U.S. patent application number 13/792380 was filed with the patent office on 2013-11-07 for antibodies against a proliferating inducing ligand (april).
This patent application is currently assigned to BIONOVION HOLDING B.V.. The applicant listed for this patent is Marco Guadagnoli, Fiona Clare Kimberley, Jan Paul Medema, Uyen Truong Phan, Hans Van Eenennaam. Invention is credited to Marco Guadagnoli, Fiona Clare Kimberley, Jan Paul Medema, Uyen Truong Phan, Hans Van Eenennaam.
Application Number | 20130295103 13/792380 |
Document ID | / |
Family ID | 42289631 |
Filed Date | 2013-11-07 |
United States Patent
Application |
20130295103 |
Kind Code |
A1 |
Medema; Jan Paul ; et
al. |
November 7, 2013 |
ANTIBODIES AGAINST A PROLIFERATING INDUCING LIGAND (APRIL)
Abstract
The present invention relates to a binding compound which binds
to human APRIL. More specifically the invention provides,
compositions of anti-APRIL specific antibodies and methods to use
such antibodies in modulating the biological activity APRIL,
particularly in inflammatory diseases, inhibition of cell
proliferation and cancer.
Inventors: |
Medema; Jan Paul;
(Amsterdam, NL) ; Van Eenennaam; Hans; (Oss,
NL) ; Guadagnoli; Marco; (Amsterdam, NL) ;
Kimberley; Fiona Clare; (Amsterdam, NL) ; Phan; Uyen
Truong; (Palo Alto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Medema; Jan Paul
Van Eenennaam; Hans
Guadagnoli; Marco
Kimberley; Fiona Clare
Phan; Uyen Truong |
Amsterdam
Oss
Amsterdam
Amsterdam
Palo Alto |
CA |
NL
NL
NL
NL
US |
|
|
Assignee: |
BIONOVION HOLDING B.V.
OSS
NL
|
Family ID: |
42289631 |
Appl. No.: |
13/792380 |
Filed: |
March 11, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13216751 |
Aug 24, 2011 |
|
|
|
13792380 |
|
|
|
|
Current U.S.
Class: |
424/139.1 ;
435/252.33; 435/320.1; 435/331; 435/69.6; 435/7.92; 530/387.3;
530/387.9; 536/23.53 |
Current CPC
Class: |
C07K 2319/30 20130101;
C07K 2319/32 20130101; A61P 29/00 20180101; A61P 35/00 20180101;
A61P 43/00 20180101; C07K 2317/73 20130101; C07K 16/241 20130101;
A61P 37/02 20180101; A61P 37/06 20180101; A61P 37/00 20180101; C07K
2317/34 20130101; G01N 33/6863 20130101; C07K 16/2878 20130101;
C07K 2317/76 20130101; C07K 2317/92 20130101; A61P 37/04 20180101;
C07K 16/2875 20130101; A61K 2039/505 20130101 |
Class at
Publication: |
424/139.1 ;
530/387.9; 530/387.3; 536/23.53; 435/320.1; 435/69.6; 435/7.92;
435/252.33; 435/331 |
International
Class: |
C07K 16/28 20060101
C07K016/28; G01N 33/68 20060101 G01N033/68 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 2, 2009 |
EP |
09154079.9 |
Apr 9, 2009 |
EP |
09157722.1 |
Claims
1. A binding compound which binds to human APRIL comprising: a. an
antibody heavy chain variable region comprising at least one CDR
selected from the group consisting of SEQ ID NOs: 9, 10, 11, 15, 16
and 17, or a variant of any said sequence; and/or b. an antibody
light chain variable region comprising at least one CDR selected
from the group consisting of SEQ ID NOs: 12, 13, 14, 18, 19 and 20,
or a variant of any said sequence.
2. The binding compound of claim 1, comprising: a. heavy chain CDRs
SEQ ID NOs: 9, 10 and 11, or variants of any said sequences; and
light chain CDRs SEQ ID NOs: 12, 13 and 14, or variants of any said
sequences; or b. heavy chain CDRs SEQ ID NOs: 15, 16 and 17 or
variants of any said sequences; and light chain CDRs SEQ ID NOs:
18, 19 and 20 or variants of any said sequences.
3. The binding compound of claim 1 selected from a binding compound
comprising a. a heavy chain variable region comprising the amino
acid sequence of SEQ ID NO: 5 and a light chain variable region
comprising the amino acid sequence selected from the group of SEQ
ID NO: 6; or b. a heavy chain variable region comprising the amino
acid sequence of SEQ ID NO: 7 and a light chain variable region
comprising the amino acid sequence selected from the group of SEQ
ID NO: 8.
4. The binding compound of claim 1, wherein any of said variant(s)
may comprise up to three amino acid modifications.
5. The binding compound of claim 1, wherein the binding compound
fully blocks the binding of APRIL with human TACI and at least
partially blocks the binding with human BCMA.
6. The binding compound of claim 5, wherein the binding compound
fully blocks the binding of human April with human BCMA.
7. The binding compound of claim 1, wherein the binding compound:
a. binds human APRIL with a K.sub.D of about 10 nM or lower; and b.
blocks binding of human TACI and/or human BCMA to human APRIL with
an IC.sub.50 of about 2 nM or lower.
8. A binding compound which binds to human APRIL wherein the
binding compound has the same epitope specificity as the compound
of claim 3.
9. A binding compound which competes for a binding epitope on human
APRIL with the binding compound of claim 3, and: a. binds human
APRIL with a K.sub.D of about 10 nM or lower; b. binds to human
APRIL with about the same K.sub.D as an antibody having a heavy
chain comprising the amino acid sequence of SEQ ID NO: 5 and a
light chain comprising the amino acid sequence of SEQ ID NO: 6; c.
binds to human APRIL with about the same K.sub.D as an antibody
having a heavy chain comprising the amino acid sequence of SEQ ID
NO: 7 and a light chain comprising the amino acid sequence of SEQ
ID NO: 8; or d. blocks binding of human TACI and/or human BCMA to
human APRIL with an IC.sub.50 of about 2 nM or lower.
10. The binding compound of claim 1, wherein the binding compound
is: a. a chimeric antibody or a fragment thereof; b. a human
antibody or a fragment thereof; c. a humanized antibody or a
fragment thereof; or d. an antibody fragment selected from the
group consisting of Fab, Fab', Fab'-SH, Fv, scFv, F(ab').sub.2,
bispecific mAb and a diabody.
11. The binding compound of claim 1, wherein the binding compound
inhibits the proliferation and survival of B-cells.
12. An isolated polynucleotide encoding the binding compound of
claim 1.
13. An expression vector comprising the isolated polynucleotide of
claim 12.
14. A host cell comprising the expression vector of claim 13.
15. A method of producing a binding compound according to claim 1
comprising: a. culturing the host cell of claim 14 in culture
medium under conditions wherein the polynucleotide is expressed,
thereby producing polypeptides comprising the light and heavy chain
variable regions; and b. recovering the polypeptides from the host
cell or culture medium.
16. A composition comprising the binding compound of claim 1 in
combination with a pharmaceutically acceptable carrier or
diluent.
17. Binding compound of claim 1 for use in therapy.
18. A method for inhibiting immune cell proliferation and/or
survival, treating cancer, treating autoimmune disease or treating
inflammatory disease comprising administrating the binding compound
of claim 1.
19. A diagnostic assay for detecting expression of human APRIL by
labeling the binding compound of claim 1 and detecting binding.
Description
INCORPORATION BY REFERENCE
[0001] This application is a continuation-in-part application of
international patent application Serial No. PCT/EP2010/052254,
filed Feb. 23, 2010, which published as PCT Publication No. WO
2010/100056 on Sep. 10, 2010, which claims benefit of European
patent application Serial Nos. 09154079.9, filed Mar. 2, 2009 and
09157722.1, filed Apr. 9, 2009.
[0002] Each of these applications and each of the documents cited
in each of these applications ("application cited documents"), and
each document referenced or cited in the application cited
documents, either in the text or during the prosecution of those
applications, as well as all arguments in support of patentability
advanced during such prosecution, are hereby incorporated herein by
reference. Various documents are also cited in this text ("herein
cited documents"). Each of the herein cited documents, and each
document cited or referenced in the herein cited documents, is
hereby incorporated herein by reference, and may be employed in the
practice of the invention.
FIELD OF THE INVENTION
[0003] The present invention relates to isolated antibodies or
fragments thereof which binds to human A PRoliferating Inducing
Ligand (APRIL), polynucleotides encoding such antibodies and host
cells producing said antibodies. The antibodies can be used to
inhibit immune cell proliferation and/or survival, to treat cancer
and to treat an inflammatory disease.
BACKGROUND OF THE INVENTION
[0004] APRIL is expressed as a type-II transmembrane protein, but
unlike most other TNF family members it is mainly processed as a
secreted protein and cleaved in the Golgi apparatus where it is
cleaved by a furin convertase to release a soluble active form
(Lopez-Fraga et al., 2001, EMBO Rep 2, 945-51,). APRIL assembles as
a non-covalently linked homo-trimer with similar structural
homology in protein fold to a number of other TNF family ligands
(Wallweber et al., 2004, Mol Biol 343, 283-90). APRIL binds two TNF
receptors: B cell maturation antigen (BCMA) and transmembrane
activator and calcium modulator and cyclophilin ligand interactor
(TACI) (reviewed in Kimberley et al., 2009, J Cell Physiol.
218(1):1-8). In addition, APRIL has recently been shown to bind
heparan sulphate proteoglycans (HSPGs) (Hendriks et al., 2005, Cell
Death Differ 12, 637-48).
[0005] APRIL shows high homology (30%) to another member of the TNF
superfamily, B cell activating factor belonging to the TNF family
(BAFF or B Lymphocyte stimulator, BLyS), with which it shares
binding to its receptors, BCMA and TACI. BAFF is also known to bind
a unique receptor, BAFF-Receptor, and through this mediates crucial
survival signals during B cell development (reviewed in Kimberley
et al., 2009, J Cell Physiol. 218(1):1-8). APRIL and BAFF have been
suggested to form mixed trimers (Roschke et al., 2002, J Immunol.
169(8):4314-21). Such mixed trimers were found to occur at a higher
prevalence in rheumatoid arthritis (RA) patients.
[0006] APRIL is predominantly expressed by immune cell subsets such
as monocytes, macrophages, dendritic cells, neutrophils, B-cells,
and T-cells, many of which also express BAFF. In addition, APRIL
can be expressed by non-immune cells such as osteoclasts,
epithelial cells and a variety of tumour tissues (reviewed in
Kimberley et al., 2009, J Cell Physiol. 218(1):1-8).
[0007] The function of APRIL was established using mouse genetic
models. hAPRIL transgenic mice develop normally, but showed
enhanced T cell survival and elevated levels of IgM antibodies
(Stein et al., 2002, J Clin Invest 109, 1587-98). In addition, T
cell independent type II responses were enhanced. Aged hAPRIL
transgenic mice displayed extreme enlargement and re-organisation
of the lymph system and enlarged spleen due to infiltration of CD5
positive B cells, a phenotype closely resembling human B-CLL
(Planelles et al., 2004, Cancer Cell 6, 399-408). APRIL deficient
mice were found to have decreased levels of IgA in circulation and
upon challenge with a T-cell dependent antigen (Castigli et al.,
2004, Proc Natl Acad Sci USA 101, 3903-8; Varfolomeev et al., 2004,
Mol Cell Biol 24, 997-1006). Next, APRIL, along with BAFF, was
demonstrated to function in class-switch recombination (CSR) of
antibodies to both IgG and IgA, independently of CD40-CD40L
signaling (Litinskiy et al., 2002, Nat Immunol 3, 822-9). In
addition, APRIL was demonstrated to be less critical than BAFF in B
cell maintenance, but was shown to have a role in B cell signaling
and drive both proliferation and survival of human and murine B
cells in-vitro (reviewed in Kimberley et al., 2009, J Cell Physiol.
218(1):1-8).
[0008] APRIL was originally identified based on its expression in
cancer cells (Hahne et al., 1998, J Exp Med 188, 1185-90). High
expression levels of APRIL mRNA were found in a panel of tumour
cell lines as well as human primary tumours such as colon, and a
lymphoid carcinoma. In addition, APRIL transfected murine
fibroblast NIH-3T3 cells were shown to grow more rapidly in
immunodeficient mice. More importantly, blocking APRIL using a
soluble APRIL receptor was shown to inhibit tumour growth of lung
and colon carcinomas (Rennert et al., 2000, J Exp Med 192,
1677-84).
[0009] Chronic Lymphocytic Leukaemia (CLL) B cells express both
APRIL and APRIL-receptors. In addition, it was shown that APRIL
protected CLL cells against spontaneous and drug-induced apoptosis
and stimulated NF-.kappa.B activation (reviewed in Kimberley et
al., 2009, J Cell Physiol. 218(1):1-8). A retrospective study under
95 CLL patients showed increased levels of APRIL in serum, which
correlated with disease progression and overall patient survival,
with a poorer prognosis for patients with high APRIL serum levels
(Planelles et al., 2007, Haematologica 92, 1284-5).
[0010] Similarly, (increased levels of) APRIL was shown to be
expressed in Hodgkin's lymphoma, Non-Hodgkin's lymphoma (NHL) and
Multiple Myeloma (MM) (reviewed in Kimberley et al., 2009, J Cell
Physiol. 218(1):1-8). A retrospective study in DLBCL patients (NHL)
showed that high APRIL expression in cancer lesions correlated with
a poor survival rate (Schwaller et al., 2007, Blood 109, 331-8).
Using NHL and MM cell-lines it was shown that treatment with APRIL
or BAFF increased survival via NF-.kappa.B activation and
up-regulation of pro-survival proteins (reviewed in Kimberley et
al., 2009, J Cell Physiol. 218(1):1-8). In accordance with this
pro-survival role of APRIL, MM cells were shown to undergo
apoptosis when cultured in the presence of TACI-Fc. Since
BAFF-receptor was less effective in enhancing apoptosis, this
indicates that APRIL, and not BAFF is primarily responsible for
enhanced survival in these cells (Abe et al., 2006, Leukemia 20,
1313-5).
[0011] APRIL was also found to be over-expressed in a number of
cell lines derived from solid tumours. Indeed, APRIL was able to
stimulate in-vitro proliferation of a number of these cell lines
(reviewed in Kimberley et al., 2009, J Cell Physiol.
218(1):1-8).
[0012] Due to its role in B cell biology APRIL also plays a role in
many autoimmune diseases. Indeed, atacicept (a commercial TACI-Fc
preparation) is already in numerous clinical trials for treatment
of several autoimmune diseases (reviewed in Gatto et al., 2008,
Curr Opin Investig Drugs. 9(11):1216-27). Increased serum levels of
APRIL and BAFF have been reported in many SLE patients (Koyama et
al., 2005, Ann Rheum Dis 64, 1065-7). A retrospective analysis
revealed that APRIL serum levels tended to correlate with
anti-dsDNA antibody titres. Evidence that APRIL may play a
functional role in SLE was obtained by testing the effect of
TACI-Fc fusion protein into lupus prone mice (Gross et al., 2000,
Nature 404, 995-9), which prevented disease development and
prolonged survival.
