U.S. patent application number 13/444216 was filed with the patent office on 2012-08-02 for method to treat using antagonistic anti-htnfsf13b human antibodies.
This patent application is currently assigned to ELI LILLY AND COMPANY. Invention is credited to Kristine Kay Kikly.
Application Number | 20120195904 13/444216 |
Document ID | / |
Family ID | 23213094 |
Filed Date | 2012-08-02 |
United States Patent
Application |
20120195904 |
Kind Code |
A1 |
Kikly; Kristine Kay |
August 2, 2012 |
METHOD TO TREAT USING ANTAGONISTIC ANTI-hTNFSF13B HUMAN
ANTIBODIES
Abstract
Human monoclonal antibodies that specifically bind to TNFSF13b
polypeptides are disclosed. These antibodies have high affinity for
hTNFSF13b (e.g., K.sub.D=10.sup.-8M or less), a slow off rate for
TNFSF13b dissociation (e.g., K.sub.off=10.sup.-3 sec.sup.-1 or
less) and neutralize TNFSF13b activity in vitro and in vivo. The
antibodies of the invention are useful in one embodiment for
inhibiting TNFSF13b activity in a human subject suffering from a
disorder in which hTNFSF13b activity is detrimental. Nucleic acids
encoding the antibodies of the present invention, as well as,
vectors and host cells for expressing them are also encompassed by
the invention.
Inventors: |
Kikly; Kristine Kay;
(Fortville, IN) |
Assignee: |
ELI LILLY AND COMPANY
Indianapolis
IN
|
Family ID: |
23213094 |
Appl. No.: |
13/444216 |
Filed: |
April 11, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12777306 |
May 11, 2010 |
8173124 |
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13444216 |
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11952363 |
Dec 7, 2007 |
7728109 |
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12777306 |
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10484790 |
Jan 22, 2004 |
7317089 |
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11952363 |
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PCT/US02/21842 |
Aug 15, 2002 |
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10484790 |
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60312808 |
Aug 16, 2001 |
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Current U.S.
Class: |
424/142.1 |
Current CPC
Class: |
C07K 2317/53 20130101;
A61P 37/00 20180101; A61K 2039/505 20130101; A61P 29/00 20180101;
A61P 35/00 20180101; C07K 2317/21 20130101; A61P 37/02 20180101;
A01K 2217/05 20130101; A61P 17/02 20180101; C07K 2317/92 20130101;
A61P 43/00 20180101; A61P 19/02 20180101; A61P 37/08 20180101; A61P
15/00 20180101; C07K 2317/34 20130101; A61P 31/04 20180101; C07K
2317/33 20130101; C07K 16/2875 20130101; A61P 11/06 20180101; A61P
17/06 20180101; C07K 2317/73 20130101; C07K 2317/76 20130101; A61P
1/04 20180101; C07K 2317/565 20130101 |
Class at
Publication: |
424/142.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 19/02 20060101 A61P019/02 |
Claims
1-41. (canceled)
42. A method of treating a human patient suffering from rheumatoid
arthritis comprising administering to the said patient an
anti-hTNFSF13b human antibody comprising SEQ ID NO: 17 and SEQ ID
NO: 19.
43. The method of treating a human patient suffering from
rheumatoid arthritis comprising administering to the said patient
an anti-hTNFSF13b human antibody comprising SEQ ID NO: 18 and SEQ
ID NO: 19.
Description
[0001] This application is a divisional of U.S. Ser. No.
12/777,306, filed 11 May 2010, which is a divisional application of
U.S. Ser. No. 11/952,363, filed 7 Dec. 2007, which is a
continuation of Ser. No. 10/484,790, filed 22 Jan. 2004, which is a
continuation of PCT/US02/21842, filed 15 Aug. 2002, which claims
the priority of U.S. provisional application No. 60/312,808 filed
16 Aug. 2001.
[0002] The TNF family ligands are known to be among the most
pleiotropic cytokines, inducing a large number of cellular
responses, including proliferation, cytotoxicity, anti-viral
activity, immunoregulatory activities, and the transcriptional
regulation of several genes. The TNF family of cytokines and
receptors has undergone a large expansion in the last few years
with the advent of massive EST sequencing. TNFSF13b is the official
name adopted by the TNF Congress for BLyS, TALL-1, BAFF, THANK,
neutrokine-.alpha., and zTNF (for review see Locksley et al. Cell
2001 104:487). Human TNFSF13b (hTNFSF13b) is a 285-amino acid type
II membrane-bound protein that possesses a N-terminal cleavage site
that allows for the existence of both soluble and membrane bound
proteins. Functionally, TNFSF13b appears to regulate B cell and
some T cell immune responses.
[0003] Studies of septic shock syndrome and other disorders arising
from overproduction of inflammatory cytokines have shown that an
afflicted host will often counter high cytokine levels by releasing
soluble cytokine receptors or by synthesizing high-affinity
anti-cytokine antibodies. Methods of treatment that mimic such
natural responses are considered as viable therapeutic approaches
for alleviating cytokine-mediated disease. Thus, there is a
well-recognized need for human antibodies that bind cytokines, such
as TNFSF13b, that are involved in the regulation of cellular immune
processes with high affinity and that have the capacity to
antagonize the activity of the targeted cytokine in vitro and in
vivo. Although international patent application WO00/50597
non-descriptively discloses antibodies directed at TNFSF13b, that
application does not specifically describe the structural or
functional characteristics of such antibodies.
[0004] The present invention provides anti-hTNFSF13b human
antibodies, or antigen-binding portions thereof. The antibodies of
the invention are characterized by high affinity binding to
TNFSF13b polypeptides, slow dissociation kinetics, and the capacity
to antagonize at least one in vitro and/or in vivo activity
associated with TNFSF13b polypeptides.
[0005] The present invention also provides anti-hTNFSF13b human
antibodies comprising a polypeptide selected from the group
consisting of a polypeptide as shown in SEQ ID NO: 2, a polypeptide
as shown in SEQ ID NO: 4, a polypeptide as shown in SEQ ID NO: 6, a
polypeptide as shown in SEQ ID NO: 8, a polypeptide as shown in SEQ
ID NO: 10, a polypeptide as shown in SEQ ID NO: 12, a polypeptide
as shown in SEQ ID NO: 14, and a polypeptide as shown in SEQ ID NO:
16.
[0006] In another embodiment, the invention provides an isolated
anti-hTNFSF13b human antibody which binds to a hTNFSF13b
polypeptide and dissociates from the hTNFSF13b polypeptide with a
K.sub.off rate constant of 1.times.10.sup.-4 s.sup.-1 or less, as
determined by surface plasmon resonance, or which inhibits TNFSF13b
induced proliferation in an in vitro neutralization assay with an
IC.sub.50 of 1.times.10.sup.-8 M or less.
[0007] In an preferred embodiment, the invention provides an
isolated anti-hTNFSF13b human antibody that has the following
characteristics:
[0008] a) inhibits TNFSF13b induced proliferation in an in vitro
neutralization assay with an IC.sub.50 of 1.times.10.sup.-8 M or
less;
[0009] b) has a heavy chain CDR3 comprising the amino acid sequence
of SEQ ID NO:16; and
[0010] c) has a light chain CDR3 comprising the amino acid sequence
of SEQ ID NO:8.
[0011] The invention also provides methods of treating or
preventing acute or chronic diseases or conditions associated with
B cell and some T cell activity including, but not limited to,
autoimmune disorders, such as systemic lupus erythematosus,
rheumatoid arthritis, and/or neoplasia comprising the
administration of an anti-hTNFSF13b human antibody of the present
invention.
[0012] In another embodiment, the present invention provides
sequences that encode the novel anti-hTNFSF13b human antibodies,
vectors comprising the polynucleotide sequences encoding
anti-hTNFSF13b human antibodies, host cells transformed with
vectors incorporating polynucleotides that encode the
anti-hTNFSF13b human antibodies, formulations comprising
anti-hTNFSF13b human antibodies and methods of making and using the
same.
[0013] In another embodiment, the present invention provides the
epitope of the antigen to which the novel anti-hTNFSF13b human
antibodies bind. Thus, the invention also provides a use of an
antibody that binds the epitope of the present invention thereby
neutralizing the TNFSF13b activity for the treatment or prevention
of acute or chronic diseases or conditions associated with B cell
and some T cell activity including, but not limited to, autoimmune
disorders, such as systemic lupus erythematosus, rheumatoid
arthritis, and/or neoplasia.
[0014] The invention also encompasses an article of manufacture
comprising a packaging material and an antibody contained within
said packaging material, wherein the antibody neutralizes TNFSF13b
activity for treatment or prevention of a human subject suffering
from a disorder in which TNFSF13b activity is detrimental, and
wherein the packaging material comprises a package insert which
indicates that the antibody neutralizes by binding an epitope of
TNFSF13b, wherein the epitope comprises lysine at position 71,
threonine at position 72, tyrosine at position 73, and glutamic
acid at position 105; and a package insert that provides for
administration of the dosage form that results in neutralizing
TNFSF13b activity.
[0015] FIG. 1. Graph illustrating the inhibition of hTNFSF13b and
IL-1 induced proliferation of T1165.17 cells by human antibody
4A5-3.1.1-B4.
[0016] FIG. 2. Graph illustrating the neutralization of hTNFSF13b
induced proliferation by human antibody 4A5-3.1.1-B4 in primary
human B cells stimulated with anti-IgM.
[0017] In order that the present invention may be more readily
understood, certain terms are first defined.
[0018] An antibody is an immunoglobulin molecule comprised of four
polypeptide chains, two heavy (H) chains (about 50-70 kDa) and two
light (L) chains (about 25 kDa) inter-connected by disulfide bonds.
Light chains are classified as kappa and lambda. Heavy chains are
classified as gamma, mu, alpha, delta, or epsilon, and define the
antibody's isotype as IgG, IgM, IgA, IgD, and IgE, respectively.
Each heavy chain is comprised of a heavy chain variable region
(abbreviated herein as HCVR) and a heavy chain constant region. The
heavy chain constant region is comprised of three domains, CH1,
CH2, and CH3 for IgG, IgD and IgA, and 4 domains, CHL CH2, CH3, CH4
for IgM and IgE. Each light chain is comprised of a light chain
variable region (abbreviated herein as LCVR) and a light chain
constant region. The light chain constant region is comprised of
one domain, CL. The HCVR and LCVR regions can be further subdivided
into regions of hypervariability, termed complementarity
determining regions (CDRs), interspersed with regions that are more
conserved, termed framework regions (FR). Each HCVR and LCVR is
composed of three CDRs and four FRs, arranged from amino-terminus
to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2,
FR3, CDR3, FR4. The assignment of amino acids to each domain is in
accordance with well known conventions. [Kabat, et al, Sequences of
Proteins of Immunological Interest, Fifth Edition, U.S. Department
of Health and Human Services, NIH Publication No. 91-3242 (1991);
Chothia, et al., J. Mol. Biol. 196:901-917 (1987); Chothia, et al.,
Nature 342:878-883 (1989)]. The functional characteristics of the
antibody to bind a particular antigen are determined by the
CDRs.
[0019] In the present disclosure the term "antibody" is intended to
refer to a monoclonal antibody per se. A monoclonal antibody can be
a human antibody, chimeric antibody, humanized antibody, Fab
fragment, Fab' fragment, F(ab')2, or single chain FV fragment.
Preferably the term "antibody" refers to human antibody.
[0020] The term "human antibody" includes antibodies having
variable and constant regions corresponding to human germline
immunoglobulin sequences. Human antibodies have several advantages
over non-human and chimeric antibodies for use in human therapy.
For example, the effector portion of a human antibody may interact
better with the other parts of the human immune system (e.g.,
destroy the target cells more efficiently by complement-dependent
cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity
(ADCC)). Another advantage is that the human immune system should
not recognize the human antibody as foreign, and, therefore the
antibody response against such an injected antibody should be less
than against a totally foreign non-human antibody or a partially
foreign chimeric antibody. In addition, injected non-human
antibodies have been reported to have a half-life in the human
circulation much shorter than the half-life of human antibodies.
