U.S. patent application number 10/818765 was filed with the patent office on 2004-10-14 for therapy of autoimmune disease in a patient with an inadequate response to tnf-alpha inhibitor.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Benyunes, Mark C..
Application Number | 20040202658 10/818765 |
Document ID | / |
Family ID | 33299824 |
Filed Date | 2004-10-14 |
United States Patent
Application |
20040202658 |
Kind Code |
A1 |
Benyunes, Mark C. |
October 14, 2004 |
Therapy of autoimmune disease in a patient with an inadequate
response to TNF-alpha inhibitor
Abstract
The present application describes therapy with antagonists which
bind to B cell surface markers, such as CD20. In particular, the
application describes the use of such antagonists to treat
autoimmune disease in a mammal who experiences an inadequate
response to a TNF.alpha.-inhibitor.
Inventors: |
Benyunes, Mark C.; (San
Francisco, CA) |
Correspondence
Address: |
GENENTECH, INC.
1 DNA WAY
SOUTH SAN FRANCISCO
CA
94080
US
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
33299824 |
Appl. No.: |
10/818765 |
Filed: |
April 6, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60461481 |
Apr 9, 2003 |
|
|
|
Current U.S.
Class: |
424/144.1 |
Current CPC
Class: |
A61P 9/04 20180101; A61P
1/00 20180101; A61P 37/00 20180101; A61P 17/02 20180101; A61P 1/16
20180101; A61P 9/10 20180101; A61P 37/06 20180101; A61P 7/06
20180101; A61P 31/04 20180101; A61P 11/00 20180101; A61P 17/06
20180101; A61K 2039/505 20130101; A61P 11/06 20180101; A61P 27/02
20180101; A61P 7/00 20180101; A61P 37/02 20180101; A61P 29/00
20180101; A61P 31/00 20180101; A61P 31/18 20180101; C07K 16/2887
20130101; A61P 13/12 20180101; A61P 9/00 20180101; A61P 1/04
20180101; A61P 21/04 20180101; C07K 2317/24 20130101; A61P 15/00
20180101; A61P 21/02 20180101; A61P 37/08 20180101; A61P 3/06
20180101; A61P 19/02 20180101; A61P 25/00 20180101; A61P 3/10
20180101; A61P 31/06 20180101; A61P 43/00 20180101; A61P 17/00
20180101 |
Class at
Publication: |
424/144.1 |
International
Class: |
A61K 039/395 |
Claims
What is claimed is:
1. A method of treating an autoimmune disease in a mammal who
experiences an inadequate response to a TNF.alpha.-inhibitor,
comprising administering to the mammal a therapeutically effective
amount of an antagonist which binds to a B cell surface marker.
2. The method of claim 1 wherein the B cell surface marker is
selected from the group consisting of CD10, CD19, CD20, CD21, CD22,
CD23, CD24, CD37, CD40, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77,
CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85 and
CD86.
3. The method of claim 1 wherein the antagonist comprises an
antibody.
4. The method of claim 3 wherein the antibody binds CD20.
5. The method of claim 1 wherein the autoimmune disease is selected
from the group consisting of arthritis, rheumatoid arthritis,
juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis,
psoriasis, dermatitis, polymyositis/dermatomyositis, toxic
epidermal necrolysis, systemic scleroderma and sclerosis, responses
associated with inflammatory bowel disease, Crohn's disease,
ulcerative colitis, respiratory distress syndrome, adult
respiratory distress syndrome (ARDS), meningitis, encephalitis,
uveitis, colitis, glomerulonephritis, allergic conditions, eczema,
asthma, conditions involving infiltration of T cells and chronic
inflammatory responses, atherosclerosis, autoimmune myocarditis,
leukocyte adhesion deficiency, systemic lupus erythematosus (SLE),
juvenile onset diabetes, multiple sclerosis, allergic
encephalomyelitis, immune responses associated with acute and
delayed hypersensitivity mediated by cytokines and T-lymphocytes,
tuberculosis, sarcoidosis, granulomatosis including Wegener's
granulomatosis, agranulocytosis, vasculitis (including ANCA),
aplastic anemia, Diamond Blackfan anemia, immune hemolytic anemia
including autoimmune hemolytic anemia (AIHA), pernicious anemia,
pure red cell aplasia (PRCA), Factor VIII deficiency, hemophilia A,
autoimmune neutropenia, pancytopenia, leukopenia, diseases
involving leukocyte diapedesis, central nervous system (CNS)
inflammatory disorders, multiple organ injury syndrome, mysathenia
gravis, antigen-antibody complex mediated diseases, anti-glomerular
basement membrane disease, anti-phospholipid antibody syndrome,
allergic neuritis, Bechet disease, Castleman's syndrome,
Goodpasture's syndrome, Lambert-Eaton Myasthenic Syndrome,
Reynaud's syndrome, Sjorgen's syndrome, Stevens-Johnson syndrome,
solid organ transplant rejection, graft versus host disease (GVHD),
pemphigoid bullous, pemphigus, autoimmune polyendocrinopathies,
Reiter's disease, stiff-man syndrome, giant cell arteritis, immune
complex nephritis, IgA nephropathy, IgM polyneuropathies or IgM
mediated neuropathy, idiopathic thrombocytopenic purpura (ITP),
thrombotic throbocytopenic purpura (TTP), autoimmune
thrombocytopenia, autoimmune disease of the testis and ovary
including autoimune orchitis and oophoritis, primary
hypothyroidism; autoimmune endocrine diseases including autoimmune
thyroiditis, chronic thyroiditis (Hashimoto's Thyroiditis),
subacute thyroiditis, idiopathic hypothyroidism, Addison's disease,
Grave's disease, autoimmune polyglandular syndromes (or
polyglandular endocrinopathy syndromes), Type I diabetes also
referred to as insulin-dependent diabetes mellitus (IDDM) and
Sheehan's syndrome; autoimmune hepatitis, lymphoid interstitial
pneumonitis (HIV), bronchiolitis obliterans (non-transplant) vs
NSIP, Guillain-Barre' Syndrome, large vessel vasculitis (including
polymyalgia rheumatica and giant cell (Takayasu's) arteritis),
medium vessel vasculitis (including Kawasaki's disease and
polyarteritis nodosa), ankylosing spondylitis, Berger's disease
(IgA nephropathy), rapidly progressive glomerulonephritis, primary
biliary cirrhosis, Celiac sprue (gluten enteropathy),
cryoglobulinemia, amyotrophic lateral sclerosis (ALS), coronary
artery disease.
6. The method of claim 1 wherein the mammal is human.
7. The method of claim 3 wherein the antibody is not conjugated
with a cytotoxic agent.
8. The method of claim 4 wherein the antibody comprises
rituximab.
9. The method of claim 4 wherein the antibody comprises humanized
2H7 v16 comprising the variable domains as in SEQ ID Nos. 1 &
2.
10. The method of claim 3 wherein the antibody is conjugated with a
cytotoxic agent.
11. The method of claim 1 which consists essentially of
administering the antagonist to the mammal.
12. A method of treating rheumatoid arthritis in a mammal who
experiences an inadequate response to a TNF.alpha.-inhibitor,
comprising administering to the mammal a therapeutically effective
amount of an antibody that binds to CD20.
13. A method of reducing the risk of a negative side effect
selected from the group consisting of an infection, heart failure
and demyelination, comprising administering to a mammal with an
autoimmune disease a therapeutically effective amount of an
antagonist which binds to a B cell surface marker.
Description
[0001] This is a non-provisional application claiming priority
under 35 USC .sctn.119 to provisional application No. 60/461,481
filed Apr. 9, 2003, the entire disclosure of which is hereby
incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention concerns therapy with antagonists
which bind to B cell surface markers, such as CD20. In particular,
the invention concerns the use of such antagonists to treat
autoimmune disease in a mammal who experiences an inadequate
response to a TNF.alpha.-inhibitor.
BACKGROUND OF THE INVENTION
[0003] Lymphocytes are one of many types of white blood cells
produced in the bone marrow during the process of hematopoiesis.
There are two major populations of lymphocytes: B lymphocytes (B
cells) and T lymphocytes (T cells). The lymphocytes of particular
interest herein are B cells.
[0004] B cells mature within the bone marrow and leave the marrow
expressing an antigen-binding antibody on their cell surface. When
a naive B cell first encounters the antigen for which its
membrane-bound antibody is specific, the cell begins to divide
rapidly and its progeny differentiate into memory B cells and
effector cells called "plasma cells". Memory B cells have a longer
life span and continue to express membrane-bound antibody with the
same specificity as the original parent cell. Plasma cells do not
produce membrane-bound antibody but instead produce the antibody in
a form that can be secreted. Secreted antibodies are the major
effector molecule of humoral immunity.
[0005] The CD20 antigen (also called human B-lymphocyte-restricted
differentiation antigen, Bp35) is a hydrophobic transmembrane
protein with a molecular weight of approximately 35 kD located on
pre-B and mature B lymphocytes (Valentine et al. J. Biol. Chem.
264(19):11282-11287 (1989); and Einfeld et al. EMBO J. 7(3):711-717
(1988)). The antigen is also expressed on greater than 90% of B
cell non-Hodgkin's lymphomas (NHL) (Anderson et al. Blood
63(6):1424-1433 (1984)), but is not found on hematopoietic stem
cells, pro-B cells, normal plasma cells or other normal tissues
(Tedder et al. J. Immunol. 135(2):973-979 (1985)). CD20 regulates
an early step(s) in the activation process for cell cycle
initiation and differentiation (Tedder et al., supra) and possibly
functions as a calcium ion channel (Tedder et al. J. Cell. Biochem.
14D:195 (1990)).
[0006] Given the expression of CD20 in B cell lymphomas, this
antigen can serve as a candidate for "targeting" of such lymphomas.
In essence, such targeting can be generalized as follows:
antibodies specific to the CD20 surface antigen of B cells are
administered to a patient. These anti-CD20 antibodies specifically
bind to the CD20 antigen of (ostensibly) both normal and malignant
B cells; the antibody bound to the CD20 surface antigen may lead to
the destruction and depletion of neoplastic B cells. Additionally,
chemical agents or radioactive labels having the potential to
destroy the tumor can be conjugated to the anti-CD20 antibody such
that the agent is specifically "delivered" to the neoplastic B
cells. Irrespective of the approach, a primary goal is to destroy
the tumor; the specific approach can be determined by the
particular anti-CD20 antibody which is utilized and, thus, the
available approaches to targeting the CD20 antigen can vary
considerably.