[0013] In addition, inhibition of APRIL and BAFF with TACI-Fc in
the CIA mouse model of rheumatoid arthritis was also found to
prevent disease progression and lower disease scores, compared with
controls (Gross et al., 2001, Immunity 15, 289-302; Wang et al.,
2001, Nat Immunol 2, 632-7). Also in another arthritis model,
synovium-SCID mouse chimeras, TACI-Fc showed a beneficial effect
(Seyler et al., 2005, J Clin Invest 115, 3083-92). Treatment with
TACI-Fc resulted in the disappearance of Germinal Centers in the
synovial tissue, decreased Ig production and decreased production
of IFN-gamma.
[0014] It was later reported that the synovial fluid of patients
with inflammatory arthritis had significantly increased APRIL
levels compared with those with patients suffering from
non-inflammatory arthritis such as osteoarthritis (Stohl et al.,
2006, Endocr Metab Immune Disord Drug Targets 6, 351-8; Tan et al.,
2003, Arthritis Rheum 48, 982-92).
[0015] Several studies focused on the presence of APRIL in the sera
of patients suffering from a wider range of systemic immune-based
rheumatic diseases (now also including Sjogren's syndrome, Reiter's
syndrome, psoriatic arthritis, polymyositis, and ankylosing
spondylitis) and found significantly increased APRIL levels in
these patients, suggesting an important role for APRIL in these
diseases as well (Jonsson et al., 1986, Scand J Rheumatol Suppl 61,
166-9; Roschke et al., 2002, J Immunol 169, 4314-21).
[0016] Finally, increased APRIL expression has also been linked to
Multiple Sclerosis (MS). APRIL expression was found to be increased
in the astrocytes of MS sufferers compared with normal controls.
This is in line with the described APRIL expression in
glioblastomas and in the serum of glioblastoma patients (Deshayes
et al., 2004, Oncogene 23, 3005-12; Roth et al., 2001, Cell Death
Differ 8, 403-10).
SUMMARY OF THE INVENTION
[0017] APRIL plays a crucial role in the survival and proliferative
capacity of several B-cell malignancies, and potentially also some
solid tumours. APRIL is also emerging as a key player in
inflammatory diseases or autoimmunity. Thus, strategies to
antagonise APRIL are a therapeutic goal for a number of these
diseases. Indeed clinical studies targeting APRIL with TACI-Fc
(Atacicept) are currently ongoing for treatment of several
autoimmune diseases. However, TACI-Fc also targets BAFF, a factor
involved in normal B-cell maintenance. Antibodies directed against
APRIL have been described in WO9614328, WO2001/60397, WO2002/94192,
WO9912965, WO2001/196528 and WO9900518. This invention describes
antibodies targeting APRIL specifically. The antibodies in this
invention fully block the binding of APRIL to TACI and at least
partially to BCMA. Some antibodies according to the invention fully
block the binding to both BCMA and TACI. Such molecules are useful
in a therapy for a number of conditions in which circulating
soluble APRIL correlates with disease activity and progression.
Since expression levels of APRIL can be used as diagnostic and
prognostic markers for different diseases, these antibodies can
also be applied in such tests.
[0018] The invention provides binding compounds which include but
are not limited to compounds such as isolated antibodies or
antibody fragments which bind to human APRIL.
[0019] In some embodiments the binding compound blocks binding to
TACI and BCMA. In some embodiments, the APRIL binding compound of
the invention includes one or more of the antibody CDRs
(Complementary Determining Regions) selected from SEQ ID NOs: 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20; and in further
embodiments, includes one or more antibody light chain CDRs of SEQ
ID NOs:12, 13, 14, 18, 19 and 20 and/or antibody heavy chain CDRs
of SEQ ID NOs: 9, 10, 11, 15, 16 and 17. In some embodiments, the
binding compound is a chimeric antibody, human antibody, humanized
antibody or a fragment thereof.
[0020] In one embodiment, the invention provides a binding compound
which bind to human APRIL comprising antibody heavy chain CDRs SEQ
ID NOs: 9, 10 and 11, or variants of any said sequences; and
antibody light chain CDRs SEQ ID NOs: 12, 13 and 14, or variants of
any said sequences.
[0021] In another embodiment, the invention provides a binding
compound which bind to human APRIL comprising antibody heavy chain
CDRs SEQ ID NOs: 15, 16 and 17 or variants of any said sequences;
and antibody light chain CDRs SEQ ID NOs: 18, 19 and 20 or variants
of any said sequences.
[0022] In another embodiment, the invention comprises a binding
compound which bind to human APRIL comprising an antibody heavy
chain variable region comprising the amino acid sequence of SEQ ID
NO: 5 and a antibody light chain variable region comprising the
amino acid sequence selected from the group of SEQ ID NO: 6.
[0023] In yet another embodiment, the invention comprises a binding
compound which bind to human APRIL comprising a antibody heavy
chain variable region comprising the amino acid sequence of SEQ ID
NO: 7 and a antibody light chain variable region comprising the
amino acid sequence of SEQ ID NO: 8.
[0024] In another embodiment the invention comprises an antibody,
wherein the heavy chain has the variable region sequence of SEQ ID
NO: 5 and is joined to a IgG1 constant region and the light chain
has the sequence of SEQ ID NO: 6 and is joined to the .kappa.
constant region. In particular, the constant region is from mouse
or human origin. More in particular, the antibody is
hAPRIL.01A.
[0025] In another embodiment the invention comprises an antibody,
wherein the heavy chain has the variable region sequence of SEQ ID
NO: 7 and is joined to a IgG1 constant region and the light chain
has the sequence of SEQ ID NO: 8 and is joined to the .kappa.
constant region. In particular, the constant region is from mouse
or human origin. More in particular, the antibody is
hAPRIL.03A.
[0026] In another embodiment the invention comprises a variant of a
binding compound which bind to human APRIL, wherein any of said
variant(s) may comprise up to three amino acid modifications in the
previous identified CDRs of each the antibody heavy and light chain
variable regions.
[0027] In another embodiment the invention comprises a variant of a
binding compound which binds to human APRIL, wherein any of said
variant(s) may comprise up to three amino acid modifications in
each of the previous identified CDRs in each of the antibody heavy
and light chain variable regions.
[0028] In another embodiment the invention comprises a variant of a
binding compound which binds to human APRIL, wherein any of said
variant(s) may comprise up to three amino acid modifications in the
previous identified CDR sequences in each of the antibody heavy and
light chain variable regions.
[0029] The invention also comprises a binding compound that fully
blocks the binding of APRIL with human TACI and at least partially
blocks the binding with human BCMA.
[0030] In another embodiment the invention comprises a binding
compound that fully blocks the binding of APRIL with human TACI and
with human BCMA.
[0031] In another embodiment the invention comprises a binding
compound which bind to human APRIL, wherein the binding compound
binds human APRIL with a K.sub.D of about 10 nM or lower; and
blocks binding of human TACI and/or human BCMA to human APRIL with
an IC.sub.50 of about 2 nM or lower.
[0032] The invention also comprises a binding compound which binds
to human APRIL wherein the binding compound has the same epitope
specificity as the antibodies described above i.e. competes for the
binding epitope of the antibodies described above.
[0033] In some embodiments the invention comprises a binding
compound which competes for a binding epitope on human APRIL with
any of the antibodies described above, and binds human APRIL with a
K.sub.D of about 10 nM or lower. In particular, the epitope on
human APRIL is the epitope which bind to the antibodies hAPRIL.01A
and hAPRIL.03A, preferably hAPRIL.01A.
[0034] In another embodiment the invention comprises a binding
compound which competes for a binding epitope on human APRIL with
any of the antibodies described above and binds to human APRIL with
about the same K.sub.D as an antibody having a heavy chain
comprising the amino acid sequence of SEQ ID NO: 5 and a light
chain comprising the amino acid sequence of SEQ ID NO: 6.
[0035] In another embodiment the invention comprises a binding
compound which competes for a binding epitope on human APRIL with
any of the compounds described above and binds to human APRIL with
about the same K.sub.D as an antibody having a heavy chain
comprising the amino acid sequence of SEQ ID NO: 7 and a light
chain comprising the amino acid sequence of SEQ ID NO: 8.
[0036] In another embodiment the invention comprises a binding
compound which competes for a binding epitope on human APRIL with
any of the antibodies described above and blocks binding of human
TACI and/or human BCMA to human APRIL with an IC.sub.50 of about 2
nM or lower.
[0037] In another embodiment the invention comprises a binding
compound which binds to the conformational human APRIL epitope
SMPSHP (preferably IRSMPSHPDRA) optionally supported by TLFR and/or
QDVTFTMGQ.
[0038] In yet another embodiment the invention comprises a binding
compound which binds to the conformational human APRIL epitope
VSREGQGRQ optionally supported by TFTMGQ.
[0039] In some embodiments the binding compound of the invention is
a chimeric antibody or a fragment thereof.
[0040] In another embodiment the binding compound of the invention
is a human antibody or a fragment thereof.
[0041] In another embodiment the binding compound of the invention
is a humanized antibody or a fragment thereof.
[0042] In another embodiment the invention comprises a binding
compound, preferably a humanized antibody, with the above
identified CDR's and a human heavy chain constant region variant
and a human light chain constant region variant, wherein each
constant region variant comprises up to 20 conservatively modified
amino acid substitutions.
[0043] In another embodiment the binding compound of the invention
is an antibody fragment selected from Fab, Fab', Fab'-SH, Fv, scFv,
F(ab').sub.2, bispecific mAb or a diabody fragment.
[0044] The invention also comprises the binding compound as
described above which inhibits the proliferation and survival of
B-cells.
[0045] The invention also comprises nucleic acids encoding the
anti-APRIL binding compound of the invention. Included in the
invention are nucleic acids encoding any one of the amino acid
sequences enclosed in SEQ ID NOS: 5 to 20. Also included within the
invention are nucleic acids comprising SEQ ID NOS 1, 2, 3 or 4. In
addition, the invention also comprises the nucleic acids encoding
the variants of the amino acid sequences as described
hereinabove.
[0046] The invention also comprises cells and expression vectors
comprising nucleic acids encoding the binding compound of the
invention.
[0047] Further, the invention comprises a method of producing a
binding compound of the invention comprising: (a) culturing the
host cell comprising a nucleic acid encoding an antibody or
antibody fragment of the invention in culture medium under
conditions wherein the nucleic acid sequence is expressed, thereby
producing polypeptides comprising the light and heavy chain
variable regions; and (b) recovering the polypeptides from the host
cell or culture medium.
[0048] The invention also comprises compositions comprising a
binding compound of the invention in combination with a
pharmaceutically acceptable carrier or diluent.
[0049] The invention also comprises a method of inhibiting the
proliferation and/or survival of an immune cell, comprising
administering to a subject in need thereof a therapeutically
effective amount of a binding compound of the invention. In one
embodiment, the method may be used to treat cancer. In another
embodiment, the method may be use to treat an autoimmune or
inflammatory disease.
[0050] In some embodiments, the invention comprises a method of
inhibiting the proliferation and/or survival of an immune cell,
comprising administering to a subject in need thereof a
therapeutically effective amount of a binding compound of the
invention, and further comprising measuring B cell proliferation
and/or survival ex vivo in a sample derived from the subject,
wherein an inhibition of the proliferation and/or survival of the B
cell indicates that the treatment should be continued.
[0051] In other embodiments, the invention comprises a method of
inhibiting the proliferation and/or survival of an immune cell,
comprising administering to a subject in need thereof a
therapeutically effective amount of a binding compound of the
invention, and further comprising measuring B cell proliferation
and/or survival ex vivo in a sample derived from the subject,
wherein an increase in B cell proliferation and/or survival
predicts the likelihood that the treatment will be successful.
[0052] The invention also comprises an immunoconjugate comprising
an anti-APRIL binding compound of the invention, linked to a
therapeutic agent such as a bacterial toxin or a radiotoxin.
Non-limiting examples of cytotoxic agents include taxol,
cytochalasin B, mitomycin, etoposide and vincristine or other
antimetabolites, alkylating agents, antibiotics and
antimitotics.
[0053] The invention also comprises a method of inhibiting the
proliferation and/or survival of an immune cell, comprising
contacting an immune cell with a binding compound of the present
invention.
[0054] In some embodiments the method comprises further
administering a second therapeutic agent or treatment modality.
[0055] In some embodiments, anti-APRIL binding compounds can be
combined with a treatment that is considered to be standard of care
in cancer or autoimmune or inflammatory disease. Rationale for such
combinations is that concurrent increased immune inhibition by
anti-APRIL will induce or facilitate initial clinical response to
standard of care treatment, induce durable clinical response and
long-term immune control of disease.
[0056] In another embodiment the binding compounds of the present
invention are used diagnostically.
[0057] In yet another embodiment the binding compounds of the
invention are used to measure B cell proliferation and/or survival
ex vivo in a sample derived from the subject, wherein an inhibition
of the proliferation and/or survival of the B cell indicates that
the treatment with the binding compound as described here above
should be continued.
[0058] In another embodiment the binding compounds according to the
invention are isolated antibodies or antibody fragments which bind
to human APRIL.
[0059] Accordingly, it is an object of the invention to not
encompass within the invention any previously known product,
process of making the product, or method of using the product such
that Applicants reserve the right and hereby disclose a disclaimer
of any previously known product, process, or method. It is further
noted that the invention does not intend to encompass within the
scope of the invention any product, process, or making of the
product or method of using the product, which does not meet the
written description and enablement requirements of the USPTO (35
U.S.C. .sctn.112, first paragraph) or the EPO (Article 83 of the
EPC), such that Applicants reserve the right and hereby disclose a
disclaimer of any previously described product, process of making
the product, or method of using the product.
[0060] It is noted that in this disclosure and particularly in the
claims and/or paragraphs, terms such as "comprises", "comprised",
"comprising" and the like can have the meaning attributed to it in
U.S. patent law; e.g., they can mean "includes", "included",
"including", and the like; and that terms such as "consisting
essentially of" and "consists essentially of" have the meaning
ascribed to them in U.S. patent law, e.g., they allow for elements
not explicitly recited, but exclude elements that are found in the
prior art or that affect a basic or novel characteristic of the
invention.
[0061] These and other embodiments are disclosed or are obvious
from and encompassed by, the following Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0062] The following detailed description, given by way of example,
but not intended to limit the invention solely to the specific
embodiments described, may best be understood in conjunction with
the accompanying drawings, in which:
[0063] FIG. 1 shows APRIL reactivity and BCMA-blocking activity of
hAPRIL.01A and hAPRIL.03A hybridoma supernatants. FIG. 1A shows
hAPRIL.01A and hAPRIL.03A binding to FLAG-hAPRIL captured by an
anti-FLAG antibody. Aprily-5 antibody was used as a positive
control. FIG. 1B demonstrates that hAPRIL.01A and hAPRIL.03A
hybridoma supernatants, and not Aprily-5 block the binding of
FLAG-hAPRIL to BCMA-Fc.
[0064] FIG. 2 shows distinct binding and receptor-blocking
characteristics of purified hAPRIL.01A and hAPRIL.03A antibodies.
FIG. 2A confirms binding of purified hAPRIL.01A and hAPRIL.03A to
FLAG-hAPRIL, captured by an anti-FLAG antibody. FIG. 2B shows that
only hAPRIL.03A binds FLAG-hAPRIL that is captured by BCMA-Fc. FIG.
2C shows that hAPRIL.01A fully blocks FLAG-hAPRIL binding to
BCMA-Fc, while hAPRIL.03A partially blocks this interaction. FIG.