Injected human antibodies will have a half-life essentially
identical to naturally occurring human antibodies, allowing smaller
and less frequent doses to be given.
[0021] The term "hTNFSF13b" refers to the human form of a member of
the tumor necrosis factor family of ligands described in
international patent applications WO98/18921 and WO00/50597
(referred to therein as neutrokine-.alpha.). The term "TNFSF13b" is
intended to encompass hTNFSF13b as well as homologs of hTNFSF13b
derived from other species. The terms "hTNFSF13b" and "TNFSF13b"
are intended to include forms thereof, which can be prepared by
standard recombinant expression methods or purchased commercially
(Research Diagnostics Inc. Catalog No. RDI-3113, rhuBAFF, Flanders,
N.J.) as well as generated synthetically.
[0022] The phrase "biological property", "biological
characteristic", and the term "activity" in reference to an
antibody of the present invention are used interchangeably herein
and include, but are not limited to, epitope affinity and
specificity (e.g., anti-hTNFSF13b human antibody binding to
hTNFSF13b), ability to antagonize the activity of the targeted
polypeptide (e.g., TNFSF13b activity), the in vivo stability of the
antibody, and the immunogenic properties of the antibody. Other
identifiable biological properties or characteristics of an
antibody recognized in the art include, for example,
cross-reactivity, (i.e., with non-human homologs of the targeted
polypeptide, or with other proteins or tissues, generally), and
ability to preserve high expression levels of protein in mammalian
cells. The aforementioned properties or characteristics can be
observed or measured using art-recognized techniques including, but
not limited to ELISA, competitive ELISA, surface plasmon resonance
analysis, in vitro and in vivo neutralization assays (e.g., Example
2), and immunohistochemistry with tissue sections from different
sources including human, primate, or any other source as the need
may be. Particular activities and biological properties of
anti-hTNFSF13b human antibodies are described in further detail in
the Examples below.
[0023] The phrase "contact position" includes an amino acid
position in the CDR1, CDR2 or CDR3 of the heavy chain variable
region or the light chain variable region of an antibody which is
occupied by an amino acid that contacts antigen. If a CDR amino
acid contacts the antigen, then that amino acid can be considered
to occupy a contact position.
[0024] "Conservative substitution" or "conservative amino acid
substitution" refers to amino acid substitutions, either from
natural mutations or human manipulation, wherein the antibodies
produced by such substitutions have substantially the same (or
improved or reduced, as may be desirable) activity(ies) as the
antibodies disclosed herein.
[0025] The term "epitope" as used herein refers to a region of a
protein molecule to which an antibody can bind. An "immunogenic
epitope" is defined as the part of a protein that elicits an
antibody response when the whole protein is the immunogen.
[0026] The term "binds" as used herein, generally refers to the
interaction of the antibody to the epitope of the antigen. More
specifically, the term "binds" relates to the affinity of the
antibody to the epitope of the antigen. Affinity is measured by
K.sub.D.
[0027] The term "inhibit" or "inhibiting" includes the generally
accepted meaning, which includes neutralizing, prohibiting,
preventing, restraining, slowing, stopping, or reversing
progression or severity of a disease or condition.
[0028] The term "neutralizing" or "antagonizing" in reference to an
anti-TNFSF13b antibody or the phrase "antibody that antagonizes
TNFSF13b activity" is intended to refer to an antibody or antibody
fragment whose binding to TNFSF13b results in inhibition of a
biological activity induced by TNFSF13b polypeptides. Inhibition of
TNFSF13b biological activity can be assessed by measuring one or
more in vitro or in vivo indicators of TNFSF13b biological activity
including, but not limited to, TNFSF13b-induced proliferation,
TNFSF13b-induced immunoglobulin secretion, TNFSF13b-induced
prevention of B cell apoptosis, or inhibition of receptor binding
in a TNFSF13b receptor binding assay. Indicators of TNFSF13b
biological activity can be assessed by one or more of the several
in vitro or in vivo assays known in the art. (see, for example,
Moore, P. A., et al., Science, 285:260-263 (1999); Schneider, P.,
et al., J. Exp. Med., 189:1747-1756 (1999); Shu, H., et al., J.
Leuko. Biol., 65:680-683 (1999); Mukhopadhyay, A., et al., J. Biol.
Chem., 274:15978-15981 (1999); Mackay, F. et al., J. Exp. Med.,
190:1697-1710 (1999); Gross, J. A., et al., Nature, 404:995-999
(2000); and Example 2). Preferably, the ability of an antibody to
neutralize or antagonize TNFSF13b activity is assessed by
inhibition of B cell proliferation as shown in Example 2.
[0029] The term "K.sub.off", as used herein, is intended to refer
to the off rate constant for dissociation of an antibody from the
antibody/antigen complex.
[0030] The term "K.sub.D", as used herein, is intended to refer to
the dissociation constant or the "off" rate divided by the "on"
rate, of a particular antibody-antigen interaction. For purposes of
the present invention K.sub.D was determined as shown in Example
4
[0031] 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 non-proteinaceous solutes. Ordinarily, an isolated
antibody is prepared by at least one purification step. In
preferred embodiments, the antibody will be purified (1) to greater
than 95% by weight of antibody as determined by the Lowry method,
and most preferably more than 99% by weight, and (2) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue, or preferably, silver stain. Preferably, an
"isolated antibody" is an antibody that is substantially free of
other antibodies having different antigenic specificities (e.g., an
isolated antibody that specifically binds hTNFSF13b substantially
free of antibodies that specifically bind antigens other than
hTNFSF13b polypeptide).
[0032] The phrase "nucleic acid molecule" includes DNA molecules
and RNA molecules. A nucleic acid molecule may be single-stranded
or double-stranded, but preferably is double-stranded DNA.
[0033] The phrase "isolated nucleic acid molecule", as used herein
in reference to nucleic acids encoding antibodies or antibody
fragments (e.g., HCVR, LCVR, CDR3) that bind hTNFSF13b polypeptide,
includes a nucleic acid molecule in which the nucleotide sequences
encoding the antibody, or antibody portion, are free of other
nucleotide sequences encoding antibodies or antibody fragments that
bind antigens other than hTNFSF13b polypeptide, which other
sequences may naturally flank the nucleic acid in human genomic
DNA. Thus, for example, an isolated nucleic acid of the invention
encoding a HCVR region of an anti-hTNFSF13b human antibody contains
no other sequences encoding HCVR regions that bind antigens other
than hTNFSF13b polypeptide.
[0034] The term "vector" includes a nucleic acid molecule capable
of transporting another nucleic acid to which it has been linked.
One type of vector is a "plasmid", which refers to a circular
double stranded DNA loop into which additional DNA segments may be
ligated. Another type of vector is a viral vector, wherein
additional DNA segments may be ligated into the viral genome.
Certain vectors are capable of autonomous replication in a host
cell into which they are introduced (e.g., bacterial vectors having
a bacterial origin of replication and episomal mammalian vectors).
Other vectors (e.g., non-episomal mammalian vectors) can be
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors are capable of directing the
expression of genes to which they are operatively linked. Such
vectors are referred to herein as "recombinant expression vectors"
(or simply, "expression vectors"). In general, expression vectors
of utility in recombinant DNA techniques are often in the form of
plasmids. In the present specification, "plasmid" and "vector" may
be used interchangeably as the plasmid is the most commonly used
form of vector. However, the invention is intended to include such
other forms of expression vectors, such as viral vectors (e.g.,
replication defective retroviruses, adenoviruses, and
adeno-associated viruses), that serve equivalent functions.
[0035] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading frame. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0036] The term "recombinant" in reference to an antibody includes
antibodies that are prepared, expressed, created or isolated by
recombinant means. Representative examples include antibodies
expressed using a recombinant expression vector transfected into a
host cell, antibodies isolated from a recombinant, combinatorial
human antibody library, antibodies isolated from an animal (e.g., a
mouse) that is transgenic for human immunoglobulin genes or
antibodies prepared, expressed, created or isolated by any means
that involves splicing of human immunoglobulin gene sequences to
other DNA sequences. Such recombinant human antibodies have
variable and constant regions derived from human germline
immunoglobulin sequences.
[0037] The phrase "recombinant host cell" (or simply "host cell")
includes a cell into which a recombinant expression vector has been
introduced. It should be understood that such terms are intended to
refer not only to the particular subject cell but to the progeny of
such a cell. Because certain modifications may occur in succeeding
generations due to either mutation or environmental influences,
such progeny may not, in fact, be identical to the parent cell, but
are still included within the scope of the term "host cell" as used
herein.
[0038] Recombinant human antibodies may also be subjected to in
vitro mutagenesis (or, when an animal transgenic for human Ig
sequences is used, in vivo somatic mutagenesis) and, thus, the
amino acid sequences of the HCVR and LCVR regions of the
recombinant antibodies are sequences that, while derived from those
related to human germline HCVR and LCVR sequences, may not
naturally exist within the human antibody germline repertoire in
vivo.
[0039] 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 can be
employed. Transfer of the human germ-line immunoglobulin gene array
in such germ-line mutant mice will result in the production of
human antibodies upon antigen challenge (see, e.g., Jakobovits, et
al., Proc. Natl. Acad. Sci. USA, 90:2551-2555, (1993); Jakobovits,
et al., Nature, 362:255-258, (1993; Bruggemann, et al., Year in
Immun., 7:33 (1993); Nature 148:1547-1553 (1994), Nature
Biotechnology 14:826 (1996); Gross, J. A., et al., Nature,
404:995-999 (2000); and U.S. Pat. Nos. 5,877,397, 5,874,299,
5,814,318, 5,789,650, 5,770,429, 5,661,016, 5,633,425, 5,625,126,
5,569,825, and 5,545,806 (each of which is incorporated herein by
reference in its entirety for all purposes)). Human antibodies can
also be produced in phage display libraries (Hoogenboom and Winter,
J. Mol. Biol., 227:381 (1992); Marks, et al., J. Mol. Biol.,
222:581 (1991)). The techniques of Cole et al. and Boerner, et al.
are also available for the preparation of human monoclonal
antibodies (Cole, et al., Monoclonal Antibodies and Cancer Therap,
Alan R. Liss, p. 77 (1985) and Boerner, et al., J. Immunol.,
147(1):86-95 (1991)).
[0040] "Container" means any receptacle and closure suitable for
storing, shipping, dispensing, and/or handling a pharmaceutical
product.
[0041] "Packaging material" means a customer-friendly device
allowing convenient administration and/or ancillary devices that
aid in delivery, education, and/or administration. The packaging
material may improve antibody administration to the patient, reduce
or improve educational instruction time for the patient, provide a
platform for improved health economic studies, and/or limit
distribution channel workload. Also, the packaging material may
include but not be limited to a paper-based package, shrink wrapped
package, see-through top packaging, trial-use coupons, educational
materials, ancillary supplies, and/or delivery device.
[0042] "Package insert" means information accompanying the product
that provides a description of how to administer the product, along
with the safety and efficacy data required to allow the physician,
pharmacist, and patient to make an informed decision regarding use
of the product, and/or patient education information. The package
insert generally is regarded as the "label" for a pharmaceutical
product.
[0043] A "subject" means a mammal; preferably a human in need of a
treatment. In regards to the present invention subjects in need of
treatment include mammals that are suffering from, or are prone to
suffer from a disorder in which TNFSF13b activity is detrimental,
for example immune diseases, including autoimmune diseases, and
inflammatory diseases. Preferred disorders include, but are not
limited to, systemic lupus erythematosus, rheumatoid arthritis,
juvenile chronic arthritis, Lyme arthritis, Crohn's disease,
ulcerative colitis, inflammatory bowel disease, asthma, allergic
diseases, psoriasis, graft versus host disease, organ transplant
rejection, acute or chronic immune disease associated with organ
transplantation, sarcoidosis, infectious diseases, parasitic
diseases, female infertility, autoimmune thrombocytopenia,
autoimmune thyroid disease, Hashimoto's disease, Sjogren's
syndrome, and cancers, particularly B or T cell lymphomas or
myelomas.
