U.S. patent application number 11/085014 was filed with the patent office on 2005-08-18 for combined use of anti-cytokine antibodies or antagonists and anti-cd20 for treatment of b cell lymphoma.
This patent application is currently assigned to Biogen Idec Inc.. Invention is credited to Hanna, Nabil.
Application Number | 20050180975 11/085014 |
Document ID | / |
Family ID | 22713760 |
Filed Date | 2005-08-18 |
United States Patent
Application |
20050180975 |
Kind Code |
A1 |
Hanna, Nabil |
August 18, 2005 |
Combined use of anti-cytokine antibodies or antagonists and
anti-CD20 for treatment of B cell lymphoma
Abstract
The present invention discloses combined therapies for treating
hematologic malignancies, including B cell lymphomas and leukemias
or solid non-hematologic tumors, comprising administration of
anti-cytokine antibodies or antagonists to inhibit the activity of
cytokines which play a role in perpetuating the activation of B
cells. The administration of such antibodies and antagonists,
particularly anti-IL10 antibodies and antagonists, is particularly
useful for avoiding or decreasing the resistance of hematologic
malignant cells or solid tumor cells to chemotherapeutic agents and
anti-CD20 or anti-CD22 antibodies. The invention also provides
combination therapies for solid tumors having B cell involvement
comprising the administration of an anti-cytokine antibody and a B
cell depleting antibody such as RITUXAN.RTM..
Inventors: |
Hanna, Nabil; (San Diego,
CA) |
Correspondence
Address: |
PILLSBURY WINTHROP SHAW PITTMAN, LLP
P.O. BOX 10500
MCLEAN
VA
22102
US
|
Assignee: |
Biogen Idec Inc.
San Diego
CA
|
Family ID: |
22713760 |
Appl. No.: |
11/085014 |
Filed: |
March 21, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11085014 |
Mar 21, 2005 |
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09822672 |
Apr 2, 2001 |
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6896885 |
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60193467 |
Mar 31, 2000 |
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Current U.S.
Class: |
424/145.1 ;
514/34; 514/49; 514/559 |
Current CPC
Class: |
A61P 35/02 20180101;
A61K 38/00 20130101; A61P 35/00 20180101; A61K 39/39541 20130101;
A61K 39/3955 20130101; A61K 2039/505 20130101; C07K 16/24 20130101;
C07K 16/244 20130101; A61K 39/39541 20130101; A61K 2300/00
20130101; A61K 39/3955 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/145.1 ;
514/049; 514/559; 514/034 |
International
Class: |
A61K 039/395; A61K
031/7072; A61K 031/203; A61K 031/704 |
Claims
1. A method of avoiding, decreasing or overcoming the resistance of
hematologic malignant cells or solid non-hematologic tumor cells to
at least one chemotherapeutic agent, comprising administering an
anti-cytokine antibody or fragment thereof or cytokine antagonist
to a patient diagnosed with a hematologic malignancy or a solid,
non-hematologic tumor prior, concurrent or after administration of
at least one chemotherapeutic agent.
2-86. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims benefit of priority from U.S.
Provisional Ser. No. 60/193,467, filed Mar. 31, 2000, and
incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The present invention concerns methods for treating
hematologic malignancies including B cell lymphomas and leukemias
with anti-cytokine agents such as antibodies and antagonists, where
the targeted cytokines play a potentiating role in the disease
process by stimulating hematologic malignant cells including B
lymphoma and leukemia cells. Treatment with anti-cytokine agents in
combination with other known therapies such as chemotherapy and
administration of therapeutic antibodies has been found to provide
a synergistic effect.
[0003] The invention also embraces the treatment of solid
non-hematologic (non-lymphoid) tumors, e.g., colorectal or liver
cancer, which tumors are characterized by B cell involvement, by
the administration of a cytokine antibody or cytokine antagonist,
in combination with treatment with an antibody to a B cell target,
e.g. CD20.
BACKGROUND OF THE INVENTION
[0004] The immune system of vertebrates (for example, primates,
which include humans, apes, monkeys, etc.) consists of a number of
organs and cell types which have evolved to: accurately and
specifically recognize foreign microorganisms ("antigen") which
invade the vertebrate-host; specifically bind to such foreign
microorganisms; and, eliminate/destroy such foreign microorganisms.
Lymphocytes, as well as other types of cells, are critical to the
immune system and to the elimination and destruction of foreign
microorganisms. Lymphocytes are produced in the thymus, spleen and
bone marrow (adult) and represent about 30% of the total white
blood cells present in the circulatory system of humans (adult).
There are two major sub-populations of lymphocytes: T cells and B
cells. T cells are responsible for cell mediated immunity, while B
cells are responsible for antibody production (humoral immunity).
However, T cells and B cells can be considered interdependent--in a
typical immune response, T cells are activated when the T cell
receptor binds to fragments of an antigen that are bound to major
histocompatability complex ("MHC") glycoproteins on the surface of
an antigen presenting cell; such activation causes release of
biological mediators ("interleukins" or "cytokines") which, in
essence, stimulate B cells to differentiate and produce antibody
("immunoglobulins") against the antigen.
[0005] Each B cell within the host expresses a different antibody
on its surface--thus one B cell will express antibody specific for
one antigen, while another B cell will express antibody specific
for a different antigen. Accordingly, B cells are quite diverse,
and this diversity is critical to the immune system. In humans,
each B cell can produce an enormous number of antibody molecules
(i.e., about 10.sup.7 to 10.sup.8). Such antibody production most
typically ceases (or substantially decreases) when the foreign
antigen has been neutralized. Occasionally, however, proliferation
of a particular B cell will continue unabated; such proliferation
can result in a cancer referred to as "B cell lymphoma."
[0006] Non-Hodgkin's lymphoma is one type of lymphoma that is
characterized by the malignant growth of B lymphocytes. According
to the American Cancer Society, an estimated 54,000 new cases will
be diagnosed, 65% of which will be classified as intermediate- or
high-grade lymphoma. Patients diagnosed with intermediate-grade
lymphoma have an average survival rate of two to five years, and
patients diagnosed with high-grade lymphoma survive an average of
six months to two years after diagnosis.
[0007] Conventional therapies have included chemotherapy and
radiation, possibly accompanied by either autologous or allogeneic
bone marrow or stem cell transplantation if a suitable donor is
available, and if the bone marrow contains too many tumor cells
upon harvesting. While patients often respond to conventional
therapies, they usually relapse within several months.
[0008] A relatively new approach to treating non-Hodgkin's lymphoma
has been to treat patients with a monoclonal antibody directed to a
protein on the surface of cancerous B cells. The antibody may be
conjugated to a toxin or radiolabel thereby affecting cell death
after binding. Alternatively, an antibody may be engineered with
human constant regions such that human antibody effector mechanisms
are generated upon antibody binding which result in apoptosis or
death of the cell.
[0009] Rituximab.RTM. (IDEC Pharmaceuticals Corporation) is one of
a new generation of monoclonal antibodies developed for the
treatment of B cell lymphomas, and in particular, non-Hodgkin's
lymphoma. Rituximab.RTM. is a genetically engineered anti-CD20
monoclonal antibody with murine light-and heavy-chain variable
regions and human gamma I heavy-chain and kappa light-chain
constant regions. Rituximab.RTM. is more effective than its murine
parent in fixing complement and mediating ADCC, and it mediates CDC
in the presence of human complement. The antibody inhibits cell
growth in the B-cell lines FL-18, Ramos, and Raji, sensitizes
chemoresistant human lymphoma cell lines to diphtheria toxin,
ricin, CDDP, doxorubicin, and etoposide, and induces apoptosis in
the DHL-4 human B-cell lymphoma line in a dose-dependent
manner.
[0010] However, many patients are refractory to or relapse
following Rituximab.RTM. therapy, as well as chemotherapy.
Therefor, there still remains a need for lymphoma treatments which
may be combined with Rituximab.RTM. therapy or chemotherapy in
order to increase the chance of remission and decrease the rate of
relapse in lymphoma patients.
[0011] Many groups have suggested using cytokines for the treatment
of various types of cancers. For instance, Wang et al. suggested
that cytokines are "directly cytotoxic to tumor cells" and showed
that interleukin-1 alpha (IL1.alpha.) potentiated the anti-tumor
effect of anti-tumor drugs against several human tumor cells in
vitro (Int. J. Cancer (Nov. 27, 1996) 68(5): 583-587). Bonvida et
al. disclose that cytokines have the potential to "enhance the
efficacy of chemotherapeutic agents" and show that recombinant
tumor necrosis factor and the chemotherapeutic agent cisplatin show
a synergistic effect against ovarian cancer cells (Gynecol. Oncol.
(September 1990) 38(3): 333-339). U.S. Pat. No. 5,716,612 teaches
that IL-4 may be used to potentiate the effect of chemotherapeutic
agents in the treatment of cancer.
[0012] However, some groups have also recognized that cytokines may
play a detrimental role in the development of some cancers. For
instance, interleukin-6 (IL6) has been known for the ability in
some instances to inhibit apoptosis of leukemic cells. (See
Yonish-Rouach et al. Wild type p53 induces apoptosis of myeloid
leukemic cells and is inhibited by interleukin-6. Nature 352:
345-347 (1991)). Recently it was shown that IL6 may play a role in
the resistance of some leukemic cells to anti-cancer
chemotherapeutic agents, and that, in vitro, anti-IL6 antibody
increases the sensitivity of cisplatin-resistance K562 cells to
cisplatin-induced apoptosis. (See Dedoussis et al. Endogenous
interleukin 6 conveys resistance to
cis-diamminedichloroplatinum-mediated apoptosis of the K562 human
leukemic cell line).
[0013] A potentiating effect on B cells has also been postulated
for EL10, the production of which has been reported to be
upregulated in some cell lines derived from B cell lymphomas (See
Cortes et al. Interleukin-10 in non-Hodgkin's lymphoma. Leuk
Lymphoma 26(3-4): 251-259 (July, 1997). However, when the serum of
NHL patients was tested for correlation between IL10 levels and
prognosis, more significance was placed on the levels of viral
IL10, which is produced from a homologous open reading frame BCFR1,
located in the genome of the Epstein Barr Virus (EBV). In fact,
another group reported at about the same time that IL10 was an
autocrine growth factor for EBV-infected lymphoma cells. (See
Beatty et al. Involvement of IL10 in the autonomous growth of
EBV-transformed B cell lines. J. Immunol. 158(9): 4045-51 (May 1,
1997)). Alternatively, others have hypothesized that IL6 and IL10
production by macrophages plays a key role in the occurrence of
lymphocytic diseases. (See U.S. Pat. No. 5,639,600).
[0014] It has also been reported that IL10 may work in combination
with IL6, IL2 and TNF-alpha to increase proliferation of
non-Hodgkin's lymphoma cells. (See Voorzanger et al.
Interleukin-(IL) 10 and IL6 are produced in vivo by non-Hodgkin's
lymphoma cells and act as cooperative growth factors. Cancer Res.
56(23): 5499-505 (Dec. 1, 1996). Also statistically significantly
higher levels of IL2, IL6, IL8, IL10, soluble IL2 receptor, soluble
transferrin receptor and neopterin were observed in NHL patients as
compared to a control group, although no single parameter was found
to be of prognostic significance. (See Stasi et al. Clinical
implications of cytokine and soluble receptor measurements in
patients with newly diagnosed non-Hodgkin's lymphoma. Eur. J.
Haemotol. 54(1): 9-17 (January, 1995).
[0015] However, there have been just as many reports in the
literature which have suggested that cytokines such as IL10 show no
correlation to disease progression, and that such cytokines may
actually be helpful in combating lymphoma rather than contributing
to the disease. For instance, when Bonnefoix et al. tested the
potential of ten cytokines (IL2, IL3, IL4, IL6, L10, IL13, G-CSF,
GM-CSF, interferon alpha and interferon gamma) to modulate the
spontaneous proliferative response of B-non-Hodgkin's lymphoma
cells of various histological subtypes, this group found that each
cytokine could be either inhibitory or stimulatory depending on the
sample, and that there was no relationship with different
histological subtypes. In fact, U.S. Pat. No. 5,770,190, herein
incorporated by reference, suggests administration of IL10 in
conjunction with chemotherapeutic agents as a treatment for acute
leukemia.