[0007] CD19 is another antigen that is expressed on the surface of
cells of the B lineage. Like CD20, CD19 is found on cells
throughout differentiation of the lineage from the stem cell stage
up to a point just prior to terminal differentiation into plasma
cells (Nadler, L. Lymphocyte Typing II 2: 3-37 and Appendix,
Renling et al. eds. (1986) by Springer Verlag). Unlike CD20
however, antibody binding to CD19 causes internalization of the
CD19 antigen. CD19 antigen is identified by the HD237-CD19 antibody
(also called the "B4" antibody) (Kiesel et al. Leukemia Research
II, 12: 1119 (1987)), among others. The CD19 antigen is present on
4-8% of peripheral blood mononuclear cells and on greater than 90%
of B cells isolated from peripheral blood, spleen, lymph node or
tonsil. CD19 is not detected on peripheral blood T cells, monocytes
or granulocytes. Virtually all non-T cell acute lymphoblastic
leukemias (ALL), B cell chronic lymphocytic leukemias (CLL) and B
cell lymphomas express CD19 detectable by the antibody B4 (Nadler
et al. J. Immunol. 131:244 (1983); and Nadler et al. in Progress in
Hematology Vol. XII pp. 187-206. Brown, E. ed. (1981) by Grune
& Stratton, Inc).
[0008] Additional antibodies which recognize differentiation
stage-specific antigens expressed by cells of the B cell lineage
have been identified. Among these are the B2 antibody directed
against the CD21 antigen; B3 antibody directed against the CD22
antigen; and the J5 antibody directed against the CD10antigen (also
called CALLA). See U.S. Pat. No. 5,595,721 issued Jan. 21, 1997
(Kaminski et al.).
[0009] The rituximab (RITUXAN.RTM.) antibody is a genetically
engineered chimeric murine/human monoclonal antibody directed
against the CD20 antigen. Rituximab is the antibody called "C2B8"
in U.S. Pat. No. 5,736,137 issued Apr. 7, 1998 (Anderson et al.).
RITUXAN.RTM. is indicated for the treatment of patients with
relapsed or refractory low-grade or follicular, CD20 positive, B
cell non-Hodgkin's lymphoma. In vitro mechanism of action studies
have demonstrated that RITUXAN.RTM. binds human complement and
lyses lymphoid B cell lines through complement-dependent
cytotoxicity (CDC) (Reff et al. Blood 83(2):435-445 (1994)).
Additionally, it has significant activity in assays for
antibody-dependent cellular cytotoxicity (ADCC). More recently,
RITUXAN.RTM. has been shown to have anti-proliferative effects in
tritiated thymidine incorporation assays and to induce apoptosis
directly, while other anti-CD19 and CD20 antibodies do not (Maloney
et al. Blood 88(10):637a (1996)). Synergy between RITUXAN.RTM. and
chemotherapies and toxins has also been observed experimentally. In
particular, RITUXAN.RTM. sensitizes drug-resistant human B cell
lymphoma cell lines to the cytotoxic effects of doxorubicin, CDDP,
VP-16, diphtheria toxin and ricin (Demidem et al. Cancer
Chemotherapy & Radiopharmaceuticals 12(3): 177-186 (1997)). In
vivo preclinical studies have shown that RITUXAN.RTM. depletes B
cells from the peripheral blood, lymph nodes, and bone marrow of
cynomolgus monkeys, presumably through complement and cell-mediated
processes (Reff et al. Blood 83(2):435-445 (1994)).
[0010] Patents and patent publications concerning CD20 antibodies
include U.S. Pat. Nos. 5,776,456, 5,736,137, 6,399,061, and
5,843,439, as well as US patent appln Nos. US 2002/0197255A1 and US
2003/0021781A1 (Anderson et al.); U.S. Pat. No. 6,455,043B1 and
WO00/09160 (Grillo-Lopez, A.); WO00/27428 (Grillo-Lopez and White);
WO00/27433 (Grillo-Lopez and Leonard); WO00/44788 (Braslawsky et
al.); WO01/10462 (Rastetter, W.); WO01/10461 (Rastetter and White);
WO01/10460 (White and Grillo-Lopez); US appln No. US2002/0006404
and WO02/04021 (Hanna and Hariharan); US appln No. US2002/0012665
A1 and WO01/74388 (Hanna, N.); US appln No. US2002/0009444A1, and
WO01/80884 (Grillo-Lopez, A.); WO01/97858 (White, C.); US appln No.
US2002/0128488A1 and WO02/34790 (Reff, M.);WO02/060955 (Braslawsky
et al.);WO2/096948 (Braslawsky et al.);WO02/079255 (Reff and
Davies); U.S. Pat. No. 6,171,586B1, and WO98/56418 (Lam et al.);
WO98/58964 (Raju, S.); WO99/22764 (Raju, S.);WO99/51642, U.S. Pat.
No. 6,194,551B1, U.S. Pat. No. 6,242,195B1, U.S. Pat. No.
6,528,624B1 and U.S. Pat. No. 6,538,124 (Idusogie et al.);
WO00/42072 (Presta, L.); WO00/67796 (Curd et al.); WO01/03734
(Grillo-Lopez et al.); US appln No. US 2002/0004587A1 and
WO01/77342 (Miller and Presta); US appln No. US2002/0197256
(Grewal, I.); U.S. Pat. Nos. 6,090,365B1, 6,287,537B1, 6,015,542,
5,843,398, and 5,595,721, (Kaminski et al.); U.S. Pat. Nos.
5,500,362, 5,677,180, 5,721,108, and 6,120,767 (Robinson et al.);
U.S. Pat No. 6,410,391B1 (Raubitschek et al.); U.S. Pat. No.
6,224,866B1 and WO00/20864 (Barbera-Guillem, E.); WO01/13945
(Barbera-Guillem, E.); WO00/67795 (Goldenberg); WO00/74718
(Goldenberg and Hansen); WO00/76542 (Golay et al.);WO01/72333
(Wolin and. Rosenblatt); U.S. Pat. No. 6,368,596B1 (Ghetie et al.);
US Appln No. US2002/0041847A1, (Goldenberg, D.); US Appln no.
US2003/0026801A1 (Weiner and Hartmann); WO02/102312 (Engleman, E.),
each of which is expressly incorporated herein by reference. See,
also, U.S. Pat. No. 5,849,898 and EP appln No. 330,191 (Seed et
al.); U.S. Pat. No. 4,861,579 and EP332,865A2 (Meyer and Weiss);
and WO95/03770 (Bhat et al.).
[0011] Publications concerning therapy with Rituximab include:
Perotta and Abuel "Response of chronic relapsing ITP of 10 years
duration to Rituximab" Abstract # 3360 Blood 10(1)(part 1-2): p.
88B (1998); Stashi et al. "Rituximab chimeric anti-CD20 monoclonal
antibody treatment for adults with chronic idopathic
thrombocytopenic purpura" Blood 98(4):952-957 (2001); Matthews, R.
"Medical Heretics" New Scientist (7 Apr., 2001); Leandro et al.
"Clinical outcome in 22 patients with rheumatoid arthritis treated
with B lymphocyte depletion" Ann Rheum Dis 61:833-888 (2002);
Leandro et al. "Lymphocyte depletion in thrumatoid arthritis: early
evidence for safety, efficacy and dose response. Arthritis and
Rheumatism 44(9): S370 (2001); Leandro et al. "An open study of B
lymphocyte depletion in systemic lupus erythematosus", Arthritis
& Rheumatism 46(1):2673-2677 (2002); Edwards and Cambridge
"Sustained improvement in rheumatoid arthritis following a protocol
designed to deplete B lymphocytes" Rhematology 40:205-211 (2001);
Edwards et al. "B-lymphocyte depletion therapy in rheumatoid
arthritis and other autoimmune disorders" Biochem. Soc. Trans.
30(4):824-828 (2002); Edwards et al. "Efficacy and safety of
Rituximab, a B-cell targeted chimeric monoclonal antibody: A
randomized, placebo controlled trial in patients with rheumatoid
arthritis. Arthritis and Rheumatism 46(9): S197 (2002); Levine and
Pestronk "IgM antibody-related polyneuropathies: B-cell depletion
chemotherapy using Rituximab" Neurology 52: 1701-1704 (1999);
DeVita et al. "Efficacy of selective B cell blockade in the
treatment of rheumatoid arthritis" Arthritis & Rheum
46:2029-2033 (2002); Hidashida et al. "Treatment of
DMARD-Refractory rheumatoid arthritis with rituximab." Presented at
the Annual Scientific Meeting of the American College of
Rheumatology; October 24-29; New Orleans, La. 2002; Tuscano, J.
"Successful treatment of Infliximab-refractory rheumatoid arthritis
with rituximab" Presented at the Annual Scientific Meeting of the
American College of Rheumatology; October 24-29; New Orleans, La.
2002.
[0012] Rhematoid arthritis (RA) is an autoimmune disorder of
unknown etiology. Most RA patients suffer a chronic course of
disease that, even with therapy, may result in progressive joint
destruction, deformity, disability and even premature death. More
than 9 million physician visits and more than 250,000
hospitalizations per year result from RA. The goals of RA therapy
are to prevent or control joint damage, prevent loss of function
and decrease pain. Initial therapy of RA usually involves
administration of one or more of the following drugs: nonsteroidal
antiinflammatory drugs (NSAIDs), glucocorticoid (via joint
injection), and low-dose prednisone. See "Guidelines for the
management of rheumatoid arthritis" Arthritis & Rheumatism
46(2): 328-346 (February, 2002). The majority of patients with
newly diagnosed RA are started with disease-modifying antirheumatic
drug (DMARD) therapy within 3 months of diagnosis. DMARDs commonly
used in RA are hydroxycloroquine, sulfasalazine, methotrexate,
leflunomide, etanercept, infliximab (plus oral and subcutaneous
methrotrexate), azathioprine, D-penicillamine, Gold (oral), Gold
(intramuscular), minocycline, cyclosporine, Staphylococcal protein
A immunoadsorption.
[0013] Because the body produces tumor necrosis factor alpha
(TNF.alpha.) during RA, TNF.alpha. inhibitors have used for therapy
of that disease.
[0014] Etanercept (ENBREL.RTM.) is an injectable drug approved in
the US for therapy of active RA. Etanercept binds to TNF.alpha. and
serves to remove most TNF.alpha. from joints and blood, thereby
preventing TNF.alpha. from promoting inflammation and other
symptoms of rheumatoid arthritis. Etanercept is an "immunoadhesin"
fusion protein consisting of the extracellular ligand binding
portion of the human 75 kD (p75) tumor necrosis factor receptor
(TNFR) linked to the Fc portion of a human IgG1. The drug has been
associated with negative side effects including serious infections
and sepsis, nervous system disorders such as multiple sclerosis
(MS). See, e.g., www.remicade-infliximab.com/pages/enbrel_embre-
l.html
[0015] Infliximab, sold under the trade name REMICADE.RTM., is an
immune-suppressing drug prescribed to treat RA and Crohn's disease.