[0044] Various aspects of the invention are described in further
detail in the following subsections.
[0045] The present invention relates to human monoclonal antibodies
that are specific for and neutralize bioactive hTNFSF13b
polypeptides. Also disclosed are antibody heavy and light chain
amino acid sequences which are highly specific for and neutralize
TNFSF13b polypeptides when they are bound to them. This high
specificity enables the anti-hTNFSF13b human antibodies, and human
monoclonal antibodies with like specificity, to be immunotherapy of
TNFSF13b associated diseases.
[0046] In one aspect, the invention provides an isolated human
antibody comprising at least one of the amino acid sequences shown
in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, or 16 and that binds a
TNFSF13b polypeptide epitope with high affinity, dissociates from a
bound TNFSF13b polypeptide with a low K.sub.off rate constant of
1.times.10.sup.-4 s.sup.-1 or less, and has the capacity to
antagonize TNFSF13b polypeptide activity. In one embodiment, the
anti-hTNFSF13b human antibody comprises a polypeptide selected from
the group consisting of: CDR1 polypeptide of the LCVR as shown in
SEQ ID NO: 4; CDR2 polypeptide of the LCVR as shown in SEQ ID NO:
6; CDR3 polypeptide of the LCVR as shown in SEQ ID NO: 8; CDR1
polypeptide of the HCVR as shown in SEQ ID NO: 12; CDR2 polypeptide
of the HCVR as shown in SEQ ID NO: 14; and CDR3 polypeptide of the
HCVR as shown in SEQ ID NO: 16. In another embodiment, the
anti-hTNFSF13b human antibody comprises at least two of the
polypeptides selected from the group consisting of: CDR1
polypeptide of the LCVR as shown in SEQ ID NO: 4; CDR2 polypeptide
of the LCVR as shown in SEQ ID NO: 6; CDR3 polypeptide of the LCVR
as shown in SEQ ID NO: 8; CDR1 polypeptide of the HCVR as shown in
SEQ ID NO: 12; CDR2 polypeptide of the HCVR as shown in SEQ ID NO:
14; and CDR3 polypeptide of the HCVR as shown in SEQ ID NO: 16. In
another embodiment, the anti-hTNFSF13b human antibody comprises at
least three of the polypeptides selected from the group consisting
of: CDR1 polypeptide of the LCVR as shown in SEQ ID NO: 4; CDR2
polypeptide of the LCVR as shown in SEQ ID NO: 6; CDR3 polypeptide
of the LCVR as shown in SEQ ID NO: 8; CDR1 polypeptide of the HCVR
as shown in SEQ ID NO: 12; CDR2 polypeptide of the HCVR as shown in
SEQ ID NO: 14; and CDR3 polypeptide of the HCVR as shown in SEQ ID
NO: 16. In another embodiment, the anti-hTNFSF13b human antibody
comprises at least four of the polypeptides selected from the group
consisting of: CDR1 polypeptide of the LCVR as shown in SEQ ID NO:
4; CDR2 polypeptide of the LCVR as shown in SEQ ID NO: 6; CDR3
polypeptide of the LCVR as shown in SEQ ID NO: 8; CDR1 polypeptide
of the HCVR as shown in SEQ ID NO: 12; CDR2 polypeptide of the HCVR
as shown in SEQ ID NO: 14; and CDR3 polypeptide of the HCVR as
shown in SEQ ID NO: 16. In another embodiment, the anti-hTNFSF13b
human antibody comprises at least five of the polypeptides selected
from the group consisting of: CDR1 polypeptide of the LCVR as shown
in SEQ ID NO: 4; CDR2 polypeptide of the LCVR as shown in SEQ ID
NO: 6; CDR3 polypeptide of the LCVR as shown in SEQ ID NO: 8; CDR1
polypeptide of the HCVR as shown in SEQ ID NO: 12; CDR2 polypeptide
of the HCVR as shown in SEQ ID NO: 14; and CDR3 polypeptide of the
HCVR as shown in SEQ ID NO: 16. In another embodiment, the
anti-hTNFSF13b human antibody comprises the polypeptides of CDR1
polypeptide of the LCVR as shown in SEQ ID NO: 4; CDR2 polypeptide
of the LCVR as shown in SEQ ID NO: 6; CDR3 polypeptide of the LCVR
as shown in SEQ ID NO: 8; CDR1 polypeptide of the HCVR as shown in
SEQ ID NO: 12; CDR2 polypeptide of the HCVR as shown in SEQ ID NO:
14; and CDR3 polypeptide of the HCVR as shown in SEQ ID NO: 16.
[0047] More preferred, the anti-hTNFSF13b human antibody comprises
a light chain variable region (LCVR) polypeptide as shown in SEQ ID
NO: 2 or a heavy chain variable region (HCVR) polypeptide as shown
in SEQ ID NO: 10. Even more preferred, the anti-hTNFSF13b human
antibody comprises the LCVR polypeptide as shown in SEQ ID NO: 2
and the HCVR polypeptide as shown in SEQ ID NO: 10.
[0048] In preferred embodiments, the isolated human antibody
dissociates from a bound TNFSF13b polypeptide with a K.sub.off rate
constant of 5.times.10.sup.-5 s.sup.-1 or less, and inhibits
TNFSF13b induced proliferation in an in vitro neutralization assay
with an IC.sub.50 of 1.times.10.sup.-7 M or less. In more preferred
embodiments, the isolated human antibody dissociates from a bound
TNFSF13b polypeptide epitope with a K.sub.off rate constant of
1.times.10.sup.-5 s.sup.-1 or less and inhibits TNFSF13b induced
proliferation in an in vitro neutralization assay with an IC.sub.50
of 1.times.10.sup.-8 M or less. In an even more preferred
embodiment, the isolated anti-TNFSF13b human antibody dissociates
from a bound hTNFSF13b polypeptide with a K.sub.off rate constant
of 5.times.10.sup.-6 5.sup.-1 or less and inhibits TNFSF13b induced
proliferation in an in vitro assay with an IC.sub.50 of
1.times.10.sup.-9 M or less. Examples of anti-hTNFSF13b human
antibodies that meet, the aforementioned kinetic and neutralization
criteria include 4A5-3.1.1-B4 antibodies.
[0049] The most preferred anti-hTNFSF13b human antibody of the
present invention is referred to herein as 4A5-3.1.1-B4.
4A5-3.1.1-B4 has LCVR and HCVR polypeptide sequences as shown in
SEQ ID NO:2 and SEQ ID NO:10, respectively. The poly-nucleotide
sequence encoding the LCVR and HCVR of 4A5-3.1.1-B4 is shown in SEQ
ID NO:1 and SEQ ID NO:9, respectively. The properties of the
anti-hTNFSF13b human antibodies of the present invention are
specifically disclosed in the Examples. Particularly notable is the
high affinity for TNFSF13b polypeptide, slow dissociation kinetics,
and high capacity to antagonize TNFSF13b polypeptide activity
demonstrated by 4A5-3.1.1-B4.
[0050] The K.sub.off of an anti-hTNFSF13b human antibody can be
determined by surface plasmon resonance as generally described in
Example 4. Generally, surface plasmon resonance analysis measures
real-time binding interactions between ligand (recombinant TNFSF13b
polypeptide immobilized on a biosensor matrix) and analyte
(antibodies in solution) by surface plasmon resonance (SPR) using
the BIAcore system (Pharmacia Biosensor, Piscataway, N.J.). SPR
analysis can also be performed by immobilizing the analyte
(antibodies on a biosensor matrix) and presenting the ligand
(recombinant TNFSF13b in solution).
[0051] In one aspect, the present invention is also directed to the
cell lines which produce the anti-hTNFSF13b human antibodies of the
present invention. The isolation of cell lines producing monoclonal
antibodies of the invention can be accomplished using routine
screening techniques known in the art. A hybridoma which produces
an anti-hTNFSF13b human antibody of the present invention has been
deposited with ATCC, (ATCC PTA-3674) as disclosed herein.
[0052] A wide variety of host expression systems can be used to
express the antibodies of the present invention including
bacterial, yeast, baculoviral, plant, and mammalian expression
systems (as well as phage display expression systems). An example
of a suitable bacterial expression vector is pUC119 (Sfi). Other
antibody expression systems are also known in the art and are
contemplated herein.
[0053] An antibody of the invention can be prepared by recombinant
expression of immunoglobulin light and heavy chain genes in a host
cell. To express an antibody recombinantly, a host cell is
transfected with one or more recombinant expression vectors
carrying DNA fragments encoding the immunoglobulin light and heavy
chains of the antibody such that the light and heavy chains are
expressed in the host cell. Preferably, the recombinant antibodies
are secreted into the medium in which the host cells are cultured,
from which medium the antibodies can be recovered. Standard
recombinant DNA methodologies are used to obtain antibody heavy and
light chain genes, incorporate these genes into recombinant
expression vectors, and introduce the vectors into host cells.
[0054] The isolated DNA encoding the HCVR region can be converted
to a full-length heavy chain gene by operatively linking the
HCVR-encoding DNA to another DNA molecule encoding heavy chain
constant regions (CH1, CH2, and CH3). The DNA sequences of human
heavy chain constant region genes are known in the art and DNA
fragments encompassing these regions can be obtained by standard
PCR amplification. The heavy chain constant region can be an IgG1,
IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region and any
allotypic variant therein as described in Kabat, (Kabat, et al,
Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No.
91-3242 (1991)), but most preferably is an IgG1, or IgG4 constant
region. Alternatively, the antibody portion can be an Fab fragment,
a Fab' fragment, F(ab')2, or a single chain FV fragment. For a Fab
fragment heavy chain gene, the HCVR-encoding DNA can be operatively
linked to another DNA molecule encoding only the heavy chain CH1
constant region.
[0055] The isolated DNA encoding the LCVR region can be converted
to a full-length light chain gene (as well as a Fab light chain
gene) by operatively linking the LCVR-encoding DNA to another DNA
molecule encoding the light chain constant region, CL. The DNA
sequences of human light chain constant region genes are known in
the art and DNA fragments encompassing these regions can be
obtained by standard PCR amplification. The light chain constant
region can be a kappa or lambda constant region.
[0056] To create a scFV gene, the HCVR- and LCVR-encoding DNA
fragments are operatively linked to another fragment encoding a
flexible linker, e.g., encoding the amino acid sequence
Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser
(G1y4-Ser).sub.3, SEQ ID NO: 20, such that the HCVR and LCVR
sequences can be expressed as a contiguous single-chain protein,
with the LCVR and HCVR regions joined by the flexible linker (see
e.g., Bird et al. Science 242:423-426 (1988); Huston, et al., Proc.
Natl. Acad. Sci. USA 85:5879-5883 (1988); McCafferty, et al.,
Nature 348:552-554 (1990)).
[0057] To express the antibodies of the invention, DNAs encoding
partial or full-length light and heavy chains, obtained as
described above, are inserted into expression vectors such that the
genes are operatively linked to transcriptional and translational
control sequences. The antibody gene is ligated into a vector such
that transcriptional and translational control sequences within the
vector serve their intended function of regulating the
transcription and translation of the antibody gene. The expression
vector and expression control sequences are chosen to be compatible
with the expression host cell used. The antibody light chain gene
and the antibody heavy chain gene can be inserted into separate
vector or, more typically, both genes are inserted into the same
expression vector. The antibody genes are inserted into the
expression vector by standard methods (e.g., ligation of
complementary restriction sites on the antibody gene fragment and
vector, or blunt end ligation if no restriction sites are present).
Additionally, or alternatively, the recombinant expression vector
can encode a signal peptide that facilitates secretion of the
anti-hTNFSF13b human antibody chain from a host cell. The
anti-hTNFSF13b human antibody chain gene can be cloned into the
vector such that the signal peptide is linked in-frame to the amino
terminus of the antibody chain gene. The signal peptide can be an
immunoglobulin signal peptide or a heterologous signal peptide
(i.e., a signal peptide from a non-immunoglobulin protein).
