Note: Descriptions are shown in the official language in which they were submitted.
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TREATMENT OF B CELL MALIGNANCIES USING
COMBINATION OF B CELL DEPLETING ANTIBODY AND IMMUNE
MODULATING ANTIBODY RELATED APPLICATIONS
Cross Reference to Related Applications
This application claims priority from U.S. Serial Nos. 60/217,706, filed
July 12, 2000, and U.S. Serial No. 09/772,938, filed January 31, 2001, and are
incorporated by reference in their entirety therein.
Field of the Invention
The invention relates to a synergistic combination antibody therapy for
treatment of B cell malignancies, especially B cell lymphomas and leukemias.
This synergistic antibody combination comprises at least one antibody having
substantial B cell depleting activity (e.g., an anti-CD19, CD20, CD22 or CD37
antibody) and an antibody.that modulates or regulates the immune system, e.g.,
by modulating B cell/T cell interactions and/or B cell activity,
differentiation or
proliferation (e.g., anti-B7, anti-CD40, anti-CD23 or anti-CD40L ). In
particular, the invention encompasses combination antibody therapies for
CD40+ malignancies, which include using anti-CD40L antibodies to prevent
CD40L from binding to CD40. These antibodies or other agents which can
inhibit CD40/CD40L interaction further can be combined with
chemotherapeutics, radiation and/or other antibodies, preferably B cell
depleting
antibodies, e.g., anti-CD19, anti-CD20, anti-CD22 and/or anti-CD40 antibodies,
or fragments thereof.
Background of Invention
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
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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.
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
anoth,~r 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' to 108). Such antibody production most typically ceases (or substantially
decreases) when the foreign antigen has been neutralized. Occasionally,
however, prolifera=tion of a particular B cell will continue unabated; such
proliferation can result in a cancer referred to as "B cell lymphoma."
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.
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
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contains too many tumor cells upon harvesting. While patients often respond to
conventional therapies, they usually relapse within several months.
It is known that B cell malignancies, e.g., B cell lymphomas and
leukemias may be successfully treated using antibodies specific to B cell
antigens that possess B cell depleting activity. Examples of B cell antibodies
that have been reported to possess actual or potential application for the
treatment of B cell malignancies include antibodies specific to CD20, CD19,
CD22, CD37 and CD40.
Also, the use anti-CD37 antibodies having B cell depleting activity have
been well reported to possess potential for treatment of B cell lymphoma. See
e.g., Presr et al., J. Clin. Oncol. 7(8): 1027-1038 (August 1989); Grossbard
et
al., Blood 8(4): 863-876 (August 15, 1992).
A. Anti-CD20 Antibodies
CD20 is a cell surface antigen expressed on more than 90% of B-cell
lymphomas, which does not shed or modulate in the neoplastic cells
(McLaughlin et al., J. ClifZ. Oncol. 16: 2825-2833 (I998b)). The CD20 antigen
is a non-glycosylated, 35 kDa B-cell membrane protein involved in
intracellular
signaling, B-cell differentiation and calcium channel mobilization (Clark et
al.,
Adv. Cancer Res. 52: 81-149 (1989); Tedder et al., Immunology Today 15: 450-
454 (1994)). The antigen appears as an early marker of the human B-cell
lineage, and is ubiquitously expressed at various antigen densities on both
normal and malignant B-cell populations. However, the antigen is absent on
fully, mature B-cells (e.g., plasma cells), early B-cell populations and stem
cells, making it a suitable target for antibody mediated therapy.
Anti-CD20 antibodies have been prepared for use both in research and
therapeutics. One anti-CD20 antibody is the monoclonal B1 antibody (U.S.
Patent No. 5,843,398). Anti-CD20 antibodies have also been prepared in the
form of radionuclides for treating B-cell lymphoma (e.g., lsil-labeled anti-
CD20
antibody), as well as a 89Sr-labeled form for the palliation of bone pain
caused
by prostate and breast cancer metastasises (Endo, Gaga To Kagaku Ryoho 26:
744-748 (1999)).
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A marine monoclonal antibody, 1F5, (an anti-CD20 antibody) was
reportedly administered by continuous intravenous infusion to B-cell lymphoma
patients. However, extremely high levels (>2 grams) of 1F5 were reportedly
required to deplete circulating tumor cells, and the results were described as
"transient" (Press et al., Blood 69: 584-591 (1987)). A potential problem with
using monoclonal antibodies in therapeutics is non-human monoclonal
antibodies (e.g., marine monoclonal antibodies) typically lack human effector
functionality, e.g., they are unable to, inter alia, mediate complement
dependent
lysis or lyse human target cells through antibody-dependent cellular toxicity
or
Fc-receptor mediated phagocytosis. Furthermore, non-human monoclonal
antibodies can be recognized by the human host as a foreign protein;
therefore,
repeated injections of such foreign antibodies can lead to the induction of
immune responses leading to harmful hypersensitivity reactions. For
marine-based monoclonal antibodies, this is often referred to as a Human
Anti-Mouse Antibody response, or "HAMA" response. Additionally, these
"foreign" antibodies can be attacked by the immune system of the host such
that
they are, in effect, neutralized before they reach their target site.
RITIIXAN~ RITUXAN~ (also known as Rituximab, MabThera~,
IDEC-C2B8 and C2B8) was the first FDA-approved monoclonal antibody and
was developed at IDEC Pharmaceuticals (see U.S. Patent Nos. 5,843,439;
5,776,456 and 5,736,137) for treatment of human B-cell lymphoma (Reff et al.,
Blood 83: 435-445 (1994)). RITUXAN~ is a chimeric, anti-CD20 monoclonal
(MAb) which is growth inhibitory and reportedly sensitizes certain lymphoma
cell lines for apoptosis by chemotherapeutic agents in vitro (Demidem et al.,
Cancer Biotherapy & Radiopharmaceuticals 12: 177- (1997)). RITUXAN~
also demonstrates anti-tumor activity when tested in vivo using marine
xenograft animal models. RITUXAN~ efficiently binds human complement,
has strong FcR binding, and can efficiently kill human lymphocytes in vitro
via
both complement dependent (CDC) and antibody-dependent (ADCC)
mechanisms (Reff et al., Blood 83: 435-445 (1994)). In macaques, the antibody
selectively depletes normal B-cells from blood and lymph nodes.
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5 RITUXAN~ has been recommended for treatment of patients with low-
grade or follicular B-cell non-Hodgkin's lymphoma (McLaughlin et al.,
Oncology (Hurztingt) 12: 1763-1777 (1998a); Maloney et al., Oracology 12: 63-
76 (1998); Leget et al., Cu~~. Opin. Oncol. 10: 548-551 (1998)). In Europe,
RITUXAN~ has been approved for therapy of relapsed stage III/IV follicular
lymphoma (White et al., Pharm. Sci. Technol. Today 2: 95-101 (1999)) and is
reportedly effective against follicular center cell lymphoma (FCC) (Nguyen et
al., Eur. J. Haematol 62: 76-82 (1999)). Other disorders treated with
RITUXAN~ include follicular centre cell lymphoma (FCC), mantle cell
lymphoma (MCL), diffuse large cell lymphoma (DLCL), and small
lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) (Nguyen et
al., 1999)). Patients with refractory or incurable NHL reportedly have
responded to a combination of RITUXAN~ and CHOP (e.g.,
cyclophosphamide, vincristine, prednisone and doxorubicin) therapies (Ohnishi
et al., Gars To Kagaku Ryola~ 25: 2223-8 (1998)). RITUXAN~ has exhibited
minimal toxicity and significant therapeutic activity in low-grade non-
Hodgkin's lymphomas (NHL) in phase I and II clinical studies (Berinstein et
al.,
Ann. Oncol. 9: 995-1001 (1998)).
RITUXAN~, which was used alone to treat B-cell NHL at weekly doses
of typically 375 mg/M2 for four weeks with relapsed or refractory low-grade or
follicular NHL, was well tolerated and had significant clinical activity (Piro
et
al., Anna. Oncol. 10: 655-61 (1999); Nguyen et al., (1999); and Coiffier et
al.,
Blood 92: 1927-1932 (1998)). However, up to 500 mg/MZ of four weekly doses
have also been administered during trials using the antibody (Maloney et al.,
Blood 90: 2188-2195 (1997)). RITUXAN~ also has been combined with
chemotherapeutics, such as CHOP (e.g., cyclophosphamide, doxorubicin,
vincristine and prednisone), to treat patients with low-grade or follicular B-
cell
non-Hodgkin's lymphoma (Czuczman et al., J. Clirz. Oracol. 17: 268-76 (1999);
and McLaughlin et al., (1998a)).
Still further, the use of anti-B7 antibodies for treatment of B cell
lymphoma was mentioned in a patent assigned to IDEC Pharmaceuticals
Corporation. However, the focus of the patent was the use thereof for treating
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diseases which immunosuppression is therapeutically beneficial. Examples
included allergic, autoimmune and transplant indications. Also mentioned was
the use of the discussed anti-B7 antibodies for treatment of B cell lymphoma.
(US Patent No. 6,113,198).
B. CD40 and CD40L
CD40 is expressed on the cell surface of mature B-cells, as well as on
leukemic and lymphocytic B-cells, and on Hodgkin's and Reed-Sternberg (RS)
cells of Hodgkin's Disease (HD) (Valle et al., Eu~. J. ImnZUnol. 19: 1463-1467
(1989); and Gruss et al., Leuk. Lymphoma 24: 393-422 (1997)). CD40 is a B-
cell receptor leading to activation and survival of normal and malignant B-
cells,
such as non-Hodgkin's follicular lymphoma (Johnson et al., Blood 82: 1848-
1857 (1993); and Metkar et al., Cancer Imrnuftol. Immunother. 47: 104 (1998)).
Signaling through the CD40 receptor protects immature B-cells and B-cell
lymphomas from IgM- or Fas-induced apoptosis (Wang et al., J. Immunology
155: 3722-3725 (1995)). Similarly, mantel cell lymphoma cells have a high
level of CD40, and the addition of exogenous CD40L enhanced their survival
and rescued them from fludarabin-induced apoptosis (Clodi et al., Brit. J.
Haenaatol. 103: 217-219 (1998)). In contrast, others have reported that CD40
stimulation may inhibit neoplastic B-cell growth both in vitro (Funakoshi et
al.,
Blood 83: 2787-2794 (1994)) and in vivo (Murphy et al., Blood 86: 1946-1953
(1995)).
Anti-CD40 antibodies (see U.S. Patent Nos. 5,874,082 and 5,667,165)
administered to mice increased the survival of mice with human B-cell
lymphomas (Funakoshi et al., (1994); and Tutt et al., J. Immufaol. 161: 3176-
3185 (1998)). Methods of treating neoplasms, including B-cell lymphomas and
EBV-induced lymphomas using anti-CD40 antibodies mimicking the effect of
CD40L and thereby delivering a death signal, are described in U.S. Patent No.
5,674,492 (I997), which is herein incorporated by reference in its entirety.
CD40 signaling has also been associated with a synergistic interaction with
CD20 (Ledbetter et al., Cir~c. Shock 44: 67-72 (1994)). Additional references
describing preparation and use of anti-CD40 antibodies include U.S. Patent
Nos. 5,874,085 (1999), 5,874,082 (1999), 5,801,227 (1998), 5,674,492 (1997)
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and 5,667,165 (1997), which are incorporated herein by reference in their
entirety.
A CD40 ligand, gp39 (also called CD40 ligand, CD40L or CD154), is
expressed on activated, but not resting, CD4+ Th cells (Spriggs et al., J.
Exp.
Med. 176: 1543-1550 (1992); Lane et al., Eur. J. InZnaunol. 22: 2573-2578
(1992); and Roy et al., J. Irnmunol. 151: 1-14 (1993)). Both CD40 and CD40L
have been cloned and characterized (Stamenkovi et al., EMBO J. 8: 1403-1410
(1989); Armitage et al., Nature 357: 80-82 (1992); Lederman et al., J. Exp.