2D demonstrates that hAPRIL.01A and hAPRIL.03A both fully block
FLAG-hAPRIL with TACI-Fc.
[0065] FIG. 3 shows the receptor-blocking ELISAs for hAPRIL.01A,
hAPRIL.03A, and 12 known commercially available monoclonal
anti-APRIL antibodies. This illustrates that hAPRIL.01A and
hAPRIL.03A are unique in their ability to block APRIL binding to
BCMA (FIG. 3A) and TACI (FIG. 3B).
[0066] FIG. 4 shows that hAPRIL.01A and hAPRIL.03A block
APRIL-driven B-cell proliferation and isotype class-switching but
do not affect BAFF-mediated processes. FIG. 4A is an in-vitro
B-cell assay which demonstrates that the described monoclonal
antibodies block known APRIL functions such as the survival and
proliferation of B cells and production of class-switched IgA
antibodies. Of significance is the demonstration that both
monoclonal antibodies block APRIL activity more effectively than
TACI-Fc, which was administered at equimolar concentration. FIG. 4B
shows that the antibodies do not affect BAFF-driven B cells
responses, while TACI-Fc blocks these processes.
[0067] FIG. 5 shows the results of targeting APRIL with hAPRIL.01A
and hAPRIL.03A (panel A) or TACI-Fc (panel B) in-vivo, in a
T-independent B cell response. Transgenic mice were challenged with
NP-Ficoll, and treated with hAPRIL.01A, hAPRIL.03A and TACI-Fc
twice per week. PBS and mouse IgG1 were used as negative controls.
The immunoglobulin titres (IgA, IgM and IgG) were measured by
ELISA. hAPRIL.01A, hAPRIL.03A and to a lesser extent TALI-Fc are
able to inhibit APRIL mediated B cell responses in the hAPRIL
transgenic mice and reduce immunoglobulin levels to that of the
WT.
[0068] FIG. 6 shows the effect of targeting APRIL with hAPRIL.01A,
hAPRIL.03A and TACI-Fc on B-cell populations in the spleen (panel
A) or peritoneal cavity (panel B). Transgenic mice were challenged
with NP-Ficoll, and treated with hAPRIL.01A, hAPRIL.03A, TACI-Fc
twice per week. PBS and mouse IgG1 were used as negative controls.
After 30 days of treatment, spleens and cells from the peritoneal
cavity were harvested and analyzed by flow cytometry. Treatment
with hAPRIL.01A or hAPRIL.03A did not affect the (sub)population of
B-cells in the spleen. In contrast, TACI-Fc strongly reduced the
total B-cell population and mature and T2 subpopulations. In the
peritoneal cavity, TACI-Fc affected the ratio of B1 vs. B2-cells,
while hAPRIL.01A and hAPRIL.03A did not affect these
subpopulations.
[0069] FIG. 7 shows the variable region sequences of hAPRIL.01A and
hAPRIL.03A. FIGS. 7A and 7B show the amino acid sequences of the
heavy and light chain variable sequence of hAPRIL.01A,
respectively. FIGS. 7C and 7D shows the amino acid sequences of the
heavy and light chain variable sequence of hAPRIL.03A,
respectively.
DETAILED DESCRIPTION
[0070] The term "antibody" refers to any form of antibody that
exhibits the desired biological activity, such as inhibiting
binding of a ligand to its receptor, or by inhibiting
ligand-induced signaling of a receptor. Thus, "antibody" is used in
the broadest sense and specifically covers, but is not limited to,
monoclonal antibodies (including full length monoclonal
antibodies), polyclonal antibodies, and multispecific antibodies
(e.g., bispecific antibodies).
[0071] "Antibody fragment" and "antibody binding fragment" mean
antigen-binding fragments and analogues of an antibody, typically
including at least a portion of the antigen binding or variable
regions (e.g. one or more CDRs) of the parental antibody. An
antibody fragment retains at least some of the binding specificity
of the parental antibody. Typically, an antibody fragment retains
at least 10% of the parental binding activity when that activity is
expressed on a molar basis. Preferably, an antibody fragment
retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of
the parental antibody's binding affinity for the target. Examples
of antibody fragments include, but are not limited to, Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; linear antibodies;
single-chain antibody molecules, e.g., sc-Fv, unibodies (technology
from Genmab); nanobodies (technology from Domantis); domain
antibodies (technology from Ablynx); and multispecific antibodies
formed from antibody fragments. Engineered antibody variants are
reviewed in Holliger and Hudson, 2005, Nat. Biotechnol. 23,
1126-1136.
[0072] A "Fab fragment" is comprised of one light chain and the
C.sub.H1 and variable regions of one heavy chain. The heavy chain
of a Fab molecule cannot form a disulfide bond with another heavy
chain molecule.
[0073] An "Fc" region contains two heavy chain fragments comprising
the C.sub.H1 and C.sub.H2 domains of an antibody. The two heavy
chain fragments are held together by two or more disulfide bonds
and by hydrophobic interactions of the CH3 domains.
[0074] A "Fab' fragment" contains one light chain and a portion of
one heavy chain that contains the V.sub.H domain and the C.sub.H1
domain and also the region between the C.sub.H1 and C.sub.H.sup.2
domains, such that an interchain disulfide bond can be formed
between the two heavy chains of two Fab' fragments to form a
F(ab').sub.2 molecule.
[0075] A "F(ab').sub.2 fragment" contains two light chains and two
heavy chains containing a portion of the constant region between
the C.sub.H1 and C.sub.H.sup.2 domains, such that an interchain
disulfide bond is formed between the two heavy chains. A
F(ab').sub.2 fragment thus is composed of two Fab' fragments that
are held together by a disulfide bond between the two heavy
chains.
[0076] The "Fv region" comprises the variable regions from both the
heavy and light chains, but lacks the constant regions.
[0077] A "single-chain Fv antibody" (or "scFv antibody") refers to
antibody fragments comprising the V.sub.H and V.sub.L domains of an
antibody, wherein these domains are present in a single polypeptide
chain. Generally, the Fv polypeptide further comprises a
polypeptide linker between the V.sub.H and V.sub.L domains which
enables the scFv to form the desired structure for antigen binding.
For a review of scFv, see Pluckthun,1994, THE PHARMACOLOGY OF
MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds.
Springer-Verlag, New York, pp. 269-315. See also, International
Patent Application Publication No. WO 88/01649 and U.S. Pat. Nos.
4,946, 778 and 5,260,203.
[0078] A "diabody" is a small antibody fragment with two
antigen-binding sites. The fragments comprises a heavy chain
variable domain (V.sub.H) connected to a light chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L or
V.sub.L-V.sub.H). By using a linker that is too short to allow
pairing between the two domains on the same chain, the domains are
forced to pair with the complementary domains of another chain and
create two antigen-binding sites. Diabodies are described more
fully in, e.g., EP 404,097; WO 93/11161; and Holliger et al., 1993,
Proc. Natl. Acad. Sci. USA 90, 6444-6448.
[0079] A "domain antibody fragment" is an immunologically
functional immunoglobulin fragment containing only the variable
region of a heavy chain or the variable region of a light chain. In
some instances, two or more V.sub.H regions are covalently joined
with a peptide linker to create a bivalent domain antibody
fragment. The two V.sub.H regions of a bivalent domain antibody
fragment may target the same or different antigens.
[0080] As used herein antibody hAPRIL.01A is a mouse antibody
wherein the heavy chain has the variable region sequence of SEQ ID
NO: 5 and is joined to a IgG1 constant region and the light chain
has the variable region sequence of SEQ ID NO: 6 and is joined to
the .kappa. constant region. Antibody hAPRIL.03A is a mouse
antibody, wherein the heavy chain has the variable region sequence
of SEQ ID NO: 7 and is joined to a IgG1 constant region and the
light chain has the variable region sequence of SEQ ID NO: 8 and is
joined to the .kappa. constant region.
[0081] An antibody fragment of the invention may comprise a
sufficient portion of the constant region to permit dimerization
(or multimerization) of heavy chains that have reduced disulfide
linkage capability, for example where at least one of the hinge
cysteines normally involved in inter-heavy chain disulfide linkage
is altered as described herein. In another embodiment, an antibody
fragment, for example one that comprises the Fc region, retains at
least one of the biological functions normally associated with the
Fc region when present in an intact antibody, such as FcRn binding,
antibody half life modulation, ADCC (antibody dependent cellular
cytotoxicity) function, and/or complement binding (for example,
where the antibody has a glycosylation profile necessary for ADCC
function or complement binding).
[0082] The term "chimeric" antibody refers to antibodies in which a
portion of the heavy and/or light chain is identical with or
homologous to corresponding sequences in antibodies derived from a
particular species or belonging to a particular antibody class or
subclass, while the remainder of the chain(s) is identical with or
homologous to corresponding sequences in antibodies derived from
another species or belonging to another antibody class or subclass,
as well as fragments of such antibodies, so long as they exhibit
the desired biological activity (See, for example, U.S. Pat. No.
4,816,567 and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81,
6851-6855).
[0083] As used herein, the term "humanized antibody" refers to
forms of antibodies that contain sequences from non-human (e.g.,
murine) antibodies as well as human antibodies. Such antibodies
contain minimal sequence derived from non-human immunoglobulin. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FR
regions are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. The humanized forms of rodent antibodies will
essentially comprise the same CDR sequences of the parental rodent
antibodies, although certain amino acid substitutions may be
included to increase affinity, increase stability of the humanized
antibody, or for other reasons. However, as CDR loop exchanges do
not uniformly result in an antibody with the same binding
properties as the antibody of origin, changes in framework residues
(FR), residues involved in CDR loop support, might also be
introduced in humanized antibodies to preserve antigen binding
affinity (Kabat et al., 1991, J. Immunol. 147, 1709).
[0084] The term "antibody" also includes "fully human" antibodies,
i.e., antibodies that comprise human immunoglobulin protein
sequences only. A fully human antibody may contain murine
carbohydrate chains if produced in a mouse, in a mouse cell, or in
a hybridoma derived from a mouse cell. Similarly, "mouse antibody"
or "rat antibody" refer to an antibody that comprises only mouse or
rat immunoglobulin sequences, respectively. A fully human antibody
may be generated in a human being, in a transgenic animal having
human immunoglobulin germline sequences, by phage display or other
molecular biological methods. Also, recombinant immunoglobulins may
also be made in transgenic mice. See Mendez et al., 1997, Nature
Genetics 15,146-156. See also Abgenix and Medarex technologies.
[0085] The antibodies of the present invention also include
antibodies with modified (or blocked) Fc regions to provide altered
effector functions. See, e.g., U.S. Pat. No. 5,624,821;
WO2003/086310; WO2005/120571; WO2006/0057702; Presta, 2006, Adv.
Drug Delivery Rev. 58:640-656. Such modification can be used to
enhance or suppress various reactions of the immune system, with
possible beneficial effects in diagnosis and therapy. Alterations
of the Fc region include amino acid changes (substitutions,
deletions and insertions), glycosylation or deglycosylation, and
adding multiple Fc. Changes to the Fc can also alter the half-life
of antibodies in therapeutic antibodies, and a longer half-life
would result in less frequent dosing, with the concomitant
increased convenience and decreased use of material. See Presta,
2005, J. Allergy Clin. Immunol. 116, 731 at 734-35.
[0086] The antibodies of the present invention also include
antibodies with intact Fc regions that provide full effector
functions, e.g. antibodies of isotype IgG1, which induce
complement-dependent cytotoxicity (CDC) or antibody dependent
cellular cytotoxicity (ADCC) in the a targeted cell.
[0087] The antibodies may also be conjugated (e.g., covalently
linked) to molecules that improve stability of the antibody during
storage or increase the half-life of the antibody in vivo. Examples
of molecules that increase the half-life are albumin (e.g., human
serum albumin) and polyethylene glycol (PEG). Albumin-linked and
PEGylated derivatives of antibodies can be prepared using
techniques well known in the art. See, e.g., Chapman, 2002, Adv.
Drug Deliv. Rev. 54, 531-545; Anderson and Tomasi, 1988, J.
Immunol. Methods 109, 37-42; Suzuki et al., 1984, Biochim. Biophys.
Acta 788, 248-255; and Brekke and Sandlie, 2003, Nature Rev. 2,
52-62.
[0088] Antibodies used in the present invention will usually bind
with at least a K.sub.D of about 10.sup.-3 M, more usually at least
10.sup.-6 M, typically at least 10.sup.-7 M, more typically at
least 10.sup.-8 M, preferably at least about 10.sup.-9 M, and more
preferably at least 10.sup.-10 M, and most preferably at least
10.sup.-11 M. See, e.g., Presta, et al., 2001, Thromb. Haemost. 85,
379-389; Yang, et al., 2001, Crit. Rev. Oncol. Hematol. 38, 17-23;
Carnahan, et al., 2003, Clin. Cancer Res. (Suppl.) 9
3982s-3990s.
[0089] Antibody affinities may be determined using standard
analysis.
[0090] The term "hypervariable region," as used herein, refers to
the amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region comprises amino acid
residues from a "complementarity determining region" or "CDR,"
defined by sequence alignment, for example residues 24-34 (L1),
50-56 (L2) and 89-97 (L3) in the light chain variable domain and
31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable
domain; see Kabat et al., 1991, Sequences of proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. and/or those residues from a
"hypervariable loop" (HVL), as defined structurally, for example,
residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain
variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the
heavy chain variable domain; see Chothia and Leskl, 1987, J. Mol.
Biol. 196, 901-917. "Framework" or "FR" residues are those variable
domain residues other than the hypervariable region residues as
herein defined.
[0091] An "isolated" antibody is one that has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials that would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In some embodiments, the
antibody will be purified (1) to greater than 95% by weight of
antibody as determined by the Lowry method, and most preferably
more than 99% by weight, (2) to a degree sufficient to obtain at
least 15 residues of N-terminal or internal amino acid sequence by
use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE
under reducing or nonreducing conditions using Coomassie blue or,
preferably, silver stain. Isolated antibody includes the antibody
in situ within recombinant cells since at least one component of
the antibody's natural environment will not be present. Ordinarily,
however, isolated antibody will be prepared by at least one
purification step.
[0092] An "isolated" nucleic acid molecule is a nucleic acid
molecule that is identified and separated from at least one
contaminant nucleic acid molecule with which it is ordinarily
associated in the natural source of the antibody nucleic acid. An
isolated nucleic acid molecule is other than in the form or setting
in which it is found in nature. Isolated nucleic acid molecules
therefore are distinguished from the nucleic acid molecule as it
exists in natural cells. However, an isolated nucleic acid molecule
includes a nucleic acid molecule contained in cells that ordinarily
express the antibody where, for example, the nucleic acid molecule
is in a chromosomal location different from that of natural
cells.
[0093] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations that typically include different
antibodies directed against different determinants (epitopes), each
monoclonal antibody is directed against a single determinant on the
antigen. The modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by the hybridoma method first described by
Kohler et al., 1975, Nature 256, 495, or may be made by recombinant
DNA methods (see, for example, U.S. Pat. No. 4,816,567). The
"monoclonal antibodies" may also be isolated from phage antibody
libraries using the techniques described in Clackson et al., 1991,
Nature 352, 624-628 and Marks et al., 1991, J. Mol. Biol. 222,
581-597, for example. The monoclonal antibodies herein specifically
include "chimeric" antibodies.
