U.S. patent application number 11/065669 was filed with the patent office on 2005-11-03 for baff, inhibitors thereof and their use in the modulation of b-cell response and treatment of autoimmune disorders.
This patent application is currently assigned to Biogen Idec MA Inc.. Invention is credited to Kalled, Susan, MacKay, Fabienne.
Application Number | 20050244411 11/065669 |
Document ID | / |
Family ID | 46150035 |
Filed Date | 2005-11-03 |
United States Patent
Application |
20050244411 |
Kind Code |
A1 |
MacKay, Fabienne ; et
al. |
November 3, 2005 |
BAFF, inhibitors thereof and their use in the modulation of B-cell
response and treatment of autoimmune disorders
Abstract
The invention provides methods for treating or preventing
disorders associated with expression of BAFF comprising BAFF and
fragments thereof, antibodies, agonists and antagonists.
Inventors: |
MacKay, Fabienne; (Vaucluse,
AU) ; Kalled, Susan; (Concord, MA) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER
LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
Biogen Idec MA Inc.
|
Family ID: |
46150035 |
Appl. No.: |
11/065669 |
Filed: |
February 24, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11065669 |
Feb 24, 2005 |
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10045574 |
Nov 7, 2001 |
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10045574 |
Nov 7, 2001 |
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09911777 |
Jul 24, 2001 |
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6869605 |
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09911777 |
Jul 24, 2001 |
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PCT/US00/01788 |
Jan 25, 2000 |
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60117169 |
Jan 25, 1999 |
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60143228 |
Jul 9, 1999 |
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Current U.S.
Class: |
424/144.1 ;
514/16.6; 514/19.3; 514/3.8 |
Current CPC
Class: |
A01K 2227/105 20130101;
A61K 2039/505 20130101; A01K 2267/0325 20130101; A01K 67/0275
20130101; C07K 14/70575 20130101; C07K 16/2875 20130101; C07K
16/2878 20130101; A61K 38/00 20130101; A01K 2267/03 20130101; A01K
2217/00 20130101; A01K 2267/0331 20130101; A01K 2207/15 20130101;
A01K 67/0278 20130101; A01K 2267/0375 20130101; A01K 2267/0368
20130101; C12N 15/8509 20130101; A61P 35/00 20180101; A01K 2217/05
20130101; A01K 2267/0381 20130101 |
Class at
Publication: |
424/144.1 ;
514/012 |
International
Class: |
A61K 039/395; A61K
038/17 |
Claims
1-59. (canceled)
60. A method of treating a mammal, the method comprising
administering to a mammal having Sjogren's syndrome a composition
comprising a BAFF blocking agent, thereby reducing immunoglobulin
production or B cell growth in the mammal.
61. The method of claim 60, wherein the mammal is a human.
62. The method of claim 60, wherein the mammal is a mouse.
63. The method of claim 62, wherein the mouse is a BAFF Tg
mouse.
64. The method of claim 60, wherein the salivary gland of the
mammal is infiltrated by MZ-like B cells.
65. The method of claim 60, wherein the BAFF blocking is selected
form the group consisting of: (a) a soluble BAFF receptor; (b) an
antibody against BAFF ligand; (c) an antibody against BAFF
receptor; and (d) an inoperative BAFF ligand molecule.
66. The method of claim 65, wherein soluble BAFF receptor comprises
an immunoglobulin Fc domain.
67. The method of claim 65, wherein the BAFF receptor is
BAFF-R.
68. The method of claim 65, wherein the BAFF receptor is TACI.
69. The method of claim 65, wherein the BAFF receptor is BCMA.
70. The method of claim 67, wherein BAFF-R is human.
71. The method of claim 65, wherein the soluble BAFF receptor
comprises a portion of SEQ ID NO:27 that binds to BAFF.
72. The method of claim 71, wherein the soluble BAFF receptor
further comprises a human immunoglobulin Fc domain.
73. The method of claim 67, wherein BAFF-R is murine.
74. The method of claim 70, wherein the soluble BAFF receptor
comprises a portion of SEQ ID NO:28 that binds to BAFF.
75. The method of claim 65, wherein the antibody against BAFF
ligand or BAFF receptor is a monoclonal antibody.
76. The method of claim 75, wherein the antibody is recombinantly
produced.
77. The method of claim 75, wherein the antibody is a chimeric
antibody.
78. The method of claim 75, wherein the antibody is a humanized
antibody.
79. The method of claim 75, wherein the antibody comprises human
constant domains.
80. The method of claim 75, wherein the antibody is a F(ab')2
fragment.
81. The method of claim 60, further comprising detecting the level
of B cell growth in the mammal.
82. The method of claim 60, further comprising detecting the level
of immunoglobulin production in the mammal.
83. The method of claim 60, further comprising detecting the levels
of B cell growth and immunoglobulin production in the mammal.
84. The method of claim 60, further comprising detecting
circulating levels of a rheumatoid factor in the mammal.
85. The method of claim 60, further comprising detecting
circulating levels of anti-DNA autoantibody in the mammal.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 09/911,777, filed Jul. 24, 2001, which claims
priority to International Application No. PCT/US00/01788 filed Jan.
25, 2000, which claims priority to U.S. Ser. No. 60/117,169 filed
on Jan. 25, 1999 and U.S. Ser. No. 60/143,228 filed Jul. 9, 2001.
The entire disclosures of the aforesaid patent applications are
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to the use of a ligand, BAFF,
a .beta.-cell activating factor belonging to the Tumor Necrosis
Family and its blocking agents to either stimulate or inhibit the
expression of B-cells and immunoglobulins. This protein and its
receptor may have anti-cancer and/or immunoregulatory applications
as well as uses for the treatment of immunosuppressive disorders
such as HIV. Specifically, the ligand and its blocking agents may
play a role in the development of hypertension and its related
disorders. Furthermore, cells transfected with the gene for this
ligand may be used in gene therapy to treat tumors, autoimmune
diseases or inherited genetic disorders involving B-cells. Blocking
agents, such as recombinant variants or antibodies specific to the
ligand or its receptor, may have immunoregulatory applications as
well. Use of BAFF as a B-cell stimulator for immune suppressed
diseases including for example uses for patients undergoing organ
transplantation (i.e. bone marrow transplant) as well as recovering
from cancer treatments to stimulate production of B-cells are
contemplated. Use of BAFF as an adjuvant and or costimulator to
boast and or restore B cells levels to approximate normal levels
are also contemplated.
BACKGROUND OF THE INVENTION
[0003] The tumor-necrosis factor (TNF)-related cytokines are
mediators of host defense and immune regulation. Members of this
family exist in membrane-anchored forms, acting locally through
cell-to-cell contact, or as secreted proteins capable of diffusing
to more distant targets. A parallel family of receptors signals the
presence of these molecules leading to the initiation of cell death
or cellular proliferation and differentiation in the target tissue.
Presently, the TNF family of ligands and receptors has at least 11
recognized receptor-ligand pairs, including: TNF:TNF-R;
LT-.alpha.:TNF-R; LT-.alpha./.beta.:LT-.beta.-R; FasL:Fas;
CD40L:CD40; CD30L:CD30; CD27L:CD27; OX40L:OX40 and 4-1BBL:4-1BB.
The DNA sequences encoding these ligands have only about 25% to
about 30% identity in even the most related cases, although the
amino acid relatedness is about 50%.
[0004] The defining feature of this family of cytokine receptors is
found in the cysteine rich extracellular domain initially revealed
by the molecular cloning of two distinct TNF receptors. This family
of genes encodes glycoproteins characteristic of Type I
transmembrane proteins with an extracellular ligand binding domain,
a single membrane spanning region and a cytoplasmic region involved
in activating cellular functions. The cysteine-rich ligand binding
region exhibits a tightly knit disulfide linked core domain, which,
depending upon the particular family member, is repeated multiple
times. Most receptors have four domains, although there may be as
few as three, or as many as six.
[0005] Proteins in the TNF family of ligands are characterized by a
short N-terminal stretch of normally short hydrophilic amino acids,
often containing several lysine or arginine residues thought to
serve as stop transfer sequences. Next follows a transmembrane
region and an extracellular region of variable length, that
separates the C-terminal receptor binding domain from the membrane.
This region is sometimes referred to as the "stalk". The C-terminal
binding region comprises the bulk of the protein, and often, but
not always, contains glycosylation sites. These genes lack the
classic signal sequences characteristic of type I membrane
proteins, type I membrane proteins with the C terminus lying
outside the cell, and a short N-terminal domain residing in the
cytoplasm. In some cases, e.g., TNF and LT-.alpha., cleavage in the
stalk region can occur early during protein processing and the
ligand is then found primarily in secreted form. Most ligands,
however, exist in a membrane form, mediating localized
signaling.
[0006] The structure of these ligands has been well-defined by
crystallographic analyses of TNF, LT-.alpha., and CD40L. TNF and
lymphotoxin-I (LT-1) are both structured into a sandwich of two
anti-parallel .beta.-pleated sheets with the "jelly roll" or Greek
key topology. The rms deviation between the C.alpha. and .beta.
residues is 0.61 C, suggesting a high degree of similarity in their
molecular topography. A structural feature emerging from molecular
studies of CD40L, TNF and LT-.alpha. is the propensity to assemble
into oligomeric complexes. Intrinsic to the oligomeric structure is
the formation of the receptor binding site at the junction between
the neighboring subunits creating a multivalent ligand. The
quaternary structures of TNF, CD40L and LT-.alpha. have been shown
to exist as trimers by analysis of their crystal structures. Many
of the amino acids conserved between the different ligands are in
stretches of the scaffold .beta.-sheet. It is likely that the basic
sandwich structure is preserved in all of these molecules, since
portions of these scaffold sequences are conserved across the
various family members. The quaternary structure may also be
maintained since the subunit conformation is likely to remain
similar.
[0007] TNF family members can best be described as master switches
in the immune system controlling both cell survival and
differentiation. Only TNF and LT.alpha. are currently recognized as
secreted cytokines contrasting with the other predominantly
membrane anchored members of the TNF family. While a membrane form
of TNF has been well-characterized and is likely to have unique
biological roles, secreted TNF functions as a general alarm
signaling to cells more distant from the site of the triggering
event. Thus TNF secretion can amplify an event leading to the
well-described changes in the vasculature lining and the
inflammatory state of cells. In contrast, the membrane bound
members of the family send signals though the TNF type receptors
only to cells in direct contact. For example T cells provide CD40
mediated "help" only to those B cells brought into direct contact
via cognate TCR interactions. Similar cell-cell contact limitations
on the ability to induce cell death apply to the well-studied Fas
system.
[0008] It appears that one can segregate the TNF ligands into three
groups based on their ability to induce cell death. First, TNF, Fas
ligand and TRAIL can efficiently induce cell death in many lines
and their receptors mostly likely have good canonical death
domains. Presumably the ligand to DR-3 (TRAMP/WSL-1) would also all
into this category. Next there are those ligands which trigger a
weaker death signal limited to few cell types and TWEAK, CD30
ligand and LTa1b2 are examples of this class. How this group can
trigger cell death in the absence of a canonical death domain is an
interesting question and suggests that a separate weaker death
signaling mechanism exists. Lastly, there are those members that
cannot efficiently deliver a death signal. Probably all groups can
have antiproliferative effects on some cell types consequent to
inducing cell differentiation e.g. CD40. Funakoshi et al.
(1994).
[0009] The TNF family has grown dramatically in recent years to
encompass at least 11 different signaling pathways involving
regulation of the immune system. The widespread expression patterns
of TWEAK and TRAIL indicate that there is still more functional
variety to be uncovered in this family. This aspect has been
especially highlighted recently in the discovery of two receptors
that affect the ability of rous sacroma and herpes simplex virus to
replicate as well as the historical observations that TNF has
anti-viral activity and pox viruses encode for decoy TNF receptors.
Brojatsch et al. (1996); Montgomery et al. (1996); Smith et al.
(1994), 76 Cell 959-962; Vassalli et al. (1992), 10 Immunol.
411-452.
[0010] TNF is a mediator of septic shock and cachexia, and is
involved in the regulation of hematopoietic cell development. It
appears to play a major role as a mediator of inflammation and
defense against bacterial, viral and parasitic infections as well
as having antitumor activity. TNF is also involved in different
autoimmune diseases. TNF may be produced by several types of cells,
including macrophages, fibroblasts, T cells and natural killer
cells. TNF binds to two different receptors, each acting through
specific intracellular signaling molecules, thus resulting in
different effects of TNF. TNF can exist either as a membrane bound
form or as a soluble secreted cytokine.
[0011] LT-I shares many activities with TNF, i.e. binding to the
TNF receptors, but unlike TNF, appears to be secreted primarily by
activated T cells and some .beta.-lymphoblastoid tumors. The
heteromeric complex of LT-.alpha. and LT-.beta. is a membrane bound
complex which binds to the LT-.beta. receptor. The LT system (LTs
and LT-R) appears to be involved in the development of peripheral
lymphoid organs since genetic disruption of LT-.beta. leads to
disorganization of T and B cells in the spleen and an absence of
lymph nodes. The LT-.beta. system is also involved in cell death of
some adenocarcinoma cell lines.
[0012] Fas-L, another member of the TNF family, is expressed
predominantly on activated T cells. It induces the death of cells
bearing its receptor, including tumor cells and HIV-infected cells,
by a mechanism known as programmed cell death or apoptosis.
Furthermore, deficiencies in either Fas or Fas-L may lead to
lymphoproliferative disorders, confirming the role of the Fas
system in the regulation of immune responses. The Fas system is
also involved in liver damage resulting from hepatitis chronic
infection and in autoimmunity in HIV-infected patients. The Fas
system is also involved in T-cell destruction in HIV patients.
TRAIL, another member of this family, also seems to be involved in
the death of a wide variety of transformed cell lines of diverse
origin.
[0013] CD40-L, another member of the TNF family, is expressed on T
cells and induces the regulation of CD40-bearing B cells.
Furthermore, alterations in the CD40-L gene result in a disease
known as X-linked hyper-IgM syndrome. The CD40 system is also
involved in different autoimmune diseases and CD40-L is known to
have antiviral properties. Although the CD40 system is involved in
the rescue of apoptotic B cells, in non-immune cells it induces
apoptosis. Many additional lymphocyte members of the TNF family are
also involved in costimulation.
[0014] Generally, the members of the TNF family have fundamental
regulatory roles in controlling the immune system and activating
acute host defense systems. Given the current progress in
manipulating members of the TNF family for therapeutic benefit, it
is likely that members of this family may provide unique means to
control disease. Some of the ligands of this family can directly
induce the apoptotic death of many transformed cells e.g. LT, TNF,
Fas ligand and TRAIL. Nagata (1997) 88 Cell 355-365. Fas and
possibly TNF and CD30 receptor activation can induce cell death in
nontransformed lymphocytes which may play an immunoregulatory
function. Amakawa et al. (1996) 84 Cell 551-562; Nagata (1997) 88
Cell 355-365; Sytwu et al. (1996); Zheng et al. (1995) 377 Nature
348-351. In general, death is triggered following the aggregation
of death domains which reside on the cytoplasmic side of the TNF
receptors. The death domain orchestrates the assembly of various
signal transduction components which result in the activation of
the caspase cascade. Nagata (1997) 88 Cell 355-365. Some receptors
lack canonical death domains, e.g. LTb receptor and CD30 (Browning
et al. (1996); Lee et al. (1996)) yet can induce cell death, albeit
more weakly. It is likely that these receptors function primarily
to induce cell differentiation and the death is an aberrant
consequence in some transformed cell lines, although this picture
is unclear as studies on the CD30 null mouse suggest a death role
in negative selection in the thymus. Amakawa et al. (1996) 84 Cell
551-562. Conversely, signaling through other pathways such as CD40
is required to maintain cell survival. Thus, there is a need to
identify and characterize additional molecules which are members of
the TNF family thereby providing additional means of controlling
diseased and manipulating the immune system.
[0015] Sjogren's syndrome (SS) is a chronic inflammatory disorder
characterized by the destruction of exocrine glands such as
salivary and lacrimal glands, leading to symptoms of dry mouth
(xerostomia) and eyes (keratoconjuncitivitis sicca). Jonsson et al.
(2000) Sjogren's syndrome in Arthritis and allied conditions
1826-1849. SS is regarded as an autoimmune disease characterized by
the presence of large mononuclear cell infiltrates in exocrine
glands, B cell hyper-reactivity and various serum autoantibodies.
Jonsson et al. (2000); Manoussakis et al. (1998) Sjogren's sydrome
in The autoimmune diseases, Academic Press, 381-404. SS can develop
alone or in association with other autoimmune disorders such as
systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA).
Jonnson et al. (2000); Manoussakis et al. (1998).
[0016] Abnormal B cell activity is a predominant feature of SS,
which is manifested by massive polyclonal B cell activation and
elevated secretion of autoantibodies such as rheumatoid factors
(RF), anti-Ro (SS-A), anti-La (SS-B) and anti-.lambda.-fodrin
autoantibodies. Jonnson et al. (2000); Manoussakis et al. (1998);
MacSween et al. (1967) Ann. Rheum. Dis. 26:402-411; Haneji et al.
(1997) Science 276; 604-607. Intense B cell activity such as
germinal center reactions occur in exocrine glands of some
patients, placing them in a high risk category for the development
of lymphomas. Jonsson et al. (2000); Stott et al. (1998) J. Clin.
Invest. 102:938-946. However, the role of B cells and
autoantibodies in the pathogenesis of SS still remains unclear.