Infliximab is a chimeric monoclonal antibody that binds to
TNF.alpha. and reduces inflammation in the body by targeting and
binding to TNF.alpha. which produces inflammation. Infliximab has
been linked to a fatal reactions such as heart failure and
infections including tuberculosis as well as demyelination
resulting in MS.
[0016] In December 2002, Abbott Laboratories received FDA approval
to market adalimumab (HUMIRA.TM.), previously known as D2E7.
Adalimumab is a human monoclonal antibody that binds to TNF.alpha.
and is approved for reducing the signs and symptoms and inhibiting
the progression of structural damage in adults with moderately to
severely active RA who have had insufficient response to one or
more traditional disease modifying DMARDs.
SUMMARY OF THE INVENTION
[0017] The present invention provides, in a first aspect, a method
of treating an autoimmune disease in a mammal who experiences an
inadequate response to a TNF.alpha.-inhibitor, comprising
administering to the mammal a therapeutically effective amount of
an antagonist which binds to a B cell surface marker.
[0018] For instance, the invention provides a method of treating
rhematoid arthritis in a mammal who experiences an inadequate
response to a TNF.alpha.-inhibitor, comprising administering to the
mammal a therapeutically effective amount of an antibody that binds
to CD20.
[0019] The invention also concerns a method of reducing the risk of
a negative side effect selected from the group consisting of an
infection, heart failure and demyelination, comprising
administering to a mammal with an autoimmune disease a
therapeutically effective amount of an antagonist which binds to a
B cell surface marker.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
[0020] For the purposes herein, "tumor necrosis factor alpha
(TNF.alpha.)" refers to a human TNF.alpha. molecule comprising the
amino acid sequence as described in Pennica et al., Nature, 312:721
(1984) or Aggarwal et al., JBC, 260:2345 (1985).
[0021] A "TNF.alpha. inhibitor" herein is an agent that inhibits,
to some extent, a biological function of TNF.alpha., generally
through binding to TNF.alpha. and neutralizing its activity.
Examples of TNF inhibitors specifically contemplated herein are
Etanercept (ENBREL.RTM.), Infliximab (REMICADE.RTM.) and Adalimumab
(HUMIRA.TM.).
[0022] The term "inadequate response to a TNF.alpha.-inhibitor"
refers to an inadequate response to previous or current treatment
with a TNF.alpha.-inhibitor because of toxicity and/or inadequate
efficacy. The inadequate response can be assessed by a clinician
skilled in treating the disease in question.
[0023] A mammal who experiences "toxicity" from previous or current
treatment with the TNF.alpha.-inhibitor experiences one or more
negative side-effects associated therewith such as infection
(especially serious infections), congestive heart failure,
demyelination (leading to multiple sclerosis), hypersensitivity,
neurologic events, autoimmunity, non-Hodgkin's lymphoma,
tuberculosis (TB), autoantibodies, etc.
[0024] A mammal who experiences "inadequate efficacy" continues to
have active disease following previous or current treatment with a
TNF.alpha.-inhibitor. For instance, the patient may have active
disease activity after 1 month or 3 months of therapy with the
TNF.alpha.-inhibitor.
[0025] By "reducing the risk of a negative side effect" is meant
reducing the risk of a side effect resulting from therapy with the
antagonist that binds to a B-cell surface marker to a lower extent
than that seen with therapy with a TNF.alpha.-inhibitor. Such side
effects include infection (especially serious infections), heart
failure, and demyelination (multiple sclerosis), etc.
[0026] 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. Exemplary B cell surface markers include the
CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, 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 is CD20.
[0027] The "CD20" antigen is a .about.35 kDa, non-glycosylated
phosphoprotein found on the surface of greater than 90% of B cells
from peripheral blood or lymphoid organs. CD20 is expressed during
early pre-B cell development and remains until plasma cell
differentiation. CD20 is present on both normal B cells as well as
malignant B cells. Other names for CD20 in the literature include
"B-lymphocyte-restricted antigen" and "Bp35". The CD20 antigen is
described in Clark et al. PNAS (USA) 82:1766 (1985), for
example.
[0028] An "autoimmune disease" herein is a disease or disorder
arising from and directed against an individual's own tissues.
Examples of autoimmune diseases or disorders include, but are not
limited to arthritis (rheumatoid arthritis, juvenile rheumatoid
arthritis, osteoarthritis, psoriatic arthritis), psoriasis,
dermatitis, polymyositis/dermatomyositis, toxic epidermal
necrolysis, systemic scleroderma and sclerosis, responses
associated with inflammatory bowel disease, Crohn's disease,
ulcerative colitis, respiratory distress syndrome, adult
respiratory distress syndrome (ARDS), meningitis, encephalitis,
uveitis, colitis, glomerulonephritis, allergic conditions, eczema,
asthma, conditions involving infiltration of T cells and chronic
inflammatory responses, atherosclerosis, autoimmune myocarditis,
leukocyte adhesion deficiency, systemic lupus erythematosus (SLE),
juvenile onset diabetes, multiple sclerosis, allergic
encephalomyelitis, immune responses associated with acute and
delayed hypersensitivity mediated by cytokines and T-lymphocytes,
tuberculosis, sarcoidosis, granulomatosis including Wegener's
granulomatosis, agranulocytosis, vasculitis (including ANCA),
aplastic anemia, Diamond Blackfan anemia, immune hemolytic anemia
including autoimmune hemolytic anemia (AIHA), pernicious anemia,
pure red cell aplasia (PRCA), Factor VIII deficiency, hemophilia A,
autoimmune neutropenia, pancytopenia, leukopenia, diseases
involving leukocyte diapedesis, central nervous system (CNS)
inflammatory disorders, multiple organ injury syndrome, mysathenia
gravis, antigen-antibody complex mediated diseases, anti-glomerular
basement membrane disease, anti-phospholipid antibody syndrome,
allergic neuritis, Bechet disease, Castleman's syndrome,
Goodpasture's syndrome, Lambert-Eaton Myasthenic Syndrome,
Reynaud's syndrome, Sjorgen's syndrome, Stevens-Johnson syndrome,
solid organ transplant rejection, graft versus host disease (GVHD),
pemphigoid bullous, pemphigus, autoimmune polyendocrinopathies,
Reiter's disease, stiff-man syndrome, giant cell arteritis, immune
complex nephritis, IgA nephropathy, IgM polyneuropathies or IgM
mediated neuropathy, idiopathic thrombocytopenic purpura (ITP),
thrombotic throbocytopenic purpura (TTP), autoimmune
thrombocytopenia, autoimmune disease of the testis and ovary
including autoimune orchitis and oophoritis, primary
hypothyroidism; autoimmune endocrine diseases including autoimmune
thyroiditis, chronic thyroiditis (Hashimoto's Thyroiditis),
subacute thyroiditis, idiopathic hypothyroidism, Addison's disease,
Grave's disease, autoimmune polyglandular syndromes (or
polyglandular endocrinopathy syndromes), Type I diabetes also
referred to as insulin-dependent diabetes mellitus (IDDM) and
Sheehan's syndrome; autoimmune hepatitis, lymphoid interstitial
pneumonitis (HIV), bronchiolitis obliterans (non-transplant) vs
NSIP, Guillain-Barre' syndrome, large vessel vasculitis (including
polymyalgia rheumatica and giant cell (Takayasu's) arteritis),
medium vessel vasculitis (including Kawasaki's disease and
polyarteritis nodosa), ankylosing spondylitis, Berger's disease
(IgA nephropathy), rapidly progressive glomerulonephritis, primary
biliary cirrhosis, Celiac sprue (gluten enteropathy),
cryoglobulinemia, amyotrophic lateral sclerosis (ALS), coronary
artery disease etc.
[0029] An "antagonist" is a molecule which, upon binding to a B
cell surface marker, destroys or depletes B cells in a mammal
and/or interferes with one or more B cell functions, e.g. by
reducing or preventing a humoral response elicited by the B cell.
The antagonist preferably is able to deplete B cells (i.e. reduce
circulating B cell levels) in a mammal treated therewith. Such
depletion may be achieved via various mechanisms such
antibody-dependent cell-mediated cytotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC), inhibition of B cell
proliferation and/or induction of B cell death (e.g. via
apoptosis). Antagonists included within the scope of the present
invention include antibodies, synthetic or native sequence peptides
and small molecule antagonists which bind to the B cell marker,
optionally conjugated with or fused to a cytotoxic agent. The
preferred antagonist comprises an antibody.
[0030] "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 Fc.gamma.RIII
only, whereas monocytes express Fc.gamma.RI, Fc.gamma.RII and
Fc.gamma.RIII. FcR expression on hematopoietic cells in summarized
is Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol
9:457-92 (1991). To assess ADCC activity of a molecule of interest,
an in vitro ADCC assay, such as that described in U.S. Pat. Nos.
5,500,362 or 5,821,337 may be performed. Useful effector cells for
such assays include peripheral blood mononuclear cells (PBMC) and
Natural Killer (NK) cells. Alternatively, or additionally, ADCC
activity of the molecule of interest may be assessed in vivo, e.g.,
in a animal model such as that disclosed in Clynes et al. PNAS
(USA) 95:652-656 (1998).
[0031] "Human effector cells" are leukocytes which express one or
more FcRs and perform effector functions. Preferably, the cells
express at least Fc.gamma.RIII and carry out ADCC effector
function. Examples of human leukocytes which mediate ADCC include
peripheral blood mononuclear cells (PBMC), natural killer (NK)
cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and
NK cells being preferred.
[0032] 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 Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma. RIII
subclasses, including allelic variants and alternatively spliced
forms of these receptors. Fc.gamma.RII receptors include
Fc.gamma.RIIA (an "activating receptor") and Fc.gamma.RIIB (an
"inhibiting receptor"), which have similar amino acid sequences
that differ primarily in the cytoplasmic domains thereof.
Activating receptor Fc.gamma.RIIA contains an immunoreceptor
tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
Inhibiting receptor Fc.gamma.RIIB contains an immunoreceptor
tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain.
(see Daron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are
reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991);
Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J.
Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to
be identified in the future, are encompassed by the term "FcR"
herein. The term also includes the neonatal receptor, FcRn, which
is responsible for the transfer of maternal IgGs to the fetus
(Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J.
Immunol. 24:249 (1994)).
