U.S. patent number RE48,370 [Application Number 16/152,031] was granted by the patent office on 2020-12-29 for levels of bcma protein expression on b cells and use in methods of treating systemic lupus erythematosus.
This patent grant is currently assigned to UNIVERSITY OF WASHINGTON, ZYMOGENETICS, INC.. The grantee listed for this patent is University of Washington, ZYMOGENETICS, INC.. Invention is credited to Stacey R. Dillon, Keith B. Elkon, Jane A. Gross.
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United States Patent |
RE48,370 |
Dillon , et al. |
December 29, 2020 |
Levels of BCMA protein expression on B cells and use in methods of
treating systemic lupus erythematosus
Abstract
The present invention provides a method of measuring the levels
of BCMA in a biological sample, specifically upon the B cell
surface. The diagnostic assays are useful in predicting an
individual's likelihood of developing or currently suffering from
an autoimmune disease, such as SLE, and for methods for treating an
individual clinically diagnosed with an autoimmune disease. This
diagnostic test serves to predict a patient's likelihood to respond
to a specific drug treatment, in particular treatment with BLyS
antagonists, either singly or in combination with other immune
suppressive drugs.
Inventors: |
Dillon; Stacey R. (Seattle,
WA), Gross; Jane A. (Seattle, WA), Elkon; Keith B.
(Seattle, WA) |
Applicant: |
Name |
City |
State |
Country |
Type |
ZYMOGENETICS, INC.
University of Washington |
Seattle
Seattle |
WA
WA |
US
US |
|
|
Assignee: |
ZYMOGENETICS, INC. (Seattle,
WA)
UNIVERSITY OF WASHINGTON (Seattle, WA)
|
Family
ID: |
41100796 |
Appl.
No.: |
16/152,031 |
Filed: |
October 4, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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12429502 |
Apr 24, 2009 |
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61047869 |
Apr 25, 2008 |
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Reissue of: |
14192911 |
Feb 28, 2014 |
9725506 |
Aug 8, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P
9/08 (20180101); A61P 1/04 (20180101); A61P
13/12 (20180101); G01N 33/564 (20130101); G01N
33/5091 (20130101); A61P 17/06 (20180101); A61P
43/00 (20180101); A61P 25/00 (20180101); A61P
37/02 (20180101); A61P 21/04 (20180101); C07K
16/24 (20130101); A61P 9/00 (20180101); C07K
14/70578 (20130101); G01N 33/564 (20130101); C07K
16/24 (20130101); G01N 33/5091 (20130101); C07K
14/70578 (20130101); A61P 29/00 (20180101); A61P
3/10 (20180101); A61P 7/04 (20180101); A61P
19/02 (20180101); A61P 37/06 (20180101); G01N
2800/065 (20130101); G01N 2800/24 (20130101); G01N
2800/285 (20130101); G01N 2800/102 (20130101); G01N
2800/347 (20130101); G01N 2800/101 (20130101); G01N
2800/065 (20130101); G01N 2800/104 (20130101); G01N
2333/70575 (20130101); G01N 2800/104 (20130101); G01N
2800/24 (20130101); G01N 2800/101 (20130101); G01N
2800/285 (20130101); G01N 2800/042 (20130101); G01N
2800/102 (20130101); G01N 2800/347 (20130101); G01N
2333/70575 (20130101); G01N 2800/042 (20130101) |
Current International
Class: |
A61K
38/00 (20060101); G01N 33/564 (20060101); G01N
33/50 (20060101); C07K 14/705 (20060101); C07K
16/24 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2006 201 471 |
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May 2006 |
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AU |
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WO 02/02641 |
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Jan 2002 |
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WO |
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WO 02/16312 |
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Feb 2002 |
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WO |
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WO 03/035846 |
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May 2003 |
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WO |
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WO 2005/000351 |
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Jan 2005 |
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WO |
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WO 2005/108986 |
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Nov 2005 |
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WO |
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WO 2006/068867 |
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Jun 2006 |
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WO |
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Other References
Dall'Era et al. Trial of Atacicept in Patients with Systemic Lupus
Erythematosus (SLE). American College of Rheumatology, 2006 Annual
Scientific Meeting, Abstract L19. cited by examiner .
Baker, Kevin P., et al., "Generation and Characterization of
LymphoStat-B, a Human Monoclonal Antibody that Antagonizes the
Bioactivities of B Lymphocyte Stimulator," Arthritis &
Rheumatism, vol. 48, No. 11, (Nov. 2003), pp. 3253-3265. cited by
applicant .
Dillon, Stacey R., et al., "An APRIL to Remember: Novel TNF Ligands
as Therapeutic Targets," Nature Reviews, vol. 5, (Mar. 2006), pp.
235-246. cited by applicant .
Claudio, E., et al., "BAFF-Induced NEMO-Independent Processing of
NF-.kappa.B2 in Maturing C Cells," Nature Immunology, (Oct. 2002),
vol. 3, No. 10, pp. 958-965. cited by applicant .
Cosman, D., "A Family of Ligands for the TNF Receptor Superfamily,"
Stem Cells, (1994), No. 12, pp. 440-455. cited by applicant .
Gras, M-P., et al., "BCMAp: An Integral Membrane Protein in the
Golgi Apparatus of Human Mature B Lymphocytes," International
Immunology, (1995), vol. 7, No. 7, pp. 1093-1106. cited by
applicant .
Hatzoglou, A., et al., "TNF Receptor Family Member BCMA (B Cell
Maturation) Associates with TNF Receptor-Associated Factor (TRAF)
1, TRAF2, and TRAF3 and Activates NF-.kappa.B, Elk-1, c-Jun
N-Terminal Kinase, and p38 Mitogen-Activated Protein Kinase," The
Journal of Immunology, (2000), vol. 165, pp. 1322-1330. cited by
applicant .
Kayagaki, N., et al., "BAFF/BLyS Receptor 3 Binds the B Cell
Survival Factor BAFF Ligand through a Discrete Surface Loop and
Promotes Processing of NF-kB2," Immunity, (Oct. 2002), vol. 10, pp.
515-524. cited by applicant .
Ryan, M.C., et al., "Antibody Targeting of B-cell Maturation
Antigen on Malignant Plasma Cells," Mol. Cancer Ther., (2007), vol.
6, No. 11, pp. 3009-3018. cited by applicant .
Smith, C.A., et al., "The TNF Receptor Superfamily of Cellular and
Viral Proteins: Activation, Costimulation, and Death," Cell, (Mar.
1994), vol. 76, pp. 959-962. cited by applicant .
Carter, R.H., et al., "Expression and Occupancy Systemic lupus of
BAFF-R on B Cells in Erythamatosus," Arthritis & Rheumatism,
vol. 52, No. 12, Dec. 2005, pp. 3943-3954. cited by applicant .
Thangarajh, M., et al., "The Expression of BAFF-Binding Receptors
is not Altered in Multiple Sclerosis or Myasthenia Gravis,"
Scandinavian Journal of Immunology, vol. 65, No. 5, May 2007, pp.
461-466. cited by applicant .
Zhang, D., et al., "Detection of the Expression Levels of B Cell
Activating Factor and its Receptors in Patients with Systemic Lupus
Erythematosus by Real-Time Fluorescence Quantitative Method,"
Annual Meeting of the American Association for Clinical Chemistry,
Orlando, FL, Jul. 24-28, 2005. cited by applicant .
Carter, R.H., et al., "Expression and Occupancy of BAFF-R on B
Cells in Systemic Lupus Erythematosus," Arthristis & Rhematism,
Dec. 2005, 52(12):3943-3954. cited by applicant .
Yang, M., et al., "B Cell Maturation Antigen, the Receptor for a
Proliferation-Inducing Ligand and B Cell-Activating Factor of the
TNF Family, Induces Antigen Presentation in B Cells," The Journal
of Immunology, 2005, 175: 2814-2824. cited by applicant .
Darce, J., et al., "Regulated Expression of BAFF-Binding Receptors
during Human B Cell Differentiation," The Journal of Immunology,
2004, vol. 103(2), pp. 689-694. cited by applicant .
Novak, A., et al., "Expression of BCMA, TACI, BAFF-R in multiple
myeloma: a mechanism for growth and survival," BLOOD , 2004, vol.
103(2), pp. 680-694. cited by applicant.
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Primary Examiner: Campell; Bruce R
Attorney, Agent or Firm: Womble Bond Dickinson (US) LLP
Parent Case Text
.[.CROSS-REFERENCE TO RELATED APPLICATIONS.]. .Iadd.CROSS REFERENCE
TO RELATED APPLICATIONS .Iaddend.
This application is a .Iadd.reissue application of U.S. Pat. No.
9,725,506, granted Aug. 8, 2017, which issued from U.S. application
Ser. No. 14/192,911 filed Feb. 28, 2014, which is a
.Iaddend.divisional of U.S. application Ser. No. 12/429,502, filed
Apr. 24, 2009, which claims the benefit of U.S. Provisional
Application No. 61/047,869 filed Apr. 25, 2008, each of which are
herein incorporated by reference in their entirety.
Claims
That which is claimed:
1. A method of reducing the systemic lupus erythematosus (SLE)
disease activity of an individual clinically diagnosed with SLE,
said method comprising: analyzing peripheral blood B cells from an
individual clinically diagnosed with SLE for the presence or
absence of elevated BCMA protein expression levels on said
peripheral blood B cells, said analyzing step comprising: (i)
measuring a first level of BCMA protein expression on the surface
of peripheral blood B cells from said individual clinically
diagnosed with SLE; (ii) comparing the first level to a second
level of BCMA protein expression on the surface of peripheral blood
B cells from a healthy individual; and (iii) identifying an
individual wherein the first level is elevated as compared to the
second level; and administering a BLyS antagonist to said
individual clinically diagnosed with SLE and having elevated BCMA
protein expression levels on said peripheral blood B cells, wherein
the BLyS antagonist is a receptor extracellular domain/Fc domain
fusion protein selected from the group consisting of TACI-Ig,
BCMA-Ig, and BAFF-R-Ig, wherein the SLE disease activity of said
individual is reduced following said administration of the BLyS
antagonist.
2. The method of claim 1 wherein said BLyS antagonist is also an
APRIL antagonist.
3. The method of claim 1, wherein said receptor-extracellular
domain/Fc domain fusion protein is TACI-Ig.
4. The method of claim 3, wherein said TACI-Ig is atacicept.
.Iadd.5. The method of claim 1, wherein said first level of BCMA
protein expression is measured by flow cytometry..Iaddend.
.Iadd.6. The method of claim 1, wherein said second level of BCMA
protein expression is measured by flow cytometry..Iaddend.
Description
REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA
EFS-WEB
The official copy of the sequence listing is submitted concurrently
with the specification as a text file via EFS-Web, in compliance
with the American Standard Code for Information Interchange
(ASCII), with a file name of 442957seqlist.txt, a creation date of
Feb. 27, 2014, and a size of 66.7 KB. The sequence listing filed
via EFS-Web is part of the specification and is hereby incorporated
in its entirety by reference herein.
BACKGROUND OF THE INVENTION
Cellular interactions which occur during an immune response are
regulated by members of several families of cell surface receptors,
including the tumor necrosis factor receptor (TNFR) family. The
TNFR family consists of a number of integral membrane glycoprotein
receptors many of which, in conjunction with their respective
ligands, regulate interactions between different hematopoietic cell
lineages (Smith et al., The TNF Receptor Superfamily of Cellular
and Viral Proteins: Activation, Costimulation and Death, 76:959-62,
1994; Cosman, Stem Cells 12:440-55, 1994).
One such receptor is BCMA, a nonglycosylated integral membrane type
I protein that is preferentially expressed in mature B lymphocytes
(Gras et al., Int. Immunol. 17:1093-106, 1995). BCMA is located on
the cell surface, as well as in a perinulear Golgi-like structure.
Overexpression of BCMA in 293 cells activates NF-kappa B, Elk-1,
the c-Jun N-terminal kinase, and the p38 mitogen-activated protein
kinase, thus producing signals for cell survival and proliferation
(Hatzoglou et al., J. Immunol., 165: 1322-30, 2000). Another such
receptor is TACI, transmembrane activator and CAML-interactor (von
Bulow and Bram, Science 228: 138-41, 1997 and WIPO Publication WO
98/39361). TACI is a membrane bound receptor having an
extracellular domain containing two cysteine-rich pseudo-repeats, a
transmembrane domain and a cytoplasmic domain that interacts with
CAML (calcium-modulator and cyclophilin ligand), an integral
membrane protein located at intracellular vesicles which is a
co-inducer of NF-AT activation when overexpressed in Jurkat cells.
A third receptor from the TNFR family expressed on the surface of B
cells is BAFF-R (Thompson et al., Science, 293: 2108-11, 2001.
Signaling of this receptor, also known as BAFF/BLyS receptor 3
(BR3), promotes processing of the transcription factor
NF-kappaB2/p100 to p52. This cascade is physiologically relevant
for survival of B cells, and therefore, the progression of B cells
to maturation (Claudio et al., Nat. Immunol., 3: 898-9, 2002;
Kayagaki et al., Immunity, 17: 515-24, 2002).
A number of BLyS and/or APRIL antagonists have been developed in
order to block the binding of these ligands to the receptor members
of the family, in order to block results of this binding which
include but should not be limited to B cell costimulation,
plasmablast and plasma cell survival, Ig class switching, enhanced
B-cell antigen presenting cell function, survival of malignant B
cells, development of B-1 cell function, B cell development beyond
the T-1 stage, and complete germinal centre formation. Some of
these molecules can also bind to and block the effect of APRIL on B
cells and other components of the immune system (Dillon et al.
(2006) Nat. Rev. Drug Dis. 5, 235-246). Molecules that have been
developed to affect B cell function by interfering with BLyS and/or
APRIL binding include BLyS antibodies such as Lymphostat-B
(Belimumab) (Baker et al, (2003) Arthritis Rheum, 48, 3253-3265 and
WO 02/02641); receptor-extracellular domain/Fc domain fusions
proteins such as TACI-Ig, including one particular embodiment,
atacicept (U.S. Patent Application No. 20060034852), BAFF-R-Fc (WO
05/0000351), and BCMA-Ig or other fusion proteins utilizing
receptor extracellular domains. A further class of BLyS and/or
APRIL antagonists include other molecules relying on BLyS binding
ability to block binding to its receptors such as AMG 623, receptor
antibodies, and other molecules disclosed in WO 03/035846 and WO
02/16312.
There remains a need in the art for further identification of cell
surface expression patterns of these TNFR family members that are
statistically associated with autoimmune disease, such as systemic
lupus erythromatosus (SLE). Such information is important for
identifying individuals who have a propensity toward developing
such autoimmune diseases, are in an active disease state, and for
identifying those that will respond favorably to BLyS and/or APRIL
antagonist treatment of these diseases. The present invention
addresses this need by providing a cell surface expression pattern
associated with autoimmune diseases and providing diagnostic tests
determining the presence of this expression pattern, namely
increased BCMA expression on B cells for those suffering from
autoimmune disease as compared to healthy controls.
SUMMARY OF THE INVENTION
The present invention provides a method of screening for levels of
TNFR family members on the B cell surface. As it has been shown
that elevated levels of BCMA are significantly associated with
autoimmune disease, such as SLE, this measurement is useful as a
diagnostic assay. Such diagnostic assays are useful in predicting
an individual's likelihood of having a condition associated with
autoimmune activity, such as SLE. The invention further provides
methods for determining appropriate treatment for an individual
with an autoimmune disease, such as SLE.
Detection of high levels of BCMA on B cells of patients exhibiting
autoimmune activity, such as those diagnosed with SLE, allows
selection of a treatment plan that is most likely to be effective
in treating the condition. These treatment plans generally involve
the use of BLyS antagonists, either singly or in combination with
another pharmaceutical such as an immune suppressive drug (like MMF
or Cellcept.RTM.) or a CD 20 antagonist (like Rituxan.RTM.).
Thus, the invention further provides methods for treating an
individual clinically diagnosed with an autoimmune condition,
generally comprising detecting high levels of BCMA on cells, as
compared to levels seen on B cells of healthy controls, and
selecting a treatment plan that is most effective for individuals
clinically diagnosed with an autoimmune disease. Detection of high
levels of BCMA on B cells also allows one to predict a patient's
likelihood to respond to a specific drug treatment, particularly
BLyS and/or APRIL antagonists. Thus, the invention further provides
methods of predicting a patient's likelihood to respond to BLyS
and/or APRIL antagonists (either singly or in combination with
other drugs) during treatment for an autoimmune condition, such as
SLE.
Very specifically, the present invention describes a method of
detecting increased BCMA protein expression on the surface of B
cells of an individual comprising measuring a first level of BCMA
protein expression in a biological sample and comparing that level
to a second level of BCMA protein expression present on the surface
of B cells of a healthy individual and determining the first level
is increased as compared to the second level, wherein said
increased BCMA protein expression is associated with an autoimmune
disease. The autoimmune disease in the present invention can be
selected from the group consisting of rheumatoid arthritis,
juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE),
lupus nephritis (LN), Wegener's disease, inflammatory bowel
disease, idiopathic thrombocytopenic purpura (ITP), thrombotic
throbocytopenic purpura (TTP), autoimmune thrombocytopenia,
multiple sclerosis, psoriasis, IgA nephropathy, IgM
polyneuropathies, myasthenia gravis, vasculitis, diabetes mellitus,
Reynaud's syndrome, Sjorgen's syndrome and glomerulonephritis. In
particular, the autoimmune disease is SLE.
The present invention also describes a method of treating an
individual clinically diagnosed with an autoimmune disease,
comprising analyzing a biological sample from an individual
clinically diagnosed with autoimmune disease for the presence or
absence of elevated BCMA protein expression levels on their B
cells, wherein the presence of elevated BCMA protein expression
levels is associated with the clinical diagnosis of autoimmune
disease; and selecting a treatment plan that is most effective for
individuals clinically diagnosed as having a condition associated
with an increased BCMA protein expression level. The treatment plan
can involve administration of a BLyS antagonist. And said BLyS
antagonist can also be an APRIL antagonist. For this method the
autoimmune disease can be selected from the group consisting of
rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus
erythematosus (SLE), lupus nephritis (LN), Wegener's disease,
inflammatory bowel disease, idiopathic thrombocytopenic purpura
(ITP), thrombotic throbocytopenic purpura (TTP), autoimmune
thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy,
IgM polyneuropathies, myasthenia gravis, vasculitis, diabetes
mellitus, Reynaud's syndrome, Sjorgen's syndrome and
glomerulonephritis. In particular, the autoimmune disease is
SLE.
Furthermore, the present invention describes methods for predicting
a patient's likelihood to respond to a drug treatment for an
autoimmune disease, comprising determining the level of BCMA
protein expression on the patient's B cells, wherein the presence
of elevated BCMA protein expression levels is predictive of the
patient's likelihood to respond to a drug treatment for the
condition. The autoimmune disease can be selected from the group
consisting of rheumatoid arthritis, juvenile rheumatoid arthritis,
systemic lupus erythematosus (SLE), lupus nephritis (LN), Wegener's
disease, inflammatory bowel disease, idiopathic thrombocytopenic
purpura (ITP), thrombotic throbocytopenic purpura (TTP), autoimmune
thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy,
IgM polyneuropathies, myasthenia gravis, vasculitis, diabetes
mellitus, Reynaud's syndrome, Sjorgen's syndrome and
glomerulonephritis. In particular, the autoimmune disease is SLE.
Additionally, the present invention method can include a drug
treatment involves administration of a BLyS antagonist and said
BLyS antagonist can also be an APRIL antagonist.
The present invention also encompasses an in vitro method of
detecting increased BCMA protein expression on the surface of B
cells of an individual, comprising measuring the level of BCMA
protein expression on the surface of B cells in a test biological
sample from the individual; comparing that level to the level of
BCMA protein expression on the surface of B cells in a sample from
a healthy control; and determining whether the level of BCMA
protein expression on the surface is B cells in the test biological
sample is increased as compared to the level in the control sample;
wherein said increased BCMA protein expression is associated with
an autoimmune disease. The autoimmune disease in this method can be
selected from the group consisting of rheumatoid arthritis,
juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE),
lupus nephritis (LN), Wegener's disease, inflammatory bowel
disease, idiopathic thrombocytopenic purpura (ITP), thrombotic
throbocytopenic purpura (TTP), autoimmune thrombocytopenia,
multiple sclerosis, psoriasis, IgA nephropathy, IgM
polyneuropathies, myasthenia gravis, vasculitis, diabetes mellitus,
Reynaud's syndrome, Sjorgen's syndrome and glomerulonephritis. In
particular, the autoimmune disease is SLE.
In a further embodiment, the present invention includes an in vitro
method of selecting a treatment plan that is most effective for
treating an individual clinically diagnosed with an autoimmune
disease, comprising analyzing in vitro a biological sample from an
individual clinically diagnosed with autoimmune disease for the
presence or absence of elevated BCMA levels on their B cells,
wherein the presence of elevated BCMA levels is associated with the
clinical diagnosis of autoimmune disease. For this method, the
treatment plan can involves the use of a BLyS antagonist and the
BLyS antagonist can also be an APRIL antagonist. The autoimmune
disease can be selected from the group consisting of rheumatoid
arthritis, juvenile rheumatoid arthritis, systemic lupus
erythematosus (SLE), lupus nephritis (LN), Wegener's disease,
inflammatory bowel disease, idiopathic thrombocytopenic purpura
(ITP), thrombotic throbocytopenic purpura (TTP), autoimmune
thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy,
IgM polyneuropathies, myasthenia gravis, vasculitis, diabetes
mellitus, Reynaud's syndrome, Sjorgen's syndrome and
glomerulonephritis.