SUMMARY OF THE INVENTION
[0017] Here we characterize the functional properties of a new
ligand of the TNF cytokine-family. The new ligand, termed BAFF (B
cell activating factor belonging to the TNF family), appears to be
expressed by T cells and dendritic cells for the purpose of B-cell
co-stimulation and may therefore play an important role in the
control of B cell function. In addition, we have generated
transgenic mice overexpressing BAFF under the control of a
liver-specific promoter. These mice have excessive numbers of
mature B cells, spontaneous germinal center reactions, secrete
autoantibodies, and have high plasma cell numbers in secondary
lymphoid organs and Ig deposition in the kidney.
[0018] The BAFF Tg mice develop as they age a secondary condition
to their lupus-like disease, showing interesting similarities with
that of SS in humans. We also identified a new and potentially
pathogenic B cell population with MZ-like features infiltrating
salivary glands of BAFF Tg mice.
[0019] Accordingly, the present invention is directed to the use of
BAFF-ligands, blocking agents and antibodies for the ligand, to
either stimulate or inhibit the growth of B-cells and the secretion
of immunoglobulin. The claimed invention may be used for
therapeutic applications in numerous diseases and disorders, as
discussed in more detail below, as well as to obtain information
about, and manipulate, the immune system and its processes.
Further, this invention can be used as a method of stimulating or
inhibiting the growth of B-cells and the secretion of
immunoglobulins. BAFF associated molecules, as described by this
invention, may also have utility in the treatment of autoimmune
diseases, disorders relating to B-cell proliferation and
maturation, BAFF ligand regulation and inflammation. The invention
may be involved in the regulation or prevention of hypertension and
hypertension-related disorders of the renal and cardiovascular
tissue.
[0020] Additional features and advantages of the invention will be
set forth in the description which follows, and in part will be
apparent from the description, or may be learned by practice of the
invention. The objectives and other advantages of the invention
will be realized and attained by the methods particularly pointed
out in the written description and claims hereof, as well as in the
appended drawings.
[0021] Thus, to achieve these and other advantages, and in
accordance with the purpose of the invention, as embodied and
broadly described herein, the invention includes a method of
effecting B-cell growth and secretion of immunoglobulins through
the administration of various BAFF ligands and related
molecules.
[0022] The invention also contemplates stimulating B-cell growth
through the use of BAFF ligands or active fragments of the
polypeptide. The polypeptide may be use alone or with a CD40 ligand
or an anti-murine antibody.
[0023] In other embodiments, the invention relates to methods of
stimulation of dendritic cell-induced B-cell growth and maturation
through the use of BAFF ligands or active fragments of BAFF. Again,
the polypeptide may be used alone or with CD40 ligand or anti-.mu.
antibodies.
[0024] In other embodiments, blocking agents of BAFF and the BAFF
receptor have been used to inhibit B-cell growth and immunoglobulin
secretion. These agents can be inoperative, recombinant BAFF, BAFF
specific antibodies, BAFF-receptor specific antibodies or an
anti-BAFF ligand molecule.
[0025] In yet other embodiments, the invention relates to the use
of BAFF, BAFF related molecules and BAFF blocking agents to treat
hypertension, hypertension related disorders, immune disorders,
autoimmune diseases, inflammation and B-cell lympho-proliferate
disorders. In another aspect, the invention relates to the use of
BAFF, BAFF-related molecules and BAFF blocking agents to treat
Sjogren's syndrome.
[0026] In a preferred embodiment, the invention relates to methods
for treating or reducing the advancement, severity or effects of
Sjogren's syndrome in a patient by administering a pharmaceutical
preparation comprising a therapeutically effective amount of a BAFF
blocking agent and a pharmaceutically acceptable carrier. In some
embodiments, the BAFF blocking agent may be a soluble BAFF receptor
molecule, an antibody directed against BAFF-ligand or an antibody
directed against a BAFF receptor. The BAFF receptor may be BCMA,
TACI, or BAFF R.
[0027] The invention encompasses the use of BAFF and BAFF-related
molecules as either agonists or antagonists in effecting immune
responses by effecting the growth and/or maturation of B-cells and
secretion of immunoglobulin.
[0028] The invention relates in other embodiments to soluble
constructs comprising BAFF which may be used to directly trigger
BAFF mediated pharmacological events. Such events may have useful
therapeutic benefits in the treatment of cancer, tumors or the
manipulation of the immune system to treat immunologic
diseases.
[0029] Additionally, in other embodiments the claimed invention
relates to antibodies directed against BAFF ligand, which can be
used, for example, for the treatment of cancers, and manipulation
of the immune system to treat immunologic disease.
[0030] In yet other embodiments the invention relates to methods of
gene therapy using the genes for BAFF.
[0031] The pharmaceutical preparations of the invention may,
optionally, include pharmaceutically acceptable carriers,
adjuvants, fillers, or other pharmaceutical compositions, and may
be administered in any of the numerous forms or routes known in the
art.
[0032] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory, and are intended to provide further explanation of
the invention as claimed.
[0033] The accompanying drawings are included to provide a further
understanding of the invention, and are incorporated in, and
constitute a part of this specification, illustrate several
embodiments of the invention, and together with the description
serve to explain the principles of the invention.
DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 (A) depicts the predicted amino acid sequence of
human [SEQ. ID. NO.: 1] and mouse BAFF [SEQ. ID. NO.:2]. The
predicted transmembrane domain (TMD, dashed line), the potential
N-linked glycosylation sites (stars) and the natural processing
site of human BAFF (arrow) are indicated. The double line above
hBAFF indicates the sequence obtained by Edman degradation of the
processed form of BAFF. (B) Depicts a comparison of the
extracellular protein sequence of BAFF [SEQ. ID. NO.: 3] and some
members of the TNF ligand family [SEQ. ID. NO.: 4 (hAPRIL); SEQ.
ID. NO.: 5 (hTNF alpha); SEQ. ID. NO.: 6 (hFasL); SEQ. ID. NO.: 7
(hLT alpha); SEQ. ID. NO.: 8 (hRANKL)]. Identical and homologous
residues are represented in black and shaded boxes, respectively.
(C) Depicts dendrogram of TNF family ligands.
[0035] FIG. 2 is a schematic characterization of recombinant BAFF
(A) Schematic representation of recombinant BAFF constructs.
Soluble recombinant BAFFs starting at Leu.sub.83 and Gln.sub.136
are expressed fused to a N-terminal Flag tag and a 6 amino acid
linker. The long form is cleaved between Arg.sub.133 and
Ala.sub.134 (arrow) in 293 T cells, to yield a processed form of
BAFF. Asn.sub.124 and Asn.sub.242 belong to N-glycosylation
consensus sites. N-linked glycan present on Asn.sub.124 is shown as
a Y. TMD: transmembrane domain. (B) Peptide N-glycanase F (PNGase
F) treatment of recombinant BAFF. Concentrated supernatants
containing Flag-tagged BAFFs and APRIL were deglycosylated and
analyzed by Western blotting using polyclonal anti-BAFF antibodies
or anti-Flag M2, as indicated. All bands except processed BAFF also
reacted with anti-Flag M2 (data not shown). (C) Full length BAFF is
processed to a soluble form. 293T cells were transiently
transfected with full length BAFF. Transfected cells and their
concentrated supernatants were analyzed by Western blotting using
polyclonal anti-BAFF antibodies. Supernatants corresponding to
10.times. the amount of cells were loaded onto the gel. (D) Size
exclusion chromatography of soluble BAFF on Superdex-200.
Concentrated supernatants containing soluble BAFF/short were
fractionated on a Superdex-200 column and the eluted fractions
analyzed by Western blotting using anti-Flag M2 antibody. The
migration positions of the molecular mass markers (in kDa) are
indicated on the left-hand side for SDS-PAGE and at the top of the
figure for size exclusion chromatography.
[0036] FIG. 3 depicts expression of BAFF (A) Northern blots (2
.mu.g poly A.sup.+ RNA per lane) of various human tissues were
probed with BAFF antisense mRNA. (B) Reverse transcriptase
amplification of BAFF, IL-2 receptor alpha chain and actin from RNA
of purified blood T cells at various time points of PHA activation,
E-rosetting negative blood cells (B cells and monocytes), in vitro
derived immature dendritic cells, 293 cells, and 293 cells
sterilely transfected with full length BAFF (293-BAFF). Control
amplifications were performed in the absence of added cDNA. IL-2
receptor alpha chain was amplified as a marker of T cell
activation.
[0037] FIG. 4 depicts BAFF binding to mature B cells. (A) Binding
of soluble BAFF to BJAB and Jurkat cell lines, and to purified
CD19.sup.+ cells of cord blood. Cells were stained with the
indicated amount (in ng/50 .mu.l) of Flag-BAFF and analyzed by flow
cytometry. (B) Binding of soluble BAFF to PBLS. PBLs were stained
with anti-CD8-FITC or with anti-CD19-FITC (horizontal axis) and
with Flag-BAFF plus M2-biotin and avidin-PE (vertical axis).
Flag-BAFF was omitted in controls.
[0038] FIG. 5 depicts BAFF costimulates B cell proliferation. (A)
Surface expression of BAFF in stably transfected 293 cells.
293-BAFF and 293 wild-type cells were stained with anti-BAFF mAb
43.9 and analyzed by flow cytometry. (B) Costimulation of PBLs by
293-BAFF cells. PBLs (10.sup.5/well) were incubated with 15.000
glutaraldehyde-fixed 293 cells (293 wt or 293-BAFF) in the presence
or absence of anti-B cell receptor antibody (anti-.mu.). Fixed 293
cells alone incorporated 100 cpm. (C) Dose dependent costimulation
of PBL proliferation by soluble BAFF in the presence of anti-.mu..
Proliferation was determined after 72 h incubation by
[.sup.3H]-thymidine incorporation. Controls include cells treated
with BAFF alone, with heat-denatured BAFF or with an irrelevant
isotype matched antibody in place of anti-.mu.. (D) Comparison of
(co)stimulatory effects of sCD40L and sBAFF on PBL proliferation.
Experiment was performed as describe in panel C. (EAFF costimulates
Ig secretion of preactivated human B cells. Purified CD19.sup.+ B
cells were activated by coculture with EL-4 T cells and activated T
cell supernatants for 5-6 d, then re-isolated and cultured for
another 7 days in the presence of medium only (-) or containing 5%
activated T cell supernatants (T-SUP) or a blend of cytokines
(IL-2, IL-4, IL-10). The columns represent means of Ig
concentrations for cultures with or without 1 .mu.g/ml BAFF.
Means.+-.SD in terms of "fold increase" were 1.23.+-.0.11 for
medium only, 2.06.+-.0.18 with T cell supernatants (4 experiments)
and 1.45.+-.0.06 with IL-2, IL-4 and IL-10 (2 experiments). These
were performed with peripheral blood (3 experiments) or cord blood
B cells (one experiment; 2.3 fold increase with T cell
supernatants, 1.5 fold increase with IL-2, IL-4 and IL-10). (F)
Dose-response curve for the effect of BAFF in cultures with T cell
supernatants, as shown in panel D. Mean.+-.SD of 3 experiments.
[0039] FIG. 6 depicts that BAFF acts as a cofactor for B cell
proliferation. The proliferation of human PBL was measured alone
(500 cpm), with the presence of BAFF ligand alone, with the
presence of goat anti-murine (mu) alone, and with both BAFF ligand
and anti-mu. The combination of both anti-mu and BAFF significantly
raised proliferation of PBL as the concentration of BAFF increased
suggesting BAFF's cofactor characteristics.
[0040] FIG. 7 depicts increased B cell numbers in BAFF Tg mice.
[0041] (A) Increased lymphocytes counts in BAFF Tg mice. The graph
compares 12 control littermates (left panel) with 12 BAFF Tg mice
(right panel). Lymphocytes counts are shown with circles and
granulocytes (including neutrophils, eosinophils, basophils) with
diamonds.
[0042] (B) Increased proportion of B cells in PBL from BAFF Tg
mice. PBL were stained with both anti-B220-FITC and anti-CD4-PE for
FACS analysis and gated on live cells using the forward side
scatter. Percentages of CD4 and B220 positive cells are indicated.
One control mouse (left) and two BAFF Tg mice (right) are shown and
the results were representative of 7 animals analysed in each
group.
[0043] (C) FACS analysis of the ratio of B to T cells in PBL. The
difference between control animals and BAFF Tg mice in (A) and (C)
was statistically significant (P<0.001).
[0044] (D) Increased MHC class II expression on B cells from BAFF
Tg mice PBL. MHC class II expression was analysed by FACS.
[0045] (E) Increased Bcl-2 expression in B cells from BAFF Tg mice
PBL. Bcl-2 expression was measured by intracytoplasmic staining and
cells were analysed by FACS. In both (D) and (E) Live cells were
gated on the forward side scatter. Four control littermates (white
bars) and 4 BAFF Tg mice are shown and are representative of at
least 12 animals analysed for each group. MFI: mean of fluorescence
intensity. The difference between control animals and BAFF Tg mice
was statistically significant (P<0.005).
[0046] (F) Increased expression of effector T cells in BAFF Tg
mice. PBL were stained with anti-CD4-Cychrome, anti-CD44-FITC and
anti-L selectin-PE. Are shown CD4.sup.+-gated cells. Percentages of
CD44.sup.hi/L-selectin.sup.lo cells are indicated. One control
mouse (left) and two BAFF Tg mice (right) are shown and the results
were representative of 8 animals analysed in each group.
[0047] FIG. 8 depicts increased B cell compartments in the spleen
but not in the bone marrow of BAFF Tg mice.
[0048] (A) FACS staining for mature B cells using both
anti-IgM-FITC and anti-B220-PE, in spleen (top panel), bone marrow
(medium panel) and MLN (bottom panel). Percentages of
B220+/IgM+mature B cells are indicated.
[0049] (B) FACS staining for preB cells (B220+/CD43-) and proB
cells (B220+/CD43+) in the bone marrow using anti-CD43-FITC,
anti-B220-Cy-chrome and anti-IgM-PE simultaneously. Are shown cells
gated on the IgM negative population. Percentages of preB cells
(B220+/CD43-) and proB cells (B220+/CD43+) cells are indicated.
[0050] For all figures (A and B) one control mouse (left) and two
BAFF Tg mice (right) are shown and results are representative of 7
animals analysed for each group.
[0051] FIG. 9 depicts increased Ig, RF and CIC levels in BAFF Tg
mice
[0052] (A) SDS-PAGE of two control sera (-) and 4 sera from BAFF Tg
mice (+) side by side with the indicated amount of a purified mouse
IgG for reference. The intensity of the albumin band in similar in
all lanes indicating that the material loaded on the gel is
equivalent for each sample. ELISA-based analysis of total mouse Ig
(B), RF (C) and CIC (D) in the sera of 19 control littermates
(white bars) and 21 BAFF Tg mice (Black bars). In the absence of a
proper RF control, the titer (log base 2) for RF is defined as the
dilution of the sera giving an O.D. 3 times higher than that of
background. The quantity of CIC is defined as the quantity of PAP
required to generate an O.D. equivalent to that obtained with the
tested serum. The difference between control animals and BAFF Tg
mice was statistically significant (P<0.001 in (B) and (C),
P<0.003 in (D)).
[0053] FIG. 10 depicts the presence of anti-ssDNA and anti-dsDNA
autoantibodies in some BAFF Tg mice.
[0054] (A) Analysis by ELISA of anti-ssDNA autoantibodies in 19
control littermates (gray bars) and 21 BAFF Tg mice (black
bars).
[0055] (B) Analysis by ELISA of anti-ssDNA autoantibodies in 5
control littermates and the 5 animals showing levels of anti-ssDNA
autoantibodies from (A).
[0056] (C) Paraffin sections of kidneys from a control mouse (left)
and a BAFF Tg mouse (right), stained with goat anti-mouse Ig-HRP.
Ig deposition is shown by a brown staining. These pictures are
representative of 6 BAFF Tg mice analysed.
[0057] FIG. 11 depicts enlarged Peyer's patches in BAFF Tg mice.
Photography of Peyers patches (indicated with an arrow) on the
small intestine of a control mouse (left) and a BAFF Tg mouse
(right). This pictures is representative of at least 12 mice
sacrificed for each group. Magnification 5.times.
[0058] FIG. 12 depicts disrupted T and B cell organization, intense
germinal center reactions, decreased number of dendritic cells and
increased number of plasma cells in the spleen of BAFF Tg mice.
[0059] A control mouse is shown in A, C, E and G and a BAFF Tg in
B, D, F, and H. B cells are blue and T cells brown (A and B).
Germinal centers are shown with an arrow (C and D). Only few
residual germinal centers are seen in control mice (C). CD11c
positive dendritic cells are brown and appear in the T cell zone,
bridging channels and the marginal zone (E). Very few are present
in BAFF Tg mice (F). Syndecan-1-positive plasma cells were only
detectable in the red pulp of BAFF Tg mice (H) but not control mice
(G).
[0060] These pictures are representative of at least 12 BAFF Tg
mice analysed and 12 control mice. The magnification is 100.times.
for all pictures except C and D which are 50.times.. B: B cell
follicle, T: PALS, WP: white pulp, RP: red pulp.
[0061] FIG. 13 depicts disrupted T and B cells organization,
intense germinal center reactions and large number of plasma cells
in the MLN of BAFF Tg mice.
[0062] The control mouse is shown in A, C, E and G and the BAFF Tg
mouse is shown in B, D, F, and H. The immunohistochemistry was
performed as described in FIG. 6. T and B cell staining is shown in
A and B, germinal centers in C and D, dendritic cells E and F and
plasma cells in G and H. GC: germinal center. Magnification
100.times..
