U.S. patent application number 14/473997 was filed with the patent office on 2015-07-23 for antibodies to il-6 and their uses.
This patent application is currently assigned to BRISTOL-MYERS SQUIBB COMPANY. The applicant listed for this patent is BRISTOL-MYERS SQUIBB COMPANY, Pfizer Inc.. Invention is credited to Peter Brams, Madhav Narasimha Devalaraja, Brigitte Devaux, Haichun Huang, David B. Passmore, Arvind Rajpal, Kristopher Toy, Lan Yang, Jun Zhang.
Application Number | 20150203574 14/473997 |
Document ID | / |
Family ID | 41431517 |
Filed Date | 2015-07-23 |
United States Patent
Application |
20150203574 |
Kind Code |
A1 |
Rajpal; Arvind ; et
al. |
July 23, 2015 |
ANTIBODIES TO IL-6 AND THEIR USES
Abstract
Antibodies and antigen-binding portions thereof that bind to
human IL-6 are provided. Also provided are nucleic acids encoding
such antibodies and antigen binding portions, methods of making
such antibodies and antigen binding portions, compositions
comprising such antibodies or antigen binding portions, and uses of
such antibodies or antigen binding portions.
Inventors: |
Rajpal; Arvind; (San
Francisco, CA) ; Devalaraja; Madhav Narasimha;
(Gaithersburg, MD) ; Toy; Kristopher; (San Jose,
CA) ; Yang; Lan; (Morgan Hill, CA) ; Huang;
Haichun; (Fremont, CA) ; Zhang; Jun; (Tracy,
CA) ; Brams; Peter; (Sacramento, CA) ; Devaux;
Brigitte; (Palo Alto, CA) ; Passmore; David B.;
(San Carlos, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pfizer Inc.
BRISTOL-MYERS SQUIBB COMPANY |
New York
Princeton |
NY
NJ |
US
US |
|
|
Assignee: |
BRISTOL-MYERS SQUIBB
COMPANY
Princeton
NJ
PFIZER INC.
New York
NY
|
Family ID: |
41431517 |
Appl. No.: |
14/473997 |
Filed: |
August 29, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13857867 |
Apr 5, 2013 |
8846037 |
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14473997 |
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13454296 |
Apr 24, 2012 |
8436158 |
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13857867 |
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12480991 |
Jun 9, 2009 |
8188235 |
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13454296 |
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61073430 |
Jun 18, 2008 |
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Current U.S.
Class: |
424/142.1 ;
435/252.3; 435/252.33; 435/252.35; 435/254.2; 435/254.21;
435/254.23; 435/320.1; 435/332; 435/419; 530/388.15; 536/23.53 |
Current CPC
Class: |
C07K 2317/56 20130101;
A61P 25/00 20180101; A61P 25/04 20180101; A61P 31/04 20180101; C07K
16/248 20130101; A61P 1/00 20180101; A61P 37/06 20180101; A61P
35/00 20180101; A61P 11/00 20180101; A61P 31/00 20180101; A61P
19/08 20180101; C07K 2317/567 20130101; A61P 9/10 20180101; A61K
2039/505 20130101; C07K 2317/21 20130101; A61P 19/02 20180101; A61P
17/06 20180101; A61P 37/02 20180101; A61P 13/12 20180101; C07K
2317/92 20130101; A61P 29/00 20180101; A61P 3/10 20180101; A61P
1/04 20180101; A61P 37/08 20180101; A61P 19/10 20180101; C07K
2317/565 20130101; A61P 37/00 20180101; A61P 1/16 20180101 |
International
Class: |
C07K 16/24 20060101
C07K016/24 |
Claims
1. An isolated human monoclonal antibody or antigen-binding portion
thereof comprising a heavy chain variable (V.sub.H) domain amino
acid sequence comprising a CDR1, CDR2, and CDR3 region selected
from the group consisting of: a) a V.sub.H CDR1 as set forth in SEQ
ID NO: 5, a V.sub.H CDR2 as set forth in SEQ ID NO:6, and a V.sub.H
CDR3 as set forth in SEQ ID NO:7; b) a V.sub.H CDR1 as set forth in
SEQ ID NO: 35, a V.sub.H CDR2 as set forth in SEQ ID NO:6, and a
V.sub.H CDR3 as set forth in SEQ ID NO:7; c) a V.sub.H CDR1 as set
forth in SEQ ID NO: 5, a V.sub.H CDR2 as set forth in SEQ ID NO:6,
and a V.sub.H CDR3 as set forth in SEQ ID NO:41; d) a V.sub.H CDR1
as set forth in SEQ ID NO: 5, a V.sub.H CDR2 as set forth in SEQ ID
NO:6, and a V.sub.H CDR3 as set forth in SEQ ID NO:44; e) a V.sub.H
CDR1 as set forth in SEQ ID NO: 5, a V.sub.H CDR2 as set forth in
SEQ ID NO:6, and a V.sub.H CDR3 as set forth in SEQ ID NO:45;
wherein said antibody binds specifically to human IL-6.
2. (canceled)
3. An isolated human antibody or antigen-binding portion thereof
comprising a light chain variable (V.sub.L) domain amino acid
sequence comprising a V.sub.L CDR1 as set forth in SEQ ID NO:16, a
V.sub.L CDR2 as set forth in SEQ ID NO:17, and a V.sub.LCDR3 as set
forth in SEQ ID NO: 18; wherein said antibody binds specifically to
human IL-6.
4-9. (canceled)
10. An isolated human monoclonal antibody or antigen-binding
portion thereof comprising V.sub.L and V.sub.H domains at least 90%
identical in amino acid sequence to the V.sub.L and V.sub.H
domains, respectively, of antibody 9C8, 9C8 N68T T83S, 9C8 E31G
N68T T83S, 9C8 I24V N68T T83S, and 22B5; wherein said antibody
binds specifically to human IL-6.
11-18. (canceled)
19. An isolated nucleic acid molecule comprising a nucleotide
sequence that encodes an antibody according to claim 1.
20. A vector comprising the nucleic acid molecule according to
claim 19, wherein the vector optionally comprises an expression
control sequence operably linked to the nucleic acid molecule.
21. A host cell comprising the vector according to claim 20.
22. A host cell comprising the nucleic acid molecule according to
claim 19.
23. A method for treating, preventing or alleviating the symptoms
of a IL-6 mediated disorder in a subject in need thereof,
comprising administering to said subject an antibody or
antigen-binding portion according to claim 1.
24-28. (canceled)
Description
CROSS-REFERENCE TO RELATED PATENTS AND PATENT APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 13/857,867, filed Apr. 5, 2013, now
U.S. Pat. No. 8,846,037; which is a divisional application of U.S.
patent application Ser. No. 13/454,296, filed Apr. 24, 2012, now
U.S. Pat. No. 8,436,158; which is a divisional application of U.S.
patent application Ser. No. 12/480,991, filed Jun. 9, 2009, now
U.S. Pat. No. 8,188,235, which claims the benefit of U.S.
Provisional Patent Application Ser. No. 61/073,430 filed Jun. 18,
2008. The disclosures of all of the aforementioned applications are
incorporated herein by reference in their entirety.
[0002] The instant application contains a Sequence Listing that has
been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII file, created
on Aug. 29, 2014, is named PCFC-596-104-Sequence-Listing.txt and is
50,115 bytes in size.
JOINT RESEARCH AGREEMENT
[0003] The disclosure and claims herein were made as a result of
activities undertaken within the scope of a joint research
agreement in effect on or before the date the claimed invention was
made between Pfizer Inc. and Medarex, Inc.
BACKGROUND
[0004] This invention relates to antibodies and antigen-binding
portions thereof that bind to human IL-6. This invention also
relates to nucleic acids encoding such antibodies and antigen
binding portions thereof; methods of making such antibodies and
antigen binding portions thereof; compositions comprising such
antibodies or antigen binding portions thereof; and uses of such
antibodies or antigen binding portions thereof.
[0005] Interleukin-6 (IL-6), which is also known as interferon B2
(IFNB2), is a pleiotropic cytokine that belongs to the family of
gp130 ligands and is produced by many cell types, including T
lymphocytes, fibroblasts and monocytes. IL-6 is produced
constitutively at low levels and is readily induced by infectious
stimuli or inflammatory cytokines. IL-6 binds to a specific
receptor IL-6R (gp80) which heterodimerizes with cell-bound or
soluble gp130 to form a functional receptor complex. Binding of
IL-6 to its receptor initiates cellular events including activation
of the JAK-STAT3 pathway and ras-mediated MAP kinase signaling.
IL-6 can elicit a diverse array of effects such as proliferation
and differentiation of B cells and monocytes, T cell activation,
hematopoiesis, osteoclast activation, keratinocyte growth, neuronal
growth, hepatocyte activation and acute phase protein induction
from hepatocytes.
[0006] IL-6 plays an important role in B cell abnormalities as
demonstrated in systemic lupus erythematosus, multiple myeloma and
lymphoproliferative disorders. Similarly, IL-6 is also implicated
in the pathogenesis of autoimmune and inflammatory diseases such as
rheumatoid arthritis and osteoarthritis. Recently, indirect
evidence suggests an association between IL-6 and chronic
obstructive pulmonary disease and insulin resistance in type 2
diabetes. IL-6 has both pro-inflammatory and anti-inflammatory
effects in the immune system, indicating that this cytokine likely
plays a central role in regulating the physiological response to
disease. Therefore, targeting IL-6 can potentially provide
therapeutic benefit in a variety of disease areas.
[0007] An increase in the production of IL-6 has been observed in a
number of diseases including: Alzheimer's disease, autoimmune
diseases, such as rheumatoid arthritis, inflammation, myocardial
infarction, Paget's disease, osteoporosis, liver fibrosis, solid
tumors (renal cell carcinoma), prostatic and bladder cancers,
neurological cancers, and B-cell malignancies (e.g., Casteleman's
disease, certain lymphomas, chronic lymphocytic leukemia, and
multiple myeloma). Research has indicated that IL-6 is linked to
the pathogenesis of many of these diseases, particularly, cancer
and rheumatoid arthritis and, therefore, blocking IL-6 should
translate into clinical benefits.
SUMMARY
[0008] An isolated antibody or antigen-binding portion thereof that
specifically binds IL-6 and may act as an IL-6 receptor antagonist,
and compositions comprising the antibody or portion are
produced.
[0009] Compositions comprising (i) the heavy and/or light chain,
the variable domains thereof, or antigen-binding portions thereof,
of the anti-IL-6 antibody, or nucleic acid molecules encoding them;
and (ii) a pharmaceutically acceptable carrier are provided. The
compositions may further comprise another component, such as a
therapeutic agent or a diagnostic agent.
[0010] Diagnostic and therapeutic methods are also provided.
Similarly, the anti-IL-6 antibodies and antigen-binding portions
thereof are provided for the manufacture of medicaments to treat
inflammatory and non-inflammatory disorders.
[0011] Vectors and host cells comprising the nucleic acid
molecules, as well as methods of recombinantly producing the
polypeptides encoded by the nucleic acid molecules are
provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIGS. 1A-1F show an alignment of the germline amino acid
sequences of the heavy and light chain variable regions compared to
the respective anti-IL-6 antibodies 9C8, 9C8 N68T T83S and 22B5
heavy and light chain variable regions (only mismatches are shown
for the 9C8, 9C8 N68T T83S, and 22B5 antibody). FIG. 1A shows the
heavy chain variable region of the 22B5 antibody. FIG. 1B shows the
light chain variable region of the 22B5 antibody. FIG. 1C shows the
heavy chain variable region of the 9C8 antibody. FIG. 1D shows the
light chain variable region of the 9C8 antibody. FIG. 1E shows the
heavy chain variable region of the 9C8 N68T T83S antibody. FIG. 1F
shows the light chain variable region of the 9C8 N68T T83S
antibody. The CDRs are underlined and mismatched gap(s) are
indicated by a pound sign (#).
[0013] FIG. 2 shows the total serum C-reactive protein (CRP) for
the vehicle and the 9C8 N68T T83S IgG.sub.2 antibody as determined
by Electrochemiluminescence Immuno-Assay. Each point represents an
average value of serum CRP from 3 cynomolgus monkeys (.+-.SE) dosed
with vehicle or anti-IL-6 antibody 9C8 N68T T83S IgG.sub.2 at 0.5
mg/kg and 5.0 mg/kg. Serum CRP was measured by Meso Scale Discovery
(MSD). LPS was administered at the 0 hour time point.
DETAILED DESCRIPTION
Definitions and General Techniques
[0014] The term "antibody" is synonymous with immunoglobulin and is
to be understood as commonly known in the art. In particular, the
term antibody is not limited by any particular method of producing
the antibody. For example, the term antibody includes, inter alia,
recombinant antibodies, monoclonal antibodies, and polyclonal
antibodies.
[0015] The basic antibody structural unit is a tetramer. Each
tetramer is composed of two identical pairs of polypeptide chains,
each pair having one "light" (about 25 kDa) and one "heavy" chain
(about 50-70 kDa). The amino-terminal portion of each chain
includes a variable region of about 100 to 120 or more amino acids
primarily responsible for antigen recognition. The carboxy-terminal
portion of each chain defines a constant region primarily
responsible for effector function. Human light chains are
classified as kappa and lambda light chains. Heavy chains are
classified as mu, delta, gamma, alpha, or epsilon, and define the
antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
Within light and heavy chains, the variable and constant regions
are joined by a "J" region of about 12 or more amino acids, with
the heavy chain also including a "D" region of about 3 or more
amino acids.