[0094] As used herein, the term "immune cell" includes cells that
are of hematopoietic origin and that play a role in the immune
response. Immune cells include lymphocytes, such as B cells and T
cells; natural killer cells; myeloid cells, such as monocytes,
macrophages, eosinophils, mast cells, basophils, and
granulocytes.
[0095] As used herein, an "immunoconjugate" refers to an anti-APRIL
antibody, or a fragment thereof, conjugated to a therapeutic
moiety, such as a bacterial toxin, a cytotoxic drug or a
radiotoxin. Toxic moieties can be conjugated to antibodies of the
invention using methods available in the art.
[0096] As used herein, a sequence "variant" refers to a sequence
that differs from the disclosed sequence at one or more amino acid
residues but which retains the biological activity of the resulting
molecule.
[0097] "Conservatively modified variants" or "conservative amino
acid substitution" refers to substitutions of amino acids are known
to those of skill in this art and may be made generally without
altering the biological activity of the resulting molecule. Those
of skill in this art recognize that, in general, single amino acid
substitutions in non-essential regions of a polypeptide do not
substantially alter biological activity (see, e.g., Watson, et al.,
Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p.
224 (4th Edition 1987)). Such exemplary substitutions are
preferably made in accordance with those set forth below as
follows:
[0098] Exemplary Conservative Amino Acid Substitutions
TABLE-US-00001 Original residue Conservative substitution Ala (A)
Gly; Ser Arg (R) Lys, His Asn (N) Gln; His Asp (D) Glu; Asn Cys (C)
Ser; Ala Gln (Q) Asn Glu (E) Asp; Gln Gly (G) Ala His (H) Asn; Gln
Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg; His Met (M) Leu;
Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser (S) Thr Thr (T) Ser
Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu
As used herein, the term "about" refers to a value that is within
an acceptable error range for the particular value as determined by
one of ordinary skill in the art, which will depend in part on how
the value is measured or determined, i.e., the limitations of the
measurement system. For example, "about" can mean within 1 or more
than 1 standard deviation per the practice in the art.
Alternatively, "about" or "comprising essentially of" can mean a
range of up to 20%. Furthermore, particularly with respect to
biological systems or processes, the terms can mean up to an order
of magnitude or up to 5-fold of a value. When particular values are
provided in the application and claims, unless otherwise stated,
the meaning of "about" or "comprising essentially of" should be
assumed to be within an acceptable error range for that particular
value.
[0099] "Specifically" binds, when referring to a ligand/receptor,
antibody/antigen, or other binding pair, indicates a binding
reaction which is determinative of the presence of the protein,
e.g., APRIL, in a heterogeneous population of proteins and/or other
biologics. Thus, under designated conditions, a specified
ligand/antigen binds to a particular receptor/antibody and does not
bind in a significant amount to other proteins present in the
sample.
[0100] "Administration" and "treatment," as it applies to an
animal, human, experimental subject, cell, tissue, organ, or
biological fluid, refers to contact of an exogenous pharmaceutical,
therapeutic, diagnostic agent, or composition to the animal, human,
subject, cell, tissue, organ, or biological fluid. "Administration"
and "treatment" can refer, e.g., to therapeutic, pharmacokinetic,
diagnostic, research, and experimental methods. Treatment of a cell
encompasses contact of a reagent to the cell, as well as contact of
a reagent to a fluid, where the fluid is in contact with the cell.
"Administration" and "treatment" also means in vitro and ex vivo
treatments, e.g., of a cell, by a reagent, diagnostic, binding
composition, or by another cell.
Monoclonal Antibodies
[0101] Monoclonal antibodies to human APRIL can be made according
to knowledge and skill in the art of injecting test subjects with
human APRIL antigen and then isolating hybridomas expressing
antibodies having the desired sequence or functional
characteristics.
[0102] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the monoclonal
antibodies). The hybridoma cells serve as a preferred source of
such DNA. Once isolated, the DNA may be placed into expression
vectors, which are then transfected into host cells such as E. coli
cells, simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. Recombinant production of antibodies will be described
in more detail below.
[0103] In a further embodiment, antibodies or antibody fragments
can be isolated from antibody phage libraries generated using the
techniques described in McCafferty et al., 1990, Nature, 348,
552-554. Clackson et al., 1991, Nature, 352, 624-628, and Marks et
al., 1991, J. Mol. Biol. 222, 581-597 describe the isolation of
murine and human antibodies, respectively, using phage libraries.
Subsequent publications describe the production of high affinity
(nM range) human antibodies by chain shuffling (Marks et al., 1992,
Bio/Technology, 10, 779-783), as well as combinatorial infection
and in vivo recombination as a strategy for constructing very large
phage libraries (Waterhouse et al., 1993, Nuc. Acids. Res. 21,
2265-2266). Thus, these techniques are viable alternatives to
traditional monoclonal antibody hybridoma techniques for isolation
of monoclonal antibodies.
Chimeric Antibodies
[0104] The antibody DNA also may be modified, for example, by
substituting the coding sequence for human heavy- and light-chain
constant domains in place of the homologous murine sequences (U.S.
Pat. No. 4,816,567; Morrison, et al., 1984, Proc. Natl Acad. Sci.
USA, 81, 6851), or by covalently joining to the immunoglobulin
coding sequence all or part of the coding sequence for
non-immunoglobulin material (e.g., protein domains). Typically such
non-immunoglobulin material is substituted for the constant domains
of an antibody, or is substituted for the variable domains of one
antigen-combining site of an antibody to create a chimeric bivalent
antibody comprising one antigen-combining site having specificity
for an antigen and another antigen-combining site having
specificity for a different antigen.
Humanized and Human Antibodies
[0105] A humanized antibody has one or more amino acid residues
from a source that is non-human. The non-human amino acid residues
are often referred to as "import" residues, and are typically taken
from an "import" variable domain. Humanization can be performed
generally following the method of Winter and co-workers (Jones et
al., 1986, Nature 321, 522-525; Riechmann et al., 1988, Nature,
332, 323-327; Verhoeyen et al., 1988, Science 239, 1534-1536), by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are antibodies wherein substantially less than an intact
human variable domain has been substituted by the corresponding
sequence from a non-human species. In practice, humanized
antibodies are typically human antibodies in which some CDR
residues and possibly some FR residues are substituted by residues
from analogous sites in non-human, for example, rodent
antibodies.
[0106] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework (FR) for the
humanized antibody (Sims et al., 1987, J. Immunol. 151, 2296;
Chothia et al., 1987, J. Mol. Biol. 196, 901). Another method uses
a particular framework derived from the consensus sequence of all
human antibodies of a particular subgroup of light or heavy chains.
The same framework may be used for several different humanized
antibodies (Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89,
4285; Presta et al., 1993, J. Immunol. 151, 2623).
[0107] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, according to a
preferred method, humanized antibodies are prepared by a process of
analysis of the parental sequences and various conceptual humanized
products using three-dimensional models of the parental and
humanized sequences. Three-dimensional immunoglobulin models are
commonly available and are familiar to those skilled in the art.
Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
CDR residues are directly and most substantially involved in
influencing antigen binding.
[0108] Humanization of antibodies is a straightforward protein
engineering task. Nearly all murine antibodies can be humanized by
CDR grafting, resulting in the retention of antigen binding. See,
Lo, Benny, K. C., editor, in Antibody Engineering: Methods and
Protocols, volume 248, Humana Press, New Jersey, 2004.
[0109] Alternatively, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (JH) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array in such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al.,
1993, Proc. Natl. Acad. Sci. USA 90, 2551; Jakobovits et al., 1993,
Nature 362, 255-258; Bruggermann et al., 1993, Year in Immunology
7, 33; and Duchosal et al., 1992, Nature 355, 258. Human antibodies
can also be derived from phage-display libraries (Hoogenboom et
al., 1991, J. Mol. Biol. 227,381; Marks et al., J. Mol. Biol. 1991,
222, 581-597; Vaughan et al., 1996, Nature Biotech 14, 309).
[0110] Amino acid sequence variants of humanized anti-APRIL
antibodies are prepared by introducing appropriate nucleotide
changes into the humanized anti-APRIL antibodies' DNAs, or by
peptide synthesis. Such variants include, for example, deletions
from, and/or insertions into, and/or substitutions of, residues
within the amino acid sequences shown for the humanized anti-APRIL
antibodies. Any combination of deletion, insertion, and
substitution is made to arrive at the final construct, provided
that the final construct possesses the desired characteristics. The
amino acid changes also may alter post-translational processes of
the humanized anti-APRIL antibodies, such as changing the number or
position of glycosylation sites.
[0111] A useful method for identification of certain residues or
regions of the humanized anti-APRIL antibodies polypeptides that
are preferred locations for mutagenesis is called "alanine scanning
mutagenesis," as described by Cunningham and Wells, 1989, Science
244, 1081-1085. Here, a residue or group of target residues are
identified (e.g., charged residues such as Arg, Asp, His, Lys, and
Glu) and replaced by a neutral or negatively charged amino acid
(most preferably alanine or polyalanine) to affect the interaction
of the amino acids with APRIL antigen. The amino acid residues
demonstrating functional sensitivity to the substitutions then are
refined by introducing further or other variants at, or for, the
sites of substitution. Thus, while the site for introducing an
amino acid sequence variation is predetermined, the nature of the
mutation per se need not be predetermined. For example, to analyze
the performance of a mutation at a given site, Ala scanning or
random mutagenesis is conducted at the target codon or region and
the expressed humanized anti-APRIL antibodies' variants are
screened for the desired activity.
[0112] Ordinarily, amino acid sequence variants of the humanized
anti-APRIL antibodies will have an amino acid sequence having at
least 75% amino acid sequence identity with the original humanized
antibody amino acid sequences of either the heavy or the light
chain more preferably at least 80%, more preferably at least 85%,
more preferably at least 90%, and most preferably at least 95%, 98%
or 99%. Identity or homology with respect to this sequence is
defined herein as the percentage of amino acid residues in the
candidate sequence that are identical with the humanized residues,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
None of N-terminal, C-terminal, or internal extensions, deletions,
or insertions into the antibody sequence shall be construed as
affecting sequence identity or homology.
[0113] Antibodies having the characteristics identified herein as
being desirable in humanized anti-APRIL antibodies can be screened
for inhibitory biologic activity in vitro or suitable binding
affinity. To screen for antibodies that bind to the BCMA or TACI
epitopes on human APRIL bound by an antibody of interest (e.g.,
those that block binding of APRIL), a routine cross-blocking assay
such as that described in Antibodies, A Laboratory Manual, Cold
Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be
performed. Antibodies that bind to the same epitope are likely to
cross-block in such assays, but not all cross-blocking antibodies
will necessarily bind at precisely the same epitope since
cross-blocking may result from steric hindrance of antibody binding
by antibodies bind at overlapping epitopes, or even nearby
non-overlapping epitopes.
[0114] Alternatively, epitope mapping, e.g., as described in Champe
et al., 1995, J. Biol. Chem. 270, 1388-1394, can be performed to
determine whether the antibody binds an epitope of interest.
"Alanine scanning mutagenesis," as described by Cunningham and
Wells, 1989, Science 244, 1081-1085, or some other form of point
mutagenesis of amino acid residues in human APRIL may also be used
to determine the functional epitope for anti-APRIL antibodies of
the present invention.
[0115] Additional antibodies binding to the same epitope as an
antibody of the present invention may be obtained, for example, by
screening of antibodies raised against APRIL for binding to the
epitope, or by immunization of an animal with a peptide comprising
a fragment of human APRIL comprising the epitope sequences (e.g.,
BCMA or TACI). Antibodies that bind to the same functional epitope
might be expected to exhibit similar biological activities, such as
blocking receptor binding, and such activities can be confirmed by
functional assays of the antibodies.
[0116] Antibody affinities may be determined using standard
analysis. Preferred binding compounds such as e.g. humanized
antibodies are those that bind human APRIL with a K.sub.d value of
no more than about 1.times.10.sup.-7; preferably no more than about
1.times.10.sup.-8; more preferably no more than about
1.times.10.sup.-9; and most preferably no more than about
1.times.10.sup.-10 or even 1.times.10.sup.-11 M.
[0117] The humanized antibody can be selected from any class of
immunoglobulins, including IgM, IgG, IgD, IgA, and IgE. Preferably,
the antibody is an IgG antibody. Any isotype of IgG can be used,
including IgG.sub.1, IgG.sub.2, IgG.sub.3, and IgG.sub.4. Variants
of the IgG isotypes are also contemplated. The humanized antibody
may comprise sequences from more than one class or isotype.
Optimization of the necessary constant domain sequences to generate
the desired biologic activity is readily achieved by screening the
antibodies in the biological assays described in the Examples.
[0118] Likewise, either class of light chain can be used in the
compositions and methods herein. Specifically, kappa, lambda, or
variants thereof are useful in the present compositions and
methods.
[0119] The antibodies and antibody fragments of the invention may
also be conjugated with cytotoxic payloads such as cytotoxic agents
or radionucleotides such as .sup.99Tc, .sup.90Y, .sup.111In,
.sup.32P, .sup.14C, .sup.125I, .sup.3H, .sup.131I, .sup.11C,
.sup.15O, .sup.13N, .sup.18F, .sup.35S, .sup.51Cr, .sup.57To,
.sup.226Ra, .sup.60Co, .sup.59Fe, .sup.57Se, .sup.152Eu, .sup.67CU,
.sup.217Ci, .sup.211At, .sup.212Pb, .sup.47Sc, .sup.109Pd,
.sup.234Th, and .sup.40K, .sup.157Gd, .sup.55Mn, .sup.52Tr and
.sup.56Fe. Such antibody conjugates may be used in immunotherapy to
selectively target and kill cells expressing a target (the antigen
for that antibody) on their surface. Exemplary cytotoxic agents
include ricin, vinca alkaloid, methotrexate, Psuedomonas exotoxin,
saporin, diphtheria toxin, cisplatin, doxorubicin, abrin toxin,
gelonin and pokeweed antiviral protein.
[0120] The antibodies and antibody fragments of the invention may
also be conjugated with fluorescent or chemiluminescent labels,
including fluorophores such as rare earth chelates, fluorescein and
its derivatives, rhodamine and its derivatives, isothiocyanate,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaladehyde,
fluorescamine, .sup.152Eu, dansyl, umbelliferone, luciferin,
luminal label, isoluminal label, an aromatic acridinium ester
label, an imidazole label, an acridimium salt label, an oxalate
ester label, an aequorin label, 2,3-dihydrophthalazinediones,
biotin/avidin, spin labels and stable free radicals.
[0121] Any method known in the art for conjugating the antibody
molecules or protein molecules of the invention to the various
moieties may be employed, including those methods described by
Hunter et al., 1962, Nature 144, 945; David et al., 1974,
Biochemistry 13,1014; Pain et al., 1981, J. Immunol. Meth. 40, 219;
and Nygren, J., 1982, Histochem. and Cytochem. 30, 407. Methods for
conjugating antibodies and proteins are conventional and well known
in the art.
Antibody Purification
[0122] When using recombinant techniques, the antibody can be
produced intracellularly, in the periplasmic space, or directly
secreted into the medium. If the antibody is produced
intracellularly, as a first step, the particulate debris, either
host cells or lysed fragments, is removed, for example, by
centrifugation or ultrafiltration. Carter et al., 1992,
Bio/Technology 10, 163-167 describe a procedure for isolating
antibodies which are secreted to the periplasmic space of E. coli.