[0063] FIG. 14 depicts enlarged and inflamed salivary glands BAFF
Tg mice. (A) Mice 15-17 months old, 4 control littermates (white
bar) and 4 BAFF Tg mice (grey bar) were sacrificed the same day for
organ collection. Both right and left submaxillary glands were
collected and weighted. The data shows the mean t standard
deviation of weight for both glands. These results are
representative of at least 4 separate groups of age-matched animals
dissected. (B) Paraffin sections of submaxillary glands from a
control littermate (left panel) and two BAFF Tg mice (middle and
right panel) were stained with H&E. Arrows indicate ducts and
acinar cells in the left and right panels. The arrow in the middie
panel is showing acinar destruction. White stars indicate
periductal infiltrates (foci). Magnification 100.times.. (C)
Paraffin sections of submaxillary glands from 7 control mice and 22
BAFF Tg mice (age 12-17 months) were prepared as shown in (B) and
scored for the disease as described in materials and methods. The
mean score of disease is indicated with a bar. *p<0.05,
**p<0.03.
[0064] FIG. 15 depicts tumor-like clusters of B-lymphoid cells in a
submaxillary tumor or infiltrates present in submaxillary glands of
BAFF Tg mice. Frozen sections of submaxillary glands (A and C) or a
submaxillary tumor (B, D and E) were stained with H&E (A, B and
C) or Biotin-labelled anti-mouse B220 (D) or anti-mouse syndecan-1
(E), follwed by Streptavidin-HRP brown staining in D and E). Arrows
in A, B, C and D indicate the tumor-like clusters, arrow in E shows
the presence of syndecan-1 positive plasma cells whereas the stars
indicate the location of tumor-like clusters. Note that tumor-like
clusters and plasma cells do not colocalize. Panels (A) and (C) are
representative of 4 animals in which these clusters were detected
as cells infiltrating submaxillary glands. Panels (B), (D) and (E)
are representative of 3 submaxillary tumors analysed. Magnification
is indicated for each panel. A germinal center (GC) is also
indicated in panel (B).
[0065] FIG. 16 depicts identification of a Mz-like B cell
population infiltrating salivary glands of BAFF Tg mice.
Submaxillary glands from 13-17 months old BAFF Tg mice and
age-matched control littermates were digested with collagenase for
purification of infiltrating lymphocytes as described in materials
and methods. Using four-color flow cytometry analysis, cells were
stained with anti-B220, anti-CD5, anti-IgM and anti-CD43. (A) shows
the gating on B220.sup.hi and B220.sup.lo/int B cells on control
(left) and BAFF Tg mice (right) as indicated. (B) shows four dot
plots for expression of IgM and CD5 on B220.sup.hi cells (left) and
B220.sup.lo/int (right) from BAFF Tg mice and control mice as
indicated. The IgM.sup.hi B cell gate on B220.sup.hi cells (left)
and the B-1a and B-1b gates on B220.sup.lo/int cells are shown. (C)
shows histograms for the expression of CD43 on gated
IgM.sup.hi/B220.sup.hi cells (left) and gated B-1a and B-1b cells
(right) from BAFF Tg mice as indicated. (D) Lymphocytes prepared
from submaxillary glands (left panel) and inguinal lymph nodes
(right panel) of a BAFF Tg mouse were stained with anti-B220,
anti-IgM and anti-L-selectin, using a four-color flow cytometry
procedure. Cells from submaxillary glands were gated on
B220.sup.hi/IgM.sup.hi (left panel) and on IgM+(right panel). A
histogram of L-selectin expression is shown in each panel. (E)
Lymphocytes from a BAFF Tg mice (right) and a control littermate
(left) were prepared as in A and stained with anti-B220, anti-IgM
and either anti-IgD, anti-CD23, anti-CD21, anti-CD1, anti-HSA,
using a three color flow cytometry procedure. Cells were gated on
B220.sup.hi and IgM.sup.hi cells as indicated on the top histograms
and expression of IgD, CD23, CD1, CD21 and HSA on these gated cells
is shown on histograms for control (left) and Tg mice (right) as
indicated. In (C), (D) and (E), mean of fluorescence intensity
(MFI) and a bar delineating the area in which the negative control
histogram is located are indicated in each histogram. (F) Schematic
representation of the common and distinct markers expressed on MZ
(right circle), B-1 (left circle) and T1 B cells (lower circle).
The phenotype of the B cells infiltrating submaxillary glands of
BAFF Tg mice is indicated by an oval with a dotted line. These
results are representative of at least 12 BAFF Tg and 7 control
mice analysed.
[0066] FIG. 17 depicts decreased saliva flow in older BAFF Tg mice.
13 BAFF Tg mice (diamonds) and 14 control littermates (circles)
were injected with pilocarpine prior to saliva collection as
described in materials and methods. Mice 13-15.5 months old are
shown in (A) and mice 8-10 months old are shown in (B). Means of
saliva flow are shown with a bar and p values are indicated in each
panel.
[0067] FIG. 18 depicts elevated levels of BAFF in sera and salivary
gland tissues from patients suffering from primary SS but lack of
correlation of these levels with levels of total IgG, RF and
presence of anti-Ro/La autoantibodies. (A) individual BAFF levels
in 39 healthy controls (square), 41 patients with primary SS
(diamond), 53 patients with SLE (circle) and 53 patients with RA
(triangle) were measured in sera by ELISA and BAFF levels plotted
on a log scale. Controls were drawn from normal healthy donors. The
horizontal black bars indicates the average for each group: normal
10.4.+-.13 (ng/ml), SS patients 53.+-.67 (ng/ml), SLE patients
12.7.+-.24.4 (ng/ml) and RA patients 23.+-.47 (ng/ml). Some
individuals did not have detectable amount of BAFF in their serum
and do not appear on the log scale, this includes 16 normals, 6 SS,
20 SLE and 10 RA patients. The dotted line delineates the range of
normal BAFF levels. *p<0.04, the p value was determined by ANOVA
t test. (B) and (C) show the correlation of BAFF levels in the sera
of patients with SS with the corresponding levels of IgG (B) and RF
(C) in each serum. r and p values calculated by ANOVA are shown.
(D) shows the levels of BAFF in patients with detected anti-Ro and
anti-La (left), anti-Ro only (middle) or no precipitin detected
(right). (E) Paraffin sections of a human labial salivary gland
biopsy from a patient with SS were stained with a rat anti-human
BAFF antibody (right) or an isotype-matched control antibody
(left). Staining of normal human labial salivary gland with rat
anti-human BAFF antibody is also shown (bottom left, magnification
100.times.). The staining was revealed using biotin-labelled
anti-rat Ig followed by Streptavidin-HRP. The staining appears
brown on the sections. Magnification 200.times.. These pictures are
representative of 4 patients with primary SS and 3 control tissues
analysed.
DETAILED DESCRIPTION OF THE INVENTION
[0068] Reference will now be made in detail to the present
preferred embodiments of the invention. This invention relates to
the use of BAFF and BAFF related molecules to effect the growth and
maturation of B-cells and the secretion of immunoglobulin. The
invention relates to the use of BAFF and BAFF related molecules to
effect responses of the immune system, as necessitated by
immune-related disorders. Additionally, this invention encompasses
the treatment of cancer and immune disorders through the use of a
BAFF, BAFF blocking agents, or BAFF related gene through gene
therapy methods.
[0069] The BAFF ligand and homologs thereof produced by hosts
transformed with the sequences of the invention, as well as native
BAFF purified by the processes known in the art, or produced from
known amino acid sequences, are useful in a variety of methods for
anticancer, antitumor and immunoregulatory applications. They are
also useful in therapy and methods directed to other diseases.
[0070] "BAFF blocking agents" refers to agents that can diminish
BAFF ligand binding to BAFF receptors, or can diminish BAFF
receptor signalling, or that can influence how the BAFF receptor
signal is interpreted within the cell.
[0071] A BAFF blocking agent that acts by diminishing
ligand-receptor binding can inhibit BAFF ligand binding by at least
20%. Examples of BAFF blocking agents include soluble BAFF
receptor-Fc molecules, anti-BAFF ligand antibodies and anti-BAFF
receptor antibodies.
[0072] "BAFF receptors" have been identified and characterized and
include TACI (see, e.g., U.S. Pat. No. 5,969,102 and WO98/39361,
incorporated herein by reference), BCMA (see, e.g., WO01/12812,
incorporated herein by reference), and BAFFR (see, e.g., Thompson
et al. (2001) Science 293:2108, incorporated herein by
reference).
[0073] Another aspect of the invention relates to the use of the
polypeptide encoded by the isolated nucleic acid encoding the
BAFF-ligand in "antisense" therapy. As used herein, "antisense"
therapy refers to administration or in situ generation of
oligonucleotides or their derivatives which specifically hybridize
under cellular conditions with the cellular mRNA and/or DNA
encoding the ligand of interest, so as to inhibit expression of the
encoded protein, i.e. by inhibiting transcription and/or
translation. The binding may be by conventional base pair
complementarity, or, for example, in the case of binding to DNA
duplexes, through specific interactions in the major groove of the
double helix. In general, "antisense" therapy refers to a range of
techniques generally employed in the art, and includes any therapy
which relies on specific binding to oligonucleotide sequences.
[0074] An antisense construct of the present invention can be
delivered, for example, as an expression plasmid, which, when
transcribed in the cell, produces RNA which is complementary to at
least a portion of the cellular mRNA which encodes Kay-ligand.
Alternatively, the antisense construct can be an oligonucleotide
probe which is generated ex vivo. Such oligonucleotide probes are
preferably modified oligonucleotides which are resistant to
endogenous nucleases, and are therefor stable in vivo. Exemplary
nucleic acids molecules for use as antisense oligonucleotides are
phosphoramidates, phosphothioate andmethylphosphonate analogs of
DNA (See, e.g., U.S. Pat. No. 5,176,996; U.S. Pat. No. 5,264,564;
and U.S. Pat. No. 5,256,775). Additionally, general approaches to
constructing oligomers useful in antisense therapy have been
reviewed, for example, by Van Der Krol et al., (1988) Biotechniues
6:958-976; and Stein et al. (1988) Cancer Res 48: 2659-2668,
specifically incorporated herein by reference.
[0075] A. BAFF-LIGAND
[0076] The BAFF-ligand of the invention, as discussed above, is a
member of the TNF family and is described in PCT application number
PCT/US98/19037 (WO99/12964) and is incorporated in its entirety
herein. The protein, fragments or homologs thereof may have wide
therapeutic and diagnostic applications.
[0077] The BAFF-ligand is present primarily in the spleen and in
peripheral blood lymphocytes, strongly indicating a regulatory role
in the immune system. Comparison of the claimed BAFF-ligand
sequences with other members of the human TNF family reveals
considerable structural similarity. All the proteins share several
regions of sequence conservation in the extracellular domain.
[0078] Although the precise three-dimensional structure of the
claimed ligand is not known, it is predicted that, as a member of
the TNF family, it may share certain structural characteristics
with other members of the family.
[0079] The novel polypeptides of the invention specifically
interact with a receptor, which has not yet been identified.
However, the peptides and methods disclosed herein enable the
identification of receptors which specifically interact with the
BAFF-ligand or fragments thereof.
[0080] The claimed invention in certain embodiments includes
methods of using peptides derived from BAFF-ligand which have the
ability to bind to their receptors. Fragments of the BAFF-ligands
can be produced in several ways, e.g., recombinantly, by PCR,
proteolytic digestion or by chemical synthesis. Internal or
terminal fragments of a polypeptide can be generated by removing
one or more nucleotides from one end or both ends of a nucleic acid
which encodes the polypeptide. Expression of the mutagenized DNA
produces polypeptide fragments.
[0081] Polypeptide fragments can also be chemically synthesized
using techniques known in the art such as conventional Merrifield
solid phase f-moc or t-boc chemistry. For example, peptides and DNA
sequences of the present invention may be arbitrarily divided into
fragments of desired length with no overlap of the fragment, or
divided into overlapping fragments of a desired length. Methods
such as these are described in more detail below.
[0082] B. Generation of Soluble Forms of BAFF-Ligand and
BAFF-Receptors
[0083] Soluble forms of the BAFF-ligand can often signal
effectively and hence can be administered as a drug which now
mimics the natural membrane form. It is possible that the
BAFF-ligand claimed herein are naturally secreted as soluble
cytokines, however, if not, one can reengineer the gene to force
secretion. To create a soluble secreted form of BAFF-ligand or BAFF
receptor, one would remove at the DNA level the N-terminus
transmembrane regions, and some portion of the stalk region, and
replace them with a type I leader or alternatively a type II leader
sequence that will allow efficient proteolytic cleavage in the
chosen expression system. A skilled artisan could vary the amount
of the stalk region retained in the secretion expression construct
to optimize both receptor binding properties and secretion
efficiency. For example, the constructs containing all possible
stalk lengths, i.e. N-terminal truncations, could be prepared such
that proteins starting at amino acids 81 to 139 would result. The
optimal length stalk sequence would result from this type of
analysis.
[0084] Soluble forms of BAFF receptors can be prepared using
techniques well-known to those of ordinary skill in the art.
Immunoglobulin Fc portions may be fused to the BAFF receptor
protein to increase the half-life of the soluble BAFF receptor.
Production of such soluble BAFF receptor proteins is described, for
example, in WO 01/12812, WO 01/24811, and PCT/US01/40626, the
entire disclosures of which are incorporated herein by
reference.
[0085] C. Generation of Antibodies Reactive with the BAFF-Ligand
and BAFF Receptors
[0086] The invention also includes antibodies specifically reactive
with the claimed BAFF-ligand or its receptors.
Anti-protein/anti-peptide antisera or monoclonal antibodies can be
made by standard protocols (See, for example, Antibodies: A
Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press:
1988)). A mammal such as a mouse, a hamster or rabbit can be
immunized with an immunogenic form of the peptide. Techniques for
conferring immunogenicity on a protein or peptide include
conjugation to carriers, or other techniques, well known in the
art.
[0087] An immunogenic portion of BAFF-ligand or its receptors can
be administered in the presence of an adjuvant. The progress of
immunization can be monitored by detection of antibody titers in
plasma or serum. Standard ELISA or other immunoassays can be used
with the immunogen as antigen to assess the levels of
antibodies.
[0088] In a preferred embodiment, the subject antibodies are
immunospecific for antigenic determinants of BAFF-ligand or its
receptors, (e.g. antigenic determinants of a polypeptide of SEQ.
ID. NO.: 2, said sequence as described in PCT application number
PCTI/US98/19037 (WO99/12964) and is incorporated in its entirety
herewith), or a closely related human or non-human mammalian
homolog (e.g. 70, 80 or 90 percent homologous, more preferably at
least 95 percent homologous). In yet a further preferred embodiment
of the present invention, the anti-BAFF-ligand or
anti-BAFF-ligand-receptor antibodies do not substantially cross
react (i.e. react specifically) with a protein which is e.g., less
than 80 percent homologous to SEQ. ID. NO.: 2 or 6 said sequence as
described in PCT application number PCT/US98/19037 (WO99/12964) and
is incorporated in its entirety herewith; preferably less than 90
percent homologous with SEQ. ID. NO.: 2 said sequence as described
in PCT application number PCT/US98/19037 (WO99/12964) and is
incorporated in its entirety herewith; and, most preferably less
than 95 percent homologous with SEQ. ID. NO.: 2 said sequence as
described in PCT application number PCT/US98/19037 (WO99/12964) and
is incorporated in its entirety herewith. By "not substantially
cross react", it is meant that the antibody has a binding affinity
for a non-homologous protein which is less than 10 percent, more
preferably less than 5 percent, and even more preferably less than
1 percent, of the binding affinity for a protein of SEQ. ID. NO.: 2
said sequence as described in PCT application number PCT/US98/19037
(WO99/12964) and is incorporated in its entirety herewith.
[0089] Production of exemplary anti-BAFF receptor antibodies is
described, for example, in WO 01/12812, WO 01/24811 and
PCT/US01/40626, the entire disclosures of which are incorporated
herein by reference.
[0090] The term antibody as used herein is intended to include
fragments thereof which are also specifically reactive with
BAFF-ligand, or its receptors. Antibodies can be fragmented using
conventional techniques and the fragments screened for utility in
the same manner as described above for whole antibodies. For
example, F(ab').sub.2 fragments can be generated by treating
antibody with pepsin. The resulting F(ab').sub.2 fragment can be
treated to reduce disulfide bridges to produce Fab' fragments. The
antibodies of the present invention are further intended to include
biospecific and chimeric molecules having anti-BAFF-ligand or
anti-BAFF-ligand-receptor activity. Thus, both monoclonal and
polyclonal antibodies (Ab) directed against BAFF-ligand,
Tumor-ligand and their receptors, and antibody fragments such as
Fab' and F(ab').sub.2, can be used to block the action of the
Ligand and their respective receptor.
[0091] Various forms of antibodies can also be made using standard
recombinant DNA techniques. Winter and Milstein (1991) Nature 349:
293-299, specifically incorporated by reference herein. For
example, chimeric antibodies can be constructed in which the
antigen binding domain from an animal antibody is linked to a human
constant domain (e.g. Cabilly et al., U.S. Pat. No. 4,816,567,
incorporated herein by reference). Chimeric antibodies may reduce
the observed immunogenic responses elicited by animal antibodies
when used in human clinical treatments.
[0092] In addition, recombinant "humanized antibodies" which
recognize BAFF-ligand or its receptors can be synthesized.
Humanized antibodies are chimeras comprising mostly human IgG
sequences into which the regions responsible for specific
antigen-binding have been inserted. Animals are immunized with the
desired antigen, the corresponding antibodies are isolated, and the
portion of the variable region sequences responsible for specific
antigen binding are removed. The animal-derived antigen binding
regions are then cloned into the appropriate position of human
antibody genes in which the antigen binding regions have been
deleted. Humanized antibodies minimize the use of heterologous
(i.e. inter species) sequences in human antibodies, and thus are
less likely to elicit immune responses in the treated subject.