Med. 175: 1091-1101 (1992); and Hollenbaugh et al., EMBO J. 11: 4313-4321
(1992)). Human CD40L is also described in U.S. Patent No. 5,945,513. Cells
transfected with the CD40L gene and expressing the CD40L protein on their
surface can trigger B-cell proliferation, and together with other stimulatory
signals, can induce antibody production (Armitage et al., (1992); and U.S.
Patent No. 5,945,513). CD40L may play an important role in the cell contact-
dependent interaction of tumor B-cells (CD40+) within the neoplastic follicles
or Reed-Sternberg cells (CD40+) in Hodgkin's Disease areas (Carbone et al.,
Arn. J. Pathol. 147: 912-922 (1995)). Anti-CD40L monoclonal antibodies
reportedly have been effectively used to inhibit the induction of murine AIDS
(MAIDS) in LP-BM5-infected mice (Green et al., Virology 241: 260-268
(1998)). However, the mechanism of CD40L-CD40 signaling leading to
survival versus cell death responses of malignant B-cells is unclear. For
example, in follicular lymphoma cells, down-regulation of a apoptosis inducing
TRAIL molecule (APO-2L) (Ribeiro et al., British J. Haematol. 103: 684-689
(1998)) and over expression of BCL-2, and in the case of B-CLL, down-
regulation of CD95 (Fas/APO-1) (Laytragoon-Lewin et al., Eur. J. Haematol.
61: 266-271 (1998)) have been proposed as mechanisms of survival. In
contrast, evidence exists in follicular lymphoma, that CD40 activation leads
to
up-regulation of TNF (Worm et al., International Inamunol. 6: 1883-1890
(1994)) CD95 molecules (Plumas et al., Blood 91: 2875-2885 (1998)).
Anti-CD40 antibodies have also been prepared to prevent or treat
antibody-mediated diseases, such as allergies and autoimmune disorders as
described in U.S. Patent No. 5,874,082 (1999). Anti-CD40 antibodies
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reportedly have been effectively combined with anti-CD20 antibodies yielding
an additive effect in inhibiting growth of non-Hodgkin's B-cell lymphomas in
cell culture (Benoit et al., Immunoplaaf~macology 35: 129-139 (1996)). In vivo
studies in mice purportedly demonstrated that anti-CD20 antibodies were more
efficacious than anti-CD40 antibodies administered individually in promoting
10- the survival of mice bearing some, but not all, lymphoma lines (Funakoshi
et
al., J. lynmunother. Emphasis Tumor Inamunol. 19: 93-101 (1996)). Anti-CD19
antibodies are reportedly also effective in vivo in the treatment of two
syngeneic
mouse B-cell lymphomas, BCL1 and A31 (Tutt et al. (1998)). Antibodies to
CD40L have also been described for use to treat disorders associated with B-
cell activation (European Patent No. 555,880 (1993)). Anti-CD40L antibodies
include monoclonal antibodies 3E4, 2H5, 2H8, 4D9-8, 4D9-9, 24-31, 24-43, 89-
76 and 89-79, as described in U.S. Patent No. 5,7474,037 (1998), and anti-
CD40L antibodies described in U.S. Patent No. 5,876,718 (1999) used to treat
graft-versus-host-disease.
C. Anti-CD22 Antibodies
The synthesis of monoclonal antibodies against CD22 and their use in
therapeutic regimens has also been reported. CD22 is a B-cell-specific
molecule involved in B-cell adhesion that may function in homotypic or
heterotypic interactions (Stamenkovic et al, Natuf~e 344:74 (1990); Wilson et
al,
J. Exp. Med. 173:137 (1991); Stamenkovic et al, Cell 66:1133 (1991)). The
CD22 protein is expressed in the cytoplasm of progenitor B and pre-B-cells
(Dorken et al, J. Inzmunol. 136:4470 (1986); Dorken et al, "Expression of
cytoplasmic CD22 in B-cell ontogeny. In Leukocyte Typing III, White Cell
Differentiation Antigens. McMichael et al, eds., Oxford University Press,
Oxford, p. 474 (1987); Schwarting et al, Blood 65:974 (1985); Mason et al,
Blood 69:836 (1987)), but is found only on the surface of mature B-cells,
being
present at the same time as surface IgD (Dorken et al, J. Imnaunol. 136:4470
(1986)). CD22 expression increases following activation and disappears with
further differentiation (Wilson et al, J. Exp. Med. 173:137 (1991); Dorken et
al,
J. Inamurtol. 136:4470 (1986)). In lymphoid tissues, CD22 is expressed by
follicular mantle and marginal zone B-cells but only weakly by germinal center
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B-cells (Dorken et al, J. Immunol. 136:4470 (1986); Ling et al, "B-cell and
plasma antigens: new and previously defined clusters" In Leukocyte Typing III.
White Cell Differentiation Antigens, McMichael et al, eds., Oxford University
Press, Oxford, p. 302 (1987)). However, in situ hybridization reveals the
strongest expression of CD22 mRNA within the germinal center and weaker
expression within the mantle zone (Wilson et al, J. Exp. Med. 173:137 (1991)).
CD22 is speculated to be involved in the regulation of B-cell activation since
the binding of CD22 mAb to B-cells irt vitro has been found to augment both
the increase in intracellular free calcium and the proliferation induced after
cross-linking of surface Ig (Pezzutto et al, J. Inamunol. 138:98 (1987);
Pezzutto
et al, J. Inamunol. 140:1791 (1988)). Other studies have determined, however,
that the augmentation of anti-Ig induced proliferation is modest (Dorken et
al, J.
Immufaol. 136:4470 (1986)). CD22 is constitutively phosphorylated, but the
level of phosphorylation is augmented after treatment of cells with PMA (Boue
et al, J. Immunol. 140:192 (1988)). Furthermore, a soluble form of CD22
inhibits the CD3-mediated activation of human T-cells, suggesting CD22 may
be important in T-cell - B-cell interactions (Stamenkovic et al, Cell 66:1133
(1991)).
Ligands that specifically bind the CD22 receptor have been reported to
have potential application in the treatment of various diseases, especially B-
cell
lymphomas and autoimmune diseases. In particular, the use of labeled and non-
labeled anti-CD22 antibodies for treatment of such diseases has been reported.
For example, Tedder et al, U.S. patent 5,484,892, that purportedly bind
CD22 with high affinity and block the interaction of CD22 with other ligands.
These monoclonal antibodies are disclosed to be useful in treating autoimmune
diseases such as glomerulonephritis, Goodpasture's syndrome, necrotizing
vasculitis, lymphadenitis, periarteritis nodosa, systemic lupus erythematosis,
arthritis, thrombocytopenia purpura, agranulocytosis, autoimmune hemolytic
anemias, and for inhibiting immune reactions against foreign antigens such as
fetal antigens during pregnancy, myasthenia gravis, insulin-resistant
diabetes,
Graves' disease and allergic responses.
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5 Also, Leung et al, U. S. Patent 5,789,557, disclose chimeric and
humanized anti-CD22 monoclonal antibodies produced by CDR grafting and
the use thereof in conjugated and unconjugated form for therapy and diagnosis
of B-cell lymphomas and leukemias. The reference discloses especially such
antibodies conjugated to cytotoxic agents, such as chemotherapeutic drugs,
10 toxins, heavy metals and radionuclides. (See U.S. Patent 5,789,554, issued
August 4, 1998, to Leung et al, and assigned to Immunomedics.)
Further, PCT applications WO 98/42378, WO 00/20864, and WO
98/41641 describe monoclonal antibodies, conjugates and fragments specific to
CD22 and therapeutic use thereof, especially for treating B-cell related
diseases.
Also, the use of anti-CD22 antibodies for treatment of autoimmune
diseases and cancer has been suggested. See, e.g., U.S. Patent 5,443,953,
issued
August 22, 1995 to Hansen et al and assigned to Immunomedics Inc. that
purports to describe anti-CD22 immunoconjugates for diagnosis and therapy,
especially for treatment of viral and bacterial infectious diseases,
cardiovascular
disease, autoimmune diseases, and cancer, and U.S. Patent 5,484,892, issued
January 16, 1998 to Tedder et al and assigned to Dana-Farber Cancer institute,
Inc. that purports to describe various monoclonal antibodies directed against
CD22, for treatment of diseases wherein retardation or blocking of CD22
adhesive function is therapeutically beneficial, particularly autoimmune
diseases.) These references suggest that an anti-CD22 antibody of fragment
may be directly or indirectly conjugated to a desired effector moiety, e.g., a
label that may be detected, such as an enzyme, fluorophore, radionuclide,
electron transfer agent during an in vitro immunoassay or in vivo imaging, or
a
therapeutic effector moiety, e.g., a toxin, drug or radioisotope.
Further, an anti-human CD22 monoclonal antibody of the IgGl isotype
is commercially available from Leinco Technologies, and reportedly is useful
for treatment of B-cell lymphomas and leukemias, including hairy cell
leukemia. (Campana, D. et al, J. Irrarraufaol. 134:1524 (1985)). Still
ft~rther,
Dorken et al, J. Irra»aunol. 150:4719 (1993) and Engel et al, J. Ifyimunol.
150:4519 (1993) both describe monoclonal antibodies specific to CD22.
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D. Anti-CD 19 Antibodies
Also, the use of anti-CD19 antibodies and fragments thereof for treating
lymphoma has been reported in the literature. For example, U. S. Patent
5,686,072, issued November 1 l, 1997, to Uhr et al, and assigned to the
University of Texas, discloses the use of anti-CD19 and anti-CD22 antibodies
and immunotoxins for treatment of leukemia lymphomas. This patent is
incorporated by reference in its entirety herein.
Further, the use of anti-CD19 antibodies for classifying the status and
prognosis of leukemias has been reported.
Thus, based on the foregoing, it is clear that numerous antibodies have
been reported to possess therapeutic potential for treatment of B cell
malignancies. Notwithstanding this fact, it is an obj ect of the invention to
provide novel antibody regimens for treatment of B cell lymphoma.
Brief Description and Obiects of the Invention
Toward that end, it is an object of the invention to provide a novel
improved antibody therapy for treatment of B cell malignancies, including
Hodgkin's and non-Hodgkin's lymphoma of any grade.
More specifically, it is an obj ect of the invention to provide a novel
antibody regimen for treatment of a B cell malignancy involving the
administration of at least one B cell depleting antibody and at least one
immunoregulatory or immunomodulatory antibody.
Even more specifically, it is an obj ect of the invention to provide a novel
antibody therapy for treatment of B cell malignancies that involves the
administration of at least one B cell depleting antibody preferably selected
from
an anti-CD20, anti-CD19, anti-CD22 or anti-CD37 antibody and at least one
immunomodulatory antibody preferably selected from an anti-B7, anti-CD23,
anti-CD40, anti-CD40L or anti-CD4 antibody.
It is another object of the invention to provide a novel therapeutic
regimen for treatment of a B cell malignancy such as non-Hodgkin's lymphoma
or chronic lymphocyte leukemia (CLL) by the administration of an antibody to
CD20 (preferably RITUXAN~) and an antibody to B7 or CD40L (respectively
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preferably Primatized anti-B7 antibodies reported in US Patent 6,113,198 to
Anderson et al, or humanized anti-CD40L antibody reported in US Patent
6,001,358, assigned to 1DEC Pharmaceuticals Corporation).
It is another object of the invention to provide novel compositions and
kits for treatment of B cell malignancies, in B cell lymphomas and leukemias,
that include at least one immunoregulatory or immunomodulatory antibody and
at least one B cell depleting antibody. Preferably, the immunoregulatory or
immunomodulatory antibody will comprise an anti-CD40, anti-CD40L or anti-
B7 antibody and the B cell depleting antibody will be specific to CD20, CD19,
CD22 or CD37. Most preferably, the composition will comprise an anti-CD40L
or anti-B7 antibody and an anti-CD20 antibody.
Another object of the invention is to provide a combination therapy for
the treatment of a B-cell lymphoma or a B-cell leukemia comprising an anti-
CD40L antibody or antibody fragment or CD40L antagonist and at least one of
the following (a) a chemotherapeutic agent or a combination of
chemotherapeutic agents, (b) radiotherapy, (c) an anti-CD20 antibody or
fragment thereof, (d) an anti-CD40 antibody or fragment thereof, (e) an anti-
CD 19 antibody or fragment thereof; (f) an anti-CD22 antibody or fragment
thereof, (g) cytokines, and combinations thereof, where antibodies may be
conjugated with a toxin or a radiolabel, or may be engineered with human
constant regions as to elicit human antibody effector mechanisms, i.e.
resulting
in apoptosis or death of targeted cells.
