U.S. patent application number 09/855158 was filed with the patent office on 2002-07-04 for methods and compositions of matter concerning april/g70, bcma, blys/agp-3, and taci.
Invention is credited to Theill, Lars Eyde, Yu, Gang.
Application Number | 20020086018 09/855158 |
Document ID | / |
Family ID | 26899121 |
Filed Date | 2002-07-04 |
United States Patent
Application |
20020086018 |
Kind Code |
A1 |
Theill, Lars Eyde ; et
al. |
July 4, 2002 |
Methods and compositions of matter concerning APRIL/G70, BCMA,
BLYS/AGP-3, and TACI
Abstract
This invention concerns interactions among APRIL/G70,
AGP-3/BLYS, BCMA, and TACI and related methods of use and
compositions of matter. It has been found that (1) sAPRIL/G70 binds
to the cell-surface receptors BCMA and TACI on T and B lymphoma
cells, resulting in stimulation of proliferation of primary human
and mouse B and T cells both in vitro and in vivo; (2) APRIL
competes with AGP3's binding to TACI and BCMA; (3) sBCMA inhibits
APRIL and AGP3 binding to its receptors; (4) sBCMA ameliorates T
cell dependent and T cell independent humoral immune responses in
vivo; (5) sTACI inhibits APRIL and AGP3 binding to its receptors
and ameliorates T cell dependent and T cell independent humoral
immune responses in vivo; and (6) BCMA exhibits similarity with
TACI within a single cysteine rich domain located N-terminal to a
potential transmembrane domain. These discoveries provides a
strategy for development of therapeutics for treatment of
autoimmune diseases, and cancer, for prevention of transplant
rejection. Disease states and disease parameters associated with
APRIL and AGP-3 may be affected by modulation of BCMA or TACI;
disease states and parameters associated with TACI can be affected
by modulation of APRIL; disease states and parameters can be
affected by modulation of any of TACI, BCMA, APRIL and AGP-3 by a
single therapeutic agent or two or more therapeutic agents
together.
Inventors: |
Theill, Lars Eyde; (Thousand
Oaks, CA) ; Yu, Gang; (Thousand Oaks, CA) |
Correspondence
Address: |
AMGEN INCORPORATED
MAIL STOP 27-4-A
ONE AMGEN CENTER DRIVE
THOUSAND OAKS
CA
91320-1799
US
|
Family ID: |
26899121 |
Appl. No.: |
09/855158 |
Filed: |
May 14, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60204039 |
May 12, 2000 |
|
|
|
60214591 |
Jun 27, 2000 |
|
|
|
Current U.S.
Class: |
424/146.1 ;
424/153.1 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 2319/00 20130101; A61P 37/02 20180101; A61P 29/00 20180101;
C07K 14/70575 20130101; A61P 17/10 20180101; A61P 1/04 20180101;
A61K 38/00 20130101; A61P 19/02 20180101; A61P 37/06 20180101 |
Class at
Publication: |
424/146.1 ;
424/153.1 |
International
Class: |
A61K 039/395 |
Claims
What is claimed is:
1. A method of inhibiting TACI activity, BCMA activity, or both in
a mammal, which comprises administering a specific binding partner
for APRIL, wherein the specific binding partner comprises a. the
consensus region of TACI (SEQ ID NO: 16); b. the consensus region
of BCMA (SEQ ID NO: 7); c. the TACI/BCMA extracellular consensus
sequence (SEQ ID NO: 13); but does not comprise the extracellular
region of TACI (SEQ ID NO: 15) or the extracellular region of BCMA
(SEQ ID NO: 6).
2. The method of claim 1, further comprising administering a
specific binding partner for AGP-3.
3. A method of treating B-cell lymphoproliferative disorders, which
comprises administering a therapeutic agent comprising a specific
binding partner selected from: a. the consensus region of TACI (SEQ
ID NO: 16); b. the consensus region of BCMA (SEQ ID NO: 7); or c.
the TACI/BCMA extracellular consensus sequence (SEQ ID NO: 13) but
not comprising the extracellular region of TACI (SEQ ID NO: 15) or
the extracellular region of BCMA (SEQ ID NO: 6).
4. A method of treating T-cell lymphoproliferative disorders, which
comprises administering a therapeutic agent comprising a specific
binding partner selected from selected from: a. the consensus
region of TACI (SEQ ID NO: 16); b. the consensus region of BCMA
(SEQ ID NO: 7); or c. the TACI/BCMA extracellular consensus
sequence (SEQ ID NO: 13) but not comprising the extracellular
region of TACI (SEQ ID NO: 15) or the extracellular region of BCMA
(SEQ ID NO: 6).
5. A method of treating one or more solid tumors, which comprises
administering a therapeutic agent comprising a specific binding
partner selected from: a. the consensus region of TACI (SEQ ID NO:
16); b. the consensus region of BCMA (SEQ ID NO: 7); or c. the
TACI/BCMA extracellular consensus sequence (SEQ ID NO: 13) but not
comprising the extracellular region of TACI (SEQ ID NO: 15) or the
extracellular region of BCMA (SEQ ID NO: 6).
6. The method of claim 5, wherein the tumor is selected from lung,
gastrointestinal, pancreatic and prostate
7. The method of any of claims 1, 3, 4, or 5, wherein the specific
binding partner is comprised within a molecule of the formula
(X.sup.1).sub.a--F.sup.1--(X.sup.2).sub.b wherein: F.sup.1 is a
vehicle; X.sup.1 and X.sup.2 are each independently selected from
--(L.sup.1).sub.c--P.sup.1,
--(L.sup.1).sub.c--P.sup.1--(L.sup.2).sub.d--- P.sup.2,
--(L.sup.1).sub.c--P.sup.1--(L.sup.2).sub.d--P.sup.2--(L.sup.3).s-
ub.e--P.sup.3, and
--(L.sup.1).sub.c--P.sup.1--(L.sup.2).sub.d--P.sup.2--(-
L.sup.3).sub.e--P.sup.3--(L.sup.4).sub.f--P.sup.4 at least one of
P.sup.1, P.sup.2, P.sup.3, and P.sup.4 is the; L.sup.1, L.sup.2,
L.sup.3, and L.sup.4 are each independently linkers; and a, b, c,
d, e, and f are each independently 0 or 1, provided that at least
one of a and b is 1.
8. The method of claim 7, wherein the molecule comprises a
structure of the formulae X.sup.1--F.sup.1 or F.sup.1--X.sup.2.
9. The method of claim 7, wherein the molecule comprises a
structure of the formula F.sup.1--(L.sup.1).sub.c--P.sup.1.
10. The method of claim 7, wherein the molecule comprises a
structure of the formula
F.sup.1--(L.sup.1).sub.c--P.sup.1--(L.sup.2).sub.d--P.sup.2 wherein
one of P.sup.1 and P.sup.2 is the consensus region of TACI (SEQ ID
NO: 16) and the other is the consensus region for BCMA (SEQ ID NO:
7).
11. The method of claim 10, wherein the vehicle is an Fc
domain.
12. The method of any of claims 1, 3, 4, or 5, wherein the specific
binding partner replaces a CDR region within an antibody
molecule.
13. A composition of matter of the formula
(X.sup.1).sub.a--F.sup.1--(X.su- p.2).sub.b wherein: F.sup.1 is a
vehicle; X.sup.1 and X.sup.2 are each independently selected from
--(L.sup.1).sub.c--P.sup.1,
--(L.sup.1).sub.c--P.sup.1--(L.sup.2).sub.d--P.sup.2,
--(L.sup.1).sub.c--P.sup.1--(L.sup.2).sub.d--P.sup.2--(L.sup.3).sub.e--P.-
sup.3, and
--(L.sup.1).sub.c--P.sup.1--(L.sup.2).sub.d--P.sup.2--(L.sup.3)-
.sub.e--P.sup.3--(L.sup.4).sub.f--P.sup.4 P.sup.1, P.sup.2,
P.sup.3, and P.sup.4 are each independently a. the consensus region
of TACI (SEQ ID NO: 16); b. the consensus region of BCMA (SEQ ID
NO: 7); or c. the TACI/BCMA extracellular consensus sequence (SEQ
ID NO: 13) but not the extracellular region of TACI (SEQ ID NO: 15)
or the extracellular region of BCMA (SEQ ID NO: 6); and a, b, c, d,
e, and f are each independently 0 or 1, provided that at least one
of a and b is 1.
14. The composition of matter of claim 13 of the formulae
X.sup.1--F.sup.1 or F.sup.1--X.sup.2.
15. The composition of matter of claim 14 of the formula
F.sup.1--(L.sup.1).sub.c--P.sup.1.
16. The composition of matter of claim 14 of the formula
F.sup.1--(L.sup.1).sub.c--P.sup.1--(L.sup.2).sub.d--P.sup.2 wherein
one of P.sup.1 and P.sup.2 is a specific binding partner for TACI
and the other is a specific binding partner for BCMA.
17. The composition of matter of claim 16, wherein the vehicle is
an Fc domain.
Description
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 60/204,039, filed May 12, 2000 and U.S.
Provisional Application Ser. No. 60/214,591, filed Jun. 27, 2000,
which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to proteins that are involved
in inflammation and immunomodulation, survival, or activation. The
invention further relates to proteins related to the tumor necrosis
factor (TNF)/nerve growth factor (NGF) superfamily and related
nucleic acids, expression vectors, host cells, and binding assays.
The specification also describes compositions and methods for the
treatment of immune-related and inflammatory, autoimmune and other
immune-related diseases or disorders, such as rheumatoid arthritis
(RA), Crohn's disease (CD), lupus, and graft versus host disease
(GvHD).
BACKGROUND OF THE INVENTION
[0003] After years of study in necrosis of tumors, tumor necrosis
factors (TNFs) .alpha. and .beta. were finally cloned in 1984. The
ensuing years witnessed the emergence of a superfamily of TNF
cytokines, including fas ligand (FasL), CD27 ligand (CD27L), CD30
ligand (CD30L), CD40 ligand (CD40L), TNF-related apoptosis-inducing
ligand (TRAIL, also designated AGP-1), osteoprotegerin binding
protein (OPG-BP or OPG ligand), 4-1BB ligand, LIGHT, APRIL, and
TALL-1. Smith et al. (1994), Cell, 76: 959-962; Lacey et al.
(1998), Cell, 93: 165-176; Chichepotiche et al. (1997), J. Biol.
Chem., 272: 32401-32410; Mauri et al. (1998), Immunity, 8: 21-30;
Hahne et al. (1998), J. Exp. Med., 188: 1185-90; Shu et al. (1999),
J. Leukocyte Biology, 65: 680-3. This family is unified by its
structure, particularly at the C-terminus. In addition, most
members known to date are expressed in immune compartments,
although some members are also expressed in other tissues or
organs, as well. Smith et al. (1994), Cell 76: 959-62. All ligand
members, with the exception of LT-.alpha., are type II
transmembrane proteins, characterized by a conserved 150 amino acid
region within C-terminal extracellular domain. Though restricted to
only 20-25% identity, the conserved 150 amino acid domain folds
into a characteristic .beta.-pleated sheet sandwich and trimerizes.
This conserved region can be proteolyticaly released, thus
generating a soluble functional form. Banner et al. (1993), Cell,
73: 431-445.
[0004] Many members within this ligand family are expressed in
lymphoid enriched tissues and play important roles in the immune
system development and modulation. Smith et al. (1994). For
example, TNF.alpha. is mainly synthesized by macrophages and is an
important mediator for inflammatory responses and immune defenses.
Tracey & Cerami (1994), Annu. Rev. Med., 45: 491-503. Fas-L,
predominantly expressed in activated T cell, modulates TCR-mediated
apoptosis of thymocyts. Nagata, S. & Suda, T. (1995) Immunology
Today, 16:39-43; Castrim et al. (1996), Immunity, 5:617-27. CD40L,
also expressed by activated T cells, provides an essential signal
for B cell survival, proliferation and immunoglobulin isotype
switching. Noelle (1996), Immunity, 4: 415-9.
[0005] The cognate receptors for most of the TNF ligand family
members have been identified. These receptors share characteristic
multiple cysteine-rich repeats within their extracellular domains,
and do not possess catalytic motifs within cytoplasmic regions.
Smith et al. (1994). The receptors signal through direct
interactions with death domain proteins (e.g. TRADD, FADD, and RIP)
or with the TRAF proteins (e.g. TRAF2, TRAF3, TRAF5, and TRAF6),
triggering divergent and overlapping signaling pathways, e.g.
apoptosis, NF-KB activation, or JNK activation. Wallach et al.
(1999), Annual Review of Immunology 17: 331-67. These signaling
events lead to cell death, proliferation, activation or
differentiation. The expression profile of each receptor member
varies. For example, TNFR1 is expressed on a broad spectrum of
tissues and cells, whereas the cell surface receptor of OPGL is
mainly restricted to the osteoclasts. Hsu et al. (1999) Proc. Natl.
Acad. Sci. USA, 96:3540-5. Such proteins are believed to play a
role in inflammatory and immune processes, suggesting their
usefulness in treating autoimmune and inflammatory disorders.
[0006] A number of research groups have recently identified TNF
family ligands with the same or substantially similar sequence, but
they have not identified the associated receptor. The ligand has
been variously named neutrokine-.alpha. (WO 98/18921, published May
7, 1998), 63954 (WO 98/27114, published Jun. 25, 1998), TL5 (EP 869
180, published Oct. 7, 1998), NTN-2 (WO 98/55620 and WO 98/55621,
published Dec. 10, 1998), TNRL1-alpha (WO 9911791, published Mar.
11, 1999), kay ligand (WO99/12964, published Mar. 18, 1999), and
AGP-3 (U.S. Provisional Application No. 60/119,906, filed Feb. 12,
1999 and No. 60/166,271, filed Nov. 18, 1999, respectively). Each
of these references is hereby incorporated by reference.
Hereinafter, this protein sequence is referred to as "AGP-3."
[0007] A recent paper has identified two previously known proteins
as receptors for AGP-3. Gross et al. (2000), Nature 404: 995-9. The
first receptor was previously identified as a lymphocyte surface
receptor named Transmembrane Activator and CAML Interactor (TACI).
