IL-2 dosing regimen (i.e., where lower total weekly doses of IL-2 axe
administered), as described elsewhere herein.
Where necessary, the subject can be administered multiple maintenance cycles
of concurrent therapy with anti-CD20 antibody and the two-level IL-2 dosing
regimen
to maintain NK cell count above an acceptable threshold level, i.e., about 150
cells/~1.
As noted above, each such maintenance cycle would comprise weekly
administration
of the anti-CD20 antibody in combination with a completed two-level IL-2
dosing
regimen (i.e., the subject completes both the first time period of higher
total weekly
dosing and the second time period of lower total weekly dosing, where the
completed
two-level IL-2 dosing regimen can further comprise a drug holiday). Further
guidance as to particular dosing regimens for the anti-CD20 antibody in
combination
with a two-level IL-2 dosing regimen are provided herein above..
The following examples are offered by way of illustration and not by way of
limitation.
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Example 1: Phase I Study of Weekly Rituximab Therapy in Combination with
Constant Total Weekly Dose of Proleukin~ IL-2 in Patients with Non-Hodgkin's
Lymphoma
The IL-2 formulation used in this study is manufactured by Chiron
Corporation of Emeryville, California, under the tradename Proleukin~ IL-2.
The IL-
2 in this formulation is a recombinantly produced, unglycosylated human II,-2
mutein, called aldesleukin, which differs from the native human IL-2 amino
acid
sequence in having the initial alanine residue eliminated and the cysteine
residue at
position 125 replaced by a serine residue (referred to as des-alanyl-1, serine-
125
human interleukin-2). This IL-2 mutein is expressed in E. coli, and
subsequently
purified by diafiltration and cation exchange chromatography as described in
TJ.S.
Patent No. 4,931,543. The IL-2 formulation marketed as Proleukin~ IL-2 is
supplied
as a sterile, white to off white preservative-free lyophilized powder in vials
containing
1.3 mg of protein (22 MILT).
The anti-CD20 antibody used in this and the following examples is Rituxan~
(rituximab; IDEC-C2B8; IDEC Pharmaceuticals Corp., San Diego, California). It
is
administered per its package insert dose (375 mg/m2 infused over 6 hours).
The primary objective of this study was to determine a maximum tolerated
weekly dose (MTD) of Proleukin~ IL-2 when administered subcutaneously as three
equivalent doses concomitantly with a weekly intravenous (IV) infusion of a
fixed
dose (375 mg/m2) of Rituxari (rituximab) for the treatment of CD20+ B-cell non-
Hodgkin's lymphoma stage III or IV. The secondary objectives were to explore
the
effect of IL-2 concomitantly with rituximab on the degree of expansion of
natural
killer (NK) cells, NK cell function as measured by antibody dependent
cytotoxicity
(ADCC), anti-tumor responses, the duration of anti-tumor responses, and the
pharmacokinetics of IL-2 and rituximab.
Study Design
This was an open-label study of escalating doses of IL-2 in combination with
375 mg/mz rituximab infused once weekly for a total of 4 doses. Multiple study
centers are anticipated. The total weekly doses of IL-2 were 13.5 million
international
units (i.e., thrice-weekly IL-2 dose of 4.5 MICT), 30.0 MIU (i.e., thrice-
weekly IL-2
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dose of 10.0 MIU), 42.0 MIU (i.e., thrice-weekly IL-2 dose of 14.0 MIU), and
54.0
MIU (i.e., thrice-weekly IL-2 dose of 18.0 MIU). These correspond to relative
total
weekly doses of about 7.9 MIU/m2, about 17.6 MIU/mz, about 24.7 MIU/mz, and
about 31.8 MIU/ma. The total weekly dose of Proleukin~ IL-2 (referred to
hereafter
in this example as IL-2) was partitioned into three equivalent doses that were
administered three times weekly (tiw) by subcutaneous injection concomitantly
with
weekly IV infusions of rituximab. Interleukin-2 treatments began 1 week after
the
first IV infusion of rituximab and continued up through the end of week S.
Patients
remained on a fixed dose of IL-2 throughout this period.
Treatments Administered and Dose Escalation Methodolo~y
Patients began treatment with 37S mg/m2 of rituximab by IV infusion on day 1
and then weekly for 3 additional weeks. Thrice-weekly subcutaneous injections
of IL-
2 started on day 8 at the assigned dose and continued a total of 4 weeks.