[0093] Construction of different classes of recombinant antibodies
can also be accomplished by making chimeric or humanized antibodies
comprising variable domains and human constant domains (CH1, CH2,
CH3) isolated from different classes of immunoglobulins. For
example, antibodies with increased antigen binding site valencies
can be recombinantly produced by cloning the antigen binding site
into vectors carrying the human: chain constant regions.
Arulanandam et al. (1993) J. Exp. Med., 177: 1439-1450,
incorporated herein by reference.
[0094] In addition, standard recombinant DNA techniques can be used
to alter the binding affinities of recombinant antibodies with
their antigens by altering amino acid residues in the vicinity of
the antigen binding sites. The antigen binding affinity of a
humanized antibody can be increased by mutagenesis based on
molecular modeling. Queen et al., (1989) Proc. Natl. Acad. Sci. 86:
10029-33 incorporated herein by reference.
[0095] D. Generation of Analogs: Production of Altered DNA and
Peptide Sequences
[0096] Analogs of the BAFF-ligand can differ from the naturally
occurring BAFF-ligand in amino acid sequence, or in ways that do
not involve sequence, or both. Non-sequence modifications include
in vivo or in vitro chemical derivatization of the BAFF-ligand.
Non-sequence modifications include, but are not limited to, changes
in acetylation, methylation, phosphorylation, carboxylation or
glycosylation.
[0097] Preferred analogs include BAFF-ligand biologically active
fragments thereof, whose sequences differ from the sequence given
in SEQ. ID NO. 2 said sequence as described in PCT application
number PCT/US98/19037 (WO99/12964) and is incorporated in its
entirety herewith, by one or more conservative amino acid
substitutions, or by one or more non-conservative amino acid
substitutions, deletions or insertions which do not abolish the
activity of BAFF-ligand. Conservative substitutions typically
include the substitution of one amino acid for another with similar
characteristics, e.g. substitutions within the following groups:
valine, glycine; glycine, alanine; valine, isoleucine, leucine;
aspartic acid, glutamic acid; asparagine, glutamine; serine,
threonine; lysine, arginine; and, phenylalanine, tyrosine.
[0098] E. Materials and Methods of the Invention
[0099] The anti-Flag M2 monoclonal antibody, biotinylated anti-Flag
M2 antibody and the anti-Flag M2 antibody coupled to agarose were
purchased from Sigma. Cell culture reagents were obtained from Life
Sciences (Basel, Switzerland) and Biowhittaker (Walkersville, Md.).
Flag-tagged soluble human APRIL (residues K.sub.110-L.sub.250) was
produced in 293 cells as described (10, 11). FITC-labeled anti-CD4,
anti-CD8 and anti-CD19 antibodies were purchased from Pharmingen
(San Diego, Calif.). Goat F(ab).sub.2 specific for the Fc.sub.5,
fragment of human IgM were purchased from Jackson ImmunoResearch
(West Grove, Pa.). Secondary antibodies were obtained from either
Pharmingen or from Jackson ImmunoResearch and used at the
recommended dilutions.
[0100] Human embryonic kidney 293 T (12) cells and fibroblast cell
lines (Table 1) were maintained in DMEM containing 10%
heat-inactivated fetal calf serum (FCS). Human embryonic kidney 293
cells were maintained in DMEM-nutrient mix F12 (1:1) supplemented
with 2% FCS. T cell lines, B cell lines, and macrophage cell lines
(Table 1) were grown in RPMI supplemented with 10% FCS. Molt-4
cells were cultiv-atedin Iscove's medium-supplemented with 10% FCS.
Epithelial cell lines were grown in MEM-alpha medium containing 10%
FCS, 0.5 mM non-essential amino acids, 10 mM Na-Hepes and 1 mM Na
pyruvate. HUVECs were maintained in M199 medium supplemented with
20% FCS, 100 .mu.g/ml of epithelial cell growth factor
(Collaborative Research, Inotech, Dottikon, Switzerland) and 100
.mu.g/ml of heparin sodium salt (Sigma). All media contained
penicillin and streptomycin antibiotics. Peripheral blood
leukocytes were isolated from heparinized blood of healthy adult
volunteers by Ficoll-Paque (Pharmacia, Uppsala, Sweden) gradient
centrifugation and cultured in RPMI, 10% FCS.
[0101] T cells were obtained from non-adherents PBLs by rosetting
with neuraminidase-treated sheep red blood cells and separated from
non-rosetting cells (mostly B cells and monocytes) by Ficoll-Paque
gradient centrifugation. Purified T cells were activated for 24 h
with phytohemagglutinin (Sigma) (1 .mu.g/ml), washed and cultured
in RPMI, 10% FCS, 20 U/ml of IL 2. CD14.sup.+ monocytes were
purified by magnetic cell sorting using anti-CD 14 antibodies, goat
anti-mouse-coated microbeads and a Minimacs.TM. device (Miltenyi
Biotech), and cultivated in the presence of GM-CSF (800 U/ml,
Leucomax.RTM., Essex Chemie, Luzern, Switzerland) and IL-4 (20
ng/ml, Lucerna Chem, Luzern, Switzerland) for 5 d, then with
GM-CSF, IL4 and TNF.alpha.(200 U/ml, Bender, Vienna, Austria) for
an additional 3 d to obtain a CD83.sup.+, dentritic cell-like
population. Human B cells of >97% purity were isolated from
peripheral blood or umbilical cord blood using anti-CD19 magnetic
beads (M450, Dynal, Oslo, Norway) as described (13).
[0102] Northern Blot Analysis
[0103] Northern blot analysis was carried out using Human Multiple
Tissue Northern Blots I and II (Clontech #7760-1 and #7759-1). The
membranes were incubated in hybridization solution (50% formamide,
2.5.times. Denhardt's, 0.2% SDS, 10 mM EDTA, 2.times.SSC, 50 mM
NaH.sub.2PO.sub.4, pH 6.5, 200 .mu.g/ml sonicated salmon sperm DNA)
for 2 h at 60.degree. C. Antisense RNA probe containing the
nucleotides corresponding to amino acids 136-285 of hBAFF was
heat-denatured and added at 2.times.10.sup.6 cpm/ml in fresh
hybridization solution. The membrane was hybridized 16 h at
62.degree. C., washed once in 2.times.SSC, 0.05% SDS (30 min at
25.degree. C.), once in 0.1.times.SSC, 0.1% SDS (20 min at
65.degree. C.) and exposed at -70.degree. C. to X-ray films.
[0104] Characterization of BAFF cDNA.
[0105] A partial sequence of human BAFF cDNA was contained in
several EST clones (e.g. GenBank Accession numbers T87299 and
AA166695) derived from fetal liver and spleen and ovarian cancer
libraries. The 5' portion of the cDNA was obtained by 5'-RACE-PCR
(Marathon-Ready cDNA, Clonetech, Palo Alto, Calif.) amplification
with oligonucleotides AP1 and JT1013
(5'-ACTGTTTCTTCTGGACCCTGAACGGC-3') [SEQ ID. NO.: 9] using the
provided cDNA library from a pool of human leukocytes as template,
as recommended by the manufacturer. The resulting PCR product was
cloned into PCR-0 blunt (Invitrogen, Nev. Leek, The Netherlands)
and subcloned as EcoRI/PstI fragment into pT7T3 Pac vector
(Pharmacia) containing EST clone T87299. Full-length hBAFF cDNA was
therefore obtained by combining 5' and 3' fragments using the
internal PstI site of BAFF. Sequence has been assigned GenBank
accession number AF116456.
[0106] A partial 617 bp sequence of murine BAFF was contained in
two overlapping EST clones (AA422749 and AA254047). A PCR fragment
spanning nucleotides 158 to 391 of this sequence was used as a
probe to screen a mouse spleen cDNA library (Stratagene, La Jolla,
Calif.).
[0107] Expression of Recombinant BAFF
[0108] Full length hBAFF was amplified using oligos JT1069
(5'-GACAAGCTTGCCACCATGGATGACTCCACA-3') [SEQ. ID. NO.: 10] and JT637
(5'-ACTAGTCACAGCAGTITCAATGC-3) [SEQ. ID. NO.: 11]. The PCR product
was cloned into PCR-0 blunt and re-subcloned as HindIII/EcoRI
fragment into PCR-3 mammalian expression vector. A short version of
soluble BAFF (amino acids Q136-L285) was amplified using oligos
JT636 (5'-CTGCAGGGTCCAGAAGAAA- CAG-3) [SEQ. ID. NO.: 12] and JT637.
A long version of soluble BAFF (aa L83-L285) was obtained from full
length BAFF using internal PstI site. Soluble BAFFs were
resubcloned as PstI/EcoRI fragments behind the haemaglutinin signal
peptide and Flag sequence of a modified PCR-3 vector, and as
PstI/SpeI fragments into a modified pQE16 bacterial expression
vector in frame with a N-terminal Flag sequence (14). Constructs
were sequenced on both strands. The establishment of stable 293
cell lines expressing the short soluble form or full length BAFF,
and the expression and purification of recombinant soluble BAFF
from bacteria and mammalian 293 cells was performed as described
(14, 15).
[0109] Reverse Transcriptase PCR
[0110] Total RNA extracted from T cells, B cells, in vitro derived
immature dendritic cells, 293 wt and 293-BAFF (full length) cells
was reverse transcribed using the Ready to Go system (Pharmacia)
according to the manufacturer's instructions. BAFF and .beta.-actin
cDNAs were detected by PCR amplification with Taq DNA polymerase
(steps of 1 min each at 94.degree. C., 55.degree. C. and 72.degree.
C. for 30 cycles) using specific oligonucleotides: for BAFF, JT1322
5'-GGAGAAGGCAACTCCAGTCA- GAAC-3' [SEQ. ID. NO.: 13] and JT1323
5'-CAATTCATCCCCAAAGACATGGAC-3' [SEQ. ID. NO.: 14]; for IL-2
receptor alpha chain, JT1368 5'-TCGGAACACAACGAAACAAGTC-3' [SEQ. ID.
NO.: 15] and JT1369 5'-CTTCTCCTTCACCTGGAAACTGACTG-3' [SEQ. ID NO.:
16]; for .beta.-actin, 5'-GGCATCGTGATGGACTCCG-3' [SEQ. ID. NO.: 17]
and 5'-GCTGGAAGGTGGACAGCGA-3- ' [SEQ. ID. NO.: 18].
[0111] Gel Permeation Chromatography
[0112] 293T cells were transiently transfected with the short form
of soluble BAFF and grown in serum-free Optimem medium for 7 d.
Conditionned supernatants were concentrated 20.times., mixed with
internal standards catalase and ovalbumin, and loaded onto a
Superdex-200 HR10/30 column. Proteins were eluted in PBS at 0.5
ml/min and fractions (0.25 ml) were precipitated with
trichloroacetic acid and analyzed by Western blotting using
anti-Flag M2 antibody. The column was calibrated with standard
proteins: ferritin (440 kDa), catalase (232 kDa), aldolase (158
kDa), bovine serum albumine (67 kDa), ovalbumine (43 kDa),
chymotrypsinogen A (25 kDa) and ribonuclease A (13.7 kDa).
[0113] PNGase F Treatment
[0114] Samples were heated in 20 .mu.l of 0.5% SDS, 1%
2-mercaptoethanol for 3 min at 95.degree. C., then cooled and
supplemented with 10% Nonidet P40 (2 .mu.l), 0.5 M sodium
phosphate, pH 7.5 (2 .mu.l) and Peptide N-glycanase F (125
units/.mu.l, 1 .mu.l, or no enzyme in controls). Samples were
incubated for 3 h at 37.degree. C. prior to analysis by Western
blotting.
[0115] EDMAN Sequencing
[0116] 293 T cells were transiently transfected with the long form
of soluble BAFF and grown in serum-free Optimem medium for 7 d.
Conditioned supernatants were concentrated 20.times., fractionated
by SDS-PAGE and blotted onto polyvinylidene difluoride membrane
(BioRad Labs, Hercules, Calif.) as previously described (16), and
then sequenced using a gas phase sequencer (ABI 120A, Perkin Elmer,
Foster City, Calif.) coupled to an analyzer (ABI 120A, Perkin
Elmer) equipped with a phenylthiohydantoin C18 2.1.times.250 mm
column. Data was analyzed using software ABI 610 (Perkin.
Elmer).
[0117] Antibodies
[0118] Polyclonal antibodies were generated by immunizing rabbits
(Eurogentec, Seraing, Belgium) with recombinant soluble BAFF.
Spleen of rats immunized with the same antigen were fused to
x63Ag8.653 mouse myeloma cells, and hybridoma were screened for
BAFF-specific IgGs. One of these monoclonal antibodies, 43.9, is an
IgG2a that specifically recognizes hBAFF.
[0119] Cells were stained in 50 .mu.l of FACS buffer (PBS, 10% FCS,
0.02% NaN.sub.3) with 50 ng (or the indicated amount) of Flag
tagged short soluble HBAFF for 20 min at 4.degree. C., followed by
anti-Flag M2 (1 .mu.g) and secondary antibody. Anti-BAFF mAb 43.9
was used at 40 .mu.g/ml. For two color FACS analysis, peripheral
blood lymphocytes were stained with Flag tagged soluble BAFF/long
(2 .mu.g/ml), followed by biotinylated anti-Flag M2 (1/400) and
PE-labeled streptavidin (1/100), followed by either FITC-labeled
anti-CD4, anti-CD8 or anti-CD19.
[0120] PBL Proliferation Assay
[0121] Peripheral blood leukocytes were incubated in 96-well plates
(10.sup.5 cells/well in 100 .mu.l RPMI supplemented with 10% FCS)
for 72 h in the presence or absence of 2 .mu.g/ml of goat
anti-human p chain antibody (Sigma) or control F(ab').sub.2 and
with the indicated concentration of native or boiled soluble
BAFF/long. Cells were pulsed for an additional 6 h with
[.sup.3H]thymidine (1 .mu.Ci/well) and harvested.
[.sup.3H]thymidine incorporation was monitored by liquid
scintillation counting. In some experiments, recombinant soluble
BAFF was replaced by 293 cells stably transfected with full length
BAFF (or 293 wt as control) that had been fixed for 5 min at
25.degree. C. in 1% paraformaldeyde. Assay was performed as
described (17). In further experiments, CD19.sup.+ cells were
isolated form PBL with magnetic beads and the remaining CD19.sup.-
cells were irradiated (3000 rads) prior to renconstitution with
CD19+cells. Proliferation assay with sBAFF was then performed as
described above.
[0122] B cell Activation Assay
[0123] Purified B cells were activated in the EL4 culture system as
described (13). Briefly, 10.sup.4 B cells mixed with
5.times.10.sup.4 irradiated murine ELA thymoma cells (clone B5)
were cultured for 5-6 d in 200 .mu.l medium containing 5% v/v of
culture supernatants from human T cells (106/ml) which had been
activated for 48 h with PHA (1 .mu.g/ml) and PMA (1 ng/ml). B cells
were then reisolated with anti-CD19 beads and cultured for another
7 d (5.times.10.sup.4 cells in 200 .mu.l, duplicate or triplicate
culture in flat bottomed 96 well plates) in medium alone or in
medium supplemented with 5% T cell supernatants, or with 50 ng/ml
IL-2 (a kind gift from the former Glaxo Institute for Molecular
Biology, Geneva) and 10 ng/ml each IL4 and IL-10 (Peprotech,
London, UK), in the presence or absence of sBAFF. The anti-Flag M2
antibody was added at a concentration of 2 .mu.g/ml and had no
effect by itself. IgM, IgG and IgA in culture supernatants were
quantitated by ELISA assays as described (13).
[0124] Human BAFF was identified by sequence homology as a possible
novel member of the TNF ligand family while we screened public
databases using an improved profile search (18). A cDNA encoding
the complete protein of 285 amino acids (aa) was obtained by
combining EST-clones (covering the 3' region) with a fragment (5'
region) amplified by PCR. The absence of a signal peptide suggested
that BAFF was a type II membrane protein that is typical of the
members of the TNF-ligand family. The protein has a predicted
cytoplasmic domain of 46 aa, a hydrophobic transmembrane region,
and an extracellular domain of 218 aa containing two potential
N-glycosylation sites (FIG. 1A). The sequence of the extracellular
domain of BAFF shows highest homology with APRIL (33% amino acid
identities, 48% homology), whereas the identity with other members
of the family such as TNF, FasL, LTa, TRAIL or RANKL is below 20%
(FIG. 1B, C). The mouse BAFF cDNA clone isolated from a spleen
library encoded a slightly longer protein (309 aa) due to an
insertion between the transmembrane region and the first of several
.beta.-strands which constitute the receptor binding domain in all
TNF ligand members (19). This .beta.-strand rich ectodomain is
almost identical in mouse and human BAFF (86% identity, 93%
homology) suggesting that the BAFF gene has been highly conserved
during evolution (FIG. 1A).
[0125] Although TNF family members are synthesized as
membrane-inserted ligands, cleavage in the stalk region between
transmembrane and receptor binding domain is frequently observed.
For example, TNF or FasL are readily cleaved from the cell surface
by metalloproteinases (20, 21). While producing several forms of
recombinant BAFF in 293T cells, we noticed that a recombinant
soluble 32 kDa form of BAFF (aa 83-285, sBAFF/long), containing the
complete stalk region and a N-terminal Flag-tag in addition to the
receptor binding domain, was extensively processed to a smaller 18
kDa fragment (FIG. 2A, B). Cleavage occurred in the stalk region
since the fragment was detectable only with antibodies raised
against the complete receptor interaction domain of BAFF but not
with anti-Flag antibodies (data not shown). Also revealed was that
only N124 (located in the stalk) but not N242 (located at the entry
of the F-.quadrature. sheet) was glycosylated, since the molecular
mass of the non-processed sBAFF/long was reduced from 32 kDa to 30
kDa upon removal of the N-linked carbohydrates with PNGase F
whereas the 18 kDa cleaved form was insensitive to this treatment.