See WO 98/39361, published Sep. 11, 1998, and von Bulow & Bram
(1997), Science, 278:138-140, each of which is hereby incorporated
by reference in its entirety. According to these references, TACI
binds an intracellular cyclophilin ligand designated CAML, which
modulates the calcium signaling pathway in lymphocytes.
[0008] The second receptor identified for AGP-3 is the so-called B
cell maturation protein (BCMA). The human BCMA gene was discovered
by molecular analysis of a t(4;16) translocation, which
characteristic of a human T cell lymphoma. Laabi et al. (1993),
EMBO J. 11: 3897-3904. BCMA mRNA was reported to be found mainly in
lymphoid tissues. Human BCMA cDNA encodes a 184 amino acids protein
(185 residues for the mouse), and the literature reports no obvious
similarity with any known protein or motif, and its function
remained unknown. The protein was reported to reside in the Golgi
apparatus (Gras et al. (1995), Intl. Immunol. 7: 1093-1106). Recent
speculation suggested that BCMA may be a distant member of the TNFR
super family. Madry et al. (1998), Intl. Immunol. 10:
1693-1702.
[0009] A ligand called APRIL or G70 is a TNF family ligand that
remains without a receptor reported in the literature. According to
the literature, APRIL is associated with prostate cancer, breast
cancer, Alzheimer's disease, immune disorders, inflammatory
disorders, and gestational abnormalities. See WO 99/00518 Jun. 26,
1997); WO 99/11791 (Sep. 5, 1997); WO 99/12965 (Sep. 12, 1997); EP
911 633 (Oct. 8, 1997); EP 919 620 (Nov. 26, 1997); WO 99/28462
(Dec. 3, 1997); WO 99/33980 (Dec. 30, 1997); WO 99/35170 (Jan. 5,
1998); and Hahne et al. (1998), J. Exp. Med. 188: 1185-90. (Each of
the foregoing references is hereby incorporated by reference in its
entirety.) A recent paper described APRIL isoforms and suggested
that APRIL causes cell death. Kelly et al. (2000), Cancer Res. 60:
1021-7. The art would benefit from identification of a receptor for
APRIL and a clarification of its activity.
SUMMARY OF THE INVENTION
[0010] It has now been found that sG70 binds to cell-surface
receptors on T and B lymphoma cells resulting in stimulation of
proliferation of primary human and mouse B and T cells both in
vitro and in vivo.
[0011] It has now been found that BCMA and TACI are cell-surface
receptors for APRIL. It has also been found that APRIL competes
with AGP3's binding to TACI and BCMA. Furthermore it is shown here
that sBCMA inhibits G70 and AGP3 binding to its receptors. sBCMA
ameliorates T cell dependent and T cell independent humoral immune
responses in vivo. In addition it has now been found that sTACI
inhibits G70 and AGP3 binding to its receptors and ameliorates T
cell dependent and T cell independent humoral immune responses in
vivo. It has also been found that BCMA exhibits similarity with
TACI within a single cysteine rich domain located N-terminal to a
potential transmembrane domain. This invention concerns novel
methods of use and compositions of matter that exploit these
discoveries. The discoveries provides a strategy for development of
therapeutics for treatment of autoimmune diseases, and cancer, for
prevention of transplant rejection.
[0012] These discoveries show that activity, disease states, and
disease parameters associated with APRIL and AGP-3 may be affected
by modulation of BCMA. Likewise, disease states and disease
parameters associated with TACI can be affected by modulation of
APRIL. Further, such disease states and disease parameters can be
affected by modulation of any of TACI, BCMA, APRIL and AGP-3
together. This discovery further suggests molecules and methods of
treatment by which more than one of TACI, BCMA, APRIL, and AGP-3
may be modulated by a single molecule.
DESCRIPTION OF THE FIGURES
[0013] FIG. 1 shows the sequence of human G70 (SEQ ID NOS: 1 and 2)
Start and stop codons are underlined.
[0014] FIGS. 2A and 2B show the DNA and amino acid sequences of
mouse APRIL/G70 (SEQ ID NOS: 3 and 4, respectively). Start and stop
codons are underlined. The amino acid sequence of FLAG-tagged
soluble mouse G70 (SEQ ID NO: 19) is also provided.
[0015] FIG. 3 shows an alignment of human (SEQ ID NO: 22) and mouse
(SEQ ID NO: 23) G70. The middle line of each row shows the
consensus sequence (SEQ ID NO: 24).
[0016] FIG. 4 shows that G70/APRIL is a potent stimulator for B and
T cell lymphoma. FIG. 4A shows dose-dependent stimulation of
proliferation of Jurkat cells (human leukemic T cells), Raji (human
Burkitt lymphoma) and K562 cells (human chronic myelogenous
leukemia cells). The proliferation of cells was determined by
incubating 3.times.10.sup.4 cells/well in 100 .mu.l medium with
indicated concentration of recombinant sG70/APRIL and
phosphate-buffered saline (PBS, no ligand) as a control. After 48
hours, the number of viable cells were measured by Celltiter 96 AQ
proliferation assay (Promega, Madison, Wis.). In FIG. 4B, U937 cell
(monocyte-like leukemia cells), NIH/3T3 (mouse embryo cell line)
and 293 (transformed human primary embryonal kidney cell line) did
not respond to sG70/APRIL stimulation.
[0017] FIGS. 5A and 5B show FACS analysis of G70/APRIL receptor
binding. G70/APRIL receptor expression was assessed on indicated
cell line using anti-Flag monoclonal antibody followed by
FITC-conjugated goat antibody to mouse IgG. A anti-mouse CD16/CD32
monoclonal antibody (Fc Block) was used to block non-specific
binding to cells.
[0018] FIG. 6 shows the effect of sG70/APRIL on human peripheral
blood B cell, T cell and granulocyte proliferation. Human
peripheral T cell (CD4+ and CD8+), B cells and granulocyte were
purified from three different donors by using RosetteSep cocktail
antibodies (Stem cell Tech. Vancouver). Purified cells were
cultured in tissue culture-treated plastic wells (Becton-Dickinson,
Lincoln park, N.J.) for 6 Days in RPMI-1640 medium supplemented
with 10% fetal calf serum, 2 mM L-glutamine and 2-ME (50 uM) in the
present different concentration of sG70/APRIL. For the B cell
proliferation assay, plastic wells were coated with purified mouse
anti-human Ig M monoclonal antibody (3 .mu.g/ml, Pharmingen, San
Diego, Calif.). The positive control for T cell stimulation is
IL-2.
[0019] FIG. 7 shows the effect of G70/APRIL on murine T-and B-cell
proliferation in vitro. T-and B-cells from the spleens of C57B1
mice were purified by selection through a murine T-cell and B-cell
enrichment columns. 1.times.10.sup.5 cells per well were cultured
in the absence or presence of various G70/APRIL for 48 hours,
pulsed during the last 18 hours with 0.5 .mu.Ci .sup.3H thymidine
and harvested to count the incorporated radioactivity.
[0020] FIG. 8 shows the effect of G70/APRIL on murine T cell
proliferation costimulated though anti-CD28 antibody. T-cells from
the spleens of C57B1 mice were purified by selection through a
murine T-cell enrichment column. 1.times.10.sup.5 T-cells per well
were treated with G70/APRIL in the absence or presence of
subliminal concentration of anti-CD28 antibody (0.9 .mu.g/ml) for
48 hours, pulsed during the last 18 hours with 0.5 .mu.Ci .sup.3H
thymidine and harvested to count the incorporated
radioactivity.
[0021] FIG. 9 shows the effect of G70/APRIL on murine T cell
proliferation costimulated though anti-CD3 antibody. T-cells from
the spleens of C57B1 mice were purified by selection through a
murine T-cell enrichment column. 1.times..sup.5 T-cells per well
were treated with G70/APRIL in the absence or presence of
subliminal concentration of anti-CD3 antibody (0.9 .mu.g/ml) for 48
hours, pulsed during the last 18 hours with 0.5 .mu.Ci .sup.3H
thymidine and harvested to count the incorporated
radioactivity.
[0022] Table 1 shows FACS analysis of spleen (Table 1A), and
mesenteric lymph nodes (Table 1B) after in vivo systemic
administration of TNF family members. Several members of TNF family
have been tested in vivo, each group have 5 mice (BDF-1, 8 weeks of
age, Dose: 1 mg/kg/day 0.2 ml for 5 days). Spleen, thymus and
mesenteric lymph nodes from three mice of each group have been
isolated for FACS analysis using a panel of T cell and B cell
surface mark antibodies. Results of FACS analysis have been
summarized as following tables.
[0023] FIGS. 10A and 10B show the sequence of human BCMA (SEQ ID
NO: 5). BCMA's extracellular domain (SEQ ID NO: 6) extends from aa
1 to aa 51 and is identified by arrows. The cysteine-rich consensus
region (SEQ ID NO: 7, described further hereinafter) is shown in
boldface. The transmembrane region (SEQ ID NO: 8) is underlined.
huBCMA-Fc (SEQ ID NO: 9). mBCMA-Fc (SEQ ID NO: 10).
[0024] FIG. 11 shows an alignment of human BCMA amino acid sequence
and murine BCMA amino acid sequence (SEQ ID NO: 11). The human
sequence is shown on the top line, the murine on the bottom line in
each row. The human-murine consensus sequence (SEQ ID NO: 12)
appears as the middle line of each row. A "+" in the consensus
sequence indicates a conservative substitution. The cysteine-rich
portion of the consensus sequence (SEQ ID NO: 13) appears in
boldface.
[0025] FIGS. 12A and 12B show the sequence of hTACI (SEQ ID NO:
14). TACI's extracellular domain (SEQ ID NO: 15) extends from aa 1
to aa 166. The cysteine-rich consensus region (SEQ ID NO: 16) is
shown in boldface, and the transmembrane region (SEQ ID NO: 17) is
underlined. hTACI-Fc (SEQ ID NO: 18).
[0026] FIG. 13 shows an alignment of cysteine rich extracellular
regions of human TACI and human BCMA. The BCMA cysteine rich
consensus region (SEQ ID NO: 20) appears as the top line, the TACI
cysteine rich consensus region (SEQ ID NO: 21) appears as the
bottom line of each row. Conserved amino acid residues are
indicated by a vertical bar (I). Related amino acid residues are
indicated with a colon (:).
[0027] FIGS. 14A, 14B and 14C show soluble mouse G70/APRIL binding
to 293 cells expressing the BCMA gene. Human 293 cells transfected
with the pmBCMA and pcDNA3 vectors were incubated with
G70/APRIL-Flag, followed by FITC-conjugated anti-Flag antibody
staining for FACS analysis. A. 293 cells transfected with pcDNA3
vector only. B. 293 cells transfected with antisense pmBCMA vector.
C. 293 cells transfected with sense pmBCMA vector.
[0028] Table 2 shows BIACore analysis of the stoichiometric binding
kinetics of APRIL and AGP-3 to BCMA and TACI. Flag-APRIL
specifically binds to murine and human BCMA with affinities of 0.25
nM and 0.29 nM, respectively, and to human TACI with an affinity of
1.48 nM. Also a longer version of Flag-tagged APRIL (aa 50-240)
binds to BCMA and TACI with high affinity similar to that of
Fc-AGP-3 (Table 2). In separate experiments, we determined that
neither APRIL nor AGP-3 bind to OPG and also that TNF.alpha., OPGL,
LIGHT, TWEAK, and TRAIL do not bind to BCMA or TACI. Hence, APRIL
and AGP-3 specifically bind to both BCMA and TACI with high
affinity.
[0029] FIG. 15 shows G70/APRIL binding to 293 cells expressing the
hTACI gene. Human 293 cells transfected with the phTACI and pcDNA3
vectors were incubated with G70/APRIL-Flag, followed by
FITC-conjugated anti-Flag antibody staining for FACS analysis. In
FIG. 15A, 293 cells were transfected with phTACI vector. In FIG.
15B, 293 cells were transfected with pcDNA3 vector only.
[0030] FIG. 16 shows G70/APRIL completely blocks AGP3 binding to
its receptor. Mouse B lymphoma cells A20 were stained with AGP3-Fc
or plus 10 fold excess G70/APRIL, CD40 ligand, TRAIL ligand and
Tweak. After washing 3 times, cells were incubated with
FITC-conjugated goat anti-human IgG-Fc secondary antibody. In FIG.
16A, 10 fold G70/APRIL completely blocked AGP3 binding to A20
cells. In FIGS. 16B, C and D. 10 fold CD40 ligand, Tweak and TRAIL
do not have that effect on AGP3 binding.
[0031] FIG. 17 shows that soluble TACI receptor (sTACI) binding
competes with G70/APRIL binding to A20 cells. A20 cells were
incubated with G70/APRIL or at same time plus 10 fold soluble TACI,
TRAIL R2 and TRAIL R3 receptor, followed by FITC-conjugated
anti-Flag antibody staining for FACS analysis.
[0032] In FIG. 17A, soluble TACI receptor partially competed in
binding G70/APRIL binding to A20 cells.
[0033] In FIGS. 17B and C, soluble TRAIL R2 and TRAIL R3 receptors
did not interfere with G70/APRIL binding.
[0034] FIG. 18 shows that soluble human BCMA-Fc receptor fusion
protein (shBCMA-Fc) and soluble human TACI-Fc receptor fusion
protein (shTACI-Fc) completely blocks soluble human AGP-3-Fc
receptor fusion protein (shAGP3-Fc) binding to A20 cells. A20 cells
were incubated shAGP3-Fc or at same time plus 10 fold shBCMA-Fc or
shTACI-Fc followed by FITC-conjugated anti-Flag antibody staining
for FACS analysis.
[0035] FIG. 19A shows shBCMA-Fc completely blocks shAGP3-Fc binding
to A20 cells. A20 cells were incubated with shAGP3-Fc with or
without 10 fold soluble hBCMA-Fc followed by FITC-conjugated
anti-Flag antibody staining for FACS analysis.