[0033] "Complement dependent cytotoxicity" or "CDC" refer to the
ability of a molecule to lyse a target in the presence of
complement. The complement activation pathway is initiated by the
binding of the first component of the complement system (C1q) 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.
[0034] "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.
[0035] Antagonists which "induce apoptosis" are those which induce
programmed cell death, e.g. of a B cell, as determined by standard
apoptosis assays, such as binding of annexin V, fragmentation of
DNA, cell shrinkage, dilation of endoplasmic reticulum, cell
fragmentation, and/or formation of membrane vesicles (called
apoptotic bodies).
[0036] 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.
[0037] "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').sub.2, and Fv fragments; diabodies; linear antibodies;
single-chain antibody molecules; and multispecific antibodies
formed from antibody fragments.
[0038] "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 (V.sub.H) followed by
a number of constant domains. Each light chain has a variable
domain at one end (V.sub.L) 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.
[0039] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
hypervariable regions both in the light chain and the heavy chain
variable domains. The more highly conserved portions of variable
domains are called the framework regions (FRs). The variable
domains of native heavy and light chains each comprise four FRs,
largely adopting a .beta.-sheet configuration, connected by three
hypervariable regions, which form loops connecting, and in some
cases forming part of, the .beta.-sheet structure. The
hypervariable regions in each chain are held together in close
proximity by the FRs and, with the hypervariable regions from the
other chain, contribute to the formation of the antigen-binding
site of antibodies (see Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)). The constant domains
are not involved directly in binding an antibody to an antigen, but
exhibit various effector functions, such as participation of the
antibody in antibody dependent cellular cytotoxicity (ADCC).
[0040] 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').sub.2 fragment that has two antigen-binding sites
and is still capable of cross-linking antigen.
[0041] "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 V.sub.H-V.sub.L 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.
[0042] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) 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 CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear at least one free thiol
group. F(ab').sub.2 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.
[0043] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa (.kappa.) and lambda (.lambda.), based on the
amino acid sequences of their constant domains.
[0044] Depending on the amino acid sequence of the constant domain
of their heavy chains, antibodies can be assigned to different
classes. There are five major classes of intact antibodies: IgA,
IgD, IgE, IgG, and IgM, and several of these may be further divided
into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and
IgA2. The heavy-chain constant domains that correspond to the
different classes of antibodies are called .alpha., .delta.,
.di-elect cons., .gamma., and .mu., respectively. The subunit
structures and three-dimensional configurations of different
classes of immunoglobulins are well known.
[0045] "Single-chain Fv" or "scFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Preferably, the Fv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the scFv to form the
desired structure for antigen binding. For a review of scFv see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994).
[0046] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (V.sub.H) connected to a light-chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L).
By using a linker that is too short to allow pairing between the
two domains on the same chain, the domains are forced to pair with
the complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for
example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.
Acad. Sci. USA, 90:6444-6448 (1993).
[0047] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations which typically include
different antibodies directed against different determinants
(epitopes), each monoclonal antibody is directed against a single
determinant on the antigen. In addition to their specificity, the
monoclonal antibodies are advantageous in that they are synthesized
by the hybridoma culture, uncontaminated by other immunoglobulins.
The modifier "monoclonal" indicates the character of the antibody
as being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method. For example, the monoclonal
antibodies to be used in accordance with the present invention may
be made by the hybridoma method first described by Kohler et al.,
Nature, 256:495 (1975), or may be made by recombinant DNA methods
(see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies"
may also be isolated from phage antibody libraries using the
techniques described in Clackson et al., Nature, 352:624-628 (1991)
and Marks et al., J. Mol. Biol., 222:581-597 (1991), for
example.
[0048] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity (U.S. Pat. No. 4,816,567; Morrison et
al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric
antibodies of interest herein include "primatized" antibodies
comprising variable domain antigen-binding sequences derived from a
non-human primate (e.g. Old World Monkey, such as baboon, rhesus or
cynomolgus monkey) and human constant region sequences (U.S. Pat.
No. 5,693,780).
[0049] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
[0050] 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; Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)) and/or those residues
from a "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2)
and 91-96 (L3) in the light chain variable domain and 26-32 (H1),
53-55 (H2) and 96-101 (H3) in the heavy chain variable domain;
Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). "Framework" or
"FR" residues are those variable domain residues other than the
hypervariable region residues as herein defined.
[0051] An antagonist "which binds" an antigen of interest, e.g. a B
cell surface marker, is one capable of binding that antigen with
sufficient affinity and/or avidity such that the antagonist is
useful as a therapeutic agent for targeting a cell expressing the
antigen.
[0052] Examples of antibodies which bind the CD20 antigen include:
"C2B8" which is now called "rituximab" ("RITUXAN.RTM.") (U.S. Pat.
No. 5,736,137, expressly incorporated herein by reference); the
yttrium-[90]-labeled 2B8 murine antibody designated "Y2B8" (U.S.
Pat. No. 5,736,137, expressly incorporated herein by reference);
murine IgG2a "B1" optionally labeled with .sup.131I to generate the
".sup.131I-B1" antibody (BEXXAR.TM.) (U.S. Pat. No. 5,595,721,
expressly incorporated herein by reference); murine monoclonal
antibody "1F5" (Press et al. Blood 69(2):584-591 (1987)); "chimeric
2H7 antibody" (U.S. Pat. No. 5,677,180, expressly incorporated
herein by reference); "humanized 2H7 v16" (see below); huMax-CD20
(Genmab, Denmark); AME-133 (Applied Molecular Evolution); and
monoclonal antibodies L27, G28-2, 93-1B3, B-C1 or NU-B2 available
from the International Leukocyte Typing Workshop (Valentine et al.,
In: Leukocyte Typing III (McMichael, Ed., p. 440, Oxford University
Press (1987)).
[0053] Examples of antibodies which bind the CD19 antigen include
the anti-CD19 antibodies in Hekman et al. Cancer Immunol.
Immunother. 32:364-372 (1991) and Vlasveld et al. Cancer Immunol.
Immunother. 40:37-47 (1995); and the B4 antibody in Kiesel et al.
Leukemia Research II, 12: 1119 (1987).
[0054] The terms "rituximab" or "RIYTUXAN.RTM." herein refer to the
genetically engineered chimeric murine/human monoclonal antibody
directed against the CD20 antigen and designated "C2B8" in U.S.
Pat. No. 5,736,137, expressly incorporated herein by reference. The
antibody is an IgG.sub.1 kappa immunoglobulin containing murine
light and heavy chain variable region sequences and human constant
region sequences. Rituximab has a binding affinity for the CD20
antigen of approximately 8.0 nM.
[0055] Purely for the purposes herein, "humanized 2H7 v16" refers
to an antibody comprising the variable light and variable heavy
sequences shown below.
[0056] Variable light-chain domain of hu2H7 v16
1 DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQ (SEQ ID NO:1)
KPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTI
SSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKR
[0057] Variable heavy-chain domain of hu2H7 v16
2 EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNM (SEQ ID NO: 2)
HWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRF
TISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYY SNSYWYFDVWGQGTLVTVSS
[0058] Preferably humanized 2H7 v16 comprises the light chain amino
acid sequence
3 DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQ (SEQ ID NO: 3)
QKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTL
TISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKRT
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;
[0059] and heavy chain amino acid sequence
4 EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHW (SEQ ID NO: 4)
VRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISV
DKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWY
FDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK.
[0060] An "isolated" antagonist is one which has been identified
and separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the antagonist, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the antagonist will be purified (1) to greater than
95% by weight of antagonist as determined by the Lowry method, and
most preferably more than 99% by weight, (2) to a degree sufficient
to obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated antagonist
includes the antagonist in situ within recombinant cells since at
least one component of the antagonist's natural environment will
not be present. Ordinarily, however, isolated antagonist will be
prepared by at least one purification step.
[0061] "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.
[0062] "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.
[0063] The expression "therapeutically effective amount" refers to
an amount of the antagonist which is effective for preventing,
ameliorating or treating the autoimmune disease in question.
[0064] The term "immunosuppressive agent" as used herein for
adjunct therapy refers to substances that act to suppress or mask
the immune system of the mammal being treated herein. This would
include substances that suppress cytokine production, downregulate
or suppress self-antigen expression, or mask the MHC antigens.
Examples of such agents include 2-amino-6-aryl-5-substituted
pyrimidines (see U.S. Pat. No. 4,665,077, the disclosure of which
is incorporated herein by reference); nonsteroidal antiinflammatory
drugs (NSAIDs); 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; methotrexate (oral or subcutaneous);
hydroxycloroquine; sulfasalazine; leflunomide; cytokine or cytokine
receptor antagonists including anti-interferon-.gamma., -.beta., or
-60 antibodies, anti-tumor necrosis factor-.alpha. antibodies
(infliximab or adalimumab), anti-TNF.alpha. immunoahesin
(etanercept), anti-tumor necrosis factor-.beta. antibodies,
anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies;
anti-LFA-1 antibodies, including anti-CD11a 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 Jul. 26, 1990); streptokinase; TGF-.beta.;
streptodomase; 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.
[0065] 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. At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32
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.
[0066] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and cyclosphosphamide
(CYTOXAN.TM.); alkyl sulfonates such as busulfan, improsulfan and
piposulfan; aziridines such as benzodopa, carboquone, meturedopa,
and uredopa; ethylenimines and methylamelamines including
altretamine, triethylenemelamine, trietylenephosphoramide,
triethylenethiophosphaoramide and trimethylolomelamine; nitrogen
mustards such as chlorambucil, 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 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, peplomycin,
potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogues such as denopterin, methotrexate, pteropterin,
trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,
azacitidine, 6-azauridine, 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; aldophosphamide glycoside; aminolevulinic acid;
amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine; diaziquone; elfornithine; elliptinium acetate;
etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;
mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin;
phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide;
procarbazine; PSK.RTM.; razoxane; 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.RTM.,
Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel
(TAXOTERE.RTM., Rhne-Poulenc Rorer, Antony, France); chlorambucil;
gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum
analogs such as cisplatin and carboplatin; vinblastine; platinum;
etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;
vincristine; vinorelbine; navelbine; novantrone; teniposide;
daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase
inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid;
esperamicins; capecitabine; and pharmaceutically acceptable salts,
acids or derivatives of any of the above. Also included in this
definition are anti-hormonal agents that act to regulate or inhibit
hormone action on tumors such as anti-estrogens including for
example tamoxifen, raloxifene, aromatase inhibiting
4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene,
LY117018, onapristone, and toremifene (Fareston); and
anti-androgens such as flutamide, nilutainide, bicalutamide,
leuprolide, and goserelin; and pharmaceutically acceptable salts,
acids or derivatives of any of the above.