Peptide sequence analysis of the 18 kDa fragment indeed showed that
cleavage occurred between R133 and A134 (FIG. 1A). R133 lies at the
end of a polybasic region which is conserved between human
(R-N-K-R) and mouse (R-N-R-R). To test whether cleavage was not
merely an artifact of expressing soluble, non-natural forms of
BAFF, membrane-bound full length BAFF was expressed in 293T cells
(FIG. 2C). The 32 kDa complete BAFF and some higher molecular mass
species (probably corresponding to non-dissociated dimers and
trimers) were readily detectable in cellular extracts, but more
than 95% of BAFF recovered from the supernatant corresponded to the
processed 18 kDa form, indicating that BAFF was also processed when
synthesized as a membrane-bound ligand.
[0126] A soluble BAFF was engineered (Q136-L285, sBAFF/short) whose
sequence started 2 aa downstream of the processing site (FIG. 1 B).
As predicted, the Flag-tag attached to the N-terminus of this
recombinant molecule was not removed (data not shown) which allowed
its purification by an anti-Flag affinity column. To test its
correct folding, the purified sBAFF/short was analyzed by gel
filtration where the protein eluted at an apparent molecular mass
of 55 kDa (FIG. 2D). The sBAFF/short correctly assembles into a
homotrimer (3.times.20 kDa) in agreement with the quaternary
structure of other TNF family members (19). Finally, unprocessed
sBAFF/long was readily expressed in bacteria, indicating that the
cleavage event was specific to eukaryotic cells.
[0127] Northern blot analysis of BAFF revealed that the 2.5 kb BAFF
mRNA was abundant in the spleen and PBLs (FIG. 3A). Thymus, heart,
placenta, small intestine and lung showed weak expression. This
restricted distribution suggested that cells present in lymphoid
tissues were the main source of BAFF. Through PCR analysis, we
found that BAFF mRNA was present in T cells and peripheral blood
monocyte-derived dendritic cells but not in B cells (FIG. 3B). Even
naive, non-stimulated T cells appeared to express some BAFF
mRNA.
[0128] A sequence tagged site (STS, SHGC-36171) was found in the
database which included the human BAFF sequence. This site maps to
human chromosome 13, in a 9 cM interval between the markers D13S286
und D13S1315. On the cytogenetic map, this interval corresponds to
13q32-34. Of the known TNF ligand family members, only RANKL
(Trance) has been localized to this chromosome (22) though quite
distant to BAFF (13q14).
[0129] In order for the ligand to exert maximal biological effects,
it was likely that the BAFF receptor (BAFF-R) would be expressed
either on the same cells or on neighboring cells present in
lymphoid tissues. Using the recombinant sBAFF as a tool to
specifically determine BAFF-R expression by FACS, we indeed found
high levels of receptor expression in various B cell lines such as
the Burkitt lymphomas Raji and BJAB (FIG. 4A, Table 1). In
contrast, cell lines of T cell, fibroblastic, epithelial and
endothelial origin were all negative. Very weak staining was
observed with the monocyte line THP-1 which, however, could be due
to Fc receptor binding. Thus, BAFF-R expression appears to be
restricted to B cell lines. The two mouse B cell lines tested were
negative using the human BAFF as a probe, although weak binding was
observed on mouse splenocytes (data not shown). The presence of
BAFF-R on B cells was corroborated by analysis of umbilical cord
and peripheral blood lymphocytes. While CD8.sup.+ and CD4.sup.+ T
cells lacked BAFF-R (FIG. 4B and data not shown), abundant staining
was observed on CD19.sup.+ B cells (FIGS. 4A and 4B), indicating
that BAFF-R is expressed on all blood B cells, including naive and
memory ones.
[0130] Since BAFF bound to blood-derived B cells, experiments were
performed to determine whether the ligand could deliver
growth-stimulatory or -inhibitory signals. Peripheral blood
lymphocytes (PBL) were stimulated with anti-IgM (.mu.) antibodies
together with fixed 293 cells stably expressing surface BAFF (FIG.
5A). The levels of [.sup.3H]thymidine incorporation induced by
anti-.mu. alone was not altered by the presence of control cells
but was increased two-fold in the presence of BAFF-transfected
cells (FIG. 5B). A dose-dependent proliferation of PBL was also
obtained when BAFF-transfected cells were replaced by purified
sBAFF (FIG. 5C), indicating that BAFF does not require membrane
attachment to exert its activity. In this experimental setup,
proliferation induced by sCD40L required concentrations exceeding 1
.mu.g/ml but was less dependent on the presence of anti-.mu. than
that mediated by BAFF (FIG. 5D). When purified CD19.sup.+ B cells
were co-cultured with irradiated autologous CD19.sup.- PBL,
costimulation of proliferation by BAFF was unaffected,
demonstrating that [.sup.3H]thymidine uptake was mainly due to B
cell proliferation and not to an indirect stimulation of another
cell type (data not shown). The observed B cell proliferation in
response to BAFF was entirely dependent on the presence of
anti-.mu. antibodies, indicating that BAFF functioned as
costimulator of B cell proliferation.
[0131] To investigate a possible effect of BAFF on immunoglobulin
secretion, purified peripheral or cord blood B cells were
preactivated by coculture with EL-4 T cells in the presence of a
cytokine mixture from supernatants of PHA/PMA stimulated T cells
(23). These B cells were reisolated to 98% purity and yielded a
two-fold increase in Ig secretion during a secondary culture in the
presence of BAFF and activated T cell cytokines as compared to
cytokines alone. A very modest effect occurred in the absence of
exogenous cytokines, and an intermediate (1.5-fold) effect was
observed in the presence of the recombinant cytokines IL-2, IL4 and
IL-10 (FIG. 5E, F).
[0132] The biochemical analysis of BAFF is also consistent with the
typical homotrimeric structure of TNF family members. Among this
family of ligands, BAFF exhibits the highest level of sequence
similarity with APRIL which we have recently characterized as a
ligand stimulating growth of various tumor cells (11). Unlike TNF
and LTO which are two family members with equally high homology
(33% identity) and whose genes are linked on chromosome 6, APRIL
and BAFF are not clustered on the same chromosome. APRIL is located
on chromosome 17 (J. L. B., unpublished data) whereas BAFF maps to
the distal arm of human chromosome 13 (13q34). Abnormalities in
this locus were characterized in Burkitt lymphomas as the second
most frequent defect (24) besides the translocation involving the
myc gene into the Ig locus (25). Considering the high expression
levels of BAFF-R on all Burkitt lymphoma cell lines analyzed (see
Table 1), this raises the intriguing possibility that some Burkitt
lymphomas may have deregulated BAFF expression, thus stimulating
growth in an autocrine manner.
[0133] The role of antigen-specific B lymphocytes during the
different stages of the immune response is highly dependent on
signals and contacts from helper T cells and antigen-presenting
cells such as dendritic cells (20). B lymphocytes first receive
these signals early on during the immune response when they
interact with T cells at the edge of the B cell follicles in
lymphoid tissues, leading to their proliferation and
differentiation into low affinity antibody forming cells (18). At
the same time some antigen-specific B cells also migrate to the B
cell follicle and contribute to the formation of germinal centers,
another site of B cell proliferation but also affinity maturation
and generation of memory B cells and high affinity plasma cells
(19).
[0134] Signals triggered by another member of the TNF super family
CD40L have been shown to be critical for the function of B
lymphocytes at multiple steps of the T cell-dependent immune
response. However, several studies clearly showed that CD40UCD40
interaction does not account for all contact-dependent T-cell help
for B cells. Indeed, CD40L-deficient T cells isolated from either
knock-out mice or patients with X-linked hyper IgM syndrome have
been shown to sucessfully induce proliferation of B cells and their
differentiation into plasma cells. Studies using blocking
antibodies against CD40L showed that a subset of surface IgD
positive B cells isolated from human tonsils proliferate and
differentiate in response to activated T cells in a
CD40-independent manner. Other members of the TNF family such as
membrane-bound TNF and CD30L have also been shown to be involved in
a CD40- and surface Ig-independent stimulation of B cells. Similar
to our results with BAFF, it has been shown that CD40-deficient B
cells can be stimulated to proliferate and differentiate into
plasma cells by helper T cells as long as the surface Ig receptors
are triggered at the same time. BAFF as well as CD30L and CD40L is
expressed by T cells but its originality resides in its expression
by dendritic cells as well as the highly specific location of its
receptor on B cells which is in contrast to CD40, CD30 and the TNF
receptor which expression has been descrided on many different
cell. This observation suggests independent and specific
BAFF-induced functions on B cells.
[0135] In support of a role for BAFF in T cell- and dendritic
cell-induced B cell growth and potential maturation, we found that
BAFF costimulates proliferation of blood-derived B cells
concomitantly with cross-linking of the B cell receptors, and,
thus, independently of CD40 signalling. Moreover, using CD19
positive B cells differentiated in vitro into a pre-plasma
cell/GC-like B cell (14), we observed a costimulatory effect of
BAFF on Ig secretion by these B cells in the presence of
supernatant from activated T cells or a blend of IL-2, IL-4 and
IL-10. Interestingly, the costimulatory effect was stronger in
presence of the activated T cell supernatant when compared to the
cytokine blend, suggesting additional soluble factors secreted by
activated T cells involved in antibody production which can
synergize with BAFF or additional BAFF itself. It is, therefore,
possible that BAFF actively contributes to the differentiation of
these GC-like B cells into plasma.
[0136] It is clear that BAFF can signal in both naive B cells as
well as GC-conunited B cells in vitro. Whether this observation
will translate or not during a normal immune response will have to
be addressed by proper in vivo experiments.
[0137] The biological responses induced in B cells by BAFF are
distinct from that of CD40L, since proliferation triggered by CD40L
was less dependent on an anti-.mu. costimulus (17) (and FIG. 5D).
Morever, CD40L can counteract apoptotic signals in B cells
following engagement of the B cell receptor (29), whereas BAFF was
not able to rescue the B cell line Ramos from anti-.mu.-mediated
apoptosis, despite the fact that Ramos cells do express BAFF-R
(Table 1; F. M. and J. L. B., unpublished observations). It is
therefore likely that CD40L and BAFF fulfill distinct functions. In
this respect, it is noteworthy that BAFF did not interact with any
of 16 recombinant receptors of the TNF family tested, including
CD40 (P.S and J.T, unpublished observations).
[0138] B cell growth was efficiently costimulated with recombinant
soluble BAFF lacking the transmembrane domain. This activity is in
contrast to several TNF family members which are active only as
membrane-bound ligand such as TRAIL, FasL and CD40L. Soluble forms
of these ligands have poor biological activity which can be
enhanced by their cross-linking, thereby mimicking the
membrane-bound ligand (15). In contrast, cross-linking Flag-tagged
sBAFF with anti-Flag antibodies or the use of membrane-bound BAFF
expressed on the surface of epithelial cells did not further
enhance the mitogenic activity of BAFF, suggesting that it can act
systemically as a secreted cytokine, like TNF does. This is in
agreement with the observation that a polybasic sequence present in
the stalk of BAFF acted as a substrate for a protease. Similar
polybasic sequences are also present at corresponding locations in
both APRIL and TWEAK and for both of them there is evidence of
proteolytic processing (30) (N.H. and J.T, unpublished
observation). Although the protease responsible for the cleavage
remains to be determined, it is unlikely to be the
metalloproteinase responsible for the release of membrane-bound TNF
as their sequence preferences differ completely (21). The
multibasic motifs in BAFF (R-N-K-R), APRIL (R-K-R-R) and Tweak
(R-P-R-R) are reminiscent of the minimal cleavage signal for furin
(R-X-K/R-R), the prototype of a proprotein convertase family
(31).
[0139] Practice of the present invention will employ, unless
indicated otherwise, conventional techniques of cell biology, cell
culture, molecular biology, microbiology, recombinant DNA, protein
chemistry, and immunology, which are within the skill of the art.
Such techniques are described in the literature. See, for example,
Molecular Cloning: A Laboratory Manual, 2nd edition. (Sambrook,
Fritsch and Maniatis, eds.), Cold Spring Harbor Laboratory Press,
1989; DNA Cloning, Volumes I and II (D. N. Glover, ed), 1985;
Oligonucleotide Synthesis, (M. J. Gait, ed.), 1984; U.S. Pat. No.
4,683,195 (Mullis et al.,); Nucleic Acid Hybridization (B. D. Hames
and S. J. Higgins, eds.), 1984; Transcription and Translation (B.
D. Hames and S. J. Higgins, eds.), 1984; Culture of Animal Cells
(R. I. Freshney, ed). Alan R. Liss, Inc., 1987; Immobilized Cells
and Enzymes, IRL Press, 1986; A Practical Guide to Molecular
Cloning (B. Perbal), 1984; Methods in Enzymology, Volumes 154 and
155 (Wu et al., eds), Academic Press, New York; Gene Transfer
Vectors for Mammalian Cells (J. H. Miller and M. P. Calos, eds.),
1987, Cold Spring Harbor Laboratory; Immunochemical Methods in Cell
and Molecular Biology (Mayer and Walker, eds.), Academic Press,
London, 1987; Handbook of Experiment Immunology, Volumes I-IV (D.
M. Weir and C. C. Blackwell, eds.), 1986; Manipulating the Mouse
Embryo, Cold Spring Harbor Laboratory Press, 1986.
[0140] The following Examples are provided to illustrate the
present invention, and should not be construed as limiting
thereof.
EXAMPLES
[0141] The following experimental procedures were utilized in
Examples 1-6.
[0142] DNA Construct for the Generation of Murine BAFF Tg Mice
[0143] Both human and murine cDNA sequences have been described
previously (Schneider et al., 1999). A PCR fragment encoding
full-length murine BAFF was generated by RT-PCR. First strand cDNA
was synthesized from mouse lung polyA+ (Clontech, Palo Alto,
Calif.) using oligo dT according to the manufacturer's protocol
(GibcoBRL, (Grand Island, N.Y.). The PCR reaction contained
1.times.Pfu buffer (Stratagene, La Jola, Calif.), 0.2 mM dNTPs, 10%
DMSO, 12.5 pM primers, 5 units Pfu enzyme (Stratagene) and the
following primers with Not1 restriction sites
5'-TAAGAATGCGGCCGCGGAATGGAT- GAGTCTGCAAA-3' [SEQ. ID. NO.: 19] and
5'-TAAGAATGCGGCCGCGGGATCACGCACTCCAGC- AA-3' [SEQ. ID. NO.: 20]. The
template was amplified for 30 cycles at 94.degree. C. for 1 min,
54.degree. C. for 2 min and 72.degree. C. for 3 min followed by a
10 min extension at 72.degree. C. This sequence corresponds to
nucleotides 214 to 1171 of the GenBank file AF119383. The PCR
fragment was digested with Not1 and then cloned into a modified
pCEP4 vector (Invitrogen, Carlsbad, Calif.). The fragment
containing murine BAFF was removed with Xba1 in order to include
the SV40 polyA addition site sequence. This fragment was cloned
into a pUC based vector where the promoter sequence was added. The
promoter, a 1 Kb blunt Bgl2-Not1 fragment containing the human ApoE
enhancer and AAT (alpha anti-trypsin) promoter was purified from
the plasmid clone 540B (a kind gift from Dr. Katherine Parker
Ponder, Washington University, St. Louis, Mo.). An EcoRV/Bgl2
fragment was purified from the final vector and used for the
generation of transgenic mice. The injected offspring of C57BL/6J
female.times.DBA/2J male F1 (BDF1) mice were backcrossed onto
C57BL/6 mice. Techniques of microinjection and generation of
transgenic mice have been previously described (Mcknights et al.,
1983).
[0144] Analytical Methods:
[0145] Serum samples were subject to reduced SDS-PAGE analysis
using a linear 12.5% gel. Total RNA from mouse liver was prepared
and processed for Northern Blot analysis using an isolation kit
from Promega (Madison, Wis.) according to the manufacturer's
guidelines. BAFF transgene-specific mRNA was detected using a probe
spanning the SV40 poly A tail of the transgene construct and
obtained by digestion of the modified pCEP4 vector with Xba1 and
BamH1. The probe recognizes a 1.8-2 Kd band corresponding to mRNA
from the BAFF transgene. PCR analysis of tail DNA from BAFF Tg mice
was carried using 12.5 pM of the following primers
5'-GCAGTTTCACAGCGATGTCCT-3' [SEQ. ID. NO.: 21] and
5'-GTCTCCGTTGCGTGAAATCTG-3' [SEQ. ID. NO.: 22] in a reaction
containing 1.times. Taq polymerase buffer (Strata gene), 0.2 nM
dNTPs, 10% DMSO and 5 units of Taq polymerase (Stratagene). A 719
bp of the transgene was amplified for 35 cycles at 94.degree. C.
for 30 sec., 54.degree. C. for 1 min. and 72.degree. C. for 1.5
min. followed by a 10 min. extension at 72.degree. C.
[0146] The presence of proteins in mouse urine was measured using
Multistix 10 SG reagent strips for urinalysis (Bayer Corporation,
Diagnostics Division, Elkhart, Ind.).
[0147] Cell-dyn and Cytofluorimetric Analysis (FACS).