[0016] It would be a benefit to lymphoma patients if therapeutic
regimens incorporating anti-cytokine antibodies could be devised
whereby such antibodies could be used to increase the sensitivity
of B lymphoma cells to other types of therapeutic drugs. It would
be particularly helpful if anti-cytokine antibodies could be
administered for the purpose of avoiding or overcoming the
resistance of B lymphoma cells in lymphoma patients to
chemotherapeutic agents, and for the purpose of potentiating the
apoptotic activity of therapeutic antibodies. Such combined
treatment regimens would add to the therapies available to lymphoma
patients and potentially decrease the rate of relapse in these
patients.
OBJECTS OF THE INVENTION
[0017] It is an object of the invention to provide a method of
avoiding, decreasing or overcoming the resistance of hematologic
malignant cells or solid non-hematologic tumor cells to at least
one chemotherapeutic agent, comprising administering an
anti-cytokine antibody or cytokine antagonist to a patient
diagnosed with a hematologic malignancy prior, concurrent or after
administration of at least one chemotherapeutic agent.
[0018] It is a more specific object of the invention to provide a
method of avoiding, decreasing or overcoming the resistance of
hematologic malignant cells to apoptosis induced by a therapeutic
agent, comprising administering an anti-cytokine antibody or
cytokine antagonist to a patient diagnosed with a hematologic
malignancy.
[0019] It is another object of the invention to provide a method of
treating a patient with a hematologic malignancy who has relapsed
following chemotherapy, comprising administering an anti-cytokine
antibody or cytokine antagonist to said patient.
[0020] It is another object of the invention to provide a method of
treating a patient having a hematologic malignancy who is
refractory to chemotherapy, comprising administering an
anti-cytokine antibody or cytokine antagonist to said patient.
[0021] It is yet another object of the invention to provide a
method of treating a patient with a hematologic malignancy who has
relapsed following therapy with a therapeutic antibody, comprising
administering an anti-cytokine antibody or cytokine antagonist to
said patient.
[0022] It is still another object of the invention to provide a
method of treating a patient with a hematologic malignancy who is
refractory to therapy with a therapeutic antibody, comprising
administering an anti-cytokine antibody or cytokine antagonist to
said patient.
[0023] It is another object of the invention to provide a method of
treating a B cell lymphoma patient comprising administering to said
patient a therapeutically effective amount of an anti-CD20 antibody
simultaneously with or consecutively with in either order an
anti-cytokine antibody.
[0024] It is another object of the invention to provide a method of
treating a solid non-hematologic (non-lymphoid) tumor wherein B
cells elicit a pro-tumor response by the administration of an
anti-cytokine antibody, e.g. an anti-IL10 antibody and at least one
B cell depleting antibody, e.g. an anti-CD20 antibody.
[0025] It is a more specific object of the invention to provide a
method of treating solid, non-lymphoid tumor involving the
digestive system, especially colorectal cancer or liver cancer by
the administration of an anti-cytokine antibody, preferably an
anti-IL10 antibody and a B cell depleting antibody, particularly a
depleting anti-CD20 antibody.
SUMMARY OF THE INVENTION
[0026] In a first aspect, the present invention relates to the
administration of anti-cytokine antibodies and cytokine
antagonists, particularly antibodies to IL10, in combination with
chemotherapy drugs and/or therapeutic antibodies to increase the
response rate and response duration in patients with hematological
malignancies such as B cell lymphomas and leukemias or solid
non-hematologic tumors, such as breast cancer, ovarian cancer,
testicular cancer and others. Thus, the present invention relates
to methods of treating hematologic malignancies such as B cell
lymphomas and leukemias by administering to a patient having a
hematologic malignancy such as B cell lymphoma or a leukemia,
antibodies directed to B cell receptors and antibodies or
antagonists which interfere with the action of certain cytokines.
In particular, the present invention relates to administration of
antibodies to B cell markers which initiate apoptosis of B lymphoma
cells, such as anti-CD20, anti-CD22, anti-CD40, anti-CD23,
anti-CD19, anti-CD37 and others identified infra, and antibodies to
or antagonists of cytokines which may interfere with apoptosis,
e.g., anti-IL10. Combined therapeutic regimens including other
treatments which would also benefit from anti-cytokine therapy,
i.e., chemotherapy, are also encompassed. The methods will find use
in particular for treating patients having hematological
malignancies such as B lymphomas or leukemias characterized by
cells that have become resistant to chemotherapeutic agents and
therapeutic antibodies.
[0027] In a second aspect, the present invention provides novel
methods of treating solid non-hematologic (non-lymphoid) tumors
having B cell involvement (but not of B cell origin), particularly
cancers wherein B cells elicit a pro-tumor response by the
administration of an antibody to a cytokine, e.g., IL10, in
conjunction with B cell specific antibody therapy, particularly a B
cell depleting antibody, and preferably CD20 antibody therapy,
optionally in combination with radiotherapy or chemotherapy.
Examples of such solid tumors include colorectal cancer, liver
cancer, breast cancer, lung cancer, prostate cancer, stomach
cancer, head and neck cancer, ovarian cancer, testicular cancer,
esophageal cancer and others. Suitable chemotherapies are discussed
infra. These cancers may comprise precancers, Stage I and II
cancers, and advanced cancers, e.g. past Stage II and including
solid tumors that have metastasized.
DETAILED DESCRIPTION OF THE INVENTION
[0028] In a first aspect, the present invention includes methods of
avoiding, decreasing or overcoming the resistance of hematologic
malignant cells including, e.g., B lymphoma and leukemia cells to
at least one chemotherapeutic agent, comprising administering an
anti-cytokine antibody or cytokine antagonist to a patient
diagnosed with B cell lymphoma.
[0029] Often, such resistance by a hematologic malignancy patient's
B cells is mediated by stimulation of the tumorigenic B cells by
one or more cytokines such that the cells fail to respond to
apoptotic signals. In such cases, the methods of the present
invention may be described as methods of avoiding, decreasing or
overcoming the resistance of such tumorigenic B cells to apoptosis,
with chemotherapeutic agents being examples of agents which may
induce apoptosis. Also encompassed are therapeutic antibodies
directed to targets on the surface of B cells, such as anti-CD19,
anti-CD20, anti-CD22, anti-CD40, and anti-CD28 and other B cell
targets identified infra.
[0030] Because resistance of B cells is often only apparent after a
patient has relapsed following, or is refractory to, a first
treatment with a therapeutic agent, the methods of the present
invention will often encompass treating patients with hematologic
malignancies such as B cell lymphoma or leukemia who have relapsed
following, or are refractory to, chemotherapy or therapy with a
therapeutic antibody. However, the anti-cytokine antibodies and
antagonists of the present invention may also be used in
conjunction with other therapies or prior to other therapies in
patients newly diagnosed with lymphoma to decrease the chance of
relapse, and increase the length and duration of the response to
therapy.
[0031] The methods of the present invention are appropriate to
treat a wide variety of hematologic malignancies, especially B cell
lymphomas and leukemias, including but not limited to low
grade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic
(SL) NHL, intermediate grade/follicular NHL, intermediate grade
diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic
NHL, high grade small non-cleaved cell NHL, bulky disease NHL and
Waldenstrom's Macroglobulinemia, chronic leukocytic leukemia, acute
myelogenous leukemia, acute lymphoblastic leukemia, chronic
lymphocytic leukemia, chronic myelogenous leukemia, lymphoblastic
leukemia, lymphocytic leukemia, monocytic leukemia, myelogenous
leukemia, and promyelocytic leukemia. It should be clear to those
of skill in the art that these lymphomas will often have different
names due to changing systems of classification, and that patients
having lymphomas and leukemias classified under different names may
also benefit from the combined therapeutic regimens of the present
invention.
[0032] For instance, a recent classification system proposed by
European and American pathologists is called the Revised European
American Lymphoma (REAL) Classification. This classification system
recognizes Mantle cell lymphoma and Marginal cell lyrnphoma among
other peripheral B-cell neoplasms, and separates some
classifications into grades based on cytology, i.e., small cell,
mixed small and large, large cell. It will be understood that all
such classified lymphomas may benefit from the combined therapies
of the present invention.
[0033] The U.S. National Cancer Institute (NCI) has in turn divided
some of the REAL classes into more clinically useful "indolent" or
"aggressive" lymphoma designations. Indolent lymphomas include
follicular cell lymphomas, separated into cytology "grades,"
diffuse small lymphocytic lymphoma/chronic lymphocytic leukemia
(CLL), lymphoplasmacytoid/Waldenstrom's Macroglobulinemia, Marginal
zone lymphoma and Hairy cell leukemia. Aggressive lymphomas include
diffuse mixed and large cell lymphoma, Burkitt's lymphomal diffuse
small non-cleaved cell lymphoma, Lymphoblastic lymphoma, Mantle
cell lymphoma and AIDS-related lymphoma. All that is required is
that the extent or duration of response to therapy be extended as a
result of administration of said anti-cytokine antibody or
antagonist. But the methods are most preferably used to treat
patients having non-Hodgkin's lymphoma (NHL), where the present
inventors have surprisingly found that administration of
anti-cytokine antibodies and antagonists has a synergistic effect.
Since the effect of cytokines and the identity of detrimental
cytokines may vary among different patients and different types of
lymphomas, and the effect of various cytokines on the resistance of
B lymphoma cells may vary with different chemotherapeutic and
immunotherapeutic agents, it is suggested that the levels of the
respective cytokines in individual patients be tested before the
patients are administered the anti-cytokine therapy.
[0034] In a second aspect, the invention provides a method of
treating solid, non-hematologic tumors wherein B cells elicit a
protein response (promote tumor growth and/or metastasis)
comprising the administration of a anti-cytokine antibody, e.g. an
anti-IL10 antibody, and an antibody to a B cell target, preferably
an anti-CD20 antibody having B cell depleting activity. However,
the invention includes the usage of antibodies to other B cell
targets identified infra. Also, this aspect further includes the
additional use of chemotherapy and/or radiotherapy.
[0035] A variety of chemotherapeutic agents have been applied to
the treatment of different types of cancers, and the methods of the
present invention will avoid, decrease or overcome the resistance
of malignant, e.g. lymnphoma, cells to at least one, but possibly
several, of these chemotherapeutic agents. In particular,
chemotherapies which may benefit by supplemental anti-cytokine
therapy include but are not limited to CHOP, ICE, Mitozantrone,
Cytarabine, DVP, ATRA, Idarubicin, hoelzer chemotherapy regime, La
La chemotherapy regime, ABVt), CEOP, 2-CdA, FLAG & IDA with or
without subsequent G-CSF treatment), VAD, M & P, C-Weekly,
ABCM, MOPP, DHAP, methotrexate, doxoribicin, daunorubicin,
tamoxifen, toremifene, and cisplatin. Other chemotherapeutic agents
are identified infra in the section relating to preferred
embodiments.
[0036] There are likely to be a variety of cytokines which play a
detrimental, stimulatory role in hematologic or non-hematologic
malignancies including leukemic and lymphoma diseases, either alone
or in cooperation with other cytokines. Thus, depending on the
patient and the disease, more than one anti-cytokine antibody or
antagonist may benefit a particular patient as a supplemental
therapy. Those cytokines include but are not limited to IL2, IL6,
IL10 and TNF-alpha. Other appropriate cytokines are identified
infra in the preferred embodiments. For non-Hodgkin's lymphoma, the
preferred anti-cytokine treatment will comprise anti-IL10
therapy.
[0037] There are several anti-IL10 antibodies which are known in
the art and may be used for the purposes of the present invention.
U.S. Pat. No. 5,871,725 describes a rat anti-human antibody
designated 19F1. Another anti-IL10 antibody, alpha-IL10, is
described in U.S. Pat. No. 5,837,293. Anti-IL10 antibodies are also
described in Tim R. Mosmann, et al., "Isolation of Monoclonal
Antibodies Specific For IL-4, IL-5, IL-6, and a New Th2-Specific
Cytokine (IL-10), Cytokine Synthesis Inhibitory Factor, By Using A
Solid Phase Radioimmunoadsorbent Assay," The Journal of Immunology,
145(9):2938-2945, Nov. 1, 1990. Antagonists may take the form of
proteins which compete for receptor binding, e.g., which lack the
ability to activate the receptor while blocking IL-10 binding, or
IL-10 binding molecules, such as antibodies. The term antibody
should be understood as encompassing antibody fragments as well as
whole antibodies, i.e., Fab, Fab.sub.2 and Fv fragments. Antibodies
may be isolated by immunizing another animal with human IL-10, but
then may be humanized using method known in the art to decrease
their immunogenicity once they are administered to a human
patient.