[0067] 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-.alpha. and -.beta.;
mullerian-inhibiting substance; mouse gonadotropin-associated
peptide; inhibin; activin; vascular endothelial growth factor;
integrin; thrombopoietin (TPO); nerve growth factors such as
NGF-.beta.; platelet-growth factor; transforming growth factors
(TGFs) such as TGF-.beta. and TGF-.beta.; insulin-like growth
factor-I and -II; erythropoietin (EPO); osteoinductive factors;
interferons such as interferon-.alpha., -.beta., and -.gamma.;
colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-1, IL-1.alpha., IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; a tumor necrosis
factor such as TNF-.alpha. or TNF-.beta.; and other polypeptide
factors including LIF and kit ligand (KL). As used herein, the term
cytokine includes proteins from natural sources or from recombinant
cell culture and biologically active equivalents of the native
sequence cytokines.
[0068] 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 Transactions, 14, pp. 375-382,
615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A
Chemical Approach to Targeted Drug Delivery," Directed Drug
Delivery, Borchardt et al, (ed.), pp. 247-267, Humana Press (1985).
The prodrugs of this invention include, but are not limited to,
phosphate-containing prodrugs, thiophosphate-containing prodrugs,
sulfate-containing prodrugs, peptide-containing prodrugs, D-amino
acid-modified prodrugs, glycosylated prodrugs,
.beta.-lactam-containing prodrugs, optionally substituted
phenoxyacetamide-containing prodrugs or optionally substituted
phenylacetamide-containing prodrugs, 5-fluorocytosine and other
5-fluorouridine prodrugs which can be converted into the more
active cytotoxic free drug. Examples of cytotoxic drugs that can be
derivatized into a prodrug form for use in this invention include,
but are not limited to, those chemotherapeutic agents described
above.
[0069] 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.
II. Production of Antagonists
[0070] The methods and articles of manufacture of the present
invention use, or incorporate, an antagonist which binds to a B
cell surface marker. Accordingly, methods for generating such
antagonists will be described here.
[0071] The B cell surface marker to be used for production of, or
screening for, antagonist(s) may be, e.g., a soluble form of the
antigen or a portion thereof, containing the desired epitope.
Alternatively, or additionally, cells expressing the B cell surface
marker at their cell surface can be used to generate, or screen
for, antagonist(s). Other forms of the B cell surface marker useful
for generating antagonists will be apparent to those skilled in the
art. Preferably, the B cell surface marker is the CD20 antigen.
[0072] While the preferred antagonist is an antibody, antagonists
other than antibodies are contemplated herein. For example, the
antagonist may comprise a small molecule antagonist optionally
fused to, or conjugated with, a cytotoxic agent (such as those
described herein). Libraries of small molecules may be screened
against the B cell surface marker of interest herein in order to
identify a small molecule which binds to that antigen. The small
molecule may further be screened for its antagonistic properties
and/or conjugated with a cytotoxic agent.
[0073] The antagonist may also be a peptide generated by rational
design or by phage display (see, e.g., WO98/35036 published 13 Aug.
1998). In one embodiment, the molecule of choice may be a "CDR
mimic" or antibody analogue designed based on the CDRs of an
antibody. While such peptides may be antagonistic by themselves,
the peptide may optionally be fused to a cytotoxic agent so as to
add or enhance antagonistic properties of the peptide.
[0074] A description follows as to exemplary techniques for the
production of the antibody antagonists used in accordance with the
present invention.
[0075] (i) Polyclonal Antibodies
[0076] Polyclonal antibodies are preferably raised in animals by
multiple subcutaneous (sc) or intraperitoneal (ip) injections of
the relevant antigen and an adjuvant. It may be useful to conjugate
the relevant antigen to a protein that is immunogenic in the
species to be immunized, e.g., keyhole limpet hemocyanin, serum
albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a
bifunctional or derivatizing agent, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R
and R.sup.1 are different alkyl groups.
[0077] Animals are immunized against the antigen, immunogenic
conjugates, or derivatives by combining, e.g., 100 .mu.g or 5 .mu.g
of the protein or conjugate (for rabbits or mice, respectively)
with 3 volumes of Freund's complete adjuvant and injecting the
solution intradermally at multiple sites. One month later the
animals are boosted with 1/5 to {fraction (1/10)} the original
amount of peptide or conjugate in Freund's complete adjuvant by
subcutaneous injection at multiple sites. Seven to 14 days later
the animals are bled and the serum is assayed for antibody titer.
Animals are boosted until the titer plateaus. Preferably, the
animal is boosted with the conjugate of the same antigen, but
conjugated to a different protein and/or through a different
cross-linking reagent. Conjugates also can be made in recombinant
cell culture as protein fusions. Also, aggregating agents such as
alum are suitably used to enhance the immune response.
[0078] (ii) Monoclonal Antibodies
[0079] 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 in minor
amounts. Thus, the modifier "monoclonal" indicates the character of
the antibody as not being a mixture of discrete antibodies.
[0080] For example, the monoclonal antibodies may be made using the
hybridoma method first described by Kohler et al., Nature, 256:495
(1975), or may be made by recombinant DNA methods (U.S. Pat. No.
4,816,567).
[0081] In the hybridoma method, a mouse or other appropriate host
animal, such as a hamster, is immunized as hereinabove described to
elicit lymphocytes that produce or are capable of producing
antibodies that will specifically bind to the protein used for
immunization. Alternatively, lymphocytes may be immunized in vitro.
Lymphocytes then are fused with myeloma cells using a suitable
fusing agent, such as polyethylene glycol, to form a hybridoma cell
(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press, 1986)).
[0082] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
[0083] Preferred myeloma cells are those that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these, preferred myeloma cell lines are murine
myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse
tumors available from the Salk Institute Cell Distribution Center,
San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from
the American Type Culture Collection, Rockville, Md. USA. Human
myeloma and mouse-human heteromyeloma cell lines also have been
described for the production of human monoclonal antibodies
(Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal
Antibody Production Techniques and Applications, pp. 51-63 (Marcel
Dekker, Inc., New York, 1987)).
[0084] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
(ELISA).
[0085] The binding affinity of the monoclonal antibody can, for
example, be determined by the Scatchard analysis of Munson et al.,
Anal. Biochem., 107:220 (1980).
[0086] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture
media for this purpose include, for example, D-MEM or RPMI-1640
medium. In addition, the hybridoma cells may be grown in vivo as
ascites tumors in an animal.
[0087] 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.
[0088] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of murine antibodies).
The hybridoma cells serve as a preferred source of such DNA. Once
isolated, the DNA may be placed into expression vectors, which are
then transfected into host cells such as E. coli cells, simian COS
cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do
not otherwise produce immunoglobulin protein, to obtain the
synthesis of monoclonal antibodies in the recombinant host cells.
Review articles on recombinant expression in bacteria of DNA
encoding the antibody include Skerra et al., Curr. Opinion in
Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Revs.,
130:151-188 (1992).
[0089] 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.,
Bio/Technology, 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.
[0090] The DNA also may be modified, for example, by substituting
the coding sequence for human heavy- and light-chain constant
domains in place of the homologous murine sequences (U.S. Pat. No.
4,816,567; Morrison, et al., Proc. Natl Acad. Sci. USA, 81:6851
(1984)), or by covalently joining to the immunoglobulin coding
sequence all or part of the coding sequence for a
non-immunoglobulin polypeptide.
[0091] Typically such non-immunoglobulin polypeptides are
substituted for the constant domains of an antibody, or they are
substituted for the variable domains of one antigen-combining site
of an antibody to create a chimeric bivalent antibody comprising
one antigen-combining site having specificity for an antigen and
another antigen-combining site having specificity for a different
antigen.
[0092] (iii) Humanized Antibodies
[0093] Methods for humanizing non-human antibodies have been
described in the art. Preferably, a humanized antibody has one or
more amino acid residues introduced into it from a source which is
non-human. These non-human amino acid residues are often referred
to as "import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by
substituting hypervariable region sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some hypervariable region residues and possibly
some FR residues are substituted by residues from analogous sites
in rodent antibodies.
[0094] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework region (FR) for the
humanized antibody (Sims et al., J. Immunol., 151:2296 (1993);
Chothia et al, J. Mol. Biol., 196:901 (1987)). Another method uses
a particular framework region derived from the consensus sequence
of all human antibodies of a particular subgroup of light or heavy
chains. The same framework may be used for several different
humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA,
89:4285 (1992); Presta et al, J. Immunol., 151:2623 (1993)).
[0095] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, according to a
preferred method, humanized antibodies are prepared by a process of
analysis of the parental sequences and various conceptual humanized
products using three-dimensional models of the parental and
humanized sequences. Three-dimensional immunoglobulin models are
commonly available and are familiar to those skilled in the art.
Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
hypervariable region residues are directly and most substantially
involved in influencing antigen binding.
[0096] (iv) Human Antibodies
[0097] 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 (J.sub.H) 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. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al.,
Nature, 362:255-258 (1993); Bruggermann et al, Year in Immuno.,
7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369 and
5,545,807.
[0098] Alternatively, phage display technology (McCafferty et al.,
Nature 348:552-553 (1990)) can be used to produce human antibodies
and antibody fragments in vitro, from immunoglobulin variable (V)
domain gene repertoires from unimmunized donors. According to this
technique, antibody V domain genes are cloned in-frame into either
a major or minor coat protein gene of a filamentous bacteriophage,
such as M13 or fd, and displayed as functional antibody fragments
on the surface of the phage particle. Because the filamentous
particle contains a single-stranded. DNA copy of the phage genome,
selections based on the functional properties of the antibody also
result in selection of the gene encoding the antibody exhibiting
those properties. Thus, the phage mimics some of the properties of
the B cell. Phage display can be performed in a variety of formats;
for their review see, e.g., Johnson, Kevin S. and Chiswell, David
J., Current Opinion in Structural Biology 3:564-571 (1993). Several
sources of V-gene segments can be used for phage display. Clackson
et al., Nature, 352:624-628 (1991) isolated a diverse array of
anti-oxazolone antibodies from a small random combinatorial library
of V genes derived from the spleens of immunized mice. A repertoire
of V genes from unimmunized human donors can be constructed and
antibodies to a diverse array of antigens (including self-antigens)
can be isolated essentially following the techniques described by
Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al.,
EMBO J. 12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and
5,573,905.
[0099] Human antibodies may also be generated by in vitro activated
B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).