[0148] Differential WBC counts of fresh EDTA anticoagulated whole
blood were performed with an Abbott Cell Dyne 3500 apparatus
(Chicago, Ill.). For FACS analysis, Fluorescein (FITC)-, Cy-chrome-
and Phycoerythrin-(PE)-labeled rat anti-mouse antibodies:
anti-B220, anti-CD4, anti-CD8, anti-CD43, anti-IgM, anti-CD5,
anti-CD25, anti-CD24, anti-CD38, anti-CD21, anti-CD44,
anti-L-selectin and hamster anti-Bcl-2/control hamster Ig kit were
purchased from Pharmingen (San Diego, Calif.). Production of
recombinant E. coli as well as mammalian cell-derived human and
mouse Flag-tagged BAFF were previously described (Schneider et al.,
1999). All antibodies were used according to the manufacturer's
specifications. PBL were purified from mouse blood as follows:
mouse blood was collected in microtubes containing EDTA and was
diluted 1/2 with PBS. Five hundred .mu.l of diluted blood was
applied on top of 1 ml of ficoll (Celardane, Hornby, Ontario,
Canada) in a 4 ml glass tube, the gradient was performed at 2000
rpm for 30 min at room temperature and the interface containing the
lymphocytes was collected and washed twice in PBS prior to FACS
staining. Spleen, bone marrow and mesenteric lymph nodes were
ground into a single cell suspension in RPMI medium (Life
Technologies, Inc., Grand Island, N.Y.) and washed in FACS buffer
(PBS supplemented with 2% fetal calf serum (JRH Biosciences,
Lenexa, Kans.). Cells were first suspended in FACS buffer
supplemented with the following blocking reagents: 10 .mu.g/ml
human Ig (Sandoz, Basel, Switzerland) and 10 .mu.g/ml anti-mouse Fc
blocking antibody (Pharmingen) and incubated 30 min on ice prior to
staining with fluorochrome-labeled antibodies. All antibodies were
diluted in FACS buffer with the blocking reagent mentioned above.
Samples were analyzed using a FACScan cytofluorometer (Becton
Dickinson).
[0149] Detection of Total Mouse Ig and Rheumatoid Factors in Mouse
Sera by ELISA Assays.
[0150] ELISA plates (Corning glass works, Corning, N.Y.) were
coated overnight at 4.degree. C. with a solution of 10 .mu.g/ml of
goat anti-total mouse Ig (Southern Biotechnology Associates, Inc.
Birmingham, Ala.) in 50 mM sodium bicarbonate buffer pH 9.6. Plates
were washed 3 times with PBS/0.1% Tween and blocked overnight with
1% gelatin in PBS. One hundred .mu.l/well of serum serial dilutions
or standard dilutions was added to the plates for 30 min at
37.degree. C. Mouse Ig were detected using 100 .mu.l/well of a 1
.mu.g/ml solution of an Alkaline Phosphatase (AP)-labeled goat
anti-total mouse Ig (Southern Biotechnology Associates) for 30 min
at 37.degree. C. After a last wash, 3 times with PBS/0.1% Tween,
the enzymatic reaction was developed using a solution of 10
.mu.g/ml of p-nitrophenyl phosphate (Boehringer Mannheim,
Indianapolis, Ind.) in 10% diethanolamine. The reaction was stopped
by adding 100 .mu.l of 3N NaOH/well. The optical density (O.D.) was
measured at 405 nm using a spectrophotometer from Molecular Devices
(Sunnyvale, Calif.). Standard curves were obtained using purified
mouse Ig purchased from Southern Biotechnology Associates. In the
case of detection of rheumatoid factors (RF), the plates were
coated with normal goat Ig (Jackson ImmunoResearch laboratories,
Inc., West Grove, Pa.) instead of goat anti-mouse Ig and detection
of mouse Ig was performed as described above. Detection of mouse
isotypes in the RF assay was done using AP-labeled goat anti-mouse
IgA, IgM, IgG2a, IgG2b and IgG3, as well as purified mouse IgA,
IgM, IgG2a, IgG2b and IgG3 for standard curves (Southern
Biotechnology Associates Inc.). All statistical comparisons were
performed by analysis of variance.
[0151] Detection of Circulating Immune Complexes (CIC) and
Precipitation of Cryoglobulins in Mouse Sera.
[0152] The assay was performed as previously described (June et
al., 1979; Singh and Tingle, 1982) with the following
modifications: ELISA plates (Corning glass works) were coated
overnight at 4.degree. C. with 5 .mu.g/ml of human C1q (Quidel, San
Diego, Calif.) in 50 mM sodium bicarbonate buffer pH 9.6. The
plates were washed 3 times with PBS/0.1% Tween. Fifty .mu.l/well of
0.3 M EDTA was added to the plates plus 50 .mu.l/well of serum
serial dilutions or solutions of known concentrations of a standard
immune complex (peroxidase-mouse anti-peroxidase (PAP) from DAKO
(Carpinteria, Calif.). The plates were incubated 30 min at
37.degree. C. The plates were washed 3 times with PBS/0.1% Tween.
Mouse Ig in the immune complexes were detected using 100 .mu.l/well
of a 1 .mu.g/ml solution of an AP-labeled goat anti-mouse Ig
(Southern Biotechnology Associates, Inc.) as described above for
the ELISA assays. Cryoglobulins were detected by incubating
overnight at 4.degree. C. mouse serum diluted 1/15 in water and
precipitates were scored visually.
[0153] Anti-Double Stranded (ds) and Single Stranded (ss) DNA
Assays.
[0154] Anti-ssDNA were performed using NUNC-immuno Plate MaxiSorp
plates (NUNC A/S, Denmark). Plates were coated overnight at
4.degree. C. first with 100 .mu.g/ml methylated BSA (Calbochem
Corp., La Jolla, Calif.), then with 50 .mu.g/ml grade I calf thymus
DNA (Sigma, St. Louis, Mo.). The calf thymus DNA was sheared by
sonication and then digested with S1 nuclease before use. For the
anti-ssDNA assay, the DNA was boiled for 10 min and chilled on ice
before use. After blocking, serial dilutions of the serum samples
were added and incubated at room temperature for 2 h.
Autoantibodies were detected with goat anti-mouse IgG-AP (Sigma)
and develop as described above for the ELISA assays. Standard
curves were obtained using known quantities of anti-DNA mAb 205,
which is specific for both ss- and dsDNA (Datta et al., 1987).
[0155] Immunohistochemistry
[0156] Spleen and lymph nodes were frozen in O.C.T. embedding
medium (Miles, Elkhart, Ind.) and mounted for cryostat sectioning.
Sections 7-10 .mu.m thick were dried and fixed in acetone. All Ab
incubations (10 .mu.g/ml) were done for 1 hr at room temperature in
a humidified box after dilution in Tris-buffered saline A (TBS-A,
0.05M Tris, 0.15M NaCl, 0.05% Tween-20 (v/v), 0.25% BSA), rinsed in
TBS-B (0.05M Tris, 0.15M NaCl, 0.05% Tween-20) and fixed 1 min in
methanol before initiating the enzymatic reaction. Horseradish
peroxidase (HRP) and alkaline phosphatase (AP) activities were
developed using the diaminobenzidine (DAB) tablet substrate kit
(Sigma) and the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue
tetrazolium (BCIP/NBT, Pierce, Rockford, Ill.), respectively.
Stained tissue sections were finally fixed 5 min in methanol and
counter stained with Giemsa (Fluka, Buchs, Switzerland).
Biotin-labeled antibodies rat anti-B220, anti-CD11c,
anti-syndecan-1 as well as unlabeled rat anti-CD4, anti-CD8.alpha.
and anti-CD8.beta. were purchased from Pharmingen. Biotin-labeled
peanut agglutinin (PNA) was obtained from Vector laboratories
(Burlingame, Calif.). (HRP)-labeled mouse anti-rat Ig and
(HRP)-streptavidin were purchased from Jackson ImmunoResearch
laboratories, Inc. and AP-labeled streptavidin from Southern
Biotechnology Associates, Inc. In the case of immunohistochemistry
on kidney tissue to detect Ig deposition, paraffin section were
used, dewaxed and blocked using diluted horse serum from Vector
(Burlingame, Calif.), followed by staining with HRP-goat anti-mouse
Ig from Jackson Immunoresearch. Detection was performed as
described above.
Example 1
[0157] BAFF Transgenic (BAFF Tg) Founder Mice have an Abnormal
Phenotype.
[0158] Full length murine BAFF was expressed in transgenic mice
using the liver specific alpha-1 antitrypsin promoter with the APO
E enhancer. The full length version was chosen with the expectation
that BAFF would be either cleaved and act systemically or if
retained in a membrane bound form that local liver specific
abnormalities would be observed possibly providing functional
clues. We obtained 13 founder mice positive for the BAFF transgene
(Table 2). Four of these mice died at a young age. Routine
pathology was carried out on mice 811 and 816 (Table 2). There was
no obvious infection in these mice; however, cardiovascular and
renal abnormalities were apparent and similar to those described
for severe hypertension (Fu, 1995) (Table 2). Hematoxylin and eosin
(H&E)-stained kidney tissue sections of founder 816 showed that
the morphology of glomeruli in that mouse was abnormal, whereas the
rest of the kidney tissue seemed normal (data not shown). Many BAFF
transgenic founder mice had proteinuria (Table 2).
Immunohistochemistry on spleen frozen tissue sections from mouse
816, revealed an abnormal and extensive B cell staining and reduced
staining for T cells and this observation was confirmed in the
progeny (see below, FIG. 12).
[0159] Using two color FACS analysis, the ratio of % B220 positive
B cells over % CD4 positive T cells was calculated. This ratio was
two to seven times higher in BAFF Tg founder mice when compared to
control negative BDF1 mice (Table 2), suggesting an increase of the
B cell population in BAFF Tg mice. We selected nine of these
founder mice to generate our different lines of transgenic mice as
underlined in Table 2. None of the remaining BAFF Tg founder mice
or the derived progeny showed any signs of ill health months after
the early death of founders 696, 700, 811 and 816, suggesting that
these 4 mice might have expressed higher levels of BAFF which
caused their death. BAFF overexpression in the liver of transgenic
mice was confirmed by Northern blot analysis (data not shown). In
all BAFF-Tg mice examined histologically, the livers showed no
abnormalities indicating that local overexpression of BAFF did not
induce any immunological or pathological events. An ELISA assay for
murine BAFF is not available; however, we showed that 2% serum from
BAFF Tg mice, but not from control mice, blocked the binding of
mammalian cell-derived mouse soluble Flag-tagged BAFF to BJAB
cells. Moreover, 5% serum from BAFF Tg mice but not from control
mice increased the proliferation of human B cells from PBL in the
presence of anti-.mu. (data not shown). These data suggest that
substantial amounts of soluble BAFF are present in the blood of
BAFF Tg.
Example 2
[0160] Peripheral Lymphocytosis in BAFF Tg Mice is Due to Elevated
B Cell Numbers
[0161] The transgenic mice population was found to have more
lymphocytes in the blood when compared to control negative
littermates, reaching values as high as 13000 lymphocytes/.mu.l of
blood (FIG. 7A). In contrast, the number of granulocytes per .mu.l
of blood in both BAFF Tg mice and control mice remained within
normal limits (FIG. 7A). Since FACS analysis, using anti-CD4 and
anti-B220 antibodies, of peripheral blood cells (PBL) from 18 BAFF
Tg mice issued from six different founder mice showed increased B/T
ratios (FIGS. 7B and 7C), the elevated lymphocyte levels resulted
from an expanded B cell subset. Likewise, using this method,
calculation of absolute numbers of CD4 circulating T cells revealed
a 50% reduction of this T cell subset in BAFF Tg mice when compared
to control mice, and the same observation was made for the CD8 T
cell subset (data not shown). All B cells from the PBL of BAFF Tg
mice have increased MHC class II and Bcl-2 expression when compared
to B cells from control mice (FIGS. 7D and 7E, respectively),
indicating some level of B cell activation in PBL of BAFF Tg mice.
T cells in the blood of BAFF Tg mice did not express the early
activation markers CD69 or CD25; however, 40 to 56% of CD4 or CD8 T
cells were activated effector T cells with a CD44.sup.hi,
L-selectin.sup.lo phenotype versus only 8% to 12% in control
littermates (FIG. 7F). Thus BAFF Tg mice clearly show signs of B
cell lymphocytosis and global B cell activation along with T cell
alterations.
Example 3
[0162] Expanded B Cell Compartments are Composed of Mature
Cells.
[0163] To see whether overexpression of BAFF in the transgenic mice
was affecting the B cell compartment centrally in the bone marrow
and peripherally in secondary lymphoid organs, we examined by FACS
the spleen, bone marrow and mesenteric lymph nodes from a total of
seven BAFF Tg mice and seven control littermates derived from four
different founder mice. The mature B cell compartment was analyzed
by staining with both anti-B220 and anti-IgM antibodies. Two
representative BAFF Tg mice and one representative control
littermate are shown in FIG. 8. The mature B cell compartment
(IgM+. B220+) was increased in both the spleen and the mesenteric
lymph nodes (FIG. 8A, top and bottom panels, respectively).
Analysis of B220+/IgM+B cells (FIG. 7A, middle panel) or the proB
cell (CD43+/B220+) and the preB cell (CD43-/B220+) compartments in
the bone marrow (FIG. 8B) showed that BAFF Tg mice and control
littermates were similar. These data indicate that overexpression
of BAFF is affecting the proliferation of mature B cells in the
periphery but not progenitor B cells in the bone marrow. Analysis
by FACS of the B cell subpopulations in the spleen, revealed an
increased proportion of marginal zone (MZ) B cells in BAFF Tg mice
when compared to control mice (Table 3). The population of
follicular B cells remained proportional in both BAFF Tg and
control mice whereas the fraction of newly formed B cells is
slightly decreased in BAFF Tg mice (Table 3). This result was also
confirmed on B220+splenic B cells using anti-CD38 versus anti-CD24
antibodies and anti-IgM versus anti-IgD antibodies and analyzing
for at the CD38.sup.hi/CD24.sup.+ and IgM.sup.hi/IgD.sup.lo for the
MZ B cell population, respectively, as previously described (Oliver
et al., 1997)(data not shown). Immunohistochemical analysis using
an anti-mouse IgM antibody revealed the expansion of the IgM-bright
MZ B cell area in the spleen of BAFF Tg mice when compared to
control mice (data not shown). All BAFF Tg B220+splenic B cells
also express higher levels of MHC class II (Table 3) and Bcl-2
(data not shown) compared to splenic B cells from control mice,
indicating that splenic B cells as well as B cells from PBL are in
an activated state.
Example 4
[0164] BAFF Tg Mice have High Levels of Total Immunoglobulins,
Rheumatoid Factors and Circulating Immune Complexes in their
Serum.
[0165] The increased B cell compartment in BAFF Tg mice suggested
that the level of total Ig in the blood of these animals might also
be increased. SDS-PAGE, analysis of serum from BAFF Tg mice and
control littermates showed that the heavy and light chains IgG
bands were at least 10 fold more intense in 3 out of 4 BAFF Tg mice
compared to the control sera (FIG. 9A). Likewise, an ELISA
determination on the sera from BAFF Tg mice show significantly
higher total Ig levels when compared to that of the control mice
(FIG. 9B).
[0166] Despite the high levels seen by SDS-PAGE, the excessively
high levels of Ig seen by ELISA determination in some mice, e.g.,
697-5, 816-8-3 and 823-20, led us to suspect the presence of
rheumatoid factors (RF) in the sera, or autoantibodies directed
against antigenic determinants on the Fc fragment of IgG (Jefferis,
1995). These antibodies could bind to the goat anti-mouse Ig used
to coat the ELISA plates and give erroneously high values. ELISA
plates were coated with normal irrelevant goat Ig and the binding
of BAFF Tg Ig to normal goat Ig was measured. FIG. 9C shows that
sera from most BAFF Tg mice contained Ig reacting with normal goat
Ig, whereas only two out of 19 control mice exhibited reactivity in
the same assay. These RF were mainly of the IgM, IgA and IgG2a
isotypes (data not shown).
[0167] Presence of RF can be associated with the presence of high
levels of circulating immune complexes (CIC) and cryoglobulin in
the blood (Jefferis, 1995). To verify whether or not BAFF Tg mice
have abnormal serum levels of CIC, a C1q-based binding assay was
used to detect CIC in the 21 BAFF Tg mice analyzed above. Only 5
BAFF Tg showed significantly high levels of CIC when compared to
control mice, nonetheless these mice corresponded to the animals
having the highest total Ig and rheumatoid factor levels (FIG. 9D).
We also observed precipitate formation when BAFF Tg mice sera were
diluted 1/15 in water but not control sera indicating the presence
of cryoglobulin in these mice (data not shown). Thus, in addition
to B cell hyperplasia, BAFF Tg mice display severe
hyperglobulinemia associated with RF and CIC.
Example 5
[0168] Some BAFF Tg Mice have High Levels of Anti-Single Stranded
(ss) and Double-Stranded (ds) DNA Autoantibodies.
[0169] Initially, we observed kidney abnormalities reminiscent of a
lupus-like disease in two of our founder mice (Table II). The
presence of anti-DNA autoantibodies have also been described in SLE
patients or the SLE-like (SWR.times.NZB)F1 (SNF1) mouse (Datta et
al., 1987). Anti-ssDNA autoantibody levels were detected in BAFF Tg
mice previously shown to have the highest level of total serum Ig
(FIG. 10A). We analyzed the serum of two BAFF Tg mice negative for
antibodies against ssDNA (697-5 and 816-1-1) and three transgenic
mice secreting anti-ssDNA antibodies (820-14, 816-8-3 and 820-7)
for the presence of anti-dsDNA antibodies in parallel with five
control littermates. BAFF Tg mice also secreted anti-dsDNA,
however, the levels of secretion did not always correlate with that
of anti-ssDNA antibodies, as serum from BAFF Tg mouse 697-5 which
did not contain detectable levels of anti-ssDNA antibodies, was
clearly positive for the presence of anti-dsDNA (FIG. 10B).