[0058] In addition to the antibody chain genes, the recombinant
expression vectors of the invention carry regulatory sequences that
control the expression of the antibody chain genes in a host cell.
Regulatory sequences comprise promoters, enhancers and other
expression control elements (e.g., polyadenylation signals) that
control the transcription or translation of the antibody chain
genes. It will be appreciated by those skilled in the art that the
design of the expression vector, including the selection of
regulatory sequences may depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, etc. Preferred regulatory sequences for mammalian host
cell expression include viral elements that direct high levels of
protein expression in mammalian cells, such as promoters and/or
enhancers derived from cytomegalovirus (CMV) (such as the CMV
promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40
promoter/enhancer), adenovirus, (e.g., the adenovirus major late
promoter (AdMLP)) and polyoma.
[0059] In addition to the antibody chain genes and regulatory
sequences, the recombinant expression vectors of the invention may
carry additional sequences, such as sequences that regulate
replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced. For example, typically the selectable marker gene
confers resistance to drugs, such as G418, hygromycin, or
methotrexate, on a host cell into which the vector has been
introduced. Preferred selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells
with methotrexate selection/amplification) and the neo gene (for
G418 selection).
[0060] For expression of the light and heavy chains, the expression
vector(s) encoding the heavy and light chains is transfected into a
host cell by standard techniques. The various forms of the term
"transfection" are intended to encompass a wide variety of
techniques commonly used for the introduction of exogenous DNA into
a prokaryotic or eukaryotic host cell, e.g., electroporation,
calcium-phosphate precipitation, DEAE-dextran transfection and the
like. Although it is theoretically possible to express the
antibodies of the invention in either prokaryotic or eukaryotic
host cells, expression of antibodies in eukaryotic cells, and most
preferably mammalian host cells, is the most preferred because such
eukaryotic cells, and in particular mammalian cells, are more
likely than prokaryotic cells to assemble and secrete a properly
folded and immunologically active antibody. Preferred mammalian
host cells for expressing the recombinant antibodies of the
invention include Chinese Hamster Ovary (CHO cells) (including
dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad.
Sci. USA, 77:4216-4220 (1980), used with a DHFR selectable marker,
e.g., as described in R. J. Kaufman and P. A. Sharp, Mol. Biol.,
159:601-621 (1982)), NSO myeloma cells, COS cells and SP2 cells.
When recombinant expression vectors encoding antibody genes are
introduced into mammalian host cells, the antibodies are produced
by culturing the host cells for a period of time sufficient to
allow for expression of the antibody in the host cells or, more
preferably, secretion of the antibody into the culture medium in
which the host cells are grown. Antibodies can be recovered from
the culture medium using standard protein purification methods.
[0061] Host cells can also be used to produce portions of intact
antibodies, such as Fab fragments of scFV molecules. It will be
understood that variations on the above procedure are within the
scope of the present invention. For example, it may be desirable to
transfect a host cell with DNA encoding either the light chain or
the heavy chain (but not both) of an antibody of this invention.
Recombinant DNA technology may also be used to remove some or all
of the DNA encoding either or both of the light and heavy chains
that is not necessary for binding to hTNFSF13b. The molecules
expressed from such truncated DNA molecules are also encompassed by
the antibodies of the invention. In a one system for recombinant
expression of an antibody of the invention, a recombinant
expression vector encoding both the antibody heavy chain and the
antibody light chain is introduced into dhfr-CHO cells by calcium
phosphate-mediated transfection. Within the recombinant expression
vector, the antibody heavy and light chain genes are each
operatively linked to enhancer/promoter regulatory elements (e.g.,
derived from SV40, CMV, adenovirus and the like, such as a CMV
enhancer/AdMLP promoter regulatory element or an SV40
enhancer/AdMLP promoter regulatory element) to drive high levels of
transcription of the genes. The recombinant expression vector also
carries a DHFR gene, which allows for selection of CHO cells that
have been transfected with the vector using methotrexate
selection/amplification. The selected transformant host cells are
culture to allow for expression of the antibody heavy and light
chains and intact antibody is recovered from the culture medium.
Standard molecular biology techniques are used to prepare the
recombinant expression vector, transfect the host cells, select for
transformants, culture the host cells and recover the antibody from
the culture medium. Antibodies or antigen-binding portions thereof
of the invention can be expressed in an animal (e.g., a mouse) that
is transgenic for human immunoglobulin genes (see e.g., Taylor, L.
D., et al. Nucl. Acids Res., 20:6287-6295 (1992)). Plant cells can
also be modified to create transgenic plants that express the
antibody or antigen binding portion thereof, of the invention.
[0062] In view of the foregoing, another aspect of the invention
pertains to nucleic acids, vectors, and host cell compositions that
can be used for recombinant expression of the antibodies and
antibody portions of the invention. Preferably, the invention
features isolated nucleic acids that encode CDRs of 4A5-3.1.1-B4,
or the full heavy and/or light chain variable region of
4A5-3.1.1-B4. Accordingly, in one embodiment, the invention
features an isolated nucleic acid encoding an antibody heavy chain
variable region that encodes the 4A5-3.1.1-B4 heavy chain CDR3
comprising the amino acid sequence of SEQ ID NO:16. Preferably, the
nucleic acid encoding the antibody heavy chain variable region
further encodes a 4A5-3.1.1-B4 heavy chain CDR2 which comprises the
amino acid sequence of SEQ ID NO:14. More preferably, the nucleic
acid encoding the antibody heavy chain variable region further
encodes a 4A5-3.1.1-B4 heavy chain CDR1 which comprises the amino
acid sequence of SEQ ID NO:12. Even more preferably, the isolated
nucleic acid encodes an antibody heavy chain variable region
comprising the amino acid sequence of SEQ ID NO:10 (the full HCVR
region of 4A5-3.1.1-B4).
[0063] In other embodiments, the invention features an isolated
nucleic acid encoding an antibody light chain variable region that
encodes the 4A5-3.1.1-B4 light chain CDR3 comprising the amino acid
sequence of SEQ ID NO:8 Preferably, the nucleic acid encoding the
antibody light chain variable region further encodes a 4A5-3.1.1-B4
light chain CDR1 which comprises the amino acid sequence of SEQ ID
NO:4. Even more preferably, the isolated nucleic acid encodes an
antibody light chain variable region comprising the amino acid
sequence of SEQ ID NO:2 (the full LCVR region of 4A5-3.1.1-B4).
[0064] In other embodiments, the invention features an isolated
nucleic acid encoding an antibody light chain variable region that
encodes the 4A5-3.1.1-B4 light chain CDR3 comprising the amino acid
sequence of SEQ ID NO:8 Preferably, the nucleic acid encoding the
antibody light chain variable region further encodes a 4A5-3.1.1-B4
light chain CDR1 which comprises the amino acid sequence of SEQ ID
NO:4. Even more preferably, the isolated nucleic acid encodes an
antibody light chain variable region comprising the amino acid
sequence of SEQ ID NO:2 (the full LCVR region of 4A5-3.1.1-B4).
[0065] In another embodiment, the invention provides an isolated
nucleic acid encoding a heavy chain CDR3 domain comprising the
amino acid sequence of SEQ ID NO:16 (i.e., the 4A5-3.1.1-B4 HCVR
CDR3). This nucleic acid can encode only the CDR3 region or, more
preferably, encodes an entire antibody heavy chain variable region
(HCVR). For example, the nucleic acid can encode a HCVR having a
CDR2 domain comprising the amino acid sequence of SEQ ID NO:14
(i.e., the 4A5-3.1.1-B4 HCVR CDR2) and a CDR1 domain comprising the
amino acid sequence of SED ID NO:12 (i.e., 4A5-3.1.1-B4 HCVR
CDR1).
[0066] In still another embodiment, the invention provides an
isolated nucleic acid encoding an antibody light chain variable
region comprising the amino acid sequence of SEQ ID NO:2 (i.e., the
4A5-3.1.1-B4 LCVR). Preferably this nucleic acid comprises the
nucleotide sequence of SEQ ID NO:1, although the skilled artisan
will appreciate that due to the degeneracy of the genetic code,
other nucleotide sequences can encode the amino acid sequence of
SEQ ID NO:2. The nucleic acid can encode only the LCVR or can also
encode an antibody light chain constant region, operatively linked
to the LCVR. In one embodiment, this nucleic acid is in a
recombinant expression vector.
[0067] In still another embodiment, the invention provides an
isolated nucleic acid encoding an antibody heavy chain variable
region comprising the amino acid sequence of SEQ ID NO:10 (i.e.,
the 4A5-3.1.1-B4 HCVR). Preferably this nucleic acid comprises the
nucleotide sequence of SEQ ID NO:9, although the skilled artisan
will appreciate that due to the degeneracy of the genetic code,
other nucleotide sequences can encode the amino acid sequence of
SEQ ID NO:10. The nucleic acid can encode only the HCVR or can also
encode a heavy chain constant region, operatively linked to the
HCVR. For example, the nucleic acid can comprise an IgG1 or IgG4
constant region. In one embodiment, this nucleic acid is in a
recombinant expression vector.
[0068] Those of ordinary skill in the art are aware that
modifications in the amino acid sequence of the antibody can result
in an antibody that display equivalent or superior functional
characteristics when compared to the original antibody. Alterations
in the antibodies of the present invention can include one or more
amino acid insertions, deletions, substitutions, truncations,
fusions, and the like, either from natural mutations or human
manipulation. The present invention encompasses antibodies
disclosed herein further comprising one or more amino acid
substitutions provided that the substituted antibodies have
substantially the same (or improved or reduced, as may be
desirable) activity(ies) as the antibodies disclosed herein.
Preferably, a CDR of the present invention has 3 or less
conservative substitutions. Preferably, a CDR of the present
invention has 2 or less conservative substitutions. Preferably, a
CDR of the present invention has one conservative substitution. The
skilled artisan will recognize that antibodies having conservative
amino acid substitutions can be prepared by a variety of techniques
known in the art. For example, a number of mutagenesis methods can
be used, including PCR assembly, Kunkel (dut-ung-) and
thiophosphate (Amersham Sculptor kit) oligonucleotide-directed
mutagenesis. Conservative substitutions of interest are shown in
Table 1 along with preferred substitutions.
TABLE-US-00001 TABLE 1 Conservative Substitutions Preferred Residue
Substitutions Substitution Ala (A) gly, val, leu, ile, ser, met,
thr Val Ara (R) lys, gln, as, his Lys Asn (N) Gln Gln Asn (D) Glu
Glu Cys (C) Ser Ser Gln (O) Asn Asn Glu (E) Asn Asp Gly (G) Ala,
ile, leu, pro, ser, met, val val Ala His (H) Asn, aln, lys, arg Arg
Ile (I) Leu, val, met, ala, phe, norleucine Leu Leu (L) norleucine,
ile, val, met, ala phe Ile Lys (K) Arg, gln, asn, his Arg Met (M)
Ala, gly, ile, leu, phe, ser, val Leu Phe (F) Leu, val, ile, ala,
trp, tyr Tyr Pro (P) Ser (S) Ala, gly, ile, leu, met, thr, val Thr
Thr (T) Ala, glv, ile, leu, met, ser, val Ser Trp (W) tyr, phe Tyr
Tyr (Y) trp, phe, thr, ser Phe Val (V) Ala, ile, leu, met, ser,
met, norleu Leu
[0069] The invention also provides recombinant expression vectors
encoding an antibody comprising a polypeptide selected from the
group consisting of a polypeptide as shown in SEQ ID NO: 2, a
polypeptide as shown in SEQ ID NO: 4, a polypeptide as shown in SEQ
ID NO: 6, a polypeptide as shown in SEQ ID NO: 8, a polypeptide as
shown in SEQ ID NO: 10, a polypeptide as shown in SEQ ID NO: 12, a
polypeptide as shown in SEQ ID NO: 14; and a polypeptide as shown
in SEQ ID NO: 16.
[0070] The invention also provides recombinant expression vectors
encoding both an antibody heavy chain and an antibody light chain.