[0100] (v) Antibody Fragments
[0101] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al, Journal of Biochemical and Biophysical Methods 24:107-117
(1992) and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
For example, the antibody fragments can be isolated from the
antibody phage libraries discussed above. Alternatively, Fab'-SH
fragments can be directly recovered from E. coli and chemically
coupled to form F(ab').sub.2 fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). According to another approach,
F(ab').sub.2 fragments can be isolated directly from recombinant
host cell culture. Other techniques for the production of antibody
fragments will be apparent to the skilled practitioner. In other
embodiments, the antibody of choice is a single chain Fv fragment
(scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No.
5,587,458. The antibody fragment may also be a "linear antibody",
e.g., as described in U.S. Pat. No. 5,641,870 for example. Such
linear antibody fragments may be monospecific or bispecific.
[0102] (vi) Bispecific Antibodies
[0103] Bispecific antibodies are antibodies that have binding
specificities for at least two different epitopes. Exemplary
bispecific antibodies may bind to two different epitopes of the B
cell surface marker. Other such antibodies may bind a first B cell
marker and further bind a second B cell surface marker.
Alternatively, an anti-B cell marker binding arm may be combined
with an arm which binds to a triggering molecule on a leukocyte
such as a T-cell receptor molecule (e.g. CD2 or CD3), or Fc
receptors for IgG (Fc.gamma.R), such as Fc.gamma.RI (CD64),
Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD16) 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-.alpha.,
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').sub.2 bispecific
antibodies).
[0104] 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).
[0105] 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 (CH1) containing the site necessary for light chain binding,
present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host organism. This
provides for great flexibility in adjusting the mutual proportions
of the three polypeptide fragments in embodiments when unequal
ratios of the three polypeptide chains used in the construction
provide the optimum yields. It is, however, possible to insert the
coding sequences for two or all three polypeptide chains in one
expression vector when the expression of at least two polypeptide
chains in equal ratios results in high yields or when the ratios
are of no particular significance.
[0106] In a preferred embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. It was found that this asymmetric
structure facilitates the separation of the desired bispecific
compound from unwanted immunoglobulin chain combinations, as the
presence of an immunoglobulin light chain in only one half of the
bispecific molecule provides for a facile way of separation. This
approach is disclosed in WO 94/04690. For further details of
generating bispecific antibodies see, for example, Suresh et al.,
Methods in Enzymology, 121:210 (1986).
[0107] According to another approach described in U.S. Pat. No.
5,731,168, the interface between a pair of antibody molecules can
be engineered to maximize the percentage of heterodimers which are
recovered from recombinant cell culture. The preferred interface
comprises at least a part of the C.sub.H3 domain of an antibody
constant domain. In this method, one or more small amino acid side
chains from the interface of the first antibody molecule are
replaced with larger side chains (e.g. tyrosine or tryptophan).
Compensatory "cavities" of identical or similar size to the large
side chain(s) are created on the interface of the second antibody
molecule by replacing large amino acid side chains with smaller
ones (e.g. alanine or threonine). This provides a mechanism for
increasing the yield of the heterodimer over other unwanted
end-products such as homodimers.
[0108] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360, WO 92/200373, and EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0109] Techniques for generating bispecific antibodies from
antibody fragments have also been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science, 229: 81 (1985) describe a
procedure wherein intact antibodies are proteolytically cleaved to
generate F(ab').sub.2 fragments. These fragments are reduced in the
presence of the dithiol complexing agent sodium arsenite to
stabilize vicinal dithiols and prevent intermolecular disulfide
formation. The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0110] Recent progress has facilitated the direct recovery of
Fab'-SH fragments from E. coli, which can be chemically coupled to
form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:
217-225 (1992) describe the production of a fully humanized
bispecific antibody F(ab').sub.2 molecule. Each Fab' fragment was
separately secreted from E. coli and subjected to directed chemical
coupling in vitro to form the bispecific antibody. The bispecific
antibody thus formed was able to bind to cells overexpressing the
ErbB2 receptor and normal human T cells, as well as trigger the
lytic activity of human cytotoxic lymphocytes against human breast
tumor targets.
[0111] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al, J. Immunol.,
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al, Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (V.sub.H) connected to a light-chain
variable domain (V.sub.L) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the
V.sub.H and V.sub.L domains of one fragment are forced to pair with
the complementary V.sub.L and V.sub.H domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for
making bispecific antibody fragments by the use of single-chain Fv
(sFv) dimers has also been reported. See Gruber et al., J.
Immunol., 152:5368 (1994).
[0112] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al. J.
Immunol. 147: 60 (1991).
[0113] III. Conjugates and Other Modifications of the
Antagonist
[0114] The antagonist used in the methods or included in the
articles of manufacture herein is optionally conjugated to a
cytotoxic agent.
[0115] Chemotherapeutic agents useful in the generation of such
antagonist-cytotoxic agent conjugates have been described
above.
[0116] Conjugates of an antagonist and one or more small molecule
toxins, such as a calicheamicin, a maytansine (U.S. Pat. No.
5,208,020), a trichothene, and CC1065 are also contemplated herein.
In one embodiment of the invention, the antagonist is conjugated to
one or more maytansine molecules (e.g. about 1 to about 10
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 (Chari et al. Cancer Research
52: 127-131 (1992)) to generate a maytansinoid-antagonist
conjugate.
[0117] Alternatively, the antagonist is conjugated to one or more
calicheamicin molecules. The calicheamicin family of antibiotics
are capable of producing double-stranded DNA breaks at
sub-picomolar concentrations. Structural analogues of calicheamicin
which may be used include, but are not limited to,
.gamma..sub.1.sup.I, .alpha..sub.2.sup.I, .alpha..sub.3.sup.I,
N-acetyl-.gamma..sub.1.sup.I, PSAG and .theta..sub.1.sup.I, (Hinman
et al. Cancer Research 53: 3336-3342 (1993) and Lode et al. Cancer
Research 58: 2925-2928 (1998)).
[0118] 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, restrictocin, phenomycin, enomycin and the
tricothecenes. See, for example, WO 93/21232 published Oct. 28,
1993.
[0119] The present invention further contemplates antagonist
conjugated with a compound with nucleolytic activity (e.g. a
ribonuclease or a DNA endonuclease such as a deoxyribonuclease;
DNase).
[0120] A variety of radioactive isotopes are available for the
production of radioconjugated antagonists. Examples include
At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.188, Sm.sup.153,
Bi.sup.212, P.sup.32 and radioactive isotopes of Lu.
[0121] Conjugates of the antagonist and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), 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 WO094/11026. The linker may
be a "cleavable linker" facilitating release of the cytotoxic drug
in the cell. For example, an acid-labile linker,
peptidase-sensitive linker, dimethyl linker or disulfide-containing
linker (Chari et al. Cancer Research 52: 127-131 (1992)) may be
used.
[0122] Alternatively, a fusion protein comprising the antagonist
and cytotoxic agent may be made, e.g. by recombinant techniques or
peptide synthesis.
[0123] In yet another embodiment, the antagonist may be conjugated
to a "receptor" (such streptavidin) for utilization in tumor
pretargeting wherein the antagonist-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g. avidin) which is conjugated to a
cytotoxic agent (e.g. a radionucleotide).
[0124] The antagonists of the present invention may also be
conjugated with a prodrug-activating enzyme which converts a
prodrug (e.g. a peptidyl chemotherapeutic agent, see WO81/01145) to
an active anti-cancer drug. See, for example, WO 88/07378 and U.S.
Pat. No. 4,975,278.
[0125] 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.
[0126] Enzymes that are useful in the method of this invention
include, but are not limited to, alkaline phosphatase useful for
converting phosphate-containing prodrugs into free drugs;
arylsulfatase useful for converting sulfate-containing prodrugs
into free drugs; cytosine deaminase useful for converting non-toxic
5-fluorocytosine into the anti-cancer drug, 5-fluorouracil;
proteases, such as serratia protease, thermolysin, subtilisin,
carboxypeptidases and cathepsins (such as cathepsins B and L), that
are useful for converting peptide-containing prodrugs into free
drugs; D-alanylcarboxypeptidases, useful for converting prodrugs
that contain D-amino acid substituents; carbohydrate-cleaving
enzymes such as .beta.-galactosidase and neuraminidase useful for
converting glycosylated prodrugs into free drugs; .beta.-lactamase
useful for converting drugs derivatized with .beta.-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.
[0127] 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)).
[0128] Other modifications of the antagonist are contemplated
herein. For example, the antagonist may be linked to one of a
variety of nonproteinaceous polymers, e.g., polyethylene glycol,
polypropylene glycol, polyoxyalkylenes, or copolymers of
polyethylene glycol and polypropylene glycol.
[0129] The antagonists disclosed herein may also be formulated as
liposomes. Liposomes containing the antagonist are prepared by
methods known in the art, such as described in Epstein et al.,
Proc. 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 WO97/38731 published Oct. 23, 1997. Liposomes with
enhanced circulation time are disclosed in U.S. Pat. No.
5,013,556.
[0130] Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of an antibody of the present invention
can be conjugated to the liposomes as described in Martin et al. J.
Biol. Chem. 257: 286-288 (1982) via a disulfide interchange
reaction. A chemotherapeutic agent is optionally contained within
the liposome. See Gabizon et al. J. National Cancer Inst.
81(19)1484 (1989).
[0131] Amino acid sequence modification(s) of protein or peptide
antagonists described herein are contemplated. For example, it may
be desirable to improve the binding affinity and/or other
biological properties of the antagonist. Amino acid sequence
variants of the antagonist are prepared by introducing appropriate
nucleotide changes into the antagonist nucleic acid, or by peptide
synthesis. Such modifications include, for example, deletions from,
and/or insertions into and/or substitutions of, residues within the
amino acid sequences of the antagonist. Any combination of
deletion, insertion, and substitution is made to arrive at the
final construct, provided that the final construct possesses the
desired characteristics. The amino acid changes also may alter
post-translational processes of the antagonist, such as changing
the number or position of glycosylation sites.
[0132] A useful method for identification of certain residues or
regions of the antagonist that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis" as described
by Cunningham and Wells Science, 244:1081-1085 (1989). Here, a
residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a
neutral or negatively charged amino acid (most preferably alanine
or polyalanine) to affect the interaction of the amino acids with
antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to analyze the performance of a
mutation at a given site, ala scanning or random mutagenesis is
conducted at the target codon or region and the expressed
antagonist variants are screened for the desired activity.
[0133] 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.
[0134] 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.