Thrice-
weekly treatment of IL-2 was defined as administration of IL-2 three times per
week
1S with at least 48 hours between injections. First injections of IL-2 were
given within
30 minutes after the start of the rituximab infusion. The following two IL-2
injections
were administered in 48-hour intervals. Weeks 2 through 4 of IL-2 subcutaneous
injections began concomitantly with rituximab infusion. Week S was IL-2
subcutaneous injections only. The thrice-weekly IL-2 dose levels studied were
4.S
million international units (MIU), 10.0 MILT, 14.0 MIU and 18.0 MIU, which
correspond to total weekly doses of 13.5 MIU, 30.0 MIU, 42.0 MIU, and 54.0
MIU.
A dose limiting toxicity (DLT) is defined as a treatment-related adverse
reaction with toxicity grading of grade 3 or grade 4 by National Cancer
Institute
(NCI) criteria (i.e., NCI Common Toxicity Criteria), with the exception of the
2S hematologic and fever toxicities, which require a toxicity grading of grade
4 to be
considered a DLT. Some specific criteria that may be encountered during the
course
of the study include grade 3 toxicities (for example, white blood cell count
(a value of
1.0- <2.0 x 103/1), platelets (a value of 2S-49 x 103/pl), hemoglobin (a value
of 6.5-
<8.0 g/dl), infection (severe, not life threatening), vomiting (6-10 episodes
in 24 hours
in the presence of sufficient anti-emetic therapy), pulmonary (dyspnea at
normal
levels of exertion), hypotension (requiring therapy and hospitalization;
resolves within
48 hours of stopping study medications), neurosensory (severe objective
sensory loss
or paresthesias that interfere with function), neuromoter (objective weakness
with
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impairment of function), fever (oral greater than 39.6-40.4°C), fatigue
(normal
activity decreased greater than 50% inability to work), weight gain on study
(at least
20.0%), local reactions (induration greater than 10 cm2; ulceration or
necrosis that is
severe or prolonged), etc.), and grade 2 toxicities (for example, cardiac
dysrhythmia
(recurrent or persistent but not requiring therapy), cardiac function (decline
of resting
ejection fraction by more than 20%), cardiac ischemia (asymptomatic ST-T wave
changes), and pericardium (pericarditis by clinical criteria). Except for what
is listed
herein, any grade 3 toxicity is considered dose limiting. Specific examples of
adverse
reactions that must be a grade 4 to be considered a DLT are absolute
neutrophil count
(ANC) <5 x 102/x,1); total white blood cell count (WBC) <1 x 103/1; hemoglobin
(Hgb) <6.5 g/dl; platelets <25 x 103/x,1; and fever (oral) greater than
40.5°C or 105°F.
Cohorts of 3 patients were enrolled at each IL-2 dose level. Adverse events of
dose-limiting toxicity (DLT) were monitored in patients through the end of
week 5. If
the current dose level of IL-2 was tolerated by all 3 patients through the end
of week 5
without any adverse events of DLT, another cohort of three patients was
enrolled at
the next higher dose level of IL-2. Subjects at this next dose level could be
enrolled
based on the DLT data at week 3 of IL-2 (week 4 of the study) and receive
rituximab,
however IL-2 was not administered until all patients within the cohort had
completed
the 5-week regimen. If one of the 3 patients experienced an adverse event of
DLT at
any time during the 5-week regimen, 3 additional patients were enrolled at
this dose
level. The dose of IL-2 was not increased to the next dose level unless the 3
additional
patients completed week S without experiencing an adverse event of DLT.
Subjects at
this next dose level could be enrolled and receive rituximab, however IL-2 was
not
administered until all patients within the cohort had completed the 5-week
regimen.
The outpatient MTD of IL-2 was considered to be the dose level immediately
below
the lowest dose level at which adverse events of DLT were observed in 2 or
more
patients.
Selection of Study Population
The primary inclusion criteria for patients enrolled in this study were:
documentation of CD20+ B cell non-Hodgkin's lymphoma stage III or IV; patients
should be relapsed or refractory to first-line treatments; Karnofsky
Performance Score
of >_ 70%; and not less than 18 years old. Patients were excluded from the
study for
the following reasons: prior treatment with IL-2; prior treatment with
rituximab for
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any indication within 3 months of study treatment; and current or prior
medical
history inconsistent with use of rituximab or IL-2.
Measurements, Safety, and Efficacy
The primary objective of the study was to determine the MTD of thrice-
weekly IL-2 when administered concomitantly with weekly doses of 375 mg/m2
rituximab. For the purpose of estimating MTD, patients could not miss more
than one
dose of IL-2 consecutively, nor miss more than 30% or more of the prescribed
IL-2
dose, and had to receive all 4 doses of rituximab in order to be included in
the analysis
(unless patients experience DLTs while on IL-2).