[0038] The appropriate dosage of anti-cytokine antibody will depend
on the cytokine targeted, the results of preliminary serum profiles
in individual patients, the type of lymphoma being treated and the
stage of disease. For anti-IL10 antibodies in the treatment of
newly diagnosed low-grade non-Hodgkin's lymphoma, the preferred
dosage may range from 0.001 mg to 100 mg/kg, preferably from about
0.1 to 100 mg/kg, and most typically about 0.4 to 20 mg/kg body
weight, depending on whether the antibody is administered
concurrently with or prior to another therapeutic agent.
Preferably, the anti-cytokine antibody is administered concurrent
or prior to a chemotherapeutic agent or other therapy, typically
from about one hour prior, to about one month prior, preferably
within one to seven days prior to administration of
chemotherapeutic or other agent.
[0039] Also included in the present invention are kits for
accomplishing the disclosed methods. A kit according to the present
invention comprises at least one anti-cytokine antibody or
antagonist which may be readily admixed or resuspended with a
pharmaceutically acceptable carrier and conveniently injected into
a lymphoma patient. In cases where the serum of a lymphoma patient
is preferably tested for cytokine profiles prior to administration
of said anti-cytokine antibody or antagonist, the kit may also or
alternatively comprise reagents and materials for testing the
relative amounts of various cytokines in the patient's serum.
[0040] Also encompassed in the present invention are combined
therapeutic methods of treating hematologic malignancies such as B
cell lymphoma and leukemias comprising administering to a patient
with a hematologic malignancy a therapeutically effective amount of
a therapeutic antibody simultaneously with or consecutively with in
either order an anti-cytokine antibody. Therapeutic antibodies are
defined as those which bind to receptors on the surface of
hematologic malignant cells, e.g., tumorigenic B cells, and mediate
their destruction or depletion when they bind, i.e., anti-CD20,
anti-CD19, anti-CD22, anti-CD21, anti-CD23, anti-CD37, and other B
cell targets identified infra. While the anti-cytokine agents of
the present invention will have some beneficial effect alone in
that they block cytokine-mediated proliferation of tumorigenic B
cells, the combined administration of the therapeutic antibodies
with the anti-cytokine agents will have a synergistic effect in
that the duration and/or extent of response will be better than the
additive effect of both types of therapies applied
independently.
[0041] While not wishing to be held to the following theory, the
present inventors believe that the synergistic effects seen by
co-administering the anti-cytokine agents of the present invention
are related to the inhibition of the targeted cytokine which may
usually have the effect to inhibit apoptosis. Accordingly, when the
anti-cytokine agents of the present invention are combined with an
agent which acts by inducing apoptosis, e.g., anti-CD20, anti-CD22,
anti-CD19, anti-CD21, anti-CD23, or anti-CD40 antibodies, the
combined administration shows a synergetic effect well beyond the
additive effect of either agent alone.
[0042] Never-the-less, this does not preclude the use of the
anti-cytokine antibodies and antagonists of the present invention
in combined therapies with other antibodies or therapeutic agents
whose efficacy is not facilitated via apoptosis. For instance,
radiolabeled antibodies facilitate the destruction of tumor cells
by binding to the B cell surface and delivering a lethal dose of
radiation. Such antibodies, as well as antibodies conjugated to
toxins, may also be used in conjunction with the anti-cytokine
agents of the present invention. Preferred radiolabeled antibodies
are those labeled with yttrium-[90] (.sup.90Y). A particularly
preferred radiolabeled antibody is Zevelin (IDEC Pharmaceuticals
Corporation), which is an anti-CD20 antibody conjugated to
.sup.90Y.
[0043] The combined therapeutic methods of the present invention
may further comprise administration of at least one
chemotherapeutic agent or regimen, where such chemotherapy
includes, by way of example, CHOP, ICE, Mitozantrone, Cytarabine,
DVP, ATRA, Idarubicin, hoelzer chemotherapy regime, La La
chemotherapy regime, ABVD, CEOP, 2-CdA, FLAG & IDA with or
without subsequent G-CSF treatment), VAD, M & P, C-Weekly,
ABCM, MOPP, DHAP, doxorubicin, cisplatin, daunorubicin, tamoxifen,
toremifene, and methotrexate as well as the additional
chemotherapeutic agents identified infra. A preferred
chemotherapeutic regimen for the treatment of non-Hodgkin's
lymphoma patients is CHOP. The anti-cytokine antibody or antagonist
is preferably administered prior to the B cell target antibody,
e.g., anti-CD20, CD22, CD19 or CD40, and/or chemotherapy, such that
proliferation of B lymphoma cells as a result of the targeted
cytokine is quelled prior to administration of the B cell
therapeutic. As described above, the target cytokine may be IL2,
IL6, IL10 or TNF-alpha among others, depending on the patient's
cytokine profile prior to treatment, but preferably the targeted
cytokine is IL10.
[0044] As mentioned above, therapeutic antibodies of the present
invention may be any antibody which targets a molecule expressed on
the surface of B cells, particularly one having B cell depleting
activity. A listing of suitable B cell targets is identified
infra.
[0045] Depending on the patient and extent of disease, the anti-B
cell target binding antibody, e.g., Rituximab.RTM. may be
administered at a dosage ranging from 0.01 to about 100 mg/kg, more
preferably from about 0.1 to 50 mg/kg, and most preferably from
about 0.4 to 20 mg/kg of body weight. Effective dosages may be
lower in combined therapeutic regimens which include anti-cytokine
agents, because the proliferative potential of B lymphoma cells
will be reduced. Again, effective doses will depend on the chosen
anti-cytokine therapy, and the relative levels of potentiating
cytokine in the patient's serum.
[0046] The combined therapies of the present invention are also
suitable for treating a wide range of lymphomas, including but not
limited to low grade/follicular non-Hodgkin's lymphoma (NHL), small
lymphocytic (SL) NHL, intermediate grade/follicular NHL,
intermediate grade diffuse NHL, high grade immunoblastic NHL, high
grade lymphoblastic NHL, high grade small non-cleaved cell NHL,
bulky disease NHL and Waldenstrom's Macroglobulinemia, chronic
leukocytic leukemia, acute myelogenous leukemia, acute
lymphoblastic leukemia, chronic lymphocytic leukemia, chronic
myelogenous leukemia, lymphoblastic leukemia, lymphocytic leukemia,
monocytic leukemia, myelogenous leukemia, and promyelocytic
leukemia. Preferred targeted diseases are non-Hodgkin's lymphoma
(NHL), and particularly low-grade, follicular NHL. Again, it may be
helpful for the serum of the lymphoma patient to be tested for
cytokine profiles prior to administration of the anti-cytokine
antibody or antagonist.
[0047] As already discussed, the combination therapies provided
herein, particularly the combined usage of an anti-cytokine
antibody, e.g. anti-IL10 and an anti-B cell target antibody, e.g.
anti-CD20, are also useful for treating solid, non-hematologic
(non-lymphoid) cancers, including by way of example, colorectal
cancer, liver cancer, and other digestive cancers, breast cancer,
esophageal cancer, head and neck cancer, lung cancer, ovarian
cancer, prostate cancer and testicular cancer. These cancers my be
in early, intermediate or advanced stages, e.g. metastasis.
[0048] The present invention also encompasses kits for
administering the therapeutic antibody and the anti-cytokine
antibody or antagonist according to the disclosed methods. Kits may
comprise more than one type of therapeutic antibody and more than
one anti-cytokine agent. Kits may also comprise reagents and
materials for testing cytokine profile prior to administration of
the therapeutic antibody and anti-cytokine antibody or
antagonist.
[0049] As noted, the invention further embraces the treatment of
solid, non-lymphoid tumors by the administration of an
anti-cytokine antibody, e.g., an anti-IL10 antibody, and a B cell
specific antibody, preferably an antibody having substantial B cell
depleting activity such as RITUXAN.RTM.. It ahs been reported that
some solid tumors apparently have B cell involvement. That is to
say that the B cells are somehow involved in promoting or
maintaining the tumorigenic state and may impede the body's immune
defense system against such tumor. With respect thereto, WO 020864
A1, incorporated by reference herein, which identifies Biocrystal
Inc. as the Applicant describes the treatment of solid,
non-lymphoid tumor using antibodies that target B cells, including
Rituxan.RTM.. It was reported therein that this treatment resulted
in pronounced anti-tumor responses, even in patients with advanced
colorectal cancer, lung cancer and liver cancer.
[0050] By contrast, the present invention provides an improved
combination therapy, wherein solid, non-lymphoid tumors are treated
by use of an anti-cytokine antibody, such as anti-IL10 and a B cell
depleting antibody, such as an anti-CD20 antibody.
[0051] This combination regimen should afford an enhanced method of
treating solid tumors, particularly those wherein B cells are
involved, but are not themselves the cancerous cells. In this
regimen, the cytokine antagonist, e.g., anti-cytokine antibody and
the B cell depleting antibody, e.g., Rituxan.RTM. will be
administered separately or together and in either order.
[0052] Additionally, this regimen may include the use of
radiotherapy, e.g., external beam irradiation, total body
irradiation, radioimmunotherapy or chemotherapy. Suitable
chemotherapies are identified infra. The radioimmunotherapy may
comprise treatment with a radiolabeled antibody that binds a target
expressed by the solid tumor.
[0053] Typically, the anti-cytokine antibody will be administered
prior to the B cell depleting antibody. It is anticipated that this
combination therapy will be suitable for treating any solid tumor
having B cell involvement. Suitable examples of solid tumors have
been identified previously. One noteworthy example is colorectal
cancer.
[0054] In this embodiment, the B cell depleting antibody and
cytokine will be administered such that it s delivered to the solid
tumor site. Preferably, the antibodies will be injected proximate
or directly at the tumor site, e.g., by intravenous injection at a
vein proximate to the tumor.
[0055] This combination regimen cell results in remission or
shrinkage of the solid tumor, e.g., a lung or colorectal tumor.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0056] In order to further describe the preferred embodiments and
full scope of the invention, the following definitions are
provided.
[0057] I. Definitions
[0058] "Cytokine antagonist" is a compound that inhibits or blocks
the expression and/or activity of a cytokine, e.g. an interleukin
or interferon or another cytokine.
[0059] A "B cell surface marker" or "B cell target" or "B cell
antigen" herein is an antigen expressed on the surface of a B cell
which can be targeted with an antagonist which binds thereto.
Exemplary B cell surface markers include the CD10, CD19, CD20,
CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD73, CD74, CDw75, CDw76,
CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85 and
CD86 leukocyte surface markers: The B cell surface marker of
particular interest is preferentially expressed on B cells compared
to other non-B cell tissues of a mammal and may be expressed on
both precursor B cells and mature B cells. In one embodiment, the
marker is one, like CD20 or CD19, which is found on B cells
throughout differentiation of the lineage from the stem cell stage
up to a point just prior to terminal differentiation into plasma
cells. The preferred B cell surface markers herein are CD19 and
CD20.
[0060] The "CD20" antigen is a -35 kDa, non-glycosylated
phosphoprotein found on the surface of greater than 90% of B cells
from peripheral blood or lymphoid organs. CD20 is expressed during
early pre-B cell development and remains until plasma cell
differentiation. CD20 is present on both normal B cells as well as
malignant B cells. Other names for CD20 in the literature include
"B-lymphocyte-restricted antigen" and "Bp35". The CD20 antigen is
described in Clark et al. PNAS (USA) 82:1766 (1985), for example.
The "CD19" antigen refers to a -90 kDa antigen identified, for
example, by the HD237-CD19 or 134 antibody (Kiesel et al. Leukemia
Research 11, 12: 1119 (1987)). Like CD20, CD19 is found on cells
throughout differentiation of the lineage from the stem cell stage
up to a point just prior to terminal differentiation into plasma
cells. Binding of an antagonist to CD 19 may cause internalization
of the CD 19 antigen.