For example, in one embodiment, the invention provides a
recombinant expression vector encoding:
[0071] a) an antibody heavy chain having a variable region
comprising the amino acid sequence of SEQ ID NO:10; and
[0072] b) an antibody light chain having a variable region
comprising the amino acid sequence of SEQ ID NO:2.
[0073] Once expressed, the whole antibodies, their dimers,
individual light and heavy chains, or other immunoglobulin forms of
the present invention can be purified according to standard
procedures of the art, including ammonium sulfate precipitation,
ion exchange, affinity, reverse phase, hydrophobic interaction
column chromatography, gel electro-phoresis and the like.
Substantially pure immunoglobulins of at least about 90 to 95%
homogeneity are preferred, and 98 to 99% or more homogeneity most
preferred, for pharmaceutical uses. Once purified, partially or to
homogeneity as desired, the polypeptides may then be used
therapeutically or prophylactically, as directed herein.
[0074] The antibodies of the present invention can be incorporated
into pharmaceutical compositions suitable for administration to a
subject. Typically, the pharmaceutical composition comprises an
antibody or antibody portion of the invention and a
pharmaceutically acceptable diluent, carrier, and/or excipient. The
pharmaceutical compositions for administration are designed to be
appropriate for the selected mode of administration, and
pharmaceutically acceptable diluents, carrier, and/or excipients
such as dispersing agents, buffers, surfactants, preservatives,
solubilizing agents, isotonicity agents, stabilizing agents and the
like are used as appropriate.
[0075] A pharmaceutical composition comprising an anti-hTNFSF13b
human antibody of the present invention can be administered to a
mammal at risk for or exhibiting autoimmunity related symptoms or
pathology such as systemic lupus erythematosus using standard
administration techniques by intravenous, intraperitoneal,
subcutaneous, pulmonary, transdermal, intramuscular, intranasal,
buccal, sublingual, or suppository administration.
[0076] The antibodies of the invention can be incorporated into a
pharmaceutical composition suitable for parenteral administration.
Peripheral systemic delivery by intravenous or intraperitoneal or
subcutaneous injection is preferred. Suitable vehicles for such
injections are straightforward. In addition, however,
administration may also be effected through the mucosal membranes
by means of nasal aerosols or suppositories. Suitable formulations
for such modes of administration are well known and typically
include surfactants that facilitate cross-membrane transfer.
[0077] The pharmaceutical compositions typically must be sterile
and stable under the conditions of manufacture and storage.
Therefore, pharmaceutical compositions may be sterile filtered
after making the formulation, or otherwise made microbiologically
acceptable. A typical composition for intravenous infusion could
have a volume as much as 250 mL of fluid, such as sterile Ringer's
solution, and 1-100 mg per mL, or more in antibody concentration.
Therapeutic agents of the invention can all be frozen or
lyophilized for storage and reconstituted in a suitable sterile
carrier prior to use. Lyophilization and reconstitution can lead to
varying degrees of antibody activity loss (e.g. with conventional
immune globulins, IgM antibodies tend to have greater activity loss
than IgG antibodies). Dosages may have to be adjusted to
compensate. The pH of the formulation will be selected to balance
antibody stability (chemical and physical) and comfort to the
patient when administered. Generally, pH between 6 and 8 is
tolerated.
[0078] TNFSF13b plays a critical role in the pathology associated
with a variety of diseases involving immune and inflammatory
factors. Therefore, a pharmaceutical composition comprising an
anti-hTNFSF13b human antibody of the invention can be used to treat
disorders in which TNFSF13b activity is detrimental, for example
immune diseases including autoimmune diseases and inflammatory
diseases. Preferred disorders include, but are not limited to,
systemic lupus erythematosus, rheumatoid arthritis, juvenile
chronic arthritis, Lyme arthritis, Crohn's disease, ulcerative
colitis, inflammatory bowel disease, asthma, allergic diseases,
psoriasis, graft versus host disease, organ transplant rejection,
acute or chronic immune disease associated with organ
transplantation, sarcoidosis, infectious diseases, parasitic
diseases, female infertility, autoimmune thrombocytopenia,
autoimmune thyroid disease, Hashimoto's disease, Sjogren's
syndrome, and cancers, particularly B or T cell lymphomas or
myelomas.
[0079] More preferably, a pharmaceutical composition comprising an
anti-hTNFSF13b human antibody and/or antibody fragment of the
invention is used to treat systemic lupus erythematosus.
[0080] The use of the antibody of an anti-hTNFSF13b human antibody
of the present invention in the manufacture of a medicament for the
treatment of at least one of the aforementioned disorders in which
TNFSF13b activity is detrimental is also contemplated herein.
[0081] In certain situations, an antibody of the invention will be
co-formulated with and/or co-adminstered with one or more
additional therapeutic agents that are used in the treatment of
autoimmune and/or inflammatory diseases. Such combination therapies
may advantageously utilize lower dosages of the administered
therapeutic agents, thus avoiding possible toxicities or
complications associated with the various monotherapies. It will be
appreciated by the skilled practitioner that when the antibodies of
the invention are used as part of a combination therapy, a lower
dosage of antibody may be desirable than when the antibody alone is
administered to a subject (e.g., a synergistic therapeutic effect
may be achieved through the use of combination therapy which, in
turn, permits use of a lower dose of the antibody to achieve the
desired therapeutic effect).
[0082] The pharmaceutical compositions of the invention may include
a "therapeutically effective amount" or a "prophylactically
effective amount" of an antibody of the invention. A
"therapeutically effective amount" refers to an amount effective,
at dosages and for periods of time necessary, to achieve the
desired therapeutic result. A therapeutically effective amount of
the antibody may vary according to factors such as the disease
state, age, sex, and weight of the individual, and the ability of
the antibody or antibody portion to elicit a desired response in
the individual. A therapeutically effective amount is also one in
which any toxic or detrimental effects of the antibody or antibody
portion are outweighed by the therapeutically beneficial effects. A
"prophylactically effective amount" refers to an amount effective,
at dosages and for periods of time necessary, to achieve the
desired prophylactic result. Typically, since a prophylactic dose
is used in subjects prior to or at an earlier stage of disease, the
prophylactically effective amount will be less than the
therapeutically effective amount.
[0083] Dosage regimens may be adjusted to provide the optimum
desired response (e.g., a therapeutic or prophylactic response).
For example, a single bolus may be administered, several divided
doses may be administered over time or the dose may be
proportionally reduced or increased as indicated by the exigencies
of the therapeutic situation.
[0084] Given their ability to bind to hTNFSF13b, antibodies, of the
invention can be used to detect TNFSF13b polypeptides (e.g, in a
biological sample, such as serum or plasma), using a conventional
immunoassay, such as an enzyme linked immunosorbent assays (ELISA),
an radioimmunoassay (RIA) or tissue immunohistochemistry. The
invention provides a method for detecting TNFSF13b in a biological
sample comprising contacting a biological sample with an antibody,
or antibody portion, of the invention and detecting either the
antibody (or antibody portion) bound to hTNFSF13b or unbound
antibody (or antibody portion), to thereby detect hTNFSF13b in the
biological sample. The antibody is directly or indirectly labeled
with a detectable substance to facilitate detection of the bound or
unbound antibody. Suitable detectable substances include various
enzymens, prosthetic groups, fluorescent materials, luminescent
materials and radioactive materials. Examples of suitable enzymes
include horseradish peroxidase, alkaline phosphatase,
.beta.-galactosidase, or acetylcholinesterase; examples of suitable
prosthetic group complexes include streptavidin/biotin and
avidin/biotin; examples of suitable fluorescent materials include
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinyl-amine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; and examples of suitable radioactive material include
125I, .sup.131I, .sup.35S, or .sup.3H.
[0085] Alternative to labeling the antibody, TNFSF13b can be
assayed in biological fluids by a competition immunoassay utilizing
TNFSF13b standards labeled with a detectable substance and an
unlabeled anti-hTNFSF13b human antibody. In this assay, the
biological sample, the labeled TNFSF13b standards and the
anti-hTNFSF13b human antibody are combined and the amount of
labeled TNFSF13b standard bound to the unlabeled antibody is
determined. The amount of TNFSF13b in the biological sample is
inversely proportional to the amount of labeled rTNFSF13b standard
bound to the anti-hTNFSF13b human antibody.
[0086] In another embodiment, the present invention provides a use
of an antibody that neutralizes TNFSF13b activity by binding an
epitope of TNFSF13b. The epitope was identified as described in
Example 10. For reference, the soluble portion of hTNFSF13b is
represented as follows:
TABLE-US-00002 Human TNFSF13b SEQ ID NO: 21 1 AVQGPEETVT QDCLQLIADS
ETPTIQKGSY TFVPWLLSFK 40 41 RGSALEEKEN KILVKETGYF FIYGQVLYTD
KTYAMGHLIQ 80 81 RKKVHVFGDE LSLVTLFRCI QNMPETLPNN SCYSAGIAKL 120
121 EEGDELQLAI PRENAQISLD GDVTFFGALK LL." 152
[0087] The hTNFSF13b amino acids involved in binding the novel
anti-hTNFSF13b human antibodies comprise at least one of the amino
acids selected from the group consisting of: threonine at position
69, lysine at position 71, threonine at position 72, tyrosine at
position 73, glutamic acid at position 105, threonine at position
106, leucine at position 107, and asparagine at position 109. In
another embodiment, the amino acids involved in binding the novel
anti-hTNFSF13b human antibodies comprise at least two of the amino
acids selected from the group consisting of: threonine at position
69, lysine at position 71, threonine at position 72, tyrosine at
position 73, glutamic acid at position 105, threonine at position
106, leucine at position 107, and asparagine at position 109. In
another embodiment, the amino acids involved in binding the novel
anti-hTNFSF13b human antibodies comprise at least three of the
amino acids selected from the group consisting of: threonine at
position 69, lysine at position 71, threonine at position 72,
tyrosine at position 73, glutamic acid at position 105, threonine
at position 106, leucine at position 107, and asparagine at
position 109. In another embodiment, the amino acids involved in
binding the novel anti-hTNFSF13b human antibodies comprise at least
four of the amino acids selected from the group consisting of:
threonine at position 69, lysine at position 71, threonine at
position 72, tyrosine at position 73, glutamic acid at position
105, threonine at position 106, leucine at position 107, and
asparagine at position 109.
[0088] In another embodiment, the amino acids involved in binding
the novel anti-hTNFSF13b human antibodies comprise lysine at
position 71, threonine at position 72, tyrosine at position 73, and
glutamic acid at position 105.
[0089] In another embodiment, the amino acids involved in binding
the novel anti-hTNFSF13b human antibodies comprise glutamic acid at
position 105 and at least one of the amino acids selected from the
group consisting of: threonine at position 69, lysine at position
71, threonine at position 72, and tyrosine at position 73. In
another embodiment, the amino acids involved in binding the novel
anti-hTNFSF13b human antibodies comprise threonine at position 106
and at least one of the amino acids selected from the group
consisting of: threonine at position 69, lysine at position 71,
threonine at position 72, and tyrosine at position 73. In another
embodiment, the amino acids involved in binding the novel
anti-hTNFSF13b human antibodies comprise leucine at position 107
and at least one of the amino acids selected from the group
consisting of: threonine at position 69, lysine at position 71,
threonine at position 72, and tyrosine at position 73. In another
embodiment, the amino acids involved in binding the novel
anti-hTNFSF13b human antibodies comprise asparagine at position 109
and at least one of the amino acids selected from the group
consisting of: lysine at position 71, threonine at position 72, and
tyrosine at position 73.
[0090] In another embodiment, the amino acids involved in binding
the novel anti-hTNFSF13b human antibodies comprise lysine at
position 71, threonine at position 72, tyrosine at position 73, and
glutamic acid at position 105.
[0091] The following examples are intended to illustrate but not to
limit the invention.