It was of importance to the success of the proposed combination therapy to
establish that NIA cell number and function and T cell number were enhanced
during the
course of the study. These cell types may be expanded after treatment with IL-
2, which
may be essential for rituximab anti-tumor activity. Therefore, measurements of
NK cell
number and function and T cell subset numbers were performed. In this manner,
lymphocyte subsets (the percent and absolute number of lymphocytes expressing
CD3
CD4, CDR, CD16+56, and CD19, and the percent of lymphocytes expressing CD20)
and NIA cell ADCC function were measured at weekly intervals throughout the
study
prior to rituximab infusion using standard protocols. NIA cell expansion
appears to be
a critical requirement for enhancement of rituximab activity, and the extent
of NK cell
expansion is a component in subsequent dosing decisions in future studies.
Other
variables observed in this study included tumor response and duration and the
pharmacokinetics of IL-2.
Efficacy was assessed in all patients as a secondary variable. An evaluable
patient was defined as: subjects must have received 4 weeks of rituximab
therapy and
70% of the prescribed Proleukin~ IL-2 dose and schedule. The response was
evaluated as follows. Tumor measurements were based upon measurements of
perpendicular diameters, using the longest diameter and its greatest
perpendicular.
Grading of tumor response is based upon the report of the International
Workshop to
Standardize Response Criteria for Non-Hodgkin's Lymphomas (see, Cheson et al.
(1999) J. Clin. Oncol. 17:1244-1253) and protocol-defined criteria as follows:
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~ Complete response (CR) - Defined as absence of clinically detectable
disease with normalization of any previously abnormal radiographic
studies, bone marrow and cerebrospinal fluid (CSF). Response must
persist for at least one month. Patients with bone marrow positive for
lymphoma prior to chemotherapy must have a repeat biopsy, which is
confirmed after a month, negative for lymphoma.
~ Partial response (PR) - Defined as at least 50% decrease in all
measurable tumor burden in the absence of new lesions and
persisting for at least ane month (applicable to measurable tumors
only).
Patients were also assessed for effects of Proleukin~' IL-2 and rituximab
therapy on the following:
~ Response duration - Defined as the time from first documented
response until progressive disease.
~ Tinae to progression - Defined as the time from study entry to
progressive disease, relapse or death.
~ Stable disease (SD) - Defined as a less than 50% reduction in
tumor burden in the absence of progressive disease.
~ Progressive disease (PD) - Defined as representing 25% or greater
increase in tumor burden or the appearance of a new site of the
disease.
~ Relapse (R) - Defined as the appearance of tumor following
documentation of a complete response.
Evaluation of efficacy by tumor response was a secondary objective. Efficacy
was also measured by the degree of expansion of NK cells; NK cell function as
measured by ADCC; anti-tumor responses; and the duration of anti-tumor
responses.
In total, 15 patients have been enrolled in the phase I clinical trial
described
above. The thrice-weekly dosing regimen was generally well tolerated through
the 14
MIU dose level. At the 18 MIU dose, 3 patients completed therapy at the full
dose.
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A fourth patient developed dose-limiting toxicity (hypersensitivity), and
further
treatment was stopped..
Thirteen patients have completed therapy and have been evaluated at week 14
for best tumor response. Overall, 7 patients responded, 4 with complete
response
(CR), and 3 with partial response (PR); 4 patients had stable disease (SD);
and 2 had
progressive disease (PD). All 7 of the responders received one of the three
highest
doses on the thrice-weekly schedule. Two CRs occurred in patients with
intermediate- or high-grade lymphoma, and 2 CR occurred in patients with low-
grade
lymphoma. The 3 PRs were seen in patients with intermediate- or high-grade,
including one with mantle-cell lymphoma.
Figure 1 shows the time course for natural killer (NK) cell count (CD16/CD56
cells) (Panel A), CD4 cell count (Panel B), and CD8 cell count (Panel C) in 11
patients undergoing concurrent therapy with weekly rituximab therapy (375
mg/m2)
and thrice-weekly doses of Proleukin~ IL-2 (recombinant human IL-2 mutein) for
treatment of non-Hodgkin's lymphoma. NK cell count was determined by flow
cytometry. Rituximab was administered by infusion over up to 6 hours on day 1
(D1),
day 8 (D8), day 15 (D15), and day 22 (D22). Proleukin~ IL-2 was administered
subcutaneously three times per week fox 4 weeks beginning on day 8. The doses
of
Proleukin~ IL-2 were 4.5 MIU (3 patients), 10 MIU (3 patients), 14 M1U (3
patients),
and 18 MIU (2 patients). The corresponding cell counts at week 10 for the 9
patients
with available data that have completed the entire dosing regimen versus their
clinical
response to therapy at week 14 are shown in Panel D (NK cell count), Panel E
(CD4
cell count) and Panel F (CD8 cell count). PD = progressive disease; SD =
stable
disease; CR/PR = complete response or partial response.