[0016] The variable regions of each heavy/light chain pair (V.sub.H
and V.sub.L), respectively, form the antigen binding site. Thus, an
intact IgG antibody, for example, has two binding sites. Except in
bifunctional or bispecific antibodies, the two binding sites are
the same.
[0017] The variable regions of the heavy and light chains exhibit
the same general structure of relatively conserved framework
regions (FR) joined by three hyper variable regions, also called
complementary determining regions or CDRs. The term "variable"
refers to the fact that certain portions of the variable domains
differ extensively in sequence among antibodies and are used in the
binding and specificity of each particular antibody for its
particular antigen. The variability, however, is not evenly
distributed throughout the variable domains of antibodies, but is
concentrated in the CDRs, which are separated by the more highly
conserved FRs. The CDRs from the two chains of each pair are
aligned by the FRs, enabling binding to a specific epitope. From
N-terminal to C-terminal, both light and heavy chains comprise the
domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of
amino acids to each domain is in accordance with the definitions of
Kabat Sequences of Proteins of Immunological Interest (National
Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia
& Lesk J. Mol. Biol. 196:901-917 (1987); Chothia et al. Nature
342:878-883 (1989), the disclosures of which are herein
incorporated by reference.
[0018] As used herein, an antibody that is referred to by number is
the same as a monoclonal antibody that is obtained from the
hybridoma of the same number. For example, anti-IL-6 monoclonal
antibody 9C8 is the same antibody as one obtained from hybridoma
9C8, or a subclone thereof.
[0019] The term "analog" or "polypeptide analog" means a
polypeptide that comprises a segment that has substantial identity
to some reference amino acid sequence and has substantially the
same function or activity as the reference amino acid sequence.
Typically, polypeptide analogs comprise one or more conservative
amino acid substitution (or insertion or deletion) with respect to
the reference sequence. Analogs can be at least 20 or 25 amino
acids long, or can be at least 50, 60, 70, 80, 90, 100, 150 or 200
amino acids long or longer, and can often be as long as the
full-length polypeptide. Some embodiments include polypeptide
analogs with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16
or 17 substitutions from the reference amino acid sequence. In some
instances, the reference amino acid sequence is a germline
sequence. Analogs of antibodies or immunoglobulin molecules can be
readily prepared by those of ordinary skill in the art.
[0020] As discussed herein, amino acid substitutions to an IL-6
antibody or antigen-binding portion thereof are those which
typically: (1) reduce susceptibility to proteolysis, (2) reduce
susceptibility to oxidation, (3) alter binding affinity for forming
protein complexes, (4) delete or create a site for glycosylation,
or (4) confer or modify other physicochemical or functional
properties of such analogs, but still retain specific binding to
IL-6.
[0021] Analogs can include various substitutions to the
normally-occurring peptide sequence. For example, single or
multiple amino acid substitutions, preferably conservative amino
acid substitutions, may be made in the normally-occurring sequence.
A conservative amino acid substitution typically does not
substantially change the structural characteristics of the parent
sequence.
[0022] The term "antigen-binding portion" of an antibody refers to
a fragment of an antibody that retains the ability to specifically
bind to an antigen (e.g., IL-6). It has been shown that the
antigen-binding function of an antibody can be performed by
fragments of a full-length antibody. Examples of binding fragments
encompassed within the term "antigen-binding portion" of an
antibody include: (i) a Fab fragment, a monovalent fragment
consisting of the V.sub.L, V.sub.H, C.sub.L and C.sub.H1 domains;
(ii) a F(ab').sub.2 fragment, a bivalent fragment comprising two
Fab fragments linked by a disulfide bridge at the hinge region;
(iii) a Fd fragment consisting of the V.sub.H and C.sub.H1 domains;
(iv) a Fv fragment consisting of the V.sub.L and V.sub.H domains of
a single arm of an antibody; (v) a domain antibody, (dAb) (Ward et
al., (1989) Nature 341:544-546), which consists of a V.sub.H
domain; and (vi) an isolated complementary determining region
(CDR). Furthermore, although the two domains of the Fv fragment,
V.sub.L and V.sub.H, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the
V.sub.L and V.sub.H regions pair to form monovalent molecules
(known as single chain Fv (scFv)). Such single chain antibodies are
also intended to be encompassed within the term "antigen-binding
portion" of an antibody. Other forms of single chain antibodies,
such as diabodies are also encompassed. Diabodies are bivalent,
bispecific antibodies in which V.sub.H and V.sub.L domains are
expressed on a single polypeptide chain, but using a linker that is
too short to allow for pairing between the two domains on the same
chain, thereby forcing the domains to pair with complementary
domains of another chain and creating two antigen binding sites. In
addition, one or more CDRs from an antibody may be incorporated
into a larger polypeptide chain, which can be covalently or
non-covalently linked to another. In embodiments having one or more
binding sites, the binding sites may be identical to one another or
may be different.
[0023] Still further, an antibody or antigen-binding portion
thereof may be part of larger immunoadhesion molecules, formed by
covalent or noncovalent association of the antibody or antibody
portion with one or more other proteins or peptides. Examples of
such immunoadhesion molecules include use of the streptavidin core
region to make a tetrameric scFv molecule and use of a cysteine
residue, a marker peptide and a C-terminal polyhistidine tag to
make bivalent and biotinylated scFv molecules. Other examples
include where one or more CDRs from an antibody are incorporated
into a molecule either covalently or noncovalently to make it an
immunoadhesin that specifically binds to an antigen of interest,
such as IL-6. In such embodiments, the CDR(s) may be incorporated
as part of a larger polypeptide chain, may be covalently linked to
another polypeptide chain, or may be incorporated noncovalently.
Antibody portions, such as Fab and F(ab').sub.2 fragments, can be
prepared from whole antibodies using any suitable technique, such
as papain or pepsin digestion, respectively, of whole antibodies.
Moreover, antibodies, antibody portions and immunoadhesion
molecules can be obtained using various recombinant DNA
techniques.
[0024] The term "chimeric antibody" means an antibody that
comprises regions from two or more different antibodies, including
antibodies from different species. For example, one or more of the
CDRs of a chimeric antibody can be derived from a human IL-6
antibody. In one example, the CDRs from a human antibody can be
combined with CDRs from a non-human antibody, such as mouse or rat.
In another example, all of the CDRs can be derived from human IL-6
antibodies. In another example, the CDRs from more than one human
IL-6 antibody can be combined in a chimeric antibody. For instance,
a chimeric antibody may comprise a CDR1 from the light chain of a
first human IL-6 antibody, a CDR2 from the light chain of a second
human IL-6 antibody and a CDR3 from the light chain of a third
human IL-6 antibody, and CDRs from the heavy chain may be derived
from one or more other IL-6 antibodies. Further, the framework
regions may be derived from one of the IL-6 antibodies from which
one or more of the CDRs are taken or from one or more different
human antibodies. Further, the term "chimeric antibody" is intended
to encompass any of the above mentioned combinations where the
combinations involved human and non-human antibodies.
[0025] The term "compete" means that a first antibody, or an
antigen-binding portion thereof, competes for binding with a second
antibody, or an antigen-binding portion thereof, where binding of
the first antibody with its cognate epitope is detectably decreased
in the presence of the second antibody compared to the binding of
the first antibody in the absence of the second antibody. The
alternative, where the binding of the second antibody to its
epitope is also detectably decreased in the presence of the first
antibody, can, but need not be the case. That is, a first antibody
can inhibit the binding of a second antibody to its epitope without
that second antibody inhibiting the binding of the first antibody
to its respective epitope. However, where each antibody detectably
inhibits the binding of the other antibody with its cognate epitope
or ligand, whether to the same, greater, or lesser extent, the
antibodies are said to "cross-compete" with each other for binding
of their respective epitope(s). Regardless of the mechanism by
which such competition or cross-competition occurs (e.g., steric
hindrance, conformational change, or binding to a common epitope,
or portion thereof), the skilled artisan would appreciate, based
upon the teachings provided herein, that such competing and/or
cross-competing antibodies can be useful for the methods disclosed
herein.
[0026] The term "conservative amino acid substitution" means an
amino acid residue is substituted by another amino acid residue
having a side chain R group with similar chemical properties (e.g.,
charge or hydrophobicity). In general, a conservative amino acid
substitution will not substantially change the functional
properties of a protein. In cases where two or more amino acid
sequences differ from each other by conservative substitutions, the
percent sequence identity may be adjusted upwards to correct for
the conservative nature of the substitution. Means for making this
adjustment are well-known to those of skill in the art. Examples of
groups of amino acids that have side chains with similar chemical
properties include 1) aliphatic side chains: glycine, alanine,
valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains:
serine and threonine; 3) amide-containing side chains: asparagine
and glutamine; 4) aromatic side chains: phenylalanine, tyrosine,
and tryptophan; 5) basic side chains: lysine, arginine, and
histidine; 6) acidic side chains: aspartic acid and glutamic acid;
and 7) sulfur-containing side chains: cysteine and methionine.
Conservative amino acids substitution groups can be, for example,
valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,
alanine-valine, glutamate-aspartate, and asparagine-glutamine.
[0027] A conservative replacement is also any change having a
positive value in the PAM250 log-likelihood matrix disclosed in
Gonnet et al., Science 256:1443-45 (1992), incorporated herein by
reference. A "moderately conservative" replacement is any change
having a non-negative value in the PAM250 log-likelihood
matrix.
[0028] "Contacting" refers to bringing an antibody or antigen
binding portion thereof and a target IL-6, or epitope thereof,
together in such a manner that the antibody can affect the
biological activity of the IL-6. Such "contacting" can be
accomplished in vitro, e.g., in a test tube, a petri dish, or the
like. In a test tube, contacting may involve only an antibody or
antigen binding portion thereof and IL-6 or epitope thereof or it
may involve whole cells. Cells may also be maintained or grown in
cell culture dishes and contacted with antibodies or antigen
binding portions thereof in that environment. In this context, the
ability of a particular antibody or antigen binding portion thereof
to affect an IL-6 related disorder, i.e., the IC.sub.50 of the
antibody, can be determined before use of the antibody in vivo with
more complex living organisms. For cells outside the organism,
multiple methods exist, and are well-known to those skilled in the
art, to contact IL-6 with the antibodies or antigen-binding
portions thereof.
[0029] The term "ELISA" refers to an enzyme-linked immunosorbent
assay. This kind of assay is well known to those of skill in the
art.
[0030] The term "epitope" includes any protein determinant capable
of specific binding to an immunoglobulin or T-cell receptor or
otherwise interacting with a molecule. Epitopic determinants
generally consist of chemically active surface groupings of
molecules such as amino acids or carbohydrate or sugar side chains
and generally have specific three dimensional structural
characteristics, as well as specific charge characteristics. An
epitope may be "linear" or "conformational." In a linear epitope,
all of the points of interaction between the protein and the
interacting molecule (such as an antibody) occur linearly along the
primary amino acid sequence of the protein. In a conformational
epitope, the points of interaction occur across amino acid residues
on the protein that are separated from one another. Once a desired
epitope on an antigen is determined, antibodies to that epitope can
be generated. An approach to achieve this is to conduct
cross-competition studies to find antibodies that competitively
bind with one another, i.e., the antibodies compete for binding to
the antigen. A high throughput process for "binning" antibodies
based upon their cross-competition is described in International
Patent Publication No. WO 03/48731.
[0031] The term "expression control sequence" as used herein means
polynucleotide sequences that are necessary to effect the
expression and processing of coding sequences to which they are
ligated. Expression control sequences include appropriate
transcription initiation, termination, promoter and enhancer
sequences; efficient RNA processing signals such as splicing and
polyadenylation signals; sequences that stabilize cytoplasmic mRNA;
sequences that enhance translation efficiency (i.e., Kozak
consensus sequence); sequences that enhance protein stability; and
when desired, sequences that enhance protein secretion. The nature
of such control sequences differs depending upon the host organism;
in prokaryotes, such control sequences generally include promoter,
ribosomal binding site, and transcription termination sequence; in
eukaryotes, generally, such control sequences include promoters and
transcription termination sequence. The term "control sequences" is
intended to include, at a minimum, all components whose presence is
essential for expression and processing, and can also include
additional components whose presence is advantageous, for example,
leader sequences and fusion partner sequences.
[0032] The term "germline" refers to the nucleotide sequences of
the antibody genes and gene segments as they are passed from
parents to offspring via the germ cells. This germline sequence is
distinguished from the nucleotide sequences encoding antibodies in
mature B cells which have been altered by recombination and
hypermutation events during the course of B cell maturation.
[0033] The term "human antibody" means any antibody in which the
variable and constant domain sequences are human sequences. The
term encompasses antibodies with sequences derived from human
genes, including those which have been changed, e.g., to decrease
possible immunogenicity, increase affinity, eliminate cysteine
residues that might cause undesirable folding, etc. The term also
encompasses such antibodies produced recombinantly in non-human
cells, which might impart glycosylation not typical of human cells.
These antibodies may be prepared in a variety of ways.
[0034] The term "humanized antibody" refers to antibodies of
non-human origin, wherein the amino acid residues that are
characteristic of antibody sequences of the non-human species are
replaced with residues found in the corresponding positions of
human antibodies. This "humanization" process can reduce the
immunogenicity in humans of the resulting antibody. Antibodies of
non-human origin can be humanized using any suitable technique well
known in the art. The antibody of interest may be engineered by
recombinant DNA techniques to substitute the CH1, CH2, CH3, hinge
domains, and/or the framework domain with the corresponding human
sequence. The term "humanized antibody" further includes within its
meaning, chimeric human antibodies and CDR-grafted antibodies.