Brief Description of the Figures
Fig. 1. Sensitivity of B-lymphoma cells to adriamycin after 4 hour
exposure.
Fig. 2. (Panel A) Anti CD40L (H~EC-131) overrides CD40L mediated
resistance to killing by ADM of B-lymphoma cells. (Panel B) Effect of
RITUXAN~ on normal and sCD40L pre-treated DHL-4 cells.
Fig. 3. (Panel A) Blocking of CD40L mediated cell survival of B-CLL
by anti-CD40L antibody (1DEC-13I). (Panel B) Blocking of CD40L mediated
survival of B-CLL by Rituxan~.
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Fig. 4. FACS analysis comprising HLA-DR expression in CD19+ CLL
cells cultured with sCD40L and not cultured with sCD40L.
Detailed Description of the Invention
The present invention provides a novel combination antibody regimen
that involves the administration of at least one immunoregulatory or
immunomodulatory antibody, e.g., an anti-B7 or anti-CD40 or anti-CD40L
antibody and at least one B cell depleting antibody, e.g., an anti-CD20, anti-
CD19, anti-CD22 or anti-CD37 antibody having substantial B cell depleting
activity.
It is believed that such combination will afford synergistic results based
on the different mechanisms by which the antibodies elicit a therapeutic
benefit.
In particular, it is theorized that the complementary mechanisms of action
will
yield a more durable and potent clinical response as it is believed that the B
cell
depleting antibody will deplete activated B cells which may be resistant to
the
action of immunoregulatory or immunomodulatory antibodies such as anti-B7
or anti-CD40L antibodies. Such activated B cells can otherwise serve as
effective antigen presenting cells for T cells as well as antibody producing
cells.
In the context of B cell malignancies, such activated B cells may include
malignant cells which unless eradicated by give rise to new cancer cells and
tumors.
Prior to discussing the invention, the following definitions are provided:
The term "antibody" as used herein is intended to include
immunoglobulins and fragments thereof which are specifically reactive to the
designated protein or peptide thereof. An antibody can include human
antibodies, primatized antibodies, chimeric antibodies, bispecific antibodies,
humanized antibodies, antibodies fused to other proteins or radiolabels, and
antibody fragments.
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.
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"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. Antibody fragments may be isolated using conventional techniques.
For example, F(abl)e fragments can be generated by treating antibodies with
pepsin. The resulting F(abl)e fragment can be treated to reduce disulfide
bridges to produce Fabl fragments.
"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.
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
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5 (FRs). The variable domains of native heavy and light chains each comprise
four FRs, largely adopting a 13-sheet configuration, connected by three
hypervariable regions, which form loops connecting, and in some cases forming
part of, the B -sheet structure. The hypervariable regions in each chain are
held
together in close proximity by the FRs and, with the hypervariable regions
from
10 the other chain, contribute to the formation of the antigen-binding site of
antibodies (see Kabat et al., Sequefzces of Pj°oteins of
InZnauhological hcte~est,
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
15 antibody in antibody dependent cellular cytotoxicity (ADCC).
Papain digestion of antibodies produces two identical antigen-binding
fragments, called "Fab" 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 cross-linking antigen.
"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.
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
residues) of the constant domains bear at least one free thiol group. F(ab')Z
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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.
The "light chains" of antibodies (immunoglobulins) from any vertebrate
species can be assigned to one of two clearly distinct types, called kappa and
lambda, based on the amino acid sequences of their constant domains.
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
I S intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may
be
further
divided into subclasses (isotypes), e.g., IgGI, IgG2, IgG3, IgG4, IgA, and
IgA2.
The
heavy-chain constant domains that correspond to the different classes of
antibodies are called alpha, delta, epsilon, gamma and mu, respectively.
Preferably, the heavy-chain constant domains will complete the gamma-1,
gamma-2, gamma-3 and gamma-4 constant region. Preferably, these constant
domains will also comprise modifications to enhance antibody stability such as
the P and E modification disclosed in U.S. Patent No. 6,011,138 incorporated
by reference in its entirety herein. The subunit structures and three
dimensional
configurations of different classes of immunoglobulins are well known.
"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, uol. 113, Rosenburg
and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
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
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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., Ps°oc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
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.
Patent 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.
By "humanized antibody" is meant an antibody derived from a non-
human antibody, typically a murine antibody, that retains or substantially
retains
the antigen-binding properties of the parent antibody, but which is less
immunogenic in humans. This may be achieved by various methods, including
(a) grafting the entire non-human variable domains onto human constant regions
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to generate chimeric antibodies; (b) grafting only the non-human
complementarity determining regions (CDRs) into human framework and
constant regions with or without retention of critical framework residues; and
(c) transplanting the entire non-human variable domains, but "cloaking" them
with a human-like section by replacement of surface residues. Such methods
are disclosed in Morrison et al., Proc. Natl. Acad. Sci. 81: 6851-5 (1984);
Morrison et al., Adv. Immuhol. 44: 65-92 (1988); Verhoeyen et al., Science
239:
1534-1536 (1988); Padlan, Molec. Inamun. 28: 489-498 (1991); and Padlan,
Molec. Immun. 31: 169-217 (1994), all of which are hereby incorporated by
reference in their entirety. Humanized anti-CD40L antibodies can be prepared
as described in U.S. Patent Application No. 081554,840 filed November 7, 1995
also incorporated herein by reference in its entirety.
By "human antibody" is meant an antibody containing entirely human
light and heavy chain as well as constant regions, produced by any of the
known
standard methods.
By "primatized antibody" is meant a recombinant antibody which has
been engineered to contain the variable heavy and light domains of a monkey
(or other primate) antibody, in particular, a cynomolgus monkey antibody, and
which contains human constant domain sequences, preferably the human
immunoglobulin gamma 1 or gamma 4 constant domain (or PE variant). The
preparation of such antibodies is described in Newman et al., Biotechnology,
10: 1458-1460 (1992); also in commonly assigned 08/379,072, 08/487,550, or
08/746,361, all of which are incorporated by reference in their entirety
herein.
These antibodies have been reported to exhibit a high degree of homology to
human antibodies, i.e., 85-98%, display human effector functions, have reduced
immunogenicity, and may exhibit high affinity to human antigens.
By "antibody fragment" is meant an fragment of an antibody such as
Fab, F(ab')2, Fab' and scFv.
By "chimeric antibody" is meant an antibody containing sequences
derived from two different antibodies, which typically are of different
species.
Most typically, chimeric antibodies comprise human and marine antibody
fragments, and generally human constant and marine variable regions.
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"B Cell Depleting Antibody" therein is an antibody or fragment that
upon administration, results in demonstrable B cell depletion. Typically, such
antibody will bind to a B cell antigen or B cell marker expressed on the
surface
of a B cell. Preferably, such antibody, after administration, typically within
about several days or less, will result in a depletion of B cell number by
about
50% or more. In a preferred embodiment, the B cell depleting antibody will be
RITUXAN~ (a chimeric anti-CD20 antibody) or one having substantially the
same or at least 20-50% the cell depleting activity of RITUXAN~.
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
CD 19, 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 CD 19 and CD20.
"Immunoregulatory Antibody" refers to an antibody that elicits an effect
on the immune system by a mechanism different from B cell depletion, e.g., by
CDL and/or ADCC activity. Examples of such include antibodies that inhibit T
cell immunity, B cell immunity, e.g. by inducing tolerance (anti-CD40L, anti-
CD40) or other immunosuppressant antibodies, e.g., those that inhibit B7 cell
signaling (anti-B7.1, anti-B7.2, anti-CD4, anti-CD23, etc.). In some
instances,
the immunoregulatory antibody may possess the ability to potentiate apoptosis.
Also, an antibody that is normally a B cell depleting antibody can be
engineered
to become immunoregulatory by substantiating human constant regions as to
take advantage of different effector mechanisms.
A "B cell surface marker" herein is an antigen expressed on the surface
of a B cell which can be targeted with an antagonist which binds thereto.
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5 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 (B7.1), CD81, CD82, CD83, CDw84,
CD85 and CD86 (B7.2) leukocyte surface markers. The B cell surface marker
of particular interest is preferentially expressed on B cells compared to
other
10 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 CD 19,
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 CD 19, CD20, CD23,
15 CD80 and CD86.
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
20 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).
The "CD19" antigen refers to a -90kDa antigen identified, for example,
by the
HD237-CD19 or B4 antibody (Kiesel et al. Leukemia Research II, 12: 1119
(1987)).
Like CD20, CD 19 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.
The "CD22" antigen refers to an antigen expressed on B cells, also
known as
"BL-CAM" and "LybB" that is involved in B cell signaling and an adhesion.
(See
Nitschke et al., Cuf~r~. Biol. 7:133 (1997); Stamenkovic et al., Nature 345:74
(1990)).
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This antigen is a membrane immunoglobulin-associated antigen that is tyrosine
phosphorylated when membrane Ig is ligated. (Engel et al., J. Etyp. Med.
181(4):1521
1586 (1995)). The gene encoding this antigen has been cloned, and its 1g
domains
characterized.
B7 antigen includes the B7.1 (CD80), B7.2 (CD81) and B7.3 antigen,
which are transmembrane antigens expressed on B cells. Antibodies which
specifically bind B7 antigens, including human B7.1 and B7.2 antigens are
known in the art. Preferred B7 antibodies comprise the primatized~ B7
antibodies disclosed by Anderson et al. in U.S. Patent No. 6,113,198, assigned
to IDEC Pharmaceuticals Corporation, as well as human and humanized B7
antibodies.
CD23 refers to the low affinity receptor for IgE expressed by B and
other cells. In the present invention, CD23 will preferably be human CD23
antigen. CD23 antibodies are also known in the art. Most preferably, in the
present invention, the CD23 antibody will be a human or chimeric anti-human
CD23 antibody comprising human IgGI or IgG3 constant domains.
A B cell "antagonist" is a molecule which, upon binding to a B cell
surface marker, destroys or depletes B cells in a mammal andlor 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.
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A CD40L antagonist is a molecule that specifically binds CD40L and
preferably antagonzes the interaction of CD40L and CD40. Examples thereof
include antibodies and antibody fragments that specifically bind CD40L,
soluble CD40, soluble CD40 fusion proteins, and small molecules that bind
CD40L. The preferred antagonist according to the invention comprises an
antibody or antibody fragment specific to CD40.
"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 US
Patent No. 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).
"Human effector cells" are leukocytes which express one or more FcRs
and
perform effector functions. Preferably, the cells express at least FcyRIII and
perform 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. The effector cells may be isolated from a native
source thereof, e.g. from blood or PBMCs as described herein.
The terms "Fc receptor" or "FcR" are used to describe a receptor that
binds to the Fc region of an antibody. 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 FcyRII
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subclasses, including allelic variants and alternatively spliced forms of
these
receptors. FcyRII receptors include FcyRIIA (an "activating receptor") and
FcyRUB (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 review M. in Daeon, 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.
Clira. 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 I~im et al.,
J.
Imrnuhol. 24:249 (1994)).
"Complement dependent cytotoxicity" or "CDC" refers 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), may be performed.
"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.
Antagonists which "induce apoptosis" are those which induce
programmed cell death, e.g. of a B cell, as determined by binding of annexin
V,
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fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell
fragmentation, and/or formation of membrane vesicles (called apoptotic
bodies).
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 (L1), 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; Rabat et al., Sequences of Proteins of
Inzmunological 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.l. 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 such
that
the antagonist is useful as a therapeutic agent for targeting a cell, i.e. a B
cell,
expressing the antigen.
An "anti-CD20 antibody" herein is an antibody that specifically binds
CD20 antigen, preferably human CD20, having measurable B cell depleting
activity, preferably having at least about 10% the B cell depleting activity
of
RITUXAN~ (see U.S. Patent No. 5,736,137, incorporated by reference herein
in its entirety).