Natural killer (NK) CD56+CD16+ cell numbers increased with thrice-weekly
IL-2 administration in all except one patient, who had disease progression
(Figure 1,
Panel A). At the beginning of week 4, 4 out of 5 responders had absolute NK
cell
counts above 572 cells/~.1, while the 4 patients with progressive disease had
NK cell
counts less than 394 cells/~,1 (individual patient data not shown; for median
cell count,
see Figure 2). For the 10 patients for whom NK cell values are currently
available at
week 10, the degree of NK CD56+CD16+ cell expansion during IL-2 therapy on the
thrice-weekly schedule was higher in responders (complete responders and
partial
responders) than in non-responders (stable disease and progressive disease
patients),
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with a median of 382 cells/~,l (range 215-494 cells/~,1) compared to 155
cells/wl
(range 40-195 cells/pl) (Figure 1, Panel D; see also Figure 2). The median NK
cell
count at week IO showed a statistically significant difference between these
groups
(p=0.01; Figure 2).
Of the three populations of cell counts collected, NK cell count appears to be
an important predictor to response with rituximab when combined with IL-2.
When
the number of NK cells is plotted against the tumor response at week 14 to
this thrice-
weekly dosing of IL-2 with weekly dosing of rituximab, it can be seen that
those
patients having stable disease or a complete response or partial response have
at least
185 cells/pl at 10 weeks post-initiation of rituximab therapy (Figure 1, Panel
D;
Figure 2).
In addition, fresh ex vivo NK-mediated natural cytolytic killing against the
NK-sensitive, LAK-resistant K562 cell line, and LAK and ADCC-mediated function
against the NK-resistant, LAK-sensitive Daudi B-cell line in the presence and
absence
IS of optimal concentrations of rituximab in a standard 4-hr 5lCr-release
cytotoxicity
assay at effectoraarget ratios of SO:I-1.56:1 have been evaluated for
responders and
non-responders in both IL-2 dosing protocols. For a description of this
cytotoxicity
assay, see, for example, Vlasveld et al. ( 1995) Cancer l~znauszol.
Imrr~unotlze~.
40(1):37-47. For this study, PBMC were isolated by Ficoll Hypague density
centrifugation from the blood of patients enrolled in the program. PBMC were
isolated prior to rituximab treatment (day l; dl), pre-IL-2 treatment (day 8;
d8), after
one week of IL-2 treatment (day 15; d15), after two weeks of IL-2 treatment
(day 22;
d22), and at subsequent time points throughout the study. PBMC were tested at
an
effector to target ratio (E:T) range of 50:1-1.56: I against a panel of SICr-
labeled target
cells comprised of K562 cells, Daudi cells, and Daudi cells in the presence of
optimal
concentrations of rituximab (2 wg/ml) to evaluate NK, LAK, and ADCC activity,
respectively. SICr release was measured after a 4-hour incubation period.
Responders in this protocol demonstrated sustained NK-mediated activity, as
noted by NK natural cytotoxicity, LAK, and ADCC-mediated killing, that was
increased progressively and was maintained at week 10, despite the fact that
IL-2
dosing was completed by the end of week 5 (Figure 3). This trend was also
observed
for patients with stable disease, though to a lesser extent than observed for
responders
(data not shown). In contrast, those who developed progressive disease
exhibited
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lower transient levels of NIA-mediated killing activity, which declined
rapidly
following cessation of IL-2 treatment (data not shown). Collectively, the NIA
cell
count data and NIA function data suggest that IL-2-mediated NK cell expansion
and
function are critical interdependent determinants of clinical response outcome
to
concurrent therapy with IL-2 and rituximab.
Thus, when undergoing concurrent therapy with IL-2 and rituximab as
outlined herein with thrice-weekly dosing for 4 weeks, those patients having
expansion of NK cell counts to greater than about 170 cells/~1 at 10 weeks
post-
initiation of therapy are predicted to be complete responders, partial
responders, or are
characterized by stable disease. In contrast, those patients having expansion
of NK
cell counts to less than about 170 cells/wl at 10 weeks post-initiation of
therapy are
predicted to have relapse or progressive disease. Thus, monitoring of NK cell
expansion in patients undergoing concurrent therapy with rituximab and IL-2
can
serve as an important diagnostic tool for a patient's prognosis with this
therapy.