5 TABLE 1 Original Exemplary Preferred Residue Substitutions
Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys
Asn (N) gln; his; asp, lys; arg gln Asp (D) glu; asn glu Cys (C)
ser; ala ser Gln (Q) asn; glu asn Glu (E) asp; gln asp Gly (G) ala
ala His (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; leu
phe; norleucine Leu (L) norleucine; ile; val; ile met; ala; phe Lys
(K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val;
ile; ala; tyr tyr Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser
Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile;
leu; met; phe; leu ala; norleucine
[0135] Substantial modifications in the biological properties of
the antagonist are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Naturally occurring residues are
divided into groups based on common side-chain properties:
[0136] (1) hydrophobic: norleucine, met, ala, val, leu, ile;
[0137] (2) neutral hydrophilic: cys, ser, thr;
[0138] (3) acidic: asp, glu;
[0139] (4) basic: asn, gln, his, lys, arg;
[0140] (5) residues that influence chain orientation: gly, pro;
and
[0141] (6) aromatic: trp, tyr, phe.
[0142] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0143] Any cysteine residue not involved in maintaining the proper
conformation of the antagonist also may be substituted, generally
with serine, to improve the oxidative stability of the molecule and
prevent aberrant crosslinking. Conversely, cysteine bond(s) may be
added to the antagonist to improve its stability (particularly
where the antagonist is an antibody fragment such as an Fv
fragment).
[0144] A particularly preferred type of substitutional variant
involves substituting one or more hypervariable region residues of
a parent antibody. Generally, the resulting variant(s) selected for
further development will have improved biological properties
relative to the parent antibody from which they are generated. A
convenient way for generating such substitutional variants is
affinity maturation using phage display. Briefly, several
hypervariable region sites (e.g. 6-7 sites) are mutated to generate
all possible amino substitutions at each site. The antibody
variants thus generated are displayed in a monovalent fashion from
filamentous phage particles as fusions to the gene III product of
M13 packaged within each particle. The phage-displayed variants are
then screened for their biological activity (e.g. binding affinity)
as herein disclosed. In order to identify candidate hypervariable
region sites for modification, alanine scanning mutagenesis can be
performed to identify hypervariable region residues contributing
significantly to antigen binding. Alternatively, or in
additionally, it may be beneficial to analyze a crystal structure
of the antigen-antibody complex to identify contact points between
the antibody and antigen. Such contact residues and neighboring
residues are candidates for substitution according to the
techniques elaborated herein. Once such variants are generated, the
panel of variants is subjected to screening as described herein and
antibodies with superior properties in one or more relevant assays
may be selected for further development.
[0145] Another type of amino acid variant of the antagonist alters
the original glycosylation pattern of the antagonist. By altering
is meant deleting one or more carbohydrate moieties found in the
antagonist, and/or adding one or more glycosylation sites that are
not present in the antagonist.
[0146] Glycosylation of polypeptides is typically either N-linked
or O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino
acid, most commonly serine or threonine, although 5-hydroxyproline
or 5-hydroxylysine may also be used.
[0147] Addition of glycosylation sites to the antagonist is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antagonist (for O-linked glycosylation sites).
[0148] Nucleic acid molecules encoding amino acid sequence variants
of the antagonist are prepared by a variety of methods known in the
art. These methods include, but are not limited to, isolation from
a natural source (in the case of naturally occurring amino acid
sequence variants) or preparation by oligonucleotide-mediated (or
site-directed) mutagenesis, PCR mutagenesis, and cassette
mutagenesis of an earlier prepared variant or a non-variant version
of the antagonist.
[0149] It may be desirable to modify the antagonist of the
invention with respect to effector function, e.g. so as to enhance
antigen-dependent cell-mediated cyotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) of the antagonist. This may
be achieved by introducing one or more amino acid substitutions in
an Fc region of an antibody antagonist. Alternatively or
additionally, cysteine residue(s) may be introduced in the Fc
region, thereby allowing interchain disulfide bond formation in
this region. The homodimeric antibody thus generated may have
improved internalization capability and/or increased
complement-mediated cell killing and antibody-dependent cellular
cytotoxicity (ADCC). See Caron et al, J. Exp Med. 176:1191-1195
(1992) and Shopes, B. J. Immunol. 148:2918-2922(1992). Homodimeric
antibodies with enhanced anti-tumor activity may also be prepared
using heterobifunctional cross-linkers as described in Wolff et al.
Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can
be engineered which has dual Fc regions and may thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al. Anti-Cancer Drug Design 3:219-230 (1989).
[0150] To increase the serum half life of the antagonist, one may
incorporate a salvage receptor binding epitope into the antagonist
(especially an antibody fragment) as described in U.S. Pat. No.
5,739,277, for example. As used herein, the term "salvage receptor
binding epitope" refers to an epitope of the Fc region of an IgG
molecule (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, or IgG.sub.4) that
is responsible for increasing the in vivo serum half-life of the
IgG molecule.
[0151] IV. Pharmaceutical Formulations
[0152] Therapeutic formulations of the antagonists used in
accordance with the present invention are prepared for storage by
mixing an antagonist having the desired degree of purity with
optional pharmaceutically acceptable carriers, excipients or
stabilizers (Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed. (1980)), in the form of lyophilized formulations or
aqueous solutions. Acceptable carriers, excipients, or stabilizers
are nontoxic to recipients at the dosages and concentrations
employed, and include buffers such as phosphate, citrate, and other
organic acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0153] Exemplary anti-CD20 antibody formulations are described in
WO98/56418, expressly incorporated herein by reference. This
publication describes a liquid multidose formulation comprising 40
mg/mL rituximab, 25 mM acetate, 150 mM trehalose, 0.9% benzyl
alcohol, 0.02% polysorbate 20 at pH 5.0 that has a minimum shelf
life of two years storage at 2-8.degree. C. Another anti-CD20
formulation of interest comprises 10mg/mL rituximab in 9.0 mg/mL
sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL
polysorbate 80, and Sterile Water for Injection, pH 6.5.
[0154] Lyophilized formulations adapted for subcutaneous
administration are described in WO97/04801. 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.
[0155] 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 cytotoxic agent, chemotherapeutic agent, cytokine
or immunosuppressive agent (e.g. one which acts on T cells, such as
cyclosporin or an antibody that binds T cells, e.g. one which binds
LFA-1). The effective amount of such other agents depends on the
amount of antagonist present in the formulation, the type of
disease or disorder or treatment, and other factors discussed
above. These are generally used in the same dosages and with
administration routes as used hereinbefore or about from 1 to 99%
of the heretofore employed dosages.
[0156] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences
16th edition, Osol, A. Ed. (1980).
[0157] 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 .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid.
[0158] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0159] V. Treatment with the Antagonist
[0160] The present invention concerns therapy of a subpopulation of
mammals, especially humans, with, or susceptible to, an autoimmune
disease, who experience an inadequate response to previous or
current treatment with a TNF.alpha.-inhibitor. Generally, the
mammal to be treated herein will be identified following therapy
with one or more treatments with one or more
TNF.alpha.-inhibitor(s) such as Etanercept (ENBREL.RTM.),
Infliximab (REMICADE.RTM.) or Adalimumab (HUMIRA.TM.), as
experiencing an inadequate response to previous or current
treatment with a TNF.alpha.-inhibitor because of toxicity and/or
inadequate efficacy. However, the invention is not limited to a
prior therapy step with such a TNF.alpha.-inhibitor; for instance,
the patient may be considered to be prone to experience a toxicity,
e.g. cardiac toxicity, with a TNF.alpha.-inhibitor before therapy
therewith has begun, or the patient may be determined to be one who
is unlikely to respond to therapy with a TNF.alpha.-inhibitor.
[0161] The various autoimmune diseases to be treated herein are
listed in the definitions section above. The preferred indications
herein are rheumatoid arthritis, psoriatic arthritis, or Crohn's
disease.
[0162] Generally, the mammal treated herein will not be suffering
from a B-cell malignancy.
[0163] According to one embodiment of the invention contemplated
herein, the therapeutic approach will reduce negative side effects
(such as infections, heart failure and demyelination) associated
with therapy with a TNF.alpha.-inhibitor.
[0164] The composition comprising an antagonist which binds to a B
cell surface marker will be formulated, dosed, and administered in
a fashion consistent with good medical practice. Factors for
consideration in this context include the particular disease or
disorder being treated, the particular mammal being treated, the
clinical condition of the individual patient, the cause of the
disease or disorder, the site of delivery of the agent, the method
of administration, the scheduling of administration, and other
factors known to medical practitioners. The therapeutically
effective amount of the antagonist to be administered will be
governed by such considerations.
[0165] As a general proposition, the therapeutically effective
amount of the antagonist administered parenterally per dose will be
in the range of about 0.1 to 20 mg/kg of patient body weight per
day, with the typical initial range of antagonist used being in the
range of about 2 to 10 mg/kg.
[0166] The preferred antagonist is an antibody, e.g. an antibody
such as RITUXAN.RTM., which is not conjugated to a cytotoxic agent.
Suitable dosages for an unconjugated antibody are, for example, in
the range from about 20 mg/m.sup.2 to about 1000 mg/m.sup.2. In one
embodiment, the dosage of the antibody differs from that presently
recommended for RITUXAN.RTM.. For example, one may administer to
the patient one or more doses of substantially less than 375
mg/m.sup.2 of the antibody, e.g. where the dose is in the range
from about 20 mg/m.sup.2 to about 250 mg/m.sup.2, for example from
about 50 mg/m.sup.2 to about 200 mg/m.sup.2.
[0167] Exemplary dosage regimens include 375 mg/m2 weekly.times.4;
or 1000 mg.times.2 (e.g. on days 1 and 15).
[0168] Moreover, one may administer one or more initial dose(s) of
the antibody followed by one or more subsequent dose(s), wherein
the mg/m.sup.2 dose of the antibody in the subsequent dose(s)
exceeds the mg/m.sup.2 dose of the antibody in the initial dose(s).
For example, the initial dose may be in the range from about 20
mg/m.sup.2 to about 250 mg/m.sup.2 (e.g. from about 50 mg/m.sup.2
to about 200 mg/m.sup.2) and the subsequent dose may be in the
range from about 250 mg/m.sup.2 to about 1000 mg/m.sup.2.
[0169] As noted above, however, these suggested amounts of
antagonist are subject to a great deal of therapeutic discretion.
The key factor in selecting an appropriate dose and scheduling is
the result obtained, as indicated above. For example, relatively
higher doses may be needed initially for the treatment of ongoing
and acute diseases. To obtain the most efficacious results,
depending on the disease or disorder, the antagonist is
administered as close to the first sign, diagnosis, appearance, or
occurrence of the disease or disorder as possible or during
remissions of the disease or disorder.
[0170] The antagonist is administered by any suitable means,
including parenteral, subcutaneous, intraperitoneal,
intrapulmonary, and intranasal, and, if desired for local
immunosuppressive treatment, intralesional administration.