Chimeric human antibodies include the V.sub.H and V.sub.L of an
antibody of a non-human species and the C.sub.H and C.sub.L domains
of a human antibody. The CDR-transplanted antibodies result from
the replacement of CDRs of the V.sub.H and V.sub.L of a human
antibody with those of the V.sub.H and V.sub.L, respectively, of an
antibody of an animal other than a human.
[0035] The term "isolated polynucleotide" as used herein means a
polynucleotide of genomic, cDNA, or synthetic origin or a
combination thereof, which by virtue of its origin the "isolated
polynucleotide" (1) is not associated with all or a portion of
polynucleotides with which the "isolated polynucleotide" is found
in nature, (2) is operably linked to a polynucleotide to which it
is not linked in nature, or (3) does not occur in nature as part of
a larger sequence.
[0036] The term "isolated protein", "isolated polypeptide" or
"isolated antibody" is a protein, polypeptide or antibody that by
virtue of its origin or source of derivation: (1) is not associated
with naturally associated components that accompany it in its
native state; (2) is free of other proteins from the same species;
(3) is expressed by a cell from a different species; or (4) does
not occur in nature. Thus, a polypeptide that is, e.g., chemically
synthesized or synthesized in a cellular system different from the
cell from which it naturally originates will be "isolated" from its
naturally associated components. A protein may also be rendered
substantially free of naturally associated components by isolation,
using any suitable protein purification technique.
[0037] Examples of isolated antibodies include an IL-6 antibody
that has been affinity purified using IL-6, and an IL-6 antibody
that has been synthesized by a cell line in vitro.
[0038] The term "K.sub.D" refers to the binding affinity
equilibrium constant of a particular antibody-antigen interaction.
An antibody is said to specifically bind an antigen when the
K.sub.D is .ltoreq.1 mM, preferably .ltoreq.100 nM, and most
preferably .ltoreq.10 nM. A K.sub.D binding affinity constant can
be measured by surface plasmon resonance, for example using the
BIACORE.TM. system as discussed in EXAMPLE 7.
[0039] The term "k.sub.off" refers to the dissociation rate
constant of a particular antibody-antigen interaction. A k.sub.off
dissociation rate constant can be measured by surface plasmon
resonance, for example using the BIACORE.TM. system as discussed in
EXAMPLE 7.
[0040] The term "naturally occurring nucleotides" as used herein
includes deoxyribonucleotides and ribonucleotides. The term
"modified nucleotides" as used herein includes, for example,
nucleotides with modified or substituted sugar groups. The term
"oligonucleotide linkages" referred to herein includes
oligonucleotide linkages such as, for example, phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phoshoraniladate, phosphoroamidate. An
oligonucleotide can include a label for detection, if desired.
[0041] "Operably linked" sequences include both expression control
sequences that are contiguous with the gene of interest and
expression control sequences that act in trans or at a distance to
control the gene of interest.
[0042] The term "percent sequence identity" in the context of
nucleic acid sequences means the residues in two sequences that are
the same when aligned for maximum correspondence. The length of
sequence identity comparison may be over at least about nine
nucleotides, usually at least about 18 nucleotides, more usually at
least about 24 nucleotides, typically at least about 28
nucleotides, more typically at least about 32 nucleotides, and at
least about 36, 48 or more nucleotides. There are a number of
different algorithms known in the art which can be used to measure
nucleotide sequence identity. For instance, polynucleotide
sequences can be compared using FASTA, Gap or Besffit, which are
programs in Wisconsin Package Version 10.0, Genetics Computer Group
(GCG), Madison, Wis. FASTA, which includes, e.g., the programs
FASTA2 and FASTA3, provides alignments and percent sequence
identity of the regions of the best overlap between the query and
search sequences (Pearson, Methods Enzymol. 183:63-98 (1990);
Pearson, Methods Mol. Biol. 132:185-219 (2000); Pearson, Methods
Enzymol. 266:227-258 (1996); Pearson, J. Mol. Biol. 276:71-84
(1998); incorporated herein by reference). Default parameters for a
particular program or algorithm are typically used. For instance,
percent sequence identity between nucleic acid sequences can be
determined using FASTA with its default parameters (a word size of
6 and the NOPAM factor for the scoring matrix) or using Gap with
its default parameters as provided in GCG Version 6.1, incorporated
herein by reference.
[0043] A reference to a nucleotide sequence encompasses its
complement unless otherwise specified. Thus, a reference to a
nucleic acid having a particular sequence should be understood to
encompass its complementary strand, with its complementary
sequence.
[0044] The term "percent sequence identity" in the context of amino
acid sequences means the residues in two sequences that are the
same when aligned for maximum correspondence. The length of
sequence identity comparison may be over at least about five amino
acids, usually at least about 20 amino acids, more usually at least
about 30 amino acids, typically at least about 50 amino acids, more
typically at least about 100 amino acids, and even more typically
about 150, 200 or more amino acids. There are a number of different
algorithms known in the art that can be used to measure amino acid
sequence identity. For instance, amino acid sequences can be
compared using FASTA, Gap or Bestfit, which are programs in
Wisconsin Package Version 10.0, Genetics Computer Group (GCG),
Madison, Wis.
[0045] Sequence identity for polypeptides is typically measured
using sequence analysis software. Protein analysis software matches
sequences using measures of similarity assigned to various
substitutions, deletions and other modifications, including
conservative amino acid substitutions. For instance, GCG contains
programs such as "Gap" and "Bestfit" which can be used with default
parameters as specified by the programs to determine sequence
homology or sequence identity between closely related polypeptides,
such as homologous polypeptides from different species of organisms
or between a wild type protein and an analog thereof. See, e.g.,
GCG Version 6.1 (University of Wisconsin, Wis.). Polypeptide
sequences also can be compared using FASTA using default or
recommended parameters, see GCG Version 6.1. FASTA (e.g., FASTA2
and FASTA3) provides alignments and percent sequence identity of
the regions of the best overlap between the query and search
sequences (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson,
Methods Mol. Biol. 132:185-219 (2000)). Another algorithm when
comparing a sequence to a database containing a large number of
sequences from different organisms is the computer program BLAST,
especially blastp or tblastn, using default parameters as supplied
with the programs. See, e.g., Altschul et al., J. Mol. Biol.
215:403-410 (1990); Altschul et al., Nucleic Acids Res. 25:3389-402
(1997).
[0046] The term "recombinant host cell" (or simply "host cell"), as
used herein, means a cell into which a recombinant expression
vector has been introduced. It should be understood that
"recombinant host cell" and "host cell" mean not only the
particular subject cell but also 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.
[0047] A protein or polypeptide is "substantially pure,"
"substantially homogeneous," or "substantially purified" when at
least about 60 to 75% of a sample exhibits a single species of
polypeptide. The polypeptide or protein may be monomeric or
multimeric. A substantially pure polypeptide or protein can
typically comprise about 50%, 60%, 70%, 80% or 90% w/w of a protein
sample, more usually about 95%, and preferably can be over 99%
pure. Protein purity or homogeneity may be indicated by any
suitable means, such as polyacrylamide gel electrophoresis of a
protein sample followed by visualizing a single polypeptide band
upon staining the gel with a stain. As one skilled in the art will
appreciate, higher resolution may be provided by using HPLC or
other means for purification.
[0048] The term "substantial similarity" or "substantial sequence
similarity," when referring to a nucleic acid or fragment thereof,
means that when optimally aligned with appropriate nucleotide
insertions or deletions with another nucleic acid (or its
complementary strand), there is nucleotide sequence identity in at
least about 85%, at least about 90%, and at least about 95%, 96%,
97%, 98% or 99% of the nucleotide bases, as measured by any
well-known algorithm of sequence identity, such as FASTA, BLAST or
Gap, as discussed above.
[0049] As applied to polypeptides, the term "substantial identity"
or "substantial similarity" means that two amino acid sequences,
when optimally aligned, such as by the programs GAP or BESTFIT
using default gap weights as supplied with the programs, share at
least 70%, 75% or 80% sequence identity, preferably at least 90% or
95% sequence identity, and more preferably at least 97%, 98% or 99%
sequence identity. In certain embodiments, residue positions that
are not identical differ by conservative amino acid
substitutions.
[0050] The term "surface plasmon resonance" refers to an optical
phenomenon that allows for the analysis of real-time biospecific
interactions by detection of alterations in protein concentrations
within a biosensor matrix, for example using the BIACORE.TM. system
(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For
further descriptions, see Jonsson U. et al., Ann. Biol. Clin.
51:19-26 (1993); Jonsson U. et al., Biotechniques 11:620-627
(1991); Jonsson B. et al., J. Mol. Recognit. 8:125-131 (1995); and
Johnsson B. et al., Anal. Biochem. 198:268-277 (1991).
[0051] "Therapeutically effective amount" refers to that amount of
the therapeutic agent being administered which will relieve to some
extent one or more of the symptoms of the disorder being treated.
In reference to the treatment of rheumatoid arthritis, a
therapeutically effective amount refers to that amount which has at
least one of the following effects: reducing the structural damage
of joints; inhibiting (that is, slowing to some extent, preferably
stopping) the accumulation of fluid in the joint area; and
relieving to some extent (or, preferably, eliminating) one or more
symptoms associated with rheumatoid arthritis.
[0052] "Treat", "treating" and "treatment" refer to a method of
alleviating or abrogating a biological disorder and/or its
attendant symptoms.
[0053] The term "utilizes" with reference to a particular gene
means that the amino acid sequence of a particular region in an
antibody was ultimately derived from that gene during B-cell
maturation. For example, the phrase "a heavy chain variable region
amino acid sequence that utilizes a human V.sub.H-3 family gene"
refers to the situation where the V.sub.H region of the antibody
was derived from the VH-3 family of gene segments during B-cell
maturation. In human B-cells, there are more than 30 distinct
functional heavy chain variable genes with which to generate
antibodies. Use of a particular heavy chain variable gene,
therefore, is indicative of a binding motif of the antibody-antigen
interaction with respect to the combined properties of binding to
the antigen and functional activity. As will be appreciated, gene
utilization analysis provides only a limited overview of antibody
structure. As human B-cells stochastically generate V-D-J heavy or
V-J kappa light chain transcripts, there are a number of secondary
processes that occur, including, without limitation, somatic
hypermutation, additions, and CDR3 extensions. See, for example,
Mendez et al. Nature Genetics 15:146-156 (1997).
[0054] The term "vector", as used herein, means a nucleic acid
molecule capable of transporting another nucleic acid to which it
has been linked. In some cases, the vector is a plasmid, i.e., a
circular double stranded piece of DNA into which additional DNA
segments may be ligated. For example, the vector is a viral vector,
wherein additional DNA segments may be ligated into the viral
genome. In another case, the 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). In another example, the 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").
[0055] The terms "label" or "labeled" refers to incorporation of
another molecule in the antibody. For example, the label is a
detectable marker, e.g., incorporation of a radiolabeled amino acid
or attachment to a polypeptide of biotinyl moieties that can be
detected by marked avidin (e.g., streptavidin containing a
fluorescent marker or enzymatic activity that can be detected by
optical or colorimetric methods). In another embodiment, the label
or marker can be therapeutic, e.g., a drug conjugate or toxin.
Various methods of labeling polypeptides and glycoproteins are
known in the art and may be used. Examples of labels for
polypeptides include, but are not limited to, the following:
radioisotopes or radionuclides (e.g., .sup.3H, .sup.14C, .sup.15N,
.sup.35S, .sup.90Y, .sup.99Tc, .sup.111In, .sup.125I, .sup.131I)
fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors),
enzymatic labels (e.g., horseradish peroxidase,
.beta.-galactosidase, luciferase, alkaline phosphatase),
chemiluminescent markers, biotinyl groups, predetermined
polypeptide epitopes recognized by a secondary reporter (e.g.,
leucine zipper pair sequences, binding sites for secondary
antibodies, metal binding domains, epitope tags), magnetic agents,
such as gadolinium chelates, toxins such as pertussis toxin, taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicine,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. In some cases, labels are attached by
spacer arms of various lengths to reduce potential steric
hindrance.
Therapeutic Methods of Use
[0056] Also provided are methods for inhibiting IL-6 activity by
administering an IL-6 antibody to a patient in need thereof. Any of
the antibodies or antigen-binding portions thereof described herein
may be used therapeutically. In a preferred embodiment, the IL-6
antibody is a human, chimeric or humanized antibody. In another
preferred embodiment, the IL-6 is human and the patient is a human
patient. Alternatively, the patient may be a mammal that expresses
an IL-6 that the IL-6 antibody cross-reacts with. The antibody may
be administered to a non-human mammal expressing IL-6 purposes or
as an animal model of human disease. Such animal models may be used
for demonstrating the therapeutic efficacy of the antibodies.
[0057] An IL-6 antibody or antibody portion thereof may be
administered to a patient who expresses abnormally high levels of
IL-6. The antibody may be administered once, or may be administered
multiple times. The antibody may be administered from three times
daily to once every six months or longer. The administering may be
on a schedule such as three times daily, twice daily, once daily,
once every two days, once every three days, once weekly, once every
two weeks, once every month, once every two months, once every
three months and once every six months. The antibody may also be
administered continuously via a minipump. The antibody may be
administered via a mucosal, buccal, intranasal, inhalable,
intravenous, subcutaneous, intramuscular, parenteral, or intratumor
route. The antibody may be administered once, at least twice or for
at least the period of time until the condition is treated,
palliated or cured. The antibody generally will be administered for
as long as the condition is present. The antibody will generally be
administered as part of a pharmaceutical composition as described
herein. The dosage of antibody will generally be in the range of
0.1 to 100 mg/kg, 0.5 to 50 mg/kg, 1 to 20 mg/kg, and 1 to 10
mg/kg. The serum concentration of the antibody may be measured by
any suitable method.