An "anti-CD22 antibody" herein is an antibody that specifically binds
CD22 antigen, preferably human CD22, having measurable B cell depleting
activity, preferably having at least about 10% the B cell depleting activity
of
RITUXAN~ (see U.S. Patent No. 5,736,137, incorporated by reference herein
in its entirety).
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5 An "anti-CD19 antibody" herein is an antibody that specifically binds
CD 19 antigen, preferably human CD 19, having measurable B cell depleting
activity, preferably having at least about 10% the B cell depleting activity
of
RITUXAN~ (see U.S. Patent No. 5,736,137, incorporated by reference herein
in its entirety).
10 An "anti-CD37 antibody" herein is an antibody that specifically binds
CD37 antigen, preferably human CD37, having measurable B cell depleting
activity, preferably having at least about 10% the B cell depleting activity
of
RITUXAN~ (see U.S. Patent No. 5,736,137, incorporated by reference herein
in its entirety).
15 An "anti-B7 antibody" herein is an antibody that specifically binds B7.I,
B7.2 or B7.3, most preferably human 87.3, that inhibits B7/CD28 interactions
and, which more does not substantially inhibit B7/CTLA-4 interactions, and
even more preferably, the particular antibodies described in U.S. Patent
6,113,898, incorporated by reference in its entirety herein. It has recently
been
20 shown that these antibodies promote apoptosis. Therefore, they are well
suited
for anti-neoplastic applications.
An "anti-CD40L antibody" is an antibody that specifically binds CD40L
(also known as CD154, gp39, TBAM), preferably one having agonistic activity.
A preferred anti-Cd40L antibody is one having the specificity of a humanized
25 antibody disclosed in U.S. Patent No. 6,011,358 (assigned to IDEC
Pharmaceuticals Corporation), incorporated by reference in its entirety
herein.
An "anti-CD4 antibody" is one that specifically binds CD4, preferably
human CD4, more preferably a primatized or humanized anti-CD4 antibody.
An "anti-CD40 antibody" is an antibody that specifically binds CD40,
preferably human CD40, such as those disclosed in U.S. Patent 5,874,085,
5,874,082, 5,801,227, 5,674,442, snf 5,667,165, all of which are incorporated
by reference herein.
Preferably, both the B cell depleting antibody and the immunoregulatory
antibody will contain human constant domains. Suitable antibodies may
include IgGl, IgG2, IgG3 and IgG4 isotypes.
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Specific examples of antibodies which bind the CD20 antigen include:
"Rituximab" ("RITUXAN~") (US Patent No. 5,736,137, expressly
incorporated herein by reference); yttrium-[90]-labeled 2B8 murine antibody
"Y2B8" (US Patent No. 5,736,B7, expressly incorporated herein by reference);
murine IgG2a "B1" optionally labeled with 1311, <e1311 B1" antibody
(BEX~~ARTM) (US Patent No. 5,595,721, expressly incorporated herein by
reference); murine monoclonal antibody "1F5" (Press et al. Blood
69(2):584-591 (1987); and "chimeric 2H7" antibody (US Patent No. 5,677,180,
expressly incorporated herein by reference).
Specific examples of antibodies which bind CD22 include
LymphocideTM reported by Immunomedics, now in clinical trials for
non-Hodgkin's lymphoma. Examples of antibodies that bind B7 antigen include
the B7 antibody reported U.S. Patent 5,885,577, issued to Linsley et al, the
anti-B7 antibody reported in U.S. Patent 5,869,050, issued in DeBoer et al,
assigned to Chiron Corporation, and the primatized~ anti-B7 antibodies
disclosed in U.S. Patent 6,113,198 to Anderson et al., all of which are
incorporated by reference in their entirety.
Specific examples of antibodies that bind CD23 are well known and
preferably include the primatized~ antibodies specific to human CD23 reported
by Reff et al., in U.S. Patent 6,011,138, issued on July 4, 1999, co-assigned
to
IDEC Pharmaceuticals Corp. and Seikakagu Corporation of Japan; those
reported by Bonnefoy et al., No. 96 12741; Rector et al. J. Irnnaunol.
55:481-488 (1985); Flores-Rumeo et al. Science 241:1038-1046 (1993); Sherr
et al. J. Immunol., 142:481-489 (1989); and Pene et al., PNAS, USA
85:6820-6824 (1988). Such antibodies axe reportedly useful for treatment of
allergy, autoimmune diseases, and inflammatory diseases.
The terms "rituximab" or "RITUXAN~" herein refer to the genetically
engineered chimeric murine/human monoclonal antibody directed against the
CD20
antigen and designated "C2B8" in US Patent No. 5,736,B7, expressly
incorporated
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herein by reference. The antibody is an IgGI kappa immunoglobulin containing
marine light and heavy chain variable region sequences and human constant
region sequences. Rituximab has a binding affinity for the CD20 antigen of
approximately 8.0 nM.
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 eight
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 ih 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.
"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.
B Cell Mali ancy
According to the present invention this includes any B cell malignancy,
e.g., B cell lymphomas and leukemias. Preferred examples include Hodgkin's
disease (all forms, e.g., relapsed Hodgkin's disease, resistant Hodgkin's
disease)
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non-Hodgkin's lymphomas (low grade, intermediate grade, high grade, and
other types). Examples include small lymphocyticB cell chronic lymphocytic
leukemia (SLL/B-CLL), lymhoplasmacytoid lymphoma (LPL), mantle cell
lymphoma (MCL), follicular lymphoma (FL), diffuse large cell lymphoma
(DLCL), Burkitt's lymphoma (BL), AIDS- related lymphomas, monocytic B
cell lymphoma, angioimmunoblastic lymphoadenopathy, small lymphocytic,
follicular, diffuse large cell, diffuse small cleaved cell, large cell
immunoblastic
lymphoblastoma, small, non-cleaved, Burkitt's and non-Burkitt's, follicular,
predominantly large cell; follicular, predominantly small cleaved cell; and
follicular, mixed small cleaved and large cell lymphomas. See, Gaidono et al.,
"Lymphomas", IN CANCER: PRINCIPLES & PRACTICE OF ONCOLOGY,
Vol. 2: 2131-2145 (DeVita et al., eds., 5th ed. 1997).
Other types of lymphoma classifications include immunocytomal
Waldenstrom's MALT-type/monocytoid B cell, mantle cell lymphoma B-
CLL/SLL, diffuse large B-cell lymphoma, follicular lymphoma, and precursor
B-LBL.
As noted, B cell malignancies further include especially leukemias such
as ALL-L3 (Burkitt's type leukemia), chronic lymphocytic leukemia (CLL),
chronic leukocytic leukemia, acute myelogenous leukemia, acute lymphoblastic
leukemia, chronic lyrnphocytic leukemia, chronic myelogenous leukemia,
lymphoblastic leukemia, lymphocytic leukemia, monocytic leukemia,
myelogenous leukemia, and promyelocytic leukemia and monocytic cell
leukemias.
The expression "therapeutically effective amount" refers to an amount
of the
antagonist which is effective for preventing, ameliorating or treating the B
cell
malignancy 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 MIiC antigens. Examples of such agents include
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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-a, (3- or 8-antibodies, anti-tumor necrosis factor-a
antibodies,
anti-tumor necrosis factor-~3 antibodies, anti-interleukin-2 antibodies and
anti-IL-2 receptor antibodies; anti-LFA-1 antibodies, including anti-CDl la
and
anti-CD18 antibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte
globulin; pan-T antibodies, preferably anti-CD3 or anti-CD4/CD4a antibodies;
soluble peptide containing a LFA-3 binding domain (WO 90/08187 published
7/26/90), streptolanase; TGF-(3; 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 T10B9.
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. At211 1131 1125 Y9o Re 186 Re
1g8 sM153 Bi212 p32 and radioactive isotopes of Lu), chemotherapeutic agents,
and toxins such as small molecule toxins or enzymatically active toxins of
bacterial, fungal, plant or animal origin, or fragments thereof.
A "chemotherapeutic agent" is a chemical compound useful in the
treatment of cancer. Examples of chemotherapeutic agents include alkylating
agents such as thiotepa and cyclosphosphaxnide (CYTOXANTM); 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
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5 trimethylolomelamime nitrogen mustards such as chiorambucil, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine,
prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine,
chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics
such
10 as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,
cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin,
chromomycins, dactinomycin, daunorubicin, detorubicin,
6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins,
15 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;
20 pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur,
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;
25 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;
30 PSK~; razoxane; sizofiran; 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 (TAXOL~, Bristol-Myers
Squibb Oncology, Princeton, NJ) and doxetaxel (Taxotere, Rhone-Poulenc
Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine;
mercaptopurine; methotrexate; platinum analogs such as cisplatin and
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carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin
C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide;
daunomycin; aminopterin; xeloda; ibandronate; CPT11; topoisomerase inhibitor
RFS 2000; difluoromethylornithine (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 antiandrogens such as flutamide, nilutamide,
bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable
salts,
acids or derivatives of any of the above.
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 -(3; mullerian-inhibiting substance; mouse
gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth
factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-13;
platelet-growth factor; transforming growth factors (TGFs) such as TGF-a and
TGF-(3; insulin-like growth factor-I and -II; erythropoietin (EPO);
osteoinductive factors; interferons such as interferon-a, -(3, and -y; colony
stimulating factors (CSFs) such as macrophage-CSF (M-CSF);
granulocytemacrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-1, IL-la, IL-2, IL-g, 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-(3;
and
other polypeptide factors including LIF and kit ligand (KL). As used herein,
the
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term cytokine includes proteins from natural sources or from recombinant cell
culture and biologically active equivalents of the native sequence cytokines.
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., Wilman,
"Prodrugs in Cancer Chemotherapy" Biochemical Society Tf°ansactions,
14, pp.
375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical
Approach to Targeted Drug Delivery," Directed DYUg 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,l3-lactam-containing prodrugs, optionally substituted
phenoxyacetamide-containing prodrugs or optionally substituted
phenylacetamide-containing prodrugs, 5-fluorocytosine and other
Sfluorouridine 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.
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.
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II. Production of Antibodies
The methods and articles of manufacture of the present invention use, or
incorporate, an antibody that has immunoregulatory activity, e.g. anti-B7,
anti-
CD23, anti-CD40L, anti-CD4 or anti-CD40 antibody, and an antibody that
binds to a B cell surface marker having B depleting activity, e.g., anti-CD20,
anti-CD22, anti-CD19, or anti-CD37 antibody. Accordingly, methods for
generating such antibodies will be described herein.
The molecule to be used for production of, or screening for, antigens)
may be, e.g., a soluble form of the antigen or a portion thereof, containing
the
desired epitope. Alternatively, or additionally, cells expressing the antigen
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. Suitable antigen sources for CD40L,
CD40,
CD19, CD20, CD22, CD23, CD37, CD4 and B7 antigen (e.g., B7.1, B7.2)
antigen for producing antibodies according to the invention are well known.
Alternatively, peptides can be synthetically prepared based upon the amino
acid
sequence. For example, With respect to CD40L, this is disclosed in Armitage et
al. (1992).
Preferably, the CD40L antibody or anti-CD40L antibody will be the
humanized anti-CD40L antibody disclosed in U.S. Patent 6,001,358, issued on
June 14, 1999, and assigned to )DEC Pharmaceuticals Corporation.
While a preferred CD40L antagonist is an antibody, antagonists other
than antibodies may also be administered. For example, the antagonist may
comprise soluble CD40, a CD40 fusion protein or 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 andlor conjugated with a cytotoxic agent.
The antagonist may also be a peptide generated by rational design or by
phage
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display (W098/35036 published 13 August 1998), for example. In one
embodiment, the molecule of choice may be a "CDR mimic" or antibody
analogue designed based on the CDRs of an antibody, for example. While the
peptide may be antagonistic by itself, the peptide may optionally be fused to
a
cytotoxic agent or to an immunoglobulin Fc region (e.g., so as to confer ADCC
and/or CDC activity on the peptide).
Exemplary techniques for the production of the antibody antagonists
used in accordance with the present invention are described.
(i) Polyclonal antibodies
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, succiic
anhydride, SOC 12, or R1N=C=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 ~g of the protein or conjugate (for
rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant
and inj ecting the solution intradermally at multiple sites. One month later
the
animals are boosted with 1/5 to 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.