[0036] FIG. 19B shows that shBCMA-Fc blocks soluble murine APRIL
(smAPRIL) binding to A20 cells. A20 cells were incubated with
smAPRIL with or without 10 fold shBCMA-Fc followed by
FITC-conjugated anti-Flag antibody staining for FACS analysis.
[0037] FIG. 20 shows serum levels of anti-KLH IgG and IgM and
anti-Pneumovax IgM in mice treated with TACI-Fc or BCMA-Fc fusion
proteins or non-fused Fc as a control. p values refer to the
comparison with the Fc-treated group. n=7. See Materials and
Methods hereinafter.
DETAILED DESCRIPTION OF THE INVENTION
[0038] Definition of Terms
[0039] The terms used throughout this specification are defined as
follows, unless otherwise limited in specific instances.
[0040] The term "comprising" means that a compound may include
additional amino acids on either or both of the N- or C-termini of
the given sequence. Of course, these additional amino acids should
not significantly interfere with the activity of the compound.
[0041] "AGF-3 activity" refers to modulation of cell growth,
survival, or activation resulting from binding by natural human
AGP-3 to TACI or BCMA, particularly in B cells. Conversely, "AGP-3
antagonist activity" refers to activity in opposition to AGP-3
activity, as would result, for example, by inhibition of binding of
AGP-3 to TACI or BCMA. Such activity can be determined, for
example, by such assays as described in "Biological activity of
AGP-3" in the Materials & Methods of PCT/US00/03653, which is
hereby incorporated by reference. Additional assays by which AGP-3
activity may be identified appear in the references WO 98/18921
(May 7, 1998); WO 98/27114 (Jun. 25, 1998); EP 869 180 (Oct. 7,
1998); WO 98/55620 and WO 98/55621 (Dec. 10, 1998); WO 99/11791
(Mar. 11, 1999); WO99/12964 (Mar. 18, 1999); and Gross et al.
(2000), Nature 404: 995-9. Any of the assays described therein may
be modified as needed by methods known to persons having ordinary
skill in the art.
[0042] "APRIL activity" refers to modulation of cell growth,
survival, or activation resulting from binding of natural human
APRIL to TACI or BCMA, particularly in T cells. Conversely, "APRIL
antagonist activity" refers to activity in opposition to APRIL
activity, as would result, for example, by inhibition of binding of
APRIL to TACI or BCMA. Such activity can be determined, for
example, by such assays as described in the Materials & Methods
hereinafter. Additional assays by which APRIL activity may be
identified appear in the references WO 99/00518 (Jun. 26, 1997); WO
99/11791 (Sep. 5, 1997); WO 99/12965 (Sep. 12, 1997); EP 911 633
(Oct. 8, 1997); EP 919 620 (Nov. 26, 1997); WO 99/28462 (Dec. 3,
1997); WO 99/33980 (Dec. 30, 1997); WO 99/35170 (Jan. 5, 1998); and
Hahne et al. (1998), J. Exp. Med. 188: 1185-90. Any of the assays
described therein and herein may be modified as needed by methods
known to persons having ordinary skill in the art.
[0043] "BCMA activity" refers to modulation of cell growth,
survival, or activation resulting from binding by natural human
APRIL or natural human AGP-3 to BCMA. Conversely, "BCMA antagonist
activity" refers to activity in opposition to BCMA activity, as
would result, for example, by inhibition of binding of AGP-3 or
APRIL to BCMA. Such activity can be determined, for example, by
such assays as described in the Materials & Methods
hereinafter. Additional assays by which BCMA activity may be
identified appear in the references WO 99/00518 (Jun. 26, 1997); WO
99/11791 (Sep. 5, 1997); WO 99/12965 (Sep. 12, 1997); EP 911 633
(Oct. 8, 1997); EP 919 620 (Nov. 26, 1997); WO 99/28462 (Dec. 3,
1997); WO 99/33980 (Dec. 30, 1997); WO 99/35170 (Jan. 5, 1998);
Hahne et al. (1998), J. Exp. Med. 188: 1185-90; WO 98/18921 (May 7,
1998); WO 98/27114 (Jun. 25, 1998); EP 869 180 (Oct. 7, 1998); WO
98/55620 and WO 98/55621 (Dec. 10, 1998); WO 99/11791 (Mar. 11,
1999); WO99/12964 (Mar. 18, 1999); and Gross et al. (2000), Nature
404: 995-9. Any of the assays described therein and herein may be
modified as needed by methods known to persons having ordinary
skill in the art.
[0044] "TACI activity" refers to modulation of cell growth,
survival, or activation resulting from binding by natural human
AGP-3 or natural human APRIL to TACI. Conversely, "TACI antagonist
activity" refers to activity in opposition to TACI activity, as
would result, for example, by inhibition of binding of AGP-3 or
APRIL to TACI. Such activity can be determined, for example, by
such assays as described in the Materials & Methods of
PCT/US00/03653, WO 98/18921 (May 7, 1998), WO 98/27114 (Jun. 25,
1998), EP 869 180 (Oct. 7, 1998), WO 98/55620 and WO 98/55621 (Dec.
10, 1998), WO 99/11791 (Mar. 11, 1999), WO99/12964 (Mar. 18, 1999),
WO 98/39361 (Sep. 11, 1998), von Bulow & Bram (1997), Science,
278:138-140, and Gross et al. (2000), Nature 404: 995-9. Any of the
assays described therein may be modified as needed by methods known
to persons having ordinary skill in the art.
[0045] The term "specific binding partner" refers to any molecule
that preferentially binds to a protein of interest, regardless of
the antagonistic or agonistic activity of the molecule toward the
protein of interest. Exemplary specific binding partners include
antibodies, solubilized receptors, peptides, modified peptides as
described hereinafter, and the like.
[0046] The term "vehicle" refers to a molecule that prevents
degradation and/or increases half-life, reduces toxicity, reduces
immunogenicity, or increases biological activity of a therapeutic
protein. Exemplary vehicles include an Fc domain (which is
preferred) as well as a linear polymer (e.g., polyethylene glycol
(PEG), polylysine, dextran, etc.); a branched-chain polymer (see,
for example, U.S. Pat. No. 4,289,872 to Denkenwalter et al., issued
Sep. 15, 1981; U.S. Pat. No. 5,229,490 to Tam, issued Jul. 20,
1993; WO 93/21259 by Frechet et al., published Oct. 28, 1993); a
lipid; a cholesterol group (such as a steroid); a carbohydrate or
oligosaccharide; or any natural or synthetic protein, polypeptide
or peptide that binds to a salvage receptor. Vehicles are further
described hereinafter.
[0047] The term "native Fc" refers to molecule or sequence
comprising the sequence of a non-antigen-binding fragment resulting
from digestion of whole antibody, whether in monomeric or
multimeric form. The original immunoglobulin source of the native
Fc is preferably of human origin and may be any of the
immunoglobulins, although IgG1 and IgG2 are preferred. Native Fc's
are made up of monomeric polypeptides that may be linked into
dimeric or multimeric forms by covalent (i.e., disulfide bonds) and
non-covalent association. The number of intermolecular disulfide
bonds between monomeric subunits of native Fc molecules ranges from
1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g.,
IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a native Fc is a
disulfide-bonded dimer resulting from papain digestion of an IgG
(see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9). The
term "native Fc" as used herein is generic to the monomeric,
dimeric, and multimeric forms.
[0048] The term "Fc variant" refers to a molecule or sequence that
is modified from a native Fc but still comprises a binding site for
the salvage receptor, FcRn. International applications WO 97/34631
(published Sep. 25, 1997) and WO 96/32478 describe exemplary Fc
variants, as well as interaction with the salvage receptor, and are
hereby incorporated by reference. Thus, the term "Fc variant"
comprises a molecule or sequence that is humanized from a non-human
native Fc. Furthermore, a native Fc comprises sites that may be
removed because they provide structural features or biological
activity that are not required for the fusion molecules of the
present invention. Thus, the term "Fc variant" comprises a molecule
or sequence that lacks one or more native Fc sites or residues that
affect or are involved in (1) disulfide bond formation, (2)
incompatibility with a selected host cell (3) N-terminal
heterogeneity upon expression in a selected host cell, (4)
glycosylation, (5) interaction with complement, (6) binding to an
Fc receptor other than a salvage receptor, or (7)
antibody-dependent cellular cytotoxicity (ADCC). Fc variants are
described in further detail in WO 00/24782, published May 4, 2000,
which is hereby incorporated by reference in its entirety.
[0049] The term "Fc domain" encompasses native Fc and Fc variant
molecules and sequences as defined above. As with Fc variants and
native Fc's, the term "Fc domain" includes molecules in monomeric
or multimeric form, whether digested from whole antibody or
produced by other means.
[0050] The term "multimer" as applied to Fc domains or molecules
comprising Fc domains refers to molecules having two or more
polypeptide chains associated covalently, noncovalently, or by both
covalent and non-covalent interactions. IgG molecules typically
form dimers; IgM, pentamers; IgD, dimers; and IgA, monomers,
dimers, trimers, or tetramers. Multimers may be formed by
exploiting the sequence and resulting activity of the native Ig
source of the Fc or by derivatizing (as defined below) such a
native Fc.
[0051] The term "dimer" as applied to Fc domains or molecules
comprising Fc domains refers to molecules having two polypeptide
chains associated covalently or non-covalently.
[0052] The terms "derivatizing" and "derivative" or "derivatized"
comprise processes and resulting compounds respectively in which
(1) the compound has a cyclic portion; for example, cross-linking
between cysteinyl residues within the compound; (2) the compound is
cross-linked or has a cross-linking site; for example, the compound
has a cysteinyl residue and thus forms cross-linked dimers in
culture or in vivo; (3) one or more peptidyl linkage is replaced by
a non-peptidyl linkage; (4) the N-terminus is replaced by
--NRR.sup.1, NRC(O)R.sup.1, --NRC(O)OR.sup.1,
--NRS(O).sub.2R.sup.1, --NHC(O)NHR, a succinimide group, or
substituted or unsubstituted benzyloxycarbonyl-NH--, wherein R and
R.sup.1 and the ring substituents are as defined hereinafter; (5)
the C-terminus is replaced by --C(O)R.sup.2 or --NR.sup.3R.sup.4
wherein R.sup.2, R.sup.3 and R.sup.4 are as defined hereinafter;
and (6) compounds in which individual amino acid moieties are
modified through treatment with agents capable of reacting with
selected side chains or terminal residues. Derivatives are further
described hereinafter.
[0053] The term "peptide" refers to molecules of 2 to 40 amino
acids, with molecules of 3 to 20 amino acids preferred and those of
6 to 15 amino acids most preferred. Exemplary peptides may be
randomly generated by any of the methods cited above, carried in a
peptide library (e.g., a phage display library), or derived by
digestion of proteins.
[0054] The term "randomized" as used to refer to peptide sequences
refers to fully random sequences (e.g., selected by phage display
methods) and sequences in which one or more residues of a naturally
occurring molecule is replaced by an amino acid residue not
appearing in that position in the naturally occurring molecule.
Exemplary methods for identifying peptide sequences include phage
display, E. coli display, ribosome display, yeast-based screening,
RNA-peptide screening, chemical screening, rational design, protein
structural analysis, and the like. Randomized peptides and methods
of generating them appear in WO 00/24782, published May 4, 2000,
which is hereby incorporated by reference in its entirety.
[0055] The term "pharmacologically active" means that a substance
so described is determined to have activity that affects a medical
parameter (e.g., T cell proliferation) or disease state (e.g.,
cancer, autoimmune disorders). Thus, pharmacologically active
compounds comprise agonistic or mimetic and antagonistic compounds
as defined below.
[0056] The terms "-mimetic" and "agonist" refer to a molecule
having biological activity comparable to a protein (e.g., APRIL,
AGP-3) that interacts with a protein of interest. These terms
further include molecules that indirectly mimic the activity of a
protein of interest, such as by potentiating the effects of the
natural ligand of the protein of interest.
[0057] The terms "antagonist" or "inhibitor" refer to a molecule
that blocks or in some way interferes with the biological activity
of the associated protein of interest, or has biological activity
comparable to a known antagonist or inhibitor of the associated
protein of interest.
[0058] Additionally, physiologically acceptable salts of the
compounds of this invention are also encompassed herein. By
"physiologically acceptable salts" is meant any salts that are
known or later discovered to be pharmaceutically acceptable. Some
specific examples are: acetate; trifluoroacetate; hydrohalides,
such as hydrochloride and hydrobromide; sulfate; citrate; tartrate;
glycolate; and oxalate.
[0059] Methods of Treatment
[0060] The present invention concerns a method of inhibiting T cell
proliferation in a mammal, which comprises administering a
therapeutic agent comprising:
[0061] a. a specific binding partner for TACI, wherein the specific
binding partner has TACI antagonist activity;
[0062] b. a specific binding partner for BCMA, wherein the specific
binding partner has BCMA antagonist activity;
[0063] c. both a and b; or
[0064] d. a specific binding partner for TACI and BCMA, wherein the
specific binding partner has TACI antagonist activity, BCMA
antagonist activity or both.
[0065] The present invention also concerns a method of inhibiting
APRIL activity in a mammal, which comprises administering a
therapeutic agent comprising a through d above.
[0066] The invention also concerns a method of inhibiting TACI
activity, BCMA activity, or both in a mammal, which comprises
administering a specific binding partner for APRIL. This method may
further comprise administering a specific binding partner for
AGP-3.
[0067] Some indications benefit from an increase in the immune
response.
[0068] Accordingly, the invention further relates to a method of
increasing T cell proliferation in a mammal, which comprises
administering a therapeutic agent comprising:
[0069] a. a specific binding partner for TACI, wherein the specific
binding partner has TACI agonist activity;
[0070] b. a specific binding partner for BCMA, wherein the specific
binding partner has BCMA agonist activity;
[0071] c. both a and b; or
[0072] d. a specific binding partner for TACI and BCMA, wherein the
specific binding partner has TACI agonist activity, BCMA agonist
activity or both.
[0073] The invention also concerns a method of increasing APRIL
activity in a mammal, which comprises administering a therapeutic
agent comprising a through d above.