Example 2: Phase II/III Clinical Trial with Weekly Rituximab Therapy for 4
Weeks
in Combination with 8-week Two-Level IL-2 Dosing Regimen of Proleukin~ IL-2 in
Patients with Non-Hodgkin's Lymphoma Who Have Previously Failed Rituximab
A phase II/III clinical trial is carried out to evaluate safety and efFicacy
of a 4-
week rituximab therapy (i.e., weeks 1-4) in combination with an 8-week two-
level IL-
2 dosing regimen of Proleukin~ TL-2 (weeks 2-9) for the treatment of CD20+ B-
cell
non-Hodgkin's lymphoma in patients who previously failed to respond to
rituximab
or relapsed within 6 months of treatment. The secondary objectives are to
further
document the effect of IL-2 concomitantly with rituximab on the degree of
expansion
of natural killer (NK) cells, NK cell function as measured by antibody
dependent
cytotoxicity (ADCC), anti-tumor responses, the duration of anti-tumor
responses, and
the pharmacokinetics of IL-2 and rituximab.
Stud~Desi; ~n
This is an open-label study employing two doses of IL-2 in combination with
375 mg/m2 rituximab infused once weekly (6-hour infusion). Patients are
administered a weekly IV infusion of a fixed dose (375 mg/m2) of Rituxan~
(rituximab) beginning on day 1 of each week for a period of 4 weeks (i.e., a
total of 4
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doses). Thus rituximab is administered on days 1, 8, 15, 22. Patients begin
concomitant administration of Proleukin~ IL-2 (hereinafter referred to as
reference
IL-2 standard) by subcutaneous injection on day 1 of the second week (i.e.,
day 8 of
the treatment period). The total weekly dose of II,-2 is partitioned into
three
equivalent doses that are administered according to a three-times-per-week
dosing
schedule, with a minimum of 48 hours between administrations, for a period of
8
weeks (i.e., total of 24 doses during weeks 2-9 of the treatment period).
During
weeks 2-5, the total weekly IL-2 dose to be administered as three equivalent
doses is
42.0 MIU (i.e., each equivalent dose is 14.0 MIU). After 4 weeks of IL-2
administration, the total weekly IL-2 dose is lowered to 30.0 MIU. Thus,
during
weeks 6-9, a total weekly IL-2 dose of 30.0 MIU is partitioned into three
equivalent
doses (i.e., each 10.0 MIU) that are administered according to the three-times-
per-
week dosing schedule. Patients are monitored for efficacy and safety of this
treatment
regimen throughout the 9-week treatment period, with follow-up determinations
occurring through week 16 (i.e., for 7 weeks beyond the last week of IL-2
administration).
Selection of Stud~pulation
Patients are eligible if they have CD20+, B-cell, non-Hodgkin's lymphoma of
low-grade or follicular histology with measurable relapsed or unresponsive
disease
after prior therapy. In addition, they must have previously received a course
of single-
agent rituximab and showed no tumor response, or had a response lasting < 6
months.
The previously administered rituximab must have included at least 75% of the
standard 4-week regimen (4 x 375 mg/m2). A record of the previous rituximab
treatment and response must be available as a source document at the site.
Other
primary inclusion and exclusion criteria are similar to those noted for the
phase I
clinical trial described in Example 2 above.
Measurements, Safety, and Efficacy
The primary objective of the study is to determine the safety and efficacy of
thrice-weekly IL-2 when administered for 8 consecutive weeks concomitantly
with
weekly doses of 375 mg/m2 rituximab. Patients must not miss more than one dose
of
IL-2 consecutively, nor miss more than 30% or more of the prescribed IL-2
dose, and
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receive all 4 doses of rituximab in order to be included in the primary
efficacy
analysis.
Efficacy is assessed by tumor response and duration of tumor response using
the procedures and criteria noted for the phase I clinical trial described in
Example 1.
Tumor response is correlated with increases in NIA cells determined by flow
cytometry. Other variables observed in this study in a subset of patients are
NK cell
function and the pharmacokinetics of IL-2, as noted for the phase I clinical
trial
described in Example 1.
Example 3: Weekly Rituximab Therapy for 8 Weeks in Combination with an 8-Week
Two-Level IL-2 Dosing Regimen of Proleukin~ IL-2 in
Patients with Aggressive Non-Hodgkin's Lymphoma
A dosing schedule similar to that outlined in Example 2 is carried out with
subjects having aggressive non-Hodgkin's Lymphoma (stage III or IV, i.e.,
intermediate- to high-grade), with the exception of extending the weekly
rituximab
therapy out to 8 weeks. In this manner, subjects are given an 8-week rituximab
therapy (i.e., weeks 1-8) in combination with an 8-week two-level IL-2 dosing
regimen of Proleukin~ IL-2 (weeks 2-9). The secondary objectives are to
further
document the effect of IL-2 concomitantly with rituximab on the degree of
expansion
of natural killer (NK) cells, NK cell function as measured by antibody
dependent
cytotoxicity (ADCC), tumor response, and the duration of tumor response.