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Monoclonal antibodies are 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
10 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., NatuYe, 256:495 (1975), or may be
15 made by
recombinant DNA methods (LJ.S. Patent No. 4,816,567).
In the hybridoma method, a mouse or other appropriate host animal,
such as a
hamster, is immunized as hereinabove described to elicit lymphocytes that
20 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
25 polyethylene
glycol, to form a hybridoma cell (Goding, Moraoclonal Antibodies: Principles
and
Practice, pp.59-103 (Academic Press, 1986)).
The hybridoma cells thus prepared are seeded and grown in a suitable
30 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
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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.
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, California USA, and SP-2 or X63-Ag8-653
cells available from the American Type Culture Collection, Manassas, Virginia,
USA. Human myeloma and mouse-human heteromyeloma cell lines also have
been described for the production of human monoclonal antibodies (I~ozbor, J.
Inununol., 133:300 1 (1984); Brodeur et al., Mon~clonal Aratibody Production
Techniques and Applieatiotas, pp. 51-63 (Marcel Dekker, Inc., New York,
1987)).
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 ifa 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 30 Scatchard analysis of Munson et al., Anal. Biochem.,
107:220 (1980).
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: Prifaciples and Practice, pp.59-103 (Academic Press,
1986)). Suitable culture media for this purpose include, for example, D-MEM or
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RPML-1640 medium. In addition, the hybridoma cells may be grown in vivo as
ascites tumors in an animal.
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.
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 ih Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Revs.,
130:151-188 (1992).
Another method of generating specific antibodies, or antibody
fragments, reactive against a CD40L, CD 19, CD22, CD20, or CD40 protein or
peptide (e.g., such as the gp39 fusion protein described in U.S. Patent No.
5,945,513) is to screen expression libraries encoding immunoglobulin genes, or
portions thereof, expressed in bacteria with a CD40L, CD19, CD20, or CD22
protein or peptide. For example, complete Fab fragments, VH regions and Fv
regions can be expressed in bacteria using phage expression libraries. See for
example, Ward et al., Nature 341: 544-546 (1989); Huse et ad., Scietzce 246:
1275-1281 (1989); and McCafferty et al., Nature 348: 552-554 (1990).
Screening such libraries with, for example, a CD40L, CD22, CD19, or CD20
peptide, can identify immunoglobulin fragments reactive with CD40L, CD22,
CD19, or CD20. Alternatively, the SCm-hu mouse (available from Genpharm)
can be used to produce antibodies or fragments thereof.
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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., BiolTechfaology,
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.
Methodologies for producing monoclonal antibodies (MAb) directed
against CD40L, including human CD40L and mouse CD40L, and suitable
monoclonal antibodies for use in the methods of the invention, are described
in
PCT Patent Application No. WO 95/06666 entitled "Anti-gp39 Antibodies and
Uses Therefor;" the teachings of which are incorporated herein by reference in
their entirety. Particularly preferred anti-human CD40L antibodies of the
invention are MAbs 24-31 and 89-76, produced respectively by hybridomas 24-
31 and 89-76. The 89-76 and 24-31 hybridomas, producing the 89-76 and 24-
31 antibodies, respectively, were deposited under the provisions of the
Budapest
Treaty with the American Type Culture Collection (ATCC), 10801 University
. Blvd., Manassas, VA 20110-2209, on Sept. 2, 1994. The 89-76 hybridoma was
assigned ATCC Accession Number HB11713 and the 24-31 hybridoma was
assigned ATCC Accession Number HB11712.
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. Patent No. 4,816,567; Morrison, et al.,
Pf~oc. Natl Acad. ScL 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.
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Typically, such non-immunoglobulin polypeptides are substituted for the
constant domains of an antibody, or they are substituted for the variable
domains of one antigencombining 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.
(iii) Humanized antibodies
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); Reichmann 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 (IJ.S. Patent 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.
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 (Suns et al., J. Immunol., 151:2296 (1993); Chothia et al.,
J. Mol. Biol, 196:901 (1987)). Another method uses a particular framework
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5 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., P~oc. Natl. Acad.
Sci.
TISA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).
It is further important that antibodies be humanized with retention of
10 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
15 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
20 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 arid most substantially involved in
25 influencing antigen binding.
(iv) Primatized Antibodies
Another highly efficient means for generating recombinant antibodies is
disclosed by Newman, Biotechnology, 10: 1455-1460 (1992). More
30 particularly, this technique results in the generation of primatized
antibodies
which contain monkey variable domains and human constant sequences. This
reference is incorporated by reference in its entirety herein. Moreover, this
technique is also described in commonly assigned U.S. Application No.
081379,072, filed on January 25, 1995, which is a continuation of U.S. Serial
35 No. 07/912,292, filed July 10, 1992, which is a continuation-in-part of
U.S.
Serial No. 07/856,281, filed March 23, 1992, which is finally a continuation-
in-
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part of U.S. Serial No. 07J735,064, filed July 25, 1991. 08J379,072 and the
parent application thereof all of which are incorporated by reference in their
entirety herein.
This technique modifies antibodies such that they are not antigenically
rejected upon administration in humans. This technique relies on immunization
of cynomolgus monkeys with human antigens or receptors. This technique was
developed to create high affinity monoclonal antibodies directed to human cell
surface antigens.
Identification of macaque antibodies to human CD40L, CD20, CD22,
CD40 or CD19 by screening of phage display libraries or monkey
heterohybridomas obtained using B lymphocytes from CD40L, CD20, CD22,
CD40, or CD19 immunized monkeys can be performed using the methods
described in commonly assigned U.S. Application No. 08J487,550, filed June 7,
1995, incorporated by reference in its entirety herein.
Antibodies generated using the methods described in these applications
have previously been reported to display human effector function, have reduced
immunogenicity, and long serum half life. The technology relies on the fact
that despite the fact that cynomolgus monkeys are phylogenetically similar to
humans, they still recognize many human proteins as foreign and therefore
mount an immune response. Moreover, because the cynomolgus monkeys are
phylogenetically close to humans, the antibodies generated in these monkeys
have been discovered to have a high degree of amino acid homology to those
produced in humans. Indeed, after sequencing macaque immunoglobulin light
and heavy chain variable region genes, it was found that the sequence of each
gene family was 85-98% homologous to its human counterpart (Newman et al.,
1992). The first antibody generated in this way, an anti-CD4 antibody, was 91-
92% homologous to the consensus sequence of human immunoglobulin
framework regions (Newman et al., 1992).
As described above, the present invention relates, in part, to the use of
monoclonal antibodies or primatized forms thereof which are specific to human
CD40L antigen and which are capable of inhibiting CD40 signaling or
inhibiting CD40/CD40L interaction. Blocking of the primary activation site
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between CD40 and CD40L with the identified antibodies (or therapeutically
effective fragments thereof), while allowing the combined antagonistic effect
on
positive co-stimulation with an agnostic effect on negative signaling will be
a
useful therapeutic approach for intervening in relapsed forms of malignancy,
especially B-cell lymphomas and leukemias. The functional activity of the
identified antibodies is defined by blocking the signals of CD40 permitting it
to
survive and avoid IgM- or Fas-induced apoptosis.
Manufacture of monoclonal antibodies which specifically bind human
CD40L, as well as primatized antibodies derived therefrom can be performed
using the methods described in U.S. Patent Nos. 6,001,358 or 5,750,105, both
assigned to IDEC Pharmaceuticals Corporation, or other known methods.
Preferably, such antibodies will possess high affinity to CD40L and therefore
may be used as immunosuppressants which inhibit the CD40L/CD40 pathway.
Similar techniques will yield monkey antibodies specific to CD20, CD19, CD22
or CD40.
Preparation of monkey monoclonal antibodies will preferably be
effected by screening of phage display libraries or by preparation of monkey
heterohybridomas using B lymphocytes obtained from CD40L (e.g., human
CD40L) immunized monkeys. The human CD40 can also be from the fusion
protein described in U.S. Patent No. 5,945,513.
As noted, the first method for generating anti-CD40L, CD19, CD20,
CD22 or CD40 antibodies involves recombinant phage display technology.
This technique is generally described supra.
Essentially, this will comprise synthesis of recombinant immunoglobulin
libraries against the target, i.e., CD19, CD22, CD20, CD40, or CD40L antigen
displayed on the surface of filamentous phage and selection of phage which
secrete antibodies having high affinity to CD40L antigen. As noted supf~a,
preferably antibodies will be selected which bind to both human CD40L and
CD40. To effect such methodology, the present inventors have created a unique
library for monkey libraries which reduces the possibility of recombination
and
improves stability.
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Essentially, to adopt phage display for use with macaque libraries, this
vector contains specific primers for PCR amplifying monkey immunoglobulin
genes. These primers are based on macaque sequences obtained while
developing the primatized technology and databases containing human .
sequences.
Suitable primers are disclosed in commonly assigned 08/379,072,
incorporated by reference herein.
The second method involves the immunization of monkeys, i.e.,
macaques, against the desired antigen target, i.e., human CD19, CD20, CD22,
CD40 or CD40L. The inherent advantage of macaques for generation of
monoclonal antibodies is discussed supra. In particular, such monkeys, z.e.,
cynomolgus monkeys, may be immunized against human antigens or receptors.
Moreover, the resultant antibodies may be used to make primatized antibodies
according to the methodology of Newman et al., (1992), and Newman et al.,
commonly assigned U.S. Serial No. 08/379,072, filed January 25, 1995, which
are incorporated by reference in their entirety.
The significant advantage of antibodies obtained from cynomolgus
monkeys is that these monkeys recognize many human proteins as foreign and
thereby provide for the formation of antibodies, some with high affinity to
desired human antigens, e.g., human surface proteins and cell receptors.
Moreover, because they are phylogenetically close to humans, the resultant
antibodies exhibit a high degree of amino acid homology to those produced in
humans. As noted above, after sequencing macaque immunoglobulin light and
heavy variable region genes, it was found that the sequence of each gene
family
was 85-88% homologous to its human counterpart (Newman et al., 1992).
Essentially, cynomolgus macaque monkeys are administered human,
CD 19, CD20, CD22, CD40, or CD40L antigen, B cells are isolated therefrom,
e.g., lymph node biopsies are taken from the animals, and B lymphocytes are
then fused with I~H6/BS (mouse x human) heteromyeloma cells using
polyethylene glycol (PEG). Heterohybridomas secreting antibodies which bind
human CD40L antigen are then identified.
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In the case of antibodies which bind to CD40L or CD40, it is desirable
that they do so in a manner which interrupts or regulates CD40 signaling
because such antibodies potentially may be used to inhibit the interaction of
CD40L with CD40, with their counter-receptors. If antibodies can be
developed against more than one epitope on CD40L or CD40, and the
antibodies are utilized together, their combined activity may potentially
provide
synergistic effects.
The disclosed invention involves the use of an animal which is primed to
produce a particular antibody (e.g., primates, such as organgutan, baboons,
macaque, and cynomolgus monkeys). Other animals which may be used to
raise antibodies to human CD40L include, but are not limited to, the
following:
mice, rats, guinea pigs, hamsters, monkeys, pigs, goats and rabbits.
Cell lines which express antibodies which specifically bind to human
CD40L antigen are then used to clone variable domain sequences for the
manufacture of primatized antibodies essentially as described in Newman et
al.,
(1992) and Newman et al., U.S. Serial No. 379,072, filed 3anuary 25, 1995,
both of which are incorporated by reference herein. Essentially, this entails
extraction of RNA therefrom, conversion to cDNA, and amplification thereof
by PCR using Ig specific primers. Suitable primers are described in Newman et
al., 1992, and in U.S. Serial No. 379,072. Similar techniques will yield cell
lines
that express antibodies specific to CD40, CD19, CD20, or CD22.
Cell lines which express antibodies which specifically bind to human
CD40L antigen are then used to clone variable domain sequences for the
manufacture of primatized antibodies essentially as described in Newman et
al.,
(1992) and Newman et al., U.S. Serial No. 379,072, filed January 25, 1995,
both of which are incorporated by reference herein. Essentially, this entails
extraction of RNA therefrom, conversion to cDNA, and amplification thereof
by PCR using Ig specific primers. Suitable primers are described in Newman et
al., 1992, and in U.S. Serial No. 379,072. Similar techniques will yield cell
lines
that express antibodies specific to CD40, CD19, CD20, or CD22.