[0058] Also provided are methods for the treatment of abnormal cell
infiltration in a mammal, including a human, comprising
administering to the mammal a therapeutically effective amount of
an IL-6 antibody or antigen binding portion thereof, as described
herein, that is effective in treating abnormal cell
infiltration.
[0059] The IL-6 antibodies or antigen-binding portions thereof can
be used to treat rheumatoid arthritis. They also can be used to
treat other diseases in which IL-6 is implicated. Examples of other
diseases that can be treated using the IL-6 antibodies or antigen
binding portions thereof include osteoarthritis, particularly the
pain associated with osteoarthritis, Castleman's disease, juvenile
idiopathic arthritis, adult-onset Still's disease, osteoporosis,
sepsis, multiple myeloma, renal cell carcinoma, and Crohn's
disease.
[0060] Some of the diseases that can be treated with the antibodies
or antigen-binding portions thereof are discussed below.
[0061] Rheumatoid arthritis (RA) is considered a chronic autoimmune
and inflammatory disease producing inflamed joints, which
eventually swell, become painful, and experience degradation of
cartilage, bone, and ligaments of the joint. A result of RA is
deformity, instability, and stiffness of the joint and scarring
within the joint. The joints deteriorate at a highly variable rate.
Many factors, including genetic predisposition, may influence the
pattern of the disease. People with rheumatoid arthritis may have a
mild course, occasional flare-ups with long periods of remission
without disease, or a steadily progressive disease, which may be
slow or rapid. Rheumatoid arthritis may start suddenly, with many
joints becoming inflamed at the same time. More often, it starts
subtly, gradually affecting different joints. Usually, the
inflammation is symmetric, with joints on both sides of the body
affected. Typically, the small joints in the fingers, toes, hands,
feet, wrists, elbows, and ankles become inflamed first, followed by
the knees and hips.
[0062] Rheumatoid arthritic pain is typically a somatic nociceptive
joint pain. Swollen wrists can pinch a nerve and result in numbness
or tingling due to carpal tunnel syndrome. Cysts may develop behind
affected knees, can rupture, causing pain and swelling in the lower
legs.
[0063] Osteoarthritis is characterized by loss of articular
cartilage and hypertrophy of bone. Onset of osteoarthritis is
usually gradual with pain being a common early symptom. As
osteoarthritis progresses, joint motion diminishes, and tenderness
and grating sensations can occur. Osteoarthritis commonly affects
the hands, feet, spine, and large weight-bearing joints, such as
the hips and knees. The diagnosis of osteoarthritis is typically
based on symptoms or by X-ray, which can show narrowing of the
joint space, increased density of subchondral bone, formation of
osteophytes at the periphery of joints, and formation of
pseudocysts in the subchondral marrow. Blood tests are performed to
exclude other conditions that can mimic osteoarthritis. In
addition, in diagnosing osteoarthritis, arthrocentesis can be
performed, whereby a sterile needle is used to remove joint fluid.
Joint fluid analysis is useful in excluding gout, infection, and
other causes of arthritis. Osteoarthritis is also known as
degenerative joint disease, degenerative arthritis, or
osteoarthritis.
[0064] Reiter's syndrome (reactive arthritis) is inflammation of
the joints and tendon attachments at the joints, often accompanied
by inflammation of the eye's conjunctiva and the mucous membranes,
such as those of the mouth and genitourinary tract, and by a
distinctive rash. Reiter's syndrome is also called reactive
arthritis because the joint inflammation appears to be a reaction
to an infection originating in the intestine or genital tract. This
syndrome is most common in men aged 20 to 40. There are two forms
of Reiter's syndrome: one occurs with sexually transmitted diseases
such as a chlamydial infection and the other usually follows an
intestinal infection such as shigellosis or salmonellosis. (Most
people who have these infections do not develop Reiter's syndrome.)
People who develop Reiter's syndrome after exposure to these
infections appear to have a genetic predisposition to this type of
reaction, related in part to the same gene found in people who have
ankylosing spondylitis.
[0065] Infectious arthritis is inflammation in a joint resulting
from bacterial, fungal, or viral infection of synovial or
periarticular tissues. Risk factors for infectious arthritis
include advanced age (i.e., greater than 60 years); alcoholism;
anemia; arthrocentesis or surgery; chronic medical illness (e.g.,
lung or liver disease); diabetes; hemophilia; immunodeficiency,
including HIV; immunosuppressive therapy, including
corticosteroids; IV drug use; malignancy; prosthetic joint implant;
renal failure; rheumatoid arthritis; sickle cell disease; skin
infections; and systemic lupus erythematosus. Patients with
rheumatoid arthritis are at particularly increased risk for
bacterial arthritis. Joint infections may be acute, with sudden
onset of joint pain and swelling (e.g., within a few hours to a few
days), or chronic, with insidious development of milder symptoms.
Acute bacterial arthritis is commonly accompanied by moderate to
severe joint pain, warmth, tenderness, and restricted motion.
Chronic bacterial arthritis is commonly accompanied by gradual
swelling, mild warmth, minimal or no redness of the joint area, and
aching pain, which may be mild.
[0066] Psoriatic arthritis is an inflammatory arthritis affecting
the joints that occurs in a minority of psoriasis patients and
increasingly in some acquired immune deficiency syndrome (AIDS)
patients. Psoriatic arthritis may be mild or may produce severe
joint deformities resembling joint changes observed in rheumatoid
arthritis. Joints that may be affected by psoriatic arthritis
include distal interphalangeal (DIP) joints of fingers and toes,
and commonly the asymmetric involvement of large and small joints
such as sacroiliacs and spine. Psoriasis of the skin or nails may
precede or follow joint involvement. The time course of psoriatic
arthritis is characterized by arthritic exacerbations and
remissions that may or may not coincide with skin exacerbations and
remissions, and progression to chronic arthritis may occur.
Diagnosis includes a diagnosis of psoriasis, a family history of
psoriasis, X-ray findings showing DIP joint involvement, asymmetric
large joint involvement, a negative blood test for rheumatoid
factor to rule out rheumatoid arthritis, and, in some patients, the
presence of HLA-B27 antigen, especially when the spine is
involved.
[0067] Polyarthritis is any type of arthritis which involves five
or more joints. Arthritis of two, three or four joints is called
oligoarthritis or pauciarthritis. Polyarthritis is most often
caused by an autoimmune disorder such as rheumatoid arthritis,
psoriatic arthritis, or lupus erythematosus, but can also be caused
by infections. Polyarthritis may be experienced at any age and is
not gender specific.
[0068] Juvenile arthritis is arthritis that begins before age 16.
There are several different types of juvenile arthritis. The most
common type is juvenile rheumatoid arthritis (JRA), also known as
juvenile idiopathic arthritis. JRA includes systemic onset JRA,
pauciarticular JRA, which involves fewer than five joints, and
polyarticular JRA, which affects five or more joints. Diagnosis of
JRA involves considering the symptoms, taking x-rays and doing
blood analyses. Specific tests that doctor may use to diagnose JRA
include complete blood counts, blood cultures for infections, bone
marrow examinations, examination of erythrocyte sedimentation rate,
rheumatoid factor antibody determination, antinuclear antibody
determination, and bone scans.
[0069] Juvenile rheumatoid arthritis is persistent or recurring
inflammation of the joints similar to rheumatoid arthritis but
beginning before age 16 and is characterized by inflammation of
joints or connective tissue. There are several types of juvenile
rheumatoid arthritis, which are determined by the symptoms that
develop during the first months of the disease and how many joints
are affected. These types include pauciarticular juvenile
rheumatoid arthritis, polyarthritis, and systemic disease (Still's
disease). In pauciarticular juvenile rheumatoid arthritis, four or
fewer joints, usually those of the leg are affected. In
polyarthritis, five or more (sometimes as many as 20 to 40) joints
are affected. In systemic disease (Still's disease), any number of
joints can be involved.
[0070] Juvenile reactive arthritis is persistent or recurring
inflammation of the joints similar to reactive arthritis but
beginning before age 16.
[0071] Juvenile psoriatic arthritis is psoriatic arthritis that
begins in a patient before the age of 16 years and is characterized
by the presence of chronic arthritis and psoriasis; or chronic
arthritis and at least two of the following: dactylitis, nail
abnormalities (e.g., pitting or onycholysis), and a family history
of psoriasis in at least one immediate relative. As in adults with
psoriatic arthritis, the arthritis may precede the skin condition.
The predominant pattern at onset of juvenile psoriatic arthritis is
an asymmetric oligoarthritis of small and large joints often with
dactylitis.
[0072] In one aspect, the IL-6 mediated disorder is characterized
by fibrosis. The term "fibrosis" as used herein refers to a
pathological condition characterized by excessive deposition and
metabolism of fibrotic material (e.g., extracellular matrix) in
response to tissue damage. In many cases, fibrosis represents a
normal repair process (i.e., wound healing) gone awry due to
chronic or excessive tissue insult leading to fibroblast or
stellate cell activation and proliferation and collagen
accumulation. Fibrosis conditions include fibroproliferative
disorders that are associated with vascular diseases, such as
cardiac disease, cerebral disease, and peripheral vascular disease,
as well as all the main tissues and organ systems such as the eye,
skin, kidney, lung, gut and liver (Wynn, Nature Reviews 4:583-594
(2004); Bataller, R and Brenner, D., J. Clin. Invest. 115:209-218
(2005)). Other sources are chemotherapeutic drugs,
radiation-induced fibrosis, and injuries and burns. While fibrosis
conditions cover a wide group of pathologies, it is believed that
for most of these conditions, the general mechanisms leading to
fibrotic tissue accumulation have many elements in common. Often
the condition is initiated in response to an influx of inflammatory
cells and perpetuated by the subsequent cytokine signaling pathways
between the infiltrating cells (e.g., macrophages, T cells) and
resident cells within the tissue (e.g., stellate, myofibroblast.
Kupffer cells). In addition, pericytes are a key fibrogenic cell
type involved in the development of scleroderma and PDGF receptor
tyrosine kinase inhibitors (RTKI) have been shown to slow the
proliferation of pericytes and suppress skin lesions in patients
with this progressive disease. In the kidney, leukocyte
infiltration plays a major role in mediating tubulointerstitial
inflammation and fibrosis in chronic kidney disease.
[0073] As used herein the term "fibrosis" is also used synonymously
with "fibroblast accumulation and collagen deposition". Fibroblasts
are connective tissue cells, which are dispersed in connective
tissue throughout the body. Fibroblasts secrete a nonrigid
extracellular matrix containing type I and/or type III collagen. In
response to an injury to a tissue, nearby fibroblasts or stellate
cells migrate into the wound, proliferate, and produce large
amounts of collagenous extracellular matrix. Collagen is a fibrous
protein rich in glycine and proline that is a major component of
the extracellular matrix and connective tissue, cartilage, and
bone. Collagen molecules are triple-stranded helical structures
called .alpha.-chains, which are wound around each other in a
ropelike helix. Collagen exists in several forms or types; of
these, type I, the most common, is found in skin, tendon, and bone;
and type III is found in skin, blood vessels, and internal organs.
Exemplary fibrosis conditions include, but are not limited to
[0074] (I) Lung diseases associated with fibrosis, e.g., idiopathic
pulmonary fibrosis, radiation induced fibrosis, chronic obstructive
pulmonary disease (COPD), scleroderma, bleomycin induced pulmonary
fibrosis, chronic asthma, silicosis, asbestos induced pulmonary
fibrosis, acute lung injury and acute respiratory distress
(including bacterial pneumonia induced, trauma induced, viral
pneumonia induced, ventilator induced, non-pulmonary sepsis
induced, and aspiration induced); [0075] (II) Chronic nephropathies
associated with injury/fibrosis (kidney fibrosis), e.g., lupus,
diabetes, scleroderma, glomerular nephritis, focal segmental
glomerular sclerosis, IgA nephropathy, hypertension, allograft,
Lupus, and Alport; [0076] (III) Gut fibrosis, e.g., scleroderma,
and radiation induced gut fibrosis; [0077] (IV) Liver fibrosis,
e.g., cirrhosis, alcohol induced liver fibrosis, nonalcoholic
steatohepatitis (NASH), biliary duct injury, primary biliary
cirrhosis, infection or viral induced liver fibrosis (e.g. chronic
HCV infection), and autoimmune hepatitis; [0078] (V) Head and neck
fibrosis, e.g., radiation induced; [0079] (VI) Corneal scarring,
e.g., LASIX.TM., corneal transplant, and trabeculectomy; [0080]
(VII) Hypertrophic scarring and keloids, e.g., burn induced and
surgical; and other fibrotic diseases, e.g., sarcoidosis,
scleroderma, spinal cord injury/fibrosis, myelofibrosis, vascular
restenosis, atherosclerosis, Wegener's granulomatosis, mixed
connective tissue disease, and Peyronie's disease.
[0081] The term "fibromyalgia" is also known as fibromyalgia
syndrome. The American College of Rheumatology (ACR) 1990
classification criteria for fibromyalgia include a history of
chronic, widespread pain for more than three months, and the
presence of pain at 11 (or more) out of 18 tender points upon
physical examination, wherein the tender points occur both above
and below the waist and on both sides of the body (see e.g., Wolfe
et al., Arthritis Rheum., 1990; 33:160-172). Fibromyalgia patients
generally display pain perception abnormalities in the form of both
allodynia (pain in response to a normally non-painful stimulus) and
hyperalgesia (an increased sensitivity to a painful stimulus). The
effects of fibromyalgia in a human patient may be assessed using
the ACR criteria, a Fibromyalgia Index Questionnaire (FIQ) total
score, indices of pain severity (e.g., VAS or Likert pain scales)
and interference, the number of tender points, or a pain threshold
assessment.