In particular, the autoimmune disease is SLE.
In a still further embodiment, the present invention includes an in
vitro method for predicting a patient's likelihood to respond to a
drug treatment for an autoimmune disease, comprising determining
the level of BCMA expression on the surface of B cells in a sample
from the patient; wherein the presence of elevated B cell levels is
predictive of the patient's likelihood to respond to a drug
treatment for the condition. The autoimmune disease can be selected
from the group consisting of rheumatoid arthritis, juvenile
rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus
nephritis (LN), Wegener's disease, inflammatory bowel disease,
idiopathic thrombocytopenic purpura (ITP), thrombotic
throbocytopenic purpura (TTP), autoimmune thrombocytopenia,
multiple sclerosis, psoriasis, IgA nephropathy, IgM
polyneuropathies, myasthenia gravis, vasculitis, diabetes mellitus,
Reynaud's syndrome, Sjorgen's syndrome and glomerulonephritis. In
particular, the autoimmune disease is SLE. The drug treatment of
the present invention can comprise a BLyS antagonist and said BLyS
antagonist can also be an APRIL antagonist.
Finally, the present invention contemplates a BLys antagonist for
use in the treatment of an autoimmune disease in a patient, wherein
said patient has elevated levels of BCMA expression on B cells. The
antagonist can be a BLyS antibody, such as Lymphostat-B. The
antagonist can also be a receptor-extracellular domain/Fc domain
fusion protein selected from the group consisting of TACI-Ig,
BCMA-Ig, and BAFF-R-Ig. In particular, the receptor-extracellular
domain/Fc domain fusion protein can be TACI-Ig, such as
atacicept.
These and other aspects of the invention will become apparent to
those persons skilled the art upon reading the details of the
invention as more fully described below.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows the levels of BAFF-R, TACI, and BCMA present on the
cells surface of naive B cells, memory B cells, and
plasmablasts.
FIG. 1B graphs the median fluorescence intensity (MFI) of BAFF-R in
total B cells, naive B cells, memory cells, and plasmablasts
comparing healthy controls (HC) and lupus patients (SLE).
FIG. 1C graphs the median fluorescence intensity (MFI) of TACI in
total B cells, naive B cells, memory cells, and plasmablasts
comparing healthy controls (HC) and lupus patients (SLE).
FIG. 1D graphs the median fluorescence intensity (MFI) of BCMA in
total B cells, naive B cells, memory cells, and plasmablasts
comparing healthy controls (HC) and lupus patients (SLE).
FIG. 2 upper level graphs the inverse relationship between BAFF-R
expression on total B cells and serum BAFF (BCMA) but the lack of a
statistically significant correlation between BAFF-R expression and
serum IgG anti-dsDNA and SLEDAI score (disease activity). FIG. 2
lower level graphs the inverse relationship between serum IgG
anti-dsDNA and the lack of correlation between serum BAFF (BCMA)
and SLEDAI score (disease activity).
FIG. 3A graphs the forward angle light scatter (FSC) measured as
MFI of the BCMA+ and BCMA- B cells in healthy controls and lupus
patients. FSC is an indication of activation in B cells. FIG. 3B
shows the results of comparing CD19 high and CD 19 low B cells for
the MFI of BCMA detection in both healthy controls and lupus
patients.
FIG. 4A shows results that indicate that BCMA+ cells are IgD+ and
IgM+ and therefore have not undergone class switching. It also
graphs the higher levels of CD86 seen in BCMA+ cells of lupus
patients and the relatively low levels of CD80 seen in both healthy
controls and lupus patients. FIG. 4B graphs the higher percentage
of IgD+ B cells seen in lupus patients as compared to healthy
controls.
FIG. 5A discloses the results of labeling the peripheral blood
mononuclear cells (PBMC) of healthy controls and lupus patients
with 5-carboxyfluorescein diacetate succinimidyl ester (CSFE) and
incubating with CpG with and without ligands. The upper level shows
plasmablast induction (CD27 high) and the lower level shows BCMA
induction. FIG. 5B graphs the experiment disclosed in 5A but
focusing on the proliferation results. FIG. 5C upper level graphs
the results from the experiment of 5A charting the increase of CD27
high cells with the increase of BCMA. The lower level reports the
MFI of BCMA in this experiment in lupus and healthy controls with
the various inductions. FIG. 5D discloses the production of IgG and
IgM in the sorted B cells of the experiment disclosed in 5A.
FIG. 6A graphs the percentage of BCMA positive cells comparing
Healthy Controls (HC) and Lupus patients in naive B cells (CD27
neg), memory cells (CD27 pos), and plasmablasts (CD27 high). FIG.
6B graphs production of CD27 high (plasmablasts) vs the production
of BCMA on the cell surface for Healthy controls and Lupus
patients.
FIG. 7 reports the MFI of the FSC of CD27 pos and CD27 neg cells
healthy controls vs. patients.
FIG. 8A discloses the percentage of CD19 high B cells found in
Healthy Controls (HC) and Lupus patients in naive B cells (CD27
neg), memory cells (CD27 pos), and plasmablasts (CD27 high). FIG.
8B charts the MFI of BCMA in CD19 high and CD19 low B cells for
healthy controls and lupus patients.
FIG. 9 graphically shows the levels of BAFF-R on the surface (by
MFI) on the surface of CD19 high and CD19 low B cells for healthy
controls and lupus patients.
FIG. 10 graphically shows the levels of TACI on the surface (by
MFI) on the surface of CD19 high and CD19 low B cells for healthy
controls and lupus patients.
BRIEF DESCRIPTION OF THE INVENTION
The present invention provides a method for screening TNFR family
members on the surface of B cells and the use of this information
for predicting the presence of autoimmune disease and predicting
the likelihood that a patient would respond to BLyS antagonist
treatment. The invention is based on the finding that the levels of
BCMA protein expression on the surface of B cells is elevated and
BLyS antagonists selectively neutralize the production of IgG by
said cells. This observation allows development of diagnostic
assays to detect the presence of increased BCMA on the B cells
surface where these higher levels are associated with autoimmune
disease, such as SLE, and also may predict the likelihood that an
individual will successfully respond to treatment methods that
neutralize such B cells, i.e., BLyS and/or APRIL antagonists.
Before the present invention is described, it is to be understood
that this invention is not limited to particular embodiments
described, as such may, of course, vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to be
limiting, since the scope of the present invention will be limited
only by the appended claims.
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
It must be noted that as used herein and in the appended claims,
the singular forms "a", "and", and "the" include plural referents
unless the context clearly dictates otherwise. Thus, for example,
reference to "a polymorphism includes a plurality of such
polymorphisms, reference to "a nucleic acid molecule" includes a
plurality of such nucleic acid molecules, and reference to "the
method" includes reference to one or more methods, method steps,
and equivalents thereof known to those skilled in the art, and so
forth.
The publications discussed herein are provided solely for their
disclosure prior to the filing date of the present application.
Nothing herein is to be construed as an admission that the present
invention is not entitled to antedate such publication by virtue of
prior invention. Further, the dates of publication provided may be
different from the actual publication dates which may need to be
independently confirmed.
Definitions
As used herein, the term "BCMA" is intended to generically refer to
both the wild-type and variant forms of the gene sequence, unless
specifically denoted otherwise. As it is commonly used in the art,
the term "gene" is intended to refer to the genomic region
encompassing 5' untranslated region(s) (UTR), exons, introns, and
3' UTR. Individual segments may be specifically referred to, e.g.
promoter, coding region, etc. Combinations of such segments that
provide for a complete BCMA protein may be referred to generically
as a protein coding sequence. The nucleotide sequence of BCMA are
publicly available (GenBank Accession number as BC058291). There
are four major haplotypes of the BCMA gene in the human genome, in
the present disclosure the term "BCMA" is meant to encompass all
four (Kawasaki et al., Genes Immun. 2:276-9, 2001).
The term "polymorphism", as used herein, refers to a difference in
the nucleotide or amino acid sequence of a given region as compared
to a nucleotide or amino acid sequence in a homologous-region of
another individual, in particular, a difference in the nucleotide
of amino acid sequence of a given region which differs between
individuals of the same species. A polymorphism is generally
defined in relation to a reference sequence. Polymorphisms include
single nucleotide differences, differences in sequence of more than
one nucleotide, and single or multiple nucleotide insertions,
inversions and deletions; as well as single amino acid differences,
differences in sequence of more than one amino acid, and single or
multiple amino acid insertions, inversions, and deletions.
The terms "polynucleotide" and "nucleic acid molecule" are used
interchangeably herein to refer to polymeric forms of nucleotides
of any length. The polynucleotides may contain
deoxyribonucleotides, ribonucleotides, and/or their analogs.
Nucleotides may have any three-dimensional structure, and may
perform any function, known or unknown. The term "polynucleotide"
includes single-, double-stranded and triple helical molecules.
"Oligonucleotide" generally refers to polynucleotides of between
about 5 and about 100 nucleotides of single- or double-stranded
DNA. However, for the purposes of this disclosure, there is no
upper limit to the length of an oligonucleotide. Oligonucleotides
are also known as oligomers or oligos and may be isolated from
genes, or chemically synthesized by methods known in the art.
The following are non-limiting embodiments of polynucleotides: a
gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes,
cDNA, recombinant polynucleotides, branched polynucleotides,
plasmids, vectors, isolated DNA of any sequence, isolated RNA of
any sequence, nucleic acid probes, and primers. A nucleic acid
molecule may also comprise modified nucleic acid molecules, such as
methylated nucleic acid molecules and nucleic acid molecule
analogs. Analogs of purines and pyrimidines are known in the art.
Nucleic acids may be naturally occurring, e.g. DNA or RNA, or may
be synthetic analogs, as known in the art. Such analogs may be
preferred for use as probes because of superior stability under
assay conditions. Modifications in the native structure, including
alterations in the backbone, sugars or heterocyclic bases, have
been shown to increase intracellular stability and binding
affinity. Among useful changes in the backbone chemistry are
phosphorothioates; phosphorodithioates, where both of the
non-bridging oxygens are substituted with sulfur;
phosphoroamidites; alkyl phosphotriesters and boranophosphates.
Achiral phosphate derivatives include 3'-O'-5'-S-phosphorothioate,
3'-S-5'-O-phosphorothioate, 3'-CH2-5'-O-phosphonate and
3'-NH-5'-O-phosphoroamidate. Peptide nucleic acids replace the
entire ribose phosphodiester backbone with a peptide linkage.
Sugar modifications are also used to enhance stability and
affinity. The .alpha.-anomer of deoxyribose may be used, where the
base is inverted with respect to the natural .beta.-anomer. The
2'-OH of the ribose sugar may be altered to form 2'-O-methyl or
2'-O-allyl sugars, which provides resistance to degradation without
comprising affinity.
Modification of the heterocyclic bases must maintain proper base
pairing. Some useful substitutions include deoxyuridine for
deoxythymidine; 5-methyl-2'-deoxycytidine and
5-bromo-2'-deoxycytidine for deoxycytidine.
5-propynyl-2'-deoxyuridine and 5-propynyl-2'-deoxycytidine have
been shown to increase affinity and biological activity when
substituted for deoxythymidine and deoxycytidine, respectively.
The terms "polypeptide" and "protein", used interchangebly herein,
refer to a polymeric form of amino acids of any length, which can
include coded and non-coded amino acids, chemically or
biochemically modified or derivatized amino acids, and polypeptides
having modified peptide backbones. The term includes fusion
proteins, including, but not limited to, fusion proteins with a
heterologous amino acid sequence, fusions with heterologous and
homologous leader sequences, with or without N-terminal methionine
residues; immunologically tagged proteins; and the like.
In the broadest sense, as used herein, the terms "autoimmune
disease," refer to a disease wherein a patient's immune system is
producing an unwanted immune response to one or more of their own
proteins. Representative examples of autoimmune diseases include
rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus
erythematosus (SLE), lupus nephritis (LN), Wegener's disease,
inflammatory bowel disease, idiopathic thrombocytopenic purpura
(ITP), thrombotic throbocytopenic purpura (TTP), autoimmune
thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy,
IgM polyneuropathies, myasthenia gravis, vasculitis, diabetes
mellitus, Reynaud's syndrome, Sjorgen's syndrome and
glomerulonephritis.
A "substantially isolated" or "isolated" polynucleotide is one that
is substantially free of the sequences with which it is associated
in nature. By substantially free is meant at least 50%, preferably
at least 70%, more preferably at least 80%, and even more
preferably at least 90% free of the materials with which it is
associated in nature. As used herein, an "isolated" polynucleotide
also refers to recombinant polynucleotides, which, by virtue of
origin or manipulation: (1) are not associated with all or a
portion of a polynucleotide with which it is associated in nature,
(2) are linked to a polynucleotide other than that to which it is
linked in nature, or (3) does not occur in nature.
Hybridization reactions can be performed under conditions of
different "stringency". Conditions that increase stringency of a
hybridization reaction of widely known and published in the art.
See, for example, Sambrook et al. (1989). Examples of relevant
conditions include (in order of increasing stringency): incubation
temperatures of 25.degree. C., 37.degree. C., 50.degree. C. and
68.degree. C.; buffer concentrations of 10.times.SSC, 6.times.SSC,
1.times.SSC, 0.1.times.SSC (where SSC is 0.15 M NaCl and 15 mM
citrate buffer) and their equivalents using other buffer systems;
formamide concentrations of 0%, 25%, 50%, and 75%; incubation times
from 5 minutes to 24 hours; 1, 2, or more washing steps; wash
incubation times of 1, 2, or 15 minutes; and wash solutions of
6.times.SSC, 1.times.SSC, 0.1.times.SSC, or deionized water.
Examples of stringent conditions are hybridization and washing at
50.degree. C. or higher and in 0.1.times.SSC (9 mM NaCl/0.9 mM
sodium citrate).
"T.sub.m" is the temperature in degrees Celsius at which 50% of a
polynucleotide duplex made of complementary strands hydrogen bonded
in anti-parallel direction by Watson-Crick base pairing dissociates
into single strands under conditions of the experiment. T.sub.m may
be predicted according to a standard formula, such as: where
[X.sup.+] is the cation concentration (usually sodium ion,
Na.sup.+) in mol/L; (% G/C) is the number of G and C residues as a
percentage of total residues in the duplex; (% F) is the percent
formamide in solution (wt/vol); and L is the number of nucleotides
in each strand of the duplex.
Stringent conditions for both DNA/DNA and DNA/RNA hybridization are
as described by Sambrook et al. Molecular Cloning, A Laboratory
Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989, herein incorporated by reference. For example,
see page 7.52 of Sambrook et al.
The term "host cell" includes an individual cell or cell culture
which can be or has been a recipient of any recombinant vector(s)
or isolated polynucleotide of the invention. Host cells include
progeny of a single host cell, and the progeny may not necessarily
be completely identical (in morphology or in total DNA complement)
to the original parent cell due to natural, accidental, or
deliberate mutation and/or change. A host cell includes cells
transfected or infected in vivo or in vitro with a recombinant
vector or a polynucleotide of the invention. A host cell which
comprises a recombinant vector of the invention is a "recombinant
host cell".
The term "binds specifically," in the context of antibody binding,
refers to high avidity and/or high affinity binding of an antibody
to a specific polypeptide i.e., epitope of a polymorphic BCMA
polypeptide. Antibody binding to an epitope on a specific
polymorphic BCMA polypeptide (also referred to herein as "a
polymorphic BCMA epitope") is preferably stronger than binding of
the same antibody to any other epitope, particularly those which
may be present in molecules in association with, or in the same
sample, as the specific polypeptide of interest, e.g., binds more
strongly to a specific BCMA polymorphic epitope than to a different
BCMA epitope so that by adjusting binding conditions the antibody
binds almost exclusively to the specific BCMA polymorphic epitope
and not to any other BCMA epitope, and not to any other BCMA
polypeptide which does not comprise the polymorphic epitope.
Antibodies which bind specifically to a polypeptide of interest may
be capable of binding other polypeptides at a weak, yet detectable,
level (e.g., 10% or less of the binding shown to the polypeptide of
interest). Such weak binding, or background binding, is readily
discernible from the specific antibody binding to the compound or
polypeptide of interest, e.g. by use of appropriate controls. In
general, antibodies of the invention which bind to a specific BCMA
polypeptide with a binding affinity of 10.sup.7 mole/l or more,
preferably 10.sup.8 mole/l or more are said to bind specifically to
the specific BCMA polypeptide. In general, an antibody with a
binding affinity of 10.sup.6 mole/liters or less is not useful in
that it will not bind an antigen at a detectable level using
conventional methodology currently used.
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
can 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.
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)). Methods of
making chimeric antibodies are known in the art.
"Humanized" forms of non-human (e. g., murine) antibodies are
chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F (ab') 2 or other antigen-binding
subsequences of antibodies) which contain minimal sequence derived
from non-human immunoglobulin.
For the most part, humanized antibodies are human immunoglobulins
(recipient antibody) in which residues from a
complementarity-determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat or rabbit having the desired
specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues which are found neither
in the recipient antibody nor in the imported CDR or framework
sequences. These modifications are made to further refine and
maximize 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 FR regions are
those of a human immunoglobulin sequence although the FR regions
may include one or more amino acid substitutions that improve
binding affinity. The number of these amino acid substitutions in
the FR are typically no more than 6 in the H chain, and in the L
chain, no more than 3. The humanized antibody optimally 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); Reichmann
et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct.
Biol., 2: 593-596 (1992). The humanized antibody includes a
PRIMATIZED antibody wherein the antigen-binding region of the
antibody is derived from an antibody produced by, e. g., immunizing
macaque monkeys with the antigen of interest. Methods of making
humanized antibodies are known in the art.
Human antibodies can also be produced using various techniques
known in the art, including phage-display libraries. Hoogenboom and
Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol.
Biol., 222: 581 (1991). The techniques of Cole et al. and Boerner
et al. are also available for the preparation of human monoclonal
antibodies. Cole et al., Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):
86-95 (1991).
"Functional fragments" of the binding antibodies of the invention
are those fragments that retain binding to BLyS, TACI, BAFF-R, or
BCMA with substantially the same affinity as the intact full chain
molecule from which they are derived and may be able to deplete B
cells as measured by in vitro or in vivo assays such as those
described herein.
Antibody "effector functions" refer to those biological activities
attributable to the Fc region (a native sequence Fc region or amino
acid sequence variant Fc region) of an antibody, and vary with the
antibody isotype. Examples of antibody effector functions include:
Clq binding and complement dependent cytotoxicity; Fc receptor
binding; antibody-dependent cell-mediated cytotoxicity (ADCC);
phagocytosis; down regulation of cell surface receptors (e. g. B
cell receptor); and B cell activation.
"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to
a form of cytotoxicity in which secreted Ig bound onto Fc receptors
(FcRs) present on certain cytotoxic cells (e. g. Natural Killer
(NK) cells, neutrophils, and macrophages) enable these cytotoxic
effector cells to bind specifically to an antigen-bearing-target
cell and subsequently kill the-target cell with cytotoxins. The
antibodies "arm" the cytotoxic cells and are absolutely required
for such killing. The primary cells for mediating ADCC, NK cells,
express FcyRIII only, whereas monocytes express FcyRI, FcyRII and
FcyRIII FcR expression on hematopoietic cells is summarized in
Table 3 on page 464 of Ravetch and Kinet, Ann. 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).
"Complement dependent cytotoxicity" or "CDC" refers to the lysis of
a target cell in the presence of complement. Activation of the
classical complement pathway is initiated by the binding of the
first component of the complement system (Clq) to antibodies (of
the appropriate subclass) which are bound to their cognate antigen.
To assess complement activation, a CDC assay, e. g. as described in
Gazzano-Santoro etal., J. Immunol. Methods 202: 163 (1996), may be
performed.
An "isolated" antibody 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 antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the antibody will be purified (1) to greater than 95%
by weight of antibody 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 antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
The terms "detectably labeled antibody" refers to an antibody (or
antibody fragment which retains binding specificity for a BCMA
polypeptide or epitope), having an attached detectable label. The
detectable label is normally attached by-chemical conjugation, but
where the label is a polypeptide, it could alternatively be
attached by genetic engineering techniques. Methods for production
of detectably labeled proteins are well known in the art.
Detectable labels may be selected from a variety of such labels
known in the art, including, but not limited to, radioisotopes,
fluorophores, paramagnetic labels, enzymes (e.g., horseradish
peroxidase), or other moieties or compounds which either emit a
detectable signal (e.g., radioactivity, fluorescence, color) or
emit a detectable signal after exposure of the label to its
substrate. Various detectable label/substrate pairs (e.g.,
horseradish peroxidase/diaminobenzidine, avidin/streptavidin,
luciferase/luciferin)), methods for labeling antibodies, and
methods for using labeled antibodies are well known in the art
(see, for example, Harlow and Lane, eds. (Antibodies: A Laboratory
Manual (1988) Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.)).
A "biological sample" encompasses a variety of sample types
obtained from an individual and can be used in a diagnostic or
monitoring assay. The definition encompasses blood and other liquid
samples of biological origin, solid tissue samples such as a biopsy
specimen or tissue cultures or cells derived there from and the
progeny thereof. The definition also includes samples that have
been manipulated in any way after their procurement, such as by
treatment with reagents, solubilization, or enrichment for certain
components, such as polynucleotides. The term "biological sample"
encompasses a clinical sample, and also includes cells in culture,
cell supernatants, cell lysates, serum, plasma, biological fluid,
and tissue samples.