Briefly, cell paste is thawed in the presence of sodium acetate (pH
3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30
min. Cell debris can be removed by centrifugation. Where the
antibody is secreted into the medium, supernatants from such
expression systems are generally first concentrated using a
commercially available protein concentration filter, for example,
an Amicon or Millipore Pellicon ultrafiltration unit. A protease
inhibitor such as PMSF may be included in any of the foregoing
steps to inhibit proteolysis and antibiotics may be included to
prevent the growth of adventitious contaminants.
[0123] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc region that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human .gamma.1, .gamma.2, or .gamma.4 heavy chains
(Lindmark et al., 1983, J. Immunol. Meth. 62, 1-13). Protein G is
recommended for all mouse isotypes and for human .gamma.3 (Guss et
al., 1986, EMBO J 5, 1567-1575). The matrix to which the affinity
ligand is attached is most often agarose, but other matrices are
available. Mechanically stable matrices such as controlled pore
glass or poly(styrenedivinyl)benzene allow for faster flow rates
and shorter processing times than can be achieved with agarose.
Where the antibody comprises a CH3 domain, the Bakerbond ABX.TM.
resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.
Other techniques for protein purification such as fractionation on
an ion-exchange column, ethanol precipitation, Reverse Phase HPLC,
chromatography on silica, chromatography on heparin SEPHAROSE.TM.
chromatography on an anion or cation exchange resin (such as a
polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium
sulfate precipitation are also available depending on the antibody
to be recovered.
[0124] In one embodiment, the glycoprotein may be purified using
adsorption onto a lectin substrate (e.g. a lectin affinity column)
to remove fucose-containing glycoprotein from the preparation and
thereby enrich for fucose-free glycoprotein.
Pharmaceutical Formulations
[0125] The invention comprises pharmaceutical formulations of an
APRIL binding compound. To prepare pharmaceutical or sterile
compositions, the antibody or fragment thereof is admixed with a
pharmaceutically acceptable carrier or excipient, see, e.g.,
Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National
Formulary, Mack Publishing Company, Easton, Pa. (1984).
Formulations of therapeutic and diagnostic agents may be prepared
by mixing with physiologically acceptable carriers, excipients, or
stabilizers in the form of, e.g., lyophilized powders, slurries,
aqueous solutions or suspensions (see, e.g., Hardman, et al., 2001,
Goodman and Gilman's The Pharmacological Basis of Therapeutics,
McGraw-Hill, New York, N.Y.; Gennaro, 2000, Remington: The Science
and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New
York, N.Y.; Avis, et al. (eds.), 1993, Pharmaceutical Dosage Forms:
Parenteral Medications, Marcel Dekker, NY; Lieberman, et al.
(eds.), 1990, Pharmaceutical Dosage Forms: Tablets, Marcel Dekker,
NY; Lieberman, et al. (eds.), 1990, Pharmaceutical Dosage Forms:
Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie, 2000,
Excipient Toxicity and Safety, Marcel Dekker, Inc., New York,
N.Y.).
[0126] Toxicity and therapeutic efficacy of the antibody
compositions, administered alone or in combination with an
immunosuppressive agent, can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., for determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio between LD.sub.50 and ED.sub.50. The data
obtained from these cell culture assays and animal studies can be
used in formulating a range of dosage for use in humans. The dosage
of such compounds lies preferably within a range of circulating
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon the
dosage form employed and the route of administration utilized.
[0127] Suitable routes of administration include parenteral
administration, such as intramuscular, intravenous, or subcutaneous
administration and oral administration. Administration of antibody
used in the pharmaceutical composition or to practice the method of
the present invention can be carried out in a variety of
conventional ways, such as oral ingestion, inhalation, topical
application or cutaneous, subcutaneous, intraperitoneal,
parenteral, intraarterial or intravenous injection. In one
embodiment, the binding compound of the invention is administered
intravenously. In another embodiment, the binding compound of the
invention is administered subcutaneously.
[0128] Alternatively, one may administer the antibody in a local
rather than systemic manner, for example, via injection of the
antibody directly into the site of action, often in a depot or
sustained release formulation. Furthermore, one may administer the
antibody in a targeted drug delivery system.
[0129] Guidance in selecting appropriate doses of antibodies,
cytokines, and small molecules are available (see, e.g.,
Wawrzynczak, 1996, Antibody Therapy, Bios Scientific Pub. Ltd,
Oxfordshire, UK; Kresina (ed.), 1991, Monoclonal Antibodies,
Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.),
1993, Monoclonal Antibodies and Peptide Therapy in Autoimmune
Diseases, Marcel Dekker, New York, N.Y.; Baert, et al., 2003, New
Engl. J. Med. 348, 601-608; Milgrom, et al., 1999, New Engl. J.
Med. 341, 1966-1973; Slamon, et al., 2001, New Engl. J. Med. 344,
783-792; Beniaminovitz, et al., 2000, New Engl. J. Med. 342,
613-619; Ghosh, et al., 2003, New Engl. J. Med. 348, 24-32; Lipsky,
et al., 2000, New Engl. J. Med. 343, 1594-1602).
[0130] Determination of the appropriate dose is made by the
clinician, e.g., using parameters or factors known or suspected in
the art to affect treatment or predicted to affect treatment.
Generally, the dose begins with an amount somewhat less than the
optimum dose and it is increased by small increments thereafter
until the desired or optimum effect is achieved relative to any
negative side effects. Important diagnostic measures include those
of symptoms of, e.g., the inflammation or level of inflammatory
cytokines produced.
[0131] A preferred dose protocol is one involving the maximal dose
or dose frequency that avoids significant undesirable side effects.
A total weekly dose is generally at least 0.05 .mu.g/kg body
weight, more generally at least 0.2 .mu.g/kg, most generally at
least 0.5 .mu.g/kg, typically at least 1 .mu.g/kg, more typically
at least 10 .mu.g/kg, most typically at least 100 .mu.g/kg,
preferably at least 0.2 mg/kg, more preferably at least 1.0 mg/kg,
most preferably at least 2.0 mg/kg, optimally at least 10 mg/kg,
more optimally at least 25 mg/kg, and most optimally at least 50
mg/kg (see, e.g., Yang, et al., 2003, New Engl. J. Med. 349,
427-434; Herold, et al., 2002, New Engl. J. Med. 346, 1692-1698;
Liu, et al., 1999, J. Neurol. Neurosurg. Psych. 67, 451-456;
Portielji, et al., 2003, Cancer Immunol. Immunother. 52, 133-144).
The desired dose of a small molecule therapeutic, e.g., a peptide
mimetic, natural product, or organic chemical, is about the same as
for an antibody or polypeptide, on a moles/kg basis.
[0132] As used herein, "inhibit" or "treat" or "treatment" includes
a postponement of development of the symptoms associated with
disease and/or a reduction in the severity of such symptoms that
will or are expected to develop with said disease. The terms
further include ameliorating existing symptoms, preventing
additional symptoms, and ameliorating or preventing the underlying
causes of such symptoms. Thus, the terms denote that a beneficial
result has been conferred on a vertebrate subject with a
disease.
[0133] As used herein, the term "therapeutically effective amount"
or "effective amount" refers to an amount of an anti-APRIL antibody
or fragment thereof, that when administered alone or in combination
with an additional therapeutic agent to a cell, tissue, or subject
is effective to prevent or ameliorate the disease or condition to
be treated. A therapeutically effective dose further refers to that
amount of the compound sufficient to result in amelioration of
symptoms, e.g., treatment, healing, prevention or amelioration of
the relevant medical condition, or an increase in rate of
treatment, healing, prevention or amelioration of such conditions.
When applied to an individual active ingredient administered alone,
a therapeutically effective dose refers to that ingredient alone.
When applied to a combination, a therapeutically effective dose
refers to combined amounts of the active ingredients that result in
the therapeutic effect, whether administered in combination,
serially or simultaneously. An effective amount of therapeutic will
decrease the symptoms typically by at least 10%; usually by at
least 20%; preferably at least about 30%; more preferably at least
40%, and most preferably by at least 50%.
[0134] Methods for co-administration or treatment with a second
therapeutic agent are well known in the art, see, e.g., Hardman, et
al. (eds.), 2001, Goodman and Gilman's The Pharmacological Basis of
Therapeutics, 10.sup.th ed., McGraw-Hill, New York, N.Y.; Poole and
Peterson (eds.), 2001, Pharmacotherapeutics for Advanced Practice:
A Practical Approach, Lippincott, Williams & Wilkins, Phila.,
Pa.; Chabner and Longo (eds.), 2001, Cancer Chemotherapy and
Biotherapy, Lippincott, Williams & Wilkins, Phila., Pa.
[0135] The pharmaceutical composition of the invention may also
contain other agent, including but not limited to a cytotoxic,
chemotherapeutic, cytostatic, anti-angiogenic or antimetabolite
agent, a tumor targeted agent, an immune stimulating or immune
modulating agent or an antibody conjugated to a cytotoxic,
cytostatic, or otherwise toxic agent. The pharmaceutical
composition can also be employed with other therapeutic modalities
such as surgery, chemotherapy and radiation.
Therapeutic uses for the Antibody and Antibody Fragments of the
Invention
[0136] The antibodies and antigen binding fragments of the
invention, which specifically bind to human APRIL, can be used to
treat several diseases in which the activity of APRIL is central to
pathology. Broadly speaking this includes cancer, auto-immunity,
inflammatory diseases and potentially multiple sclerosis, a CNS
disease.
Cancer
[0137] The antibody or antigen binding fragments of the invention
which specifically bind APRIL can be used to treat cancer.
Preferred cancers whose growth and survival may be inhibited by the
invention include any cancers known to express APRIL and depend on
this for proliferative signals. Non-limiting examples of such
cancers include several B cell malignancies, such as Chronic
Lymphocytic Leukaemia (CLL), Multiple Myeloma, Hodgkin's lymphoma
and Non-Hodgkin's lymphoma including Burkitt's lumphoma and diffuse
large B cell lymphoma, and also potentially several solid tumors
such as glioblastomas, where APRIL expression has been
reported.
[0138] The binding compounds of the invention may be used alone or
in combination with other anti-cancer agents, such as
chemotherapeutic reagents or other biological agents. Additionally
the invention includes refractory or recurrent malignancies or
treatment of metastases derived from any of these malignancies.
Autoimmune Disease
[0139] The binding compounds of the invention may be used to treat
several autoimmune diseases, where the expression of APRIL has been
sown to play a role in pathology. Examples of such diseases are
Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE) and
Sjogren's syndrome. In addition, higher then normal titres of APRIL
were found in the serum of multiple sclerosis patients and also
increased levels found in their astrocytes. Thus, APRIL is a
contributing factor to disease pathology and therapeutic blockage
of APRIL in MS may be beneficial.
Non-Therapeutic uses for the Antibody and Antibody Fragments of the
Invention
[0140] The non-therapeutic uses for these antibodies include flow
cytometry, western blotting, enzyme linked immunosorbant assay
(ELISA), immunohistochemistry.
[0141] The antibodies of this invention may also be used as an
affinity purification reagent via immobilization to a sepharose
column.
[0142] The antibody may also be useful in diagnostic assays, e.g.,
for detecting expression of APRIL in specific cells, tissues, or
serum. For diagnostic applications, the antibody typically will be
labeled (either directly or indirectly) with a detectable moiety.
Numerous labels are available which can be generally grouped into
the following categories: biotin, fluorochromes, radionucleotides,
enzymes, iodine, and biosynthetic labels.
[0143] The antibodies of the present invention may be employed in
any known assay method, such as competitive binding assays, direct
and indirect sandwich assays, and immunoprecipitation assays. Zola,
Monoclonal Antibodies. A Manual of Techniques, pp. 147-158 (CRC
Press, Inc. 1987).
[0144] The antibody may also be used for in vivo diagnostic assays.
Generally, the antibody is labeled with a radionuclide so that the
antigen or cells expressing it can be localized using
immunoscintiography or positron emission tomography.
[0145] The invention will now be further described by way of the
following non-limiting examples.
EXAMPLES
Example 1
Immunization and Selection of Anti-APRIL Antibodies
[0146] Immunization of Mice with APRIL cDNA
[0147] To generate antibodies against the human APRIL protein, a
cDNA encoding the full length open reading frame of APRIL was
subcloned into the pCI-neo vector (Promega, Madison, Wis.).
Expression of the obtained vector was checked by transient
transfection of pCI-neo-hAPRIL in 293 cells (American Type Culture
Collection, Manassas, Va.) and immunoblotting with mouse
anti-hAPRIL IgG1 Aprily-5 (1:5,000) (Alexis, San Diego, Calif.),
followed by goat anti-mouse IgG1-HRP (1:2,000) (Southern
Biotechnology, Birmingham, Ala.).
[0148] Mice were immunized by gene gun immunization using a Helios
Gene gun (BioRad, Hercules, Calif.) and DNA coated gold bullets
(BioRad) following manufacturer's instructions. Briefly, 1 .mu.m
gold particles were coated with pCI-neo-hAPRIL cDNA and commercial
expression vectors for mouse Flt3L and mouse GM-CSF in a 2:1:1
ratio (both from Aldevron, Fargo, N. Dak.). A total of 1 .mu.g of
plasmid DNA was used to coat 500 .mu.g of gold bullets.
[0149] Specifically, 7-8 weeks old female BALB/C mice were
immunized in the ears with a gene gun, receiving 4 or 5 cycles of a
shot in both ears. Approximately, a 1:3,200 anti-hAPRIL titer was
detected by ELISA in mouse serum after three DNA immunizations In
the ELISA, all incubation steps were followed by a wash step with
PBST (PBS with 0.1% Tween 20) 3 times. Maxisorp 96-well
immunoplates (Nunc, Rochester, N.Y.) were coated with rabbit
anti-FLAG polyclonal antibody (50 ng/well in PBS) (Sigma, St.
Louis, Mo.) overnight at 4.degree. C. and blocked with 10% Goat
serum/PBST for 1 hour at RT. Plates were incubated with supernatant
(1:4 in PBS) from 293T cells transiently transfected with CMV
promoter driven secreted form of FLAG-hAPRIL (pCR3-hAPRIL) for 1 h
at RT, followed by incubations with mouse sera dilutions and
1:2,000 HRP-conjugated goat anti-mouse IgG (Southern Biotechnology)
for 1 hour each at RT. After the final PBST wash, anti-hAPRIL
immunoreactivity was visualized with 100 .mu.l OptiEIA TMB
substrate (BD Biosciences, Franklin Lake, N.J.). Reactions were
stopped with 100 .mu.l 0.5 M H.sub.2SO.sub.4 and absorbances were
read at 460 and 620 nm. Mice that demonstrated reactivity against
hAPRIL were immunized for a final, fourth time and sacrificed four
days later. Erythrocyte-depleted spleen cell populations were
prepared as described previously (Steenbakkers et al., 1992, J.
Immunol. Meth. 152: 69-77; Steenbakkers et al., 1994, Mol. Biol.
Rep. 19: 125-134) and frozen at -140.degree. C.