Eligible subjects are administered a weekly IV infusion of a fixed dose
(375 mg/m2) of Rituxari (rituximab) beginning on day 1 of each week for a
period of
8 weeks (i.e., a total of 8 doses). Thus rituximab is administered on days 1,
8, 15, 22,
29, 36, 43, and 50. Subjects begin concommitant administration of Proleukin~
IL-2
(hereinafter referred to as reference IL-2 standard) by subcutaneous injection
on day 1
of the second week (i.e., day 8 of the treatment period). The total weekly
doses of IL-
2 are partitioned into three equivalent doses that are administered according
to a
three-times-per-week dosing schedule, with a minimum of 48 hours between
administrations, for a period of 8 weeks (i.e., total of 24 doses during weeks
2-9 of the
treatment period). During weeks 2-5, the total weekly IL-2 dose to be
administered as
three equivalent doses is 42.0 MIU (i.e., each equivalent dose is 14.0 MIL>].
After 4
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weeks of IL-2 administration, the total weekly IL-2 dose is lowered to 30.0
MIU.
Thus, during weeks 6-9, a total weekly IL-2 dose of 30.0 MIU is partitioned
into three
equivalent doses (i.e., each 10.0 MIIJ) that are administered according to the
three-
times-per-week dosing schedule. Subjects are monitored for efficacy and safety
of
this treatment regimen throughout the 9-week treatment period, with follow-up
determinations occurring through week 16 (i.e., for 7 weeks beyond the last
week of
IL-2 administration).
Example 4: Phase I Clinical Trial with Weekly Rituximab Therapy for 4 Weeks in
Combination with 4-week Constant Total Weekly Dose of Monomeric IL-2
in Patients with Non-Hodgkin's Lymphoma
A phase I clinical trial is carried out to examine the use of a monomeric
formulation of IL-2, L2-7001, for the treatment of CD20+ B-cell non-Hodgkin's
lymphoma. The particular monomeric IL-2 formulation to be used is L2-7001.
This
liquid formulation comprises the same human TL-2 mutein (aldesleukin) as
Proleukin~
IL-2 with the exception of the final purification steps prior to its
formulation. As
noted in Example I above, this IL-2 mutein is expressed from E. coli. The
initial
purification steps to obtain aldesleukin are similar for the two formulations.
See U.S.
Patent No. 4,931,543. In both cases, the recombinantly produced IL-2 mutein
occurs
as refractile bodies within the host cells. Following cell disruption, the
refractile
bodies are isolated and initially purified using size exclusion chromatography
and RP-
HPLC. The remaining purification steps for the IL-2 mutein used in L2-7001 are
as
follows. The resulting protein precipitate is solubilized by guanidine
hydrochloride,
then processed by diafiltration, ion exchange chromatography, and subsequent
diafiltration to obtain the final purified IL-2 mutein for use in making the
L2-7001
formulation. In contrast, when this IL-2 mutein is used in Proleukin~ IL-2,
the
protein precipitate resulting from the initial purification steps is
solubilized byl%
SDS, then processed by size exclusion chromatography and dialfiltration. The
purif ed IL-2 mutein is then formulated into L2-7001 according to the method
disclosed in the copending application entitled "Stabilized Liquid Polypeptide-
Contaihircg Phaf~naaceutical Compositions," filed October 3, 2000, and
assigned U.S.
Application Serial No. 09/677,643.
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This is an MTD dose-fording study similar to that described for
Proleukin° IL-
2. In this study, a constant total weekly dose of L2-7001 is administered over
a 4-
week period in combination with 4 weekly doses of rituximab at its recommended
dose (i.e., 375 mg/m2). The total weekly IL-2 doses are partitioned into three
equivalent doses that are administered according to a three-times-a-week
dosing
schedule, with a minimum of 48 hours between administrations. The dose
escalation
methodology is similar to that described before. The initial escalating total
weekly
doses of L2-7001 are 540 fig, 810 fig, 1080 ~,g, and 1500 ~g as determined
from AUC
data for pharmacokinetics of L2-7001. See Table 4 in Example 8 below. Study
design and data collected are similar to those described in Example 1 above.
Safety
and efficacy are evaluated as noted in the clinical trials described above.
Example 5: Weekly Rituximab Therapy for 4 Weeks in Combination with 8-week
Two-Dose Regimen of L2-7001 in Patients with Non-Hodgkin's Lymphoma
Who Have Previously Failed Rituximab
As an alternative to the dosing regimen outlined in Example 2, eligible
subjects are administered the monomeric IL-2 formulation L2-7001, instead of
Proleukin~ IL-2. In this manner, subjects are administered a weekly IV
infusion of a
fixed dose (375 mg/m2) of Rituxari (rituximab) beginning on day 1 of each week
for
a period of 4 weeks (i.e., a total of 4 doses). Thus rituximab is administered
on days
1, 8, 15, 22. Subjects begin concomitant administration of L2-7001
(hereinafter
referred to as IL-2 in this example) by subcutaneous injection on day 1 of the
second
week (i.e., day 8 of the treatment period). The total weekly dose of IL-2 is
partitioned
into three equivalent doses that axe administered according to a three-times-
per-week
dosing schedule, with a minimum of 48 hours between administrations, for a
period of
8 weeks (i.e., total of 24 doses during weeks 2-9 of the treatment period).