EXAMPLE 1
Generation of Anti-hTNFSF13b Human Monoclonal Antibodies
[0092] Monoclonal antibodies were generated using the
HuMAb-Mouse.TM. technology at Medarex by immunizing the mice with
soluble hTNFSF13b (amino acids 133-285, purchased from RDI,
Flanders, N.J.). Both the HCo7 and HCo12 mice were used. Mice were
immunized with 15 .mu.g to 50 .mu.g soluble hTNFSF13b in RIBI,
Freund's complete adjuvant or Freund's incomplete adjuvant. Eight
mice producing serum antibody titers to hTNFSF13b were injected
i.v. with 10 .mu.g hTNFSF13b in PBS. The spleen was harvested three
days later from each mouse and fused with myeloma cells according
to the method described in Zola (Zola, H. Monoclonal antibodies: A
Manual of Techniques. CRC Press, Boca Raton, Fla. (1987)).
[0093] Hybridomas were tested for binding to hTNFSF13b and to make
sure they were expressing human immunoglobulin heavy and light
chains. Antibody binding to hTNFSF13b was detected by ELISA as
follows:
[0094] Plates were coated with 50 .mu.l of 5 .mu.g/ml hTNFSF13b in
PBS overnight at 4.degree. C. Plates were then emptied and blocked
with 100 .mu.l PBS+0.05% Tween 20 (PBST)+5% chicken serum for 1
hour at room temperature. After washing three times with PBST, the
plates were drained and 100 .mu.l diluted secondary reagents
(HRP-HuIgGFc, Jackson cat#109-036-098 or HRP-HuKappa, Bethyl
cat#A80-115P; 1:5000 in blocking buffer) was added per well. After
an 1 hour incubation at room temperature plates were washed three
times as described above. Plates were developed using 10 ml citrate
phosphate buffer pH 4.0, 80 .mu.l ABTS, 8 .mu.l H.sub.2O.sub.2 per
plate. After incubating 30 min. to 1 hour at room temperature,
absorbance of the plates was read A415-A490. Hybridomas that showed
binding to hTNFSF13b and that were huIgG heavy chain and human
kappa light chain were selected for subcloning.
[0095] Cell culture media of subcloned hybridomas was concentrated
in Amicon ProFlux M12 tangential filtration systems using an Amicon
S3Y30 UF membranes. The concentrated media was passed over
protein-A Sephaose columns (5 to 20 ml column) at a flow rate of 5
ml/min. The columns were washed with buffer A (PBS, pH 7.4) until
the absorbance returned to baseline and the bound polypeptides were
eluted with 50 mM citric acid, pH 3.2. Fractions were immediately
neutralized with 1M Tris, pH 8.0. Fractions were then analyzed by
SDS-PAGE. Fractions containing antibody were pooled and
concentrated using an Ultrafree.TM. centrifugal filter unit
(Millipore, 10 kDa molecular weight cut-off).
EXAMPLE 2
Functional Activity of Anti-hTNFSF13b Human Antibodies
[0096] Neutralizing activity of the anti-hTNFSF13b human antibodies
of the invention was measured using a murine 1'-1 dependent B cell
line, T1165.17. The cells were washed three times with assay media
(RPMI1640 containing 10% FBS, 1 mM sodium pyruvate,
5.times.10.sup.-5 M 2-mercaptoethanol and penicillin, streptomycin
and fungizone) to remove IL-1. The cells were resuspended at
100,000 cells/ml in assay media containing 2.5 ng/ml soluble
huTNFSF13b and plated at 5000 cells/well in a 96 well plate and
incubated at 37.degree. C. in 5% CO.sub.2. Supernatants from ELISA
positive hybridomas were included at a 1:4 dilution. Forty-eight
hours later, 20 .mu.l of Promega.TM. CellTiter 96.TM. Aqueous One
Solution (Madison, Wis.) was added and the plate incubated for 5
more hours at 37.degree. C. in 5% CO.sub.2. Absorbance was read at
A490, to measure proliferation. An example of neutralization
activity for one of the hybridoma supernatants, 4A5-3.1.1-B4, is
shown in FIG. 1. As a control, the antibodies were added to IL-1
stimulated cells. There was no evidence of inhibition of IL-1
stimulated proliferation, only the hTNFSF13b stimulated
proliferation.
[0097] The neutralizing antibodies were tested for the ability to
inhibit TNFSF13b augmented primary human B cell proliferation in
response to anti-IgM stimulation. Primary human B cells were
isolated from human blood using CD19 positive selection using the
MACS magnetic isolation system (Miltenyi Biotec, Auburn, Calif.).
The B cells were added to wells of a 96-well plate at
2.times.10.sup.5 cells per well in complete RPMI containing 10% FCS
(complete RPMI is RPMI1640 containing 10 mM L-glutamine, 100 U/ml
penicillin, 100 .mu.g/ml streptomycin, 1 mM sodium puruvate, 0.1 mM
non-essential amino acids, and 1.times.10.sup.-5 M
.beta.-mercaptoethanol). Some of the wells were coated with 10
.mu.g/ml anti-human IgM in PBS (BD PharMingen, Clone G20-127),
overnight at 4.degree. C. and washed four times with PBS before
use. Some of the cells were stimulated with soluble hTNFSF13b (25
ng/ml) in the presence or absence of neutralizing anti-hTNFSF13b
antibody (2.5 .mu.g/ml). FIG. 2 illustrates the ability of
4A5-3.1.1-B4 to neutralize the stimulatory effect of hTNFSF13b.
EXAMPLE 3
Characterization of Monoclonal Antibodies
[0098] All of the neutralizing anti-hTNFSF13b antibodies were
either human IgG1 or human IgG4. They were also assayed for their
ability to bind to hTNFSF13b in a denatured state, i.e., hTNFSF13b
separated on SDS-PAGE and blotted onto nitrocellulose. All of the
neutralizing antibodies failed to bind hTNFSF13b in a Western blot
while several of the non-neutralizing antibodies were able to do
so.
[0099] Experiments utilizing the BIACore.TM. system were performed
to determine if non-neutralizing antibodies and neutralizing
antibodies bound to the same site on hTNFSF13b. First, 4A5-3.1.1-B4
was coated onto a chip followed by injection of hTNFSF13b and then
a saturating amount of non-neutralizing antibody. Once saturation
was achieved, a high concentration of 4A5-3.1.1-B4 was run over the
chip. Eleven of the non-neutralizing monoclonal antibodies were
unable to compete for the same binding site as 4A5-3.1.1-B4. One
non-neutralizing hybridoma was able to block the binding of
4A5-3.1.1-B4 by approximately 45%, indicating that it may have an
epitope near the 4A5-3.1.1-B4 epitope.
[0100] Using the same experimental design, it was also determined
that the neutralizing mAb, 4A5-3.1.1-B4, could compete for the same
binding site as one of the receptors for hTNFSF13b, TACI. These
experiments suggest that TACI-Fc and 4A5-3.1.1-B4 may have
overlapping epitopes on hTNFSF13b.
[0101] 4A5-3.1.1-B4 was immobilized on a solid phase by passing the
antibody solution over an IMAC resin loaded with Co.sup.+2.
Following binding, the cobalt was oxidized to the +3 state by
incubation of the resin with a dilute peroxide solution. After
washing the resin, native hTNFSF13b and hTNFSF13b that was modified
(by reduction/alkylation or by thermal denaturation) was passed
over the column. After washing, the bound protein was eluted with
an acidic solution and the eluted proteins were analyzed by MALDI
MS. 4A5-3.1.1-B4 bound native recombinant hTNFSF13b, but did not
bind either the chemically or thermally modified hTNFSF13b.
Therefore, the 4A5-3.1.1-B4 appears to recognize a conformational
epitope on soluble hTNFSF13b.
[0102] Recombinant soluble hTNFSF13b (RDI) was incubated with
4A5-3.1.1-B4 or anti-TNFSF13b rabbit polyclonal antibody (MoBiTec,
Marco Island, Fla.; against amino acids 254 to 269 of hTNFSF13b) on
ice for 2 hours and the protein mixture was applied to a
size-exclusion HPLC (two, tandem TosoHaas TSK-GEL G3000PW columns)
equilibrated in PBS at a flow rate of 0.25 ml/min. Proteins were
eluted with PBS. As controls, antibody solutions and the solution
of hTNFSF13b were analyzed separately. Human TNFSF13b eluted from
the size exclusion column in a position consistent with a trimer of
TNFSF13b molecules. The elution of trimeric hTNFSF13b shifted to an
earlier timepoint in the presence of 4A5-3.1.1-B4 but not in the
presence of anti-TNFSF13b polyclonal antibodies indicating the
binding of trimeric hTNFSF13b to the 4A5-3.1.1-B4 antibody. This
data suggests that the neutralizing mAb 4A5-3.1.1-B4 binds to a
conformational epitope on hTNFSF13b.
EXAMPLE 4
Affinity Measurement of Monoclonal Antibodies by BIAcore
[0103] The affinity of various anti-hTNFSF13b human antibodies for
hTNFSF13b was measured using a BIAcore 2000 instrument system. The
system utilizes the optical properties of Surface Plasmon Resonance
to detect alteration in protein concentration of interacting
molecules within a dextran biosensor matrix. Except where noted,
all reagents and materials were purchased from BIAcore AB (Upsala,
Sweden). All measurements were performed at 25.degree. C. Samples
were dissolved in HBS-EP buffer (150 mM NaCl, 3 mM EDTA, 0.005%
(w/v) surfactant P-20, and 10 mM HEPES, pH 7.4). Goat anti-mouse
IgG (Fc specific; Jackson Immunoresearch, West Grove, Pa.) was
immobilized on flow cell 1 on a CM5 sensor chip using the amine
coupling kit. Goat anti-human IgG (Fc specific; Jackson
Immunoresearch) was immobilized on flow cell 2 also by amine
coupling. Both antibodies were immobilized to reach 700 response
units each.
[0104] Binding of recombinant hTNFSF13b (Research Diagnostics,
Inc., Flanders, N.J.) was evaluated using multiple analytical
cycles. Each cycle was performed at a flow rate of 30 .mu.l/min.
and consisted of the following steps: injection of 150 .mu.l of
4A5-3.1.1-B4 at 20 .mu.g/ml, injection of 250 .mu.l of hTNFSF13b
(starting at 50 nM and using 2 fold serial dilutions for each
cycle) followed by 15 minutes for dissociation, and regeneration
using 90 .mu.l of 10 mM glycine HCl, pH 1.5.
[0105] Association and dissociation rates for each cycle were
evaluated using a Langmuir 1:1 binding model in the BlAevaluation
software. The K.sub.D of 4A5-3.1.1-B4 for hTNFSF13b was determined
to be 38 pM.
EXAMPLE 5
Cloning and Sequencing of Heavy and Light Chain Antigen Binding
Regions
[0106] The variable region for the heavy and light chain for the
neutralizing human mAb 4A5-3.1.1-B4 were cloned and sequenced using
the following protocols. mRNA was prepared from 2.times.10.sup.6
hybridoma cells using the Micro-Fast Track protocol (Invitrogen)
supplied with the kit. cDNA was prepared from 200 .mu.l of ethanol
precipitate of mRNA using cDNA Cycle kit (Invitrogen) by spinning
the aliquot of mRNA for 30 min. at 14,000 rpm at 4.degree. C.
followed by washing the pellet with 70% ethanol. The air dried
pellet was resuspended in 11.5 .mu.l of sterile water and cDNA was
prepared following the kit's instructions. The optional second
round of cDNA systhesis was omitted but the cDNA was cleaned using
the pheno/chlorform extraction step and ethanol precipitation. The
cDNA pellet was resuspended in 30 .mu.l of TE for use in PCR.
[0107] The PCR reactions were set up with degenerate primers at the
5' end of the variable region for the heavy and light chain paired
with 3' primers in the constant region. For each 50 ul reaction, 1
ul of cDNA was used. The reaction was set up as directed for use
with Pfut followed by 20 cycles. The PCR products were checked by
running 5 .mu.l of each reaction on a 1% agarose gel. The positive
reactions were cloned using the Zero Blunt TOPO PCR cloning kit
(Invitrogen). Minipreps from the positive clones were sequenced and
analyzed for productive gene rearrangements. Results from
independent PCR reactions and sequencing of multiple clones
revealed the sequences as described below.