[0150] Selection of Anti-APRIL Antibody Producing B Cells
[0151] To select B cell clones producing anti-APRIL antibodies,
1.5.times.10.sup.7 erythrocyte-depleted splenocytes were subjected
to two rounds of negative panning on 2.3.times.10.sup.7
Dynabeads.RTM. M-450 tosyl-activated beads (Invitrogen, Carlsbad,
Calif.) coated with anti-FLAG M2 antibody (Sigma). 50 .mu.g
anti-FLAG M2 antibody was coated per 1.times.10.sup.8 beads in 500
.mu.l according to manufacturer's instructions. Beads and
splenocyte suspension were incubated for 30 minutes on ice and
resuspended in cold DMEM F12/P/S/10% BCS. Unbound splenocytes were
separated from the beads using the Dyna1 MPC (Magnetic Particle
Concentrator) (Invitrogen). For the positive panning, splenocytes
were incubated with 2.3.times.10.sup.7 beads coated with anti-FLAG
M2 bound to FLAG-hAPRIL for 30 minutes on ice. Beads and unbound
splenocytes were separated as described above with a total of 12
washes.
[0152] Antigen-specific B-cells were cultured as described by
Steenbakkers et al., 1994, Mol. Biol. Rep. 19: 125-134. Briefly,
selected B-cells were mixed with 7.5% (v/v) T-cell supernatant and
50,000 irradiated (2,500 RAD) EL-4 B5 nursing cells in a final
volume of 200 .mu.l DMEM F12/P/S/10% BCS in a 96-well flat-bottom
tissue culture plates. On day eight, supernatants were screened for
hAPRIL reactivity by ELISA as described above. 21 APRIL-reactive
supernatants were identified and tested for their ability to
inhibit the interaction of APRIL with BCMA-Fc. In the ELISA, all
incubation steps were followed by a wash step with PBST (PBS with
0.1% Tween 20) 3 times. A Maxisorp 96-well immunoplate was coated
with BCMA-Fc (50 ng/well in PBS) (R&D Systems, Minneapolis,
Minn.) overnight at 4.degree. C. and blocked with 10% Goat
serum/PBST for 1 hour at RT. FLAG-hAPRIL containing supernatants
were pre-incubated with antibody-containing B-cell supernatants for
1 hour at RT and then added to the BCMA-Fc coated plate for 1 hour
at RT. Bound FLAG-hAPRIL was detected by incubation with 1 .mu.g/ml
anti-FLAG BioM2-biotin antibody (Sigma) and 1:2,000
Streptavidin-HRP (Southern Biotechnology) for 1 hour each at RT.
After the final PBST wash, APRIL-bound BCMA-Fc was visualized with
100 .mu.l OptiEIA TMB substrate (BD Biosciences). Reactions were
stopped with 100 .mu.l 0.5 M H.sub.2SO.sub.4, and absorbances were
read at 460 and 620 nm.
[0153] Subsequently, 8 B-cell clones were immortalized by
mini-electrofusion following published procedures (Steenbakkers et
al., 1992, J. Immunol. Meth. 152, 69-77; Steenbakkers et al., 1994,
Mol. Biol. Rep. 19, 125-34). Specifically, B-cells were mixed with
10.sup.6 NS-1 myeloma cells, and serum was removed by washing with
DMEM F12 media. Cells were treated with pronase solution for three
minutes and washed with fusion medium. Electrofusions were
performed in a 50 .mu.l fusion chamber by an alternating electric
field of 30 s, 2 MHz, 400 V/cm followed by a square, high field
pulse of 10 .mu.s, 3 kV/cm and again by an alternating electric
field of 30 s, 2 MHz, 400 V/cm. Contents of the chamber were
transferred to hybridoma selective medium and plated in a 96-well
plate under limiting dilution conditions. On day 14 following the
fusions, hybridoma supernatants were screened for APRIL reactivity
and BCMA-blocking activity, as described above. Two distinct
anti-hAPRIL hybridomas, named hAPRIL.01A and hAPRIL.03A were
isolated and subcloned by limited dilution to safeguard their
integrity. hAPRIL reactivity and BCMA-blocking activity of
hAPRIL.01A and hAPRIL.03A antibodies were confirmed with hybridoma
supernatants (see FIG. 1).
Example 2
Purification and Characterization of Anti-APRIL Antibodies
[0154] Stabilization of Anti-APRIL Producing Hybridomas and
Purification of Anti-APRIL Antibodies
[0155] Clonal cell populations were obtained for each hybridoma by
multiple rounds of limiting dilutions (six for hAPRIL.01A and four
for hAPRIL.03A). Stable hybridomas were cultured in serum-free
media using CELLine bioreactors (Integra-Biosciences, Chur,
Switzerland) according to manufacturer's instructions. Following
7-10 days in culture, supernatants were harvested and filtered
through a 0.22 .mu.M nitrocellulose membrane. Supernatants were
diluted 1:1 in high salt binding buffer (1 M Glycine/2M NaCl, pH
9.0), and antibodies were purified with Protein G HiTrap 5 ml
columns (GE Healthcare, Piscataway, N.J.). After PBS wash of the
column, antibodies were eluted with 0.1 M Glycine pH 2.7 and
neutralized with 3 M Tris. Buffer was exchanged for PBS using PD-10
gel-filtration columns (GE Healthcare). Antibodies were
concentrated with Amicon Ultra-15 centrifugal filter units
(Millipore, Billerica, Mass.) and quantified using
spectrophotometry.
[0156] Using a mouse monoclonal antibody isotyping test kit
(Serotec, Raleigh, N.C.), the (sub)-isotype of both hAPRIL.01A and
hAPRIL.03A antibodies was determined to be IgG1 Kappa.
[0157] Binding Analysis
[0158] Protein-based ELISA experiments using purified hAPRIL.01A
and hAPRIL.03A antibodies were performed to determine apparent
binding affinities (reported as EC.sub.50 values). Binding was
compared to mouse anti-hAPRIL IgG1 Aprily-5 (Alexis). Maxisorp
96-well immunoplates (Nunc) were coated with either rabbit
anti-FLAG polyclonal antibody (Sigma) or BCMA-Fc (R&D Systems)
at 50 ng/well in PBS overnight at 4.degree. C. and blocked with 10%
Goat serum/PBST for 1 hour at RT. Plates were washed with PBST 3
times and incubated with supernatant (1:4 in PBS) containing
FLAG-hAPRIL for 1 hour at RT. Plates were again washed with PBST 3
times and incubated with hAPRIL.01A, hAPRIL.03A, and Aprily-5
antibodies (10 .mu.g/ml high test with 10-fold dilutions in
triplicates) for 1 h at RT. After three washes with PBST, bound
antibodies were detected with goat anti-mouse IgG-HRP (1:2,000)
(Southern Biotechnology) for 1 hour at RT. Plate was washed three
times with PBST, and APRIL-reactivity was visualized with OptiEIA
TMB substrate (Becton Dickinson). The concentration for
half-maximal binding is reported as a measure of relative binding
affinity. When FLAG-hAPRIL was captured by the anti-FLAG antibody
(FIG. 2A), EC.sub.50 values for hAPRIL.01A, hAPRIL.03A and Aprily-5
were calculated as 2.2, 1.4, and 1.7 nM, respectively. When
FLAG-hAPRIL was captured by BCMA-Fc (FIG. 2B), hAPRIL.01A antibody
binding was not observed, suggesting that the APRIL-BCMA
interaction blocked the hAPRIL.01A epitope. In contrast, binding of
hAPRIL.03A to the APRIL-BCMA complex was observed. Antibody
detection of the receptor-ligand complex may prove useful in
diagnostic assays and for research purposes to follow the clearance
of soluble APRIL.
[0159] Kinetic Analysis by Bio-Light Interferometry (ForteBio)
[0160] To further characterize the binding characteristics of the
antibodies, each was profiled using bio-light interferometry on the
Octet system (ForteBio, Menlo Park, Calif.) to elucidate binding
kinetics and calculate equilibrium binding constants. This assay
was performed by coupling purified hAPRIL.01A and hAPRIL.03A
antibodies to amine-reactive biosensors (Fortebio) using standard
amine chemistry. Recombinant human APRIL (R&D Systems) binding
to and dissociation from the biosensors was then observed at two
concentrations, 1 and 2 .mu.g/ml. Specifically, amine-reactive
biosensors were pre-wetted by immersing them in wells containing
0.1M MES pH=5 for 2 minutes. The biosensors were then activated
using a 0.1M NHS/0.4M EDC mixture for 5 minutes. hAPRIL.01A and
hAPRIL.03A antibodies were coupled by immersing the biosensors in a
solution of 5 .mu.g/mL of the antibody for 18 minutes. The
biosensor surface was quenched using a solution of 1M ethanolamine
pH 8.5 for 7 minutes. Biosensors were equilibrated in PBS for 5
minutes. Association of recombinant APRIL was observed by placing
the biosensors in wells containing either 1 or 2 .mu.g/ml APRIL and
monitoring interferometry for 20 minutes. Dissociation was measured
after transfer of the biosensors into PBS and monitoring of the
interferometry signal for 20 minutes. The observed on and off rates
(k.sub.obs and k.sub.d) were fit using a 1:1 binding global fit
model, and the equilibrium binding constant K.sub.D was calculated
(see Table 1).
TABLE-US-00002 TABLE 1 Binding characteristics of humanized anti-
hAPRIL antibodies of the invention K.sub.obs K.sub.dissoc K.sub.D
mAb M.sup.-1s.sup.-1 s.sup.-1 M hAPRIL.01A 4.89E+04 3.69E-05
7.53E-10 hAPRIL.03A 7.54E+04 4.21E-05 5.58E-10
[0161] Receptor Blockade
[0162] Blocking abilities of hAPRIL.01A and hAPRIL.03A were
confirmed using purified antibodies. Maxisorp 96-well plates were
coated with either BCMA-Fc (R&D Systems) or TACI-Fc (R&D
Systems) at 50 ng/well overnight at 4.degree. C. and blocked with
10% Goat serum/PBST for 1 hour at RT. FLAG-hAPRIL containing
supernatants were pre-incubated with hAPRIL.01A, hAPRIL.03A, and
Aprily-5 antibodies (10 .mu.g/ml high test with 10-fold dilutions
in triplicates) for 1 h at RT. Plates were washed with PBST 3
times, and bound FLAG-hAPRIL was detected by incubation with 1
.mu.g/ml anti-FLAG BioM2-biotin antibody (Sigma) and 1:2,000
Streptavidin-HRP (Southern Biotechnology) for 1 hour each at RT.
After the final PBST wash, APRIL-bound BCMA-Fc was visualized with
OptiEIA TMB substrate (BD Biosciences). As shown in FIGS. 2C and
2D, hAPRIL.01A fully blocks FLAG-hAPRIL binding to BCMA-Fc and
TACI-Fc, whereas hAPRIL.03A fully blocks FLAG-hAPRIL binding to
TACI-Fc, while only partially blocking the hAPRIL-BCMA-Fc
interaction. Aprily-5 does not block FLAG-hAPRIL binding to either
BCMA-Fc or TACI-Fc. The concentration of half-maximum inhibition
(IC.sub.50) was determined for hAPRIL.01A as 1.2 and 0.4 nM for
BCMA-Fc and TACI-Fc, respectively. The IC.sub.50 for hAPRIL.03A to
TACI-Fc was determined as 1.3 nM.
[0163] Commercial Antibodies
[0164] Commercially available anti-APRIL antibodies were obtained
as described in Table 2.
TABLE-US-00003 TABLE 2 Commercially available anti-human APRIL
monoclonal antibodies Antibody Company Cat no. Aprily-1 Alexis
ALX-804-148-C100 Aprily-2 Alexis ALX-804-844-C100 Aprily-5 Alexis
ALX-804-801-C100 Aprily-8 Alexis ALX-804-149-C100 Sacha-1 Alexis
ALX-804-141-C100 Sacha-2 Alexis ALX-804-804-C100 anti-CD256, clone
T3-6 BioLegend 318502 mouse anti-human APRIL LifeSpan Biosciences
LS-C18658 mouse anti-human APRIL LifeSpan Biosciences LS-C18659
mouse anti-human APRIL LifeSpan Biosciences LS-C18687 TNFSF13
monoclonal anti- Tebu-bio H00008741-M01 body (M01), clone H4-E8
TNFSF13 monoclonal anti- (ABNOVA) H00008741-M02 body (M02), clone
G3 Human APRIL/TNFSF13 R and D MAB884 MAb (Clone 101115)
[0165] To study whether the blocking characteristics of hAPRIL.01A
and hAPRIL.03A are unique, all known commercially available
anti-APRIL antibodies were tested for their ability to block the
interaction of FLAG-hAPRIL to BCMA-Fc and TACI-Fc (FIGS. 3A and
3B). Blockade of receptor binding was studied using an ELISA. An
ELISA plate was coated with 50 with 100 .mu.l of BCMA-Fc at 1
.mu.g/ml or with 100 .mu.l of TACI-Fc at a concentration of 2
.mu.g/ml in coating buffer and incubated overnight at 4.degree. C.
The plate was then washed with PBS/0.2% Tween and then incubated
with for 1 hour at 37.degree. C. with 100 .mu.l PBS/5% BSA per
well. The plate was then washed four times with PBS/0.2% Tween. In
a separate plate APRIL monoclonal antibodies were pre-mixed with
APRIL supernatant and incubated for 30 minutes on ice. Conditioned
medium containing soluble APRIL was diluted 1 in 4 and mixed with
an equal volume of PBS containing the antibodies titrated in
doubling dilutions starting with 5 .mu.g/ml. 100 .mu.l of the
pre-incubated mix was transferred to the ELISA plate and incubated
for 2 hours at 37.degree. C. The plate was then washed four times
with PBS/0.2% Tween. Anti-Flag-HRP antibody was then diluted in PBS
at a concentration of 1:1000 and then 100 .mu.l of this added to
each well and incubated for 1 hour at 37.degree. C. The plate was
then washed four times with PBS/0.2% Tween and then 100 .mu.l of
ABTS added to each well (the ABTS was diluted to the ratio 10 ml of
reagent plus 5 .mu.l of H.sub.2O.sub.2 made immediately before
addition). The colour was allowed to develop and then the OD at 405
nm read on an ELISA plate reader. Human IgG1 was used as a control
protein to coat the plate as this is the same isotype as the
Fc-fusion proteins and controlled for APRIL sticking to the plate
non-specifically. As is apparent from FIG. 3, none of the
commercially available antibodies was able to block the binding of
FLAG-APRIL to either TACI-Fc or BCMA-Fc, whereas hAPRIL.01A and
hAPRIL.03A do inhibit (partially) the binding to TACI-Fc and
BCMA-Fc.
[0166] Species Cross-Reactivity
[0167] Binding of hAPRIL.01A and hAPRIL.03A to mouse APRIL was also
examined by BIAcore, but no binding of either antibody was
observed. The antibodies appear only to bind human APRIL.
Example 3
Functional Profiling of Murine Anti-Human APRIL Antibodies
[0168] Mouse B Cell Response to APRIL
[0169] In order to show that the antibodies of this invention can
functionally block APRIL in-vitro a mouse B cell assays was used to
examine two APRIL driven responses in B cells--proliferation and
IgA production.