During
weeks 2-5, the total weekly IL-2 dose to be administered as three equivalent
doses is
810 ~g (i.e., each equivalent dose is 270 ~,g). After 4 weeks of IL-2
administration,
the total weekly IL-2 dose is lowered to 540 q.g. Thus, during weeks 6-9, a
total
weekly IL-2 dose of 540 ~g is partitioned into three equivalent doses (i.e.,
each 180
fig) that are administered according to the three-times-per-week dosing
schedule.
Subjects are monitored for efficacy and safety of this treatment regimen
throughout
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the 9-week treatment period, with follow-up determinations occurring through
week
16 (i.e., for 7 weeks beyond the last week of IL-2 administration).
Example 6: Phase II/III Clinical Trial with Weekly Rituximab Therapy for 8
Weeks
in Combination with 8-week Two-Dose Regimen of L2-7001 in
Patients with Aggressive Non-Hodgkin's Lymphoma
As an alternative to the dosing regimen outlined in Example 3, eligible
subjects are administered the monomeric IL-2 formulation L2-7001, instead of
Proleukin~ IL-2. a weekly IV infusion of a fixed dose (375 mg/m2) of Rituxan~
(rituximab) beginning on day 1 of each week for a period of 8 weeks (i.e., a
total of 8
doses). Thus rituximab is administered on days l, 8, 15, 22, 29, 36, 43, and
50.
Patients begin concommitant administration of L2-7001 (hereinafter referred to
as IL-
2) by subcutaneous injection on day 1 of the second week (i.e., day 8 of the
treatment
. period). The total weekly doses of IL-2 are partitioned into three
equivalent doses
that are administered according to a three-times-per-week dosing schedule,
with a
minimum of 48 hours between administrations, for a period of 8 weeks (i.e.,
total of
24 doses during weeks 2-9 of the treatment period). During weeks 2-5, the
total
weekly IL-2 dose to be administered as three equivalent doses is 810 ~.g
(i.e., each
equivalent dose is 270 fig). After 4 weeks of IL-2 administration, the total
weekly IL-
2 dose is lowered to 540 fig. Thus, during weeks 6-9, a total weekly IL-2 dose
of 540
~g is partitioned into three equivalent doses (i.e., each 180 fig) that are
administered
according to the three-times-per-week dosing schedule. Subjects are monitored
for
efficacy and safety of this treatment regimen throughout the 9-week treatment
period,
with follow-up determinations occurring through week 16 (i.e., for 7 weeks
beyond
the last week of IL-2 administration).
Example 7: Calculating Equivalent Doses for Proleukin~ IL-2
in Different Units of Measure
The foregoing doses of Proleukin~ IL-2 used in the phase I and phase II
clinical trials represent absolute doses in MILT. One can readily determine
the
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corresponding relative dose in MIU/m2 as the average person is approximately
1.7 ma.
Similarly, one can determine the corresponding absolute dose in microgram
units
given that Proleukin~ IL-2 has a biological activity of about 15 MIU per mg of
this
IL-2 product. Table 2 provides equivalent total weekly doses for Proleukin~ IL-
2 in
different units of measure.
Table 2: Equivalent total weekly doses for Proleukin~ IL-2 in different units
of
measure.
MIU/mz MIU Micrograms (p,g)
10.6 18.0 1200.00
14.7 25.0 1666.67
17.6 30.0 2000.00
20.6 35.0 2333.33
22.9 39.0 2600.00
24,7 42.0 2800.00
29.4 50.0 3333.33
31.8 54.0 3600.00
Example 8: Calculation IL-2 Serum Concentration-Time Curves for Pharmaceutical
Formulations of IL-2
The area under the serum concentration-time curve (AUC) of Proleukin~ IL-2
administered subcutaneously (SC) at 4.5 million international units (MIU)
(equivalent
to approximately 300 pg protein) was determined using data from an unpublished
HIVstudy. Serum concentration time profiles were measured in 8 IL-2 naive, HIV
patients following an initial exposure to IL-2 dosing in this study. For each
patient,
the AUC was calculated using the linear trapezoidal rule up to the last
measurable
concentrations and extrapolated to 24 hours (Winnonlin software version 3.1,
Pharsight Corporation, California). The average AUCo_24, SD, and the lower and
upper 95% confidence limits at 4.5 MItJ dose are presented in Table 3.