As used herein, the terms "treatment", "treating", and the like,
refer to obtaining a desired pharmacologic and/or physiologic
effect. The effect may be prophylactic in terms of completely or
partially preventing a disease or symptom thereof and/or may be
therapeutic in terms of a partial or complete cure for a disease
and/or adverse affect attributable to the disease. "Treatment", as
used herein, covers any treatment of a disease in a mammal,
particularly in a human, and includes: (a) preventing the disease
from occurring in a subject which may be predisposed to the disease
but has not yet been diagnosed as having it; (b) inhibiting the
disease, i.e., arresting its development; and (c) relieving the
disease, i.e., causing regression of the disease.
"Immunosuppressive drugs" are any molecules that interfere with the
immune system and blunt its response to foreign or self antigens.
Cyclophosphamide (CYC) and mycophenolate mofetil (MMF) are two such
kinds of molecules. This term is intended to encompass any drug or
molecule useful as a therapeutic agent in downregulating the immune
system.
A "fusion protein" and a "fusion polypeptide" refer to a
polypeptide having two portions covalently linked together, where
each of the portions is a polypeptide having a different property.
The property may be a biological property, such as activity in
vitro or in vivo. The property may also be a simple chemical or
physical property, such as binding to a target molecule, catalysis
of a reaction, etc. The two portions may be linked directly by a
single peptide bond or through a peptide linker containing one or
more amino acid residues. Generally, the two portions and the
linker will be in reading frame with each other.
A "conjugate" refers to any hybrid molecule, including fusion
proteins and as well as molecules that contain both amino acid or
protein portions and non-protein portions. Conjugates may be
synthesized by a variety of techniques known in the art including,
for example, recombinant DNA techniques, solid phase synthesis,
solution phase synthesis, organic chemical synthetic techniques or
a combination of these techniques. The choice of synthesis will
depend upon the particular molecule to be generated. For example, a
hybrid molecule not entirely "protein" in nature may be synthesized
by a combination of recombinant techniques and solution phase
techniques.
As used herein, the term "Fc-fusion protein" designates
antibody-like molecules which combine the binding specificity of a
heterologous protein with the effector functions of immunoglobulin
constant domains. Structurally, the Fc-fusion proteins comprise a
fusion of an amino acid sequence with the desired binding
specificity which is other than the antigen recognition and binding
site of an antibody (i. e., is "heterologous"), and an
immunoglobulin constant domain sequence. The Fc-fusion protein
molecule typically includes a contiguous amino acid sequence
comprising at least the binding site of a receptor or a ligand. The
immunoglobulin constant domain sequence in the Fc-fusion protein
can be obtained from any immunoglobulin, such as IgG-1, IgG-2,
IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD
or IgM. For example, useful Fc-fusion proteins according to this
invention are polypeptides that comprise the BLyS binding portions
of a BLyS receptor without the transmembrane or cytoplasmic
sequences of the BLyS receptor. In one embodiment, the
extracellular domain of BAFF-R, TACI or BCMA is fused to a constant
domain of an immunoglobulin sequence.
The terms "individual," "subject," and "patient," used
interchangeably herein, refer to a mammal, including, but not
limited to, murines, simians, humans, mammalian farm animals,
mammalian sport animals, and mammalian pets.
The term "mammal" 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 herein is human.
Detection of BCMA Polypeptides
The present invention provides for detection of BCMA polypeptides.
The term "BCMA polypeptide" encompasses an amino acid sequence
encoded by an open reading frame (ORF) of a known BCMA
polynucleotide, including the full-length native polypeptide and
fragments thereof, particularly biologically active fragments
and/or fragments corresponding to functional domains, e.g. a region
or domain having biological activity, etc.; antigenic fragments
thereof, and including fusions of the subject polypeptides to other
proteins or parts thereof. The amino acid sequences of BCMA
polypeptides have been disclosed. (See e.g. Laabi et al., Nucleic
Acids Research 22: 1147-1154, 1994; Laabi et al., EMBO J., 11:
3897-3904 (1992); Gras et al., Int. Immunology, 7: 1093-1106
(1995); and Madry et al., Int. Immunology, 10: 1693-1702 (1998).
The BCMA polypeptides of the invention can be isolated from a
variety of sources, such as from human tissue types or from another
source, or prepared by recombinant and/or synthetic methods. A
polymorphism in a BCMA polypeptide is generally defined relative to
a reference sequence.
As used herein, "polymorphic BCMA polypeptide" refers to an amino
acid sequence of a recombinant or non-recombinant polypeptide
having an amino acid sequence of i) a native polymorphic BCMA
polypeptide, ii) a fragment of a polymorphic BCMA polypeptide, iii)
polypeptide analogs of a polymorphic BCMA polypeptide, iv) variants
of a polymorphic BCMA polypeptide; v) an immunologically active
fragment of a polymorphic BCMA polypeptide; and vi) fusion proteins
comprising a polymorphic BCMA polypeptide. Polymorphic BCMA
polypeptides of the invention can be obtained from a biological
sample, or from any source whether natural, synthetic,
semi-synthetic or recombinant.
The term "polymorphic BCMA polypeptide" or "BCMA polypeptide"
encompasses a polypeptide comprising from at least about 5 amino
acids, at least about 10 amino acids, at least about 15 amino
acids, at least about 25 amino acids, at least about 50 amino
acids, at least about 75 amino acids, at least about 100 amino
acids, at least about 200 amino acids, at least about 300 amino
acids, at least about 400 amino acids, or up to the entire
polypeptide of a polymorphic BCMA polypeptide. In some embodiments,
a polymorphic BCMA polypeptide exhibits biological activity, e.g.,
the polypeptide causes proliferation of B-cells and production of
immunoglobulin in an in vitro assay. Other assays for BCMA
biological activity are known in the art and can be used to
determine whether a polymorphic BCMA polypeptide exhibits
biological activity and, if desired, to quantitate BCMA biological
activity. BCMA biological assays are described in various
publications, e.g., Moore et al., supra.
BCMA polypeptides can be obtained by any known method, or a
combination of such methods, including isolation from natural
sources; production by chemical synthesis; and production by
standard recombinant techniques. BCMA polypeptides can be isolated
from a biological source, using affinity chromatography, e.g.,
using antibodies specific for a BCMA polypeptide are immobilized on
a solid support. The polypeptides may be expressed in prokaryotes
or eukaryotes in accordance with conventional ways, depending upon
the purpose for expression. For large scale production of the
protein, a unicellular organism, such as E. coli, B. subtilis, S.
cerevisiae, insect cells in combination with baculovirus vectors,
or cells of a higher organism such as vertebrates, particularly
mammals, e.g. COS 7 cells, CHO cells, HEK293 cells, and the like,
may be used as the expression host cells. In some situations, it is
desirable to express the gene in eukaryotic cells, where the
protein will benefit from native folding and post-translational
modifications. The polypeptide can then be isolated from cell
culture supernatant or from cell lysates using affinity
chromatography methods or anion exchange/size exclusion
chromatography methods, as described above.
With the availability of the protein or fragments thereof in large
amounts, by employing an expression host, the protein may be
isolated and purified in accordance with conventional ways. A
lysate may be prepared of the expression host and the lysate
purified using HPLC, exclusion chromatography, gel electrophoresis,
affinity chromatography, or other purification technique. The
isolated proteins can be used to produce antibodies, which are in
turn, used to detect the presence of that protein using standard
assay systems, e.g., ELISA or FACS analysis.
Preparation of BCMA Polypeptides
In addition to the plurality of uses described in greater detail in
following sections, the BCMA nucleic acid compositions are used in
the preparation of all or a portion of the BCMA polypeptides, as
described above. The polynucleotides (including cDNA or the
full-length gene) are used to express a partial or complete gene
product. Constructs comprising the subject polynucleotides can be
generated synthetically. Alternatively, single-step assembly of a
gene and entire plasmid from large numbers of
oligodeoxyribonucleotides is described by, e.g., Stemmer et al.,
Gene (Amsterdam) (1995) 164(1):49-53. In this method, assembly PCR
(the synthesis of long DNA sequences from large numbers of
oligodeoxyribonucleotides (oligos)) is described. The method is
derived from DNA shuffling (Stemmer, Nature (1994) 370:389-391),
and does not rely on DNA ligase, but instead relies on DNA
polymerase to build increasingly longer DNA fragments during the
assembly process. Appropriate polynucleotide constructs are
purified using standard recombinant DNA techniques as described in,
for example, Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd Ed., (1989) Cold Spring Harbor Press, Cold Spring
Harbor, N.Y., and under current regulations described in United
States Dept. of HHS, National Institute of Health (NIH) Guidelines
for Recombinant DNA Research.
Polynucleotide molecules comprising a polynucleotide sequence
provided herein are propagated by placing the molecule in a vector.
Viral and non-viral vectors are used, including plasmids. The
choice of plasmid will depend on the type of cell in which
propagation is desired and the purpose of propagation. Certain
vectors are useful for amplifying and making large amounts of the
desired DNA sequence. Other vectors are suitable for expression in
cells in culture. Still other vectors are suitable for transfer and
expression in cells in a whole animal or person. The choice of
appropriate vector is well within the skill of the art. Many such
vectors are available commercially. The partial or full-length
polynucleotide is inserted into a vector typically by means of DNA
ligase attachment to a cleaved restriction enzyme site in the
vector. Alternatively, the desired nucleotide sequence can be
inserted by homologous recombination in vivo. Typically this is
accomplished by attaching regions of homology to the vector on the
flanks of the desired nucleotide sequence. Regions of homology are
added by ligation of oligonucleotides, or by polymerase chain
reaction using primers comprising both the region of homology and a
portion of the desired nucleotide sequence, for example.
For expression, an expression cassette or system may be employed.
The gene product encoded by a polynucleotide of the invention is
expressed in any convenient expression system, including, for
example, bacterial, yeast, insect, amphibian and mammalian systems.
Suitable vectors and host cells are described in U.S. Pat. No.
5,654,173. In the expression vector, a BCMA polypeptide-encoding
polynucleotide is linked to a regulatory sequence as appropriate to
obtain the desired expression properties. These-can include
promoters (attached either at the 5' end of the sense strand or at
the 3' end of the antisense strand), enhancers, terminators,
operators, repressors, and inducers. The promoters can be regulated
or constitutive. In some situations it may be desirable to use
conditionally active promoters, such as tissue-specific or
developmental stage-specific promoters. These are linked to the
desired nucleotide sequence using the techniques described above
for linkage to vectors. Any techniques known in the art can be
used. In other words, the expression vector will provide a
transcriptional and translational initiation region, which may be
inducible or constitutive, where the coding region is operably
linked under the transcriptional control of the transcriptional
initiation region, and a transcriptional and translational
termination region. These control regions may be native to the BCMA
gene, or may be derived from exogenous sources.
Expression vectors generally have convenient restriction sites
located near the promoter sequence to provide for the insertion of
nucleic acid sequences encoding heterologous proteins. A selectable
marker operative in the expression host may be present. Expression
vectors may be used for the production of fusion proteins, where
the exogenous fusion peptide provides additional functionality,
i.e. increased protein synthesis, stability, reactivity with
defined antisera, an enzyme marker, e.g. .beta.-galactosidase,
etc.
Expression cassettes may be prepared comprising a transcription
initiation region, the gene or fragment thereof, and a
transcriptional termination region. Of particular interest is the
use of sequences that allow for the expression of functional
epitopes or domains, usually at least about 8 amino acids in
length, more usually at least about 15 amino acids in length, to
about 25 amino acids, and up to the complete open reading frame of
the gene. After introduction of the DNA, the cells containing the
construct may be selected by means of a selectable marker, the
cells expanded and then used for expression.
BCMA polypeptides may be expressed in prokaryotes or eukaryotes in
accordance with conventional ways, depending upon the purpose for
expression. For large scale production of the protein, a
unicellular organism, such as E. coli, B. subtilis, S. cerevisiae,
insect cells in combination with baculovirus vectors, or cells of a
higher organism such as vertebrates, particularly mammals, e.g. COS
7 cells, HEK 293, CHO, Xenopus Oocytes, etc., may be used as the
expression host cells. In some situations, it is desirable to
express a polymorphic BCMA nucleic acid molecule in eukaryotic
cells, where the polymorphic BCMA protein will benefit from native
folding and post-translational modifications. Small peptides can
also be synthesized in the laboratory. Polypeptides that are
subsets of the complete BCMA sequence may be used to identify and
investigate parts of the protein important for function.
Specific expression systems of interest include bacterial, yeast,
insect cell and mammalian cell derived expression systems.
Representative systems from each of these categories is are
provided below:
Bacteria. Expression systems in bacteria include those described in
Chang et al., Nature (1978) 275:615; Goeddel et al., Nature (1979)
281:544; Goeddel et al., Nucleic Acids Res. (1980) 8:4057; EP 0
036,776; U.S. Pat. No. 4,551,433; DeBoer et al., Proc. Natl. Acad.
Sci. (USA) (1983) 80:21-25; and Siebenlist et al., Cell (1980)
20:269.
Yeast. Expression systems in yeast include those described in
Hinnen et al., Proc. Natl. Acad. Sci. (USA) (1978) 75:1929; Ito et
al., J. Bacteriol. (1983) 153:163; Kurtz et al., Mol. Cell. Biol.
(1986) 6:142; Kunze et al., J. Basic Microbiol. (1985)25:141;
Gleeson et al., J. Gen. Microbiol. (1986) 132:3459; Roggenkamp et
al., Mol. Gen. Genet. (1986) 202:302; Das et al., J. Bacteriol.
(1984) 158:1165; De Louvencourt et al., J. Bacteriol. (1983) 154:
737; Van den Berg et al., Bio/Technology (1990)8:135; Kunze et al.,
J. Basic Microbiol. (1985)25:141; Cregg et al., Mol. Cell. Biol.
(1985) 5:3376; U.S. Pat. Nos. 4,837,148 and 4,929,555; Beach and
Nurse, Nature (1981) 300:706; Davidow et al., Curr. Genet. (1985)
10:380; Gaillardin et al., Curr. Genet. (1985) 10:49; Ballance et
al., Biochem. Biophys. Res. Commun. (1983) 112:284-289; Tilburn et
al., Gene (1983) 26:205-221; Yelton et al., Proc. Natl. Acad. Sci.
(USA) (1984) 81:1470-1474; Kelly and Hynes, EMBO J. (1985)
4:475479; EP 0 244,234; and WO 91/00357.
Insect Cells. Expression of heterologous genes in insects is
accomplished as described in U.S. Pat. No. 4,745,051; Friesen et
al., "The Regulation of Baculovirus Gene Expression", in: The
Molecular Biology Of Baculoviruses (1986) (W. Doerfler, ed.); EP 0
127,839; EP 0 155,476; and Vlak et al., J. Gen. Virol. (1988)
69:765-776; Miller et al., Ann. Rev. Microbiol. (1988) 42:177;
Carbonell et al., Gene (1988) 73:409; Maeda et al., Nature (1985)
315:592-594; Lebacq-Verheyden et al., Mol. Cell. Biol. (1988)
8:3129; Smith et al., Proc. Natl. Acad. Sci. (USA) (1985) 82:8844;
Miyajima et al., Gene (1987) 58:273; and Martin et al., DNA (1988)
7:99. Numerous baculoviral strains and variants and corresponding
permissive insect host cells from hosts are described in Luckow et
al., Bio/Technology (1988) 6:47-55, Miller et al., Generic
Engineering (1986) 8:277-279, and Maeda et al., Nature (1985)
315:592-594.
Mammalian Cells. Mammalian expression is accomplished as described
in Dijkema et al., EMBO J. (1985) 4:761, Gorman et al., Proc. Natl.
Acad. Sci. (USA) (1982) 79:6777, Boshart et al., Cell (1985) 41:521
and U.S. Pat. No. 4,399,216. Other features of mammalian expression
are facilitated as described in Ham and Wallace, Meth. Enz. (1979)
58:44, Barnes and Sato, Anal. Biochem. (1980) 102:255, U.S. Pat.
Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, WO 90/103430, WO
87/00195, and U.S. Pat. No. RE 30,985.
When any of the above host cells, or other appropriate host cells
or organisms, are used to replicate and/or express the
polynucleotides or nucleic acids of the invention, the resulting
replicated nucleic acid, RNA, expressed protein or polypeptide, is
within the scope of the invention as a product of the host cell or
organism. The product is recovered by any appropriate means known
in the art.
Once the gene corresponding to a selected polynucleotide is
identified, its expression can be regulated-in the cell to which
the gene is native. For example, an endogenous gene of a cell can
be regulated by an exogenous regulatory sequence inserted into the
genome of the cell at location sufficient to at least enhance
expressed of the gene in the cell. The regulatory sequence may be
designed to integrate into the genome via homologous recombination,
as disclosed in U.S. Pat. Nos. 5,641,670 and 5,733,761, the
disclosures of which are herein incorporated by reference, or may
be designed to integrate into the genome via non-homologous
recombination, as described in WO 99/15650, the disclosure of which
is herein incorporated by reference. As such, also encompassed in
the subject invention is the production of BCMA proteins without
manipulation of the encoding nucleic acid itself, but instead
through integration of a regulatory sequence into the genome of
cell that already includes a gene encoding the desired protein, as
described in the above incorporated patent documents.
Preparation of Antibodies Specific for BCMA Polypeptides
The invention further can encompass the use of antibodies,
particularly isolated antibodies, that are specific for BCMA
polypeptides. The antibodies of the invention are useful in a
variety of diagnostic assays or treatments, as described in further
detail below. For example, an antibody can be used to detect and/or
measure the levels of a BCMA polypeptide in a biological
sample.
Isolated BCMA polypeptides of the invention are useful for the
production of antibodies, where short fragments provide for
antibodies specific for the particular polypeptide, and larger
fragments or the entire protein allow for the production of
antibodies over the surface of the polypeptide. Accordingly, the
methods of the present invention can utilize isolated antibodies
which specifically bind a BCMA polypeptide, or antigenic fragment
thereof. Antibodies may be raised to the wild-type or variant
forms. Antibodies may be raised to isolated peptides corresponding
to these domains, or to the native protein. Antibodies may be
raised to polypeptides and/or peptide fragments of BCMA from any
mammalian species. As one non-limiting example, an enzyme-linked
immunosorbent assay (ELISA) can be used to determine the
specificity of a given monoclonal antibody for a BCMA
polypeptide.
The BCMA polypeptides are useful for the production of antibodies,
where short fragments provide for antibodies specific for the
particular polypeptide, and larger fragments or the entire protein
allow for the production of antibodies over the surface of the
polypeptide. As used herein, the term "antibodies" includes
antibodies of any isotype, fragments of antibodies which retain
specific binding to antigen, including, but not limited to, Fab,
Fv, scFv, and Fd fragments, fusion proteins comprising such
antibody fragments, detectably labeled antibodies, and chimeric
antibodies. "Antibody specificity", in the context of
antibody-antigen interactions, is a term well understood in the
art, and indicates that a given antibody binds to a given antigen,
wherein the binding can be inhibited by that antigen or an epitope
thereof which is recognized by the antibody, and does not
substantially bind to unrelated antigens. Methods of determining
specific antibody binding are well known to those skilled in the
art, and can be used to determine the specificity of antibodies for
a BCMA polypeptide. In specific embodiments, the BCMA antibody
binds to the extracellular domain of BCMA. See, for example, Carter
et al. (2007) Mol Can Ther 6:3009-18. In still further embodiments,
the BCMA antibody binds to the extracellular domain of BCMA and
further blocks BCMA activity. Methods for determining if a BCMA
antibody blocks BCMA activity are known.
Antibodies are prepared in accordance with conventional ways, where
the expressed is polypeptide or protein is used as an immunogen, by
itself or conjugated to known immunogenic carriers, e.g. KLH, pre-S
HBsAg, other viral or eukaryotic proteins, or the like. Various
adjuvants may be employed, with a series of injections, as
appropriate. For monoclonal antibodies, after one or more booster
injections, the spleen is isolated, the lymphocytes immortalized by
cell fusion, and then screened for high affinity antibody binding.
The immortalized cells, i.e. hybridomas, producing the desired
antibodies may then be expanded. For further description, see
Monoclonal Antibodies: A Laboratory Manual, Harlow and Lane eds.,
Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1988. If
desired, the mRNA encoding the heavy and light chains may be
isolated and mutagenized by cloning in E. coli, and the heavy and
light chains mixed to further enhance the affinity of the antibody.
Alternatives to in vivo immunization as a method of raising
antibodies include binding to phage display libraries, usually in
conjunction with in vitro affinity maturation.
Antibodies may be attached, directly or indirectly (e.g., via a
linker molecule) to a solid support for use in a diagnostic assay
to determine and/or measure the presence of BCMA polypeptide in a
biological sample. Attachment is generally covalent, although it
need not be. Solid supports include, but are not limited to, beads
(e.g., polystyrene beads, magnetic beads, and the like); plastic
surfaces (e.g., polystyrene or polycarbonate multi-well plates
typically used in an ELISA or radioimmunoassay (RIA), and the
like); sheets, e.g., nylon, nitrocellulose, and the like; and
chips, e.g., SiO.sub.2 chips such as those used in microarrays.
Accordingly, the invention further provides assay devices
comprising antibodies attached to a solid support.