[0170] All cell lines were maintained at 37.degree. C. with 5%
CO.sub.2. Mouse splenocytes and purified B cells were grown in
RPMI-1640 (Gibco) supplemented with 8% FCS, 2 mM Glutamine and
Beta-mercaptoethanol at 50 .mu.M, and supplemented with penicillin
and streptomycin at a concentration of 10 .mu.g/ml. Splenic mouse B
cells were isolated from wild-type mice using magnetic activated
cell separation (MACS) columns with CD45R/B220 MACS beads (Miltenyi
Biotec, Utrecht, The Netherlands). The cells were cultured in
96-well round-bottomed microtiter plates at a density of
2.times.10.sup.5/well in a final volume of 200 .mu.l. For all
assays conditioned medium containing the various forms of soluble
APRIL were normalised for expression levels prior to use. To
measure proliferation, cells were treated with anti-IgM (Jackson
ImmunoResearch) and soluble APRIL in conditioned medium or as
purified protein at a final concentration of 1 .mu.g/ml.
Cross-linking anti-Flag monoclonal antibody was added to the well
at a final concentration of 1 .mu.g/ml. The cells were incubated at
37.degree. C. and after 48 hours pulsed with 0.3 .mu.Ci (0.011 MBq)
of tritiated thymidine ([6-.sup.3H] Thymidine, GE Healthcare, The
Netherlands) for 18 hours, before harvesting. To measure IgA
production, mouse B cells were cultured and treated with APRIL, as
above. Following incubation for 6 days, supernatant was collected
and assayed for IgA content by ELISA. Briefly, ELISA plates were
coated with 2 .mu.g/ml anti-mouse-Ig (Southern Biotech), blocked
with PBS/1% BSA and incubated with the collected supernatant. Bound
IgA was then detected with HRP labelled anti-mouse-IgA (Southern
Biotech, Uithoorn, the Netherlands). As a control, cells were
treated with 10 .mu.g/ml LPS (Invivogen) plus 1 ng/ml of human
TGF.beta. (Sigma-Aldrich). As shown in FIG. 4A, hAPRIL.01A and to a
lesser extent hAPRIL.03A are able to inhibit APRIL induced
class-switch recombination as was determined by the reduced IgA
secretion from mouse splenic B-cells. TACI-Fc as a control
inhibited the IgA secretion, while mouse IgG1 and human Ig did not
affect the APRIL-induced IgA secretion from splenic B-cells. In
addition, hAPRIL.01A and hAPRIL.03A were demonstrated to inhibit
APRIL-induced mouse splenic B-cell proliferation. To establish the
specificity of the antibodies, the effect of hAPRIL.01A and
hAPRIL.03A on BAFF-induced IgA secretion and proliferation was
studied. As shown in FIG. 4B, neither hAPRIL.01A nor hAPRIL.03A
inhibited BAFF induced IgA secretion and proliferation, while
TACI-Fc as a control inhibited both processes.
[0171] In-Vivo Experiment to Block APRIL Function
[0172] To demonstrate an in-vivo blocking effect of the antibodies
on APRIL function, we examined the ability of the antibodies to
block the NP-Ficoll induced humoral response in mice. The mice used
were 8-10 week old APRIL transgenic (TG) mice and wildtype (WT)
littermates, both on a C57BL/6 background. The APRIL transgenic
mice express human APRIL under the Lck-distal promoter, which
directs transgene expression to mature thymocytes and peripheral T
lymphocytes (Stein et al., 2002, J Clin Invest 109, 1587-98). The
mice were bred in the animal facility of the Academic Medical
Center and the experiment was approved by the institutional ethical
committee. The mice were divided into several groups and treated as
follows: five APRIL WT mice were treated with PBS (200 .mu.l) and 5
groups of five APRIL transgenic mice were treated with the
following molecules: hAPRIL.01A or hAPRIL.03A or TACI-Fc or
subisotype-matched control antibody msIgG1_k (200 .mu.g/mouse in
200 .mu.l PBS) or PBS. Treatment of the mice was started 3 days
before the NP-Ficoll immunization (day 0; 100 .mu.l i.p. with 250
.mu.g of the immunogen)--injections were continued twice a week for
28 days. Blood was collected via tail vein at day -1, 3, 7, 14 and
28. Anti-(4-hydroxy-nitrophenacetyl) (NP)-specific antibodies (IgM,
IgG and IgA) were assayed in 6 independent ELISA using diluted sera
(1:100 for IgA; 1:500 for IgG and 1:2,000 for IgM) as previously
described (Hardenberg et al., Immunol Cell Biol, 86(6), 530-4,
(2008)). Briefly 96-well ELISA plates (Greiner) were coated with
NP-BSA at 5 .mu.g/ml (Biosearch Technologies) in sodium carbonate
buffer (pH 9.6) overnight at 4.degree. C. The wells were blocked
with 1% BSA for 1 hr at 37.degree. C. and incubated with diluted
sera for 2 hrs at room temperature. HRP-conjugated isotype specific
antibodies (Goat anti-mouse IgG, IgA and IgM--from Southern
Biotech) were used as revealing antibodies. All dilutions were made
in PBS/BSA 1%/Tween 20 0.05%. One way ANOVA test was used to check
statistical significance between the groups TG (PBS) vs TG
(hAPRIL.01A) and TG (PBS) vs TG (hAPRIL.03A). As apparent from FIG.
5, both hAPRIL.01A and hAPRIL.03A inhibited the T-cell independent
B-cell responses in vivo. TACI-Fc inhibited this response less
efficient. PBS and mouse IgG1 as an isotype-matched control, did
not affect the IgA, IgM and IgG anti-NP response.
[0173] To examine the long-term effect of hAPRIL.01A and hAPRIL.03A
on B cell populations mice were treated as described above. On day
30, mice were sacrificed and the spleen and peritoneal exudate
cavity (PEC) analysed for B cell expression by flow cytometry.
Briefly, splenocytes and lymphocytes from the PEC were separated
from red blood cells by one wash with erythrocyte lysis buffer and
then counted. Cells were washed and resuspended in PBS/1% BSA and
seeded in 96-well round-bottomed plates at a density of
5.times.10.sup.5 per well. Next, cells were stained with the
following antibodies at the recommended concentrations: B220-FITC
(BD bioscience) and CD3-APC (ebioscience); IgD-FITC (BD bioscience)
and IgM-PE (BD bioscience); IgD-FITC (BD bioscience), CD3-APC
(ebioscience) and CD43-PE (BD bioscience). Antibodies were
incubated for 40 minutes, washed three times with PBS/1% BSA and
then analysed by flow cytometry using the FACSCalibur (Becton
Dickenson). B220.sup.+ B-cells, mature B-cells
(IgD.sup.+IgM.sup.int) and T2 B-cells (IgD.sup.+IgM.sup.+) in
spleen were quantified (see FIG. 6A). In addition, B1
(CD43.sup.+IgD.sup.int) and B2 (CD43.sup.-IgD.sup.+) subpopulations
were quantified in PEC (see FIG. 6B). The decrease in B cells in
response to TACI-Fc treatment is evident from both the spleen and
the PEC, indicating that long term administration of TACI-Fc may
have a detrimental effect on normal B cell populations. This is not
seen with hAPRIL.01A and hAPRIL.03A antibodies, suggesting that in
cases where APRIL but not BAFF is the primary cause of pathology,
the antibodies of this invention may show less side-effects than
TACI-Fc.
Example 4
Anti-APRIL Antibodies Sequences
[0174] Cloning of Immunoglobulin cDNAs
[0175] Degenerate primer PCR-based methods were used to determine
the DNA sequences encoding the variable regions for the mouse
antibodies that are expressed by hybridomas hAPRIL.01A and
hAPRIL.03A. Total RNA was isolated from 5.times.10.sup.6 hybridomas
cells using TRIZOL (Invitrogen), and gene specific cDNAs for the
heavy and light chains were synthesized using the iScript Select
cDNA synthesis kit (Biorad) according to the manufacturer's
instructions. The V.sub.H and V.sub.L genes were PCR-amplified
using a Novagen-based Ig-primer set (Novagen, San Diego, Calif.)
and Taq polymerase (Invitrogen). All PCR products that matched the
expected amplicon size of 500 bp were cloned into pCR4 TOPO vector
(Invitrogen), and the constructs were transformed in DH5.alpha. E.
coli (Invitrogen) according to the manufacturer's instructions.
Clones were screened by colony PCR using universal M13 forward and
reverse primers, and two clones from each reaction were selected
for DNA sequencing analysis. Sequences were searched against
databases of germline and rearranged IgV variable region sequences
using the website for NCBI Ig-Blast BLASTN 2.2.16. Blast results
for hAPRIL.01A and hAPRIL.03A showed one in-frame V.sub.H sequence
and one in frame V.sub.L sequence for each antibody. The amino acid
sequences were confirmed by mass spectrometry. The sequences are
disclosed in the attached Sequence Listing, FIG. 7 and listed in
Table 3.
TABLE-US-00004 TABLE 3 Sequence ID numbers for murine anti-human
APRIL antibodies of this invention SEQ ID NO: Description 1
hAPRIL.01A heavy chain variable region (DNA) 2 hAPRIL.01A light
chain variable region (DNA) 3 hAPRIL.03A heavy chain variable
region (DNA) 4 hAPRIL.03A light chain variable region (DNA) 5
hAPRIL.01A heavy chain variable region (AA) 6 hAPRIL.01A light
chain variable region (AA) 7 hAPRIL.03A heavy chain variable region
(AA) 8 hAPRIL.03A light chain variable region (AA) 9 hAPRIL.01A
heavy chain CDR1 (AA) 10 hAPRIL.01A heavy chain CDR2 (AA) 11
hAPRIL.01A heavy chain CDR3 (AA) 12 hAPRIL.01A light chain CDR1
(AA) 13 hAPRIL.01A light chain CDR2 (AA) 14 hAPRIL.01A light chain
CDR3 (AA) 15 hAPRIL.03A heavy chain CDR1 (AA) 16 hAPRIL.03A heavy
chain CDR2 (AA) 17 hAPRIL.03A heavy chain CDR3 (AA) 18 hAPRIL.03A
light chain CDR1 (AA) 19 hAPRIL.03A light chain CDR2 (AA) 20
hAPRIL.03A light chain CDR3 (AA)
Example 5
Epitope Mapping using Pepscan Method
[0176] Synthesis of Peptides and Pepscan Screening
[0177] The synthetic linear and CLIPS peptides were synthesized and
screened using credit-card format mini PEPSCAN cards (455-well
plate with 3 ul wells) as described by Slootstra et al. (Slootstra
et al., 1996, Mol. Diversity 1, 87-96) and Timmerman et al.
(Timmerman et al., 2007, J. Mol. Recognit. 20, 283-299). The
binding of antibodies (hAPRIL.01A and hAPRIL.03A) to each peptide
was tested in a PEPSCAN-based enzyme-linked immuno assay (ELISA).
The 455-well creditcard-format polypropylene cards, containing the
covalently linked peptides, were incubated with sample (for example
1 ug/ml antibody diluted in a PBS solution containing 5% horse
serum (vol/vol) and 5% ovalbumin (weight/vol)) and 1% Tween 80
(4.degree. C., overnight). After washing the peptides were
incubated with an anti-antibody peroxidase (dilution 1/1000, for
example rabbit anti-mouse peroxidase, Southern Biotech) (1 hour,
25.degree. C.), and subsequently, after washing the peroxidase
substrate 2,2'-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and
2, ul/ml 3% H2O2 were added. After 1 hour the color development was
measured. The color development of the ELISA was quantified with a
CCD-camera and an image processing system. The setup consists of a
CCD-camera and a 55 mm lens (Sony CCD Video Camara XC-77RR, Nikon
micro-nikkor 55 mm f/2.8 lens), a camera adaptor (Sony Camara
adaptor DC-77RR) and Image Processing Software.
[0178] Synthesis Peptides
[0179] A total of 4225, primarily, CLIPS peptides were synthesized.
The target sequence used, 147 amino acids, with loops according to
alignment with 1XU2.pdb underlined: [0180]
RAVLTQKQKKQHSVLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTF-
TMGQVVSREGQGRQETLFRCIRSMPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHGTFLGFVKL
(SEQ ID NO:21). Loops on "top" side of protein: QKKQHSVLHL (SEQ ID
NO:22), ALRRGRGL (SEQ ID NO:23), QAQGYGVRI (SEQ ID NO:24), QDAGVYLL
(SEQ ID NO:25), SREGQGRQETV (SEQ ID NO:26), FHLHQGDILSV (SEQ ID
NO:27) and loops on "bottom" side of protein: INATSKDDSDVTE (SEQ ID
NO:28), VLFQDVTFTMG (SEQ ID NO:29), IRSMPSHPDRAYNSC (SEQ ID NO:30),
IIPRARAKL (SEQ ID NO:31), NLSPHGTFLGF (SEQ ID NO:32). The
interconnecting regions are mostly sheets. Note that the "top" and
"bottom" side are chosen arbitrarily.
[0181] The following CLIPS topologies were used: T2 CLIPS couples
to the side-chain of two cysteines to form a single loop topology,
while T3 CLIPS couples to the side-chain of three cysteines to form
double loop topology, while T2T2 CLIPS first T2 couples to two
cysteines (labeled C), and second T2 couples to two cysteines and
finally T2T3 CLIPS T2 couples to two cysteines and T3 couples to
three cysteines.
[0182] In total 20 different sets of peptides were synthesized:
[0183] 191-1 (set-1): All overlapping 35-mer sequences covering the
complete 147 AA target sequence were synthesized. In this set the
different loops, when present in the sequence, as defined above
were constrained in double loop or sheet-like topology through two
T2 CLIPS. [0184] 191-2 (set-2) A total of nine sheets were
identified. All 9.times.9 combinations were synthesized to mimic
double sheet conformations. The sequence GSG was used as a linker.
[0185] 191-3 (set-3) The same as set-2 as explained above but with
a shorter sheet length. [0186] 191-6 (set-4) All overlapping linear
35-mer sequences covering the complete 147 AA target sequence were
synthesized. [0187] 191-7 (set-5) All overlapping linear 15-mer
sequences covering the complete 147 AA target sequence were
synthesized. [0188] 191-8 (set-6a) Short linear sequences (of
varying length) only covering the loop regions of the complete 147
AA target sequence were synthesized. [0189] 191-16 (set-6b)
Different peptides were selected from the five "bottom" loops.
These were recombined in a 9.times.9 matrix onto the T3 CLIPS to
form double looped topologies with "bottom" loops of two different
lengths. [0190] 191-17 (set-7) All overlapping 135 different 15-mer
sequences were synthesized with a cysteine at position 1, 8 and 15.
The three cysteine were coupled to a T3 CLIPS. [0191] 191-18
(set-9) Long versions of the six "top" loops and long versions of
the four "bottom" loops were recombined with each other on the T3
CLIPS. [0192] 191-19 (set-10) Six+Six+Four different sized loops of
the "top" loop region were all recombined with each other on the T3
CLIPS. [0193] 191-20 (set-11,17,18,19,20) 33 different sequences
broadly covering the "top" or "bottom" loops were recombined with
other on the T3 CLIPS. These sets of peptides are in sets 11, 17,
18, 19 and 20. Reason for this "scattering" is the card layout.
[0194] 191-22 (set-12) Different sized loops of all "top" and
"bottom" loops were synthesized as single loops on T2 CLIPS. [0195]
191-23 (set-13) All overlapping single looped 15-mer sequences
covering the complete target protein were synthesized on T2 CLIPS.