Therefore, BAFF Tg mice showing the most severe hyperglobulinemia
secrete pathological levels of anti-DNA autoantibodies.
Additionally, and also reminiscent of a lupus-like problem in these
mice we detected immunoglobulin deposition in the kidney of six
BAFF Tg mice analyzed (FIG. 10C), three of these mice did not
secrete detectable levels anti-DNA antibodies (data not shown).
Example 6
[0170] BAFF Tg Mice have Enlarged B Cell Follicles, Numerous
Germinal Centers, Reduced Dendritic Cell Numbers and Increased
Plasma Cell Numbers in both the Spleen and Mesenteric Lymph Nodes
(MLN).
[0171] BAFF Tg mice had large spleens, MLN (data not shown) and
Peyer's patches (FIG. 11). Immunohistochemistry showed the presence
of enlarged B cell follicles and reduced peripheral arteriolar
lymphoid sheets (PALS or T cell area) in BAFF Tg mice (FIG. 12B).
Interestingly, few germinal centers were observed in non-immunized
control littermates (and is typical of this colony in general) and
those present were small (FIG. 12C), whereas BAFF Tg mice possessed
numerous germinal centers in the absence of immunization (FIG.
12D). Staining with anti-CD11c for dendritic cells in the T cell
zone and the marginal zone of control mice (FIG. 12E) was
considerably reduced in BAFF Tg mice (FIG. 12F).
Syndecan-1-positive plasma cells were almost undetectable in the
spleen from control littermates (FIG. 12G), yet the red pulp of
BAFF Tg mice was strongly positive for syndecan-1 (FIG. 12H). Very
similar observations were made for the MLN (FIG. 13). In the MLN of
BAFF Tg mice the B cell areas were dramatically expanded (FIG. 13B)
in contrast to the normal node where B cell follicles were easily
recognizable at the periphery of the node under the capsule with a
typical paracortical T cell zone (FIG. 13A). The medulla of MLN
from BAFF Tg mice were filled with syndecan-1 positive cells which
presumably are plasma cells (FIG. 13H). In conclusion, analysis of
secondary lymphoid organs in BAFF Tg mice was consistent with the
expanded B cell phenotype showing multiple cellular abnormalities
and intense immune activity.
[0172] The Following Experimental Procedures were used in Examples
7-13.
[0173] Mice and Reagents.
[0174] Full length murine BAFF was expressed in transgenic mice
using the liver-specific alpha 1 antitrypsin promoter with the Apo
E enhancer as previously described. (MacKay et al. (1999) J. Exp.
Med. 190:1697-1710). C57BL/6 mice were purchased from ARC (Perth,
Australia). BAFF transgenic mice are maintained as heterozygotes
for the transgene by backcrossing onto C57BL/6 mice. BAFF Tg mice
are screened for the presence of the transgene, both by PCR and
southern blot analysis using genomic DNA isolated from 2-3 mm long
tail snips. (MacKay et al.). We used animals from two separate
lines of BAFF Tg mice issued after 10-12 backcrossings to C57BL/6.
Age-matched negative littermates were used as controls. Animals
between 8 and 17 months of age were used. Animals were housed under
conventional barrier protection and handled in accordance with the
Animal Experimentation and Ethic Committee (AEEC), which complies
with the Australian code of practice for the care and use of
animals for scientific purposes. Flag-tagged soluble human BAFF
(amino acids (aa) 83-285) was expressed by E. coli and purified as
described previously. (Schneider et al. (1999) J. Exp. Med.
189:1747-1756.) Anti-human BAFF antibodies Buffy-2 (rat IgM),
Buffy-5 (Rat IgG1) and A21G3.3 (mouse IgG1, K) were obtained after
immunization of rats or mice with recombinant soluble human BAFF as
previously detailed. (Schneider et al.). These antibodies recognize
the TNF homology domain of soluble BAFF. The antibody A21G3.3 was
purified as follows: 500 ml of medium from hybridoma cultures were
diluted 1 to 1 with 0.1 M Sodium Phosphate (pH 7.2) buffer
containing 150 mM of NaCl. The diluted media was loaded onto a
protein-L column (Clontech, Palo Alto, Calif.) at 1 ml/minute and
eluted with 0.1 M Glycine (pH 2.8). The eluted solution was
neutralized with 1 M Sodium Phosphate buffer (pH 7.2). The peak
fractions were confirmed by SDS-PAGE. Centricon Plus-20 (Millipore,
Bedford, Mass.) was used to exchange the buffer to PBS and to
concentrate the purified antibody. The purified A21G3.3 antibody
was labelled with 10.times. mole of EZ link sulfo-NHS-LC Biotin
(Pierce, Rockford, Ill.) and incubated at room temperature for 30
minutes. The biotinylation reaction was stopped with 150 mM
glycine. The sample was further purified with a desalting column to
remove the free biotin (Amersham Pharmacia, Uppsala, Sweden). Flow
cytometry.
[0175] Mice were sacrificed and spleen and submaxillary glands were
collected. Spleens and lymph nodes were dissociated by grinding
between frosted glass slides (Menzel-Glaser, Braunschweig,
Germany). Cells were filtered through a 70 .mu.m nylon Cell
Strainer (Falcon, Becton Dickinson, Franklin Lakes, N.J.) and
erythrocytes (only for spleen) removed by osmotic lysis with red
blood cell lysis solution (8.34 mg/ml ammonium chloride, 0.84 mg/ml
sodium bicarbonate, 1 mM EDTA, pH 8.0). Submaxillary glands were
cut into pieces of 2-3 millimeters and incubated in a sterile
collagenase solution (Roche Diagnostics, Mannheim, Germany), 1
mg/ml in PBS (Ca.sup.2+ and Mg.sup.2+ free), for 1 h at 37.degree.
C., until leukocytes were released from the tissue. After
digestion, the cell suspension was filtered through a 70 .mu.m
nylon cell strainer (Falcon) and filtered cells were washed twice
with PBS. Leukocytes obtained from spleens or submaxillary glands
were resuspended in FACS buffer (1% BSA, 0.05% sodium azide in PBS)
at a concentration of 5.times.10.sup.6 cells/ml. Surface staining
was done using various combinations of FITC-, PE-, Cy5- and
Cychome.TM.-labelled antibodies. Fluorescent-labelled anti-mouse
antibodies anti-CD4 (L3T4), anti-CD8a (Ly-2) anti-CD45R/B220
(RA3-6B2), anti-Ly6-G (GR1), anti-IgD (11-26c.2a), anti-CD11b
(Mac1), anti-Ly55 (NK1.1, NKR-P1C), anti-IgM (R6-60.2), anti-CD23
(IgE Fc Receptor, clone B3B4), anti-CD24 (Heat Stable Antigen,
30F1), anti-CD43 (S7), anti-L-selectin (MEL-14), anti-CD1 (1B1) and
anti-CD21 (7G6) were supplied by BD PharMingen (San Diego, Calif.).
Cy5-conjugated anti-IgM antibody was purchased from Jackson
ImmunoResearch laboratories Inc. (West Grove, Pa.). FITC-labelled
antibodies were used diluted 1/100 whereas other
fluorochrome-labelled antibodies were used at 1/200 final dilution.
For flow cytometry we acquired 30,000-100,000 events per sample.
Data was collected on a FACSCalibur flow cytometer and analysed
using CELLQues.TM. software (Becton Dickinson).
[0176] Immunohistochemistry.
[0177] Spleen (removed comma) and submaxillary glands were
collected from both control and BAFF Tg mice. Tissues were either
frozen in OCT compound (Tissue-Tek, Sakura Finetek, Torrance,
Calif., USA) or fixed in 10% buffered formalin and embedded in
paraffin. Biopsies of human parotid glands were processed into
paraffin blocks by pathologists at the Flinders Medical center in
Adelaide. Paraffin sections, 5 .mu.m thick, were re-hydrated in
successive baths of xylene, 100% ethanol and H.sub.2O. Slides were
cooked under pressure in citrate buffer (8.2 mM trisodium citrate,
1.7 mM citrate acid, pH 6.0). Slides were either stained with
hematoxylin-eosin (H&E) for histologic examination or used for
immunohistochemical staining. Prior to immunohistochemical staining
tissue sections were pre-incubated with human Ig (Sandoz, Basel,
Switzerland) 10 .mu.g/ml in TBS-Triton (0.5% Triton) to block
non-specific binding and washed twice with TBS-Triton. Sections
were incubated with rat anti-human BAFF (Buffy-2) or an
isotype-matched control rat antibody (Jackson ImmunoResearch
Laboratories, Inc.), 5 .mu.g/ml for 30 min at room temperature, and
washed with TBS-Triton. Slides were then incubated with
biotin-labelled rabbit anti-rat Ig (1/100, DAKO (Australia) PTY
LTD, Botany, Australia) for 30 min at room temperature, followed by
horseradish peroxidase-labelled (HRP)-Streptavidin (Jackson
ImmunoResearch Laboratories Inc.) 30 min and visualized using the
substrate 3,3' diaminobenzidine (DAB) (Vector laboratories, Inc.,
Burlingame, Calif.). Sections were counterstained using hematoxylin
(Sigma) and Scott's blueing solution and dehydrated in successive
baths of H.sub.2O, 100% ethanol and xylene. Slides were mounted
with cover slips and Eukitt mounting solution (Calibrated
Instruments Inc., Hawthorne, N.Y.). Endogenous peroxidase activity
was blocked using 2% hydrogen peroxide in methanol for 20 min
before staining with the primary antibody. Frozen sections of
spleen and submaxillary glands were subjected to
immunohistochemical analysis as previously described (32).
Biotin-labelled anti-mouse B220 and anti-mouse Syndecan-1 were
purchased from BD PharMingen and the staining was detected using
HRP-streptavidin (Jackson ImmunoResearch Laboratories Inc.) and
visualized using DAB. All slides were observed under a Leica light
microscope and images were captured using a Leica DC 200 camera
(Leica, Bannockburn, Ill.). Scoring of SS disease activity in
mice.
[0178] Tissue sections of mouse submaxillary glands stained with
H&E were examined at 100.times. under the microscope and scored
as previously described. (White et al. (1974) J. Immunol.
112:178-185). The degree of inflammatory infiltrates is graded as
follows: 1 indicated that 1 to 5 foci of mononuclear cells were
seen (more than 20 cells per focus), 2 indicated that more than 5
foci of mononuclear cells were seen but without significant
parenchymal destruction, 3 indicated that multiple confluent foci
were seen with moderate degeneration of parenchymal tissue and 4
indicated extensive infiltration of the gland with mononuclear
cells and extensive parenchymal destruction.
[0179] Measurement of Salivary Flow.
[0180] Mice were anaesthetized and injected ip with 50 .mu.g of
sterile pilocarpine in PBS (Sigma, St Louis, Mo.) per 100 g body
weight. After 4 min, saliva was collected for 5 min on a cotton
swab. The weight of the cotton swab was measured before and after
saliva collection. The amount of saliva collected was normalized to
0 g of saliva per g of body weight. Patients with primary SS and
sera.
[0181] Sera were collected from 41 patients followed between 1995
and 2001 at Flinders Medical Centre who fulfilled at least four of
six European consensus criteria for the diagnosis of primary SS.
(Vitali et al. (1993) Arthritis Rheum. 36:340-347. No patient was
treated with corticosteroids or immunosupressive agents. Control
sera were collected from 39 healthy donors. Labial salivary gland
biopsies with lymphocyte focus scores of >1 per 4 mm.sup.2 of
salivary gland tissue were obtained from 4 patients with primary SS
(Chisholm, et al. (1968) J. Clin. Pathol. 21:656-660), and
histologically normal labial salivary gland tissues obtained from 3
controls.
[0182] ELISA Assays for Detection of Human BAFF in Sera from
Patients with Primary SS.
[0183] Sera from patients were diluted 1/10 and pre-cleared from
human Ig on protein-A-Sepharose beads (10% beads (pelleted beads)
v/v, Amersham Pharmacia) overnight at 4.degree. C. ELISA plates
(NUNC Nalge International, Rochester, N.Y.) were coated with 2
.mu.g/ml rat anti-human BAFF antibody (Buffy-5), overnight at
4.degree. C. Following blocking, serial dilutions of the
pre-cleared sera were added, followed by the detection antibody,
biotin-conjugated mouse anti-human BAFF (0.5 .mu.g/ml, clone
A21G3.3). Alkaline phosphatase (AP)-labelled streptavidin (AP-SA,
Jackson ImmunoResearch Laboratories Inc.) and the corresponding AP
substrate Sigma 104 (Sigma) were used for detection. The reaction
was stopped using 3N NaOH. Plates were read at an OD of 405 nm, and
a standard curve was generated using known quantities of
recombinant human BAFF diluted in human serum and treated as
described above for patients' samples.
[0184] Statistical Analyses were Done using the StatView Software
and ANOVA.
Example 7
[0185] BAFF Tg Mice Develop a Sjogren-Like Syndrome with Age.
[0186] Following routine dissections of more than 50 BAFF Tg mice
and over 20 age-matched littermate controls, we observed that many
mice over age 13 months had enlarged salivary (submaxillary) glands
(FIG. 14A). Histological preparations from 22 BAFF Tg mice 12-17
months-old and 7 age-matched control mice were examined and scored
for disease severity. The scoring system used (White et al.) takes
into consideration the number of foci of infiltrating leukocytes in
the gland and the extent of destruction of the parenchymal
tissue.
[0187] A massive destruction of epithelial duct/acinar cells with
small periductal foci, as well as large leukocytic infiltrates, was
observed in submaxillary glands from BAFF tg animals (FIG. 14B,
middle and right panel). Although small foci of infiltrating
leukocytes were detected in the submaxillary glands of age-matched
control mice, disease grades remained between 0 and 2, while the
majority of BAFF tg mice scored 3 or greater. Overall, our analysis
revealed that 40% of BAFF Tg mice over 12 months of age exhibited
severe disease (grade 3 or above, FIG. 14C), an incidence which is
significantly greater than that observed for control mice (FIG.
14C). Furthermore, the pathology observed in the BAFF tg mice was
independent of the sex of the animals (data not shown). Lacrimal
glands were not examined as mice did not develop obvious ocular
disorders (keratoconjonctivitis).
Example 8
[0188] Presence of Tumor-Like Infiltrates in Salivary Glands of
BAFF Tg Mice.
[0189] Interestingly. 3 BAFF Tg mice between 13 and 15 months-old,
developed a large submaxillary tumor (over 1 cm of diameter), while
no such tumors were observed in age-matched controls. Histological
analysis revealed that these tumors contained hyperplastic lymphoid
tissue composed mainly of activated B-lymphocytes (FIG. 15D) and
numerous germinal centers (FIG. 15B). It is not known whether the
tumors arose from infiltrates within submaxillary glands or from
neighbouring lymph nodes, as no remaining glandular tissue was
evident by histology. However, as only one submaxillary gland was
found in these mice, it is likely that the tumors arose from
infiltrates within the gland which ultimately overwhelmed and
destroyed all glandular tissue. Unusual aggregates of cells
organised in diffused foci (small lymphocytes in sinusoids) (FIG.
15A, B and C) were detected in these tumors (FIG. 15B), as well as
in the large leukocyte infiltrates found in salivary glands of 4
independent BAFF Tg mice (two examples are shown in FIGS. 15A and
C). Cells in these infiltrates were B-lymphoid cells expressing
high levels of B220 (FIG. 15D), however they were not plasma cells
since they were not positive for the plasma cell marker, syndecan-1
(FIG. 15E). A consulting pathologist indicated that because no
lympho-epithelial aggregates were seen, the infiltrating B cells
could not be definitively classified as malignant. Studies to
examine the clonality of these unusual B cells are in progress to
determine whether they are simply tumor-like clusters of lymphoid
cells that do not meet criteria for malignancy or cells with real
neoplastic potential.
Example 9
[0190] Submaxillary Glands from Older BAFF Tg Mice are Infiltrated
by a Large Number of B-lymphocytes.
[0191] Seven BAFF Tg mice that had significantly larger
submaxillary glands compared to age-matched control mice were
selected for a detailed assessment of infiltrating cells. One gland
from each mouse was digested with collagenase, after which
mononuclear cells were purified and analysed by flow cytometry.
Absolute counts of various cell types showed a consistent and
significant increase in the number of B-lymphocytes infiltrating
submaxillary glands of aging BAFF Tg mice (Table 1). Numbers of T
cells, NK cells, macrophages and granulocytes also increased,
however a large variation was seen between animals (Table 1).
Hematoxylin and eosin staining done on the second gland collected
from each Tg animal confirmed that the disease score on these
tissues was at least 3 (data not shown). Mice with severe lesions
in their submaxillary glands (disease grade above 3) did not
secrete anti-Ro/SSA and/or anti-La/SSB autoantibodies, which are
often associated with human SS (data not shown).