A single antibody or a battery of different antibodies can then be
used to create an assay device. Such an assay device can be
prepared using conventional technology known to those skilled in
the art. The antibody can be purified and isolated using known
techniques and bound to a support surface using known procedures.
The resulting surface having antibody bound thereon can be used to
assay a test sample, e.g., a biological sample, in vitro to
determine if the sample contains one or more types of BCMA
polypeptides. For example, antibodies which bind only to a specific
BCMA epitope can be attached to the surface of a material.
Alternatively, a plurality of specific antibodies, which may be
arranged in an array, wherein antibodies specific for two or more
different BCMA epitopes are attached to the solid support, can be
used. A test sample is brought into contact with the antibodies
bound to the surface of material. Specific binding can be detected
using any known method. If specific binding is not detected, it can
be deduced that the sample does not contain the specific BCMA
epitope. As one non-limiting example of how specific binding can be
detected, once the test sample has been contacted with the
antibodies bound to the solid support, a second, detectably-labeled
antibody can be added, which recognizes a BCMA epitope distinct
from the epitope recognized by the solid support-bound
antibody.
A variety of other reagents may be included in the assays to detect
BCMA polypeptides described herein. These include reagents such as
salts, neutral proteins, e.g. albumin, detergents, etc., that are
used to facilitate optimal protein-protein binding, and/or reduce
non-specific or background interactions. Reagents that improve the
efficiency of the assay, such as protease inhibitors,
anti-microbial agents, etc. may be used. The components are added
in any order that provides for the requisite binding. Incubations
are performed at any suitable temperature, typically between
4.degree. C. and 40.degree. C. Incubation periods are selected for
optimum activity, but may also be optimized to facilitate rapid
high-throughput screening. Typically between 0.1 and 1 hour will be
sufficient.
Bispecific antibodies are antibodies that have binding
specificities for at least two different epitopes. Exemplary
bispecific antibodies may bind to two different epitopes of the B
cell surface marker. Other such antibodies may bind a first B cell
marker and further bind a second B cell surface marker.
Alternatively, an anti-B cell marker binding arm may be combined
with an arm which binds to a triggering molecule on a leukocyte
such as a T-cell receptor molecule (e. g. CD2 or CD3), or Fc
receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and
FcyRIII (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-, vinca alkaloid, ricin A chain,
methotrexate or radioactive isotope hapten).
Bispecific antibodies can be prepared as full length antibodies or
antibody fragments (e. g. F (ab') 2 bispecific antibodies). Methods
for making bispecific antibodies are known in the art. Traditional
production of full length bispecific antibodies is based on the
coexpression of two immunoglobulin heavy chain-light chain pairs,
where the two chains have different specificities (Millstein et
al., Nature, 305: 537-539 (1983)).
Because of the random assortment of immunoglobulin heavy and light
chains, these hybridomas (quadromas) produce a potential mixture of
10 different antibody molecules, of which only one has the correct
bispecific structure. Purification of the correct molecule, which
is usually done by affinity chromatography steps, is rather
cumbersome, and the product yields are low. Similar procedures are
disclosed in WO93/08829, and in Traunecker et al., EMBO J., 10:
3655-3659 (1991).
According to a different approach, antibody variable domains with
the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences. The
fusion preferably is with an immunoglobulin heavy chain constant
domain, comprising at least part of the hinge, CH2, and CH3
regions. It is preferred to have the first heavy-chain constant
region (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.
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). According to another
approach described in U.S. Pat. No. 5,731,168, the interface
between a pair of antibody molecules can be engineered to maximize
the percentage of heterodimers which are recovered from recombinant
cell culture. The preferred interface comprises at least a part of
the CH3 domain of an antibody constant domain. In this method, one
or more small amino acid side chains from the interface of the
first antibody molecule are replaced with larger side chains (e. g.
tyrosine or tryptophan). Compensatory "cavities" of identical or
similar size to the large side chain(s) are created on the
interface of the second antibody molecule by replacing large amino
acid side chains with smaller ones (e. g. alanine or threonine).
This provides a mechanism for increasing the yield of the
heterodimer over other unwanted end-products such as
homodimers.
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.
Techniques for generating bispecific antibodies from antibody
fragments have also been described in the literature. For example,
bispecific antibodies can be prepared using chemical linkage.
Brennan et al, Science, 229: 81 (1985) describe a procedure wherein
intact antibodies are proteolytically cleaved to generate F (ab') 2
fragments. These fragments are reduced in the presence of the
dithiol complexing agent sodium arsenite to stabilize vicinal
dithiols and prevent intermolecular disulfide formation. The Fab'
fragments generated are then converted to thionitrobenzoate (TNB)
derivatives. One of the Fab'-TNB derivatives is then reconverted to
the Fab'-thiol by reduction with mercapto-ethylamine and is mixed
with an equimolar amount of the other Fab'-TNB derivative to form
the bispecific antibody. The bispecific antibodies produced can be
used as agents for the selective immobilization of enzymes.
Recent progress has facilitated the direct recovery of Fab'-SH
fragments from E. coli, which can be chemically coupled to form
bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225
(1992) describe the production of a fully humanized bispecific
antibody F (ab') 2 molecule. Each Fab' fragment was separately
secreted from E. coli and subjected to directed chemical coupling
in vitro to form the bispecific antibody. The bispecific antibody
thus formed was able to bind to cells overexpressing the ErbB2
receptor and normal human T cells, as well as trigger the lytic
activity of human cytotoxic lymphocytes against human breast tumor
targets.
Various techniques for making and isolating bispecific antibody
fragments directly from recombinant cell culture have also been
described. For example, bispecific antibodies have been produced
using leucine zippers. Kostelny et al., J. Immunol., 148 (5):
1547-1553 (1992). The leucine zipper peptides from the Fos and Jun
proteins were linked to the Fab' portions of two different
antibodies by gene fusion.
The antibody homodimers were reduced at the hinge region to form
monomers and then re-oxidized to form the antibody heterodimers.
This method can also be utilized for the production of antibody
homodimers.
The "diabody" technology described by Hollinger et al, Proc. Natl.
Acad. Sci. USA, 90: 6444-6448 (1993) has provided an alternative
mechanism for making bispecific antibody fragments. The fragments
comprise a heavy-chain variable domain (VH) connected to a
light-chain variable domain (VL) by a linker which is too short to
allow pairing between the two domains on the same chain.
Accordingly, the VH and VL domains of one fragment are forced to
pair with the complementary VL and VH domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for
making bispecific antibody fragments by the use of single-chain Fv
(sFv) dimers has also been reported. See Gruber et al., J.
Immunology, 152: 5368 (1994). Antibodies with more than two
valencies are also contemplated. For example, trispecific
antibodies can be prepared. Tuttet al., J. Immunol. 147:
60(1991).
Diagnostic Assays
The invention further provides methods for detecting the presence
of and/or a level of BCMA mRNA in a biological sample; and methods
for detecting the presence of and/or a level of BCMA polypeptide in
a biological sample.
In other embodiments, a method is provide for detecting a level of
BCMA mRNA in a biological sample derived from an individual,
comprising analyzing a polynucleotide sample from an individual for
the level of BCMA polypeptide-encoding mRNA. The level of BCMA mRNA
may be associated with autoimmune disease.
In still other embodiments, a method is provided for detecting the
presence of and/or the level of a BCMA polypeptide in a biological
sample.
A number of methods are available for determining the expression
level of a BCMA nucleic acid molecule, e.g., a BCMA mRNA, or BCMA
polypeptide in a particular sample. Diagnosis may be performed by a
number of methods to determine the absence or presence or altered
amounts of normal or abnormal BCMA mRNA in a patient sample. For
example, detection may utilize staining of cells or histological
sections with labeled antibodies, performed in accordance with
conventional methods. Cells are permeabilized to stain cytoplasmic
molecules. The antibodies of interest are added to the cell sample,
and incubated for a period of time sufficient to allow binding to
the epitope, usually at least about 10 minutes. The antibody may be
labeled with radioisotopes, enzymes, fluorescers, chemiluminescers,
or other labels for direct detection. Alternatively, a second stage
antibody or reagent is used to amplify the signal. Such reagents
are well known in the art. For example, the primary antibody may be
conjugated to biotin, with horseradish peroxidase-conjugated avidin
added as a second stage reagent. Alternatively, the secondary
antibody conjugated to a fluorescent compound, e.g. fluorescein,
rhodamine, Texas red, etc. Final detection uses a substrate that
undergoes a color change in the presence of the peroxidase. The
absence or presence of antibody binding may be determined by
various methods, including flow cytometry of dissociated cells,
microscopy, radiography, scintillation counting, etc. The presence
and/or the level of a BCMA polypeptide may also be detected and/or
quantitated in any way known to one of ordinary skill.
In addition, a test can include measurements of the expression of
BCMA mRNA. Biochemical studies may be performed to determine
whether a sequence polymorphism in a BCMA coding region or control
regions is associated with disease. Disease associated
polymorphisms may include deletion or truncation of the gene,
mutations that alter expression level, that affect the activity of
the protein, etc.
Changes in the promoter or enhancer sequence that may affect
expression levels of BCMA can be compared to expression levels of
the normal allele by various methods known in the art. Methods for
determining promoter or enhancer strength include quantitation of
the expressed natural protein; insertion of the variant control
element into a vector with a reporter gene such as
.beta.-galactosidase, luciferase, chloramphenicol
acetyltransferase, etc. that provides for convenient quantitation;
and the like.
Diagnostic methods of the subject invention in which the level of
BCMA gene expression is of interest will typically involve
comparison of the BCMA nucleic acid or protein abundance of a
sample of interest with that of a control value to determine any
relative differences, where the difference may be measured
qualitatively and/or quantitatively, which differences are then
related to the presence or absence of an abnormal BCMA gene
expression pattern. A variety of different methods for determine
the nucleic acid abundance in a sample are known to those of skill
in the art, where particular methods of interest include those
described in: Pietu et al., Genome Res. (June 1996) 6: 492-503;
Zhao et al., Gene (Apr. 24, 1995) 156: 207-213; Soares, Curr. Opin.
Biotechnol. (October 1997) 8: 542-546; Raval, J. Pharmacol Toxicol
Methods (November 1994) 32: 125-127; Chalifour et al., Anal.
Biochem (Feb. 1, 1994) 216: 299-304; Stolz & Tuan, Mol.
Biotechnol. (December 19960 6: 225-230; Hong et al., Bioscience
Reports (1982) 2: 907; and McGraw, Anal. Biochem. (1984) 143: 298.
Also of interest are the methods disclosed in WO 97/27317, the
disclosure of which is herein incorporated by reference.
By a gene whose expression level is "correlated with" or
"associated with" a particular physiologic state, it is intended a
gene whose expression shows a statistically significant correlation
with the physiologic state. The strength of the correlation between
the expression level of a differentially expressed gene and the
presence or absence of a particular physiologic state may be
determined by a statistical test of significance. Methods for
determining the strength of a correlation between the expression
level of a differentially-expressed gene and a particular
physiologic state by assigning a statistical score to the
correlation are reviewed in Holloway et al. (2002) Nature Genetics
Suppl. 32:481-89, Churchill (2002) Nature Genetics Suppl.
32:490-95, Quackenbush (2002) Nature Genetics Suppl. 32: 496-501;
Slonim (2002) Nature Genetics Suppl. 32:502-08; and Chuaqui et al.
(2002) Nature Genetics Suppl. 32:509-514; each of which is herein
incorporated by reference in its entirety.
Additional tests that have been associated with autoimmune disease
severity or progression can be combined with the BCMA test
described above to render a full diagnosis or outlook result.
For example, the American College of Rheumatology has developed 11
criteria to diagnose SLE, which span the clinical spectrum of SLE
in aspects of skin, systemic, and laboratory tests. These criteria
include malar rash, discoid rash, sensitivity to sun light, oral
ulcers, arthritis, serositis, kidney and central nervous system
inflammation, blood alterations, and the presence of antinuclear
antibodies. A patient must meet four of these criteria in order to
be classified as a SLE patient. (Tan et al. (1982) Arthritis
Rheumatol. 25:1271-1277). SLE is usually confirmed by tests
including, but not limited to, blood tests to detect anti-nuclear
antibodies; blood and urine tests to assess kidney function;
complement tests to detect the presence of low levels of complement
that are often associated with SLE; a sedimentation rate (ESR) or
C-reactive protein (CRP) to measure inflammation levels; X-rays to
assess lung damage and EKGs to assess heart damage.
Monitoring Effects of Drug Treatment
Monitoring the influence of agents (e.g., drugs, compounds) on the
expression or BCMA protein (e.g., modulation of transcriptional
activation) can be applied not only in basic drug screening, but
also in clinical trials. For example, the effectiveness of an agent
determined by a screening assay as described herein to decrease
BCMA gene expression, or protein levels, can be monitored in
clinical trials of subjects exhibiting decreased BCMA gene
expression or protein levels. In such clinical trials, the
expression or activity of a BCMA gene, and preferably, other genes
that have been implicated in, for example, a disorder associated
with levels of BCMA protein can be used as a "read out" or markers
of the phenotype of a particular cell, in the present case, B
cells.
In some embodiments, the present invention provides a method for
monitoring the effectiveness of treatment of a subject with an
agent (e.g., an agonist, antagonist, peptidomimetic, protein,
peptide, nucleic acid, small molecule, or other drug) comprising
the steps of (i) obtaining a pre-administration sample from a
subject prior to administration of the agent; (ii) detecting the
level of expression of a BCMA protein or mRNA, in the
pre-administration sample; (iii) obtaining one or more
post-administration samples from the subject, (iv) detecting the
level of expression or activity of the BCMA protein or mRNA in the
post-administration samples; (v) comparing the level of expression
or activity of the BCMA protein or mRNA in the pre-administration
sample with the BCMA protein or mRNA in the post administration
sample or samples; and (vi) altering the administration of the
agent to the subject accordingly. According to such an embodiment,
BCMA expression or activity may be used as an indicator of the
effectiveness of an agent, even in the absence of an observable
phenotypic response.
The basal expression level of BCMA in different tissue may be
determined by analysis of tissue samples from individuals typed for
the presence or absence of a specific polymorphism. Any convenient
method may be use, e.g. ELISA, RIA, etc. for protein quantitation,
northern blot or other hybridization analysis, quantitative RT-PCR,
etc. for mRNA quantitation. The tissue specific expression is
correlated with the genotype.
The alteration of BCMA expression in response to a modifier is
determined by administering or combining the candidate modifier
with an expression system, e.g. animal, cell, in vitro
transcription assay, etc. The effect of the modifier on BCMA
transcription and/or steady state mRNA levels is determined. As
with the basal expression levels, tissue specific interactions are
of interest. Correlations are made between the ability of an
expression modifier to affect BCMA levels, and the presence of the
provided polymorphisms. A panel of different modifiers, cell types,
etc. may be screened in order to determine the effect under a
number of different conditions.
Treatment Methods
The present invention provides a method of treating an individual
clinically diagnosed with a condition associated with increased
BCMA levels on the B cell surface. The methods generally comprises
analyzing a biological sample to measure BCMA levels and comparing
such levels to those present in healthy controls. A treatment plan
that is most effective for individuals clinically diagnosed as
having a condition associated with increased BCMA levels, such as
autoimmune disease, is then selected. Thus, the invention further
provides a method for predicting a patient's likelihood to respond
to a drug treatment for a condition associated with increased BCMA
levels, comprising determining a patient's expression of a BCMA
gene, wherein the presence of a increased BCMA levels associated
with an autoimmune condition, such as SLE, and is predictive of the
patient's likelihood to respond to a drug treatment for the
condition.
Thus, another aspect of the invention provides methods for
tailoring an individual's therapeutic treatment with BCMA
expression according to that individual's drug response.
Pharmacogenomics allows a clinician or physician to target
prophylactic or therapeutic treatments to patients who will most
benefit from the treatment and to avoid treatment of patients who
will experience toxic drug-related side effects.
Autoimmune Diseases
The following is a non-limiting list of the possible autoimmune
diseases that treatment thereof could be aided by the use of the
BCMA measuring assay presently disclosed. B-cell regulated
autoimmune diseases include arthritis (rheumatoid arthritis,
juvenile rheumatoid arthritis, osteoarthritis, psoriatic
arthritis), psoriasis, dermatitis including atopic dermatitis;
chronic autoimmune urticaria, polymyositis/dermatomyositis, toxic
epidermal necrolysis, systemic scleroderma and sclerosis, responses
associated with inflammatory bowel disease (IBD) (Crohn's disease,
ulcerative colitis), respiratory distress syndrome, adult
respiratory distress syndrome (ARDS), meningitis, allergic
rhinitis, 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), lupus (including
nephritis, non-renal, discoid, alopecia), juvenile onset diabetes,
multiple sclerosis, allergic encephalomyelitis, immune responses
associated with acute and delayed hyper-sensitivity mediated by
cytokines and T-lymphocytes, tuberculosis, sarcoidosis,
granulomatosis including Wegener's granulomatosis, agranulocytosis,
vasculitis (including ANCA), aplastic anemia, Coombs positive
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, CNS inflammatory disorders,
multiple organ injury syndrome, myasthenia 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 (including pretreatment for high panel reactive antibody
titers, IgA deposit in tissues, etc), graft versus host disease
(GVHD), pemphigoid bullous, pemphigus (all including vulgaris,
foliaceus), 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, ALS, and coronary
artery disease.
BLyS and/or APRIL Antagonists
If high levels of BCMA on a B cell surface of a patient suffering
from an autoimmune disease are seen, this suggests the likelihood
that the patient will respond favorably to inhibition of BLyS
and/or APRIL. Thus, the present invention also comprises BLyS
and/or APRIL antagonists that are used for the treatment of
autoimmune diseases wherein the patient has elevated levels of BCMA
protein expression on the surface of their B cells. The following
are representative examples of BLyS and/or APRIL antagonists that
could be utilized to treat such patients. For the purposes of
functioning as a BLyS and/or APRIL antagonist, the extracellular
domain of any of the TNFR family receptors is a polypeptide
essentially free of the transmembrane or cytoplasmic domains that
generally retains the ability to bind BLyS. Specifically, the
extracellular domain of TACI can comprise amino acids 1 to 154 of
the TACI polypeptide sequence (SEQ ID NO: 2). Additionally, the ECD
can be fragments or variants of this sequence, such as ECD forms of
TACI as described in von Bulow et al., supra, WO 98/39361, WO
00/40716, WO 01/85782, WO 01/87979, and WO 01/81417. In particular,
these ECD forms can comprise amino acids 1-106 of SEQ ID NO:2,
amino acids 1-142 of SEQ ID NO:2, amino acids 30-154 of SEQ ID
NO:2, amino acids 30-106 of SEQ ID NO:2, amino acids 30-110 of SEQ
ID NO:2, amino acids 30-119 of SEQ ID NO:2, amino acids 1-166 of
SEQ ID NO:2, amino acids 1-165 of SEQ ID NO:2, amino acids 1-114 of
SEQ ID NO: 2, amino acids 1-119 of SEQ ID NO:2, amino acids 1-120
of SEQ ID NO:2, and amino acids 1-126 of SEQ ID NO:2. In addition,
the TACI ECD can comprise those molecules having only one cysteine
rich domain.
ECD forms of BAFF-R include those comprising amino acids 1-71 of
the BAFF-R polypeptide sequence (SEQ ID NO: 4). Additionally, the
ECD can be fragments or variants of this sequence such as ECD forms
of BAFF-R as described in WO 02/24909, WO 03/14294, and WO
02/38766. In particular, these ECD forms can comprise amino acids
1-77 of SEQ ID NO: 4, amino acids 7-77 of SEQ ID NO:4, amino acids
1-69 of SEQ ID NO:4, amino acids 7-69 of SEQ ID NO:4, amino acids
2-62 of SEQ ID NO:4, amino acids 2-71 of SEQ ID NO:4, amino acids
1-61 of SEQ ID NO:4 and amino acids 2-63 of SEQ ID NO:4, amino
acids 1-45 of SEQ ID NO:4, amino acids 1-39 of SEQ ID NO:4, amino
acids 7-39 of SEQ ID NO:4, amino acids 1-17 of SEQ ID NO:4, amino
acids 39-64 of SEQ ID NO:4, amino acids 19-35 of SEQ ID NO:4, and
amino acids 17-42 of SEQ ID NO:4. In addition, the BAFF-R ECD can
comprise those molecules having a cysteine rich domain.
ECD forms of BCMA include those comprising amino acids 1-48 of the
BCMA polypeptide sequence (SEQ ID NO: 6). Additionally, the ECD can
be fragments or variants of this sequence, such as ECD forms of
BCMA as described in WO 00/40716 and WO 05/075511. In particular,
these ECD forms can comprise amino acids 1-150 of SEQ ID NO:6,
amino acids 1-48 of SEQ ID NO:6, amino acids 1-41 of SEQ ID NO:6,
amino acids 8-41 of SEQ ID NO:6, amino acids 8-37 of SEQ ID NO:6,
amino acids 8-88 of SEQ ID NO:6, amino acids 41-88 of SEQ ID NO:6,
amino acids 1-54 of SEQ ID NO:6, amino acids 4-55 of SEQ ID NO:6,
amino acids 4-51 of SEQ ID NO:6, and amino acids 21-53 of SEQ ID
NO:6. In addition, the BCMA ECD can comprise those molecules having
only a partial cysteine rich domain.