[0074] The inventors contemplate carrying out the foregoing methods
of treatment with any of several different types of molecules,
including small molecules, antibodies, and engineered peptides and
fusion molecules described hereinafter. These molecules may also be
used in assays to identify cells and tissues that express AGP-3,
TACI, APRIL, or BCMA. The invention further concerns nucleic acids,
vectors, and host cells useful in preparing such molecules.
[0075] The invention further concerns methods of identifying
compounds that are useful in the aforementioned methods of use.
Such compounds include nucleic acids, peptides, proteins,
carbohydrates, lipids or small molecular weight organic molecules
and may act either as agonists or antagonists of BCMA, TACI, AGP-3
or APRIL-protein activity.
[0076] AGP-3, APRIL, BCMA, and TACI are believed to play a role in
regulation of immune function. Accordingly, these molecules, their
soluble forms, and agonists and antagonists thereof may be useful
for the diagnosis and/or treatment of inflammation and immune
function diseases. Indications for antagonists include, but are not
limited to the following:
[0077] infections such as bacterial, fungal, protozoan and viral
infections, especially HIV-1 or HIV-2;
[0078] diarrhorea;
[0079] psoriasis;
[0080] inflammation;
[0081] allergies;
[0082] atopic dermatitis;
[0083] respiratory allergic diseases such as asthma, allergic
rhinitis, hypersensitivity lung disease, hypersensitivity
pneumonitis, eosinophilic pneumonia (e.g. Loeffler's syndrome,
chronic eosinophilic pneumonia, interstitial lung disease (ILD),
such as idiopathic pulmonary fibrosis or ILD associated with
rheumatoid arthritis, systemic lupus erythematosus, ankylosing
spondylitis, systemic sclerosis, Sjogren's syndrome, polymyositis
or dermatomyositis);
[0084] systemic anaphylaxis or hypersensitivity responses;
[0085] drug allergy;
[0086] insect sting allergy;
[0087] inflammatory bowel disease, such as Crohn's disease and
ulcerative colitis;
[0088] spondyloarthropathy;
[0089] scleroderma;
[0090] psoriasis;
[0091] inflammatory dermatosis such as dermatitis, eczema, atopic
dermatitis, allergic contact dermatitis, urticaria, vasculitis
(e.g. necrotizing, cutaneous and hypersensitivity vasculitis),
eosinphilic myositis and eosinophilic fasciitis;
[0092] autoimmune diseases such as rheumatoid arthritis, psoriatic
arthritis, multiple sclerosis, systemic lupus erythematosus,
myasthenia gravis, juvenile onset diabetes, glomerulonephritis,
autoimmune thyroiditis and Behcet's disease;
[0093] graft rejection, including allograft rejection or
graft-versus-host disease;
[0094] cancers with leukocyte infiltration of the skin or
organs;
[0095] reperfusion injury;
[0096] atherosclerosis;
[0097] certain haematologic malignancies;
[0098] shock, including septic shock and endotoxic shock.
[0099] Agonists can be used for treating:
[0100] immunosuppression e.g. in AIDS patients or individuals
undergoing radiation therapy, chemotherapy, therapy for autoimmune
disease or other drug therapy, and immunosuppression due congenital
deficiency in receptor function or other causes; and
[0101] infectious diseases such as parasitic diseases, including
helminth infections, such as nematodes (round worms).
[0102] Compositions of Matter
[0103] Any number of molecules may serve as specific binding
partners within the present invention. Of particular interest are
antibodies, peptides, and Fc-peptide fusion molecules.
[0104] Antibodies.
[0105] The invention also provides for an antibody or antigen
binding domain thereof, or a fragment, variant, or derivative
thereof, which binds to an epitope on any of the target molecules
(APRIL, AGP-3, TACI, or BCMA) and has partial or complete agonist
or antagonist activity. Preferably, the target molecule is
mammalian, more preferably human, and may be in soluble or cell
surface associated forms, or fragments, derivatives and variants
thereof.
[0106] A number of methods for antibody generation are known in the
art. All such methods are useful in generating molecules useful in
accordance with the present invention. Conventionally, an antibody
may be prepared by immunizing an animal with the target molecule
(e.g., murine or human BCMA or TACI) or with an immunogenic
fragment, derivative or variant thereof. In addition, an animal may
be immunized with cells transfected with a vector containing a
nucleic acid molecule encoding the target molecule such that the
target molecule is expressed and associated with the surface of the
transfected cells. Alternatively, specific binding partners that
are antibodies may be obtained by screening a library comprising
antibody or antigen binding domain sequences for binding to the
target molecule. Such a library is conveniently prepared in
bacteriophage as protein or peptide fusions to a bacteriophage coat
protein which are expressed on the surface of assembled phage
particles and the encoding DNA sequences contained within the phage
particles (so-called "phage display library"). In one example, a
phage display library contains DNA sequences encoding human
antibodies, such as variable light and heavy chains. Sequences
binding to the target molecule may be further evolved by multiple
rounds of mutagenesis and screening.
[0107] Specific binding partners that are antibodies or antigen
binding domains may be tetrameric glycoproteins similar to native
antibodies, or they may be single chain antibodies; for example,
Fv, Fab, Fab' or F(ab)' fragments, bispecific antibodies,
heteroantibodies, or other fragments, variants, or derivatives
thereof, which are capable of binding the target molecule and
partially or completely neutralize the target molecule activity.
Antibodies or antigen binding domains may be produced in hybridoma
cell lines (antibody-producing cells such as spleen cells fused to
mouse myeloma cells, for example) or may be produced in
heterologous cell lines transfected with nucleic acid molecules
encoding said antibody or antigen binding domain.
[0108] Antibodies of the invention include polyclonal monospecific
polyclonal, monoclonal, recombinant, chimeric, humanized, fully
human, single chain and/or bispecific antibodies. Antibody
fragments include those portions of an antibody that bind to an
epitope on a target molecule. Examples of such fragments include
Fab F(ab'), F(ab)', Fv, and sFv fragments. The antibodies may be
generated by enzymatic cleavage of full-length antibodies or by
recombinant DNA techniques, such as expression of recombinant
plasmids containing nucleic acid sequences encoding antibody
variable regions.
[0109] Polyclonal antibodies are heterogeneous populations of
antibody molecules derived from the sera of animals immunized with
an antigen. An antigen is a molecule or a portion of a molecule
capable of being bound by an antibody which is additionally capable
of inducing an animal to produce antibody capable of binding to an
epitope of that antigen. An antigen can have one or more epitope.
The specific reaction referred to above is meant to indicate that
the antigen will react, in a highly selective manner, with its
corresponding antibody and not with the multitude of other
antibodies which can be evoked by other antigens.
[0110] Polyclonal antibodies directed toward a target molecule
generally are raised in animals (e.g., rabbits or mice) by multiple
subcutaneous or intraperitoneal injections of the target molecule
and an adjuvant. In accordance with the invention, it may be useful
to conjugate the target molecule, or a variant, fragment, or
derivative thereof to a carrier protein that is immunogenic in the
species to be immunized, such as keyhole limpet heocyanin, serum,
albumin, bovine thyroglobulin, or soybean trypsin inhibitor. Also,
aggregating agents such as alum are used to enhance the immune
response. After immunization, the animals are bled and the serum is
assayed for anti-target antibody titer.
[0111] Monoclonal antibodies (mAbs) contain a substantially
homogeneous population of antibodies specific to antigens, which
population contains substantially similar epitope binding sites.
Such antibodies may be of any immunoglobulin class including IgG,
IgM, IgE, IgA, IgD and any subclass thereof. A hybridoma producing
a monoclonal antibody of the present invention may be cultivated in
vitro, in situ, or in vivo. Production of high titers in vivo or in
situ is a preferred method of production.
[0112] Monoclonal antibodies directed toward the target molecule
are produced using any method which provides for the production of
antibody molecules by continuous cell lines in culture. Examples of
suitable methods for preparing monoclonal antibodies include
hybridoma methods of Kohler et al., Nature 256 495-497 (1975), and
the human B-cell hybridoma method, Kozbor, J. Immunol. 133, 3001
(1984); Brodeur et al., Monoclonal Antibody Production Techniques
and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987);
and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory (1988); the contents of which references are
incorporated entirely herein by reference.
[0113] Preferred specific binding partners include monoclonal
antibodies which will inhibit partially or completely the binding
of the human target molecule to its cognate ligand or receptor or
an antibody having substantially the same specific binding
characteristics, as well as fragments and regions thereof.
Preferred methods for determining monoclonal antibody specificity
and affinity by competitive inhibition can be found in Harlow et
al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1988), Colligan et al., eds.,
Current Protocols in Immunology, Greene Publishing Assoc. and Wiley
Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol.,
92:589-601 (1983). Each of these references is incorporated herein
by reference in its entirety.
[0114] Also provided by the invention are hybridoma cell lines
which produce monoclonal antibodies reactive with target
polypeptides.
[0115] Chimeric antibodies are molecules in which different
portions are derived from different animal species, such as those
having a variable region derived from a murine monoclonal antibody
and a human immunoglobulin constant region. Chimeric antibodies are
primarily used to reduce immunogenicity in application and to
increase yields in production, for example, where murine monoclonal
antibodies have higher yields from hybridomas but higher
immunogenicity in humans, such that human/murine chimeric
monoclonal antibodies are used.
[0116] Chimeric antibodies and methods for their production are
known in the art. Cabilly et al., Proc. Natl. Acad. Sci. USA,
81:3273-3277 (1984); Morrison et al., Proc. Natl. Acad. Sci. USA,
81:6851-6855 (1984); Boulianne et al., Nature 312:643-646 (1984);
Neuberger et al., Nature, 314:268-270 (1985); Liu et al., Proc.
Natl. Acad. Sci. USA, 84:3439-3443 (1987); and Harlow and Lane
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
(1988). These references are incorporated herein by reference in
their entirety.
[0117] A chimeric monoclonal antibody of the invention may be used
as a therapeutic agent. In such a chimeric antibody, a portion of
the heavy and/or light chain is identical with or homologous to
corresponding sequence in antibodies derived from a particular
species or belonging to one particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequence in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity (see U.S. Pat. No. 4,816,567; Morrison
et al., Proc. Natl. Acad. Sci., 81, 6851-6855 (1985).
[0118] As used herein, the term "chimeric antibody" includes
monovalent, divalent or polyvalent immunoglobulins. A monovalent
chimeric antibody is a dimer (HL) formed by a chimeric H chain
associated through disulfide bridges with a chimeric L chain. A
divalent chimeric antibody is tetramer (H.sub.2L.sub.2) formed by
two HL dimers associated through at least one disulfide bridge. A
polyvalent chimeric antibody can also be produced, for example, by
employing a C.sub.H region that aggregates (e.g., from an IgM H
chain, or .mu. chain).
[0119] Murine and chimeric antibodies, fragments and regions of the
present invention may comprise individual heavy (H) and/or light
(L) immunoglobulin chains. A chimeric H chain comprises an antigen
binding region derived from the H chain of a non-human antibody
specific for the target molecule, which is linked to at least a
portion of a human H chain C region (C.sub.H), such as C.sub.1, or
CH.sub.2.
[0120] A chimeric L chain according to the present invention
comprises an antigen binding region derived from the L chain of a
non-human antibody specific for the target molecule, linked to at
least a portion of a human L chain C region (C.sub.L).
[0121] Specific binding partners, such as antibodies, fragments, or
derivatives, having chimeric H chains and L chains of the same or
different variable region binding specificity, can also be prepared
by appropriate association of the individual polypeptide chains,
according to known method steps, t., according to Ausubel et al.,
eds. Current Protocols in Molecular Biology. Wiley Interscience,
N.Y. (1993), and Harlow et al., Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1988). The contents of these references are incorporated entirely
herein by reference. With this approach, hosts expressing chimeric
H chains (or their derivatives) are separately cultured from hosts
expressing chimeric L chains (or their derivatives), and the
immunoglobulin chains are separately recovered and then associated.
Alternatively, the hosts can be co-cultured and the chains allowed
to associate spontaneously in the culture medium, followed by
recovery of the assembled immunoglobulin, fragment or
derivative.
[0122] As an example, the antigen binding region of the specific
binding partner (such as a chimeric antibody) of the present
invention is preferably derived from a non-human antibody specific
for the human analog of the target molecule. Preferred sources for
the DNA encoding such a non-human antibody include cell lines which
produce antibodies, such as hybrid cell lines commonly known as
hybridomas.
[0123] The invention also provides for fragments, variants and
derivatives, and fusions of anti-target antibodies, wherein the
terms "fragments", "variants", "derivatives" and "fusions" are
defined herein. The invention encompasses fragments, variants,
derivatives, and fusions of anti-target antibodies which are
functionally similar to the unmodified antibody, that is, they
retain at least one of the activities of the unmodified antibody.
In addition to the modifications set forth above, also included is
the addition of genetic sequences coding for cytotoxic proteins
such as plant and bacterial toxins. The fragments, variants,
derivatives and fusions of the antibodies can be produced from any
of the hosts of this invention.
[0124] Suitable fragments include, for example, Fab, Fab',
F(ab').sub.2, Fv and scFv. These fragments lack the Fc fragment of
an intact antibody, clear more rapidly from the circulation, and
can have less non-specific tissue binding than an intact antibody.
See Wahl et al., J. Nucl. Med., 24:316-325 (1983). These fragments
are produced from intact antibodies using methods well known in the
art, for example by proteolytic cleavage with enzymes such as
papain (to produce Fab fragments) or pepsin (to produce
F(ab').sub.2 fragments). The identification of these antigen
binding regions and/or epitopes recognized by monoclonal antibodies
of the present invention provides the information necessary to
generate additional monoclonal antibodies with similar binding
characteristics and therapeutic or diagnostic utility that parallel
the embodiments of this invention.
[0125] Variants of specific binding partners are also provided. In
one embodiment, variants of antibodies and antigen binding domains
comprise changes in light and/or heavy chain amino acid sequences
that are naturally occurring or are introduced by in vitro
engineering of native sequences using recombinant DNA techniques.
Naturally occurring variants include "somatic" variants which are
generated in vivo in the corresponding germ line nucleotide
sequences during the generation of an antibody response to a
foreign antigen.