[0196] 191-24 (set-14) Six different 9-mer sequences covering the
"top" loops were recombined with each in a 6.times.6.times.6 triple
looped matrix on T2T3 CLIPS combination. [0197] 191-25 (set-15) The
same set of overlapping peptides as set-1. All overlapping 35-mer
sequences covering the complete 147AA target sequence were
synthesized. In this set the different loops, when present in the
sequence, as defined above were constrained into triple loop
topology through T3T2 CLIPS. [0198] 191-26 (set-16) Six different
9-mer sequences covering the "bottom" loops were recombined with
each in a 6.times.6.times.6 triple looped matrix on T2T3 CLIPS
combination.
[0199] Data Analysis and Epitope Determination
[0200] Each antibody was tested on all 4225 peptides and their
binding values were ranked. Clearly re-occurring sequences in most
the top binders (.about.top 1%) were considered as epitope
candidates. Two additional supporting analyses were done. Firstly,
it was investigated if multiple identified parts can form one
discontinuous epitope. This was done through the homologous
structure 1XU2.pdb. Secondly, it was investigated if each of
multiple identified binding parts was recognized without support of
the other part. These two parameters, co-localization on the 3D
structure and independent recognition, were used to support that a
conformational and discontinuous epitope was identified. For
hAPRIL.01A it was determined that it binds to IRSMPSHPDRA (SEQ ID
NO:33), with the core region being SMPSHP (SEQ ID NO:34). The TLFR
(SEQ ID NO:35) and/or QDVTFTMGQ (SEQ ID NO:36) (core region is
VTFTM (SEQ ID NO:37)) motifs were shown to support the binding of
hAPRIL.01A. hAPRIL.03A was shown to bind VSREGQGRQ (SEQ ID NO:38)
motif, with core region being EGQ. The TFTMGQ (SEQ ID NO:39) motif
was shown to support binding of hAPRIL.03A.
[0201] The invention is further described by the following numbered
paragraphs:
[0202] 1. A binding compound which binds to human APRIL comprising:
[0203] a. an antibody heavy chain variable region comprising at
least one CDR selected from the group consisting of SEQ ID NOs: 9,
10, 11, 15, 16 and 17, or a variant of any said sequence; and/or
[0204] b. an antibody light chain variable region comprising at
least one CDR selected from the group consisting of SEQ ID NOs: 12,
13, 14, 18, 19 and 20, or a variant of any said sequence. [0205] 2.
The binding compound of paragraph 1, comprising: [0206] a. heavy
chain CDRs SEQ ID NOs: 9, 10 and 11, or variants of any said
sequences; and light chain CDRs SEQ ID NOs: 12, 13 and 14, or
variants of any said sequences; or [0207] b. heavy chain CDRs SEQ
ID NOs: 15, 16 and 17 or variants of any said sequences; and light
chain CDRs SEQ ID NOs: 18, 19 and 20 or variants of any said
sequences. [0208] 3. The binding compound of paragraph 1 selected
from a binding compound comprising [0209] a. a heavy chain variable
region comprising the amino acid sequence of SEQ ID NO: 5 and a
light chain variable region comprising the amino acid sequence
selected from the group of SEQ ID NO: 6; or [0210] b. a heavy chain
variable region comprising the amino acid sequence of SEQ ID NO: 7
and a light chain variable region comprising the amino acid
sequence selected from the group of SEQ ID NO: 8. [0211] 4. The
binding compound of paragraphs 1-3, wherein any of said variant(s)
may comprise up to three amino acid modifications. [0212] 5. The
binding compound of any of the above paragraphs, wherein the
binding compound fully blocks the binding of APRIL with human TACI
and at least partially blocks the binding with human BCMA. [0213]
6. The binding compound of paragraph 5, wherein the binding
compound fully blocks the binding of human April with human BCMA.
[0214] 7. The binding compound of any of the above paragraphs,
wherein the binding compound: [0215] a. binds human APRIL with a
K.sub.D of about 10 nM or lower; and [0216] b. blocks binding of
human TACI and/or human BCMA to human APRIL with an IC.sub.50 of
about 2 nM or lower. [0217] 8. A binding compound which binds to
human APRIL wherein the binding compound has the same epitope
specificity as the compound selected from paragraph 3. [0218] 9. A
binding compound which competes for a binding epitope on human
APRIL with any of the binding compounds of paragraph 3, and has one
of the following characteristics: [0219] a. binds human APRIL with
a K.sub.D of about 10 nM or lower; [0220] b. binds to human APRIL
with about the same K.sub.D as an antibody having a heavy chain
comprising the amino acid sequence of SEQ ID NO: 5 and a light
chain comprising the amino acid sequence of SEQ ID NO: 6; [0221] c.
binds to human APRIL with about the same K.sub.D as an antibody
having a heavy chain comprising the amino acid sequence of SEQ ID
NO: 7 and a light chain comprising the amino acid sequence of SEQ
ID NO: 8; [0222] d. blocks binding of human TACI and/or human BCMA
to human APRIL with an IC.sub.50 of about 2 nM or lower. [0223] 10.
The binding compound of any of the above paragraphs, wherein the
binding compound is: [0224] a. a chimeric antibody or a fragment
thereof; [0225] b. a human antibody or a fragment thereof; [0226]
c. a humanized antibody or a fragment thereof; or [0227] d. an
antibody fragment selected from the group consisting of Fab, Fab',
Fab'-SH, Fv, scFv, F(ab').sub.2, bispecific mAb and a diabody.
[0228] 11. The binding compound of any of the above paragraphs,
wherein the binding compound inhibits the proliferation and
survival of B-cells. [0229] 12. An isolated polynucleotide encoding
the binding compound of any one of paragraphs 1 to 11. [0230] 13.
An expression vector comprising the isolated polynucleotide of
paragraph 12. [0231] 14. A host cell comprising the expression
vector of paragraph 13. [0232] 15. A method of producing a binding
compound according to any one of paragraphs 1 to 11 comprising:
[0233] a. culturing the host cell of paragraph 14 in culture medium
under conditions wherein the polynucleotide is expressed, thereby
producing polypeptides comprising the light and heavy chain
variable regions; and [0234] b. recovering the polypeptides from
the host cell or culture medium. [0235] 16. A composition
comprising the binding compound of any one of paragraphs 1 to 11 in
combination with a pharmaceutically acceptable carrier or diluent.
[0236] 17. Binding compound of any one of paragraphs 1 to 11 for
use in therapy. [0237] 18. The use of the binding compound of any
one of paragraphs 1 to 11 for [0238] a. inhibition of immune cell
proliferation and/or survival; [0239] b. treatment of cancer;
[0240] c. treatment of an autoimmune disease; or [0241] d.
treatment of an inflammatory disease. [0242] 19. The use of the
binding compound of any one of paragraphs 1 to 11 in a diagnostic
method.
Sequence CWU 1
1
391363DNAMus musculus 1gaggtccagt tgcagcagtc tggacctgag ctggtaaagc
ctggggcttc agtgaagatg 60tcctgcaagg cttctggata cacattcact agctatgtga
tgcactgggt gaagcagaag 120cctgggcagg gccttgagtg gattggatat
attaatcctt ataatgatgc tcctaaatac 180aatgagaagt tcaaaggcaa
ggccacagtg acttcagaca agtcctccgg cacagcctac 240atggagctca
gcagcctgac ctctgaggac tctgcggtct attactgtgc aaggggcttg
300ggttacgccc tttactatgc tatggactac tggggtcaag gaacctcagt
caccgtctcc 360tca 3632321DNAMus musculus 2gacattgtga tgacccagtc
tcaaaaattc aagtccacat cagtaggaga cagggtcagc 60gtcacctgca aggccagtca
gaatgtgggt aataatgtag cctggtatca acagaaagca 120gggcaatctc
ctaaagcact gatttcctcg gcatccaacc gtgacagtgg agtccctgat
180cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa
tgtgcagtct 240gaagacttgg cagactattt ctgtcagcaa tataacatct
atccattcac gttcggctcg 300gggacaaagt tggaaataaa a 3213366DNAMus
musculus 3caggttactc tgaaagagtc tggccctggg atattgcagc cctcccagac
cctcagtctg 60acttgttctt tctctgggtt ttcactgagc acttatggta taggagtagg
ctggattcgt 120cagccttcag ggaagggtct ggagtggctg gcacacattt
ggtggaatga taataagtac 180tataacacag ccctgaagag ccggctcaca
atctccaagg atacctccaa caaccaggta 240ttcctcaaga tcgccagtgt
ggacactgca gatactgcca catactactg tgctcgaata 300gctgggggta
actacgacta tgctatggac cactggggtc aaggaacctc agtcaccgtc 360tcctca
3664324DNAMus musculus 4caaattgttc tcacccagtc tccagcaatc atgtctacat
ctcctgggga gaaggtcacc 60ttgacctgca gtgccagctc aagtgtaagt tctacctact
tgtactggta ccagcagaag 120ccaggatcct cccccaaact ctggatttat
agcacatcca acctggcttc tggagtccct 180gctcgcttca gtggcagtgg
gtctgggacc tcttactctc tcacaatcag cagcatggag 240gctgaggatg
ctgcctctta tttctgccat cagtggagta gttacccacc tacgttcggt
300gctgggacca agctggagct gaaa 3245121PRTMus musculus 5Glu Val Gln
Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser
Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25
30 Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Ala Pro Lys Tyr Asn Glu
Lys Phe 50 55 60 Lys Gly Lys Ala Thr Val Thr Ser Asp Lys Ser Ser
Gly Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Leu Gly Tyr Ala Leu
Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ser Val Thr
Val Ser Ser 115 120 6107PRTMus musculus 6Asp Ile Val Met Thr Gln
Ser Gln Lys Phe Lys Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser
Val Thr Cys Lys Ala Ser Gln Asn Val Gly Asn Asn 20 25 30 Val Ala
Trp Tyr Gln Gln Lys Ala Gly Gln Ser Pro Lys Ala Leu Ile 35 40 45
Ser Ser Ala Ser Asn Arg Asp Ser Gly Val Pro Asp Arg Phe Thr Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln
Ser 65 70 75 80 Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ile
Tyr Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Glu Leu Glu Ile Lys
100 105 7122PRTMus musculus 7Gln Val Thr Leu Lys Glu Ser Gly Pro
Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser
Phe Ser Gly Phe Ser Leu Ser Thr Tyr 20 25 30 Gly Ile Gly Val Gly
Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala
His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Asn Thr Ala 50 55 60 Leu
Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Asn Asn Gln Val 65 70
75 80 Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr
Tyr 85 90 95 Cys Ala Arg Ile Ala Gly Gly Asn Tyr Asp Tyr Ala Met
Asp His Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115
120 8108PRTMus musculus 8Gln Ile Val Leu Thr Gln Ser Pro Ala Ile
Met Ser Thr Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Leu Thr Cys Ser
Ala Ser Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu Tyr Trp Tyr Gln
Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Ser Thr
Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80
Ala Glu Asp Ala Ala Ser Tyr Phe Cys His Gln Trp Ser Ser Tyr Pro 85
90 95 Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105
95PRTMus musculus 9Ser Tyr Val Met His 1 5 1017PRTMus musculus
10Tyr Ile Asn Pro Tyr Asn Asp Ala Pro Lys Tyr Asn Glu Lys Phe Lys 1
5 10 15 Gly 1112PRTMus musculus 11Gly Leu Gly Tyr Ala Leu Tyr Tyr
Ala Met Asp Tyr 1 5 10 1211PRTMus musculus 12Lys Ala Ser Gln Asn
Val Gly Asn Asn Val Ala 1 5 10 137PRTMus musculus 13Ser Ala Ser Asn
Arg Asp Ser 1 5 149PRTMus musculus 14Gln Gln Tyr Asn Ile Tyr Pro
Phe Thr 1 5 157PRTMus musculus 15Thr Tyr Gly Ile Gly Val Gly 1 5
1616PRTMus musculus 16His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Asn
Thr Ala Leu Lys Ser 1 5 10 15 1712PRTMus musculus 17Ile Ala Gly Gly
Asn Tyr Asp Tyr Ala Met Asp His 1 5 10 1812PRTMus musculus 18Ser
Ala Ser Ser Ser Val Ser Ser Thr Tyr Leu Tyr 1 5 10 197PRTMus
musculus 19Ser Thr Ser Asn Leu Ala Ser 1 5 209PRTMus musculus 20His
Gln Trp Ser Ser Tyr Pro Pro Thr 1 5 21147PRTHomo sapiens 21Arg Ala
Val Leu Thr Gln Lys Gln Lys Lys Gln His Ser Val Leu His 1 5 10 15
Leu Val Pro Ile Asn Ala Thr Ser Lys Asp Asp Ser Asp Val Thr Glu 20
25 30 Val Met Trp Gln Pro Ala Leu Arg Arg Gly Arg Gly Leu Gln Ala
Gln 35 40 45 Gly Tyr Gly Val Arg Ile Gln Asp Ala Gly Val Tyr Leu
Leu Tyr Ser 50 55 60 Gln Val Leu Phe Gln Asp Val Thr Phe Thr Met
Gly Gln Val Val Ser 65 70 75 80 Arg Glu Gly Gln Gly Arg Gln Glu Thr
Leu Phe Arg Cys Ile Arg Ser 85 90 95 Met Pro Ser His Pro Asp Arg
Ala Tyr Asn Ser Cys Tyr Ser Ala Gly 100 105 110 Val Phe His Leu His
Gln Gly Asp Ile Leu Ser Val Ile Ile Pro Arg 115 120 125 Ala Arg Ala
Lys Leu Asn Leu Ser Pro His Gly Thr Phe Leu Gly Phe 130 135 140 Val
Lys Leu 145 2210PRTHomo sapiens 22Gln Lys Lys Gln His Ser Val Leu
His Leu 1 5 10 238PRTHomo sapiens 23Ala Leu Arg Arg Gly Arg Gly Leu
1 5 249PRTHomo sapiens 24Gln Ala Gln Gly Tyr Gly Val Arg Ile 1 5
258PRTHomo sapiens 25Gln Asp Ala Gly Val Tyr Leu Leu 1 5
2611PRTHomo sapiens 26Ser Arg Glu Gly Gln Gly Arg Gln Glu Thr Val 1
5 10 2711PRTHomo sapiens 27Phe His Leu His Gln Gly Asp Ile Leu Ser
Val 1 5 10 2813PRTHomo sapiens 28Ile Asn Ala Thr Ser Lys Asp Asp
Ser Asp Val Thr Glu 1 5 10 2911PRTHomo sapiens 29Val Leu Phe Gln
Asp Val Thr Phe Thr Met Gly 1 5 10 3015PRTHomo sapiens 30Ile Arg
Ser Met Pro Ser His Pro Asp Arg Ala Tyr Asn Ser Cys 1 5 10 15
319PRTHomo sapiens 31Ile Ile Pro Arg Ala Arg Ala Lys Leu 1 5
3211PRTHomo sapiens 32Asn Leu Ser Pro His Gly Thr Phe Leu Gly Phe 1
5 10 3311PRTHomo sapiens 33Ile Arg Ser Met Pro Ser His Pro Asp Arg
Ala 1 5 10 346PRTHomo sapiens 34Ser Met Pro Ser His Pro 1 5
354PRTHomo sapiens 35Thr Leu Phe Arg 1 369PRTHomo sapiens 36Gln Asp
Val Thr Phe Thr Met Gly Gln 1 5 375PRTHomo sapiens 37Val Thr Phe
Thr Met 1 5 389PRTHomo sapiens 38Val Ser Arg Glu Gly Gln Gly Arg
Gln 1 5 396PRTHomo sapiens 39Thr Phe Thr Met Gly Gln 1 5
* * * * *