[0061] A "hematologic malignancy" includes any malignancy
associated with cells in the bloodstream. Examples thereof include
B and T cell lymphomas, leukemias including but not limited to low
grade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic
(SL) NHL, intermediate grade/follicular NHL, intermediate grade
diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic
NHL, high grade small non-cleaved cell NHL, bulky disease NHL and
Waldenstrom's Macroglobulinemia, chronic leukocytic leukemia, acute
myelogenous leukemia, acute lymphoblastic leukemia, chronic
lymphocytic leukemia, chronic myelogenous leukemia, lymphoblastic
leukemia, lymphocytic leukemia, monocytic leukemia, myelogenous
leukemia, and promyelocytic leukemia. It should be clear to those
of skill in the art that these lymphomas will often have different
names due to changing systems of classification (as previously
discussed), and that patients having lymphomas and leukemias
classified under different names may also benefit from the combined
therapeutic regimens of the present invention.
[0062] A solid, non-hematologic (non-lymphoid) tumor refers to a
non-hematologic malignancy having B cell involvement, i.e., where B
cells are involved in a "protumor" response. Such solid tumors are
characterized by palpable tumors, typically at least 0.5 mm in
diameter, more typically at least 1.0 mm in diameter. Examples
thereof include colorectal cancer, liver cancer, breast cancer,
lung cancer, head and neck cancer, stomach cancer, testicular
cancer, prostate cancer, ovarian cancer, uterine cancer and others.
These cancers may be in the early stages (precancer), intermediate
(Stages I and II) or advanced, including solid tumors that have
metastasized. These solid tumors will preferably be cancers wherein
B cells elicit a protumor response, i.e. the presence of B cells is
involved in tumor development, maintenance or metastasis.
[0063] A B cell "antagonist" is a molecule which, upon binding to a
B cell surface marker, destroys or depletes B cells in a mammal
and/or interferes with one or more B cell functions, e.g. by
reducing or preventing a humoral response elicited by the B cell.
The antagonist preferably is able to deplete B cells (i.e. reduce
circulating B cell levels) in a mammal treated therewith. Such
depletion may be achieved via various mechanisms such
antibody-dependent cell mediated cytotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC), inhibition of B cell
proliferation and/or induction of B cell death (e.g. via
apoptosis). Antagonists included within the scope of the present
invention include antibodies, synthetic or native sequence peptides
and small molecule antagonists which bind to the B cell marker,
optionally conjugated with or fused to a cytotoxic agent. The
preferred antagonist comprises an antibody, more preferably a B
cell depleting antibody.
[0064] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC"
refer to a cell-mediated reaction in which nonspecific cytotoxic
cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK)
cells, neutrophils, and macrophages) recognize bound antibody on a
target cell and subsequently cause lysis of the target cell. The
primary cells for mediating ADCC, NK cells, express FcyRIII only,
whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression
on hematopoietic cells in summarized is Table 3 on page 464 of
Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess
ADCC activity of a molecule of interest, an in vitro ADCC assay,
such as that described in U.S. Pat. Nos. 5,500,362 or 5,821,337 may
be performed. Useful effector cells for such assays include
peripheral blood mononuclear cells (PBMC) and Natural Killer (NK)
cells. Alternatively, or additionally, ADCC activity of the
molecule of interest may be assessed in vivo, e.g., in a animal
model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656
(1998).
[0065] "Human effector cells" are leukocytes which express one or
more FcRs and perform effector functions. Preferably, the cells
express at least FcyRIII and carry out ADCC effector function.
Examples of human leukocytes which mediate ADCC include peripheral
blood mononuclear cells (PBMC), natural killer (NK) cells,
monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK
cells being preferred.
[0066] The terms "Fc receptor" or "FCR"are used to describe a
receptor that binds to the Fc region of an antibody.
[0067] The preferred FcR is a native sequence human FcR. Moreover,
a preferred FcR is one which binds an IgG antibody (a gamma
receptor) and includes receptors of the FcyRI, FcyRII, and Fcy RIII
subclasses, including allelic variants and alternatively spliced
forms of these receptors. FcyRII receptors include FcyRIIA (an
"activating receptor") and FcyRIIB (an "inhibiting receptor"),
which have similar amino acid sequences that differ primarily in
the cytoplasmic domains thereof. Activating receptor FcyRIIA
contains an immunoreceptor tyrosine-based activation motif (ITAM)
in its cytoplasmic domain. Inhibiting receptor FcyRIIB contains an
immunoreceptor tyrosine-based inhibition motif (ITIM) in its
cytoplasmic domain. (see Daeron, Annu. Rev. Immunol. 15:203-234
(1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol
9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de
Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs,
including those to be identified in the future, are encompassed by
the term "FCR" herein. The term also includes the neonatal
receptor, FcRn, which is responsible for the transfer of maternal
IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim
et al., J. Immunol. 24:249 (1994)).
[0068] "Complement dependent cytotoxicity" or "CDC" refer to the
ability of a molecule to lyse a target in the presence of
complement. The complement activation pathway is initiated by the
binding of the first component of the complement system (Clq) to a
molecule (e.g. an antibody) complexed with a cognate antigen. To
assess complement activation, a CDC assay, e.g. as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), maybe
performed.
[0069] "Growth inhibitory" antagonists are those which prevent or
reduce proliferation of a cell expressing an antigen to which the
antagonist binds. For example, the antagonist may prevent or reduce
proliferation of B cells in vitro and/or in vivo.
[0070] Antagonists which "induce apoptosis" are those which induce
programmed cell death, e.g. of a B cell, as determined by standard
apoptosis assays, such as binding of annexin V, fragmentation of
DNA, cell shrinkage, dilation of endoplasmic reticulum, cell
fragmentation, and/or formation of membrane vesicles (called
apoptotic bodies).
[0071] The term "antibody" herein is used in the broadest sense and
specifically covers intact monoclonal antibodies, polyclonal
antibodies, multispecific antibodies (e.g. bispecific antibodies)
formed from at least two intact antibodies, and antibody fragments
so long as they exhibit the desired biological activity.
[0072] "Antibody fragments" comprise a portion of an intact
antibody, preferably comprising the antigen-binding or variable
region thereof. Examples of antibody fragments include Fab, Fab',
F(ab')2, and Fv fragments; diabodies; linear antibodies;
single-chain antibody molecules; and multispecific antibodies
formed from antibody fragments.
[0073] "Native antibodies" are usually heterotetrameric
glycoproteins of about 150,000 daltons, composed of two identical
light (L) chains and two identical heavy (H) chains. Each light
chain is linked to a heavy chain by one covalent disulfide bond,
while the number of disulfide linkages varies among the heavy
chains of different immunoglobulin isotypes. Each heavy and light
chain also has regularly spaced intrachain disulfide bridges. Each
heavy chain has at one end a variable domain (VH) followed by a
number of constant domains. Each light chain has a variable domain
at one end (VL) and a constant domain at its other end; the
constant domain of the light chain is aligned with the first
constant domain of the heavy chain, and the light-chain variable
domain is aligned with the variable domain of the heavy chain.
Particular amino acid residues are believed to form an interface
between the light chain and heavy chain variable domains.
[0074] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
hypervariable regions both in the light chain and the heavy chain
variable domains. The more highly conserved portions of variable
domains are called the framework regions (FRs). The variable
domains of native heavy and light chains each comprise four FRs,
largely adopting a P-sheet configuration, connected by three
hypervariable regions, which form loops connecting, and in some
cases forming part of, the (3 sheet structure. The hypervariable
regions in each chain are held together in close proximity by the
FRs and, with the hypervariable regions from the other chain,
contribute to the formation of the antigen-binding site of
antibodies (see Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)). The constant domains
are not involved directly in binding an antibody to an antigen, but
exhibit various effector functions, such as participation of the
antibody in antibody dependent cellular cytotoxicity (ADCC).
[0075] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fob" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab'2 fragment that has two antigen-binding sites and is
still capable of crosslinking antigen.
[0076] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and antigen-binding site. This region
consists of a dimer of one heavy chain and one light chain variable
domain in tight, non-covalent association. It is in this
configuration that the three hypervariable regions of each variable
domain interact to define an antigen-binding site on the surface of
the VH-VL dimer. Collectively, the six hypervariable regions confer
antigen binding specificity to the antibody. However, even a single
variable domain (or half of an Fv comprising only three
hypervariable regions specific for an antigen) has the ability to
recognize and bind antigen, although at a lower affinity than the
entire binding site.
[0077] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CHI) of the heavy chain.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CHI domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear at least one free thiol
group. F(ab')Z antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0078] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa (x) and lambda (k), based on the amino acid
sequences of their constant domains.
[0079] Depending on the amino acid sequence of the constant domain
of their heavy chains, antibodies can be assigned to different
classes. There are five major classes of intact antibodies: IgA,
IgD, IgE, IgG, and IgM, and several of these may be further divided
into subclasses (isotypes), e.g., IgG 1, IgG2, IgG3, IgG4, IgA, and
IgA2. The heavy-chain constant domains that correspond to the
different classes of antibodies are called a, 8, s, y, and R,
respectively. The subunit structures and three-dimensional
configurations of different classes of immunoglobulins are well
known.
[0080] "Single-chain Fv" or "scFv" antibody fragments comprise the
VH and VL domains of antibody, wherein these domains are present in
a single polypeptide chain. Preferably, the Fv polypeptide further
comprises a polypeptide linker between the VH and VL domains which
enables the scFv to form the desired structure for antigen binding.
For a review of scFv see Pluckthun in The Pharmacology of
Monoclonal Antibodies, vol 113, Rosenburg and Moore, eds.,
Springer-Verlag, New York, pp. 269-315 (1994).
[0081] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VL) in the same polypeptide chain (VH-VL). By using a linker that
is too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen binding sites.
Diabodies are described more fully in, for example, EP 404,097; WO
93/11161; and Hollinger et al., Proc. Nad. Acad. Sci. USA,
90:6444.-6448 (1993).
[0082] The term "monoclonal antibody" as used herein refers to 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. Furthermore, in contrast to conventional
(polyclonal) antibody preparations which typically include
different antibodies directed against different determinants
(epitopes), each monoclonal antibody is directed against a single
determinant on the antigen. In addition to their specificity, the
monoclonal antibodies are advantageous in that they are synthesized
by the hybridoma culture, uncontaminated by other immunoglobulins.
The modifier "monoclonal" indicates the 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.,
Nature, 256:495 (1975), or may be made by recombinant DNA methods
(see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies"
may also be isolated from phage antibody libraries using the
techniques described in Clackson et al., Nature, 352:624-628 (1991)
and Marks et al., J. Mol. Biol., 222:581-597 (1991), for
example.
[0083] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity (U.S. Pat. No. 4,816,567; Morrison et
al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric
antibodies of interest herein include "primatized" antibodies
comprising variable domain antigen binding sequences derived from a
non-human primate (e.g. Old World Monkey, such as baboon, rhesus or
cynomolgus monkey) and human constant region sequences (U.S. Pat.
No. 5,693,780).
[0084] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (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. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. 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. 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 loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
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., Nature
321:522-525 (1986); Riechlnann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
[0085] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region comprises amino acid
residues from a "complementarity determining region" or "CDR" (e.g.
residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain
variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the
heavy chain variable domain; Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)) and/or those residues
from a "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2)
and 91-96 (L3) in the light chain variable domain and 26-32 (H1),
53-55 (H2) and 96- 101 (H3) in the heavy chain variable domain;
Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). "Framework" or
"FR" residues are those variable domain residues other than the
hypervariable region residues as herein defined. An antagonist
"which binds" an antigen of interest, e.g. a B cell surface marker,
is one capable of binding that antigen with sufficient affinity
and/or avidity such that the antagonist is useful as a therapeutic
agent for targeting a cell expressing the antigen.
[0086] Examples of antibodies which bind the CD20 antigen include:
"C2B8" which is now called "rituximab" ("RITUXAN.RTM.") (U.S. Pat.
No. 5,736,137, expressly incorporated herein by reference); the
yttrium-[90]-labeled 2138 murine antibody designated "Y2B8" (U.S.