[0126] Variants of antibodies and antigen binding domains are also
prepared by mutagenesis techniques known in the art. In one
example, amino acid changes may be introduced at random throughout
an antibody coding region and the resulting variants may be
screened for a desired activity, such as binding affinity for the
target molecule. Alternatively, amino acid changes may be
introduced in selected regions of an antibody, such as in the light
and/or heavy chain CDRs, and framework regions, and the resulting
antibodies may be screened for binding to the target molecule or
some other activity. Amino acid changes encompass one or more amino
acid substitutions in a CDR, ranging from a single amino acid
difference to the introduction of all possible permutations of
amino acids within a given CDR, such as CDR3. In another method,
the contribution of each residue within a CDR to target binding may
be assessed by substituting at least one residue within the CDR
with alanine (Lewis et al. (1995), Mol. Immunol. 32:1065-72).
Residues which are not optimal for binding to the target molecule
may then be changed in order to determine a more optimum sequence.
Also encompassed are variants generated by insertion of amino acids
to increase the size of a CDR, such as CDR3. For example, most
light chain CDR3 sequences are nine amino acids in length. Light
chain CDR3 sequences in an antibody which are shorter than nine
residues may be optimized for binding to the target molecule by
insertion of appropriate amino acids to increase the length of the
CDR.
[0127] In one embodiment, antibody or antigen binding domain
variants comprise one or more amino acid changes in one or more of
the heavy or light chain CDR1, CDR2 or CDR3 and optionally one or
more of the heavy or light chain framework regions FR1, FR2 or FR3.
Amino acid changes comprise substitutions, deletions and/or
insertions of amino acid residues.
[0128] Variants may also be prepared by "chain shuffling" of either
light or heavy chains. Marks et al. (1992), Biotechnology 10:
779-83. Typically, a single light (or heavy) chain is combined with
a library having a repertoire of heavy (or light) chains and the
resulting population is screened for a desired activity, such as
binding to the target molecule. This technique permits screening of
a greater sample of different heavy (or light) chains in
combination with a single light (or heavy) chain than is possible
with libraries comprising repertoires of both heavy and light
chains.
[0129] The specific binding partners of the invention can be
bispecific. Bispecific specific binding partners of this invention
can be of several configurations. For example, bispecific
antibodies resemble single antibodies (or antibody fragments) but
have two different antigen binding sites (variable regions).
Bispecific antibodies can be produced by chemical techniques (see
e.g., Kranz et al., Proc. Natl. Acad. Sci. USA, 78:5807 (1981)), by
"polydoma" techniques (see U.S. Pat. No. 4,474,893 to Reading) or
by recombinant DNA techniques. For example, a bispecific antibody
in accordance with this invention may bind to APRIL and AGP-3. As
another example, a bispecific antibody may bind to TACI and
BCMA.
[0130] The specific binding partners of the invention may also be
heteroantibodies. Heteroantibodies are two or more antibodies, or
antibody binding fragments (Fab) linked together, each antibody or
fragment having a different specificity.
[0131] The invention also relates to "humanized" antibodies.
Methods for humanizing non-human antibodies are well known in the
art. Generally, a humanized antibody has one or more amino acid
residues introduced into a human antibody from a source which is
non-human. In general, non-human residues will be present in CDRs.
Humanization can be performed following methods known in the art
(Jones et al., Nature 321, 522-525 (1986); Riechmann et al.,
Nature, 332, 323-327 (1988); Verhoeyen et al., Science 239,
1534-1536 (1988)), by substituting rodent
complementarily-determinin- g regions (CDRs) for the corresponding
regions of a human antibody.
[0132] The specific binding partners of the invention, including
chimeric, CDR-grafted, and humanized antibodies can be produced by
recombinant methods known in the art. Nucleic acids encoding the
antibodies are introduced into host cells and expressed using
materials and procedures described herein and known in the art. In
a preferred embodiment, the antibodies are produced in mammalian
host cells, such as CHO cells. Fully human antibodies may be
produced by expression of recombinant DNA transfected into host
cells or by expression in hybridoma cells as described above.
[0133] Techniques for creating recombinant DNA versions of the
antigen-binding regions of antibody molecules which bypass the
generation of monoclonal antibodies are encompassed within the
practice of this invention. To do so, antibody-specific messenger
RNA molecules are extracted from immune system cells taken from an
immunized animal, and transcribed into complementary DNA (cDNA).
The cDNA is then cloned into a bacterial expression system. One
example of such a technique suitable for the practice of this
invention uses a bacteriophage lambda vector system having a leader
sequence that causes the expressed Fab protein to migrate to the
periplasmic space (between the bacterial cell membrane and the cell
wall) or to be secreted. One can rapidly generate and screen great
numbers of functional Fab fragments for those which bind the
antigen. Such target molecule specific binding partners (Fab
fragments with specificity for the target molecule) are
specifically encompassed within the term "antibody" as it is
defined, discussed, and claimed herein.
[0134] Also within the scope of the invention are techniques
developed for the production of chimeric antibodies by splicing the
genes from a mouse antibody molecule of appropriate
antigen-specificity together with genes from a human antibody
molecule of appropriate biological activity, such as the ability to
activate human complement and mediate ADCC. (Morrison et al., Proc.
Natl. Acad. Sci., 81:6851 (1984); Neuberger et al., Nature, 312:604
(1984)). One example is the replacement of a Fc region with that of
a different isotype. Specific binding partners such as antibodies
produced by this technique are within the scope of the
invention.
[0135] In a preferred embodiment of the invention, the antibodies
are fully human antibodies. Thus encompassed by the invention are
antibodies that bind target molecules and are encoded by nucleic
acid sequences which are naturally occurring somatic variants of
human germline immunoglobulin nucleic acid sequence, and fragments,
synthetic variants, derivatives and fusions thereof. Such
antibodies may be produced by any method known in the art.
Exemplary methods include immunization with a target antigen (any
target polypeptide capable of elicing an immune response, and
optionally conjugated to a carrier) of transgenic animals (e.g.,
mice) that are capable of producing a repertoire of human
antibodies in the absence of endogenous immunoglobulin production.
See, for example, Jakobovits et al., Proc. Natl. Acad. Sci., 90
2551-2555 (1993); Jakobovits et al., Nature, 362, 255-258 (1993);
Bruggermann et al., Year in Immunol., 7, 33 (1993).
[0136] Alternatively, human antibodies may be generated through the
in vitro screening of phage display antibody libraries. See
Hoogenboom et al, J. Mol. Biol., 227, 381 (1991); Marks et al., J.
Mol. Biol., 222, 581 (1991), incorporated herein by reference.
Various antibody-containing phage display libraries have been
described and may be readily prepared by one skilled in the art.
Libraries may contain a diversity of human antibody sequences, such
as human Fab, Fv, and scFv fragments, that may be screened against
an appropriate target. As described further below, phage display
libraries may comprise peptides or proteins other than antibodies
which may be screened to identify specific binding partners of the
target molecule.
[0137] An anti-idiotypic (anti-Id) antibody is an antibody which
recognizes unique determinants generally associated with the
antigen-binding site of an antibody. An Id antibody can be prepared
by immunizing an animal of the same species and genetic type (e.g.,
mouse strain) as the source of the monoclonal antibody with the
monoclonal antibody to which an anti-Id is being prepared. The
immunized animal will recognize and respond to the idiotypic
determinants of the immunizing antibody by producing an antibody to
these idiotypic determinants (the anti-Id antibody). See, for
example, U.S. Pat. No. 4,699,880, which is herein entirely
incorporated by reference. The anti-Id antibody may also be used as
an "immunogen" to induce an immune response in yet another animal,
producing a so-called anti-anti-Id antibody. The anti-anti-Id may
be epitopically identical to the original monoclonal antibody which
induced the anti-Id. Thus, by using antibodies to the idiotypic
determinants of a mAb, it is possible to identify other clones
expressing antibodies of identical specificity.
[0138] Peptides and Peptide Fusion Molecules.
[0139] The patent application WO 00/24782, published May 4, 2000,
mentioned previously herein describes in detail various peptide
generation techniques. That patent application further describes
various derivatives and fusion molecules.
[0140] In particular, a peptide used as a specific binding partner
may be comprised within a molecule of the formula
(X.sup.1).sub.a--F.sup.1--(X.sup.2).sub.b
[0141] wherein:
[0142] F.sup.1 is a vehicle;
[0143] X.sup.1 and X.sup.2 are each independently selected from
--(L.sup.1).sub.c--P.sup.1,
--(L.sup.1).sub.c--P.sup.1--(L.sup.2).sub.d--- P.sup.2,
--(L.sup.1).sub.c--P.sup.1--(L.sup.2).sub.d--P.sup.2--(L.sup.3).s-
ub.e--P.sup.3, and
--(L.sup.1).sub.c--P.sup.1--(L.sup.2).sub.d--P.sup.2--(-
L.sup.3).sub.e--P.sup.3--(L.sup.4).sub.f--P.sup.4
[0144] P.sup.1, P.sup.2, P.sup.3, and P.sup.4 are each
independently peptide sequences, wherein at least one is a specific
binding partner;
[0145] L.sup.1, L.sup.2, L.sup.3, and L.sup.4 are each
independently linkers; and
[0146] a, b, c, d, e, and f are each independently 0 or 1, provided
that at least one of a and b is 1.
[0147] Preferably, such a molecule comprises a structure of the
formulae
X.sup.1--F.sup.1
[0148] or
F.sup.1--X.sup.2.
[0149] A more preferred molecule comprises a structure of the
formula
F.sup.1--(L.sup.1).sub.c--P.sup.1.
[0150] or a structure of the formula
F.sup.1--(L.sup.1).sub.c--P.sup.1--(L.sup.2).sub.d--P.sup.2
[0151] wherein P.sup.1 and/or P.sup.2 is a specific binding partner
for TACI or BCMA. Such molecules facilitate modulation of both TACI
and BCMA; for example, one of P.sup.1 and P.sup.2 is a specific
binding partner for TACI and the other is a specific binding
partner for BCMA. Conversely, in a ligand inhibitor, one of P.sup.1
and P.sup.2 is a specific binding partner for APRIL and the other
is a specific binding partner for AGP-3.
[0152] For all of these molecules, the preferred vehicle is an Fc
domain. Among Fc domains, IgG Fc, particularly IgG1, are
preferred.
[0153] The Fc domains, linkers, and processes of preparation of the
foregoing molecules is described in WO 00/24782, published May 4,
2000.
[0154] Soluble Receptor Fragments
[0155] Another class of specific binding partners are soluble
receptor fragments. Of particular interest are the fragments
identified in the figures:
[0156] a. the extracellular region of TACI (SEQ ID NO: 15).
[0157] b. the extracellular region of BCMA (SEQ ID NO: 6).
[0158] c. the consensus region of TACI (SEQ ID NO: 16).
[0159] d. the consensus region of BCMA (SEQ ID NO: 7).
[0160] e. the TACI/BCMA extracellular consensus sequence (SEQ ID
NO: 13).
[0161] These molecules have the heretofore unrecognized advantage
of binding both APRIL and AGP-3. Like the aforementioned peptides,
these specific binding partners may also be covalently linked to a
vehicle, preferably an Fc domain.
[0162] Muteins
[0163] Additional useful peptide sequences may result from
conservative and/or non-conservative modifications of the amino
acid sequences of the aforementioned antibodies, peptides,
Fc-fusion peptides, and receptor fragments.
[0164] Conservative modifications will produce molecules having
functional and chemical characteristics similar to those of the
molecule from which such modifications are made. In contrast,
substantial modifications in the functional and/or chemical
characteristics of the molecules may be accomplished by selecting
substitutions in the amino acid sequence that differ significantly
in their effect on maintaining (a) the structure of the molecular
backbone in the area of the substitution, for example, as a sheet
or helical conformation, (b) the charge or hydrophobicity of the
molecule at the target site, or (c) the size of the molecule.
[0165] For example, a "conservative amino acid substitution" may
involve a substitution of a native amino acid residue with a
nonnative residue such that there is little or no effect on the
polarity or charge of the amino acid residue at that position.
Furthermore, any native residue in the polypeptide may also be
substituted with alanine, as has been previously described for
"alanine scanning mutagenesis" (see, for example, MacLennan et al.,
1998, Acta Physiol. Scand. Suppl. 643:55-67; Sasaki et al., 1998,
Adv. Biophys. 35:1-24, which discuss alanine scanning
mutagenesis).
[0166] Desired amino acid substitutions (whether conservative or
non-conservative) can be determined by those skilled in the art at
the time such substitutions are desired. For example, amino acid
substitutions can be used to identify important residues of the
molecule sequence, or to increase or decrease the affinity of the
molecules described herein. Exemplary amino acid substitutions are
set forth in Table 3.
1TABLE 3 Amino Acid Substitutions Original Exemplary Preferred
Residues Substitutions Substitutions Ala (A) Val, Leu, Ile Val Arg
(R) Lys, Gln, Asn Lys Asn (N) Gln Gln Asp (D) Glu Glu Cys (C) Ser,
Ala Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro, Ala Ala His
(H) Asn, Gln, Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Leu Phe,
Norleucine Leu (L) Norleucine, Ile, Val, Ile Met, Ala, Phe Lys (K)
Arg, 1,4 Diamino- Arg butyric Acid, Gln, Asn Met (M) Leu, Phe, Ile
Leu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro (P) Ala Gly Ser (S)
Thr, Ala, Cys Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp,
Phe, Thr, Ser Phe Val (V) Ile, Met, Leu, Phe, Leu Ala,
Norleucine
[0167] In certain embodiments, conservative amino acid
substitutions also encompass non-naturally occurring amino acid
residues which are typically incorporated by chemical peptide
synthesis rather than by synthesis in biological systems.
[0168] As noted in the foregoing section "Definition of Terms,"
naturally occurring residues may be divided into classes based on
common sidechain properties that may be useful for modifications of
sequence. For example, non-conservative substitutions may involve
the exchange of a member of one of these classes for a member from
another class. Such substituted residues may be introduced into
regions of the molecule that are homologous with non-human
orthologs, or into the non-homologous regions of the molecule. In
addition, one may also make modifications using P or G for the
purpose of influencing chain orientation.