The AUCo_24 value of Proleukin~ IL-2 administered SC at doses equivalent to
18 MIU (1200 fig) was estimated using data from three different studies where
this
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IL-2 product was administered SC. Two are published studies, one in HIV
patients
(N=3) (Piscitelli et al. (1996) PharrnacotheraPy 16(5):754-759) and one in
cancer
patients (N=7) (Kirchner et al. (I998) Br. J. Clin. Pha~macol. 46:5-10). The
third is
an unpublished study in which serum concentration time data were available
from 6
cancer patients after SC doses of IL-2. The similarity of the AUC in cancer
and HIV
patients was previously established (unpublished data). The actual doses
administered in these three studies ranged between 18 and 34 MIU. For the two
published trials, the AUC up to 24 hours (AUCo_24) values were normalized to
18
MIU dose by multiplying the AUC with the quotient of 18 and actual dose in
MIU.
For example, if the AUCo_24 for a 20 MIU dose was calculated to be 400, the
normalized AUCo_2ø would be 400*18/20=360. For the unpublished cancer-patient
study, individual AUC values were calculated from the serum concentration time
data
using the linear trapezoidal rule up to the last measurable concentrations and
extrapolated to 24 hours (Winnonlin software version 3.1, Pharsight
Corporation,
I S California) then were normalized to 18 MIU dose as noted above. The
overall mean
and SD for all three studies was calculated as the weighted average of the
means and
variances, respectively, using equations 1 and 2.
- UXl +nzX2 +n3X3J
1. XP -
(n,+nz+n3)
(n, -I~si +(nz -l~sz +(n3 -1~s3
2. SDP =
(n,+nz+n3-3)
Where nl,nz,n3,X1,X2,X3 and si,s2,s3 are the number of subjects, means, and
2S variances for each of the three studies, respectively. XP and SDP are
estimates of
the overall mean and standard deviation. The overall average AUC, SD, and the
lower and upper 9S% confidence limits at 18 MILT are also presented in Table
2.
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Table 3: Average (~ SD) AUCo_z4 obtained after initial exposure to a single
dose
administration of Proleukin~ IL-2 administered subcutaneously.
Proleukin~ AUCo_aa SD LL of 95% UL of 95%
IL-2 (IU*hr/ml) CI' CI'
Dose
(MIU/~,g)
4.5 / 300 56 15 26 86
6.0 / 400 71 24 117
7.5 / 500 86 31.5 22.5 148.5
18 / 1200 375 139 97 653
~ Upper (UL) and lower (LL) limits of the 95% confidence intervals (CI). 95%
CI were calculated as fhe mean t Z
SD.
Z Values for 6.0 MIU are estimated based on actual values for 4.5 MIU and 7.5
MILD.
Similar to Proleukin~ IL-2, L2-7001, a liquid formulation of monomeric IL-2,
was administered to HIV patients at doses ranging from 50 to 180 ~,g
(unpublished
data). The exposures obtained from this study as measured by AUC are shown in
Table 4. These exposure values were within the range of the exposure values
generated using Proleukin~ IL-2 (Table 3).
Table 4: Average (~ SD) AUCo_24 obtained after an initial exposure to a single
dose
administration of the monomeric IL-2 formulation L2-7001.
L2-7001 AUCo_24 SD
Dose (IU*hr/ml)
(MIU/p
g)
0.75/50 65 12
1.3 5/90 120 3 9
2.0/135 156 45
2.7/180 300 108
The IL-2 exposure data (AUC) was obtained from the published literature
where recombinant human native IL-2 was administered SC to 8 cancer patients
at
doses ranging from 0.1 MU to 3.0 MCT. The reported average (%CV) AUCs for the
0.3, 1, and 3 MU dose levels were 120 (38), 177 (36), and 359 (46) U*hr/ml
(Gustavson (1998) J. Biol. Respov~se Modifiers 1998:440-449). As indicated in
Thompson et al. 1987 Cancer Research 47:4202-4207, the units measured in this
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study were normalized to BRMP units (Rossio et al. (1986) Lyn2phokine Research
5
(suppl 1):513-S18), which was adopted later as international units (IU) by WHO
(Gearing and Thorpe (1988) J. Inununological Methods 114:3-9). The AUC values
generated under the study conditions also agree well with the established
Proleukin~
IL-2 exposure.
All publications and patent applications mentioned in the specification are
indicative of the level of those skilled in the art to which this invention
pertains. All
publications and patent applications are herein incorporated by reference to
the same
extent as if each individual publication or patent application was
specifically and
individually indicated to be incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more
than routine experimentation, many equivalents to the specific embodiments of
the
invention described herein. Such equivalents are intended to be encompassed by
the
following claims.
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