Pat. No. 5,736,137, expressly incorporated herein by reference);
murine IgG2a "131" optionally labeled with 1311 to generate the
"131I-B1" antibody (BEXXAR.TM.) (U.S. Pat. No. 5,595,721, expressly
incorporated herein by reference); murine monoclonal antibody "1F5"
(Press et al. Blood 69(2):584-591 (1987)); "chimeric 2H7" antibody
(U.S. Pat. No. 5,677,180, expressly incorporated herein by
reference); and monoclonal antibodies L27, G28-2, 93-1133, B-C1 or
NU-B2 available from the International Leukocyte Typing Workshop
(Valentine et al., In: Leuikocyte Typing III (McMichael, Ed., p.
440, Oxford University Press (1987)). Examples of antibodies which
bind the CD 19 antigen include the anti-CD 19 antibodies in Hekman
et al., Cancer Immunol. Immunother. 32:364-372 (1991) and Vlasveld
et al. Cancer Immunol. Immunother. 40:37-47(1995); and the B4
antibody in Kiesel et al. Leukemia Research 11, 12: 1119
(1987).
[0087] The terms "rituximab" or "RITUXAN.RTM." herein refer to the
genetically engineered chimeric murine/human monoclonal antibody
directed against the CD20 antigen and designated "C2B8" in U.S.
Pat. No. 5,736,137, expressly incorporated herein by reference. The
antibody is an IgG, kappa immunoglobulin containing murine light
and heavy chain variable region sequences and human constant region
sequences. Rituximab has a binding affinity for the CD20 antigen of
approximately 8.O nM.
[0088] An "isolated" antagonist is one which has been identified
and separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the antagonist, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the antagonist will be purified (1) to greater than
95% by weight of antagonist as determined by the Lowry method, and
most preferably more than 99% by weight, (2) to a degree sufficient
to obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated antagonist
includes the antagonist in situ within recombinant cells since at
least one component of the antagonist's natural environment will
not be present. Ordinarily, however, isolated antagonist will be
prepared by at least one purification step. "Mammal" for purposes
of treatment refers to any animal classified as a mammal, including
humans, domestic and farm animals, and zoo, sports, or pet animals,
such as dogs, horses, cats, cows, etc. Preferably, the mammal is
human.
[0089] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment
include those already with the disease or disorder as well as those
in which the disease or disorder is to be prevented. Hence, the
mammal may have been diagnosed as having the disease or disorder or
may be predisposed or susceptible to the disease.
[0090] The expression "therapeutically effective amount" refers to
an amount of the antagonist which is effective for preventing,
ameliorating or treating the autoimmune disease in question. The
term "immunosuppressive agent" as used herein for adjunct therapy
refers to substances that act to suppress or mask the immune system
of the mammal being treated herein. This would include substances
that suppress cytokine production, downregulate or suppress
self-antigen expression, or mask the MHC antigens.
[0091] Examples of such agents include 2-amino-6-aryl-5-substituted
pyrimidines (see U.S. Pat. No. 4,665,077, the disclosure of which
is incorporated herein by reference); azathioprine;
cyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde
(which masks the MHC antigens, as described in U.S. Pat. No.
4,120,649); anti-idiotypic antibodies for MHC antigens and MHC
fragments; cyclosporin A; steroids such as glucocorticosteroids,
e.g., prednisone, methylprednisolone, and dexamethasone; cytokine
or cytokine receptor antagonists including anti-interferon-y, -(3,
or-a antibodies, anti-tumomecrosis factor-a antibodies,
anti-tumornecrosis factor-(i antibodies, anti-interleukin-2
antibodies and anti-IL-2 receptor antibodies; anti-LFA-1
antibodies, including anti-CD 11a and anti-CD18 antibodies;
anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-T
antibodies, preferably antiCD3 or anti-CD4/CD4a antibodies; soluble
peptide containing a LFA-3 binding domain (WO 90/08187 published
Jul. 26, 1990); streptokinase; TGF-0; streptodornase; RNA or DNA
from the host; FK506; RS-61443; deoxyspergualin; rapamycin; T-cell
receptor (Cohen et al., U.S. Pat. No. 5,114,721); T-cell receptor
fragments (Offner et al., Science 251: 430-432 (1991); WO 90/11294;
laneway, Nature, 341: 482 (1989); and WO 91/01133); and T cell
receptor antibodies (EP 340,109) such as TLOB9.
[0092] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g. I.sup.131, Y.sup.90, Ar.sup.211,
P.sup.32, Re.sup.188, Re.sup.186, Sm.sup.153, B.sup.212 and
others), chemotherapeutic agents, and toxins such as small molecule
toxins or enzymatically active toxins of bacterial, fungal, plant
or animal origin, or fragments thereof.
[0093] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and cyclosphosphamide
(CYTOXAN.TM.); alkyl sulfonates such as busulfan, improsulfan and
piposulfan; aziridines such as benzodopa, carboquone, meturedopa,
and uredopa; ethylenimines and methylamelamines including
altretamine, triethylenemelamine, trietylenephosphoramide,
triethylenethiophosphaoramide and trimethylolomelamine; nitrogen
mustards such as chlorambucil, chlomaphazine, cholophosphamide,
estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide
hydrochloride, melphalan, novembiehin, phenesterine, prednimustine,
trofosfamide, uracil mustard; nitrosureas such as carmustine,
chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;
antibiotics such as aclacinomysins, actinomycin, authramycin,
azaserine, bleomycins, cactinomycin, calicheamicin, carabicin,
carminomycin, carzinophilin, chromoinycins, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin,
epirubicin, esorubicin, idambicin, marcellomycin, mitomycins,
mycophenolic acid, nogalamycin, olivomycins, peplomycin,
potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogues such as denopterin, methotrexate, pteropterin,
trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,
azacitidine, 6-azauridine, carrnofur, cytarabine, dideoxyuridine,
doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine; diaziquone; elfornithine; elliptinium acetate;
etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;
mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin;
phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide;
procarbazine; PSK.RTM.; razoxane; sizofrran; spirogermanium;
tenuazonic acid; triaziquone; 2,2',2'-trichlorotriethylamine;
urethan; vindesine; dacarbazine; mannomustine; mitobronitol;
mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");
cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOLO,
Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel
(TAXOTEW, Rh6ne-Poulenc Rorer, Antony, France); chlorambucil;
gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum
analogs such as cisplatin and carboplatin; vinblastine; platinum;
etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;
vincristine; vinorelbine; navelbine; novantrone; teniposide;
daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase
inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid;
esperamicins; capecitabine; and pharmaceutically acceptable salts,
acids or derivatives of any of the above. Also included in this
definition are anti-hormonal agents that act to regulate or inhibit
hormone action on tumors such as anti-estrogens including for
example tamoxifen, raloxifene, aromatase inhibiting
4(5)-imidazoles, 4 hydroxytamoxifen, trioxifene, keoxifene,
LY117018, onapristone, and toremifene (Fareston); and
anti-androgens such as flutamide, nilutamide, bicalutamide,
leuprolide, and goserelin; and pharmaceutically acceptable salts,
acids or derivatives of any of the above.
[0094] The term "cytokine" is a generic term for proteins released
by one cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormone such as human growth hormone, N-methionyl human
growth hormone, and bovine growth hormone; parathyroid hormone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein
hormones such as follicle stimulating hormone (FSH), thyroid
stimulating hormone (TSH), and luteinizing hormone (LH); hepatic
growth factor; fibroblast growth factor; prolactin; placental
lactogen; tumor necrosis factor-a and -0; mullerian-inhibiting
substance; mouse gonadotropin-associated peptide; inhibin; activin;
vascular endothelial growth factor; integrin; thrombopoietin (TPO);
nerve growth factors such as NGF-P; platelet growth factor;
transforming growth factors (TGFs) such as TGF-a and TGF-0;
insulin-like growth factor-I and -II; erythropoietin (EPO);
osteoinductive factors; interferons such as interferon-a, -P, and
-y; colony stimulating factors (CSFs) such as macrophage-CSF
(M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF
(GCSF); interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; a tumor necrosis
factor such as TNF-a or TNF-P; and other polypeptide factors
including LIF and kit ligand (KL). As used herein, the term
cytokine includes proteins from natural sources or from recombinant
cell culture and biologically active equivalents of the native
sequence cytokines.
[0095] The term "prodrug" as used in this application refers to a
precursor or derivative form of a pharmaceutically active substance
that is less cytotoxic to tumor cells compared to the parent drug
and is capable of being enzymatically activated or converted into
the more active parent form. See, e.g., Wihnan, "Prodrugs in Cancer
Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382,
615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A
Chemical Approach to Targeted Drug Delivery," Directed Drug
Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press
(1985). The prodrugs of this invention include, but are not limited
to, phosphate-containing prodrugs, thiophosphate-containing
prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,
D-amino acid-modified prodrugs, glycosylated prodrugs,
(3-lactam-containing prodrugs, optionally substituted
phenoxyacetamide-containing prodrugs or optionally substituted
phenylacetamide-containing prodrugs, 5 fluorocytosine and other
5-fluorouridine prodrugs which can be converted into the more
active cytotoxic free drug. Examples of cytotoxic drugs that can be
derivatized into a prodrug form for use in this invention include,
but are not limited to, those chemotherapeutic agents described
above.
[0096] A "liposome" is a small vesicle composed of various types of
lipids, phospholipids and/or surfactant which is useful for
delivery of a drug (such as the antagonists disclosed herein and,
optionally, a chemotherapeutic agent) to a mammal. The components
of the liposome are commonly arranged in a bilayer formation,
similar to the lipid arrangement of biological membranes. The term
"package insert" is used to refer to instructions customarily
included in commercial packages of therapeutic products, that
contain information about the indications, usage, dosage,
administration, contraindications and/or warnings concerning the
use of such therapeutic products.
[0097] II. Production of Antagonists
[0098] The methods and articles of manufacture of the present
invention use, or incorporate, an antagonist which binds to a B
cell surface marker and/or a cytokine. Accordingly, methods for
generating such antagonists will be described here. The B cell
surface marker or cytokine to be used for production of, or
screening for, antagonist(s) may be, e.g., a soluble form of the
antigen or a portion thereof, containing the desired epitope.
Alternatively, or additionally, cells expressing the B cell surface
marker at their cell surface can be used to generate, or screen
for, antagonist(s). Other forms of the B cell surface marker useful
for generating antagonists will be apparent to those skilled in the
art. Preferably, the B cell surface marker is the CD19 or CD20
antigen. Preferably, the cytokine is IL-10.
[0099] While the preferred antagonist is an antibody, antagonists
other than antibodies are contemplated herein. For example, the
antagonist may comprise a small molecule antagonist optionally
fused to, or conjugated with, a cytotoxic agent (such as those
described herein). Libraries of small molecules may be screened
against the B cell surface marker of interest herein in order to
identify a small molecule which binds to that antigen. The small
molecule may further be screened for its antagonistic properties
and/or conjugated with a cytotoxic agent.
[0100] The antagonist may also be a peptide generated by rational
design or by phage display (see, e.g., W098/35036 published 13 Aug.
1998). In one embodiment, the molecule of choice may be a "CDR
mimic" or antibody analogue designed based on the CDRs of an
antibody. While such peptides may be antagonistic by themselves,
the peptide may optionally be fused to a cytotoxic agent so as to
add or enhance antagonistic properties of the peptide.
[0101] A description follows as to exemplary techniques for the
production of the antibody antagonists used in accordance with the
present invention.
[0102] Polyclotial Antibodies
[0103] Polyclonal antibodies are preferably raised in animals by
multiple subcutaneous (sc) or intraperitoneal (ip) injections of
the relevant antigen and an adjuvant. It may be useful to conjugate
the relevant antigen to a protein that is immunogenic in the
species to be immunized, e.g., keyhole limpet hemocyanin, serum
albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a
bifunctional or derivatizing agent, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOC12, or R1N.dbd.C.dbd.NR, where R and RI are
different alkyl groups. Animals are immunized against the antigen,
immunogenic conjugates, or derivatives by combining, e.g., 100 pg
or 5 wg of the protein or conjugate (for rabbits or mice,
respectively) with 3 volumes of Freund's complete adjuvant and
injecting the solution intradermally at multiple sites. One month
later the animals are boosted with 1/5 to {fraction (1/10)} the
original amount of peptide or conjugate in Freund's complete
adjuvant by subcutaneous injection at multiple sites. Seven to 14
days later the animals are bled and the serum is assayed for
antibody titer. Animals are boosted until the titer plateaus.