Cell lines which express antibodies which specifically bind to human
CD40L antigen are then used to clone variable domain sequences for the
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5 manufacture of primatized antibodies essentially as described in Newman et
al.,
(1992) and Newman et al., U.S. Serial No. 379,072, filed January 25, 1995,
both of which are incorporated by reference herein. Essentially, this entails
extraction of RNA therefrom, conversion to cDNA, and amplification thereof
by PCR using Ig specific primers. Suitable primers are described in Newman et
10 al., 1992, and in U.S. Serial No. 379,072. Similar techniques will yield
cell lines
that express antibodies specific to CD40, CD19, CD20, or CD22.
The cloned monkey variable genes are then inserted into an expression
vector which contains human heavy and light chain constant region genes.
Preferably, this is effected using a proprietary expression vector of IDEC,
Inc.,
15 referred to as NEOSPLA. This vector contains the cytomegalovirus
promoter/enhancer, the mouse beta globin major promoter, the SV40 origin of
replication, the bovine growth hormone polyadenylation sequence, neomycin
phosphotransferase exon 1 and exon 2, human immunoglobulin kappa or
lambda constant region, the dihydrofolate reductase gene, the human
20 immunoglobulin gamma 1 or gamma 4 PE constant region and leader sequence.
This vector has been found to result in very high level expression of
primatized
antibodies upon incorporation of monkey variable region genes, transfection in
CHO cells, followed by selection in 0418 containing medium and methotrexate
amplification.
25 For example, this expression system has been previously disclosed to
result in primatized antibodies having high avidity (I~d < 10-1° M)
against CD4
and other human cell surface receptors. Moreover, the antibodies have been
found to exhibit the same affinity, specificity and functional activity as the
original monkey antibody. This vector system is substantially disclosed in
30 commonly assigned U.S. Serial No. 379,072, incorporated by reference herein
as well as U.S. Serial No. 08/149,099, filed an November 3, 1993, also
incorporated by reference in its entirety herein. This system provides for
high
expression levels, i.e., > 30 pg/cell/day. Of course, the same methods can be
used to produce cell lines that produce antibodies specific to CD 19, CD20,
35 CD22, or CD40.
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(v) Huznazz azztibodies
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 PH) 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. LISA, 90:255 1 (1993); Jakobovits et
al.,
Natuz~e, 362:255-258 (1993); Bruggermann et al., Yeas' in inzznuno., 7:33
(1993);
and US Patent 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 izz vitz°o, 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., Cuz~rent Opinion izz Structuz~al Biology 3:564-57 1
(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
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array of antigens (including self antigens) can be isolated essentially
following
the techniques described by Marks et al., J.MoI. Biol, 222:581-597 (1991), or
Griffith et al., EMBO J. 12:725-734 (1993). See, also, US Patent Nos.
5,565,332 and 5,573,905.
Human antibodies may also be generated by in vitro activated B cells
(see US
Patents 20 5,567,610 and 5,229,275). A preferred means of generating human
antibodies using SCm mice is disclosed in commonly-owned, co-pending
applications.
(vi) Antibody fragments
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., .Iournal of Biochemical and
Biophysical Methods 24:107-117 (1992) and Brennan et al., Scienee, 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'-SH
fragments can be directly recovered from E. coli and chemically coupled to
form F(ab')2 fragments (Carter et al., BiolTeeJZnology 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; US
Patent No. 5,571,894; and US Patent No. 5,587,458. The antibody fragment
may also be a "linear antibody", e.g., as described in US Patent 5,641,870 for
example. Such linear antibody fragments may be monospecific or bispecific.
(vii) Bispecific antibodies
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
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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)2 bispecific antibodies).
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).
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
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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.
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
94104690. For further details of generating bispecific antibodies see, for
example, Suresh et al., Methods ih Enz~mology, 121:210 (1986).
According to another approach described in US Patent 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 chains) 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.
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5 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 (US Patent No. 4,676,980), and for
10 treatment of HIV infection (WO 91/00360, WO 921200373, 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 US Patent No. 4,676,980, along with a number of cross-linking
techniques.
15 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
20 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
25 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.
Recent progress has facilitated the direct recovery of Fab'-SH fragments
from E. coli, which can be chemically coupled to form bispecific antibodies.
30 Shalaby et al., J.Exp. Med., 175:2 17-225 (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
ifa
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
35 cells, as well as trigger the lytic activity of human cytotoxic lymphocytes
against human breast tumor targets.
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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.
I~ostelny 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.,
1'roc.Natl. Acael. 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. Accordingly, the VH and 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. Irnmunol., 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).
III. Conjugates and Other Modifications of the Antagonist
The antibodies 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
antibody-cytotoxic
agent conjugates have been described above.
Conjugates of an antibody and one or more small molecule toxins, such
as a calicheamicin, a maytansine (IIS Patent No. 5,208,020), a trichothene,
and
CC 1065 are also contemplated herein. In one preferred embodiment of the
invention, the antagonist is conjugated to one or more maytansine molecules
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(e.g. about 1 to about 10 maytansine molecules 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 (Charm et al. Cafzcer
Research 52:127-131(1992)) to generate a maytansinoid-antagonist conjugate.
Alternatively, the antibody may be 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, yli, oczI,
a,3I,
N-acetyl-yli, PSAG and OIl (Hinman et al. Cazzcer Researcla 53:3336-3342
(1993) and Lode et al, Cancer Reseaf-ch 58: 2925-2928 (1998)).
Enzymatically active toxins and fragments thereof which 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, Aleurites fordii proteins, dianthin proteins, Phytolaca
americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin,
restri.ctocin,
phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232
published October 28, 1993.
The present invention further contemplates antibody 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 Atzll, h31, hzs~ ~,90~ Reis6~
RE188, Smls3, Bizlz, P3z and radioactive isotopes of Lu.
Conjugates of the antibody and cytotoxic agent may be made using a
variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyriyldithiol)
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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), aldehydes
(such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)
hexanediamine), bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-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
(Charm et al. Cancer Research 52:127-131 (1992)) may be used.
Alternatively, a fusion protein comprising the antibody and cytotoxic
agent may be made, e.g. by recombinant techniques or peptide synthesis.
In yet another embodiment, the antibody 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 antibodies of the present invention may also be conjugated with a
prodrug activating enzyme which converts a prodrug (e.g. a peptidyl
chemotherapeutic agent, see WO81/01145) to an active anti-cancer drug. See,
for example, WO 88/07378 and U.S. Patent No. 4,975,278.
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.
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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-toxic5-fluorocytosine into the anti-cancer drug, 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;
carbohydratecleaving enzymes such as 13-galactosidase and neuraminidase
useful for converting glycosylated prodrugs into free drugs; 13-lactamase
useful
for converting drugs derivatized with 13-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.
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)).
Other modifications of the antibody are contemplated herein. For
example, the antibody 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.
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5 The antibodies 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. Natl. 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 October 23, 1997.
10 Liposomes with enhanced circulation time are disclosed in U.S. Patent No.
5,013,556.
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).
15 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. Claena. 257:286-288 (1982) via a disulfide interchange reaction. A
chemotherapeutic agent is optionally contained within the liposome. See
20 Gabizon et al. J.National Cancerlnst. 81(19)1484 (1989).
Amino acid sequence modifications) 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 antibody. Amino
acid sequence variants of the antibody are prepared by introducing appropriate
25 nucleotide changes into the antibody encoding 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 arnve at the final construct, provided that the final construct
possesses
30 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.
A useful method for identification of certain residues or regions of the
antibody that are preferred locations for mutagenesis is called "alanine
scanning
35 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
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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.
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 fusion to the N- or C-terminus of the antagonist of an enzyme, or
a
polypeptide which increases the serum half life of the antagonist.
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. 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.
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Table 1
Original Residuexemplary SubstitutionsPreferred Substitutions
la (A) al; leu; ile al
g (R) lys; gin; asn lys
sn (1~ . gln; his; asp, lys; gln
arg
sp (D) glu; asn glu
Cys (C) ser; ala ser
Gln (Q asn; glu asn
Glu (E) asp; gin asp
Gly (G) ala ala
is (H) asn; gin; lys; arg arg
1e (I) leu; val; met; ala; leu
he; norleucine
eu (L) orleucine; ile; val; ile
et; ala; phe
ys (K) arg; g 1 n; asn arg
et (M) leu; phe; ile leu
he (F) leu; val; ile; ala; yr
tyr
ro (P) ala ala
Ser (S) hr hr
Thr (T) ser ser
rp (W) yr; phe yr
yr (Y) rp; phe; thr; ser he
al (V) '1e; leu; met; phe; leu
ala; norleucine
Substantial modifications in the biological properties of the antibody 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
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chain. Naturally occurnng residues are divided into groups based on common
side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophiuic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gln, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
Non-conservative substitutions will entail exchanging a member of one
of these classes for another class.
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
bonds) may be added to the antagonist to improve its stability (particularly
where the antagonist is an antibody fragment such as an Fv fragment).
A particularly preferred type of substitutional variant involves
substituting one or more hypervariable region residues of a parent antibody
(e.g.
a humanized or human antibody). Generally, the resulting variants 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 identified hypervariable region residues contributing
significantly to antigen binding. Alternatively, or in addition, it may be
beneficial to analyze a crystal structure of the antigen-antibody complex to
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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.
Another type of amino acid variant of the antibody 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.
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 seine or threonine, although 5-hydroxyproline or 5-hydroxylysine
may also be used.
Addition of glycosylation sites to the antibody 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 seine or threonine residues to the sequence of the original
antagonist (for O-linked glycosylation sites).
Nucleic acid molecules encoding amino acid sequence variants of the
antibody are prepared by a variety of methods known in the art. These methods
include, but axe not limited to, isolation from a natural source (in the case
of
naturally occurnng amino acid sequence variants) or preparation by
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5 oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis,
and
cassette mutagenesis of an earlier prepared variant or a non-variant version
of
the antagonist.
It may be desirable to modify the antibodies used in the invention to
improve effector function, e.g. so as to enhance antigen-dependent
10 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 residues) may be introduced in the Fc region,
thereby
allowing interchain disulfide bond formation in this region. The homodimeric
15 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 heterobifunctional
20 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 3:2 19-230 (1989).
To increase the serum half life of the antibody, one may incorporate a
25 salvage receptor binding epitope into the antagonist (especially an
antibody
fragment) as described in US Patent 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., IgGI, IgG2, IgG3, or IgG4) that is responsible for increasing
the
30 in vivo serum half life of the IgG molecule.
IV. Pharmaceutical Formulations
Therapeutic formulations comprising antagonists used in accordance
with the present invention are prepared for storage by mixing an antagonist
35 having the desired degree of purity with optional pharmaceutically
acceptable
carriers, excipients or stabilizers (Renaington 's Pharmaceutical Scieraces
16th
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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 TWEENTM, PLURONICSTM or
polyethylene glycol (PEG).
The immunomodulatory antibody and the B cell depleting antibody may
be in the same formulation or may be administered in difficult formulations.
The composition may further include other non-antibody antagonists, e.g.,
CD40L or B7 antagonists. Examples there of include soluble CD40, B7 and
fusions thereof. Administration can be concurrent or sequential, and may be
effective in either order.
Exemplary anti-CD20 antibody formulations are described in
W098/56418, expressly incorporated herein by reference. This publication
describes a liquid multidose formulation comprising 40 mglmL 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°C.
Another
anti-CD20 formulation of interest comprises 1 OmglmL rituximab in 9.0 mg/mL
sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7mglniL polysorbate
80, and Sterile Water for Injection, pH 6.5.
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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.
The formulation herein may also contain more than one active
compound as 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 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.
The active ingredients may also be entrapped in microcapsules prepared,
for example, by 30 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
Renzington 's Plzarnaaceutical Sciences 16th edition, Osol, A. Ed. (1980).