[0082] Although chronic, widespread pain is a hallmark symptom of
fibromyalgia, patients typically also exhibit other symptoms,
including one or more of the following: fatigue, sleep
disturbances, migraine or tension headaches, irritable bowel
syndrome, changes in urinary frequency, morning stiffness, numbness
and tingling, dysmenorrhea, multiple chemical sensitivities,
difficulty concentrating, and circulatory problems that affect the
small blood vessels of the skin (Raynaud's phenomenon). As with
many conditions that cause chronic pain, fibromyalgia patients may
also experience fibromyalgia-induced anxiety, depression, or both.
Some fibromyalgia patients find that cold, damp weather, emotional
stress, overexertion, and other factors exacerbate their
symptoms.
[0083] Pain associated with fibromyalgia refers to any pain
associated with fibromyalgia syndrome, including the chronic,
widespread pain that is a hallmark of fibromyalgia and pain
associated with other symptoms of fibromyalgia.
[0084] Ankylosing spondylitis is a rheumatic disease that causes
arthritis of the spine and sacroiliac joints. It varies from
intermittent episodes of back pain that occur throughout life to a
severe chronic disease that attacks the spine, peripheral joints
and other body organs. Typically, the first symptoms are frequent
pain and stiffness in the lower back and buttocks, which comes on
gradually over the course of a few weeks or months. The pain is
usually dull and diffuse, rather than localized. Ankylosing
spondylitis is typically diagnosed with a thorough physical exam
including x-rays, individual medical history, and a family history
of ankylosing spondylitis, as well as blood work including a test
for HLA-B27.
[0085] Psoriasis is a chronic inflammatory skin disorder that may
afflict people of all ages. Clinically, psoriasis most frequently
affects the skin of the elbows, knees, scalp, lumbosacral areas,
intergluteal cleft, or glans penis. Skin affected by psoriasis
typically contains one or more dry lesions comprised of a
well-demarcated, pink to salmon-colored plaque covered by loosely
adherent scales that are characteristically silver-white in color.
In about 30% of psoriasis patients, the nails are also affected by,
for example, pitting or onycholysis. All forms of psoriasis are
contemplated, including psoriasis annularis and psoriasis annulata,
which are also known as psoriasis circinata; psoriasis arthropica;
psoriasis diffusa or diffused psoriasis; exfoliative psoriasis;
flexural psoriasis; psoriasis geographica; psoriasis gyrate;
psoriasis nummularis; palmar psoriasis; psoriasis punctata; and a
rare variant form known as generalized pustular psoriasis of
Zambusch or simply just pustular psoriasis. Morphologically,
established lesions of psoriasis have well known histologic
characteristics such as epidermal thickening and parakeratotic
scale. Pathologically, psoriasis is currently believed to be a
T-cell mediated autoimmune disorder. Onset of psoriasis is usually
gradual and the typical time course is characterized by chronic
remissions and recurrences and, occasionally, acute exacerbations.
Diagnosis of psoriasis is made by evaluating a patient's clinical
signs and symptoms and family history of psoriasis. Diagnosing
psoriasis by just visually inspecting the patient's skin lesions is
rarely difficult and usually this is all that is required for a
complete diagnosis. Occasionally, however, a skin biopsy is
subjected to a histologic analysis to look for signs of
psoriasis.
[0086] Systemic lupus erythematosus ("SLE"), also called
disseminated lupus erythematosus, is a chronic inflammatory
connective tissue disorder of unknown cause that can involve
joints, kidneys, serous surfaces, and vessel walls and that occurs
predominantly in young women but also in children. Ninety percent
of SLE cases occur in women. SLE may begin abruptly with fever,
simulating acute infection, or may develop insidiously over months
or years with episodes of fever and malaise. Vascular headaches,
epilepsy, or psychoses may be initial findings. Manifestations
referable to any organ system may appear. Articular symptoms,
ranging from intermittent arthralgias to acute polyarthritis, occur
in approximately 90% of patients and may exist for years before
other manifestations appear. In long-standing disease, capsular
insertional erosions at the metacarpophalangeal joints with marked
secondary joint deformity may occur without x-ray evidence of
obvious marginal erosions (Jaccoud's arthritis). However, most
lupus polyarthritis is nondestructive and nondeforming.
[0087] Systemic lupus erythematosus is rare under the age of 5, and
most children with SLE develop the disease during adolescence.
Signs and symptoms of juvenile SLE are similar to those in adults.
However, children have a particularly high level of transition from
the discoid to the systemic disease.
[0088] Gout (also known as gouty arthritis) is recurrent acute or
chronic arthritis of peripheral joints results from a build-up in
the body of too much uric acid, which forms crystals that deposit
in joints and cause inflammation. During an acute attack of gout
there is swelling, inflammation, and extreme pain in a joint,
frequently that of the big toe. Chronic gout can set in after
several years of attacks, permanently damaging and deforming joints
and destroying cells of the kidney. Most cases occur in men and the
first attack rarely occurs before the age of 30.
[0089] Undifferentiated Spondyloarthropathy (USpA) is a term used
to describe symptoms and signs of spondylitis in someone who does
not meet the criteria for a definitive diagnosis of ankylosing
spondylitis or related disease. A number of well-established
syndromes are included within the spondyloarthropathy family
including ankylosing spondylitis, psoriatic arthritis, the
arthritis of inflammatory bowel disease, Reiter's syndrome, chronic
reactive arthritis and enthesitis related juvenile arthritis. Over
time, some people with USpA will develop a well-defined form of
spondylitis such as ankylosing spondylitis.
[0090] Juvenile-onset spondyloarthritis (JSpA), also known as
Juvenile Spondyloarthropathy, is the medical term for a group of
childhood rheumatic diseases, which cause arthritis before the age
of 16 and may span through adult life. The juvenile
spondyloarthropathies include undifferentiated spondyloarthropathy,
juvenile ankylosing spondylitis, juvenile psoriatic arthritis, the
arthritis associated with inflammatory bowel disease
(enteropathogenic arthritis), reactive arthritis, (Reiter's
syndrome is one type of reactive arthritis), and the SEA syndrome
(seronegativity, enthesopathy, arthropathy). JSpA typically causes
pain and inflammation in the joints in the lower part of the body,
for example, the pelvis, hips, knees and ankles. Other areas of the
body can also be affected, such as the spine, eyes, skin and
bowels. Fatigue and lethargy can also occur.
[0091] Crohn's disease is a nonspecific chronic transmural
inflammatory disease that most commonly affects the distal ileum
and colon but may occur in any part of the GI tract. Chronic
diarrhea with abdominal pain, fever, anorexia, weight loss, and a
right lower quadrant mass or fullness are the most common symptoms
of Crohn's disease. Less common symptoms include poor appetite,
fever, night sweats, rectal pain, and rectal bleeding. Crohn's
disease may affect the colon, the rectum, and the small intestine
and, in rare instances, also the stomach, mouth, and esophagus. The
most common patterns of Crohn's disease pathology are (1)
inflammation characterized by right lower quadrant abdominal pain
and tenderness; (2) recurrent partial obstruction caused by
intestinal stenosis and leading to severe colic, abdominal
distention, constipation, and vomiting; (3) diffuse jejunoileitis,
with inflammation and obstruction resulting in malnutrition and
chronic debility; and (4) abdominal fistulas and abscesses, usually
late developments, often causing fever, painful abdominal masses,
and generalized wasting. Crohn's disease should be suspected in a
patient with the inflammatory or obstructive symptoms described
above and in a patient without prominent GI symptoms but with
perianal fistulas or abscesses or with otherwise unexplained
arthritis, erythema nodosum, fever, anemia, or stunted growth (in a
child). Laboratory findings are nonspecific and may include anemia,
leukocytosis, hypoalbuminemia, and increased levels of acute-phase
reactants reflected in elevated ESR, C-reactive protein, or
orosomucoids. Elevated alkaline phosphatase and .gamma.-glutamyl
transpeptidase accompanying colonic disease often reflect primary
sclerosing cholangitis. Diagnosis is usually made by x-ray.
[0092] In advanced cases, the string sign may be seen with marked
ileal strictures and separation of bowel loops. In earlier cases,
x-ray diagnosis may sometimes be difficult, but air double-contrast
barium enema and enteroclysis may show superficial aphthous and
linear ulcers. Colonoscopy and biopsy may help confirm the
diagnosis of Crohn's colitis and allow direct visualization and
biopsy of the terminal ileum. Upper GI endoscopy may identify
gastroduodenal involvement in Crohn's disease patients with upper
GI symptoms.
[0093] Ulcerative colitis is a chronic, inflammatory, and
ulcerative disease arising in the colonic mucosa, characterized
most often by bloody diarrhea. Bloody diarrhea of varied intensity
and duration is interspersed with asymptomatic intervals are the
most common symptoms of ulcerative colitis. Usually an attack
begins insidiously, with increased urgency to defecate, mild lower
abdominal cramps, and blood and mucus in the stools. However, an
attack may be acute and fulminant, with sudden violent diarrhea,
high fever, signs of peritonitis, and profound toxemia. Some cases
develop following a documented infection (e.g., amebiasis,
bacillary dysentery). When ulceration is confined to the
rectosigmoid, the stool may be normal or hard and dry, but rectal
discharges of mucus loaded with red blood cells and white blood
cells accompany or occur between bowel movements. Systemic symptoms
are mild or absent. If ulceration extends proximally, stools become
looser and the patient may have >10 bowel movements/day, often
with severe cramps and distressing rectal tenesmus, without respite
at night. The stools may be watery, may contain mucus, and
frequently consist almost entirely of blood and pus. Malaise,
fever, anemia, anorexia, weight loss, leukocytosis, and
hypoalbuminemia may be present with extensive active ulcerative
colitis. The patient's history and stool examination permit a
presumptive diagnosis of ulcerative colitis that should always be
confirmed by sigmoidoscopy, which provides a direct, immediate
indication of disease activity. In early cases, the mucous membrane
is finely granular and friable, with loss of the normal vascular
pattern and often with scattered hemorrhagic areas; minimal trauma
(friability) causes bleeding in multiple pinpoint spots. The mucosa
soon breaks down into a red, spongy surface dotted with many tiny
blood- and pus-oozing ulcers. As the mucosa becomes progressively
involved, the inflammation and hemorrhage extend into the bowel
muscle. Large mucosal ulcers with copious purulent exudate
characterize severe disease. Islands of relatively normal or
hyperplastic inflammatory mucosa (pseudopolyps) project above areas
of ulcerated mucosa. Biopsies may be nonspecific and sometimes
cannot exclude acute infectious (self-limited) colitis; however,
features that suggest chronicity (e.g., distorted crypt
architecture, crypt atrophy, a chronic inflammatory infiltrate)
support the diagnosis of ulcerative colitis. Even during
asymptomatic intervals, the sigmoidoscopic appearance is rarely
normal; some degree of friability or granularity almost always
persists. There is loss of the normal vascular pattern, and biopsy
shows evidence of chronic inflammation. Plain x-rays of the abdomen
sometimes help to judge the severity and proximal extent of the
colitis by showing loss of haustration, mucosal edema, and absence
of formed stool in the diseased bowel. Later in the course of
disease, however, the entire colon should be evaluated to determine
the extent of involvement. Total colonoscopy is the most sensitive
and widely used method, although barium enema can be informative.
Colonoscopy with biopsy is mandatory to evaluate the nature of a
stricture. Biopsy may also help distinguish ulcerative colitis from
Crohn's disease if the inflammation is highly focal or if a
granuloma is seen.
[0094] Irritable bowel syndrome (IBS) is a motility disorder
involving the entire GI tract, causing recurring upper and lower GI
symptoms, including variable degrees of abdominal pain,
constipation and/or diarrhea, and abdominal bloating. The cause of
irritable bowel syndrome (IBS) is unknown. No anatomic cause can be
found. Emotional factors, diet, drugs, or hormones may precipitate
or aggravate heightened GI motility. Features of IBS are pain
relieved by defecation, an alternating pattern of bowel habits,
abdominal distention, mucus in the stool, and sensation of
incomplete evacuation after defecation. In general, the character
and location of pain, precipitating factors, and defecatory pattern
are distinct for each patient. Patients with IBS may also have
extraintestinal symptoms (e.g., fibromyalgia, headaches,
dyspareunia, temporomandibular joint syndrome). Two major clinical
types of IBS have been described. In constipation-predominant IBS,
constipation is common, but bowel habits vary. Most patients have
pain over at least one area of the colon, associated with periodic
constipation alternating with a more normal stool frequency. Stool
often contains clear or white mucus. The pain is either colicky,
coming in bouts, or a continuous dull ache; it may be relieved by a
bowel movement. Eating commonly triggers symptoms. Bloating,
flatulence, nausea, dyspepsia, and pyrosis can also occur.
Diarrhea-predominant IBS is characterized by precipitous diarrhea
that occurs immediately on rising or during or immediately after
eating. Nocturnal diarrhea is unusual. Pain, bloating, and rectal
urgency are common, and incontinence may occur. Diagnosis of IBS is
based on characteristic bowel patterns, time and character of pain,
and exclusion of other disease processes through physical
examination and routine diagnostic tests. Due to a lack of a
readily identifiable structural or biochemical abnormality in this
syndrome, the medical community has developed a consensus
definition and criteria, known as the Rome criteria, to aid in
diagnosis of IBS. According to the Rome criteria, IBS is indicated
by abdominal pain or discomfort which is (1) relieved by defection
and/or (2) associated with a change in frequency or consistency of
stools, plus two or more of the following: altered stool frequency,
altered stool formation, altered stool passage, passage of mucus,
and bloating or feeling of abdominal distention. Palpation of the
abdomen may reveal tenderness, particularly in the left lower
quadrant, at times associated with a palpable, tender sigmoid. A
routine digital rectal examination should be performed on all
patients, and a pelvic examination on women.