Preferably, the animal is boosted with the conjugate of the same
antigen, but conjugated to a different protein and/or through a
different cross-linking reagent. Conjugates also can be made in
recombinant cell culture as protein fusions. Also, aggregating
agents such as alum are suitably used to enhance the immune
response.
[0104] (ii) Monoclonal Antibodies
[0105] Monoclonal antibodies are obtained from a population of
substantially homogeneous antibodies, Le., the individual
antibodies comprising the population are identical except for
possible naturally occurring mutations that may be present in minor
amounts. Thus, the modifier "monoclonal" indicates the character of
the antibody as not being a mixture of discrete antibodies. For
example, the monoclonal antibodies may be made using the hybridoma
method first described by Kohler et al., Nature, 256:495 (1975), or
may be made by recombinant DNA methods (U.S. Pat. No.
4,816,567).
[0106] In the hybridoma method, a mouse or other appropriate host
animal, such as a hamster, is immunized as hereinabove described to
elicit lymphocytes that produce or are capable of producing
antibodies that will specifically bind to the protein used for
immunization. Alternatively, lymphocytes may be immunized in vitro.
Lymphocytes then are fused with myeloma cells using a suitable
fusing agent, such as polyethylene glycol, to form a hybridoma cell
[Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103
(Academic Press, 1986)].
[0107] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
[0108] Preferred myeloma cells are those that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these, preferred myeloma cell lines are murine
myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse
tumors available from the Salk Institute Cell Distribution Center,
San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from
the American Type Culture Collection, Rockville, Md. USA. Human
myeloma and mouse human heteromyeloma cell lines also have been
described for the production of human monoclonal antibodies
[Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal
Antibody Production Techniques and Applications, pp. 51-63 (Marcel
Dekker, Inc., New York, 1987)].
[0109] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
(ELISA). The binding affinity of the monoclonal antibody can, for
example, be determined by the Scatchard analysis of Munson et al.,
Anal. Biochem., 107:220 (1980).
[0110] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media
for this purpose include, for example, D-MEM or RPMI-1640 medium.
In addition, the hybridoma cells may be grown in vivo as ascites
tumors in an animal.
[0111] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0112] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of murine antibodies).
The hybridoma cells serve as a preferred source of such DNA. Once
isolated, the DNA may be placed into expression vectors, which are
then transfected into host cells such as E. coli cells, simian COS
cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do
not otherwise produce immunoglobulin protein, to obtain the
synthesis of monoclonal antibodies in the recombinant host cells.
Review articles on recombinant expression in bacteria of DNA
encoding the antibody include Skerra et al., Curr. Opinion in
Immunol., 5:256-262 (1993) and Phickthun, Immunol. Revs.,
130:151-188 (1992).
[0113] In a further embodiment, antibodies or antibody fragments
can be isolated from antibody phage libraries generated using the
techniques described in McCafferty et al., Nature, 348:552-554
(1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et
al., J. Mol. Biol., 222:581-597 (1991) 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.,
BiolTechnology, 10:779-783 (1992)), as well as combinatorial
infection and in vivo recombination as a strategy for constructing
very large phage libraries (Waterhouse et al., Nuc. Acids. Res.,
21:2265-2266 (1993)). Thus, these techniques are viable
alternatives to traditional monoclonal antibody hybridoma
techniques for isolation of monoclonal antibodies.
[0114] The DNA also may be modified, for example, by substituting
the coding sequence for human heavy- and light-chain constant
domains in place of the homologous murine sequences (U.S. Pat. No.
4,816,567; Morrison, et al, Proc. Natl Acad. Sci. USA, 81:6851
(1984)), or by covalently joining to the immunoglobulin coding
sequence all or part of the coding sequence for a
non-immunoglobulin polypeptide. Typically such non-immunoglobulin
polypeptides are substituted for the constant domains of an
antibody, or they are substituted for the variable domains of one
antigen-combining site of an antibody to create a chimeric bivalent
antibody comprising one antigen-combining site having specificity
for an antigen and another antigen combining site having
specificity for a different antigen.
[0115] (iii) Humanized Antibodies
[0116] Methods for humanizing non-human antibodies have been
described in the art. Preferably, a humanized antibody has one or
more amino acid residues introduced into it from a source which is
non-human. These non-human amino acid residues are often referred
to as "import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al, Nature, 332:323-327
(1988); Verhoeyen et aL, Science, 239:1534-1536 (1988)), by
substituting hypervariable region sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)
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 hypervariable region residues and possibly
some FR residues are substituted by residues from analogous sites
in rodent antibodies.
[0117] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework region (FR) for the
humanized antibody (Sims et al, J. Immunol, 151:2296 (1993);
Chothia et al., J. Mol. Biol, 196:901 (1987)). Another method uses
a particular framework region derived from the consensus sequence
of all human antibodies of a particular subgroup of light or heavy
chains. The same framework may be used for several different
humanized antibodies (Carter et aL, Proc. Nad. Acad. Sci. USA,
89:4285 (1992); Presta et al., J. Immunol, 151:2623 (1993)).
[0118] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, according to a
preferred method, humanized antibodies are prepared by a process of
analysis of the parental sequences and various conceptual humanized
products using three dimensional models of the parental and
humanized sequences. Three-dimensional immunoglobulin models are
commonly available and are familiar to those skilled in the art.
Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
hypervariable region residues are directly and most substantially
involved in influencing antigen binding.
[0119] (iv) Human Antibodies
[0120] As an alternative to humanization, human antibodies can be
generated. For example, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (JH) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array in such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al.,
Proc. Mad. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al.,
Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno.,
7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369 and 5,545,807.
Alternatively, phage display technology (McCafferty et al., Nature
348:552-553 (1990)) can be used to produce human antibodies and
antibody fragments in vitro, from immunoglobulin variable (V)
domain gene repertoires from unimmunized donors. According to this
technique, antibody V domain genes are cloned in-frame into either
a major or minor coat protein gene of a filamentous bacteriophage,
such as M13 or fd, and displayed as functional antibody fragments
on the surface of the phage particle. Because the filamentous
particle contains a single-stranded DNA copy of the phage genome,
selections based on the functional properties of the antibody also
result in selection of the gene encoding the antibody exhibiting
those properties. Thus, the phage mimics some of the properties of
the B cell. Phage display can be performed in a variety of formats;
for their review see, e.g. Johnson, Kevin S. and Chiswell, David
J., Current Opinion in Structural Biology 3:564-571(1993). Several
sources of V-gene segments can be used for phage display. Clackson
et al., Nature, 352: 624-628 (1991) isolated a diverse array of
anti-oxazolone antibodies from a small random combinatorial library
of V genes derived from the spleens of immunized mice. A repertoire
of V genes from unimmunized human donors can be constructed and
antibodies to a diverse array of antigens (including self-antigens)
can be isolated essentially following the techniques described by
Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al.,
EMBO J. 12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and
5,573,905. Human antibodies may also be generated by in vitro
activated B cells (see U.S. Pat. No. 5,567,610 and 5,229,275).
[0121] (v) Antibody Fragments
[0122] 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., Journal of Biochemical and Biophysical Methods 24:107-117
(1992) and Brennan et al., Science, 229:81 (1985)). 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'-Sli
fragments can be directly recovered from E. coli and chemically
coupled to form F(ab')2 fragments [Carter et aL, Bio/Technology
10:163-167 (1992)]. 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. In other embodiments,
the antibody of choice is a single chain Fv fragment (scFv). See WO
93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458. The
antibody fragment may also be a "linear antibody", e.g., as
described in U.S. Pat. No. 5,641,870 for example. Such linear
antibody fragments may be monospecific or bispecific.
[0123] (vi) Bispecific Antibodies
[0124] Bispecific antibodies are antibodies that have binding
specificities for at least two different epitopes. Exemplary
bispecific antibodies may bind to two different epitopes of the B
cell surface marker. Other such antibodies may bind a first B cell
marker and further bind a second B cell surface marker.
Alternatively, an anti-B cell marker binding arm may be combined
with an arm which binds to a triggering molecule on a leukocyte
such as a T-cell receptor molecule (e.g. CD2 or CD3), or Fc
receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and
FcyRIII (CD 16) so as to focus cellular defense mechanisms to the B
cell. Bispecific antibodies may also be used to localize cytotoxic
agents to the B cell. These antibodies possess a B cell
marker-binding arm and an arm which binds the cytotoxic agent (e.g.
saporin, anti-interferon-a, vinca alkaloid, ricin A chain,
methotrexate or radioactive isotope hapten). Bispecific antibodies
can be prepared as full length antibodies or antibody fragments
(e.g. F(ab')Z bispecific antibodies).
[0125] Methods for making bispecific antibodies are known in the
art. Traditional production of full length bispecific antibodies is
based on the coexpression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(Millstein et al., Nature, 305:537-539 (1983)). Because of the
random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different
antibody molecules, of which only one has the correct bispecific
structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and
the product yields are low. Similar procedures are disclosed in WO
93/08829, and in Traunecker et al., EMBO J, 10:3655-3659
(1991).
[0126] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences. The
fusion preferably is with an immunoglobulin heavy chain constant
domain, comprising at least part of the hinge, CH2, and CH3
regions. It is preferred to have the first heavy-chain constant
region (CHI) containing the site necessary for light chain binding,
present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host organism. This
provides for great flexibility in adjusting the mutual proportions
of the three polypeptide fragments in embodiments when unequal
ratios of the three polypeptide chains used in the construction
provide the optimum yields. It is, however, possible to insert the
coding sequences for two or all three polypeptide chains in one
expression vector when the expression of at least two polypeptide
chains in equal ratios results in high yields or when the ratios
are of no particular significance.
[0127] In a preferred embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain light chain pair (providing a second binding
specificity) in the other arm. It was found that this asymmetric
structure facilitates the separation of the desired bispecific
compound from unwanted immunoglobulin chain combinations, as the
presence of an immunoglobulin light chain in only one half of the
bispecific molecule provides for a facile way of separation. This
approach is disclosed in WO 94/04690. For further details of
generating bispecific antibodies see, for example, Suresh et al.,
Methods in Enzymology, 121:210 (1986).
[0128] According to another approach described in U.S. Pat. No.
5,731,168, the interface between a pair of antibody molecules can
be engineered to maximize the percentage of heterodimers which are
recovered from recombinant cell culture. The preferred interface
comprises at least a part of the CH3 domain of an antibody constant
domain. In this method, one or more small amino acid side chains
from the interface of the first antibody molecule are replaced with
larger side chains (e.g. tyrosine or tryptophan). Compensatory
"cavities" of identical or similar size to the large side chain(s)
are created on the interface of the second antibody molecule by
replacing large amino acid side chains with smaller ones (e.g.
alanine or threonine). This provides a mechanism for increasing the
yield of the heterodimer over other unwanted end-products such as
homodimers.
[0129] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360, WO 92/200373, and EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0130] Techniques for generating bispecific antibodies from
antibody fragments have also been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science, 229:81 (1985) describe a
procedure wherein intact antibodies are proteolytically cleaved to
generate F(ab')2 fragments. These fragments are reduced in the
presence of the dithiol complexing agent sodium arsenite to
stabilize vicinal dithiols and prevent intermolecular disulfide
formation. The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0131] Recent progress has facilitated the direct recovery of
Fab'-SH fragments from E. coli, which can be chemically coupled to
form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:
217-225 (1992) describe the production of a fully humanized
bispecific antibody F(ab')2 molecule. Each Fab' fragment was
separately secreted from E. coli and subjected to directed chemical
coupling in vitro to form the bispecific antibody. The bispecific
antibody thus formed was able to bind to cells overexpressing the
ErbB2 receptor and normal human T cells, as well as trigger the
lytic activity of human cytotoxic lymphocytes against human breast
tumor targets.
[0132] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (VH) connected to a light-chain
variable domain (VL) by a linker which is too short to allow
pairing between the two domains on the same chain.
[0133] Accordingly, the VH arid VL domains of one fragment are
forced to pair with the complementary VL and VH domains of another
fragment, thereby forming two antigen-binding sites. Another
strategy for making bispecific antibody fragments by the use of
single-chain Fv (sFv) dimers has also been reported. See Gruber et
al., J. Immunol.,152:5368 (1994). Antibodies with more than two
valencies are contemplated. For example, trispecific antibodies can
be prepared. Tutt et al. J. Immunol. 147: 60 (1991).