Parenteral infusions include intramuscular, intravenous,
intraarterial, intraperitoneal, or subcutaneous administration. In
addition, the antagonist may suitably be administered by pulse
infusion, e.g., with declining doses of the antagonist. Preferably
the dosing is given by injections, most preferably intravenous or
subcutaneous injections, depending in part on whether the
administration is brief or chronic.
[0171] One may administer other compounds, such as cytotoxic
agents, chemotherapeutic agents, immunosuppressive agents and/or
cytokines with the antagonists herein. The combined administration
includes coadministration, using separate formulations or a single
pharmaceutical formulation, and consecutive administration in
either order, wherein preferably there is a time period while both
(or all) active agents simultaneously exert their biological
activities. For RA, and other autoimmune diseases, the antagonist
(e.g. CD20 antibody) may be combined with any one or more of
disease-modifying antirheumatic drugs (DMARDs) such as
hydroxycloroquine, sulfasalazine, methotrexate, leflunomide,
azathioprine, D-penicillamine, Gold (oral), Gold (intramuscular),
minocycline, cyclosporine, Staphylococcal protein A
immunoadsorption; intravenous immunoglobulin (IVIG); nonsteroidal
antiinflammatory drugs (NSAIDs); glucocorticoid (e.g. via joint
injection); corticosteroid (e.g. methylprednisolone and/or
prednisone); folate etc. Preferably, a TNF.alpha.-inhibitor is not
administered to the mammal during the period of treatment with the
CD20 antagonist.
[0172] Aside from administration of protein antagonists to the
patient the present application contemplates administration of
antagonists by gene therapy. Such administration of nucleic acid
encoding the antagonist is encompassed by the expression
"administering a therapeutically effective amount of an
antagonist". See, for example, WO96/07321 published Mar. 14, 1996
concerning the use of gene therapy to generate intracellular
antibodies.
[0173] There are two major approaches to getting the nucleic acid
(optionally contained in a vector) into the patient's cells; in
vivo and ex vivo. For in vivo delivery the nucleic acid is injected
directly into the patient, usually at the site where the antagonist
is required. For ex vivo treatment, the patient's cells are
removed, the nucleic acid is introduced into these isolated cells
and the modified cells are administered to the patient either
directly or, for example, encapsulated within porous membranes
which are implanted into the patient (see, e.g. U.S. Pat. Nos.
4,892,538 and 5,283,187). There are a variety of techniques
available for introducing nucleic acids into viable cells. The
techniques vary depending upon whether the nucleic acid is
transferred into cultured cells in vitro, or in vivo in the cells
of the intended host. Techniques suitable for the transfer of
nucleic acid into mammalian cells in vitro include the use of
liposomes, electroporation, microinjection, cell fusion,
DEAE-dextran, the calcium phosphate precipitation method, etc. A
commonly used vector for ex vivo delivery of the gene is a
retrovirus.
[0174] The currently preferred in vivo nucleic acid transfer
techniques include transfection with viral vectors (such as
adenovirus, Herpes simplex I virus, or adeno-associated virus) and
lipid-based systems (useful lipids for lipid-mediated transfer of
the gene are DOTMA, DOPE and DC-Chol, for example). In some
situations it is desirable to provide the nucleic acid source with
an agent that targets the target cells, such as an antibody
specific for a cell surface membrane protein or the target cell, a
ligand for a receptor on the target cell, etc. Where liposomes are
employed, proteins which bind to a cell surface membrane protein
associated with endocytosis may be used for targeting and/or to
facilitate uptake, e.g. capsid proteins or fragments thereof tropic
for a particular cell type, antibodies for proteins which undergo
internalization in cycling, and proteins that target intracellular
localization and enhance intracellular half-life. The technique of
receptor-mediated endocytosis is described, for example, by Wu et
al, J. Biol. Chem. 262:4429-4432 (1987); and Wagner et al., Proc.
Natl. Acad. Sci. USA 87:3410-3414 (1990). For review of the
currently known gene marking and gene therapy protocols see
Anderson et al., Science 256:808-813 (1992). See also WO 93/25673
and the references cited therein.
[0175] 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.
EXAMPLE 1
[0176] A patient with active rheumatoid arthritis who has an
inadequate response to one or more TNF.alpha.-inhibitor therapies
is treated with an antibody that binds the B-cell surface antigen,
CD20.
[0177] Candidates for therapy according to this example include
those who have experienced an inadequate response to previous or
current treatment with etanercept, infliximab and/or adalimumab
because of toxicity or inadequate efficacy (etanercept for
.gtoreq.3 months at 25 mg twice a week or at least 4 infusions of
infliximab at .gtoreq.3 mg/kg).
[0178] Patients may have swollen joint count (SJC).gtoreq.8 (66
joint count), and tender joint count (TJC).gtoreq.8 (68 joint
count) at screening and randomization; either CRP.gtoreq.1.5 mg/dl
(15 mg/L) or ESR.gtoreq.28 mm/h; and/or radiographic evidence of at
least one joint with definite erosion attributable to rheumatoid
arthritis, as determined by the central reading site (any joint of
the hands, wrists or feet can be considered with the exception of
the DIP joints of the hands).
[0179] The CD20 antibody used for therapy may be Rituximab
(commercially available from Genentech, Inc.) or humanized 2H7
v16.
[0180] Patients are treated with a therapeutically effective dose
of the CD20 antibody, for instance, 1000 mg i.v. on Days 1 and 15,
or 375 mg/m2 i.v. weekly.times.4.
[0181] Patients may also receive concomitant MTX (10-25 mg/week per
oral (p.o.) or parenteral), together with a corticosteroid regimen
consisting of methylprednisolone 100 mg i.v. 30 minutes prior to
infusions of the CD20 antibody and prednisone 60 mg p.o. on Days
2-7, 30 mg p.o. Days 8-14, returning to baseline dose by Day 16.
Patients may also receive folate (5 mg/week) given as either a
single dose or as divided daily doses. Patients optionally continue
to receive any background corticosteroid (.ltoreq.10 mg/d
prednisone or equivalent) throughout the treatment period.
[0182] The primary endpoint may be the proportion of patients with
an ACR20 response at Week 24 using a Cochran-Mantel-Haenszel (CMH)
test for comparing group differences, adjusted for rheumatoid
factor and region.
[0183] Potential secondary endpoints include:
[0184] 1. Proportion of patients with ACR50 and 70 responses at
Week 24. These may be analyzed as specified for the primary
endpoint.
[0185] 2. Change in Disease Activity Score (DAS) from screening to
Week 24. These may be assessed using an ANOVA model with baseline
DAS, rheumatoid factor, and treatment as terms in the model.
[0186] 3. Categorical DAS responders (EULAR response) at Week 24.
These may be assessed using a CMH test adjusted for rheumatoid
factor.
[0187] 4. Changes from screening in ACR core set (SJC, TJC,
patient's and physician's global assessments, HAQ, pain, CRP, and
ESR). Descriptive statistics may be reported for these
parameters.
[0188] 5. Changes from screening in SF-36. Descriptive statistics
may be reported for the 8 domain scores and the mental and physical
component scores. In addition, the mental and physical component
scores may be further categorized and analyzed.
[0189] 6. Change in modified Sharp radiographic total score,
erosion score, and joint space narrowing score. These may be
analyzed using continuous or categorical methodology, as
appropriate.
[0190] Exploratory endpoints and analysis may involve:
[0191] ACR(20/50/70 and ACR n) and change in DAS responses over
Weeks 8, 12, 16, 20, 24 and beyond will be assessed using a binary
or continuous repeated measures model, as appropriate. Exploratory
radiographic analyses including proportion of patients with no
erosive progression may be assessed at weeks 24 and beyond.
[0192] Further exploratory endpoints (for example complete clinical
response, disease free period) will be analyzed descriptively as
part of the extended observation period.
[0193] Changes from Screen in FACIT-F fatigue will be analyzed with
descriptive statistics.
[0194] Therapy of RA with the CD20 antibody in patients with an
inadequate response to TNF-.alpha. inhibitor therapy as described
above will result in a beneficial clinical response according to
any one or more of the endpoints noted above.
Sequence CWU 1
1
4 1 107 PRT Artificial sequence humanized sequence 1 Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser 20 25 30 Tyr
Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro 35 40 45
Leu Ile Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ser Arg 50 55
60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65
70 75 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp
80 85 90 Ser Phe Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile 95 100 105 Lys Arg 2 122 PRT Artificial sequence humanized
sequence 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr
Phe Thr 20 25 30 Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu 35 40 45 Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly
Asp Thr Ser Tyr 50 55 60 Asn Gln Lys Phe Lys Gly Arg Phe Thr Ile
Ser Val Asp Lys Ser 65 70 75 Lys Asn Thr Leu Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp 80 85 90 Thr Ala Val Tyr Tyr Cys Ala Arg
Val Val Tyr Tyr Ser Asn Ser 95 100 105 Tyr Trp Tyr Phe Asp Val Trp
Gly Gln Gly Thr Leu Val Thr Val 110 115 120 Ser Ser 3 213 PRT
Artificial sequence humanized sequence 3 Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Ser Ser Val Ser 20 25 30 Tyr Met His Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro 35 40 45 Leu Ile Tyr
Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ser Arg 50 55 60 Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp 80 85 90
Ser Phe Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 95 100
105 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser 110
115 120 Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
125 130 135 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val
Asp 140 145 150 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr
Glu Gln 155 160 165 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr
Leu Thr Leu 170 175 180 Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
Ala Cys Glu Val 185 190 195 Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser Phe Asn Arg 200 205 210 Gly Glu Cys 4 452 PRT Artificial
sequence humanized sequence 4 Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Tyr Thr Phe Thr 20 25 30 Ser Tyr Asn Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp Val Gly Ala Ile
Tyr Pro Gly Asn Gly Asp Thr Ser Tyr 50 55 60 Asn Gln Lys Phe Lys
Gly Arg Phe Thr Ile Ser Val Asp Lys Ser 65 70 75 Lys Asn Thr Leu
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90 Thr Ala Val
Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser Asn Ser 95 100 105 Tyr Trp
Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val 110 115 120 Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 125 130 135
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 140 145
150 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 155
160 165 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
170 175 180 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser 185 190 195 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys 200 205 210 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys 215 220 225 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp 260 265 270 Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln 290 295 300 Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His 305 310 315 Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 320 325 330 Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 335 340 345 Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 350 355 360 Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 365 370 375 Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 380 385 390
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 395 400
405 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 410
415 420 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
425 430 435 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro 440 445 450 Gly Lys
* * * * *
References