Human antibody 4A5-3.1.1-B4 light chain sequences (CDRs are in
bold).
TABLE-US-00003 ##STR00001##
Human antibody 4A5-3.1.1-B4 heavy chain sequences (CDRs are in
bold, signal sequence is italicized).
TABLE-US-00004 ##STR00002##
EXAMPLE 6
Species Crossreactivity of Anti-hTNFSF13b Human Antibodies with
Non-Human TNFSF13b
[0108] In order to determine the species crossreactivity of the
neutralizing mAbs, an ELISA was set up utilizing 4A5-3.1.1-B4 as
both the capture and detecting mAb. Human recombinant TNFSF13b was
used as the standard curve. Human TNFSF13b could be detected in the
culture supernatant from CHO cells transfected with a vector
expressing hTNFSF13b, supernatants from cultured human monocytes or
human serum or plasma. Supernatants from CHO cells expressing
murine TNFSF13b were tested for reactivity in the ELISA and were
negative. 4A5-3.1.1-B4 was also unable to immunopreciptate murine
TNFSF13b but was able to immunoprecipitate human TNFSF13b. Murine
TNFSF13b was used in the proliferation assay described in Example
2. Using this proliferation assay, 4A5-3.1.1-B4 was unable to
neutralize the proliferation induced by murine TNFSF13b. This
indicates that 4A5-3.1.1-B4 is unable to recognize murine
TNFSF13b.
EXAMPLE 7
Amino Acid sequence of Heavy Chain 4A5-3.1.1-B4
[0109] Below is the amino acid sequence of the heavy chain
4A5-3.1.1-B4 antibody which comprises the HCVR and the IgG4
constant region. The human IgG4 constant region has a serine at
position 231. However, this position at 231 was substituted from a
serine to a proline which introduces a structural change in the
hinge region for obtaining optimal inter-chain disulfide bonds.
This reduces the generation of half antibodies. Half antibodies are
formed from one heavy chain and one light chain.
TABLE-US-00005 (SEQ ID NO: 17) 1 QVQLQQWGAG LLKPSETLSL TCAVYGGSFS
GYYWSWIRQP PGKGLEWIGE 51 INHSGSTNYN PSLKSRVTIS VDTSKNQFSL
KLSSVTAADT AVYYCARGYY 101 DILTGYYYYF DYWGQGTLVT VSSASTKGPS
VFPLAPCSRS TSESTAALGC 151 LVKDYFPEPV TVSWNSGALT SGVHTFPAVL
QSSGLYSLSS VVTVPSSSLG 201 TKTYTCNVDH KPSNTKVDKR VESKYGPPCP
PCPAPEFLGG PSVFLFPPKP 251 KDTLMISRTP EVTCVVVDVS QEDPEVQFNW
YVDGVEVHNA KTKPREEQFN 301 STYRVVSVLT VLHQDWLNGK EYKCKVSNKG
LPSSIEKTIS KAKGQPREPQ 351 VYTLPPSQEE MTKNQVSLTC LVKGFYPSDI
AVEWESNGQP ENNYKTTPPV 401 LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV
MHEALHNHYT QKSLSLSLGK"
[0110] In addition, an alanine for phenylalanine substitution at
position 237 and an alanine or glutamic acid substitituion for
leucine at position 238 can be made to lessen the effector function
of the antibody.
EXAMPLE 8
Amino Acid Sequence of Heavy Chain 4A5-3.1.1-B4
[0111] Below is the amino acid sequence of the heavy chain
4A5-3.1.1-B4 antibody which comprises the HCVR and the IgG1
constant region.
TABLE-US-00006 (SEQ ID NO: 18) 1 QVQLQQWGAG LLKPSETLSL TCAVYGGSFS
GYYWSWIRQP PGKGLEWIGE 51 INHSGSTNYN PSLKSRVTIS VDTSKNQFSL
KLSSVTAADT AVYYCARGYY 101 DILTGYYYYF DYWGQGTLVT VSSASTKGPS
VFPLAPSSKS TSGGTAALGC 151 LVKDYFPEPV TVSWNSGALT SGVHTFPAVL
QSSGLYSLSS VVTVPSSSLG 201 TQTYICNVNH KPSNTKVDKK VEPKSCDKTH
TCPPCPAPEL LGGPSVFLFP 251 PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK
FNWYVDGVEV HNAKTKPREE 301 QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS
NKALPAPIEK TISKAKGQPR 351 EPQVYTLPPS RDELTKNQVS LTCLVKGFYP
SDIAVEWESN GQPENNYKTT 401 PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS
CSVMHEALHN HYTQKSLSLS 451 PGK
EXAMPLE 9
Amino Acid sequence of Light Chain 4A5-3.1.1-B4
[0112] Below is the amino acid sequence of the light chain
4A5-3.1.1-B4 antibody which comprises the LCVR and the kappa
constant region.
TABLE-US-00007 (SEQ ID NO: 19) 1 EIVLTQSPAT LSLSPGERAT LSCRASQSVS
RYLAWYQQKP GQAPRLLIYD 51 ASNRATGIPA RFSGSGSGTD STLTISSLEP
EDFAVYYCQQ RSNWPRTFGQ 101 GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA
SVVCLLNNFY PREAKVQWKV 151 DNALQSGNSQ ESVTEQDSKD STYSLSNTLT
LSKADYEKHK VYACEVTHQG 201 LSSPVTKSFN RGEC
EXAMPLE 10
Identification of the Epitope for 4A5-3.1.1-B4
[0113] The epitope to which 4A5-3.1.1-B4 bound and neutralized
human TNFSF13b was determined Human and murine TNFSF13b sequences
were aligned as shown below:
TABLE-US-00008 Mouse TNFSF13b 1 AFQGPEETEQ DVDLSAPPAP CLPGCRHSQH
DDNGMNLRNI IQDCLQLIA 49 SEQ ID NO: 22 Human TNFSF13b 1 AVQGPEE---
---------- ---------- --------TV TQDCLQLIA 18 SEQ ID NO: 21 Mouse
TNFSF13b 50 DSDTPTIRKG TYTFVPWLLS FKRGNALEEK ENKIVVRQTG YFFIYSQVLY
99 Human TNFSF13b 19 DSETPTIQKG SYTFVPWLLS FKRGSALEEK ENKILVKETG
YFFIYGQVLY 68 Mouse TNFSF13b 100 TDPIFAMGHV IQRKKVHVFG DELSLVTLFR
CIQNMPKTLP NNSCYSAGIA 149 Human TNFSF13 69 TDKTYAMGHL IQRKKVHVFG
DELSLVTLFR CIQNMPETLP NNSCYSAGIA 118 Mouse TNFSF13b 150 RLEEGDEIQL
AIPRENAQIS RNGDDTFFGA LKLL. 183 Human TNFSF13b 119 KLEEGDELQL
AIPRENAQIS LDGDVTFFGA LKLL." 152
[0114] A homology model was created for human TNFSF13b based on the
known crystal structure for several TNF family members. Exposed
residues that are different between mouse and human TNFSF13b are
potential binding sites for 4A5-3.1.1-B4 since 4A5-3.1.1-B4
neutralizes human but not mouse TNFSF13b.
[0115] Three potential epitopes were identified: 1) K71, T72, Y73,
E105; 2) Q26, S29, L139, D140; and 3) L53, K55, E56, K119.
Mutagenesis was performed to make chimeric molecules by changing
the amino acid sequence from human to mouse. Chimera A was L139R,
D140N; Chimera B was K71P, T72I, Y73F; Chimera C was K71P, T72I,
Y73F, E105K; Chimera D was L53V, K55R, E56Q; Chimera E was
E105K.
[0116] Using the proliferation assay as described in Example 2, all
of the chimeras were tested for functional activity and
neutralization by 4A5-3.1.1-B4. Initial assays were performed using
supernatants from 293 transient transfections for each of the
chimeras and both human TNFSF13b and murine TNFSF13b parent
molecules. All of the chimeras induced similar proliferation
indicating that the chimeras produced were functional. Using 6
ug/ml of 4A5-3.1.1-B4, 100% neutralization was observed with human
TNFSF13b and chimeras A, B, D and E. No neutralization was observed
for murine TNFSF13b or chimera C. Purified TNFSF13b mutants were
produced for chimera A, B, and C and the assay was repeated using
11 ng/ml of each parent TNFSF13b or chimera TNFSF13b and 1 ug/ml of
4A5-3.1.1-B4. The results showed 100% neutralization was observed
with human TNFSF13b and chimera A, 88% neutralization with chimera
B, and no neutralization was observed for murine TNFSF13b or
chimera C.
EXAMPLE 11
In Vivo Studies using 4A5-3.1.1-B4
[0117] Transgenic mice overexpressing soluble human TNFSF13b are
generated using established techniques as described by Hogan, B. et
al. (1986) Manipulating the Mouse Embryo: A Laboratory Manual. Cold
Spring Harbor Laboratory, NY] as modified by Fox and Solter (Mol.
Cell. Biol. 8: 5470, 1988). Briefly, a DNA fragment encompassing
the hTNFSF13b gene is microinjected into the male pronuclei of
newly fertilized one-cell-stage embryos (zygotes) of the FVB/N
strain. The embryos are cultured in vitro overnight to allow
development to the two-cell-stage. Two-cell embryos are then
transplanted into the oviducts of pseudopregnant CD-1 strain mice
to allow development to term. To test for the presence of the
transgene in the newborn mice, a small piece of toe is removed from
each animal and digested with proteinase K to release the nucleic
acids. A sample of the toe extract is subsequently subjected to PCR
analysis to identify transgene-containing mice.
[0118] The hTNFSF13b transgenic mice had a dramatic increase in
peripheral B cells, generally about three fold compared to age and
sex matched littermates. There was a slight increase in peripheral
T cells as well. The hTNFSF13b transgenic mice were treated with
4A5-3.1.1-B4 to determine if neutralization of hTNFSF13b would
result in a reduction in B cell numbers back to normal levels. At
15 weeks old, female hTNFSF13b mice were injected subcutaneously
twice a week for three weeks with either 25 ug of 4A5-3.1.1-B4 or
isotype control antibody. Four days after the last injection of
antibody, the mice were sacrificed and the spleen removed for
analysis. B and T cell numbers were calculated by determining the
percentage of CD19+ cells, for B cells, and CD3+ cells, for T cells
using flow cytometry and absolute white blood cell count for each
spleen. The results are shown below demonstrate that in vivo
administration of 4A5-3.1.1-B4 to hTNFSF13b transgenic mice is able
to restore the normal numbers of T and B cells (average.+-.standard
deviation)
TABLE-US-00009 B cells T cells Treatment Group (.times.10.sup.6)
(.times.10.sup.6) Wild type littermates 29 .+-. 11 46 .+-. 15
Transgenic + Isotype mAb 122 .+-. 30 75 .+-. 14 Transgenic + 4A5
mAb 29 .+-. 5 46 .+-. 12
Sequences of the Present Invention:
TABLE-US-00010 [0119] SEQ ID NO: 1 .fwdarw. polynucleotide sequence
encoding light chain variable region
GAAATTGTGTTGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGG
GGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCCGCTACT
TAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGG
GTCTGGGACAGACTCCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATT
TTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCGGACGTTCGGC
CAAGGGACCAAGGTGGAAATCAAACGAACT SEQ ID NO: 2 .fwdarw. amino acid
sequence encoding light chain variable region
EIVLTQSPATLSLSPGERATLSCRASQSVSRYLAWYQQKPGQAPRLLIYD
ASNRATGIPARFSGSGSGTDSTLTISSLEPEDFAVYYCQQRSNWPRTFGQ GTKVEIKRT SEQ ID
NO: 3 .fwdarw. polynucleotide sequence encoding light chain CDR1
AGGGCCAGTCAGAGTGTTAGCCGCTACTTAGCC SEQ ID NO: 4 .fwdarw. amino acid
sequence encoding light chain CDR1 RASQSVSRYLA SEQ ID NO: 5
.fwdarw. polynucleotide sequence encoding light chain CDR2
GATGCATCCAACAGGGCCACT SEQ ID NO: 6 .fwdarw. amino acid sequence
encoding light chain CDR2 DASNRAT SEQ ID NO: 7 .fwdarw.