[0169] In making such modifications, the hydropathic index of amino
acids may be considered. Each amino acid has been assigned a
hydropathic index on the basis of their hydrophobicity and charge
characteristics, these are: isoleucine (+4.5); valine (+4.2);
leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine
(-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline
(-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);
aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine
(-4.5).
[0170] The importance of the hydropathic amino acid index in
conferring interactive biological function on a protein is
understood in the art. Kyte et al. J. Mol. Biol., 157: 105-131
(1982). It is known that certain amino acids may be substituted for
other amino acids having a similar hydropathic index or score and
still retain a similar biological activity. In making changes based
upon the hydropathic index, the substitution of amino acids whose
hydropathic indices are within .+-.2 is preferred, those which are
within .+-.1 are particularly preferred, and those within .+-.0.5
are even more particularly preferred.
[0171] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. The greatest local average hydrophilicity of a
protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with its immunogenicity and antigenicity, i.e.,
with a biological property of the protein.
[0172] The following hydrophilicity values have been assigned to
amino acid residues: arginine (+3.0); lysine (+3.0); aspartate
(+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine
(+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline
(.+-.0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0);
methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine
(-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
In making changes based upon similar hydrophilicity values, the
substitution of amino acids whose hydrophilicity values are within
-2 is preferred, those which are within .+-.1 are particularly
preferred, and those within .+-.0.5 are even more particularly
preferred. One may also identify epitopes from primary amino acid
sequences on the basis of hydrophilicity. These regions are also
referred to as "epitopic core regions."
[0173] A skilled artisan will be able to determine suitable
variants of the polypeptide as set forth in the foregoing sequences
using well known techniques. For identifying suitable areas of the
molecule that may be changed without destroying activity, one
skilled in the art may target areas not believed to be important
for activity. For example, when similar polypeptides with similar
activities from the same species or from other species are known,
one skilled in the art may compare the amino acid sequence of a
molecule to similar molecules. With such a comparison, one can
identify residues and portions of the molecules that are conserved
among similar polypeptides. It will be appreciated that changes in
areas of a molecule that are not conserved relative to such similar
molecules would be less likely to adversely affect the biological
activity and/or structure of the molecule. One skilled in the art
would also know that, even in relatively conserved regions, one may
substitute chemically similar amino acids for the naturally
occurring residues while retaining activity (conservative amino
acid residue substitutions). Therefore, even areas that may be
important for biological activity or for structure may be subject
to conservative amino acid substitutions without destroying the
biological activity or without adversely affecting the molecule
structure.
[0174] Additionally, one skilled in the art can review
structure-function studies identifying residues in similar
molecules that are important for activity or structure. In view of
such a comparison, one can predict the importance of amino acid
residues in a molecule that correspond to amino acid residues that
are important for activity or structure in similar molecules. One
skilled in the art may opt for chemically similar amino acid
substitutions for such predicted important amino acid residues of
the molecules.
[0175] One skilled in the art can also analyze the
three-dimensional structure and amino acid sequence in relation to
that structure in similar polymolecules. In view of that
information, one skilled in the art may predict the alignment of
amino acid residues of a molecule with respect to its three
dimensional structure. One skilled in the art may choose not to
make radical changes to amino acid residues predicted to be on the
surface of the protein, since such residues may be involved in
important interactions with other molecules. Moreover, one skilled
in the art may generate test variants containing a single amino
acid substitution at each desired amino acid residue. The variants
can then be screened using activity assays know to those skilled in
the art. Such data could be used to gather information about
suitable variants. For example, if one discovered that a change to
a particular amino acid residue resulted in destroyed, undesirably
reduced, or unsuitable activity, variants with such a change would
be avoided. In other words, based on information gathered from such
routine experiments, one skilled in the art can readily determine
the amino acids where further substitutions should be avoided
either alone or in combination with other mutations.
[0176] A number of scientific publications have been devoted to the
prediction of secondary structure. See Moult J., Curr. Op. in
Biotech., 7(4): 422-427 (1996), Chou et al., Biochemistry, 13(2):
222-245 (1974); Chou et al., Biochemistry, 113(2): 211-222 (1974);
Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol., 47: 45-148
(1978); Chou et al., Ann. Rev. Biochem., 47: 251-276 and Chou et
al., Biophys. T. 26: 367-384 (1979). Moreover, computer programs
are currently available to assist with predicting secondary
structure. One method of predicting secondary structure is based
upon homology modeling. For example, two polypeptides or proteins
which have a sequence identity of greater than 30%, or similarity
greater than 40% often have similar structural topologies. The
recent growth of the protein structural data base (PDB) has
provided enhanced predictability of secondary structure, including
the potential number of folds within a polypeptide's or protein's
structure. See Holm et al., Nucl. Acid. Res., 27(1): 244-247
(1999). It has been suggested (Brenner et al., Curr. Op. Struct.
Biol., 7(3): 369-376 (1997)) that there are a limited number of
folds in a given polypeptide or-protein and that once a critical
number of structures have been resolved, structural prediction will
gain dramatically in accuracy.
[0177] Additional methods of predicting secondary structure include
"threading" (ones, D., Curr. Opin. Struct. Biol., 7(3): 377-87
(1997); Sippl et al., Structure, 4(1): 15-9 (1996)), "profile
analysis" (Bowie et al., Science, 253: 164-170 (1991); Gribskov et
al., Meth. Enzym., 183: 146-159 (1990); Gribskov et al., Proc. Nat.
Acad. Sci., 84(13): 4355-8 (1987)), and "evolutionary linkage" (See
Home, supra, and Brenner, supra).
[0178] Production of Specific Binding Partners
[0179] When the specific binding partner to be prepared is a
proteinaceous specific binding partner, such as an antibody or an
antigen binding domain or an Fc-peptide fusion molecule, various
biological or chemical methods for producing said partner are
available.
[0180] Biological methods are preferable for producing sufficient
quantities of a specific binding partner for therapeutic use.
Standard recombinant DNA techniques are particularly useful for the
production of antibodies and antigen binding domains of the
invention. Exemplary expression vectors, host cells and methods for
recovery of the expressed product are described below.
[0181] A nucleic acid molecule encoding an antibody or antigen
binding domain is inserted into an appropriate expression vector
using standard ligation techniques. The vector is typically
selected to be functional in the particular host cell employed
(i.e., the vector is compatible with the host cell machinery such
that amplification of the gene and/or expression of the gene can
occur). A nucleic acid molecule encoding an antibody may be
amplified/expressed in prokaryotic, yeast, insect (baculovirus
systems) and/or eukaryotic host cells. Selection of the host cell
will depend in part on whether an antibody is to be
post-transitionally modified (e.g., glycosylated and/or
phosphorylated). If so, yeast, insect, or mammalian host cells are
preferable. For a review of expression vectors, see Meth. Enz. v.
185, (D.V. Goeddel, ed.), Academic Press Inc., San Diego, Calif.
(1990).
[0182] Typically, expression vectors used in any host cells will
contain one or more of the following components: a promoter, one or
more enhancer sequences, an origin of replication, a
transcriptional termination sequence, a complete intron sequence
containing a donor and acceptor splice site, a leader sequence for
secretion, a ribosome binding site, a polyadenylation sequence, a
polylinker region for inserting the nucleic acid encoding the
polypeptide to be expressed, and a selectable marker element. Each
of these sequences is discussed in more detail below.
[0183] The vector components may be homologous (i.e., from the same
species and/or strain as the host cell), heterologous (i.e., from a
species other than the host cell species or strain), hybrid (i.e.,
a combination of different sequences from more than one source),
synthetic, or native sequences which normally function to regulate
immunoglobulin expression. As such, a source of vector components
may be any prokaryotic or eukaryotic organism, any vertebrate or
invertebrate organism, or any plant, provided that the components
are functional in, and can be activated by, the host cell
machinery.
[0184] An origin of replication is selected based upon the type of
host cell being used for expression. For example, the origin of
replication from the plasmid pBR322 (Product No. 303-3s, New
England Biolabs, Beverly, Mass.) is suitable for most Gram-negative
bacteria while various origins from SV40, polyoma, adenovirus,
vesicular stomatitus virus (VSV) or papillomaviruses (such as HPV
or BPV) are useful for cloning vectors in mammalian cells.
Generally, the origin of replication component is not needed for
mammalian expression vectors (for example, the SV40 origin is often
used only because it contains the early promoter).
[0185] A transcription termination sequence is typically located 3'
of the end of a polypeptide coding regions and serves to terminate
transcription. Usually, a transcription termination sequence in
prokaryotic cells is a G-C rich fragment followed by a poly T
sequence. While the sequence is easily cloned from a library or
even purchased commercially as part of a vector, it can also be
readily synthesized using methods for nucleic acid synthesis such
as those described above.
[0186] A selectable marker gene element encodes a protein necessary
for the survival and growth of a host cell grown in a selective
culture medium. Typical selection marker genes encode proteins that
(a) confer resistance to antibiotics or other toxins, e.g.,
ampicillin, tetracycline, or kanamycin for prokaryotic host cells,
(b) complement auxotrophic deficiencies of the cell; or (c) supply
critical nutrients not available from complex media. Preferred
selectable markers are the kanamycin resistance gene, the
ampicillin resistance gene, and the tetracycline resistance gene. A
neomycin resistance gene may also be used for selection in
prokaryotic and eukaryotic host cells.
[0187] Other selection genes may be used to amplify the gene which
will be expressed. Amplification is the process wherein genes which
are in greater demand for the production of a protein critical for
growth are reiterated in tandem within the chromosomes of
successive generations of recombinant cells. Examples of suitable
selectable markers for mammalian cells include dihydrofolate
reductase (DHFR) and thymidine kinase. The mammalian cell
transformants are placed under selection pressure which only the
transformants are uniquely adapted to survive by virtue of the
marker present in the vector. Selection pressure is imposed by
culturing the transformed cells under conditions in which the
concentration of selection partner in the medium is successively
changed, thereby leading to amplification of both the selection
gene and the DNA that encodes an antibody. As a result, increased
quantities of an antibody are synthesized from the amplified
DNA.
[0188] A ribosome binding site is usually necessary for translation
initiation of mRNA and is characterized by a Shine-Dalgarno
sequence (prokaryotes) or a Kozak sequence (eukaryotes). The
element is typically located 3' to the promoter and 5' to the
coding sequence of the polypeptide to be expressed. The
Shine-Dalgarno sequence is varied but is typically a polypurine
(i.e., having a high A-G content). Many Shine-Dalgarno sequences
have been identified, each of which can be readily synthesized
using methods set forth above and used in a prokaryotic vector.
[0189] A leader, or signal, sequence is used to direct secretion of
a polypeptide. A signal sequence may be positioned within or
directly at the 5' end of a polypeptide coding region. Many signal
sequences have been identified and may be selected based upon the
host cell used for expression. In the present invention, a signal
sequence may be homologous (naturally occurring) or heterologous to
a nucleic acid sequence encoding an antibody or antigen binding
domain. A heterologous signal sequence selected should be one that
is recognized and processed, i.e., cleaved, by a signal peptidase,
by the host cell. For prokaryotic host cells that do not recognize
and process a native immunoglobulin signal sequence, the signal
sequence is substituted by a prokaryotic signal sequence selected,
for example, from the group of the alkaline phosphatase,
penicillinase, or heat-stable enterotoxin II leaders. For yeast
secretion, a native immunoglobulin signal sequence may be
substituted by the yeast invertase, alpha factor, or acid
phosphatase leaders. In mammalian cell expression the native signal
sequence is satisfactory, although other mammalian signal sequences
may be suitable.
[0190] In most cases, secretion of an antibody or antigen binding
domain from a host cell will result in the removal of the signal
peptide from the antibody. Thus the mature antibody will lack any
leader or signal sequence.
[0191] In some cases, such as where glycosylation is desired in a
eukaryotic host cell expression system, one may manipulate the
various presequences to improve glycosylation or yield. For
example, one may alter the peptidase cleavage site of a particular
signal peptide, or add prosequences, which also may affect
glycosylation. The final protein product may have, in the -1
position (relative to the first amino acid of the mature protein)
one or more additional amino acids incident to expression, which
may not have been totally removed. For example, the final protein
product may have one or two amino acid found in the peptidase
cleavage site, attached to the N-terminus. Alternatively, use of
some enzyme cleavage sites may result in a slightly truncated form
of the desired polypeptide, if the enzyme cuts at such area within
the mature polypeptide.
[0192] The expression vectors of the present invention will
typically contain a promoter that is recognized by the host
organism and operably linked to a nucleic acid molecule encoding an
antibody or antigen binding domain. Either a native or heterologous
promoter may be used depending the host cell used for expression
and the yield of protein desired.
[0193] Promoters suitable for use with prokaryotic hosts include
the beta-lactamase and lactose promoter systems; alkaline
phosphatase, a tryptophan (trp) promoter system; and hybrid
promoters such as the tac promoter. Other known bacterial promoters
are also suitable. Their sequences have been published, thereby
enabling one skilled in the art to ligate them to the desired DNA
sequence(s), using linkers or adapters as needed to supply any
required restriction sites.
[0194] Suitable promoters for use with yeast hosts are also well
known in the art. Yeast enhancers are advantageously used with
yeast promoters. Suitable promoters for use with mammalian host
cells are well known and include those obtained from the genomes of
viruses such as polyoma virus, fowlpox virus, adenovirus (such as
Adenovirus 2), bovine papilloma virus, avian sarcoma virus,
cytomegalovirus, a retrovirus, hepatitis-B virus and most
preferably Simian Virus 40 (SV40). Other suitable mammalian
promoters include heterologous mammalian promoters, e.g.,
heat-shock promoters and the actin promoter.
[0195] Additional promoters which may be used for expressing the
specific binding partners of the invention include, but are not
limited to: the SV40 early promoter region (Benoist and Chambon
(1981), Nature, 290:304-310); the CMV promoter; the promoter
contained in the 3' long terminal repeat of Rous sarcoma virus
(Yamamoto et al. (1980), Cell, 22: 787-97); the herpes thymidine
kinase promoter (Wagner et al. (1981), Proc. Natl. Acad. Sci.