In a further embodiment, the BLyS binding region of a BLyS receptor
(e. g., an extracellular domain or fragment thereof of BAFF-R, BCMA
or TACI) can be fused to an Fc portion of an immunoglobulin
molecule to facilitate its solubility in vivo. According to one
embodiment, the BLyS and/or APRIL antagonist binds to a BLyS
polypeptide with a binding affinity of 100 nM or less. According to
another embodiment, the BLyS and/or APRIL antagonist binds to a
BLyS polypeptide with a binding affinity of 10 nM or less.
According to yet another embodiment, the BLyS and/or APRIL
antagonist binds to a BLyS polypeptide with a binding affinity of 1
nM or less.
In another example, BLyS and/or APRIL antagonists include BLyS
binding polypeptides that are not native sequences or variants
thereof. Some examples of such polypeptides are those having the
sequence of Formula I, Formula II, Formula III as described in WO
05/000351. In particular, some binding polypeptides include
ECFDLLVRAWVPCSVLK (SEQ ID NO:13), ECFDLLVRHWVPCGLLR (SEQ ID NO:14),
ECFDLLVRRWVPCEMLG (SEQ ID NO:15), ECFDLLVRSWVPCHMLR (SEQ ID NO:16),
ECFDLLVRHWVACGLLR (SEQ ID NO:17), or sequences listed in FIG. 32 of
WO 05/000351.
Alternatively, the BLyS and/or APRIL antagonist can bind an
extracellular domain of native sequence TACI, BAFF-R, or BCMA at
its BLyS binding region to partially or fully block, inhibit or
neutralize BLyS binding in vitro, in situ, or in vivo. For example,
such indirect antagonist is a TACI antibody that binds in a region
of TACI such that the binding of BLyS is sterically hindered. For
example, binding at amino acids 72-109 or a neighboring region is
believed to block BLyS binding. It could also be advantageous to
block APRIL binding to this molecule, which is believed to occur in
the region of amino acids 82-222. Another BLyS and/or APRIL
antagonist is a BAFF-R antibody that binds in a region of BAFF-R
such that binding of human BAFF-R to BLyS is sterically hindered.
For example, binding at amino acids 23-38 or amino acids 17-42 or a
neighboring region is believed to block BLyS binding. Finally, a
further indirect antagonist would be a BCMA antibody that binds in
a region of BCMA such that the binding of BLyS is sterically
hindered. For example, binding at amino acids 5-43 or a neighboring
region is believed to block BLyS (or APRIL) binding.
In some embodiments, a BLyS and/or APRIL antagonist according to
this invention includes BLyS antibodies. The term "antibody" when
referring to is used in the broadest sense and specifically covers,
for example, monoclonal antibodies, polyclonal antibodies,
antibodies with poly-epitopic specificity, single chain antibodies,
and fragments of antibodies. According to some embodiments, a
polypeptide of this invention is fused into an antibody framework,
for example, in the variable region or in a CDR such that the
antibody can bind to and inhibit BLyS binding to TACI, BAFF-R, or
BCMA or inhibits BLyS signaling. The antibodies comprising a
polypeptide of this invention can be chimeric, humanized, or human.
The antibodies comprising a polypeptide of this invention can be an
antibody fragment. Alternatively, an antibody of this invention can
be produced by immunizing an animal with a polypeptide of this
invention. Thus, an antibody directed against a polypeptide of this
invention is contemplated.
In particular, antibodies specific for BLyS that bind within a
region of human BLyS (SEQ ID NO: 8) comprising residues 162-275
and/or a neighboring amino acid of amino acids selected from the
group consisting of 162, 163, 206, 211, 231, 233, 264 and 265 of
human BLyS are contemplated. The binding of the antibodies are such
that the antibody sterically hinders BLyS binding to one or more of
its receptors. Such antibodies are described in WO 02/02641 and WO
03/055979. A particularly preferred antibody is the one described
as Lymphostat-B (Baker et al. (2003) Arthritis Rheum, 48,
3253-3265).
Other Immunosuppressive Drugs
The present method contemplates the use of other immunosuppressive
drugs either singly or in combination with a BLyS, APRIL or BCMA
inhibitor. These other drugs include, but are not limited to,
immunosuppressive agents such as calcineurin inhibitors (e.g.,
cyclosporin A or FK506), steroids (e.g., methyl prednisone or
prednisone), or immunosuppressive agents that arrest the growth of
immune cells(e.g., rapamycin), anti-CD40 pathway inhibitors (e.g.,
anti-CD40 antibodies, anti-CD40 ligand antibodies and small
molecule inhibitors of the CD40 pathway), transplant salvage
pathway inhibitors (e.g., mycophenolate mofetil (MMF)), IL-2
receptor antagonists (e.g., Zeonpax.COPYRGT. from Hoffmann-1a Roche
Inc., and Simulet from Novartis, Inc.), or analogs thereof,
cyclophosphamide, thalidomide, azathioprine, monoclonal antibodies
(e.g., Daclizumab (anti-interleukin (IL)-2), Infliximab (anti-tumor
necrosis factor), MEDI-205 (anti-CD2), abx-cb1 (anti-CD147)), and
polyclonal antibodies (e.g., ATG (anti-thymocyte globulin)).
In one embodiment, a therapeutically effective amount of a BCMA
antagonist (such as an antibody that binds the extracellular domain
of BCMA) can be administered to a subject in combination with a
therapeutically effective amount of rituximab. Such methods allow
for the improved depletion and/or neutralization of B cells from
said subject. Co-administration of such compounds can occur
simultaneously or consecutively and through any effective route of
administration.
As used herein, "a therapeutically effective amount" of an agent of
interest is an amount which, when administered to a subject, is
sufficient to achieve a desired effect, such as the depletion
and/or neutralization of B-cells in a subject being treated with
that composition.
In some embodiments a "therapeutically effective amount" or
"effective amount" of a pharmaceutical composition containing a
BCMA antagonist or an anti-CD 20 agent such as rituximab is from
about 0.1 to about 200 mg/kg body weight in single or divided
doses; for example from about 1 to about 100 mg/kg, from about 2 to
about 50 mg/kg, from about 3 to about 25 mg/kg, or from about 5 to
about 10 mg/kg. When animal assays are used, a dosage is
administered to provide a target tissue concentration similar to
that which has been shown to be effective in the animal assays. It
is recognized that the method of treatment may comprise a single
administration of a therapeutically effective amount or multiple
administrations of a therapeutically effective amount of the BCMA
antagonist (such as an antibody that binds the extracellular domain
of BCMA) and the anti-CD 20 agent such as rituximab.
The term "anti-CD 20 agent" encompasses any molecule that binds to
CD-20 and in the most preferred embodiment targets the cell
associated with the CD-20 protein for killing. Such molecules
include anti-CD-20 antibodies, such as RITUXAN.RTM. and follow-on
versions of that agent such as ocrelizumab, a humanized version of
that antibody, ofatumumbab (HuMax-CD20.RTM. a fully human anti-CD
20 agent), DXL625 (a second generation anti-CD20 monoclonal), GA101
(a third generation anti-CD20 agent that has an altered Fc region),
the anti-CD20 molecules described in U.S. Application No.
20060121032, the anti-CD20 molecules described in U.S. Application
No. 200700202059, the anti-CD20 molecules described in U.S.
Application No. 20070014720, the anti-CD20 molecules described in
U.S. Application No. 20060251652, the anti-CD20 molecules described
in U.S. Application No. 20050069545, the anti-CD20 molecules
described in U.S. Application No. 20040167319, TRU-015 (a small
molecule immunopharmaceutical molecule that targets CD 20), as well
as conjugated molecules such as ibritumomab (ZEVALIN.RTM.).
Pharmaceutical Formulations
Therapeutic formulations of the BLyS and/or APRIL antagonists such
as BLyS-binding antibodies used in accordance with the present
invention are prepared for storage by mixing an antibody having the
desired degree of purity with optional pharmaceutically acceptable
carriers, excipients or stabilizers (Remitgtorz's Phamamaceutical
Science 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 olyvinylpyrrolidone; 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,
PLURONICS.TM. or polyethylene glycol (PEG)).
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 antibody 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 described herein or about from 1 to 99% of
the heretofore employed dosages.
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).
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 .ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,
degradable lactic acid-glycolic acid copolymers such as the LUPRON
DEPOT (injectable microspheres composed of lactic acid-glycolic
acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid.
The formulations to be used for in vivo administration must be
sterile. This is readily accomplished by filtration through sterile
filtration membranes.
EXPERIMENTAL
The following examples are put forth so as to provide those of
ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Celsius, and pressure
is at or near atmospheric.
EXAMPLE 1
Measurement of BCMA Levels on B Cells of Autoimmune Patients
Peripheral blood B cells were obtained by negative selection from
16 healthy controls (HC) and 15 SLE patients with whose disease
activity was assessed by analysis of disease activity by SLEDAI. B
cells were sub-grouped as CD19+CD27- (naive), CD19+CD27+ (memory)
and CD19+CD27high (plasmablasts). BAFF-R, TACI, and BCMA expression
were compared by flow cytometry on each subset of B cells from SLE
and healthy controls (HC) were compared by flow cytometry. The
levels of serum BAFF-BLyS, APRIL and autoantibodies were quantified
by ELISAs. In some experiments, BCMA-positive and -negative B cells
were separated by flow cytometric sorting.
Expression Pattern of BAFF-R, TACI and BCMA in the Human Peripheral
Blood B Cells
Peripheral blood B cells were stained with anti-BAFF-R, anti-TACI
and anti-BCMA antibodies. It has been shown that the expressions of
BAFF receptors change with B cell maturation; hence, naive (CD27-),
memory B cells (CD27+) and plasmablasts (CD27high) were all
analyzed separately. BAFF-R and TACI are detected in all the
subsets but BAFF-R expression is low in plasmablasts and TACI
expression is lower in naive B cells (FIG. 1A). Even though it has
been reported that BCMA expression is limited only in the plasma
cells and germinal center B cells, we could also see the BCMA+
population in naive and memory B cells from peripheral blood (FIG.
1A). This difference might be due to the different specificity of
antibodies to the various transcript variants because BCMA was
reported as having five transcript variants which are results of
alternative splicing. Average of BCMA+ B cells are 24% in naive B
cells and 20.8% in memory B cells, which is lower than that of
plasmablasts (37.9%) (FIG. 6A)
Expression of BAFF-R, TACI and BCMA on the B Cells in Controls and
SLE Patients
B cells from 15 patients with SLE and 16 non-autoimmune individuals
were examined for expression of BAFF receptors. In the analysis of
BAFF-R expression of the SLE and control groups, SLE patients
showed significantly decreased BAFF-R expression in all three B
cell subsets (MFI of control vs SLE, naive=1387 vs 929, p=0.002;
memory=1290 vs 840, p=0.003; plasmablasts=130 vs 85, p=0.077, FIG.
1B). TACI expressions of both two groups are not significantly
different in all three subsets (MFI of control vs SLE, naive=18.8
vs 24.4, p=0.144; memory=73.2 vs 60.4, p=0.121; plasmablasts=39.0
vs 32.1, p=0.110, FIG. 1C).
The B cells from SLE had higher surface BCMA expression than normal
controls (MFI of control vs SLE--17.8 vs 27.9, p=0.038; FIG. 1D).
The BCMA expression of SLE in memory B cells is higher than that of
normal controls (BCMA MFI of naive B cells=19.5 vs 29.5, p=0.008,
FIG. 1D). But the BCMA expression of naive and plasmablasts are not
significantly different.
Relationships of BAFF Receptors and Serum BAFF/APRIL Level,
Auto-Antibodies, Disease Activity.
There was an inverse correlation between BAFF-R expression of total
B cells and serum BAFF (p=0.0491) but there was no significant
correlation between BAFF-R expression and serum APRIL (FIG. 2).
BAFF-R expression was inversely correlated with anti-dsDNA weakly
(p=0.064) but not statistically significant. There was no
correlation between BAFF-R and disease activity assessed by SLEDAI
score (p=0.342, FIG. 2). MFI of BCMA positive memory cell is
inversely correlated with serum IgG anti-dsDNA antibody levels
(p=0.0119, FIG. 2) even though there's no significant correlation
between BCMA and disease activity (SLEDAI) or serum BAFF level.
Characteristics of BCMA Positive B Cells
Lupus patients have increased BCMA expression on the naive and
memory B cells. We examined the characteristics of BCMA+ B cells to
see the relations with autoimmunity. Mean FSC of BCMA+ and BCMA-
cells in the each B cell subset was analyzed at first. Notably,
lupus patients have the higher mean FSC than normal control
indicating that the increased proportion of activated B cells in
lupus as expected. BCMA+ B cells had higher mean FSC than BCMA- B
cells in naive B cells from both lupus and healthy controls (FIG.
3A). In memory B cells, BCMA+ B cells from healthy controls and
lupus patients had significantly higher mean FSC than BCMA- B cells
(FIG. 3A). These data might indicate that BCMA positive B cells in
naive and memory B cells are more activated B cells than BCMA
negative B cells. This was true in both normal and SLE patients and
there were no differences between them.
We also analyzed BCMA expression on the CD19high population which
was reported as increased in the lupus patients. We couldn't find
significant differences between healthy controls and lupus patients
in CD19high population in our study (Sup. FIG. 2) but CD19high B
cells have much higher BCMA expressions than CD191ow B cells in
both healthy controls and lupus patients (FIG. 3B). Higher CD19
expression in BCMA+ B cells might be related with higher
sensitivity to many immunologic responses including BCR signaling
because CD19 is a BCR co-receptor that augments BCR signaling and
partner of multiple receptors like CD21, CD40, CD38, CD72, VLA-4
and Fc.gamma.RIIB.
Most of the BCMA+ B cells are IgD+ and IgM+ in both lupus and
healthy people, which means BCMA+ B cells are not class switched
(FIG. 4A). Therefore, CD27- BCMA+ B cells might be included into
the naive B cell population. CD27+BCMA+ B cells also shows IgD+ and
these cells could be un-class-switched memory B cells. Furthermore,
lupus patients have higher percentage of IgD+ B cells than healthy
control (FIG. 4B).
B cells of lupus patients are usually more activated than healthy
people and it was reported that CD86 expression is much higher in
lupus B cells. CD80 and CD86 expression of BCMA+ B cells were
examined in lupus and healthy people. Lupus patients have much
higher CD86 expressions and the percentage of CD86+ cells on the
BCMA+ cells are higher than BCMA- cells. Healthy controls have
lower CD86 expressions but percentage of CD86+ cells on the BCMA+
cells are higher than BCMA- cells. CD80 expressions were not high
in both lupus and healthy controls (FIG. 4A). BAFF and APRIL plus
CPG increased BCMA expression on all the subsets of B cells and
induced expansion of plasmablasts and naive B cells.
B cells isolated from PBMC of lupus patient and healthy control
were labeled with CFSE (Caroxy-fluorescein diacetate) and incubated
with CPG (oligodeoxynucleotides containing a CpG motif) with or
without BAFF, APRIL or HT. After 4 day incubation, BCMA expression
on each subset of B cells and their proliferation were analyzed
(FIG. 5A and FIG. 5C). CPG alone didn't increase BCMA expression.
BCMA expressions on all the subsets of B cells were increased
significantly by CPG+BAFF and CPG+HT, especially on CD27high
plasmablasts. CPG+APRIL also increased BCMA expression slightly.
BAFF, APRIL, and HT induced strong proliferation of CD27 high B
cells and moderate proliferation of CD27- B cells (FIG. 5B). But
CD27+ population was not expanded. Decrease of CD27+ memory B
population could be due to CD27+ cells were differentiated to
CD27high plasma cells or due to the lack of proliferation CD27+ B
cells.
Ig Secretion and Auto-Antibody Production by BCMA+ and BCMA- B
Cells
B cells are isolated from PBMC of lupus and healthy people by
magnetic sorting (B cell enrichment kit. Stemcell, British
Columbia, Canada). BCMA+ B cells are sorted from isolated B cells
by FACS sorting. The sorted BCMA+ B cells are incubated with or
without anti-CD40/IL4, anti-CD40/IL4/CPG, anti-CD40/IL4/BCR for 5
days in the 96-well culture plate. The IgM and IgG concentration
and anti-dsDNA activity of supernatants were analyzed by ELISA.
Summary of Results:
Whereas BAFF-R expression on SLE B cells was significantly lower
compared to its levels on HC B cells (MFI of control vs SLE,
naive=1387 vs 929, p=0.002; memory=1290 vs 840, p=0.003), BCMA
expression was substantially higher on SLE B cells (MFI of control
vs SLE: 18 vs 28, p=0.038). This was most pronounced in the memory
B cell subset (MFI of control vs SLE=19 vs 29, p=0.008). BCMA
levels were not correlated with either disease severity (SLEDAI) or
with serum BLyS or APRIL levels. BCMA+ cells also tended to be
larger than BCMA-negative cells, and had higher CD19 and CD86
expression, indicating a greater degree of activation. To examine
the functional implications of differential BCMA expression,
purified BCMA-positive or BCMA-negative B cells were incubated with
BLyS, APRIL or BLyS/APRIL heterotrimers (HT) with or without the
TLR9 stimulator, CpG. Whereas none of the ligands induced
proliferation of the BCMA+ B cells on their own, the combination of
CpG with each individual ligand induced substantial BCMA+ B cell
proliferation.
Conclusions:
BCMA expression on B cells from patients with SLE is significantly
increased, especially on memory B cells. BCMA positive cells have a
more activated phenotype and produce higher amounts of
immunoglobulin and autoantibodies. All 3 ligands, BLyS, APRIL or HT
potently activate BCMA positive cells in the presence of CpG. These
findings may help to explain the observed expansion of DNA reactive
SLE B cells in the presence of BLyS and APRIL.
EXAMPLE 2
Combination Treatment with BCMA Antibodies and Rituximab in Human
CD20 Transgenic Mice for the Depletion and/or Neutralization of B
Cells
BCMA expression is important for the survival of plasma cells. Gene
targeting has confirmed that survival of long lived bone marrow
derived from plasma cells was reduced in BCMA deficient mice. Since
CD20 is not expressed on long lived plasma cells in the bone
marrow, targeting these cells with blocking or cytotoxic antibodies
to BCMA should deplete these cells which may contribute a
significant component of the anti-CD20 resistant autoantibody
producing cells in patients with SLE. Combining such treatment with
rituximab will provide further beneficial results.
Rituximab does not bind to mouse CD20 and therefore cannot be used
to deplete B cells in normal mice. Also, there are no commercially
available anti-mouse CD20 mAbs effective for in vivo B cell
depletion. Gong et al. (J. Immunol 174:817-826; 2005) utilized a
strain of human CD20 transgenic mice (generated using bacterial
artificial chromosome/BAC technology) that they could treat with
anti-human CD20 mAbs (rituximab and ocrelizumab) and induce B cell
depletion or neutralization similar to that observed in humans
treated with these mAbs. This example utilizes a strain of hCD20 Tg
mice disclosed in Ahuja et al., J. Immunol 179:3351-3361; 2007.
General approach: Use optimal doses of each therapeutic (BCMA
antibody antagonists which binds to the extracellar domain of BCMA
and rituximab), either sequentially or in combination and evaluate
B cell depletion and/or neutralization. Assess B cell depletion at
an optimal timepoint (3 weeks), but include additional groups to
evaluate B cell recovery following treatment cessation (.about.20
weeks).
Materials and Methods
TABLE-US-00001 TABLE 1 Final dose Volume/ Concen- solution mouse
Treatment tration (mg/mL) (mL) Route Schedule PBS -- -- 0.2 SC
M/W/F .times. 3 weeks BCMA Therapeu- SC M/W/F .times. 3 antibody
tically weeks (9 effective doses/mouse) dose Rituxan stock = 10.0
0.2-0.25 IP once a week (Rituximab) 10 mg/mL for 2 or 3 10 mg/kg
weeks (three weekly doses for Groups 3 and 4; two weekly doses for
Group 5)
Animal Care, Acclimation, and Housing
Room temperature is maintained at 70-74.degree. F. and humidity
maintained at 30%-70%. A light/dark cycle of 12 hours is used,
except when room lights may be turned on during the dark cycle to
accommodate study-related activities. Each animal is offered rodent
chow (irradiated 5056, Pico Lab, Richmond, Ind.) and water ad
libitum. Procedures in this study are designed to avoid or minimize
discomfort, distress, or pain to animals. Treatment of study
animals is in accordance with conditions specified in the Guide for
the Care and Use of Laboratory Animals (ILAR publication, 1996,
National Academy Press). The animals are randomly assigned to the
various treatment groups.
Treatment Groups: The period of dosing is designated as the dosing
phase. The first day of dosing is designated "Day 1." The day prior
to Day 1 is "Day -1." The day following the dosing phase terminal
sacrifice is the first day of the recovery phase.
TABLE-US-00002 TABLE 2 Sac Sac 1.sup.st Treatment 2.sup.nd
Treatment Day Week Group (route) (route) Dose schedule 23 ~20 1 PBS
(SC) PBS (SC) 3 times weekly 5 6 2 BCMA antibody -- 3 times weekly
for 3 5 5 Therapeutically weeks (total of 9 effective dose doses)
(SC) 3 Rituximab -- once weekly for 3 5 5 10 mg/kg (IP) weeks
(total of 3 doses) 4 Rituximab BCMA antibody Stagger treatments 5 6
10 mg/kg (IP) (SC) by 2 days; continue Therapeutically both
treatments for effective dose 3 wks 5 BCMA antibody Rituximab BCMA
antibody 5 6 (SC) 10 mg/kg (IP) for 3 weeks (9 doses),
Therapeutically Rituximab for last 2 effective dose weeks of BCMA
antibody dosing period (2 doses)
Rituximab is administered intraperitoneally (IP). The BCMA antibody
and vehicle (PBS) is administered via subcutaneous (SC) injection
within the subscapular region three times weekly (9 total doses).