[0095] Irritable bowel disease (IBD), also known as inflammatory
bowel disease, is characterized by chronic inflammation at various
sites in the GI tract. IBD comprises two known clinical subtypes,
Crohn's Disease (CD) and ulcerative colitis (UC). Certain
differences in disease patterns justify a distinction between
Crohn's disease and ulcerative colitis.
[0096] Pain associated with IBD and IBS may present as either
chronic or acute pain. For example a feature of IBS is acute pain
relieved by defecation while chronic abdominal pain is typical of
Crohn's disease. While pain associated with IBD and IBS may occur
extraintestinal or extravisceral generally these ailments produce
visceral pain. Visceral pain is pain associated with the viscera,
which encompasses the organs of the abdominal cavity. These organs
include the sex organs, spleen intestines, colon, rectum and other
organs of the digestive system. Visceral pain has five important
clinical characteristics: (1) it is not evoked from all viscera
(organs such as liver, kidney, most solid viscera, and lung
parenchyma are not sensitive to pain); (2) it is not always linked
to visceral injury; (3) it is diffuse and poorly localized; (4) it
is referred to other locations; and (5) it is accompanied with
motor and autonomic reflexes, such as the nausea, vomiting, and
lower-back muscle tension that occurs in renal colic.
[0097] Pain is an important physiological protective mechanism
designed to warn of danger from potentially injurious stimuli from
the external environment. The system operates through a specific
set of primary sensory neurones and is activated by noxious stimuli
via peripheral transducing mechanisms (see Millan, 1999, Prog.
Neurobiol., 57, 1-164 for a review). These sensory fibers are known
as nociceptors and are characteristically small diameter axons with
slow conduction velocities. Nociceptors encode the intensity,
duration and quality of noxious stimulus and by virtue of their
topographically organized projection to the spinal cord, the
location of the stimulus. The nociceptors are found on nociceptive
nerve fibers of which there are two main types, A-delta fibers
(myelinated) and C fibers (non-myelinated). The activity generated
by nociceptor input is transferred, after complex processing in the
dorsal horn, either directly, or via brain stem relay nuclei, to
the ventrobasal thalamus and then on to the cortex, where the
sensation of pain is generated.
[0098] Pain may generally be classified as acute or chronic. Acute
pain begins suddenly and is short-lived (usually twelve weeks or
less). It is usually associated with a specific cause such as a
specific injury and is often sharp and severe. It is the kind of
pain that can occur after specific injuries resulting from surgery,
dental work, a strain or a sprain. Acute pain does not generally
result in any persistent psychological response. In contrast,
chronic pain is long-term pain, typically persisting for more than
three months and leading to significant psychological and emotional
problems. Common examples of chronic pain are neuropathic pain
(e.g. painful diabetic neuropathy, postherpetic neuralgia), carpal
tunnel syndrome, back pain, headache, cancer pain, arthritic pain
and chronic post-surgical pain.
[0099] When a substantial injury occurs to body tissue, via disease
or trauma, the characteristics of nociceptor activation are altered
and there is sensitisation in the periphery, locally around the
injury and centrally where the nociceptors terminate. These effects
lead to a heightened sensation of pain. In acute pain these
mechanisms can be useful, in promoting protective behaviors which
may better enable repair processes to take place. The normal
expectation would be that sensitivity returns to normal once the
injury has healed. However, in many chronic pain states, the
hypersensitivity far outlasts the healing process and is often due
to nervous system injury. This injury often leads to abnormalities
in sensory nerve fibers associated with maladaptation and aberrant
activity (Woolf & Salter, 2000, Science, 288, 1765-1768).
[0100] Clinically, pain is present when discomfort and abnormal
sensitivity feature among the patient's symptoms. Patients tend to
be quite heterogeneous and may present with various pain symptoms.
Such symptoms include: 1) spontaneous pain which may be dull,
burning, or stabbing; 2) exaggerated pain responses to noxious
stimuli (hyperalgesia); and 3) pain produced by normally innocuous
stimuli (allodynia--Meyer et al., 1994, Textbook of Pain, 13-44).
Although patients suffering from various forms of acute and chronic
pain may have similar symptoms, the underlying mechanisms may be
different and may, therefore, require different treatment
strategies. Pain can also therefore be divided into a number of
different subtypes according to differing pathophysiology,
including nociceptive, inflammatory and neuropathic pain.
[0101] Nociceptive pain is induced by tissue injury or by intense
stimuli with the potential to cause injury. Pain afferents are
activated by transduction of stimuli by nociceptors at the site of
injury and activate neurons in the spinal cord at the level of
their termination. This is then relayed up the spinal tracts to the
brain where pain is perceived (Meyer et al., 1994, Textbook of
Pain, 13-44). The activation of nociceptors activates two types of
afferent nerve fibers. Myelinated A-delta fibers transmit rapidly
and are responsible for sharp and stabbing pain sensations, while
unmyelinated C fibers transmit at a slower rate and convey a dull
or aching pain. Moderate to severe acute nociceptive pain is a
prominent feature of pain from central nervous system trauma,
strains/sprains, burns, myocardial infarction and acute
pancreatitis, post-operative pain (pain following any type of
surgical procedure), posttraumatic pain, renal colic, cancer pain
and back pain. Cancer pain may be chronic pain such as tumor
related pain (e.g. bone pain, headache, facial pain or visceral
pain) or pain associated with cancer therapy (e.g. postchemotherapy
syndrome, chronic postsurgical pain syndrome or post radiation
syndrome). Cancer pain may also occur in response to chemotherapy,
immunotherapy, hormonal therapy or radiotherapy. Back pain may be
due to herniated or ruptured intravertebral discs or abnormalities
of the lumber facet joints, sacroiliac joints, paraspinal muscles
or the posterior longitudinal ligament. Back pain may resolve
naturally but in some patients, where it lasts over 12 weeks, it
becomes a chronic condition which can be particularly
debilitating.
[0102] Neuropathic pain is currently defined as pain initiated or
caused by a primary lesion or dysfunction in the nervous system.
Nerve damage can be caused by trauma and disease and thus the term
`neuropathic pain` encompasses many disorders with diverse
aetiologies. These include, but are not limited to, peripheral
neuropathy, diabetic neuropathy, post herpetic neuralgia,
trigeminal neuralgia, back pain, cancer neuropathy, HIV neuropathy,
phantom limb pain, carpal tunnel syndrome, central post-stroke pain
and pain associated with chronic alcoholism, hypothyroidism,
uremia, multiple sclerosis, spinal cord injury, Parkinson's
disease, epilepsy and vitamin deficiency. Neuropathic pain is
pathological as it has no protective role. It is often present well
after the original cause has dissipated, commonly lasting for
years, significantly decreasing a patient's quality of life (Woolf
and Mannion, 1999, Lancet, 353, 1959-1964). The symptoms of
neuropathic pain are difficult to treat, as they are often
heterogeneous even between patients with the same disease (Woolf
& Decosterd, 1999, Pain Supp., 6, S141-S147; Woolf and Mannion,
1999, Lancet, 353, 1959-1964). They include spontaneous pain, which
can be continuous, and paroxysmal or abnormal evoked pain, such as
hyperalgesia (increased sensitivity to a noxious stimulus) and
allodynia (sensitivity to a normally innocuous stimulus).
[0103] The inflammatory process is a complex series of biochemical
and cellular events, activated in response to tissue injury or the
presence of foreign substances, which results in swelling and pain
(Levine and Taiwo, 1994, Textbook of Pain, 45-56). Arthritic pain
is the most common inflammatory pain. Rheumatoid disease is one of
the commonest chronic inflammatory conditions in developed
countries and rheumatoid arthritis is a common cause of disability.
The exact aetiology of rheumatoid arthritis is unknown, but current
hypotheses suggest that both genetic and microbiological factors
may be important (Grennan & Jayson, 1994, Textbook of Pain,
397-407). It has been estimated that almost 16 million Americans
have symptomatic osteoarthritis (OA) or degenerative joint disease,
most of whom are over 60 years of age, and this is expected to
increase to 40 million as the age of the population increases,
making this a public health problem of enormous magnitude (Houge
& Mersfelder, 2002, Ann Pharmacother., 36, 679-686; McCarthy et
al., 1994, Textbook of Pain, 387-395). Most patients with
osteoarthritis seek medical attention because of the associated
pain. Arthritis has a significant impact on psychosocial and
physical function and is known to be the leading cause of
disability in later life. Ankylosing spondylitis is also a
rheumatic disease that causes arthritis of the spine and sacroiliac
joints. It varies from intermittent episodes of back pain that
occur throughout life to a severe chronic disease that attacks the
spine, peripheral joints and other body organs.
[0104] Another type of inflammatory pain is visceral pain which
includes pain associated with inflammatory bowel disease (IBD).
Visceral pain is pain associated with the viscera, which encompass
the organs of the abdominal cavity. These organs include the sex
organs, spleen and part of the digestive system. Pain associated
with the viscera can be divided into digestive visceral pain and
non-digestive visceral pain. Commonly encountered gastrointestinal
(GI) disorders that cause pain include functional bowel disorder
(FBD) and inflammatory bowel disease (IBD). These GI disorders
include a wide range of disease states that are currently only
moderately controlled, including, in respect of FBD,
gastro-esophageal reflux, dyspepsia, irritable bowel syndrome (IBS)
and functional abdominal pain syndrome (FAPS), and, in respect of
IBD, Crohn's disease, ileitis and ulcerative colitis, all of which
regularly produce visceral pain. Other types of visceral pain
include the pain associated with dysmenorrhea, cystitis and
pancreatitis and pelvic pain.
[0105] It should be noted that some types of pain have multiple
aetiologies and thus can be classified in more than one area, e.g.
back pain and cancer pain have both nociceptive and neuropathic
components.
[0106] Other types of pain include: [0107] pain resulting from
musculo-skeletal disorders, including myalgia, fibromyalgia,
spondylitis, sero-negative (non-rheumatoid) arthropathies,
non-articular rheumatism, dystrophinopathy, glycogenolysis,
polymyositis and pyomyositis; [0108] heart and vascular pain,
including pain caused by angina, myocardical infarction, mitral
stenosis, pericarditis, Raynaud's phenomenon, scleredoma and
skeletal muscle ischemia; [0109] head pain, such as migraine
(including migraine with aura and migraine without aura), cluster
headache, tension-type headache mixed headache and headache
associated with vascular disorders; and [0110] orofacial pain,
including dental pain, otic pain, burning mouth syndrome and
temporomandibular myofascial pain.
[0111] IL-6 antibodies or antigen-binding portions thereof can be
used in combination with one or more other therapeutic agents. For
example, an antibody or antigen-binding portions thereof can be
used with a COX-2 inhibitor, such as celecoxib, for the treatment
of diseases such as rheumatoid arthritis, osteoarthritis and pain.
IL-6 antibodies or antigen-binding portions thereof and the other
therapeutic agents can be administered to the patient in the same
dosage form or in different dosage forms. Moreover, they can be
administered at the same time or at different times. Below are some
examples of therapeutic agents that can be used in combination with
anti-IL-6 antibodies or antigen-binding portions thereof.
Rheumatoid Arthritis
[0112] IL-6 antibodies and antigen binding portions thereof may
also be used in co-therapies. Suitable antiinflammatory co-therapy
compounds include: cyclosporine, zoledronic acid, efalizumab,
alefacept, etodolac, lornoxicam, OM-89, valdecoxib, tocilizumab,
abatacept, meloxicam, etanercept, nambumetone, rimexolone,
153Sm-EDTMP, prosorba, imidazole salicylate, oprelvekin, hylauronic
acid, naproxen, piroxicam, diacerein, lumericoxib, tacrolimus,
aceclofenac, actarit, tenoxicam, rosiglitazone, deflazacort,
adalimumab, leflunomide, risedronate sodium, misoprostol and
diclofenac, SK-1306X, infliximab, anakinra, celecoxib, diclofenac,
etoricoxib and felbinac, reumacon, golimumab, denosumab,
ofatumumab, 10rT1 antibody, pelubiprofen, licofelone, temsirolimus,
eculizumab, iguratimod, and prednisone. Other suitable
antiinflammatories include CP-481715, ABN-912, MLN-3897,
HuMax-IL-15, RA-1, paclitaxel, Org-37663, Org 39141, AED-9056,
AMG-108, fontolizumab, pegsunercept, pralnacasan, apilimod,
GW-274150, AT-001, 681323 (GSK) K-832, R-1503, ocrelizumab, DE-096,
Cpn10, THC+CBD (GW Pharma), 856553 (GSK), ReN-1869, immunoglobulin,
mm-093, amelubant, SCIO-469, ABT-874, LenkoVAX, LY-2127399,
TRU-015, KC-706, amoxapinet and dipyridamole, TAK-715, PG 760564,
VX-702, prednisolone and dipyridamole, PMX-53, belimumab,
prinaberel, CF-101, tgAAV-TNFR:Fc, R-788, prednisolone and SSRI,
CP-690550 and PMI-001.