U.S.A., 78: 1444-5); the regulatory sequences of the
metallothionine gene (Brinster et al. (1982), Nature, 296: 39-42):
prokaryotic expression vectors such as the beta-lactamase promoter
(Villa-Kamaroff et al. (1978), Proc. Natl. Acad. Sci. U.S.A., 75:
3727-31); or the tac promoter (DeBoer, et al. (1983), Proc. Natl.
Acad. Sci. U.S.A., 80: 21-25). Also of interest are the following
animal transcriptional control regions, which exhibit tissue
specificity and have been utilized in transgenic animals: the
elastase I gene control region which is active in pancreatic acinar
cells (Swift et al. (1984), Cell, 38: 639-46; Ornitz et al. (1986),
Cold Spring Harbor Symp. Quant. Biol. 50: 399-409; MacDonald
(1987), Hepatology, 7: 425-515); the insulin gene control region
which is active in pancreatic beta cells (Hanahan (1985), Nature,
315: 115-122); the immunoglobulin gene control region which is
active in lymphoid cells (Grosschedl et al. (1984), Cell. 38:
647-58; Adames et al. (1985), Nature, 318: 533-8; Alexander et al.
(1987), Mol. Cell. Biol., 7: 1436-44); the mouse mammary tumor
virus control region which is active in testicular, breast,
lymphoid and mast cells (Leder et al. (1986), Cell, 45: 485-95),
albumin gene control region which is active in liver (Pinkert et
al. (1987), Genes and Devel., 1: 268-76); the alphafetoprotein gene
control region which is active in liver (Krumlauf et al. (1987),
Mol. Cell. Biol., 5: 1639-48; Hammer et al. (1987), Science, 235:
53-58); the alpha 1-antitrypsin gene control region which is active
in the liver (Kelsey et al. (1987), Genes and Devel., 1: 161-171);
the beta-globin gene control region which is active in myeloid
cells (Mogram et al. (1985), Nature, 315: 338-340; Kollias et al.
(1986), Cell, 46: 89-94); the myelin basic protein gene control
region which is active in oligodendrocyte cells in the brain
(Readhead et al. (1987), Cell. 48: 703- 712); the myosin light
chain-2 gene control region which is active in skeletal muscle
(Sani (1985), Nature, 314: 283-286); and the gonadotropic releasing
hormone gene control region which is active in the hypothalamus
(Mason et al. (1986), Science, 234: 1372-8).
[0196] An enhancer sequence may be inserted into the vector to
increase transcription in eucaryotic host cells. Several enhancer
sequences available from mammalian genes are known (e.g., globin,
elastase, albumin, alpha-feto-protein and insulin). Typically,
however, an enhancer from a virus will be used. The SV40 enhancer,
the cytomegalovirus early promoter enhancer, the polyoma enhancer,
and adenovirus enhancers are exemplary enhancing elements for the
activation of eukaryotic promoters. While an enhancer may be
spliced into the vector at a position 5' or 3' to the polypeptide
coding region, it is typically located at a site 5' from the
promoter.
[0197] Preferred vectors for practicing this invention are those
which are compatible with bacterial, insect, and mammalian host
cells. Such vectors include, inter alia, pCRII, pCR3, and pcDNA3.1
(Invitrogen Company, San Diego, Calif.), pBSII (Stratagene Company,
La Jolla, Calif.), pET15 (Novagen, Madison, Wis.), pGEX (Pharmacia
Biotech, Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.),
pETL (BlueBacII; Invitrogen), pDSR-alpha (PCT Publication No.
WO90/14363) and pFastBacDual (Gibco/BRL, Grand Island, N.Y.).
[0198] Additional possible vectors include, but are not limited to,
cosmids, plasmids or modified viruses, but the vector system must
be compatible with the selected host cell. Such vectors include,
but are not limited to plasmids such as Bluescript.RTM. plasmid
derivatives (a high copy number ColE1-based phagemid, Stratagene
Cloning Systems Inc., La Jolla Calif.), PCR cloning plasmids
designed for cloning Taq-amplified PCR products (e.g., TOPO.TM. TA
Cloning.RTM. Kit, PCR2.1.RTM. plasmid derivatives, Invitrogen,
Carlsbad, Calif.), and mammalian, yeast or virus vectors such as a
baculovirus expression system (pBacPAK plasmid derivatives,
Clontech, Palo Alto, Calif.). The recombinant molecules can be
introduced into host cells via transformation, transfection,
infection, electroporation, or other known techniques.
[0199] Host cells of the invention may be prokaryotic host cells
(such as E. coli) or eukaryotic host cells (such as a yeast cell,
an insect cell, or a vertebrate cell). Prokaryotic host cells such
as E. coli produce unglycosylated protein; for example,
unglyclosylated shBCMA and unglycosylated shTACI, which may possess
advantages over the glycosylated eukaryotic molecules. The host
cell, when cultured under appropriate conditions, expresses an
antibody or antigen binding domain of the invention which can
subsequently be collected from the culture medium (if the host cell
secretes it into the medium) or directly from the host cell
producing it (if it is not secreted). Selection of an appropriate
host cell will depend upon various factors, such as desired
expression levels, polypeptide modifications that are desirable or
necessary for activity, such as glycosylation or phosphorylation,
and ease of folding into a biologically active molecule.
[0200] A number of suitable host cells are known in the art and
many are available from the American Type Culture Collection
(ATCC), Manassas, Va. Examples include mammalian cells, such as
Chinese hamster ovary cells (CHO) (ATCC No. CCL61) CHO DHFR-cells
(Urlaub et al. (1980), Proc. Natl. Acad. Sci. USA 97,4216-20),
human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573),
or 3T3 cells (ATCC No. CCL92). The selection of suitable mammalian
host cells and methods for transformation, culture, amplification,
screening and product production and purification are known in the
art. Other suitable mammalian cell lines, are the monkey COS-1
(ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651), and the
CV-1 cell line (ATCC No. CCL70). Further exemplary mammalian host
cells include primate cell lines and rodent cell lines, including
transformed cell lines. Normal diploid cells, cell strains derived
from in vitro culture of primary tissue, as well as primary
explants, are also suitable. Candidate cells may be genotypically
deficient in the selection gene, or may contain a dominantly acting
selection gene. Other suitable mammalian cell lines include but are
not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929
cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK
hamster cell lines, which are available from the American Type
Culture Collection, Manassas, Va.). Each of these cell lines is
known by and available to those skilled in the art of protein
expression.
[0201] Similarly useful as host cells suitable for the present
invention are bacterial cells. For example, the various strains of
E. coli (e.g., HB101, (ATCC No. 33694) DH5Qc, DH10, and MC1061
(ATCC No. 53338)) are well-known as host cells in the field of
biotechnology. Various strains of Pseudomonas spp., B. subtilis,
other Bacillus spp., Streptomyces spp., and the like may also be
employed in this method.
[0202] Many strains of yeast cells known to those skilled in the
art are also available as host cells for expression of the
polypeptides of the present invention. Preferred yeast cells
include, for example, Saccharomyces cerivisae.
[0203] Additionally, where desired, insect cell systems may be
utilized in the methods of the present invention. Such systems are
described for example in Kitts et al. (1993), Biotechniques, 14:
810-7, Lucklow (1993), Curr. Opin. Biotechnol., 4: 564-72, and
Lucklow et al. (1993), T. Virol., 67: 4566-79. Preferred insect
cells are Sf-9 and Hi5 (Invitrogen, Carlsbad, Calif.).
[0204] Transformation or transfection of a nucleic acid molecule
encoding a specific binding partner into a selected host cell may
be accomplished by well known methods including methods such as
calcium chloride, electroporation, microinjection, lipofection or
the DEAE-dextran method. The method selected will in part be a
function of the type of host cell to be used. These methods and
other suitable methods are well known to the skilled artisan, and
are set forth, for example, in Sambrook et al., supra.
[0205] One may also use transgenic animals to express glycosylated
specific binding partners, such as antibodies and antigen binding
domain. For example, one may use a transgenic milk-producing animal
(a cow or goat, for example) and obtain glycosylated binding
partners in the animal milk. Alternatively, one may use plants to
produce glycosylated specific binding partners.
[0206] Host cells comprising (as by transformation or transfection)
an expression vector encoding a specific binding partner of the
target molecule may be cultured using standard media well known to
the skilled artisan. The media will usually contain all nutrients
necessary for the growth and survival of the cells. Suitable media
for culturing E. coli cells are for example, Luria Broth (LB)
and/or Terrific Broth (TB). Suitable media for culturing eukaryotic
cells are RPMI 1640, MEM, DMEM, all of which may be supplemented
with serum and/or growth factors as required by the particular cell
line being cultured. A suitable medium for insect cultures is
Grace's medium supplemented with yeastolate, lactalbumin
hydrolysate, and/or fetal calf serum as necessary.
[0207] Typically, an antibiotic or other compound useful for
selective growth of transfected or transformed cells is added as a
supplement to the media. The compound to be used will be dictated
by the selectable marker element present on the plasmid with which
the host cell was transformed. For example, where the selectable
marker element is kanamycin resistance, the compound added to the
culture medium will be kanamycin. Other compounds for selective
growth include ampicillin, tetracycline and neomycin.
[0208] The amount of an antibody or antigen binding domain produced
by a host cell can be evaluated using standard methods known in the
art. Such methods include, without limitation, Western blot
analysis, SDS-polyacrylamide gel electrophoresis, non-denaturing
gel electrophoresis, HPLC separation, immunoprecipitation, and/or
activity assays.
[0209] Purification of a specific binding partner that has been
secreted into the cell media can be accomplished using a variety of
techniques including affinity, immunoaffinity or ion exchange
chromatography, molecular sieve chromatography, preparative gel
electrophoresis or isoelectric focusing, chromatofocusing, and high
pressure liquid chromatography. For example, antibodies comprising
a Fc region may be conveniently purified by affinity chromatography
with Protein A, which selectively binds the Fc region. Modified
forms of an antibody or antigen binding domain may be prepared with
affinity tags, such as hexahistidine or other small peptide such as
FLAG (Eastman Kodak Co., New Haven, Conn.) or myc (Invitrogen) at
either its carboxyl or amino terminus and purified by a one-step
affinity column. For example, polyhistidine binds with great
affinity and specificity to nickel, thus an affinity column of
nickel (such as the Qiagen.RTM. nickel columns) can be used for
purification of polyhistidine-tagged specific binding partners. See
for example, Ausubel et al., eds. (1993), Current Protocols in
Molecular Biology, Section 10.11.8, John Wiley & Sons, New
York. In some instances, more than one purification step may be
required.
[0210] Specific binding partners of the invention which are
expressed in procaryotic host cells may be present in soluble form
either in the periplasmic space or in the cytoplasm or in an
insoluble form as part of intracellular inclusion bodies. Specific
binding partners can be extracted from the host cell using any
standard technique known to the skilled artisan. For example, the
host cells can be lysed to release the contents of the
periplasm/cytoplasm by French press, homogenization, and/or
sonication followed by centrifugation.
[0211] Soluble forms of an antibody or antigen binding domain
present either in the cytoplasm or released from the periplasmic
space may be further purified using methods known in the art, for
example Fab fragments are released from the bacterial periplasmic
space by osmotic shock techniques.
[0212] If an antibody or antigen binding domain has formed
inclusion bodies, they can often bind to the inner and/or outer
cellular membranes and thus will be found primarily in the pellet
material after centrifugation. The pellet material can then be
treated at pH extremes or with chaotropic partner such as a
detergent, guanidine, guanidine derivatives, urea, or urea
derivatives in the presence of a reducing partner such as
dithiothreitol at alkaline pH or tris carboxyethyl phosphine at
acid pH to release, break apart, and solubilize the inclusion
bodies. The soluble specific binding partner can then be analyzed
using gel electrophoresis, immunoprecipitation or the like. If it
is desired to isolate a solublized antibody or antigen binding
domain, isolation may be accomplished using standard methods such
as those set forth below and in Marston et al. (1990), Meth. Enz.,
182: 264-75.
[0213] In some cases, an antibody or antigen binding domain may not
be biologically active upon isolation. Various methods for
"refolding" or converting the polypeptide to its tertiary structure
and generating disulfide linkages, can be used to restore
biological activity. Such methods include exposing the solubilized
polypeptide to a pH usually above 7 and in the presence of a
particular concentration of a chaotrope. The selection of chaotrope
is very similar to the choices used for inclusion body
solubilization, but usually the chaotrope is used at a lower
concentration and is not necessarily the same as chaotropes used
for the solubilization. In most cases the refolding/oxidation
solution will also contain a reducing partner or the reducing
partner plus its oxidized form in a specific ratio to generate a
particular redox potential allowing for disulfide shuffling to
occur in the formation of the protein's cysteine bridge(s). Some of
the commonly used redox couples include cysteine/cystamine,
glutathione (GSH)/dithiobis GSH, cupric chloride, dithiothreitol
(DTT)/dithiane DTT, and 2-mercaptoethanol (bME) dithio-b(ME). In
many instances, a cosolvent may be used or may be needed to
increase the efficiency of the refolding and the more common
repartners used for this purpose include glycerol, polyethylene
glycol of various molecular weights, arginine and the like.
[0214] Specific binding partners of the invention may also be
prepared by chemical synthesis methods (such as solid phase peptide
synthesis) using techniques known in the art such as those set
forth by Merrifield et al. (1963), J. Am. Chem. Soc., 85: 2149;
Houghten et al. (1985), Proc Natl Acad. Sci. USA 82: 5132; and
Stewart and Young (1984), Solid Phase Peptide Synthesis, Pierce
Chemical Co., Rockford, Ill. Such polypeptides may be synthesized
with or without a methionine on the amino terminus. Chemically
synthesized antibodies and antigen binding domains may be oxidized
using methods set forth in these references to form disulfide
bridges. Antibodies so prepared will retain at least one biological
activity associated with a native or recombinantly produced
antibody or antigen binding domain.
[0215] The invention will now be further described by specific
experimental examples. These examples are meant to be illustrative
rather than limiting.