[0134] III. Conjugates and Other Modifications of the
Antagonist
[0135] The antagonists used in the methods or included in the
articles of manufacture herein are optionally conjugated to a
cytotoxic agent. Chemotherapeutic agents useful in the generation
of such antagonist-cytotoxic agent conjugates have been described
above.
[0136] Conjugates of an antagonist and one or more small molecule
toxins, such as a calicheamicin, a maytansine (U.S. Pat. No.
5,208,020), a trichothene, and CC 1065 are also contemplated
herein. In one embodiment of the invention, the antagonist is
conjugated to one or more maytansine molecules (e.g. about 1 to
about 10 maytansinemolecules per antagonist molecule). Maytansine
may, for example, be converted to May-SS-Me which may be reduced to
May-SH3 and reacted with modified antagonist (Chari et al. Cancer
Research 52: 127-131 (1992)) to generate a maytansinoid-antagonist
conjugate.
[0137] Alternatively, the antagonist is conjugated to one or more
calicheamicin molecules. The calicheamicin family of antibiotics
are capable of producing double-stranded DNA breaks at
sub-picomolar concentrations. Structural analogues of calicheamicin
which may be used include, but are not limited to, `yJ1, a21, a31,
N-acetyl-yl`, PSAG and 011 (Hinman et al. Cancer Research 53:
3336-3342 (1993) and Lode et al. Cancer Research 58: 2925-2928
(1998)).
[0138] Enzymatically active toxins and fragments thereofwhich can
be used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
4leuritesfordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin and the
tricothecenes. See, for example, WO 93/21232 published Oct. 28,
1993.
[0139] The present invention further contemplates antagonist
conjugated with a compound with nucleolytic activity (e.g. a
ribonuclease or a DNA endonuclease such as a deoxyribonuclease;
DNase). A variety of radioactive isotopes are available for the
production of radioconjugated antagonists. Examples include
Ate',113',1125, Y9o Re 186, Re 188, Sm153, Bi212 P32 and
radioactive isotopes of Lu. Conjugates of the antagonist and
cytotoxic agent may be made using a variety of bifunctional protein
coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)
propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)
cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional
derivatives of imidoesters (such as dimethyl adipimidate HCL),
active esters (such as disuccinimidyl suberate), aidehydes (such as
glutareldehyde), bis azido compounds (such as bis (p-azidobenzoyl)
hexanediamine), bis-diazonium derivatives (such as
bis-(pdiazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin can be prepared as described in Vitetta et al. Science
238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antagonist. See W094/11026. The linker may
be a "cleavable linker" facilitating release of the cytotoxic drug
in the cell. For example, an acid-labile linker,
peptidase-sensitive linker, dimethyl linker or disulfide-containing
linker (Chari et aL Cancer Research 52: 127-131 (1992)) may be
used. Alternatively, a fusion protein comprising the antagonist and
cytotoxic agent may be made, e.g. by recombinant techniques or
peptide synthesis.
[0140] In yet another embodiment, the antagonist may be conjugated
to a "receptor" (such streptavidin) for utilization in tumor
pretargeting wherein the antagonist-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g. avidin) which is conjugated to a
cytotoxic agent (e.g. a radionucleotide). The antagonists of the
present invention may also be conjugated with a prodrug-activating
enzyme which converts a prodrug (e.g. a peptidyl chemotherapeutic
agent, see W081/01145) to an active anti-cancer drug. See, for
example, WO 88/07378 and U.S. Pat. No. 4,975,278.
[0141] The enzyme component of such conjugates includes any enzyme
capable of acting on a prodrug in such a way so as to covert it
into its more active, cytotoxic form. Enzymes that are useful in
the method of this invention include, but are not limited to,
alkaline phosphatase useful for converting phosphate-containing
prodrugs into free drugs; arylsulfatase useful for converting
sulfate containing prodrugs into free drugs; cytosine deaminase
useful for converting non-toxic 5-fluorocytosine into the
anti-cancer drug, 5-fluorouracil; proteases, such as serratia
protease, thermolysin, subtilisin, carboxypeptidases and cathepsins
(such as cathepsins B and L), that are useful for converting
peptide-containing prodrugs into free drugs;
D-alanylcarboxypeptidases, useful for converting prodrugs that
contain D-amino acid substituents; carbohydrate cleaving enzymes
such as li-galactosidase and neuraminidase useful for converting
glycosylated prodrugs into free drugs; (3-lactamase useful for
converting drugs derivatized with (3-lactams into free drugs; and
penicillin amidases, such as penicillin V amidase or penicillin G
amidase, useful for converting drugs derivatized at their amine
nitrogens with phenoxyacetyl or phenylacetyl groups, respectively,
into free drugs. Alternatively, antibodies with enzymatic activity,
also known in the art as "abzymes", can be used to convert the
prodrugs of the invention into free active drugs (see, e.g.,
Massey, Nature 328: 457-458 (1987)). Antagonist-abzyme conjugates
can be prepared as described herein for delivery of the abzyme to a
tumor cell population.
[0142] The enzymes of this invention can be covalently bound to the
antagonist by techniques well known in the art such as the use of
the heterobifunctional crosslinking reagents discussed above.
Alternatively, fusion proteins comprising at least the antigen
binding region of an antagonist of the invention linked to at least
a functionally active portion of an enzyme of the invention can be
constructed using recombinant DNA techniques well known in the art
[see, e.g., Neuberger et al., Nature, 312: 604-608 (1984)].
[0143] Other modifications of the antagonist are contemplated
herein. For example, the antagonist may be linked to one of a
variety of nonproteinaceous polymers, e.g., polyethylene glycol,
polypropylene glycol, polyoxyalkylenes, or copolymers of
polyethylene glycol and polypropylene glycol. The antagonists
disclosed herein may also be formulated as liposomes. Liposomes
containing the antagonist are prepared by methods known in the art,
such as described in Epstein et al., Proc. Mad. Acad Sci. USA,
82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030
(1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and W097/38731
published Oct. 23, 1997. Liposomes with enhanced circulation time
are disclosed in U.S. Pat. No. 5,013,556.
[0144] Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of an antibody of the present invention
can be conjugated to the liposomes as described in Martin et al.,
J. Biol. Chem. 257: 286-288 (1982) via a disulfide interchange
reaction. A chemotherapeutic agent is optionally contained within
the liposome. See Gabizon et al. J. National Cancer Inst.81(19)1484
(1989). Amino acid sequence modification(s) of protein or peptide
antagonists described herein are contemplated. For example, it may
be desirable to improve the binding affinity and/or other
biological properties of the antagonist.
[0145] Amino acid sequence variants of the antagonist are prepared
by introducing appropriate nucleotide changes into the antagonist
nucleic acid, or by peptide synthesis. Such modifications include,
for example, deletions from, and/or insertions into and/or
substitutions of, residues within the amino acid sequences of the
antagonist. Any combination of deletion, insertion, and
substitution is made to arrive at the fmal construct, provided that
the final construct possesses the desired characteristics. The
amino acid changes also may alter post-translational processes of
the antagonist, such as changing the number or position of
glycosylation sites.
[0146] A useful method for identification of certain residues or
regions of the antagonist that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis" as described
by Cunningham and Wells Science, 244:1081-1085 (1989). Here, a
residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a
neutral or negatively charged amino acid (most preferably alanine
or polyalanine) to affect the interaction of the amino acids with
antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to analyze the performance of a
mutation at a given site, ala scanning or random mutagenesis is
conducted at the target codon or region and the expressed
antagonist variants are screened for the desired activity.
[0147] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antagonist with an
N-terminal methionyl residue or the antagonist fused to a cytotoxic
polypeptide. Other insertional variants of the antagonist molecule
include the fuision to the N- or C-terminus of the antagonist of an
enzyme, or a polypeptide which increases the serum half-life of the
antagonist.
[0148] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
antagonist molecule replaced by different residue. The sites of
greatest interest for substitutional mutagenesis of antibody
antagonists include the hypervariable regions, but FR alterations
are also contemplated.
[0149] Conservative substitutions are shown in Table 1 under the
heading of "preferred substitutions". If such substitutions result
in a change in biological activity, then more substantial changes,
denominated "exemplary substitutions" in Table 1, or as further
described below in reference to amino acid classes, may be
introduced and the products screened.
1 TABLE 1 Original Exemplary Preferred Residue Substitutions
Substitutions Ala (A) val; leu; ile val Arg (R) lys; gin; asn lys
Asn (N) gin; his; asp, lys; arg gln Asp (D) glu; asn glu Cys (C)
ser; ala ser Gin (Q) asn; glu asn Glu (E) asp; gin asp Gly (G) ala
ala His (H) asn; gin; lys; arg arg Ile (I) leu; val; met; ala; ICU
phe; norleucine Lea (L) norleucine; ile; val; ile met; ala; phe Lys
(K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val;
ile; ala; tyr tyr Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser
TIP (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile;
leu; met; phe; ICU ala; norleucine
[0150] Substantial modifications in the biological properties of
the antagonist are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Naturally occurring residues are
divided into groups based on common side-chain properties:
[0151] (1) hydrophobic: norleucine, met, ala, val, leu, ile;
[0152] (2) neutral hydrophilic: cys, ser, thr;
[0153] (3) acidic: asp, glu;
[0154] (4) basic: asn, gln, his, lys, arg;
[0155] (5) residues that influence chain orientation: gly, pro;
and
[0156] (6) aromatic: trp, tyr, phe.
[0157] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0158] Any cysteine residue not involved in maintaining the proper
conformation of the antagonist also may be substituted, generally
with serine, to improve the oxidative stability of the molecule and
prevent aberrant crosslinking. Conversely, cysteine bond(s) may be
added to the antagonist to improve its stability (particularly
where the antagonist is an antibody fragment such as an Fv
fragment).
[0159] A particularly preferred type of substitutional variant
involves substituting one or more hypervariable region residues of
a parent antibody. Generally, the resulting variant(s) selected for
further development will have improved biological properties
relative to the parent antibody from which they are generated. A
convenient way for generating such substitutional variants is
affinity maturation using phage display. Briefly, several
hypervariable region sites (e.g. 6-7 sites) are mutated to generate
all possible amino substitutions at each site. The antibody
variants thus generated are displayed in a monovalent fashion from
filamentous phage particles as fusions to the gene III product of
M13 packaged within each particle. The phage-displayed variants are
then screened for their biological activity (e.g. binding affinity)
as herein disclosed. In order to identify candidate hypervariable
region sites for modification, alanine scanning mutagenesis can be
performed to identify hypervariable region residues contributing
significantly to antigen binding. Alternatively, or in
additionally, it may be beneficial to analyze a crystal structure
of the antigen-antibody complex to identify contact points between
the antibody and antigen. Such contact residues and neighboring
residues are candidates for substitution according to the
techniques elaborated herein. Once such variants are generated, the
panel of variants is subjected to screening as described herein and
antibodies with superior properties in one or more relevant assays
may be selected for further development.
[0160] Another type of amino acid variant of the antagonist alters
the original glycosylation pattern of the antagonist. By altering
is meant deleting one or more carbohydrate moieties found in the
antagonist, and/or adding one or more glycosylation sites that are
not present in the antagonist.
[0161] Glycosylation of polypeptides is typically either N-linked
or O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino
acid, most commonly serine or threonine, although 5-hydroxyproline
or 5-hydroxylysine may also be used. Addition of glycosylation
sites to the antagonist is conveniently accomplished by altering
the amino acid sequence such that it contains one or more of the
above-described tripeptide sequences (for N-linked glycosylation
sites). The alteration may also be made by the addition of, or
substitution by, one or more serine or threonine residues to the
sequence of the original antagonist (for O-linked glycosylation
sites).
[0162] Nucleic acid molecules encoding amino acid sequence variants
of the antagonist are prepared by a variety of methods known in the
art. These methods include, but are not limited to, isolation from
a natural source (in the case of naturally occurring amino acid
sequence variants) or preparation by oligonucleotide-mediated (or
site directed) mutagenesis, PCR mutagenesis, and cassette
mutagenesis of an earlier prepared variant or a non-variant version
of the antagonist.