Sustained-release preparations may be 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,
noir degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid
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copolymers such as the LUPRON DEPOTTM (injectable microspheres
composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. The formulations to be used for in vivo
administration must be sterile. This is readily accomplished by filtration
through
sterile filtration membranes.
V. Treatment with the B Cell Depleting Antibody and
Immunoregulatory Antibody
A composition comprising B cell depleting antibody and/or an
immunoregulatory antibody will be formulated, dosed, and administered in a
fashion consistent with good medical practice. Factors for consideration in
this
context include the particular B cell malignancy 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.
As noted previously, the B cell depleting antibody and the
immunoregulatory antibody may be in the same or in different formulations.
These antagonist formulations can be administered separately or concurrently,
and in either order. Preferably, the B cell depleting antibody specific to the
B
cell antigen target, e.g., CD20, CD19, CD22, CD37 or CD22, will be
administered separately from the immunoregulatory antibody, e.g., an anti-
CD40L antibody, anti-CD40 antibody, or anti-B7 antibody. Preferably, the
CD40L antibody will be the humanized anti-CD40L antibody disclosed in U.S.
Patent 6,001,358 and the anti-B7 antibody the primatized antibody disclosed in
US Patent 6,113,898. As noted, this antibody has recently been show to possess
apoptotic activity. Also the preferred CD40L antibody has been shown to have
efficacy in treatment of both T and B cell autoimmune diseases. Also, unlike
another humanized anti-CD40L antibody (Sc~) reported by Biogen, this
antibody is not known to cause any adverse toxicity.
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As a general proposition, the therapeutically effective amount of an
antibody administered parenterally per dose will typically be in the range of
about 0.1 to 500 mg/kg of patient body weight per day, with the typical
initial
range of antagonist used being in the range of about 2 to 100 mg/kg.
The preferred B cell depleting antibody is RITUXAN~. Suitable
dosages for such antibody are, for example, in the range from about 20mg1m2 to
about 1000mg/m2. The dosage of the antibody may be the same or different
from that presently recommended for RITUXAN~ for the treatment of non-
Hodgkin's lymphoma. For example, one may administer to the patient one or
more doses of substantially less than 375mg1m2 of the antibody, e.g. where the
dose is in the range from about 20mg/m2 to about 250mg/m2, for example from
about SOmg/m2 to about 200mg/m2.
Moreover, one may administer one or more initial doses) of the antibody
followed by one or more subsequent dose(s), wherein the mglm2 dose of the
antibody in the subsequent doses) exceeds the mg/mz dose of the antibody in
the
initial dose(s). For example, the initial dose may be in the range from about
20mg/m2 to about 250mg1m2 (e.g. from about 50mg1m2 to about 200mg/m2) and
the subsequent dose may be in the range from about 250mg/mz to about
1 OOOmg/mz.
As noted above, however, these suggested amounts of both
immunoregulatory and B cell depleting antibody 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. 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 particular
B
cell malignancy, 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.
The antibodies are administered by any suitable means, including
parenteral,
subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired
for
local
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5 immunosuppressive treatment, intralesional administration. Parenteral
infusions
include intramuscular, intravenous, intraarterial, intraperitoneal, or
subcutaneous administration. In addition, the antibody may suitably be
administered by pulse infusion, e.g., with declining doses of the antibody.
Preferably the dosing is given by injections, most preferably intravenous or
10 subcutaneous injections, depending in part on whether the administration is
brief or chronic.
One additionally may administer other compounds, such as
chemotherapeutic agents, immunosuppressive agents and/or cytokines with the
antibodies herein. The combined administration includes co-administration,
15 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.
Aside from administration of antibodies to the patient the present
20 application contemplates administration of antibodies by gene therapy. Such
administration of nucleic acid encoding the antibodies is encompassed by the
expression "administering a therapeutically effective amount of an
antagonist".
See, for example, W096/07321 published March 14, 1996 concerning the use of
gene therapy to generate intracellular antibodies.
25 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
30 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. Patent 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
35 cultured cells i~a vitro, or ifi vivo in the cells of the intended host.
Techniques
suitable for the transfer of nucleic acid into mammalian cells in vitro
include the
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use of liposomes, electroporation, microinjection, cell fusion, DEAF-dextran,
the calcium phosphate precipitation method, etc. A commonly used vector for
ex vivo delivery of the gene is a retrovirus.
The currently preferred ifa 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 andJor 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., .l. Biol. Chem 262:4429-4432 (1987); and Wagner et al., Proc. Natl.
Acad. Sci. USA 87:3410-3414(1990). For review of the currently known gene
marking and gene therapy protocols see Anderson et al., SciefZCe 256:808-8 13
(1992). See also WO 93/25673 and the references cited therein.
VI. Articles of Manufacture
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 may be 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
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solution bag or a vial having a stopper pierceable by a hypodermic injection
needle). As whole, there may be one or several compositions. At least one
active agent in one of those compositions is an antibody having B cell
depleting
activity and at least one antibody is an immunoregulatory antibody such as an
anti-CD40L, anti-CD40, anti-CD23, anti-CD4 or anti-B7 antibody. The label or
package insert indicates that the composition is used for treating a patient
having or predisposed to B cell malignancy, such as those listed hereinabove.
The article of manufacture may further comprise a second container comprising
a pharmaceutically acceptable 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.
Further details of the invention are illustrated by the following
non-limiting Examples. The disclosures of all citations in the specification
are
expressly incorporated herein by reference.
The antibodies of the invention may be administered to a human or other
animal in accordance with the aforementioned methods of treatment in an
amount sufficient to produce such effect to a therapeutic ox prophylactic
degree.
Such antibodies of the invention can be administered to such human or other
animal in a conventional dosage form prepared by combining the antibody of
the invention with a conventional pharmaceutically acceptable earner or
diluent
according to known techniques. It will be recognized by one of skill in the
art
that the form and character of the pharmaceutically acceptable carrier or
diluent
is dictated by the amount of active ingredient with which it is to be
combined,
the route of administration and other well-known variables.
The routine of administration of the antibody (or fragment thereof) of
the invention may be oral, parenteral, by inhalation or topical. The term
parenteral as used herein includes intravenous, intraperitoneal,
intramuscular,
subcutaneous, rectal or vaginal administration. The subcutaneous and
intramuscular forms of parenteral administration are generally preferred.
The daily parenteral and oral dosage regimes for employing compounds
of the invention to prophylactically or therapeutically induce
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immunosuppression, or to therapeutically treat carcinogenic tumors will
generally be in the range of about 0.05 to 100, but preferably about 0.5 to
10,
milligrams per kilogram body weight per day.
The antibodies of the invention may also be administered by inhalation.
By "inhalation" is meant intranasal and oral inhalation administration.
Appropriate dosage forms for such administration, such as an aerosol
formulation or a metered dose inhaler, may be prepared by conventional
techniques. The preferred dosage amount of a compound of the invention to be
employed is generally within the range of about 10 to 100 milligrams.
The antibodies of the invention may also be administered topically. By
topical administration is meant non-systemic administration and includes the
application of an antibody (or fragment thereof) compound of the invention
externally to the epidermis, to the buccal cavity and instillation of such an
antibody into the ear, eye and nose, and where it does not significantly enter
the
blood stream. By systemic administration is meant oral, intravenous,
intraperitoneal and intramuscular administration. The amount of an antibody
required for therapeutic or prophylactic effect will, of course, vary with the
antibody chosen, the nature and severity of the condition being treated and
the
animal.
EXAMPLES
Example 1
Properties o~lymphonaa cells. DHT 4 cells
The concept that anti-CD40L antibody could block CD40L-CD40
mediated survival of malignant B-cells from chemotherapy induced
toxicity/apoptosis was tested in vitro using IDEC-131, and the B-lymphoma cell
line, DHL-4 (Roos et al., Leuk. Res. 10: 195-202 (196)) exposed to adriamycin
(ADM). IDEC-131 is a humanized version of the murine, monoclonal
anti-human CD40L antibody, 24-31.
Initially, the minimum concentration of ADM cytotoxic to DHL-4 cells
was determined by exposing DHL-4 cells for 4 hours to different concentrations
of ADM. The cell cytotoxicity of DHL-4 cells after 5 days in culture was
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measured by Alamar Blue, a dye-reduction assay by live cells (see
Gazzano-Santoro et al., J. Immunol. lhletla. 202: 163-171 (1997)). Briefly, 1
x
105 DHL-4 cells in growth medium (RMPI-1640 plus 10% Fetal Calf Serum)
were incubated with varying concentrations of ADM (1 x 10-6 M to 1 x 10-8 M)
in cell culture tubes at 37 ° C. for 4 hours. After incubation, cells
were washed,
re-suspended in growth medium at 1 x 105 cells/ml concentration and 200 ~,1 of
cell suspension was added to each well of 96-well flat-bottom plate. Plates
were
incubated at 37 °C. and tested for cytotoxicity at different time
points. During
the last 18 hours of incubation, 50 p l of redox dye Alamar Blue (Biosource
International, Cat. #DAL 1100) was added to each well. Following incubation,
plates were cooled by incubating at room temperature for 10 minutes on a
shaker, and the intracellular reduction of the dye was determined.
Fluorescence
was read using a 96-well fluorometer with excitation at 530 nm and emission at
590 nm. The results are expressed as relative fluorescence units (RFU). The
percentage of cytotoxicity was calculated as follows:
[1- (average RFU of test sample = Average RFU of control cells)] x
100%. Titration curve of ADM cytotoxicity was established and minimal
concentrations of the drug for cytotoxicity was selected for subsequent
assays.
The results, as displayed in Fig. 1, shows cell cytotoxicity of DHL-4
cells cultured for 5 days after being exposed to ADM (2 x 10-~ M and 4 x 10-g.
M
of ADM) for 4 hours prior to culture. Cells were washed once after exposure
and cultured in growth medium for 5 days and cytotoxicity determined by
Alamar Blue dye-uptake assay, as described above. Additionally, the DHL-4
cells were characterized for the membrane expression of selected CD molecules
by flow cytometry. DHL-4 cells have been found to express CD19, CD20,
CD40 molecules, but no expression of CD40L was detected.
Example 2
Anti -CD40L antibody overf-ides CD40L mediated resistance to killira~y
to killira.~, by adriajny iya of-lynaphoma cells
Fig. 2A shows the effect of an anti-CD40L antibody on CD40L-CD40
mediated resistance of DHL-4 cells to cell death induced by ADM. DHL-4
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5 cells (0.5 x 106 cells/ml) were incubated in the presence of 10 ~g/ml of
soluble
CD40L (sCD40L, P. A. Brams, E. A. Padlan, I~. Hariharan, K. Slater, J.
Leonard, R. Noelle, and R. Newman, "A humanized anti-human CD 154
monoclonal antibody blocks CD 154-CD40 mediated human B cell activation,"
(manuscript submitteel)) for 1 hour at 37°C. After 1 hour of
incubation, low
10 concentrations of ADM (2 x 10-~ M - 4 x 10-8 M) were added and incubated
for
another 4 hours in the presence or absence of CD40L (10 ~.g/ml). Following
exposure to ADM, cells were washed and resuspended in growth medium at 0.5
x 106 cells/ml concentration, and 100 ~,l of cell suspension added to each
well of
96-well flat bottom plate, in duplicate, with or without sCD40L. sCD40L (10
15 ~,glml) was added to cultures that have been continuously exposed to sCD40L
during ADM treatment and to cultures that had no sCD40L during ADM
exposure. In addition, mEC-131 at 10 ~,g/ml was added to cultures to
determine its effect on DHL-4 cells incubated with sCD40L and ADM. After 5
days, the cytotoxicity was measured by Alamar Blue dye-uptake assay, as
20 described.
Data show that sCD40L prolonged survival of DHL-4 cells after ADM
treatment, whereas, as expected, increased cytotoxicity was observed in cells
that were exposed to ADM in the absence of sCD40L. Furthermore, addition of
anti-CD40L antibody (IDEC-131) reversed CD40L mediated cell survival,
25 leading to increase in cell cytotoxicity (Fig. 2A).