WORKING EXAMPLES
[0216] Materials and Methods
[0217] Isolation of BCMA and TACI cDNA
[0218] Mouse and human BCMA cDNA were isolated by PCR using the
mouse BCMA sense primer
2 5'-CACAATACCTGTGGCCCTCTTAAGAG-3'(SEQ ID NO: 25),
[0219] and antisense primer
3 5'-TGGTAAACGGTCATCCTAACGACATC-3'(SEQ ID NO:26),
[0220] the human BCMA sense primer
4 5'-TTACTTGTCCTTCCAGGCTGTTCT-3'(SEQ ID NO: 27),
[0221] and antisense primer
5 5'-CATAGAAACCAAGGAAGTTTCTACC-3'(SEQ ID NO:28).
[0222] For isolation of human TACI cDNA, the sense primer
6 5'-AGCATCCTGAGTAATGAGTGGCCTGG-3'(SEQ ID NO: 29)
[0223] and antisense primer
7 5'-GTGATGACGACCTACAGCTGCACTGGG-3'(SEQ ID NO: 30)
[0224] were used. Poly (A)+ RNA from the mouse B lymphoma cell line
-A20 and human lymph Node were reverse-transcribed and cDNA were
synthesized by using the Smart RACE cDNA amplification Kit
(Clontech, palo Alto, Calif.). The full-length cDNA of mouse and
human BCMA genes as well as human TACI gene were cloned into pcDNA3
vector for mammalian cell expression (Invitrogen, Carlsbad,
Calif.).
[0225] Recombinant Proteins
[0226] Soluble murine APRIL-Flag protein was generated by fusing
Flag sequence in frame to the N-terminus of APRIL amino acid
101-239.
[0227] Soluble mAPRIL-Flag protein was expressed in E. coli and the
refolded protein was affinity-purified by anti-Flag M2 antibody
column. Fc-tagged AGP3 protein was generated by fusing OPG signal
peptide followed by human IgG-.cndot.1 Fc in frame to the
N-terminus of AGP3 amino acid 128-285. The protein was expressed in
baculovirus and purified with protein A sepharose column. Soluble
TACI protein (amino acid 1-165) and BCMA protein (amino acid 4-55)
followed by human IgG-.gamma.1 Fc in frame was expressed in E.
coli. The inclusion bodies formed were solubilized. The refolded
protein was purified by cation exchange chromatography.
[0228] In vivo Study
[0229] B6 mice (6-8 weeks old) were purchased from Charles River
Laboratories and murine APRIL-Flag and other TNF proteins were
injected i.p. of 1 mg/kg/day for 5 days. On day 7, cells from mouse
spleens and mesenteric lymph nodes were collected and B and T cell
activation and differentiation was analyzed by FACS using specific
monoclonal antibodies staining.
[0230] Cell Lines and Proliferation Assays
[0231] 293 human kidney epithelial cells, Raji Burkitt lymphoma,
human T lymphoblastoma Jurkat cells and A20, mouse B lymphoma cell
line were purchased from the American Type Culture Collection
(Rockville, Md.).Raji, Jurkat and A20 cells were maintained in a
complete medium of RPMI-1640 (life Technologies) supplemented with
10%fetal bovine serum (HyClone, Logan, Utah) and 25 mM HEPES. 293
cells were cultured in Dulbecco's modified Eagle's medium (Life
Technologies) with 10% fetal bovine serum. The proliferation of
cells were determined by incubating 5.times.10.sup.4 cells/well in
100 .mu.L medium with the indicated concentration of APRIL-flag
protein using the celltiter 96 AQ proliferation assay (Promega
Corp., Madison, Wis.) following the manufacturer's instructions.
Alternatively, cells were pulsed for 18 h with .sup.3H thymidine
(0.5 .mu.Ci/well), after harvesting cells, .sup.3H thymidine
incorporation was monitored by liquid scintillation counting.
[0232] Transfection and Flow Cytometric Analysis
[0233] For 293 cell expressing BCMA and TACI receptor,
2.times.10.sup.6 293 cells were plated into 6 well plate, cells
were transfected with lipofectAMINE 2000 following the
manufacturer's procedure (Life Technologies), 48 h after
transfection, cells were collected and incubated at 4 C with 1
.mu.g/ml APRIL-Flage ligand or Blys (AGP3)-Fc ligand for 60 min,
after washing 3 times with PBS (containing 2% FBS), cells were
stained with FITC-conjugated secondary antibody for 30 min, then
washed 3 times with PBS and fluorescence was analyzed by FACS
scanner (Becton Dickinson, Mountain View, Calif.).
[0234] Determination of the Binding Affinities of APRIL and TALL-1
for BCMA and TACI
[0235] Biomolecular interaction analysis (BIA) was performed using
a BIACORE 2000 (Biacore AB, Uppsala, Sweden). The receptors,
BCMA-Fc and TACI-Fc (2 .mu.g/ml in 10 mM sodium acetate, pH 4.5),
were immobilized on Sensor Chip CM5 using the BIACORE standard
amine coupling procedure. An immobilization level of approximately
120 RU's was achieved. The analytes, Flag-APRIL and Fc-AGP-3 were
diluted between 100 nM-0.01 nM in running buffer (10 mM HEPES, 0.5
M NaCl, 3 mM EDTA, 0.005% Tween 20, 2 mg/ml CM dextran, pH 6.8).
The analytes were injected over an immobilized receptor surface for
2 minutes at 50 .mu.l/min and allowed to dissociate for 10 minutes.
Bound protein was removed by a 1minute injection of 50 mM HCl.
Binding affinities were determined using a 1:1 Langmuir model (BIA
Evaluation software Version 3.1.2, BIACORE).
[0236] T Cell Co-stimulation Assay
[0237] T cells from the spleens of C57 BI/6 mice were purified by
negative selection through a murine T cell enrichment column
(R&D Systems). T cells (1.times.10.sup.5 per well) were
cultured in the absence or presence of various APRIL-Flag protein
for 48 hr. Alternatively, 96 well plates were precoated with
subliminal quantities of anti-CD3 antibody, T cells were treated
with APRIL-Flag protein for 72 hr, pulsed during the last 18 hr
with 1 .mu.Ci of .sup.3H thymidine and harvested to count the
incorporation radioactivity.
[0238] B Cell Proliferation and Ig Secretion
[0239] Mouse B cell were negatively selected from spleens by mouse
B cell recovery column (Cedarlane, Hornby, Ontario Canada).
1.times.10.sup.6/ml were seeded in 96-well flat bottom tissue
culture plates in medium (RPMI-1640, 5% FBS, 5.times.10.sup.-5M 2
ME, affinity-purified goat anti-mouse IgM 2.5 .mu.g/ml Pharmingen,
San Diego). B cells were then treated with APRIL-Flag protein plus
different concentration of soluble BCMA-Fc protein for 72 hr and
culture received 1 .mu.Ci of .sup.3H thymidine during the last 18
hr. proliferation of B cell was quantitated by measuring the
incorporation of radioactivity.
[0240] For analysis of Ig secretion from B cells, purified B cells
5.times.10.sup.5/ml were cultured in 96-well flat bottom tissue
culture plates in the presence of APRIL-Flag for six days. The
culture supernatant were harvested and IgG, IgM and IgA levels were
determined by an isotype specific sandwich ELISA technique. Ig
concentration in test samples were determined by comparing
triplicate test values with isotype control standard.
[0241] Induction and Detection of Anti-keyhole Limpet Hemocyanin
(KLH) and Anti-Pneumovax Antibodies.
[0242] Mice (Balb/c females of 9-11 wk and 19-21 g, Charles River
Laboratories, Wilmington, Mass.) were immunized on day 0 with 100
.mu.g of KLH (Pierce, Rockford, Ill.) in CFA s.c. or with 115 .mu.g
of Pneumovax (Merck, West Point, Pa.) i.p. Starting on day 0, mice
received 7 daily i.p. injections of 5 mg/Kg of either TACI-Fc or
BCMA-Fc fusion proteins or non-fused Fc and were then bled on day
7. Anti-KLH and anti-Pneumovax IgG and IgM were measured in serum
by ELISA. Briefly, for the measurement of anti-KLH antibodies,
plates were coated with KLH in PBS, blocked, and added with
dilutions of standard and test samples. Captured anti-KLH IgG or
IgM were revealed using anti-IgG or anti-IgM biotinylated
antibodies and neutravidin-conjugated HRP. For the measurement of
anti-Pneumovax IgM, plates were coated with Pneumovax using
poly-L-lysine, blocked, and added with dilutions of standard and
test samples. Captured anti-Pneumovax IgM were revealed using an
anti-IgM biotinylated antibody and neutravidin-conjugated HRP.
Results were compared with the Student t test.
[0243] Results
[0244] G70/APRIL in vitro Function
[0245] Human and mouse G70 also called APRIL was isolated and
characterized (FIGS. 1, 2, and 3). FLAG-tagged soluble mouse G70
(smG70) was produced in E. coli purified and refolded (FIG. 2).
Soluble G70 (smG70) specifically stimulates B and T cell lymphoma
cell proliferation in a dose-dependent manner (FIG. 4).
Furthermore, soluble G70:
[0246] 1) specifically binds to cell-surface receptors expressed on
human B and T lymphoma cells (FIG. 5);
[0247] 2) specifically stimulates proliferation of purified human
peripheral blood B and T cells (FIG. 6);
[0248] 3) stimulates proliferation of purified murine spleen B and
T cells in a dose-dependent manner (FIG. 7);
[0249] 4) acts synergistically with anti-CD28 antibody to stimulate
proliferation of purified murine T cells (FIG. 8);
[0250] 5) has a strong costimulatory activity on purified murine T
cells (FIG. 9) in the presence of sub-optimal concentration of the
T cell receptor activator: anti-CD3 antibody.
[0251] G70/APRIL in vivo Function
[0252] A series of experiments were performed to elucidate soluble
G70/APRIL's biological activity in normal mice in vivo. Each group
consisted of 5 mice (BDF-1, 8 weeks of age, dosed at 1 mg/kg/day,
0.2 ml for 5 days). Spleen, thymus and mesenteric lymph nodes from
three mice of each group was used for FACS analysis using a panel
of T cell and B cell surface marker antibodies and all the mice
were analyzed by standard necropsy and pathological analysis.
[0253] Spleen (Table 1A): murine soluble G70 caused an average
about 60% decrease in the percentage of CD3.sup.+ T cells. In
addition, there was an average 5-fold increase in T-helper cells
activation and an average 22-fold increase in cytotoxic T cell
activation as measured by IL-2 receptor expression. In addition the
percentage of immature B cells increased about 2-fold while the
percentage of mature B-cells increased 3- to 4-fold. The total
percentage of lymphocytes (T+B) was unchanged compared to
control.
[0254] Mesenteric Lymph Nodes (Table 1B): soluble G70 treated mice
had an average of 25% decrease in the percentage of T cells. There
was an average 3-fold increase in % activated T-helper cells and
36-fold increase in activated cytotoxic T-cells as measured by
CD25/IL-2 receptor expression. In addition the percentage of
immature B cells was increased on average 2-fold whereas mature B
cells were up on average 4-fold.
[0255] In summary our preliminary observations indicate that
G70/APRIL stimulates both T and B cells in the spleen and
mesenteric lymph nodes. Pathological analysis revealed that soluble
G70 treated mice have slightly enlarged spleens of normal
morphology.
[0256] G70/APRIL is a Ligand for BCMA and TACI
[0257] G70/APRIL is related to the TNF ligand family member
AGP3/BlyS. The TNFR receptor family member TACI (FIG. 12) was
recently shown to be a receptor for AGP3 ([A-570A patent
application ser. no.]). Furthermore, TACI has a match to the orphan
TNFR receptor family member BCMA (FIG. 10) in a conserved
extracellular cysteine rich domain (FIG. 13). These observations
together prompted us to investigate whether G70/APRIL is a ligand
for BCMA and TACI and to test whether in addition to TACI AGP3/BlyS
is also a ligand for BCMA.
[0258] Soluble mouse G70 specifically binds to 293 cells expressing
exogenous BCMA (FIG. 14). G70 also binds to 293 cells expressing
TACI (FIG. 15). Furthermore soluble G70 specifically blocks
AGP3/BLyS binding to cell-surface receptors located on mouse B
lymphoma cells (FIG. 16). This suggest that G70 and AGP3 both binds
to BCMA and TACI.
[0259] smBCMA-Fc and shTACI-Fc prevent G70 and AGP3 Ligand Binding
to Cell-surface Receptors
[0260] Soluble BCMA (smBCMA-Fc; FIG. 10) and soluble TACI
(shTACI-Fc) were produced in E. coli purified to homogeneity and
refolded.
[0261] Soluble TACI receptor specifically prevents G70 from binding
to mouse B cells. (FIG. 17). Furthermore, shBCMA-Fc and shTACI-Fc
both prevent binding of AGP3 to B cells (FIG. 18; and FIG. 19A).
Soluble hBCMA-Fc also ameliorates G70 binding to A20 cells (FIG.
19B).
[0262] In summary: 1) both G70 and AGP3 binds the orphan TNFR
receptor family members TACI and BCMA; 2) soluble BCMA and TACI
both effectively inhibits G70 and AGP3 from binding to B cells; 3)
G70 and AGP3 competes for binding to cell-surface receptors.
[0263] Effects of TACI-Fc and BCMA-Fc Treatment on the Production
of Anti-KLH and Anti-Pneumovax Antibodies.
[0264] Treatment with either TACI-Fc or BCMA-Fc significantly
inhibited the production of anti-KLH and anti-Pneumovax antibodies.
Serum levels of both anti-KLH IgG and IgM were approximately 25%
and 19% lower, respectively, in the TACI-Fc-treated mice than
controls (FIG. 20). Serum anti-KLH IgG and IgM were approximately
52% and 66% lower, respectively, in the BCMA-Fc-treated mice than
controls (FIG. 20). Serum levels of anti-Pneumovax IgM were also
lower in the TACI-Fc- and BCMA-Fc-treated mice than controls (24%
and 42%, respectively, FIG. 20).
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