Osteoarthritis
[0113] IL-6 antibodies and antigen binding portions thereof may
further be co-administered for the treatment of osteoarthritis with
one or more agents useful for treating one or more indicia of
osteoarthritis. Examples of agents useful for treating one or more
indicia of osteoarthritis to be used in combination with anti-IL-6
antibodies or antigen-binding portions thereof include matrix
metalloproteinase (MMP) inhibitors, aggrecanase inhibitors,
inducible nitric oxide (iNOS) inhibitors, inhibitors of
insulin-like growth factor (IGF) expression or activity, inhibitors
of fibroblast growth factor (FGF) expression or activity,
inhibitors of CD 44 expression or activity, inhibitors of
interleukin (IL) expression or activity, inhibitors of tumor
necrosis factor alpha (TNF-alpha) expression or activity,
inhibitors of tumor necrosis factor-inducible protein 6 (TSG-6)
expression or activity, inhibitors of Bikunin expression or
activity, inhibitors of beta-secretase (BACE), inhibitors of
PACE-4, inhibitors of nuclear receptor rev-ErbA alpha (NR1D1)
expression or activity, inhibitors of endothelial differentiation
sphingolipid G-protein-coupled receptor 1 (EDG-1) expression or
activity, inhibitors of proteinase-activated receptor (PAR)
expression or activity, inhibitors of cartilage-derived
retinoic-acid-sensitive protein (CD-RAP) expression or activity,
inhibitors of protein kinase C zeta (PKCz), inhibitors of resistin
expression or activity, inhibitors of a disintegrin and
metalloproteinase 8 (ADAMS), inhibitors of complement component 1 s
subcomponent (C1s) expression or activity, inhibitors of formyl
peptide receptor-like 1 (FPRL1) expression or activity.
[0114] Additional examples of agents useful in combination with
IL-6 antibodies and antigen binding portions thereof include
inhibitors of MMP-2, -3, -9, or -13; inhibitors of aggrecanase-1 or
-2; inhibitors of IGF-1 or -2 expression or activity; inhibitors of
FGF-2, -18, or -9 expression or activity; and inhibitors of IL-1,
-4 or -6 expression or activity.
[0115] Further examples of agents useful in combination with IL-6
antibodies and antigen binding portions thereof include IGF-1 or -2
antibodies; FGF receptor-2 or -3 antagonists, CD 44 antibodies,
IL-1, -4 or -6 antibodies, TNF-alpha antibodies; TSG-6 antibodies;
bikunin antibodies; NR1D1 antagonists; EDG-1 antagonists; PAR
antagonists, CD-RAP antibodies, resistin antibodies, C1s
antibodies, and FPRL1 antibodies.
Pain
[0116] IL-6 antibodies or antigen binding portions thereof can be
administered in combination with one or more additional
pharmacologically active compounds for the treatment of pain. The
compounds may be administered at the same time in a single dosage
form or separately in dosage forms that can be the same or
different. Alternatively, the compounds can be administered
sequentially. The pharmaceutically acceptable salts of the
pharmacologically active compounds may also be used in the
combinations.
[0117] Examples of compounds that can be administered with IL-6
antibodies or antigen binding portions thereof include:
[0118] Cyclooxygenase-2 (COX-2) selective inhibitors such as
celecoxib, rofecoxib, parecoxib, valdecoxib, deracoxib, etoricoxib,
and lumiracoxib; opioid analgesics such as morphine, hydromorphone,
oxymorphone, fentanyl, codeine, dihydrocodeine, oxycodone,
hydrocodone, buprenorphine, tramadol, and nalbuphine; nonsteroidal
antiinflammatory drugs (NSAIDs) such as aspirin, diclofenac,
diflunisal, ibuprofen, fenoprofen, naproxen, nepafenac, and
acetaminophen; Phosphodiesterase V inhibitors (PDEV) such as
sildenafil; alpha-2-delta ligands such as gabapentin and
pregabalin; and local anaesthetics such as benzocaine, lidocaine,
ropivacaine, menthol, camphor and methyl salicylate.
[0119] Examples of other types of compounds and classes of
compounds that can be used in combination with IL-6 antibodies and
antigen binding portions thereof include: barbiturate sedatives;
benzodiazepines; Histamine H.sub.1 antagonists having a sedative
action; sedatives; skeletal muscle relaxants; N-methyl-D-aspartic
acid (NMDA) receptor antagonists; alpha-adrenergics; tricyclic
antidepressants; anticonvulsants such as carbamazepine; tachykinin
(NK) antagonists, particularly NK-3, NK-2 or NK-1 antagonists;
muscarinic antagonists; neuroleptics; vanilloid receptor agonists
or antagonists; beta-adrenergics; corticosteroids; Serotonin (5-HT)
receptor agonists or antagonists such as a 5-HT.sub.1B/1D,
5-HT.sub.2A, and 5-HT.sub.3 receptor antagonists; cholinergic
(nicotinic) analgesics; cannabinoids; metabotropic glutamate
subtype 1 receptor (mGluR1) antagonists; serotonin reuptake
inhibitors such as sertraline; noradrenaline (norepinephrine)
reuptake inhibitors such as reboxetine, in particular
(S,S)-reboxetine; dual serotonin-noradrenaline reuptake inhibitors
such as duloxetine; inducible nitric oxide synthase (iNOS)
inhibitors such as S-[2-[(1-iminoethyl)amino]ethyl]L-homocysteine,
S-[2-[(1-iminoethyl)-amino]ethyl]-4,4-dioxo-L-cysteine,
S-[2-[(1-iminoethyl)amino]ethyl]-2-methyl-L-cysteine,
(2S,5Z)-2-amino-2-methyl-7-[(1-iminoethyl)amino]-5-heptenoic acid,
2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)-butyl]thio]-5-chloro-3-pyri-
dinecarbonitrile;
2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-4-chlorobenzonit-
rile,
(2S,4R)-2-amino-4-[[2-chloro-5-(trifluoromethyl)phenyl]thio]-5-thiaz-
olebutanol,
2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-6-(trifluorometh-
yl)-3 pyridinecarbonitrile,
2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-5-chlorobenzonit-
rile,
N-[4-[2-(3-chlorobenzylamino)ethyl]phenyl]thiophene-2-carboxamidine,
and guanidinoethyldisulfide; acetylcholinesterase inhibitors;
prostaglandin E.sub.2 subtype 4 (EP4) antagonists such as
N-[({2-[4-(2-ethyl-4,6-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl)phenyl]ethy-
l}amino)-carbonyl]-4-methylbenzenesulfonamide or
4-[(1S)-1-({[5-chloro-2-(3-fluorophenoxyl)pyridin-3-yl]carbonyl}amino)eth-
yl]benzoic acid; leukotriene B4 antagonists such as
1-(3-biphenyl-4-ylmethyl-4-hydroxy-chroman-7-yl)-cyclopentanecarboxylic
acid; 5-lipoxygenase inhibitors; and sodium channel blockers.
Fibromyalgia Syndrome
[0120] Products: analgesics such as acetaminophen, naproxen sodium,
ibuprofen, tramadol, trazodone; cyclobenzaprine; aspirin,
celecoxib, valdecoxib, indomethacin, and other NSAIDs;
antidepressants such as tricyclic antidepressants and selective
serotonin reuptake inhibitors, for example antidepressants such as
amitriptyline, imipramine, nortriptyline, doxepin, fluoxetine,
sertraline, and paroxetine; muscle relaxants such as
cyclobenzaprine; sleeping aids such as zolpidem.
[0121] Classes: norepinephrine-serotonin reuptake inhibitors (NSRIs
and SNRIs); norepinephrine reuptake inhibitor (NRIs); selective
serotonin reuptake inhibitors (SSRIs); tricyclic antidepressants;
selective cyclooxygenase-2 (COX-2) inhibitors; nonsteroidal
anti-inflammatory drugs (NSAIDs); analgesics.
Ankylosing Spondylitis
[0122] Products: analgesics such as acetaminophen, naproxen sodium,
ibuprofen, tramadol, aspirin, celecoxib, valdecoxib, indomethacin,
and other NSAIDs; disease-modifying antirheumatic drugs (DMARDs)
such as sulfasalazine or methotrexate; corticosteroids; and tumor
necrosis factor (TNF) blockers such as etanercept and
infliximab.
[0123] Classes: analgesics; NSAIDs; COX-2 inhibitors; DMARDs;
corticosteroids; TNF blockers.
Psoriasis
[0124] Products: phototherapy, including psoralen ultraviolet A
(psoralen UVA or PUVA) therapy, narrow-band ultraviolet B (UVB)
therapy, and combination light therapy; topical corticosteroids;
vitamin D analogs such as calcipotriene; anthralin; topical
retinoids (i.e., vitamin A derivatives) such as acitretin and
tazarotene; clobetasol propionate; methotrexate; azathioprine;
cyclosporine; hydroxyurea; and immune-modulating drugs such as
alefacept, efalizumab, and etanercept.
[0125] Classes: phototherapy; corticosteroids; vitamin D analogs;
vitamin A derivatives.
Gout
[0126] Products: NSAIDs such as acetaminophen, naproxen sodium,
ibuprofen, tramadol, aspirin, celecoxib, valdecoxib, and
indomethacin; and corticosteroids such as prednisone.
[0127] Classes: analgesics; NSAIDs; COX-2 inhibitors; and
corticosteroids.
Crohn's Disease
[0128] Products: analgesics such as acetaminophen, naproxen sodium,
ibuprofen, tramadol, aspirin, celecoxib, valdecoxib, indomethacin,
and other NSAIDs; anti-inflammatory drugs; sulfasalazine,
mesalamine, balsalazide, and olsalazine; and corticosteroids such
as prednisone and budesonide; immunosuppressant drugs such as
azathioprine, mercaptopurine, TNF blockers such as infliximab and
adalimumab, methotrexate, and cyclosporine; antibiotics such as
metronidazole and ciprofloxacin; anti-diarrheals such as
loperamide; and laxatives.
[0129] Classes: analgesics; NSAIDs; COX-2 inhibitors;
anti-inflammatory drugs; TNF blockers; antibiotics;
anti-diarrheals; and laxatives.
Ulcerative Colitis
[0130] Classes: analgesics such as acetaminophen, naproxen sodium,
ibuprofen, tramadol, aspirin, celecoxib, valdecoxib, indomethacin,
and other NSAIDs; anti-inflammatory drugs; sulfasalazine,
mesalamine, balsalazide, and olsalazine; corticosteroids;
immunosuppressant drugs such as azathioprine, mercaptopurine, TNF
blockers such as infliximab and adalimumab, methotrexate, and
cyclosporine; anti-diarrheals such as loperamide; and
laxatives.
[0131] Classes: NSAIDs; COX-2 inhibitors; anti-inflammatory drugs;
TNF blockers; corticosteroids; immunosuppressants; Janus kinase-3
(Jak-3) inhibitors; TNF blockers; anti-diarrheals; and
laxatives.
Irritable Bowel Syndrome
[0132] Products: anti-diarrheals such as loperamide; laxatives;
anticholinergic drugs; antidepressants such as tricyclic
antidepressants and selective serotonin reuptake inhibitors, for
example antidepressants such as amitriptyline, imipramine,
nortriptyline, doxepin, fluoxetine, sertraline, and paroxetine;
alosetron; and tegaserod.
[0133] Classes: anti-diarrheals; laxatives; anticholinergic drugs;
norepinephrine-serotonin reuptake inhibitors (NSRIs and SNRIs);
norepinephrine reuptake inhibitor (NRIs); selective serotonin
reuptake inhibitors (SSRIs); tricyclic antidepressants.
Pharmaceutical Compositions and Administration
[0134] Also provided are pharmaceutical compositions for the
treatment of abnormal cell infiltration in a mammal, including a
human, comprising an amount of an IL-6 antibody or antigen binding
portion thereof, as described herein, that is effective in treating
abnormal cell infiltration, and a pharmaceutically acceptable
carrier. The compositions provide a therapeutic benefit to patients
with one of more of a variety of inflammatory and autoimmune
diseases, such as rheumatoid arthritis, atherosclerosis,
granulomatous diseases, multiple sclerosis, asthma and cancer.
[0135] IL-6 antibodies and antigen-binding portions thereof can be
incorporated into pharmaceutical compositions suitable for
administration to a subject. Typically, the pharmaceutical
composition comprises an IL-6 antibody or antigen-binding portion
thereof and a pharmaceutically acceptable carrier. As used herein,
"pharmaceutically acceptable carrier" means any and all solvents,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, and the like that are
physiologically compatible. Some examples of pharmaceutically
acceptable carriers are water, saline, phosphate buffered saline,
dextrose, glycerol, ethanol and the like, as well as combinations
thereof. In many cases, it will be preferable to include isotonic
agents, for example, sugars, polyalcohols such as mannitol,
sorbitol, or sodium chloride in the composition. Additional
examples of pharmaceutically acceptable substances are wetting
agents or minor amounts of auxiliary substances such as wetting or
emulsifying agents, preservatives or buffers, which enhance the
shelf life or effectiveness of the antibody.