1TABLE 1 Analysis of leukocytes present in salivary glands of BAFF
transgenic mice and control littermates by flow cytometry. Total
CD4+ CD8+ Mac-1+ Age Leukocytes B cells T cells T cells NK cells
cells GR1+ cells (months) (.times.10.sup.6) (.times.10.sup.6)
(.times.10.sup.6) (.times.10.sup.6) (.times.10.sup.6)
(.times.10.sup.6) (.times.10.sup.6) Control Littermates Control 1
14 8.4 0.048 -- ND 0.04 0.22 ND Control 2 14 5.6 0.045 -- ND 0.03
0.17 ND Control 3 14 5.9 0.028 -- ND 0.02 0.23 ND Control 4 15 1.5
0.0014 0.0082 0.0011 0.02 0.57 0.062 Mean .+-. SD 14.25 .+-. 0.5
5.35 .+-. 2.8 0.03 .+-. 0.02 0.027 .+-. 0.009 0.29 .+-. 0.18 BAFF
Tg mice BAFF Tg 1 14 30 1.1 -- ND 0.27 0.7 ND BAFF Tg 2 14 31.7
0.14 -- ND 0.31 1.03 ND BAFF Tg 3 16 42.3 0.6 -- ND 0.02 0.23 ND
BAFF Tg 4 8 6 0.35 0.039 0.028 0.13 0.29 0.45 BAFF Tg 5 16 11 4.6
0.084 1.6 0.64 0.27 4.3 BAFF Tg 6 17 2.5 0.03 0.027 0.022 0.087
0.17 0.23 BAFF Tg 7 16 22 9.3 0.34 1.6 1.4 0.5 8.7 Mean .+-. SD
14.4 .+-. 3 21 .+-. 14 2.3 .+-. 3.5 0.41 .+-. 0.5 0.46 .+-. 0.31 p
values P < 0.05 P < 0.05 Animals were sacrificed and
submaxillary glands dissected. Tissues were digested and leukocytes
purified as described in materials and methods. The Total number of
leukocytes per gland was counted using an hemocytometer. Cells were
stained with anti-B220 (B cells), anti-CD4 and anti-CD8 (T cells),
anti-NK1.1 (NK cells), anti-Mac1 (macrophages) and anti-GR1
(granulocytes) fluorochrome-labelled antibodies. Flow cytometry
analysis provided percentage values for each subpopulation # of
cells and absolute numbers were calculated relative to the total
number of cells collected per gland. ND: not detected, --: not
done. Only significant p values are indicated.
Example 10
[0192] A Large Proportion of B Cells Infiltrating Submaxillary
Glands of BAFF Tg Mice have a Marginal Zone (MZ)-Like
Phenotype.
[0193] A flow cytometric profiling analysis of B cells infiltrating
the submaxillary glands of BAFF Tg mice revealed two B cell subsets
based on their level of expression of B220 (FIG. 16A). Analysis of
the B-1 B cell population in the B220 low/intermediate
(B220.sup.lo/int) gate revealed a 3-fold increase of B-1b
(CD5.sup.-) B cells and presence of B-1a (CD5.sup.+) B cells not
detected in control mice (FIG. 16B). In the B220 high (B220.sup.hi)
gate, cells were further analysed based on their level of IgM
expression. The IgM dull (IgM.sup.dull) subset of B cells is
present in both control and BAFF Tg mice, however this population
is larger in BAFF Tg mice compared to control animals (FIG. 16B).
The IgM.sup.dull subset in both control and BAFF Tg mice contains
B-2-like cells, which are CD21.sup.lo/int, IgD.sup.+ and CD23.sup.+
(data not shown). The B220.sup.hi/IgM.sup.hi B cell subset is
greatly increased in BAFF Tg mice compared to control mice (FIG.
16B). Moreover, the IgM.sup.hi B cells found in BAFF Tg mice are
phenotypically different than IgM.sup.hi B cells from control mice.
Indeed, the small IgM.sup.hi B cell subset detected in control mice
had characteristics of B-2 B cells (CD21.sup.lo/int, IgD.sup.+ and
CD23+, add HSA and L-selectin (FIG. 16D, 16E), whereas the large
IgM.sup.hi B cell subset of BAFF Tg mice had no counterpart in
control mice and resembled MZ B cells in many respects. These cells
were IgD.sup.lo, CD21.sup.hi, CD23.sup.lol-, CD1.sup.hi, HSA.sup.++
(FIG. 16E) and L-selectin.sup.lo (FIG. 16D). These cells also
express lower levels of CD43 when compared to B-1 B cells (FIG.
16C). We therefore-refer to these-cells as M-Z-like-B-cells-because
their profile is closer to that of MZ B cells than to other
phenotypically-related B cell subsets such as B-1 and splenic
transitional T1 B cells (FIG. 16F). (Wells et al. (1994) J.
Immunol. 153:5503-5515; Amano et al. (1998) J. Immunol.
161:1710-1717). However, these cells also share some similarities
with B-1b cells, such as low expression of CD43.
Example 11
[0194] Older BAFF Tg Mice Exhibit Impaired Saliva Production.
[0195] The inflammation seen in the salivary glands of BAFF Tg mice
was reminiscent of the inflammation described for SS patients.
Therefore, we aimed to determine if the inflammation in BAFF Tg
mice caused an impairment of normal saliva production. To this end,
13 BAFF Tg mice and 14 control littermates between 8 and 15.5
months of age received pilocarpine, a known stimulant for salivary
flow, 4 min prior to the collection and measurement of saliva. We
found that mice between 13 and 15 months of age had a significantly
reduced production of saliva when compared to age-matched control
animals (FIG. 17A). However, this difference was not seen with mice
between 8 and 10 months of age (FIG. 17B). Interestingly, reduction
in saliva flow correlated with the highest numbers of B-lymphocytes
detected in submaxillary glands of these Tg animals (data not
shown).
Example 12
[0196] Elevated Levels of BAFF Detected in the Serum of Patients
Suffering from Primary Sjogren's Syndrome.
[0197] As the Sjogren's-like pathology observed in some BAFF Tg
mice showed similar features to human SS, we measured BAFF levels
in sera of patients suffering from this disease. Sera from 41
patients with primary SS and 39 healthy individuals were analysed
using a human BAFF-specific ELISA assay. This assay showed that at
least 15 patients out of 41 (36%) clearly had higher levels of
serum BAFF when compared to healthy individuals (FIG. 18A). Two
patients had very high BAFF levels (over 200 ng/ml, FIG. 18A). BAFF
levels in patients' sera did not correlate with the levels of total
IgG or RF (FIG. 18B and C, respectively). These BAFF levels also
did not correlate with the presence of precipitins such as anti-Ro
and/or anti-La (FIG. 18D). We confirmed human BAFF levels in
patients' sera using BAFF-specific immunoprecipitation procedures
followed by western blotting techniques (data not shown).
Example 13
[0198] Detection of BAFF Expressing Cells in Tissue Sections from
Labial Glands of Patients with SS.
[0199] We examined the expression of BAFF at the site of abnormal
lymphocyte infiltrates in biopsies of labial glands from SS
patients. A strong BAFF+ signal was detected on leukocytic
infiltrates within patient tissues, although clearly not all cells
were positive for BAFF within the infiltrate (FIG. 18E, right
panel). The positive signal detected was specific since it could be
competed away by the addition of recombinant human BAFF (data not
shown). The antibody did not stain normal labial salivary gland
tissue (FIG. 18E, bottom left). Preliminary experiments using
2-color staining on tissues indicate that infiltrating
macrophages/monocytes are the likely source of BAFF in these
inflamed tissues (data not shown).
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Sequence CWU 1
1
26 1 285 PRT Homo sapiens 1 Met Asp Asp Ser Thr Glu Arg Glu Gln Ser
Arg Leu Thr Ser Cys Leu 1 5 10 15 Lys Lys Arg Glu Glu Met Lys Leu
Lys Glu Cys Val Ser Ile Leu Pro 20 25 30 Arg Lys Glu Ser Pro Ser
Val Arg Ser Ser Lys Asp Gly Lys Leu Leu 35 40 45 Ala Ala Thr Leu
Leu Leu Ala Leu Leu Ser Cys Cys Leu Thr Val Val 50 55 60 Ser Phe
Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg 65 70 75 80
Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly 85
90 95 Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly
Leu 100 105 110 Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser
Ser Gln Asn 115 120 125 Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu
Glu Thr Val Thr Gln 130 135 140 Asp Cys Leu Gln Leu Ile Ala Asp Ser
Glu Thr Pro Thr Ile Gln Lys 145 150 155 160 Gly Ser Tyr Thr Phe Val
Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser 165 170 175 Ala Leu Glu Glu
Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr 180 185 190 Phe Phe
Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met 195 200 205
Gly His Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu 210
215 220 Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr
Leu 225 230 235 240 Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys
Leu Glu Glu Gly 245 250 255 Asp Glu Leu Gln Leu Ala Ile Pro Arg Glu
Asn Ala Gln Ile Ser Leu 260 265 270 Asp Gly Asp Val Thr Phe Phe Gly
Ala Leu Lys Leu Leu 275 280 285 2 309 PRT Mus sp. 2 Met Asp Glu Ser
Ala Lys Thr Leu Pro Pro Pro Cys Leu Cys Phe Cys 1 5 10 15 Ser Glu
Lys Gly Glu Asp Met Lys Val Gly Tyr Asp Pro Ile Thr Pro 20 25 30
Gln Lys Glu Glu Gly Ala Trp Phe Gly Ile Cys Arg Asp Gly Arg Leu 35
40 45 Leu Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Ser Ser Phe Thr
Ala 50 55 60 Met Ser Leu Tyr Gln Leu Ala Ala Leu Gln Ala Asp Leu
Met Asn Leu 65 70 75 80 Arg Met Glu Leu Gln Ser Tyr Arg Gly Ser Ala
Thr Pro Ala Ala Ala 85 90 95 Gly Ala Pro Glu Leu Thr Ala Gly Val
Lys Leu Leu Thr Pro Ala Ala 100 105 110 Pro Arg Pro His Asn Ser Ser
Arg Gly His Arg Asn Arg Arg Ala Phe 115 120 125 Gln Gly Pro Glu Glu
Thr Glu Gln Asp Val Asp Leu Ser Ala Pro Pro 130 135 140 Ala Pro Cys
Leu Pro Gly Cys Arg His Ser Gln His Asp Asp Asn Gly 145 150 155 160
Met Asn Leu Arg Asn Ile Ile Gln Asp Cys Leu Gln Leu Ile Ala Asp 165
170 175 Ser Asp Thr Pro Thr Ile Arg Lys Gly Thr Tyr Thr Phe Val Pro
Trp 180 185 190 Leu Leu Ser Phe Lys Arg Gly Asn Ala Leu Glu Glu Lys
Glu Asn Lys 195 200 205 Ile Val Val Arg Gln Thr Gly Tyr Phe Phe Ile
Tyr Ser Gln Val Leu 210 215 220 Tyr Thr Asp Pro Ile Phe Ala Met Gly
His Val Ile Gln Arg Lys Lys 225 230 235 240 Val His Val Phe Gly Asp
Glu Leu Ser Leu Val Thr Leu Phe Arg Cys 245 250 255 Ile Gln Asn Met
Pro Lys Thr Leu Pro Asn Asn Ser Cys Tyr Ser Ala 260 265 270 Gly Ile
Ala Arg Leu Glu Glu Gly Asp Glu Ile Gln Leu Ala Ile Pro 275 280 285
Arg Glu Asn Ala Gln Ile Ser Arg Asn Gly Asp Asp Thr Phe Phe Gly 290
295 300 Ala Leu Lys Leu Leu 305 3 102 PRT Homo sapiens 3 Val Thr
Gln Asp Cys Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr 1 5 10 15
Ile Gln Lys Gly Ser Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys 20
25 30 Arg Gly Ser Ala Leu Glu Glu Lys Tyr Gly Gln Val Leu Tyr Thr
Asp 35 40 45 Lys Thr Tyr Ala Met Gly His Leu Ile Gln Arg Lys Lys
Val His Val 50 55 60 Phe Gly Asp Glu Leu Ser Asn Asn Ser Cys Tyr
Ser Ala Gly Ile Ala 65 70 75 80 Lys Leu Glu Glu Gly Asp Glu Leu Gln
Leu Ala Ile Pro Arg Glu Asn 85 90 95 Ala Gln Ile Ser Leu Asp 100 4
96 PRT Homo sapiens 4 Lys Gln His Ser Val Leu His Leu Val Pro Ile
Asn Ala Thr Ser Lys 1 5 10 15 Asp Asp Ser Asp Val Thr Glu Val Met
Trp Gln Pro Ala Leu Arg Arg 20 25 30 Gly Arg Gly Leu Gln Ala Gln
Tyr Ser Gln Val Leu Phe Gln Asp Val 35 40 45 Thr Phe Thr Met Gly
Gln Val Val Ser Arg Glu Gly Gln Gly Arg Ala 50 55 60 Tyr Asn Ser
Cys Tyr Ser Ala Gly Val Phe His Leu His Gln Gly Asp 65 70 75 80 Ile
Leu Ser Val Ile Ile Pro Arg Ala Arg Ala Lys Leu Asn Leu Ser 85 90
95 5 104 PRT Homo sapiens 5 Ser Asp Lys Pro Val Ala His Val Val Ala
Asn Pro Gln Ala Glu Gly 1 5 10 15 Gln Leu Gln Trp Leu Asn Arg Arg
Ala Asn Ala Leu Leu Ala Asn Gly 20 25 30 Val Tyr Ser Gln Val Leu
Phe Lys Gly Gln Gly Cys Pro Ser Thr His 35 40 45 Val Leu Leu Thr
His Thr Ile Ser Arg Ile Ala Val Ser Tyr Gln Thr 50 55 60 Glu Gly
Ala Glu Ala Lys Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly 65 70 75 80
Val Phe Gln Leu Glu Lys Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg 85
90 95 Pro Asp Tyr Leu Asp Phe Ala Glu 100 6 97 PRT Homo sapiens 6
Glu Leu Arg Lys Val Ala His Leu Thr Gly Lys Ser Asn Ser Arg Ser 1 5
10 15 Met Pro Leu Glu Trp Glu Asp Thr Tyr Gly Ile Val Leu Leu Ser
Gly 20 25 30 Val Lys Tyr Ser Lys Val Tyr Phe Arg Gly Gln Ser Cys
Asn Asn Leu 35 40 45 Pro Leu Ser His Lys Val Tyr Met Arg Asn Ser
Lys Tyr Pro Gln Met 50 55 60 Trp Ala Arg Ser Ser Tyr Leu Gly Ala
Val Phe Asn Leu Thr Ser Ala 65 70 75 80 Asp His Leu Tyr Val Asn Val
Ser Glu Leu Ser Leu Val Asn Phe Glu 85 90 95 Glu 7 102 PRT Homo
sapiens 7 Thr Leu Lys Pro Ala Ala His Leu Ile Gly Asp Pro Ser Lys
Gln Asn 1 5 10 15 Ser Leu Leu Trp Arg Ala Asn Thr Asp Arg Ala Phe
Leu Gln Asp Gly 20 25 30 Phe Tyr Ser Gln Val Val Phe Ser Gly Lys
Ala Tyr Ser Pro Lys Ala 35 40 45 Thr Ser Ser Pro Leu Tyr Leu Ala
His Glu Val Gln Leu Phe Ser Ser 50 55 60 Gln Tyr Pro Phe Pro Trp
Leu His Ser Met Tyr His Gly Ala Ala Phe 65 70 75 80 Gln Leu Thr Gln
Gly Asp Gln Leu Ser Thr His Thr Asp Gly Ile Pro 85 90 95 His Leu
Val Leu Ser Phe 100 8 109 PRT Homo sapiens 8 Glu Ala Gln Pro Phe
Ala His Leu Thr Ile Asn Ala Thr Asp Ile Pro 1 5 10 15 Ser Gly Ser
His Lys Val Ser Leu Ser Ser Trp Tyr His Asp Arg Gly 20 25 30 Trp
Gly Lys Ile Ser Asn Met Tyr Ala Asn Ile Cys Phe Arg His His 35 40
45 Glu Thr Ser Gly Asp Leu Ala Thr Glu Tyr Leu Gln Leu Met Val Tyr
50 55 60 Val Thr Lys Thr Ser Ile Lys Ile Pro Ser Glu Phe His Phe
Tyr Ser 65 70 75 80 Ile Asn Val Gly Gly Phe Phe Lys Leu Arg Ser Gly
Glu Glu Ile Ser 85 90 95 Ile Glu Val Ser Asn Pro Ser Leu Leu Asp
Pro Asp Gln 100 105 9 26 DNA Homo sapiens 9 actgtttctt ctggaccctg
aacggc 26 10 30 DNA Homo sapiens 10 gacaagcttg ccaccatgga
tgactccaca 30 11 23 DNA Homo sapiens 11 actagtcaca gcagtttcaa tgc
23 12 22 DNA Homo sapiens 12 ctgcagggtc cagaagaaac ag 22 13 24 DNA
Homo sapiens 13 ggagaaggca actccagtca gaac 24 14 24 DNA Homo
sapiens 14 caattcatcc ccaaagacat ggac 24 15 22 DNA Homo sapiens 15
tcggaacaca acgaaacaag tc 22 16 26 DNA Homo sapiens 16 cttctccttc
acctggaaac tgactg 26 17 19 DNA Homo sapiens 17 ggcatcgtga tggactccg
19 18 19 DNA Homo sapiens 18 gctggaaggt ggacagcga 19 19 35 DNA Homo
sapiens 19 taagaatgcg gccgcggaat ggatgagtct gcaaa 35 20 35 DNA Homo
sapiens 20 taagaatgcg gccgcgggat cacgcactcc agcaa 35 21 21 DNA Homo
sapiens 21 gcagtttcac agcgatgtcc t 21 22 21 DNA Homo sapiens 22
gtctccgttg cgtgaaatct g 21 23 4 PRT Artificial Sequence Description
of Artificial Sequence Illustrative motif 23 Arg Asn Lys Arg 1 24 4
PRT Artificial Sequence Description of Artificial Sequence
Illustrative motif 24 Arg Lys Arg Arg 1 25 4 PRT Artificial
Sequence Description of Artificial Sequence Illustrative motif 25
Arg Pro Arg Arg 1 26 4 PRT Artificial Sequence Description of
Artificial Sequence Illustrative motif 26 Arg Xaa Xaa Arg 1
* * * * *