The addition of mEC-131 alone had no effect on DHL-4 cells treated
with sCD40L, which indicates that the antibody, by itself, does not have any
direct inhibitory or cytotoxic activities on DHL-4 cells (Fig. 2B). DHL-4
cells
pre-incubated with and without sCD40L were cultured in the presence of
30 different concentrations of mEC-131, RITUXAN~, the anti-CD20 antibody
CE9.1, and anti-CD4 antibodies (Anderson et al., Clin. InZmunol. c~
ImnaufaopatlZOl. 84: 73-84 (1997)). After 5 days, the
cytotoxicity/proliferation
of DHL-4 cells was determined by Alamar Blue assay, as described above. Fig.
2B shows no effect on the proliferation or the cytotoxicity of DHL-4 cells by
35 IDEC-131, whereas RITUXAN~, as expected, inhibited cell proliferation and
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induced cytotoxicity. No effect was seen in the DHL-4 cells cultured with
anti-CD4 antibodies.
Example 3
CD40L-CD40 s~nalirzg~revents ap~tosis ofB-lyjn laoma
cells by anti-CD20 antibody. RITZIXAN~
The effect of CD40L-CD40 mediated signaling on anti-CD20 antibody
induced apoptosis of B-lymphoma cells was determined using an in vitro
system involving DHL-4 cells and the surface cross-linking of RITUXAN~.
DHL-4 cells (0.5 to 1 x 106 cells/ml) were cultured with sCD40L (10 ~g/ml) at
37° C. After overnight culture, cells were harvested and incubated with
10
~,glml of RITU~~AN~ or the control antibody (CE9.1; an anti-CD4 antibody)
with or without sCD40L (10 ~,g/ml) on ice. After 1 hour of incubation, cells
were centrifuged to remove unbound antibodies, and resuspended at 1 x 106
cells/ml in growth medium (5% FCS-RPMI) and cultured in tissue culture
tubes. The cells surface bound antibodies were cross-linked by spiking F(ab')2
fragments of goat anti-human Ig-Fcy specific antibodies at 15 ~.glml, and the
cultures were incubated at 37° C. until assayed for apoptosis.
Apoptosis was
detected using a flow cytometry caspase-3 assay. Cultured cells were harvested
at 4 and 24 hours, washed and fixed at 4° C. using Cytofix
(Cytofix/CytopermTM
Kit, Pharmingen Cat. #2075KK). After 20 min of fixation, cells were washed
and 15 p,1 of affinity purified PE-conjugated polyclonal rabbit anti-caspase-3
antibody (Pharmingen, Cat. # 67345) and 50 ~l of cytoperm (Pharmingen; Cat.
#2075KK) were added. Cells were incubated on ice in the dark for 30 min.
After incubation cells were washed once and resuspended in cytoperm. Flow
cytometry data was acquired on FACScan and analyzed using WinList software
from Verity Software House.
Table I shows resistance of RITUXANC~ induced apoptosis in DHL-4
lymphoma cells by exposure to sCD40L. In these studies, activation of
caspase-3 Was used as the surrogate marker since our previous studies revealed
good correlation between caspase-3 and Tunel assay. Cross-linking of
RITU~AN~ on the DHL-4 cell surface in the presence of sCD40L decreased
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levels of apoptosis, whereas cells not exposed to sCD40L apoptosed. In
comparison, cultures incubated in the presence of an antibody of the same
isotype, control antibody (CE9.1), resulted in no apoptosis of the cells.
Thus,
the data suggests that sCD40L induced signaling of CD40 pathway can lead to
development of RITUXAN~ mediated killing of B-lymphoma cells.
Table I:
Resistance of RITUXAN~ mediated apoptosis of DHL-4 cells by
sCD40L
Culture Conditions % Apoptosis
(1VHI~~a~
4 Hours 24 Hours
DHT-4 cells exposed to sCD40L
Cells only 3.35 (17.42)4.94 (7.62)
Cells + RITUXAN 1.97 (1.97)4.54 (6.54)
Cells + RITUXAN + anti- 21.17 (17.39)9.62 (13.44)
hu.IgG.F(ab')a
Cells + CE9.1 2.31 (13.25)4.15 (7.85)
Cells + CE9.1 + anti-hu.IgG.F(ab')2 2.09 (22.14)4.14 (9.57)
Cells + anti-hu.IgG.F(ab')2 1.93 (12.57)5.13 (8.02)
DHL-4 cells not exposed
to sCD40L
Cells only 4.36 (14.34)5.08 (17.62)
Cells + RITUXAN 5.67 (10.66)1.08 (17.92)
Cells + RITUXAN + anti- 74.82 (22.80)30.63 (26.84)
hu.IgG.F(ab')2
Cells + CE9.1 5.99 (14.00)3.05 (18.24)
Cells + CE9.1 + anti-hu.I-G.F(ab')2 5.96 (12.11)2.24 (18.19)
Cells + anti-hu.IgG.F(ab')Z 6.09 (12.27)1.85 (17.27)
~a' Percent positive cells with caspase-3 activity and its mean fluorescent
intensity in log scale.
Example 4
Effect of IDEGl31 on the survival o chronic lytnphocytic leukemia ~CLL) cells
To determine the effect of IDEC-131 on the growth and survival of
B-CLL cells in vitro, B-CLL cells were cultured with and without IDEC-131 in
the presence of CD40L in vitro. Peripheral blood mononuclear cells (PBMC)
were isolated from a CLL patient's blood using a Ficoll-Hypaque gradient
centrifugation. Viability was determined by Trypan blue dye exclusion and was
>98%. Flow cytometric analysis revealed that >70% of the lymphocytes were
CD 19+1CD20+. CLL cells (PBMC) were cultured in CLL growth medium
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(e.g., RPMI-1640 medium supplemented with 5% FCS or 2% of autologous
donor plasma, supplemented with 2 mM L-Glutamine and 100 LTlml
Penicillin-Streptomycin). In addition, for some experiments, CD 19+ B-cells
were purified using CD 19+ DynabeadsTM as per manufacture's instructions
(Dynal, Cat. #111.03/111.04) and cultured as above. CLL or purified B-CLL
cells cultured in growth medium mostly under went spontaneous apoptotic cell
death. However, culturing these cells in the presence of sCD40L extended their
viability in cultures. Table II indicates the cell viability of CD 19+ B-CLL
cells
grown in the presence or absence of sCD40L (5 ~g/ml) at different time points
and indicates the longer survival of CLL cells. B-CLL cells from Patient #1
cultured with sCD40L had >_ 60% viability for greater than 2 weeks, whereas
cells grown in the absence of sCD40L had less than 10% viability.
Table II:
Survival of B-CLL cells in the presence of sCD40L
B-CLL Time %Viability~a~
Sam 1e (Hours) -) CD40L (+)
CD40L
Patient #1 0 _>90 _>90
48 88 90
96 46 77
144 30 72
Patient #2 0 _>90 _>90
72 40 72
96 31 65
144 17 51
~a~ equals
the percent
viability
determined
by Trypan
blue dye exclusion.
Fig. 3A shows the effect of IDEC-131 on the growth and survival of B-
CLL cells after 7 days in culture. Purified B-CLL cells from a CLL patient (2
x
106 cells/ml) were divided into two culture tubes. Cells in one tube were
mixed
with sCD40L (5 ~g/ml) in equal volume of growth medium, whereas the other
tube was incubated with equal volume of growth medium as control. After 1
hour of incubation at 37° C., cells were gently mixed and 100 ~l of
cell
suspension media added to each well of a 96-well flat bottom plate in
duplicate
with and without varying concentrations of IDEC-131 (10 ~g/ml to 0.3 ~,glml).
Seven days later, cell survival/death in culture was determined by Alamar Blue
assay, as described above. Data showed cell survival in cultures with sCD40L.
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The addition of IDEC-131 into culture resulted in increased cell death, which
indicated a reversal of cell survival or a sensitization to cell death.
Additionally, RITUXAN~ administered at the same concentration as the
IDEC-131 produced less of lower effect than IDEC-131 on cell death (Fig. 3B).
Example 5
CD40L-CD40 mediated up-re~ztlatiora ofHLA DR molecules in B-CLL
To determine whether the CD40L-CD40 signal transduction pathway is
intact, CLL cells from CLL patients were cultured (5 x 105 cellslml) with and
without 5 ~,g/ml of CD40L at 37 ° C. At 48 hours and 144 hours, the
class II
molecule, HLA-DR expression, was determined on CD 19~ cells by flow
cytometry using standard procedures. Briefly, cultured lymphocytes were
harvested at different time points and analyzed for surface expression of
molecules using antibodies coupled to either fluorescein (FITC) or
phycoerythrin (PE) for single or double staining using a FACScan
(Becton-Dickinson) flow cytometer. To stain for flow cytometry, 1 x 106 cells
in culture tubes were incubated with appropriate antibodies as follows:
anti-CD45-FITC to gate lymphocyte population on a scatter plot; anti-CD 19-PE
(Pharmingen, Cat. # 30655) or anti-CD20-FITC (Pharmingen; Cat. #33264)
antibodies to determine the CD19+ and/or CD20+ B-cells; anti-CD3-FITC
antibodies (Pharmingen; Cat. #30104) to gate-off the T cells; anti-CD 19-RPE
and anti-HLA-DR-FITC antibodies (Pharmingen; Cat. #32384) to determine the
Pclass II expression on CD19+ cells. Cells were washed once by centrifugation
(at 200 x g, for 6 min.) with 2 ml cold PBS and incubated with antibody for 30
min. on ice, after which the cells washed once, fixed in 0.5% paraformaldehyde
and stored at 4°C. until analyzed. Flow cytometry data was acquired on
FAGsan and analyzed using WinList software (Verity Software House). The
machine was set to autogating to allow examination of quadrants containing
cells that were single stained with either RPE or FITC, unstained or doubly
stained. Fig. 4 shows the comparison of HLA-DR expression in CD 19~ CLL
cells cultured with sCD40L and those cells not cultured with sCD40L. A higher
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5 level of HLA-DR expression was detected on B-CLL cells cultured in the
presence of sCD40L (Table III).
Table III
CD40L-CD40 mediated up-regulation of HLA-DR molecule in B-CLL
S ample Time HLA-DR+~a>
%Positive
MFI
Control 48 hrs 81 92
144 hrs 88 1655
Cells + sCD40L 48 hrs 88 101
144 hrs 95 2943
(a) CD19'~ B-cells that are positive for HLA-DR molecules and its mean
10 fluorescent intensity (MIF)
(b)
Example 6
Preparation of IDEC-131 and RITUXAN~
For treatment of a CD40~ malignancy, IDEC-131 at about IO to about
15 50 mg/ml in a formulation buffer 10 mM Na-citrate, 150 mM NaCl, 0.02%
Polysorbate 80 at pH 6.5 is infused intravenously (iv) to a subject. IDEC-131
is
administered before, after or in conjunction with RITUXAN~. The
RITUXANO dosage infused ranges from about 3 to about 10 mg/kg of subject
weight.
Example 7
Preparation ofIDEC 131 arid CHOP
For treatment of CD40+ malignancies responsive to CHOP (e.g.,
Hodgkin's Disease, Non-Hodgkin's lymphoma and chronic lymphocytic
leukemia, as well as salvage therapy for malignancies wherein cells are
CD40~),
IDEC-131 is infused at a dosage ranging from about 3 to about 10 mg per kg of
patient weight immediately prior to the initiation of the CHOP cycle.
IDEC-131 administration will be repeated prior to each CHOP cycle for a total
of 4 to 8 cycles.
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Example 8
Administf~ation ofanti- CD40L o~ anti-B7 ifa conabination with RITUXAN~
to tf~eat B-Bell l~a~homa in a subiect
Combination therapies are particularly useful as salvage therapies or for
treating relapsed or aggressive forms of CD40+ malignancies (e.g., Hodgkin's
Disease, Non-Hodgkin's lymphoma and CLL). When mEC-131 is to be
administered in combination With CHOP and RITUXAN~, mEC-131 is
administered as discussed above in Example 6, followed by the schedule
specified for CHOP-B7EC-131 administration in Example 7. Alternatively, the
same regimen is effected wherein mEC-131 (anti-CD40L) is substantially
within an anti-B7 antibody.
All references discussed above are hereby incorporated by reference in
their entirety.
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