The first dose is administered on Day 1 (D1). In combination
treatment groups, and where relevant, all animals receive the IP
injection first, followed by administration of the SC injection
within a period of 60 minutes. GROUP 4: rituximab 1.sup.st, then
BCMA antibody; GROUP 5: BCMA antibody 1.sup.st, then rituximab.
Dose volumes are adjusted weekly according to individual animal
body weights.
Summary of Study Endpoints:
Whole blood (150 .mu.L; EDTA) is collected and flow cytometry
analysis performed for T and B cell subsets. Serum (.about.100
.mu.L whole blood placed in serum separator tubes) is collected at
various timepoints for later analysis of total IgG.sub.1,
IgG.sub.2a, IgM, IgE and IgA by Luminex assay. At sac, spleens are
collected and processed for flow analysis and later IHC/histology.
Splenectomy is performed under isoflurane anesthesia prior to blood
collection. Whole spleen is weighed and recorded before sectioning.
At sac, major peripheral lymph nodes (inguinal, axillary, brachial,
cervical, and mesenteric) are collected for possible future IHC and
histology. The lymph nodes are fixed with formalin or Zinc Tris
buffer and stored in 70% alcohol for possible future use. For
histology: 1/3 spleen and 1 LLN (left LN) are placed into ZnTris
and the same tissues (right LN) is collected into 10% NBF.
All animals in Groups 1-5 have serum (100 .mu.L blood in serum
separator tubes) and whole blood (150 .mu.L in EDTA tubes)
collected on day -5 via retro-orbital vein. Serum Collection: A
minimum of 100 .mu.L of whole blood is placed into serum separator
tubes and allowed to clot for a minimum of 15 minutes. Blood is
then spun at 4000 rpm for 10 minutes. A minimum of 50 .mu.L of
serum is aliquoted into a second container. Aliquots of serum are
stored at .ltoreq.60.degree. C.
Whole Blood in EDTA Collection: A minimum of 150 .mu.L of blood is
placed into a microtainer tube containing EDTA. The tubes are
gently inverted a minimum of 20 times. Whole blood in EDTA samples
are stored at room temperature until processed for flow
cytometry.
All animals that are sacrificed are anesthetized with isoflurane.
All sacrificed animals have blood, serum, spleen and major
peripheral lymph nodes collected. The spleen is collected under
isoflurane anesthesia prior to the blood sample to avoid altering
splenocyte subsets. The whole spleen is weighed. The spleen is cut
into 3 sections (cranial, middle & caudal), to be processed as
shown in Table 3.
TABLE-US-00003 TABLE 3 Collection of Spleen Samples Spleen
section.sup.1 Media Use Storage Cranial Zinc-Tris Buffer IHC Room
Temperature Middle RPMI + 10% FBS Flow Cytometry 4 degrees Celsius
Caudal 10% NBF Histology Room Temperature
Lymph Node Collection: Major peripheral lymph nodes (inguinal,
axillary, brachial, cervical and mesenteric) are collected for
histology/IHC in cassettes. a. Cassettes containing Left Lymph
Nodes (excluding mesenteric LN) for IHC are placed into Zinc Tris
buffer. b. Cassettes containing Right Lymph Nodes (excluding
mesenteric LN) for histology are placed into 10% NBF. c. Cassettes
containing Mesenteric Lymph Nodes are collected into 10% NBF by
taking the entire intestinal tract (from stomach to just above the
rectum) whole and unflushed. All samples are stored at room
temperature.
TABLE-US-00004 TABLE 4 Collection of Lymph Nodes LN.sup.1 Media
Potential Use Storage Left LNs Zinc-Tris Buffer IHC Room
Temperature Mesenteric 10% NBF Histology Room Temperature Right LNs
10% NBF Histology Room Temperature
Whole Blood Immunophenotyping: Briefly, whole blood is collected
into BD Microtainer.TM. tubes containing K.sub.2EDTA anticoagulant.
A 50 .mu.L aliquot of whole blood is incubated with the appropriate
working antibody cocktail (see Table 5) and red blood cells are
lysed. Prior to sample acquisition on the flow cytometer, Flow
Count.TM. fluorescent microspheres are added to each sample tube
for calculating absolute cell concentrations. Data acquisition is
conducted on a BD FACSCalibur flow cytometer equipped with a 15 mW
air-cooled Argon ion laser with 488 nm emission and a red-diode
laser with 635 nm emission.
TABLE-US-00005 TABLE 5 Whole blood four-color monoclonal antibody
panel Antibody Panel Cell Type Identified CD45/B220/ Total
lymphocytes [CD45+] IgD/IgM Total B lymphocyte [CD45+/B220+] Mature
B lymphocytes [CD45+/B220+/IgD+/IgM-] Immature B lympho-
[CD45+/B220+/IgD-/IgM+] cytes Mature Naive B
[CD45+/B220+/IgD+/IgM+] lymphocytes CD3/B220/ Total T lymphocytes
[CD3+] CD19/hu CD20 Total B lymphocyte [CD3-/B220+] B
lymphocytes.sup.1 [B220+/CD19+/huCD20-] B lymphocytes.sup.2
[B220+/CD19+/huCD20+] .sup.1The phenotype [B220+/CD19+/huCD20-]
describes a population of B cells expressing both B220 and murine
CD19 surface antigen but not the human CD20 transgene. .sup.2The
phenotype [B220+/CD19+/huCD20+] describes a population of B cells
expressing both B220 and murine CD19 surface antigen including the
human CD20 transgene.
Spleen Immunophenotyping: Briefly, single cells are isolated and
incubated with the appropriate antibody cocktails (see Table 6).
Instrument calibration and data acquisition is conducted as for
whole blood immunophenotyping.
TABLE-US-00006 TABLE 6 Spleen four-color monoclonal antibody panel
ANTIBODY PANEL B CELL SUBSET IDENTIFIED IgD/IgM/B220/ F(I) Mature
[B220+/IgM.sup.low/IgD+] hCD20 F(II) Less Mature [B220+/IgM+/IgD+]
F(III) Less Mature [B220+/IgM+/IgD.sup.low] CD23/CD21/B220/ MZ
(marginal zone) [B220+/CD21+/CD23.sup.low] CD45 FO (follicular)
[B220+/CD21.sup.low/CD23+] NF Newly Formed [B220+/CD21-/CD23-]
IgM/CD21/B220/ M (Mature) [B220+/IgM.sup.low/CD21.sup.low] CD45 T2
(Transitional 2)/ [B220+/IgM+/CD21+] MZ T1 (Transitional 1)
[B220+/IgM+/CD21-]
IHC Analysis
Tissues: Test tissues includes samples from both spleens and lymph
nodes. For the spleen samples, transverse sections of spleen
(cranial and caudal pieces) from each animal is included. The
cranial spleen sections (Zinc Tris-fixed) are stained with rat
monoclonal antibody to CD45R/B220, CD138, or CD5 alone. A subset of
tissue sections will also be stained with rat isotype IgG as a
negative control. The caudal spleen sections (formalin-fixed) is
stained with biotinylated PNA (if necessary to visualize GC;
H&E may suffice) and H&E. A subset of tissue sections is
also stained with biotinylated parathyroid hormone-related protein
(PTHrP) as a negative control.
The lymph nodes examined from each animal include the inguinal,
axillary, brachial, cervical, and mesenteric. The lymph nodes are
fixed with formalin or zinc Tris and held in 70% alcohol for
possible future use.
Antibodies: The antibodies used included three rat monoclonal
antibodies to mouse CD45R/B220 (clone RA3-6B2, isotype: rat IgG2a,
k; 0.5 mg/mL, #557390, BD Biosciences, San Jose, Calif.), CD5
(Ly-1, clone 53-7.3, 0.5 mg/mL, #553017, BD Biosciences), and CD138
(clone Syndecan-1, 0.5 mg/mL, #553712, BD Biosciences).
Statistical Analyses: Statistical analyses of group differences in
organ weights, B cell counts, and immunoglobulin levels were
conducted using analysis of variance (ANOVA).
Collection of the data outlined above will allow the percent change
in absolute concentration of B cells in the mouse peripheral blood
samples at various time points for the stated treatment group to be
determined. Alternatively, the data can be expressed as a change in
the percent of positive lymphocytes or B220+ cells. Such data will
demonstrate that that the combination of anti-CD 20 agents and BCMA
antagonists, such as RITUXAN.RTM. and a BCMA antibody, result in a
depletion (in some embodiments a synergistic depletion) and/or
neutralization of B cells levels compared to the level of reduction
with RITUXAN.RTM. and BCMA antagonists alone at many time
points.
While the present invention has been described with reference to
the specific embodiments thereof, it is to be understood by those
skilled in the art that various changes may be made and an
equivalence may be substituted without departing from the true
spirit and scope of the invention. In addition, many modifications
may be made to adapt a particular situation, material, composition
of matter, process, process step or steps, to the object, spirit
and scope of the present invention. All such modifications are
intended to be within the scope of the claims appended hereto.
SEQUENCE LISTINGS
1
2611377DNAhomo sapiensCDS(14)...(892) 1agcatcctga gta atg agt ggc
ctg ggc cgg agc agg cga ggt ggc cgg 49 Met Ser Gly Leu Gly Arg Ser
Arg Arg Gly Gly Arg 1 5 10agc cgt gtg gac cag gag gag cgc ttt cca
cag ggc ctg tgg acg ggg 97Ser Arg Val Asp Gln Glu Glu Arg Phe Pro
Gln Gly Leu Trp Thr Gly 15 20 25gtg gct atg aga tcc tgc ccc gaa gag
cag tac tgg gat cct ctg ctg 145Val Ala Met Arg Ser Cys Pro Glu Glu
Gln Tyr Trp Asp Pro Leu Leu 30 35 40ggt acc tgc atg tcc tgc aaa acc
att tgc aac cat cag agc cag cgc 193Gly Thr Cys Met Ser Cys Lys Thr
Ile Cys Asn His Gln Ser Gln Arg 45 50 55 60acc tgt gca gcc ttc tgc
agg tca ctc agc tgc cgc aag gag caa ggc 241Thr Cys Ala Ala Phe Cys
Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly 65 70 75aag ttc tat gac cat
ctc ctg agg gac tgc atc agc tgt gcc tcc atc 289Lys Phe Tyr Asp His
Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile 80 85 90tgt gga cag cac
cct aag caa tgt gca tac ttc tgt gag aac aag ctc 337Cys Gly Gln His
Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu 95 100 105agg agc
cca gtg aac ctt cca cca gag ctc agg aga cag cgg agt gga 385Arg Ser
Pro Val Asn Leu Pro Pro Glu Leu Arg Arg Gln Arg Ser Gly 110 115
120gaa gtt gaa aac aat tca gac aac tcg gga agg tac caa gga ttg gag
433Glu Val Glu Asn Asn Ser Asp Asn Ser Gly Arg Tyr Gln Gly Leu
Glu125 130 135 140cac aga ggc tca gaa gca agt cca gct ctc ccg ggg
ctg aag ctg agt 481His Arg Gly Ser Glu Ala Ser Pro Ala Leu Pro Gly
Leu Lys Leu Ser 145 150 155gca gat cag gtg gcc ctg gtc tac agc acg
ctg ggg ctc tgc ctg tgt 529Ala Asp Gln Val Ala Leu Val Tyr Ser Thr
Leu Gly Leu Cys Leu Cys 160 165 170gcc gtc ctc tgc tgc ttc ctg gtg
gcg gtg gcc tgc ttc ctc aag aag 577Ala Val Leu Cys Cys Phe Leu Val
Ala Val Ala Cys Phe Leu Lys Lys 175 180 185agg ggg gat ccc tgc tcc
tgc cag ccc cgc tca agg ccc cgt caa agt 625Arg Gly Asp Pro Cys Ser
Cys Gln Pro Arg Ser Arg Pro Arg Gln Ser 190 195 200ccg gcc aag tct
tcc cag gat cac gcg atg gaa gcc ggc agc cct gtg 673Pro Ala Lys Ser
Ser Gln Asp His Ala Met Glu Ala Gly Ser Pro Val205 210 215 220agc
aca tcc ccc gag cca gtg gag acc tgc agc ttc tgc ttc cct gag 721Ser
Thr Ser Pro Glu Pro Val Glu Thr Cys Ser Phe Cys Phe Pro Glu 225 230
235tgc agg gcg ccc acg cag gag agc gca gtc acg cct ggg acc ccc gac
769Cys Arg Ala Pro Thr Gln Glu Ser Ala Val Thr Pro Gly Thr Pro Asp
240 245 250ccc act tgt gct gga agg tgg ggg tgc cac acc agg acc aca
gtc ctg 817Pro Thr Cys Ala Gly Arg Trp Gly Cys His Thr Arg Thr Thr
Val Leu 255 260 265cag cct tgc cca cac atc cca gac agt ggc ctt ggc
att gtg tgt gtg 865Gln Pro Cys Pro His Ile Pro Asp Ser Gly Leu Gly
Ile Val Cys Val 270 275 280cct gcc cag gag ggg ggc cca ggt gca
taaatggggg tcagggaggg 912Pro Ala Gln Glu Gly Gly Pro Gly Ala285
290aaaggaggag ggagagagat ggagaggagg ggagagagaa agagaggtgg
ggagagggga 972gagagatatg aggagagaga gacagaggag gcagaaaggg
agagaaacag aggagacaga 1032gagggagaga gagacagagg gagagagaga
cagaggggaa gagaggcaga gagggaaaga 1092ggcagagaag gaaagagaca
ggcagagaag gagagaggca gagagggaga gaggcagaga 1152gggagagagg
cagagagaca gagagggaga gagggacaga gagagataga gcaggaggtc
1212ggggcactct gagtcccagt tcccagtgca gctgtaggtc gtcatcacct
aaccacacgt 1272gcaataaagt cctcgtgcct gctgctcaca gcccccgaga
gcccctcctc ctggagaata 1332aaacctttgg cagctgccct tcctcaaaaa
aaaaaaaaaa aaaaa 13772293PRThomo sapiens 2Met Ser Gly Leu Gly Arg
Ser Arg Arg Gly Gly Arg Ser Arg Val Asp1 5 10 15Gln Glu Glu Arg Phe
Pro Gln Gly Leu Trp Thr Gly Val Ala Met Arg 20 25 30Ser Cys Pro Glu
Glu Gln Tyr Trp Asp Pro Leu Leu Gly Thr Cys Met 35 40 45Ser Cys Lys
Thr Ile Cys Asn His Gln Ser Gln Arg Thr Cys Ala Ala 50 55 60Phe Cys
Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp65 70 75
80His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His
85 90 95Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser Pro
Val 100 105 110Asn Leu Pro Pro Glu Leu Arg Arg Gln Arg Ser Gly Glu
Val Glu Asn 115 120 125Asn Ser Asp Asn Ser Gly Arg Tyr Gln Gly Leu
Glu His Arg Gly Ser 130 135 140Glu Ala Ser Pro Ala Leu Pro Gly Leu
Lys Leu Ser Ala Asp Gln Val145 150 155 160Ala Leu Val Tyr Ser Thr
Leu Gly Leu Cys Leu Cys Ala Val Leu Cys 165 170 175Cys Phe Leu Val
Ala Val Ala Cys Phe Leu Lys Lys Arg Gly Asp Pro 180 185 190Cys Ser
Cys Gln Pro Arg Ser Arg Pro Arg Gln Ser Pro Ala Lys Ser 195 200
205Ser Gln Asp His Ala Met Glu Ala Gly Ser Pro Val Ser Thr Ser Pro
210 215 220Glu Pro Val Glu Thr Cys Ser Phe Cys Phe Pro Glu Cys Arg
Ala Pro225 230 235 240Thr Gln Glu Ser Ala Val Thr Pro Gly Thr Pro
Asp Pro Thr Cys Ala 245 250 255Gly Arg Trp Gly Cys His Thr Arg Thr
Thr Val Leu Gln Pro Cys Pro 260 265 270His Ile Pro Asp Ser Gly Leu
Gly Ile Val Cys Val Pro Ala Gln Glu 275 280 285Gly Gly Pro Gly Ala
2903586DNAhomo sapiensCDS(27)...(578) 3gcagcttgtg cggcggcgtc ggcacc
atg agg cga ggg ccc cgg agc ctg cgg 53 Met Arg Arg Gly Pro Arg Ser
Leu Arg 1 5ggc agg gac gcg cca gcc ccc acg ccc tgc gtc ccg gcc gag
tgc ttc 101Gly Arg Asp Ala Pro Ala Pro Thr Pro Cys Val Pro Ala Glu
Cys Phe 10 15 20 25gac ctg ctg gtc cgc cac tgc gtg gcc tgc ggg ctc
ctg cgc acg ccg 149Asp Leu Leu Val Arg His Cys Val Ala Cys Gly Leu
Leu Arg Thr Pro 30 35 40cgg ccg aaa ccg gcc ggg gcc agc agc cct gcg
ccc agg acg gcg ctg 197Arg Pro Lys Pro Ala Gly Ala Ser Ser Pro Ala
Pro Arg Thr Ala Leu 45 50 55cag ccg cag gag tcg gtg ggc gcg ggg gcc
ggc gag gcg gcg ctg ccc 245Gln Pro Gln Glu Ser Val Gly Ala Gly Ala
Gly Glu Ala Ala Leu Pro 60 65 70ctg ccc ggg ctg ctc ttt ggc gcc ccc
gcg ctg ctg ggc ctg gca ctg 293Leu Pro Gly Leu Leu Phe Gly Ala Pro
Ala Leu Leu Gly Leu Ala Leu 75 80 85gtc ctg gcg ctg gtc ctg gtg ggt
ctg gtg agc tgg agg cgg cga cag 341Val Leu Ala Leu Val Leu Val Gly
Leu Val Ser Trp Arg Arg Arg Gln 90 95 100 105cgg cgg ctt cgc ggc
gcg tcc tcc gca gag gcc ccc gac gga gac aag 389Arg Arg Leu Arg Gly
Ala Ser Ser Ala Glu Ala Pro Asp Gly Asp Lys 110 115 120gac gcc cca
gag ccc ctg gac aag gtc atc att ctg tct ccg gga atc 437Asp Ala Pro
Glu Pro Leu Asp Lys Val Ile Ile Leu Ser Pro Gly Ile 125 130 135tct
gat gcc aca gct cct gcc tgg cct cct cct ggg gaa gac cca gga 485Ser
Asp Ala Thr Ala Pro Ala Trp Pro Pro Pro Gly Glu Asp Pro Gly 140 145
150acc acc cca cct ggc cac agt gtc cct gtg cca gcc aca gag ctg ggc
533Thr Thr Pro Pro Gly His Ser Val Pro Val Pro Ala Thr Glu Leu Gly
155 160 165tcc act gaa ctg gtg acc acc aag acg gcc ggc cct gag caa
caa 578Ser Thr Glu Leu Val Thr Thr Lys Thr Ala Gly Pro Glu Gln
Gln170 175 180tagcaggg 5864184PRThomo sapiens 4Met Arg Arg Gly Pro
Arg Ser Leu Arg Gly Arg Asp Ala Pro Ala Pro1 5 10 15Thr Pro Cys Val
Pro Ala Glu Cys Phe Asp Leu Leu Val Arg His Cys 20 25 30Val Ala Cys
Gly Leu Leu Arg Thr Pro Arg Pro Lys Pro Ala Gly Ala 35 40 45Ser Ser
Pro Ala Pro Arg Thr Ala Leu Gln Pro Gln Glu Ser Val Gly 50 55 60Ala
Gly Ala Gly Glu Ala Ala Leu Pro Leu Pro Gly Leu Leu Phe Gly65 70 75
80Ala Pro Ala Leu Leu Gly Leu Ala Leu Val Leu Ala Leu Val Leu Val
85 90 95Gly Leu Val Ser Trp Arg Arg Arg Gln Arg Arg Leu Arg Gly Ala
Ser 100 105 110Ser Ala Glu Ala Pro Asp Gly Asp Lys Asp Ala Pro Glu
Pro Leu Asp 115 120 125Lys Val Ile Ile Leu Ser Pro Gly Ile Ser Asp
Ala Thr Ala Pro Ala 130 135 140Trp Pro Pro Pro Gly Glu Asp Pro Gly
Thr Thr Pro Pro Gly His Ser145 150 155 160Val Pro Val Pro Ala Thr
Glu Leu Gly Ser Thr Glu Leu Val Thr Thr 165 170 175Lys Thr Ala Gly
Pro Glu Gln Gln 1805995DNAhomo sapiensCDS(219)...(770) 5aagactcaaa
cttagaaact tgaattagat gtggtattca aatccttacg tgccgcgaag 60acacagacag
cccccgtaag aacccacgaa gcaggcgaag ttcattgttc tcaacattct
120agctgctctt gctgcatttg ctctggaatt cttgtagaga tattacttgt
ccttccaggc 180tgttctttct gtagctccct tgttttcttt ttgtgatc atg ttg cag
atg gct ggg 236 Met Leu Gln Met Ala Gly 1 5cag tgc tcc caa aat gaa
tat ttt gac agt ttg ttg cat gct tgc ata 284Gln Cys Ser Gln Asn Glu
Tyr Phe Asp Ser Leu Leu His Ala Cys Ile 10 15 20cct tgt caa ctt cga
tgt tct tct aat act cct cct cta aca tgt cag 332Pro Cys Gln Leu Arg
Cys Ser Ser Asn Thr Pro Pro Leu Thr Cys Gln 25 30 35cgt tat tgt aat
gca agt gtg acc aat tca gtg aaa gga acg aat gcg 380Arg Tyr Cys Asn
Ala Ser Val Thr Asn Ser Val Lys Gly Thr Asn Ala 40 45 50att ctc tgg
acc tgt ttg gga ctg agc tta ata att tct ttg gca gtt 428Ile Leu Trp
Thr Cys Leu Gly Leu Ser Leu Ile Ile Ser Leu Ala Val 55 60 65 70ttc
gtg cta atg ttt ttg cta agg aag ata agc tct gaa cca tta aag 476Phe
Val Leu Met Phe Leu Leu Arg Lys Ile Ser Ser Glu Pro Leu Lys 75 80
85gac gag ttt aaa aac aca gga tca ggt ctc ctg ggc atg gct aac att
524Asp Glu Phe Lys Asn Thr Gly Ser Gly Leu Leu Gly Met Ala Asn Ile
90 95 100gac ctg gaa aag agc agg act ggt gat gaa att att ctt ccg
aga ggc 572Asp Leu Glu Lys Ser Arg Thr Gly Asp Glu Ile Ile Leu Pro
Arg Gly 105 110 115ctc gag tac acg gtg gaa gaa tgc acc tgt gaa gac
tgc atc aag agc 620Leu Glu Tyr Thr Val Glu Glu Cys Thr Cys Glu Asp
Cys Ile Lys Ser 120 125 130aaa ccg aag gtc gac tct gac cat tgc ttt
cca ctc cca gct atg gag 668Lys Pro Lys Val Asp Ser Asp His Cys Phe
Pro Leu Pro Ala Met Glu135 140 145 150gaa ggc gca acc att ctt gtc