[0163] It may be desirable to modify the antagonist of the
invention with respect to effector function, e.g. so as to enhance
antigen-dependent cell-mediated cyotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) of the antagonist. This may
be achieved by introducing one or more amino acid substitutions in
an Fc region of an antibody antagonist. Alternatively or
additionally, cysteine residue(s) may be introduced in the Fc
region, thereby allowing interchain disulfide bond formation in
this region. The homodimeric antibody thus generated may have
improved internalization capability and/or increased
complement-mediated cell killing and antibody-dependent cellular
cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195
(1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992). Homodimeric
antibodies with enhanced anti-tumor activity may also be prepared
using heterobifunetional cross-linkers as described in Wolff et al.
Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can
be engineered which has dual Fc regions and may thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al. Anti-Cancer Drug Design 3:219-230 (1989).
[0164] To increase the serum half life of the antagonist, one may
incorporate a salvage receptor binding epitope into the antagonist
(especially an antibody fragment) as described in U.S. Pat. No.
5,739,277, for example. As used herein, the term "salvage receptor
binding epitope" refers to an epitope of the Fc region of an IgG
molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for
increasing the in vivo serum half-life of the IgG molecule.
[0165] IV. Pharmaceutical Formulations
[0166] Therapeutic formulations of the antagonists used in
accordance with the present invention are prepared for storage by
mixing an antagonist or antagonists having the desired degree of
purity with optional pharmaceutically acceptable carriers,
excipients or stabilizers (Remington's Pharmaceutical Sciences 16th
edition, Osol, A. Ed. (1980)), in the form of lyophilized
formulations or aqueous solutions. Acceptable carriers, excipients,
or stabilizers are nontoxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, and other organic acids; antioxidants including ascorbic
acid and methionine; preservatives (such as octadecyldimethylbenzyl
ammonium chloride; hexamethonium chloride; benzalkonium chloride,
benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens such as methyl or propyl paraben; catechol; resorcinol;
cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less
than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0167] Exemplary anti-CD20 antibody formulations are described in
W098/56418, expressly incorporated herein by reference. This
publication describes a liquid multidose formulation comprising 40
mg/mL rituximab, 25 mM acetate, 150 mM trehalose, 0.9% benzyl
alcohol, 0.02% polysorbate 20 at pH 5.0 that has a minimum shelf
life of two years storage at 2-8.degree. C. Another anti-CD20
formulation of interest comprises 1 Omg/mL rituximab in 9.0 mg/mL
sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL
polysorbate 80, and Sterile Water for Injection, pH 6.5.
Lyophilized formulations adapted for subcutaneous administration
are described in W097/04801. Such lyophilized formulations may be
reconstituted with a suitable diluent to a high protein
concentration and the reconstituted formulation may be administered
subcutaneously to the mammal to be treated herein.
[0168] The formulation herein may also contain more than one active
compound zi.; necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other. For example, it may be desirable to
further provide a cytotoxic agent, chemotherapeutic agent, cytokine
or immunosuppressive agent (e.g. one which acts on T cells, such as
cyclosporin or an antibody that binds T cells, e.g. one which binds
LFA-1). The effective amount of such other agents depends on the
amount of antagonist present in the formulation, the type of
disease or disorder or treatment, and other factors discussed
above. These are generally used in the same dosages and with
administration routes as used hereinbefore or about from 1 to 99%
of the heretofore employed dosages.
[0169] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly(methylmethacylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmacelitical Sciences 16th edition, Osol, A. Ed.
(1980).
[0170] Sustained-release preparations maybe prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antagonist,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid.
[0171] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0172] V. Treatment with the Antagonist
[0173] A composition comprising an antagonist which binds to a B
cell surface antigen and a composition which contains a cytokine
antagonist, e.g. an antibody, wherein both may be in the same
composition will be formulated, dosed, and administered in a
fashion consistent with good medical practice. Preferably, the
anti-cytokine will comprise an anti-IL10 antibody and the B cell
antagonist will comprise a B cell depleting antibody, preferably an
anti-CD20 antibody such as Rituxan.RTM.. Factors for consideration
in this context include the particular disease or disorder being
treated, the particular mammal being treated, the clinical
condition of the individual patient, the cause of the disease or
disorder, the site of delivery of the agent, the method of
administration, the scheduling of administration, and other factors
known to medical practitioners. The therapeutically effective
amount of the antagonist to be administered will be governed by
such considerations.
[0174] As a general proposition, the therapeutically effective
amount of the antagonist administered parenterally per dose will be
in the range of about 0.1 to 20 mg/kg of patient body weight per
day, with the typical initial range of antagonist used being in the
range of about 2 to 10 mg/kg.
[0175] The preferred antagonist is an antibody, e.g. an antibody
such as RITUXAN.RTM., which is not conjugated to a cytotoxic agent.
Suitable dosages for an unconjugated antibody are, for example, in
the range from about 20 mg/mz to about I OOOmg/m2. In one
embodiment, the dosage of the antibody differs from that presently
recommended for RITUXAN.RTM.. For example, one may administer to
the patient one or more doses of substantially less than 375 mg/m2
of the antibody, e.g. where the dose is in the range from about 20
mg/mz to about 250 mg/m2, for example from about 50 mg/m2 to about
200 mg/m2.
[0176] Moreover, one may administer one or more initial dose(s) of
the antibody followed by one or more subsequent dose(s), wherein
the mg/m2 dose of the antibody in the subsequent dose(s) exceeds
the mg/m2 dose of the antibody in the initial dose(s). For example,
the initial dose may be in the range from about 20 mg/m2 to about
250 mg/m2 (e.g. from about 50 mg/m2 to about 200 mg/mz) and the
subsequent dose may be in the range from about 250 mg/m2 to about
1000 mg/m2.
[0177] As noted above, however, these suggested amounts of
antagonist are subject to a great deal of therapeutic discretion.
The key factor in selecting an appropriate dose and scheduling is
the result obtained, as indicated above.
[0178] For example, relatively higher doses may be needed initially
for the treatment of ongoing and acute diseases. To obtain the most
efficacious results, depending on the disease or disorder, the
antagonist is administered as close to the first sign, diagnosis,
appearance, or occurrence of the disease or disorder as possible or
during remissions of the disease or disorder.
[0179] The antagonist is administered by any suitable means,
including parenteral, subcutaneous, intraperitoneal,
intrapulmonary, and intranasal, and, if desired for local
immunosuppressive treatment, intralesional administration.
Parenteral infusions include intramuscular, intravenous,
intraarterial, intraperitoneal, or subcutaneous administration.
[0180] In addition, the antagonist may suitably be administered by
pulse infusion, e.g., with declining doses of the antagonist.
Preferably the dosing is given by injections, most preferably
intravenous or subcutaneous injections, depending in part on
whether the administration is brief or chronic.
[0181] One may administer other compounds, such as cytotoxic
agents, chemotherapeutic agents, immunosuppressive agents and/or
cytokines with the antagonists herein. The combined administration
includes coadministration, using separate formulations or a single
pharmaceutical formulation, and consecutive administration in
either order, wherein preferably there is a time period while both
(or all) active agents simultaneously exert their biological
activities.
[0182] Aside from administration of protein antagonists to the
patient the present application contemplates administration of
antagonists by gene therapy. Such administration of nucleic acid
encoding the antagonist is encompassed by the expression
"administering a therapeutically effective amount of an
antagonist". See, for example, W096/07321 published Mar. 14, 1996
concerning the use of gene therapy to generate intracellular
antibodies.
[0183] There are two major approaches to getting the nucleic acid
(optionally contained in a vector) into the patient's cells; in
vivo and ex vivo. For in vivo delivery the nucleic acid is injected
directly into the patient, usually at the site where the antagonist
is required. For ex vivo treatment, the patient's cells are
removed, the nucleic acid is introduced into these isolated cells
and the modified cells are administered to the patient either
directly or, for example, encapsulated within porous membranes
which are implanted into the patient (see, e.g. U.S. Pat. Nos.
4,892,538 and 5,283,187). There are a variety of techniques
available for introducing nucleic acids into viable cells. The
techniques vary depending upon whether the nucleic acid is
transferred into cultured cells in vitro, or in vivo in the cells
of the intended host. Techniques suitable for the transfer of
nucleic acid into mammalian cells in vitro include the use of
liposomes, electroporation, microinjection, cell fusion,
DEAE-dextran, the calcium phosphate precipitation method, etc. A
commonly used vector for ex vivo delivery of the gene is a
retrovirus.
[0184] The currently preferred in vivo nucleic acid transfer
techniques include transfection with viral vectors (such as
adenovirus, Herpes simplex I virus, or adeno-associated virus) and
lipid-based systems (useful lipids for lipid mediated transfer of
the gene are DOTMA, DOPE and DC-Chol, for example). In some
situations it is desirable to provide the nucleic acid source with
an agent that targets the target cells, such as an antibody
specific for a cell surface membrane protein or the target cell, a
ligand for a receptor on the target cell, etc. Where liposomes are
employed, proteins which bind to a cell surface membrane protein
associated with endocytosis may be used for targeting and/or to
facilitate uptake, e.g. capsid proteins or fragments thereof tropic
for a particular cell type, antibodies for proteins which undergo
internalization in cycling, and proteins that target intracellular
localization and enhance intracellular half-life. The technique of
receptor-mediated endocytosis is described, for example, by Wu et
aL, J. Biol. Chem. 262:4429-4432 (1987); and Wagner et al., Proc.
Nad. Acad. Sci. USA 87:3410-3414 (1990). For review of the
currently known gene marking and gene therapy protocols see
Anderson et al, Science 256:808-813 (1992). See also WO 93/25673
and the references cited therein.
[0185] VI. Articles of Manufacture
[0186] In another embodiment of the invention, an article of
manufacture containing materials useful for the treatment of the
diseases or disorders described above is provided. The article of
manufacture comprises a container and a label or package insert on
or associated with the container. Suitable containers include, for
example, bottles, vials, syringes, etc. The containers maybe formed
from a variety of materials such as glass or plastic. The container
holds or contains a composition which is effective for treating the
disease or disorder of choice and may have a sterile access port
(for example the container may be an intravenous solution bag or a
vial having a stopper pierceable by a hypodermic injection needle).
At least one active agent in the composition is the antagonist
which binds a B cell surface marker. Preferably CD20, and an
anti-cytokine antibody, e.g. an anti-LIO antibody. The label or
package insert indicates that the composition is used for treating
a patient having or predisposed to an autoimmune disease, such as
those listed herein. The article of manufacture may further
comprise a second container comprising a pharmaceutically-accepta-
ble diluent buffer, such as bacteriostatic water for injection
(BWFI), phosphate-buffered saline, Ringer's solution and dextrose
solution. It may further include other materials desirable from a
commercial and user standpoint, including other buffers, diluents,
filters, needles, and syringes.
[0187] Further details of the invention are illustrated by the
following non-limiting Examples. The disclosure of all citations in
the specification are expressly incorporated by reference.
EXAMPLES
Example 1
[0188] Treatment of Non-Hodgkin's Lymphoma
[0189] A patient with non-Hodgkin's lymphoma is intravenously
administered an anti-IL10 antibody at a dosage of 50 mg/m.sup.2 IV
weekly for four weeks. Thereafter, the patient is administered
RITUXAN.RTM. intravenously according to the following dosage
schedules:
[0190] (A) 50 mg/m.sup.2 IV day 1 150 mg/m.sup.2 IV days on 8, 15
& 22
[0191] (B) 150 mg/m.sup.2 IV day 1 375 mg/m.sup.2 IV on days 8, 15
& 22
[0192] (C) 375 mg/m.sup.2 IV on days 1, 8, 15 & 22
[0193] This same patient is administered CHOP chemotherapy
according to the regimen described in U.S. Pat. No. 5,736,137.
[0194] After treatment, the patient is monitored to evaluate the
effect on lymphoma status, e.g., number and size of tumors.
Example 2
[0195] Treatment of Solid Tumor in Advanced Stage
[0196] A patient having an advanced colorectal cancer characterized
by B cell involvement is treated concurrently with an anti-IL10
antibody and RITUXAN.RTM. at the same dosages as in Example 1.
[0197] After treatment the patient is evaluated to determine
whether such treatment has resulted in an anti-tumor response,
e.g., based on tumor shrinkage, lower tumor antigen expression or
other means of evaluating disease prognosis.
* * * * *