polynucleotide sequence encoding light chain CDR3
CAGCAGCGTAGCAACTGGCCTCGGACG SEQ ID NO: 8 .fwdarw. amino acid
sequence encoding light chain CDR3 QQRSNWPRT SEQ ID NO: 9 .fwdarw.
polynucleotide sequence encoding heavy chain variable region ATGAAA
CACCTGTGGTTCTTCCTCCTCCTGGTGGCAGCTCCCAGATGGGTCCTGTC
CCAGGTGCAACTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGA
CCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTAC
TGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGA
AATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAG
TCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAACTGAGC
TCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGGGTATTA
CGATATTTTGACTGGTTATTATTACTACTTTGACTACTGGGGCCAGGGAA
CCCTGGTCACCGTCTCCTCA SEQ ID NO: 10 .fwdarw. amino acid sequence
encoding heavy chain variable region
MKHLWFFLLLVAAPRWVLSQVQLQQWGAGLLKPSETLSLTCAVYGGSFSG
YYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLK
LSSVTAADTAVYYCARGYYDILTGYYYYFDYWGQGTLVTVSS SEQ ID NO: 11 .fwdarw.
polynucleotide sequence encoding heavy chain CDR1
GGTGGGTCCTTCAGTGGTTACTACTGGAGC SEQ ID NO: 12 .fwdarw. amino acid
sequence encoding heavy chain CDR1 GGSFSGYYWS SEQ ID NO: 13
.fwdarw. polynucleotide sequence encoding heavy chain CDR2
GAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGT SEQ ID NO: 14
.fwdarw. amino acid sequence encoding heavy chain CDR2
EINHSGSTNYNPSLKS SEQ ID NO: 15 .fwdarw. polynucleotide sequence
encoding heavy chain CDR3
GGGTATTACGATATTTTGACTGGTTATTATTACTACTTTGACTAC SEQ ID NO: 16
.fwdarw. amino acid sequence encoding heavy chain CDR3
GYYDILTGTTTTFDY
Sequence CWU 1
1
261327DNAHomo sapiens 1gaaattgtgt tgacgcagtc tccagccacc ctgtctttgt
ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc cgctacttag
cctggtacca gcagaaacct 120ggccaggctc ccaggctcct catctatgat
gcatccaaca gggccactgg catcccagcc 180aggttcagtg gcagtgggtc
tgggacagac tccactctca ccatcagcag cctagagcct 240gaagattttg
cagtttatta ctgtcagcag cgtagcaact ggcctcggac gttcggccaa
300gggaccaagg tggaaatcaa acgaact 3272109PRTHomo sapiens 2Glu Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Tyr 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Ser Thr Leu Thr Ile Ser Ser Leu
Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser
Asn Trp Pro Arg 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg Thr 100 105333DNAHomo sapiens 3agggccagtc agagtgttag ccgctactta
gcc 33411PRTHomo sapiens 4Arg Ala Ser Gln Ser Val Ser Arg Tyr Leu
Ala1 5 10521DNAHomo sapiens 5gatgcatcca acagggccac t 2167PRTHomo
sapiens 6Asp Ala Ser Asn Arg Ala Thr1 5727DNAHomo sapiens
7cagcagcgta gcaactggcc tcggacg 2789PRTHomo sapiens 8Gln Gln Arg Ser
Asn Trp Pro Arg Thr1 59426DNAHomo sapienssig_peptide(1)..(57)
9atgaaacacc tgtggttctt cctcctcctg gtggcagctc ccagatgggt cctgtcccag
60gtgcaactac agcagtgggg cgcaggactg ttgaagcctt cggagaccct gtccctcacc
120tgcgctgtct atggtgggtc cttcagtggt tactactgga gctggatccg
ccagccccca 180gggaaggggc tggagtggat tggggaaatc aatcatagtg
gaagcaccaa ctacaacccg 240tccctcaaga gtcgagtcac catatcagta
gacacgtcca agaaccagtt ctccctgaaa 300ctgagctctg tgaccgccgc
ggacacggct gtgtattact gtgcgagagg gtattacgat 360attttgactg
gttattatta ctactttgac tactggggcc agggaaccct ggtcaccgtc 420tcctca
42610142PRTHomo sapiensSIGNAL(1)..(19) 10Met Lys His Leu Trp Phe
Phe Leu Leu Leu Val Ala Ala Pro Arg Trp1 5 10 15Val Leu Ser Gln Val
Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys 20 25 30Pro Ser Glu Thr
Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe 35 40 45Ser Gly Tyr
Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu 50 55 60Glu Trp
Ile Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro65 70 75
80Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln
85 90 95Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr 100 105 110Tyr Cys Ala Arg Gly Tyr Tyr Asp Ile Leu Thr Gly Tyr
Tyr Tyr Tyr 115 120 125Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 130 135 1401130DNAHomo sapiens 11ggtgggtcct tcagtggtta
ctactggagc 301210PRTHomo sapiens 12Gly Gly Ser Phe Ser Gly Tyr Tyr
Trp Ser1 5 101348DNAHomo sapiens 13gaaatcaatc atagtggaag caccaactac
aacccgtccc tcaagagt 481416PRTHomo sapiens 14Glu Ile Asn His Ser Gly
Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser1 5 10 151545DNAHomo sapiens
15gggtattacg atattttgac tggttattat tactactttg actac 451615PRTHomo
sapiens 16Gly Tyr Tyr Asp Ile Leu Thr Gly Tyr Tyr Tyr Tyr Phe Asp
Tyr1 5 10 1517450PRTHomo sapiens 17Gln Val Gln Leu Gln Gln Trp Gly
Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala
Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30Tyr Trp Ser Trp Ile Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Asn His
Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr
Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu
Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg
Gly Tyr Tyr Asp Ile Leu Thr Gly Tyr Tyr Tyr Tyr Phe Asp Tyr 100 105
110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
115 120 125Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
Glu Ser 130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser
Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val 195 200 205Asp His Lys
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 210 215 220Tyr
Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly225 230
235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser Gln Glu 260 265 270Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 325 330 335Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345
350Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Arg Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln Glu Gly
Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440 445Gly Lys
45018453PRTHomo sapiens 18Gln Val Gln Leu Gln Gln Trp Gly Ala Gly
Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr
Gly Gly Ser Phe Ser Gly Tyr 20 25 30Tyr Trp Ser Trp Ile Arg Gln Pro
Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Asn His Ser Gly
Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser
Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Gly Tyr
Tyr Asp Ile Leu Thr Gly Tyr Tyr Tyr Tyr Phe Asp Tyr 100 105 110Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120
125Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220Ser Cys Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu225 230 235
240Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
245 250 255Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val 260 265 270Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val 275 280 285Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser 290 295 300Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu305 310 315 320Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350Gln
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360
365Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
370 375 380Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr385 390 395 400Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu 405 410 415Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser 420 425 430Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445Leu Ser Pro Gly Lys
45019214PRTHomo sapiens 19Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Ser Arg Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn Arg Ala
Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Ser Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Arg 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Asn Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 2102015PRTHomo sapiens 20Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser1 5 10 1521152PRTHomo sapiens 21Ala Val Gln
Gly Pro Glu Glu Thr Val Thr Gln Asp Cys Leu Gln Leu1 5 10 15Ile Ala
Asp Ser Glu Thr Pro Thr Ile Gln Lys Gly Ser Tyr Thr Phe 20 25 30Val
Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu Glu Lys 35 40
45Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile Tyr Gly
50 55 60Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His Leu Ile
Gln65 70 75 80Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu
Val Thr Leu 85 90 95Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro
Asn Asn Ser Cys 100 105 110Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu
Gly Asp Glu Leu Gln Leu 115 120 125Ala Ile Pro Arg Glu Asn Ala Gln
Ile Ser Leu Asp Gly Asp Val Thr 130 135 140Phe Phe Gly Ala Leu Lys
Leu Leu145 15022183PRTMus musculus 22Ala Phe Gln Gly Pro Glu Glu
Thr Glu Gln Asp Val Asp Leu Ser Ala1 5 10 15Pro Pro Ala Pro Cys Leu
Pro Gly Cys Arg His Ser Gln His Asp Asp 20 25 30Asn Gly Met Asn Leu
Arg Asn Ile Ile Gln Asp Cys Leu Gln Leu Ile 35 40 45Ala Asp Ser Asp
Thr Pro Thr Ile Arg Lys Gly Thr Tyr Thr Phe Val 50 55 60Pro Trp Leu
Leu Ser Phe Lys Arg Gly Asn Ala Leu Glu Glu Lys Glu65 70 75 80Asn
Lys Ile Val Val Arg Gln Thr Gly Tyr Phe Phe Ile Tyr Ser Gln 85 90
95Val Leu Tyr Thr Asp Pro Ile Phe Ala Met Gly His Val Ile Gln Arg
100 105 110Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu Val Thr
Leu Phe 115 120 125Arg Cys Ile Gln Asn Met Pro Lys Thr Leu Pro Asn
Asn Ser Cys Tyr 130 135 140Ser Ala Gly Ile Ala Arg Leu Glu Glu Gly
Asp Glu Ile Gln Leu Ala145 150 155 160Ile Pro Arg Glu Asn Ala Gln
Ile Ser Arg Asn Gly Asp Asp Thr Phe 165 170 175Phe Gly Ala Leu Lys
Leu Leu 18023119PRTHomo sapiens 23Glu Ile Val Leu Thr Gln Ser Pro
Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Ser Val Ser Arg Tyr 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn
Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Ser Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp
Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Arg 85 90 95Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105
110Pro Ser Val Phe Ile Phe Pro 11524357DNAHomo sapiens 24gaaattgtgt
tgacgcagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca gagtgttagc cgctacttag cctggtacca gcagaaacct
120ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg
catcccagcc 180aggttcagtg gcagtgggtc tgggacagac tccactctca
ccatcagcag cctagagcct 240gaagattttg cagtttatta ctgtcagcag
cgtagcaact ggcctcggac gttcggccaa 300gggaccaagg tggaaatcaa
acgaactgtg gctgcaccat ctgtcttcat cttcccg 35725154PRTHomo sapiens
25Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp1
5 10 15Val Leu Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu
Lys 20 25 30Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly
Ser Phe 35 40 45Ser Gly Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly
Lys Gly Leu 50 55 60Glu Trp Ile Gly Glu Ile Asn His Ser Gly Ser Thr
Asn Tyr Asn Pro65 70 75 80Ser Leu Lys Ser Arg Val Thr Ile Ser Val
Asp Thr Ser Lys Asn Gln 85 90 95Phe Ser Leu Lys Leu Ser Ser Val Thr
Ala Ala Asp Thr Ala Val Tyr 100 105 110Tyr Cys Ala Arg Gly Tyr Tyr
Asp Ile Leu Thr Gly Tyr Tyr Tyr Tyr 115 120 125Phe Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser 130 135 140Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala145 15026462DNAHomo sapiens 26atgaaacacc
tgtggttctt cctcctcctg gtggcagctc ccagatgggt cctgtcccag 60gtgcaactac
agcagtgggg cgcaggactg ttgaagcctt cggagaccct gtccctcacc
120tgcgctgtct atggtgggtc cttcagtggt tactactgga gctggatccg
ccagccccca 180gggaaggggc tggagtggat tggggaaatc aatcatagtg
gaagcaccaa ctacaacccg 240tccctcaaga gtcgagtcac catatcagta
gacacgtcca agaaccagtt ctccctgaaa 300ctgagctctg tgaccgccgc
ggacacggct gtgtattact gtgcgagagg gtattacgat 360attttgactg
gttattatta ctactttgac tactggggcc agggaaccct ggtcaccgtc
420tcctcagcct ccaccaaggg cccatcggtc ttccccctgg ca 462
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