acc acg aaa acg aat gac tat tgc aag 716Glu Gly Ala Thr Ile Leu Val
Thr Thr Lys Thr Asn Asp Tyr Cys Lys 155 160 165agc ctg cca gct gct
ttg agt gct acg gag ata gag aaa tca att tct 764Ser Leu Pro Ala Ala
Leu Ser Ala Thr Glu Ile Glu Lys Ser Ile Ser 170 175 180gct agg
taattaacca tttcgactcg agcagtgcca ctttaaaaat cttttgtcag 820Ala
Argaatagatgat gtgtcagatc tctttaggat gactgtattt ttcagttgcc
gatacagctt 880tttgtcctct aactgtggaa actctttatg ttagatatat
ttctctaggt tactgttggg 940agcttaatgg tagaaacttc cttggtttca
tgattaaagt cttttttttt cctga 9956184PRThomo sapiens 6Met Leu Gln Met
Ala Gly Gln Cys Ser Gln Asn Glu Tyr Phe Asp Ser1 5 10 15Leu Leu His
Ala Cys Ile Pro Cys Gln Leu Arg Cys Ser Ser Asn Thr 20 25 30Pro Pro
Leu Thr Cys Gln Arg Tyr Cys Asn Ala Ser Val Thr Asn Ser 35 40 45Val
Lys Gly Thr Asn Ala Ile Leu Trp Thr Cys Leu Gly Leu Ser Leu 50 55
60Ile Ile Ser Leu Ala Val Phe Val Leu Met Phe Leu Leu Arg Lys Ile65
70 75 80Ser Ser Glu Pro Leu Lys Asp Glu Phe Lys Asn Thr Gly Ser Gly
Leu 85 90 95Leu Gly Met Ala Asn Ile Asp Leu Glu Lys Ser Arg Thr Gly
Asp Glu 100 105 110Ile Ile Leu Pro Arg Gly Leu Glu Tyr Thr Val Glu
Glu Cys Thr Cys 115 120 125Glu Asp Cys Ile Lys Ser Lys Pro Lys Val
Asp Ser Asp His Cys Phe 130 135 140Pro Leu Pro Ala Met Glu Glu Gly
Ala Thr Ile Leu Val Thr Thr Lys145 150 155 160Thr Asn Asp Tyr Cys
Lys Ser Leu Pro Ala Ala Leu Ser Ala Thr Glu 165 170 175Ile Glu Lys
Ser Ile Ser Ala Arg 18071149DNAhomo sapiensCDS(173)...(1023)
7gaattcggca cgaggcagaa aggagaaaat tcaggataac tctcctgagg ggtgagccaa
60gccctgccat gtagtgcacg caggacatca acaaacacag ataacaggaa atgatccatt
120ccctgtggtc acttattcta aaggccccaa ccttcaaagt tcaagtagtg at atg
gat 178 Met Asp 1gac tcc aca gaa agg gag cag tca cgc ctt act tct
tgc ctt aag aaa 226Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser
Cys Leu Lys Lys 5 10 15aga gaa gaa atg aaa ctg aag gag tgt gtt tcc
atc ctc cca cgg aag 274Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser
Ile Leu Pro Arg Lys 20 25 30gaa agc ccc tct gtc cga tcc tcc aaa gac
gga aag ctg ctg gct gca 322Glu Ser Pro Ser Val Arg Ser Ser Lys Asp
Gly Lys Leu Leu Ala Ala 35 40 45 50acc ttg ctg ctg gca ctg ctg tct
tgc tgc ctc acg gtg gtg tct ttc 370Thr Leu Leu Leu Ala Leu Leu Ser
Cys Cys Leu Thr Val Val Ser Phe 55 60 65tac cag gtg gcc gcc ctg caa
ggg gac ctg gcc agc ctc cgg gca gag 418Tyr Gln Val Ala Ala Leu Gln
Gly Asp Leu Ala Ser Leu Arg Ala Glu 70 75 80ctg cag ggc cac cac gcg
gag aag ctg cca gca gga gca gga gcc ccc 466Leu Gln Gly His His Ala
Glu Lys Leu Pro Ala Gly Ala Gly Ala Pro 85 90 95aag gcc ggc ctg gag
gaa gct cca gct gtc acc gcg gga ctg aaa atc 514Lys Ala Gly Leu Glu
Glu Ala Pro Ala Val Thr Ala Gly Leu Lys Ile 100 105 110ttt gaa cca
cca gct cca gga gaa ggc aac tcc agt cag aac agc aga 562Phe Glu Pro
Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn Ser Arg115 120 125
130aat aag cgt gcc gtt cag ggt cca gaa gaa aca gtc act caa gac tgc
610Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln Asp Cys
135 140 145ttg caa ctg att gca gac agt gaa aca cca act ata caa aaa
gga tct 658Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys
Gly Ser 150 155 160tac aca ttt gtt cca tgg ctt ctc agc ttt aaa agg
gga agt gcc cta 706Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg
Gly Ser Ala Leu 165 170 175gaa gaa aaa gag aat aaa ata ttg gtc aaa
gaa act ggt tac ttt ttt 754Glu Glu Lys Glu Asn Lys Ile Leu Val Lys
Glu Thr Gly Tyr Phe Phe 180 185 190ata tat ggt cag gtt tta tat act
gat aag acc tac gcc atg gga cat 802Ile Tyr Gly Gln Val Leu Tyr Thr
Asp Lys Thr Tyr Ala Met Gly His195 200 205 210cta att cag agg aag
aag gtc cat gtc ttt ggg gat gaa ttg agt ctg 850Leu Ile Gln Arg Lys
Lys Val His Val Phe Gly Asp Glu Leu Ser Leu 215 220 225gtg act ttg
ttt cga tgt att caa aat atg cct gaa aca cta ccc aat 898Val Thr Leu
Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn 230 235 240aat
tcc tgc tat tca gct ggc att gca aaa ctg gaa gaa gga gat gaa 946Asn
Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu 245 250
255ctc caa ctt gca ata cca aga gaa aat gca caa ata tca ctg gat gga
994Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly
260 265 270gat gtc aca ttt ttt ggt gca ttg aaa ct gctgtgacct
acttacacca 1043Asp Val Thr Phe Phe Gly Ala Leu Lys275 280tgtctgtagc
tattttcctc cctttctctg tacctctaag aagaaagaat ctaactgaaa
1103ataccaaaaa aaaaaaaaaa aaaaaaaaaa ccctcgagcg gccgcc
11498283PRThomo sapiens 8Met Asp Asp Ser Thr Glu Arg Glu Gln Ser
Arg Leu Thr Ser Cys Leu1 5
10 15Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu
Pro 20 25 30Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly Lys
Leu Leu 35 40 45Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu
Thr Val Val 50 55 60Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu
Ala Ser Leu Arg65 70 75 80Ala Glu Leu Gln Gly His His Ala Glu Lys
Leu Pro Ala Gly Ala Gly 85 90 95Ala Pro Lys Ala Gly Leu Glu Glu Ala
Pro Ala Val Thr Ala Gly Leu 100 105 110Lys Ile Phe Glu Pro Pro Ala
Pro Gly Glu Gly Asn Ser Ser Gln Asn 115 120 125Ser Arg Asn Lys Arg
Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln 130 135 140Asp Cys Leu
Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys145 150 155
160Gly Ser Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser
165 170 175Ala Leu Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr
Gly Tyr 180 185 190Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys
Thr Tyr Ala Met 195 200 205Gly His Leu Ile Gln Arg Lys Lys Val His
Val Phe Gly Asp Glu Leu 210 215 220Ser Leu Val Thr Leu Phe Arg Cys
Ile Gln Asn Met Pro Glu Thr Leu225 230 235 240Pro Asn Asn Ser Cys
Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly 245 250 255Asp Glu Leu
Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu 260 265 270Asp
Gly Asp Val Thr Phe Phe Gly Ala Leu Lys 275 28091680DNAmus
musculusCDS(164)...(1093) 9tactcactat agggctcgag cggccgcccg
ggcaggtgct cctgggggaa cccagccctg 60ccatgctctg agggcagtct cccaggacac
agatgacagg aaatgaccca cccctgtggt 120cacttactcc aaaggcctag
accttcaaag tgctcctcgt gga atg gat gag tct 175 Met Asp Glu Ser 1gca
aag acc ctg cca cca ccg tgc ctc tgt ttt tgc tcc gag aaa gga 223Ala
Lys Thr Leu Pro Pro Pro Cys Leu Cys Phe Cys Ser Glu Lys Gly 5 10 15
20gaa gat atg aaa gtg gga tat gat ccc atc act ccg cag aag gag gag
271Glu Asp Met Lys Val Gly Tyr Asp Pro Ile Thr Pro Gln Lys Glu Glu
25 30 35ggt gcc tgg ttt ggg atc tgc agg gat gga agg ctg ctg gct gct
acc 319Gly Ala Trp Phe Gly Ile Cys Arg Asp Gly Arg Leu Leu Ala Ala
Thr 40 45 50ctc ctg ctg gcc ctg ttg tcc agc agt ttc aca gcg atg tcc
ttg tac 367Leu Leu Leu Ala Leu Leu Ser Ser Ser Phe Thr Ala Met Ser
Leu Tyr 55 60 65cag ttg gct gcc ttg caa gca gac ctg atg aac ctg cgc
atg gag ctg 415Gln Leu Ala Ala Leu Gln Ala Asp Leu Met Asn Leu Arg
Met Glu Leu 70 75 80cag agc tac cga ggt tca gca aca cca gcc gcc gcg
ggt gct cca gag 463Gln Ser Tyr Arg Gly Ser Ala Thr Pro Ala Ala Ala
Gly Ala Pro Glu 85 90 95 100ttg acc gct gga gtc aaa ctc ctg aca ccg
gca gct cct cga ccc cac 511Leu Thr Ala Gly Val Lys Leu Leu Thr Pro
Ala Ala Pro Arg Pro His 105 110 115aac tcc agc cgc ggc cac agg aac
aga cgc gct ttc cag gga cca gag 559Asn Ser Ser Arg Gly His Arg Asn
Arg Arg Ala Phe Gln Gly Pro Glu 120 125 130gaa aca gaa caa gat gta
gac ctc tca gct cct cct gca cca tgc ctg 607Glu Thr Glu Gln Asp Val
Asp Leu Ser Ala Pro Pro Ala Pro Cys Leu 135 140 145cct gga tgc cgc
cat tct caa cat gat gat aat gga atg aac ctc aga 655Pro Gly Cys Arg
His Ser Gln His Asp Asp Asn Gly Met Asn Leu Arg 150 155 160aac atc
att caa gac tgt ctg cag ctg att gca gac agc gac acg ccg 703Asn Ile
Ile Gln Asp Cys Leu Gln Leu Ile Ala Asp Ser Asp Thr Pro165 170 175
180act ata cga aaa gga act tac aca ttt gtt cca tgg ctt ctc agc ttt
751Thr Ile Arg Lys Gly Thr Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe
185 190 195aaa aga gga aat gcc ttg gag gag aaa gag aac aaa ata gtg
gtg agg 799Lys Arg Gly Asn Ala Leu Glu Glu Lys Glu Asn Lys Ile Val
Val Arg 200 205 210caa aca ggc tat ttc ttc atc tac agc cag gtt cta
tac acg gac ccc 847Gln Thr Gly Tyr Phe Phe Ile Tyr Ser Gln Val Leu
Tyr Thr Asp Pro 215 220 225atc ttt gct atg ggt cat gtc atc cag agg
aag aaa gta cac gtc ttt 895Ile Phe Ala Met Gly His Val Ile Gln Arg
Lys Lys Val His Val Phe 230 235 240ggg gac gag ctg agc ctg gtg acc
ctg ttc cga tgt att cag aat atg 943Gly Asp Glu Leu Ser Leu Val Thr
Leu Phe Arg Cys Ile Gln Asn Met245 250 255 260ccc aaa aca ctg ccc
aac aat tcc tgc tac tcg gct ggc atc gcg agg 991Pro Lys Thr Leu Pro
Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Arg 265 270 275ctg gaa gaa
gga gat gag att cag ctt gca att cct cgg gag aat gca 1039Leu Glu Glu
Gly Asp Glu Ile Gln Leu Ala Ile Pro Arg Glu Asn Ala 280 285 290cag
att tca cgc aac gga gac gac acc ttc ttt ggt gcc cta aaa ctg 1087Gln
Ile Ser Arg Asn Gly Asp Asp Thr Phe Phe Gly Ala Leu Lys Leu 295 300
305ctg taa ctcacttgct ggagtgcgtg atccccttcc ctcgtcttct ctgtacctcc
1143Leu *gagggagaaa cagacgactg gaaaaactaa aagatgggga aagccgtcag
cgaaagtttt 1203ctcgtgaccc gttgaatctg atccaaacca ggaaatataa
cagacagcca caaccgaagt 1263gtgccatgtg agttatgaga aacggagccc
gcgctcagaa agaccggatg aggaagaccg 1323ttttctccag tcctttgcca
acacgcaccg caaccttgct ttttgccttg ggtgacacat 1383gttcagaatg
cagggagatt tccttgtttt gcgatttgcc atgagaagag ggcccacaac
1443tgcaggtcac tgaagcattc acgctaagtc tcaggattta ctctcccttc
tcatgctaag 1503tacacacacg ctcttttcca ggtaatacta tgggatacta
tggaaaggtt gtttgttttt 1563aaatctagaa gtcttgaact ggcaatagac
aaaaatcctt ataaattcaa gtgtaaaata 1623aacttaatta aaaaggtaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 168010309PRTmus musculus
10Met Asp Glu Ser Ala Lys Thr Leu Pro Pro Pro Cys Leu Cys Phe Cys1
5 10 15Ser Glu Lys Gly Glu Asp Met Lys Val Gly Tyr Asp Pro Ile Thr
Pro 20 25 30Gln Lys Glu Glu Gly Ala Trp Phe Gly Ile Cys Arg Asp Gly
Arg Leu 35 40 45Leu Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Ser Ser
Phe Thr Ala 50 55 60Met Ser Leu Tyr Gln Leu Ala Ala Leu Gln Ala Asp
Leu Met Asn Leu65 70 75 80Arg Met Glu Leu Gln Ser Tyr Arg Gly Ser
Ala Thr Pro Ala Ala Ala 85 90 95Gly Ala Pro Glu Leu Thr Ala Gly Val
Lys Leu Leu Thr Pro Ala Ala 100 105 110Pro Arg Pro His Asn Ser Ser
Arg Gly His Arg Asn Arg Arg Ala Phe 115 120 125Gln Gly Pro Glu Glu
Thr Glu Gln Asp Val Asp Leu Ser Ala Pro Pro 130 135 140Ala Pro Cys
Leu Pro Gly Cys Arg His Ser Gln His Asp Asp Asn Gly145 150 155
160Met Asn Leu Arg Asn Ile Ile Gln Asp Cys Leu Gln Leu Ile Ala Asp
165 170 175Ser Asp Thr Pro Thr Ile Arg Lys Gly Thr Tyr Thr Phe Val
Pro Trp 180 185 190Leu Leu Ser Phe Lys Arg Gly Asn Ala Leu Glu Glu
Lys Glu Asn Lys 195 200 205Ile Val Val Arg Gln Thr Gly Tyr Phe Phe
Ile Tyr Ser Gln Val Leu 210 215 220Tyr Thr Asp Pro Ile Phe Ala Met
Gly His Val Ile Gln Arg Lys Lys225 230 235 240Val His Val Phe Gly
Asp Glu Leu Ser Leu Val Thr Leu Phe Arg Cys 245 250 255Ile Gln Asn
Met Pro Lys Thr Leu Pro Asn Asn Ser Cys Tyr Ser Ala 260 265 270Gly
Ile Ala Arg Leu Glu Glu Gly Asp Glu Ile Gln Leu Ala Ile Pro 275 280
285Arg Glu Asn Ala Gln Ile Ser Arg Asn Gly Asp Asp Thr Phe Phe Gly
290 295 300Ala Leu Lys Leu Leu30511185PRThomo sapiens 11Met Arg Arg
Gly Pro Arg Ser Leu Arg Gly Arg Asp Ala Pro Ala Pro1 5 10 15Thr Pro
Cys Val Pro Ala Glu Cys Phe Asp Leu Leu Val Arg His Cys 20 25 30Val
Ala Cys Gly Leu Leu Arg Thr Pro Arg Pro Lys Pro Ala Gly Ala 35 40
45Ala Ser Ser Pro Ala Pro Arg Thr Ala Leu Gln Pro Gln Glu Ser Val
50 55 60Gly Ala Gly Ala Gly Glu Ala Ala Leu Pro Leu Pro Gly Leu Leu
Phe65 70 75 80Gly Ala Pro Ala Leu Leu Gly Leu Ala Leu Val Leu Ala
Leu Val Leu 85 90 95Val Gly Leu Val Ser Trp Arg Arg Arg Gln Arg Arg
Leu Arg Gly Ala 100 105 110Ser Ser Ala Glu Ala Pro Asp Gly Asp Lys
Asp Ala Pro Glu Pro Leu 115 120 125Asp Lys Val Ile Ile Leu Ser Pro
Gly Ile Ser Asp Ala Thr Ala Pro 130 135 140Ala Trp Pro Pro Pro Gly
Glu Asp Pro Gly Thr Thr Pro Pro Gly His145 150 155 160Ser Val Pro
Val Pro Ala Thr Glu Leu Gly Ser Thr Glu Leu Val Thr 165 170 175Thr
Lys Thr Ala Gly Pro Glu Gln Gln 180 18512247PRThomo sapiens 12Met
Ser Gly Leu Gly Arg Ser Arg Arg Gly Gly Arg Ser Arg Val Asp1 5 10
15Gln Glu Glu Arg Trp Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe
20 25 30Tyr Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys
Gly 35 40 45Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu
Arg Ser 50 55 60Pro Val Asn Leu Pro Pro Glu Leu Arg Arg Gln Arg Ser
Gly Glu Val65 70 75 80Glu Asn Asn Ser Asp Asn Ser Gly Arg Tyr Gln
Gly Leu Glu His Arg 85 90 95Gly Ser Glu Ala Ser Pro Ala Leu Pro Gly
Leu Lys Leu Ser Ala Asp 100 105 110Gln Val Ala Leu Val Tyr Ser Thr
Leu Gly Leu Cys Leu Cys Ala Val 115 120 125Leu Cys Cys Phe Leu Val
Ala Val Ala Cys Phe Leu Lys Lys Arg Gly 130 135 140Asp Pro Cys Ser
Cys Gln Pro Arg Ser Arg Pro Arg Gln Ser Pro Ala145 150 155 160Lys
Ser Ser Gln Asp His Ala Met Glu Ala Gly Ser Pro Val Ser Thr 165 170
175Ser Pro Glu Pro Val Glu Thr Cys Ser Phe Cys Phe Pro Glu Cys Arg
180 185 190Ala Pro Thr Gln Glu Ser Ala Val Thr Pro Gly Thr Pro Asp
Pro Thr 195 200 205Cys Ala Gly Arg Trp Gly Cys His Thr Arg Thr Thr
Val Leu Gln Pro 210 215 220Cys Pro His Ile Pro Asp Ser Gly Leu Gly
Ile Val Cys Val Pro Ala225 230 235 240Gln Glu Gly Gly Pro Gly Ala
2451317PRTArtificial SequenceBLyS binding polypeptide 13Glu Cys Phe
Asp Leu Leu Val Arg Ala Trp Val Pro Cys Ser Val Leu1 5 10
15Lys1417PRTArtificial SequenceBLyS binding polypeptide 14Glu Cys
Phe Asp Leu Leu Val Arg His Trp Val Pro Cys Gly Leu Leu1 5 10
15Arg1517PRTArtificial SequenceBLyS binding polypeptide 15Glu Cys
Phe Asp Leu Leu Val Arg Arg Trp Val Pro Cys Glu Met Leu1 5 10
15Gly1617PRTArtificial SequenceBLyS binding polypeptide 16Glu Cys
Phe Asp Leu Leu Val Arg Ser Trp Val Pro Cys His Met Leu1 5 10
15Arg1717PRTArtificial SequenceBLyS binding polypeptide 17Glu Cys
Phe Asp Leu Leu Val Arg His Trp Val Ala Cys Gly Leu Leu1 5 10
15Arg181214DNAArtificial SequenceTACI-Fc fusion
proteinCDS(17)...(1192) 18tattaggccg gccacc atg gat gca atg aag aga
ggg ctc tgc tgt gtg ctg 52 Met Asp Ala Met Lys Arg Gly Leu Cys Cys
Val Leu 1 5 10ctg ctg tgt ggc gcc gtc ttc gtt tcg ctc agc cag gaa
atc cat gcc 100Leu Leu Cys Gly Ala Val Phe Val Ser Leu Ser Gln Glu
Ile His Ala 15 20 25gag ttg aga cgc ttc cgt aga gct atg aga tcc tgc
ccc gaa gag cag 148Glu Leu Arg Arg Phe Arg Arg Ala Met Arg Ser Cys
Pro Glu Glu Gln 30 35 40tac tgg gat cct ctg ctg ggt acc tgc atg tcc
tgc aaa acc att tgc 196Tyr Trp Asp Pro Leu Leu Gly Thr Cys Met Ser
Cys Lys Thr Ile Cys 45 50 55 60aac cat cag agc cag cgc acc tgt gca
gcc ttc tgc agg tca ctc agc 244Asn His Gln Ser Gln Arg Thr Cys Ala
Ala Phe Cys Arg Ser Leu Ser 65 70 75tgc cgc aag gag caa ggc aag ttc
tat gac cat ctc ctg agg gac tgc 292Cys Arg Lys Glu Gln Gly Lys Phe
Tyr Asp His Leu Leu Arg Asp Cys 80 85 90atc agc tgt gcc tcc atc tgt
gga cag cac cct aag caa tgt gca tac 340Ile Ser Cys Ala Ser Ile Cys
Gly Gln His Pro Lys Gln Cys Ala Tyr 95 100 105ttc tgt gag aac aag
ctc agg agc cca gtg aac ctt cca cca gag ctc 388Phe Cys Glu Asn Lys
Leu Arg Ser Pro Val Asn Leu Pro Pro Glu Leu 110 115 120agg aga cag
cgg agt gga gaa gtt gaa aac aat tca gac aac tcg gga 436Arg Arg Gln
Arg Ser Gly Glu Val Glu Asn Asn Ser Asp Asn Ser Gly125 130 135
140agg tac caa gga ttg gag cac aga ggc tca gaa gca agt cca gct ctc
484Arg Tyr Gln Gly Leu Glu His Arg Gly Ser Glu Ala Ser Pro Ala Leu
145 150 155cca ggt ctc aag gag ccc aaa tct tca gac aaa act cac aca
tgc cca 532Pro Gly Leu Lys Glu Pro Lys Ser Ser Asp Lys Thr His Thr
Cys Pro 160 165 170ccg tgc cca gca cct gaa gcc gag ggg gca ccg tca
gtc ttc ctc ttc 580Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser
Val Phe Leu Phe 175 180 185ccc cca aaa ccc aag gac acc ctc atg atc
tcc cgg acc cct gag gtc 628Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val 190 195 200aca tgc gtg gtg gtg gac gtg agc
cac gaa gac cct gag gtc aag ttc 676Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe205 210 215 220aac tgg tac gtg gac
ggc gtg gag gtg cat aat gcc aag aca aag ccg 724Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 225 230 235cgg gag gag
cag tac aac agc acg tac cgt gtg gtc agc gtc ctc acc 772Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 240 245 250gtc
ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtc 820Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 255 260
265tcc aac aaa gcc ctc cca tcc tcc atc gag aaa acc atc tcc aaa gcc
868Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
270 275 280aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc cca
tcc cgg 916Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg285 290 295 300gat gag ctg acc aag aac cag gtc agc ctg acc
tgc ctg gtc aaa ggc 964Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly 305 310 315ttc tat ccc agc gac atc gcc gtg gag
tgg gag agc aat ggg cag ccg 1012Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro 320 325 330gag aac aac tac aag acc acg
cct ccc gtg ctg gac tcc gac ggc tcc 1060Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser 335 340 345ttc ttc ctc tac agc
aag ctc acc gtg gac aag agc agg tgg cag cag 1108Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 350 355 360ggg aac gtc
ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac 1156Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His365 370 375
380tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa taatctagag
1202Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 385
390gcgcgccaat ta 121419392PRTArtificial SequenceTACI-Fc fusion
protein 19Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu
Cys Gly1 5 10 15Ala Val Phe Val Ser Leu Ser Gln Glu Ile His Ala Glu
Leu Arg Arg 20 25
30Phe Arg Arg Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro
35 40 45Leu Leu Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln
Ser 50 55 60Gln Arg Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg
Lys Glu65 70 75 80Gln Gly Lys Phe Tyr Asp His Leu Leu Arg Asp Cys
Ile Ser Cys Ala 85 90 95Ser Ile Cys Gly Gln His Pro Lys Gln Cys Ala
Tyr Phe Cys Glu Asn 100 105 110Lys Leu Arg Ser Pro Val Asn Leu Pro
Pro Glu Leu Arg Arg Gln Arg 115 120 125Ser Gly Glu Val Glu Asn Asn
Ser Asp Asn Ser Gly Arg Tyr Gln Gly 130 135 140Leu Glu His Arg Gly
Ser Glu Ala Ser Pro Ala Leu Pro Gly Leu Lys145 150 155 160Glu Pro
Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 165 170
175Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
180 185 190Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val 195 200 205Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val 210 215 220Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln225 230 235 240Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln 245 250 255Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 260 265 270Leu Pro Ser
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 275 280 285Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 290 295
300Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser305 310 315 320Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr 325 330 335Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr 340 345 350Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe 355 360 365Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys 370 375 380Ser Leu Ser Leu
Ser Pro Gly Lys385 390201070DNAArtificial SequenceTACI-Fc fusion
proteinCDS(17)...(1048) 20tattaggccg gccacc atg gat gca atg aag aga
ggg ctc tgc tgt gtg ctg 52 Met Asp Ala Met Lys Arg Gly Leu Cys Cys
Val Leu 1 5 10ctg ctg tgt ggc gcc gtc ttc gtt tcg ctc agc cag gaa
atc cat gcc 100Leu Leu Cys Gly Ala Val Phe Val Ser Leu Ser Gln Glu
Ile His Ala 15 20 25gag ttg aga cgc ttc cgt aga gct atg aga tcc tgc
ccc gaa gag cag 148Glu Leu Arg Arg Phe Arg Arg Ala Met Arg Ser Cys
Pro Glu Glu Gln 30 35 40tac tgg gat cct ctg ctg ggt acc tgc atg tcc
tgc aaa acc att tgc 196Tyr Trp Asp Pro Leu Leu Gly Thr Cys Met Ser
Cys Lys Thr Ile Cys 45 50 55 60aac cat cag agc cag cgc acc tgt gca
gcc ttc tgc agg tca ctc agc 244Asn His Gln Ser Gln Arg Thr Cys Ala
Ala Phe Cys Arg Ser Leu Ser 65 70 75tgc cgc aag gag caa ggc aag ttc
tat gac cat ctc ctg agg gac tgc 292Cys Arg Lys Glu Gln Gly Lys Phe
Tyr Asp His Leu Leu Arg Asp Cys 80 85 90atc agc tgt gcc tcc atc tgt
gga cag cac cct aag caa tgt gca tac 340Ile Ser Cys Ala Ser Ile Cys
Gly Gln His Pro Lys Gln Cys Ala Tyr 95 100 105ttc tgt gag aac gag
ccc aaa tct tca gac aaa act cac aca tgc cca 388Phe Cys Glu Asn Glu
Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 110 115 120ccg tgc cca
gca cct gaa gcc gag ggg gca ccg tca gtc ttc ctc ttc 436Pro Cys Pro
Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe125 130 135
140ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc
484Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
145 150 155aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc
aag ttc 532Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe 160 165 170aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc
aag aca aag ccg 580Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro 175 180 185cgg gag gag cag tac aac agc acg tac cgt
gtg gtc agc gtc ctc acc 628Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr 190 195 200gtc ctg cac cag gac tgg ctg aat
ggc aag gag tac aag tgc aag gtc 676Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val205 210 215 220tcc aac aaa gcc ctc
cca tcc tcc atc gag aaa acc atc tcc aaa gcc 724Ser Asn Lys Ala Leu
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 225 230 235aaa ggg cag
ccc cga gaa cca cag gtg tac acc ctg ccc cca tcc cgg 772Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 240 245 250gat
gag ctg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc 820Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 255 260
265ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg
868Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
270 275 280gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac
ggc tcc 916Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser285 290 295 300ttc ttc ctc tac agc aag ctc acc gtg gac aag
agc agg tgg cag cag 964Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln 305 310 315ggg aac gtc ttc tca tgc tcc gtg atg
cat gag gct ctg cac aac cac 1012Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His 320 325 330tac acg cag aag agc ctc tcc
ctg tct ccg ggt aaa taatctagag 1058Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 335 340gcgcgccaat ta 107021344PRTArtificial
SequenceTACI-Fc fusion protein 21Met Asp Ala Met Lys Arg Gly Leu
Cys Cys Val Leu Leu Leu Cys Gly1 5 10 15Ala Val Phe Val Ser Leu Ser
Gln Glu Ile His Ala Glu Leu Arg Arg 20 25 30Phe Arg Arg Ala Met Arg
Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro 35 40 45Leu Leu Gly Thr Cys
Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser 50 55 60Gln Arg Thr Cys
Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu65 70 75 80Gln Gly
Lys Phe Tyr Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala 85 90 95Ser
Ile Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn 100 105
110Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
115 120 125Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro 130 135 140Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val145 150 155 160Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val 165 170 175Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln 180 185 190Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln 195 200 205Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 210 215 220Leu
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro225 230
235 240Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr 245 250 255Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser 260 265 270Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr 275 280 285Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr 290 295 300Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe305 310 315 320Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 325 330 335Ser Leu
Ser Leu Ser Pro Gly Lys 340221082DNAArtificial SequenceTACI-Fc
fusion proteinCDS(17)...(1060) 22tattaggccg gccacc atg gat gca atg
aag aga ggg ctc tgc tgt gtg ctg 52 Met Asp Ala Met Lys Arg Gly Leu
Cys Cys Val Leu 1 5 10ctg ctg tgt ggc gcc gtc ttc gtt tcg ctc agc
cag gaa atc cat gcc 100Leu Leu Cys Gly Ala Val Phe Val Ser Leu Ser
Gln Glu Ile His Ala 15 20 25gag ttg aga cgc ttc cgt aga gct atg aga
tcc tgc ccc gaa gag cag 148Glu Leu Arg Arg Phe Arg Arg Ala Met Arg
Ser Cys Pro Glu Glu Gln 30 35 40tac tgg gat cct ctg ctg ggt acc tgc
atg tcc tgc aaa acc att tgc 196Tyr Trp Asp Pro Leu Leu Gly Thr Cys
Met Ser Cys Lys Thr Ile Cys 45 50 55 60aac cat cag agc cag cgc acc
tgt gca gcc ttc tgc agg tca ctc agc 244Asn His Gln Ser Gln Arg Thr
Cys Ala Ala Phe Cys Arg Ser Leu Ser 65 70 75tgc cgc aag gag caa ggc
aag ttc tat gac cat ctc ctg agg gac tgc 292Cys Arg Lys Glu Gln Gly
Lys Phe Tyr Asp His Leu Leu Arg Asp Cys 80 85 90atc agc tgt gcc tcc
atc tgt gga cag cac cct aag caa tgt gca tac 340Ile Ser Cys Ala Ser
Ile Cys Gly Gln His Pro Lys Gln Cys Ala Tyr 95 100 105ttc tgt gag
aac aag ctc agg agc gag ccc aaa tct tca gac aaa act 388Phe Cys Glu
Asn Lys Leu Arg Ser Glu Pro Lys Ser Ser Asp Lys Thr 110 115 120cac
aca tgc cca ccg tgc cca gca cct gaa gcc gag ggg gca ccg tca 436His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser125 130
135 140gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc
cgg 484Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg 145 150 155acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac
gaa gac cct 532Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro 160 165 170gag gtc aag ttc aac tgg tac gtg gac ggc gtg
gag gtg cat aat gcc 580Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala 175 180 185aag aca aag ccg cgg gag gag cag tac
aac agc acg tac cgt gtg gtc 628Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val 190 195 200agc gtc ctc acc gtc ctg cac
cag gac tgg ctg aat ggc aag gag tac 676Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr205 210 215 220aag tgc aag gtc
tcc aac aaa gcc ctc cca tcc tcc atc gag aaa acc 724Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu Lys Thr 225 230 235atc tcc
aaa gcc aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg 772Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 240 245
250ccc cca tcc cgg gat gag ctg acc aag aac cag gtc agc ctg acc tgc
820Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
255 260 265ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg
gag agc 868Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser 270 275 280aat ggg cag ccg gag aac aac tac aag acc acg cct
ccc gtg ctg gac 916Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp285 290 295 300tcc gac ggc tcc ttc ttc ctc tac agc
aag ctc acc gtg gac aag agc 964Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser 305 310 315agg tgg cag cag ggg aac gtc
ttc tca tgc tcc gtg atg cat gag gct 1012Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala 320 325 330ctg cac aac cac tac
acg cag aag agc ctc tcc ctg tct ccg ggt aaa 1060Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 335 340 345taatctagag
gcgcgccaat ta 108223348PRTArtificial SequenceTACI-Fc fusion protein
23Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly1
5 10 15Ala Val Phe Val Ser Leu Ser Gln Glu Ile His Ala Glu Leu Arg
Arg 20 25 30Phe Arg Arg Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp
Asp Pro 35 40 45Leu Leu Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn
His Gln Ser 50 55 60Gln Arg Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser
Cys Arg Lys Glu65 70 75 80Gln Gly Lys Phe Tyr Asp His Leu Leu Arg
Asp Cys Ile Ser Cys Ala 85 90 95Ser Ile Cys Gly Gln His Pro Lys Gln
Cys Ala Tyr Phe Cys Glu Asn 100 105 110Lys Leu Arg Ser Glu Pro Lys
Ser Ser Asp Lys Thr His Thr Cys Pro 115 120 125Pro Cys Pro Ala Pro
Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe 130 135 140Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val145 150 155
160Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
165 170 175Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro 180 185 190Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr 195 200 205Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val 210 215 220Ser Asn Lys Ala Leu Pro Ser Ser
Ile Glu Lys Thr Ile Ser Lys Ala225 230 235 240Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 245 250 255Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 260 265 270Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 275 280
285Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
290 295 300Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln305 310 315 320Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His 325 330 335Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 340 345241109DNAArtificial SequenceTACI-Fc fusion
proteinCDS(17)...(1090) 24tattaggccg gccacc atg gat gca atg aag aga
ggg ctc tgc tgt gtg ctg 52 Met Asp Ala Met Lys Arg Gly Leu Cys Cys
Val Leu 1 5 10ctg ctg tgt ggc gcc gtc ttc gtt tcg ctc agc cag gaa
atc cat gcc 100Leu Leu Cys Gly Ala Val Phe Val Ser Leu Ser Gln Glu
Ile His Ala 15 20 25gag ttg aga cgc ttc cgt aga gct atg aga tcc tgc
ccc gaa gag cag 148Glu Leu Arg Arg Phe Arg Arg Ala Met Arg Ser Cys
Pro Glu Glu Gln 30 35 40tac tgg gat cct ctg ctg ggt acc tgc atg tcc
tgc aaa acc att tgc 196Tyr Trp Asp Pro Leu Leu Gly Thr Cys Met Ser
Cys Lys Thr Ile Cys 45 50 55 60aac cat cag agc cag cgc acc tgt gca
gcc ttc tgc agg tca ctc agc 244Asn His Gln Ser Gln Arg Thr Cys Ala
Ala Phe Cys Arg Ser Leu Ser 65 70 75tgc cgc aag gag caa ggc aag ttc
tat gac cat ctc ctg agg gac tgc 292Cys Arg Lys Glu Gln Gly Lys Phe
Tyr Asp His Leu Leu Arg Asp Cys 80 85 90atc agc tgt gcc tcc atc tgt
gga cag cac cct aag caa tgt gca tac 340Ile Ser Cys Ala Ser Ile Cys
Gly Gln His Pro Lys Gln Cys Ala Tyr 95 100 105ttc tgt gag aac aag
ctc agg agc cca gtg aac ctt cca cca gag ctc 388Phe Cys Glu Asn Lys
Leu Arg Ser Pro Val Asn Leu Pro Pro Glu Leu 110 115 120agg gag ccc
aaa tct tca gac aaa act cac aca tgc cca ccg tgc cca 436Arg Glu Pro
Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro125 130 135
140gca cct gaa gcc gag ggg gca ccg tca gtc ttc ctc ttc ccc cca
aaa
484Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys
145 150 155ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc aca
tgc gtg 532Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val 160 165 170gtg gtg gac gtg agc cac gaa gac cct gag gtc aag
ttc aac tgg tac 580Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr 175 180 185gtg gac ggc gtg gag gtg cat aat gcc aag
aca aag ccg cgg gag gag 628Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu 190 195 200cag tac aac agc acg tac cgt gtg
gtc agc gtc ctc acc gtc ctg cac 676Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His205 210 215 220cag gac tgg ctg aat
ggc aag gag tac aag tgc aag gtc tcc aac aaa 724Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 225 230 235gcc ctc cca
tcc tcc atc gag aaa acc atc tcc aaa gcc aaa ggg cag 772Ala Leu Pro
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 240 245 250ccc
cga gaa cca cag gtg tac acc ctg ccc cca tcc cgg gat gag ctg 820Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 255 260
265acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc
868Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
270 275 280agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag
aac aac 916Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn285 290 295 300tac aag acc acg cct ccc gtg ctg gac tcc gac
ggc tcc ttc ttc ctc 964Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu 305 310 315tac agc aag ctc acc gtg gac aag agc
agg tgg cag cag ggg aac gtc 1012Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val 320 325 330ttc tca tgc tcc gtg atg cat
gag gct ctg cac aac cac tac acg cag 1060Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln 335 340 345aag agc ctc tcc ctg
tct ccg ggt aaa taa tctagaggcg cgccaatta 1109Lys Ser Leu Ser Leu
Ser Pro Gly Lys * 350 35525357PRTArtificial SequenceTACI-Fc fusion
protein 25Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu
Cys Gly1 5 10 15Ala Val Phe Val Ser Leu Ser Gln Glu Ile His Ala Glu
Leu Arg Arg 20 25 30Phe Arg Arg Ala Met Arg Ser Cys Pro Glu Glu Gln
Tyr Trp Asp Pro 35 40 45Leu Leu Gly Thr Cys Met Ser Cys Lys Thr Ile
Cys Asn His Gln Ser 50 55 60Gln Arg Thr Cys Ala Ala Phe Cys Arg Ser
Leu Ser Cys Arg Lys Glu65 70 75 80Gln Gly Lys Phe Tyr Asp His Leu
Leu Arg Asp Cys Ile Ser Cys Ala 85 90 95Ser Ile Cys Gly Gln His Pro
Lys Gln Cys Ala Tyr Phe Cys Glu Asn 100 105 110Lys Leu Arg Ser Pro
Val Asn Leu Pro Pro Glu Leu Arg Glu Pro Lys 115 120 125Ser Ser Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 130 135 140Glu
Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr145 150
155 160Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val 165 170 175Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val 180 185 190Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser 195 200 205Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu 210 215 220Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ser225 230 235 240Ser Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 245 250 255Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 260 265
270Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr 290 295 300Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu305 310 315 320Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser 325 330 335Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser 340 345 350Leu Ser Pro Gly Lys
35526311PRTArtificial SequenceBAFF-R-Fc fusion protein 26Met Ser
Ala Leu Leu Ile Leu Ala Leu Val Gly Ala Ala Val Ala Ser1 5 10 15Thr
Arg Arg Gly Pro Arg Ser Leu Arg Gly Arg Asp Ala Pro Ala Pro 20 25
30Thr Pro Cys Val Pro Ala Glu Cys Phe Asp Leu Leu Val Arg His Cys
35 40 45Val Ala Cys Gly Leu Leu Arg Thr Pro Arg Pro Lys Pro Ala Gly
Ala 50 55 60Ser Ser Pro Ala Pro Arg Thr Ala Leu Gln Pro Gln Glu Ser
Gln Val65 70 75 80Thr Asp Lys Ala Ala His Tyr Thr Leu Cys Pro Pro
Cys Pro Ala Pro 85 90 95Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys 100 105 110Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val 115 120 125Ala Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp 130 135 140Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr145 150 155 160Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 165 170
175Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
180 185 190Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg 195 200 205Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys 210 215 220Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp225 230 235 240Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys 245 250 255Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 260 265 270Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 275 280 285Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 290 295
300Leu Ser Leu Ser Pro Gly Lys305 310
* * * * *