[0136] The compositions of this invention may be in a variety of
forms, for example, liquid, semi-solid and solid dosage forms, such
as liquid solutions (e.g., injectable and infusible solutions),
dispersions or suspensions, tablets, pills, powders, liposomes and
suppositories. The form depends on the intended mode of
administration and therapeutic application. Typical compositions
are in the form of injectable or infusible solutions, such as
compositions similar to those used for passive immunization of
humans. In one case the mode of administration is parenteral (e.g.,
intravenous, subcutaneous, intraperitoneal, intramuscular). In
another case, the antibody is administered by intravenous infusion
or injection. In another case, the antibody is administered by
intramuscular or subcutaneous injection. Formulations for injection
may be presented in unit dosage form, e.g., in ampoules or in
multi-dose containers, with or without an added preservative. The
compositions may take such forms as suspensions, solutions, or
emulsions in oily or aqueous vehicles, and may contain formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the active ingredient may be in powder form for
constitution with a suitable vehicle, e.g., sterile pyrogen-free
water, before use. Sterile injectable solutions can be prepared by
incorporating the IL-6 antibody in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filter sterilization. In
one case, the antibody is administered in a formulation as a
sterile aqueous solution having a pH that ranges from about 5.0 to
about 6.5 and comprising from about 1 mg/ml to about 200 mg/ml of
antibody, from about 1 millimolar to about 100 millimolar of
histidine buffer, from about 0.01 mg/ml to about 10 mg/ml of
polysorbate 80 or polysorbate 20, from about 100 millimolar to
about 400 millimolar of a non-reducing sugar selected from but not
limited to trehalose or sucrose, from about 0.01 millimolar to
about 1.0 millimolar of disodium EDTA dihydrate and optionally
comprise a pharmaceutically acceptable antioxidant in addition to a
chelating agent. Suitable antioxidants include, but are not limited
to, methionine, sodium thiosulfate, catalase, and platinum. For
example, the composition may contain methionine in a concentration
that ranges from 1 mM to about 100 mM, and in particular, is about
27 mM. In some cases, a formulation contains 5 mg/ml of antibody in
a buffer of 20 mM sodium citrate, pH 5.5, 140 mM NaCl, and 0.2
mg/ml polysorbate 80. It is to be noted that dosage values may vary
with the type and severity of the condition to be alleviated. In
the case of sterile powders for the preparation of sterile
injectable solutions, the suitable methods of preparation include
vacuum drying and freeze-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof. The proper fluidity of a
solution can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prolonged
absorption of injectable compositions can be brought about by
including in the composition an agent that delays absorption, for
example, monostearate salts and gelatin.
[0137] IL-6 antibodies or antigen-binding portions thereof can be
administered by a variety of methods, although for many therapeutic
applications, the route/mode of administration can be subcutaneous,
intramuscular, or intravenous infusion. As will be appreciated by
the skilled artisan, the route and/or mode of administration will
vary depending upon the desired results.
[0138] In certain cases, the IL-6 antibody compositions may be
prepared with a carrier that will protect the antibody against
rapid release, such as a controlled release formulation, including
implants, transdermal patches, and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Many methods for
the preparation of such formulations may be used. See, e.g.,
Sustained and Controlled Release Drug Delivery Systems, J. R.
Robinson, ed., Marcel Dekker, Inc., New York, 1978, which is
incorporated herein by reference.
[0139] Additional active compounds also can be incorporated into
the compositions. In some cases, an inhibitory IL-6 antibody is
co-formulated with and/or co-administered with one or more
additional therapeutic agents. These agents include, without
limitation, antibodies that bind other targets, anti-tumor agents,
anti-angiogenesis agents, signal transduction inhibitors,
anti-proliferative agents, chemotherapeutic agents, or peptide
analogues that inhibit IL-6. Such combination therapies may require
lower dosages of the inhibitory IL-6 antibody as well as the
co-administered agents, thus avoiding possible toxicities or
complications associated with the various monotherapies.
[0140] The compositions may include a "therapeutically effective
amount" or a "prophylactically effective amount" of an antibody or
antigen-binding portion. 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 or antigen-binding
portion 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 antigen-binding
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 may be less than the
therapeutically effective amount.
[0141] Dosage regimens can be adjusted to provide the optimum
desired response (e.g., a therapeutic or prophylactic response).
For example, a single bolus can be administered, several divided
doses can be administered over time or the dose can be
proportionally reduced or increased as indicated by the exigencies
of the therapeutic situation. It is especially advantageous to
formulate parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the mammalian subjects to be treated; each unit
containing a predetermined quantity of IL-6 antibody or antigen
binding portion thereof calculated to produce the desired
therapeutic effect in association with the required pharmaceutical
carrier.
[0142] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of an IL-6 antibody or antibody
portion is 0.025 to 50 mg/kg, 0.1 to 50 mg/kg, 0.1-25, 0.1 to 10 or
0.1 to 3 mg/kg. In one case, the IL-6 antibody or antibody portion
thereof is administered in a formulation as a sterile aqueous
solution having a pH that ranges from about 5.0 to about 6.5 and
comprising from about 1 mg/ml to about 200 mg/ml of antibody, from
about 1 millimolar to about 100 millimolar of histidine buffer,
from about 0.01 mg/ml to about 10 mg/ml of polysorbate 80, from
about 100 millimolar to about 400 millimolar of trehalose, and from
about 0.01 millimolar to about 1.0 millimolar of disodium EDTA
dihydrate. It is to be noted that dosage values may vary with the
type and severity of the condition to be alleviated. It is to be
further understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions, and that dosage
ranges set forth herein are exemplary only and are not intended to
limit the scope or practice of the claimed compositions.
[0143] Another aspect provided herein are kits comprising an IL-6
antibody or antigen-binding portion or a composition comprising
such an antibody or antigen-binding portion. A kit may include, in
addition to the antibody or composition, diagnostic or therapeutic
agents. A kit can also include instructions for use in a diagnostic
or therapeutic method. In one case, the kit includes the antibody
or a composition comprising it and a diagnostic agent that can be
used in a method described herein. In another case, the kit
includes the antibody or a composition comprising it and one or
more therapeutic agents that can be used in a method described
herein.
Diagnostic Methods of Use
[0144] Another aspect provided herein are diagnostic methods. The
anti-IL-6 antibodies or antigen binding portion thereof can be used
to detect IL-6 in a biological sample in vitro or in vivo. One
aspect provides a method for diagnosing the presence or location of
IL-6-expressing cells in a subject in need thereof, comprising the
steps of injecting the antibody into the subject, determining the
expression of IL-6 in the subject by localizing where the antibody
has bound, comparing the expression in the subject with that of a
normal reference subject or standard, and diagnosing the presence
or location of the cells. The anti-IL-6 antibodies may also be used
as a marker for inflammation and/or for the infiltration of immune
cells, such as monocytes and T cells, into a tissue.
[0145] The anti-IL-6 antibodies can be used in any suitable
immunoassay, including, without limitation, an ELISA, a RIA, flow
cytometry, tissue immunohistochemistry, a Western blot or an
immunoprecipitation. The anti-IL-6 antibodies or antigen binding
portion thereof can be used to detect IL-6 from humans. In another
case, the anti-IL-6 antibodies can be used to detect IL-6 from
cynomolgus monkeys, rhesus monkeys and rodents, such as mice and
rats.
[0146] Methods for detecting IL-6 in a biological sample generally
comprise contacting the biological sample with an anti-IL-6
antibody or antigen binding portions thereof and detecting the
bound antibody. In one case, the anti-IL-6 antibody or antigen
binding portion thereof is directly labeled with a detectable
label. In another case, the anti-IL-6 antibody (the first antibody)
is unlabeled and a second antibody or other molecule that can bind
the anti-IL-6 antibody is labeled. A second antibody is chosen that
is able to specifically bind the particular species and class of
the first antibody. For example, if the anti-IL-6 antibody is a
human IgG, then the secondary antibody could be an anti-human-IgG.
Other molecules that can bind to antibodies include, without
limitation, Protein A and Protein G, both of which are available
commercially, e.g., from Pierce Chemical Co.
[0147] Suitable labels for the antibody or secondary antibody are
disclosed herein and include various enzymes, 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,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; and examples of suitable radioactive material include
.sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0148] IL-6 can also be assayed in a biological sample by a
competition immunoassay utilizing IL-6 standards labeled with a
detectable substance and an unlabeled anti-IL-6 antibody. In this
assay, the biological sample, the labeled IL-6 standards and the
anti-IL-6 antibody are combined and the amount of labeled IL-6
standard bound to the unlabeled antibody is determined. The amount
of IL-6 in the biological sample is inversely proportional to the
amount of labeled IL-6 standard bound to the anti-IL-6
antibody.
[0149] One can use such immunoassays for a number of purposes. For
example, the anti-IL-6 antibodies or antigen binding portions
thereof can be used to detect IL-6 in cultured cells. In one case,
the anti-IL-6 antibodies or antigen binding portions thereof are
used to determine the amount of IL-6 on the surface of cells that
have been treated with various compounds. This method can be used
to identify compounds that modulate IL-6 protein levels. According
to this method, one sample of cells is treated with a test compound
for a period of time while another sample is left untreated. If the
total IL-6 expression is to be measured, the cells are lysed and
the total IL-6 expression is measured using any suitable
immunoassay. The total IL-6 expression in the treated versus the
untreated cells is compared to determine the effect of the test
compound.
[0150] Immunoassays for measuring total IL-6 expression include
flow cytometry and immunohistochemistry. If the cell surface IL-6
expression is to be measured, the cells are not lysed, and the cell
surface levels of IL-6 are measured using one of the immunoassays
described above. A preferred immunoassay for determining cell
surface levels of IL-6 includes the steps of labeling the cell
surface proteins with a detectable label, such as biotin or
.sup.125I, immunoprecipitating the IL-6 with an anti-IL-6 antibody
and then detecting the labeled IL-6.
[0151] Another immunoassay for determining the localization of
IL-6, e.g., cell surface levels, is by using immunohistochemistry.
An immunoassay to detect cell surface levels of IL-6 includes
binding of an anti-IL-6 antibody labeled with an appropriate
fluorophore, such as fluorescein or phycoerythrin, and detecting
the primary antibody using flow cytometry. In another example, the
anti-IL-6 antibody is unlabeled and a second antibody or other
molecule that can bind the anti-IL-6 antibody is labeled. Methods
such as ELISA, RIA, flow cytometry, Western blot,
immunohistochemistry, cell surface labeling of integral membrane
proteins and immunoprecipitation are well known in the art. See,
e.g., Harlow and Lane. In addition, the immunoassays can be scaled
up for high throughput screening in order to test a large number of
compounds for either activation or inhibitors of IL-6.
[0152] The anti-IL-6 antibodies or antigen binding portions thereof
also can be used to determine the levels of IL-6 in a tissue or in
cells derived from the tissue. In some examples, the tissue is a
diseased tissue or a tissue biopsy. The tissue or biopsy can be
used in an immunoassay to determine, e.g., total IL-6 expression,
cell surface levels of IL-6 or localization of IL-6 by the methods
discussed above. Such methods can be used to determine whether a
tissue expresses high levels of IL-6, which could be indicative
that the tissue is a target for treatment with anti-IL-6
antibody.
[0153] IL-6 antibodies and antigen-binding portions thereof also
can be used in vivo to identify tissues and organs that express
IL-6. In some cases, the anti-IL-6 antibodies are used to identify
IL-6-expressing cells. Human anti-IL-6 antibodies may safely be
used in vivo without eliciting a substantial immune response to the
antibody upon administration, unlike antibodies of non-human origin
or with humanized or chimeric antibodies.
[0154] The method comprises the steps of administering a detectably
labeled anti-IL-6 antibody or a composition comprising them to a
patient in need of such a diagnostic test and subjecting the
patient to imaging analysis to determine the location of the
IL-6-expressing tissues. Imaging analysis is well known in the
medical art, and includes, without limitation, x-ray analysis,
magnetic resonance imaging (MRI) or computed tomography (CT). The
antibody can be labeled with any agent suitable for in vivo
imaging, for example a contrast agent, such as barium, which can be
used for x-ray analysis, or a magnetic contrast agent, such as a
gadolinium chelate, which can be used for MRI or CT. Other labeling
agents include, without limitation, radioisotopes, such as
.sup.99Tc. In another case, the anti-IL-6 antibody will be
unlabeled and will be imaged by administering a second antibody or
other molecule that is detectable and that can bind the anti-IL-6
antibody. In one example, a biopsy is obtained from the patient to
determine whether the tissue of interest expresses IL-6.
[0155] The detectably labeled anti-IL-6 may comprise a fluorophore.
In certain cases, the fluorophore is selected from the group
consisting of a near-infrared fluorescent dye, dinitrophenyl,
fluorescein and derivatives thereof, rhodamine, derivatives of
rhodamine, phycoerythrin, phycocyanin, allophycocyanin,
o-phthaldehyde and fluorescamine, Texas red, Rhodamine green,
Oregon green, Cascade blue, phycoerythrin, CY3, CY5, CY2, CY7,
coumarin, infrared 40, MR 200, IRD 40, Alexa Fluor, Cascade Blue,
Tetramethylrhodamine, Pacific Blue, SYBR, and BODIPY. In another
example, the fluorophore includes one of the following compounds
with their emission maxima indicated in nm in parentheses, Cy2.TM.
(506), GFP (Red Shifted) (507), YO-PRO.RTM.-1 (509), YOYO.RTM.-1
(509), Calcein (517), FITC (518), FluorX.RTM. (519), Alexa.RTM.
(520), Rhodamine 110 (520), 5-FAM (522), Oregon Green.RTM. 500
(522), Oregon Green.RTM. 488 (524), RiboGreen.RTM. (525), Rhodamine
Green.RTM. (527), Rhodamine 123 (529), Magnesium Green.RTM. (531),
Calcium Green.RTM. (533), TO-PRO.RTM.-1 (533), TOTO.RTM.-1 (533),
JOE (548), BODIPY.RTM. 530/550 (550), Dil (565), BODIPY.RTM. (568),
BODIPY.RTM. 558/568 (568), BODIPY.RTM. 564/570 (570), Cy3.RTM.
(570), Alexa.RTM. 546 (570), TRITC (572), Magnesium Orange.RTM.
(575), Phycoerythrin R&B (575), Rhodamine Phalloidin (575),
Calcium Orange.RTM. (576), Pyronin Y (580), Rhodamine B (580),
TAMRA (582), Rhodamine Red.RTM. (590), Cy3.5.RTM. (596), ROX (608),
Calcium Crimson.TM. (615), Alexa.RTM. 594 (615), Texas Red.RTM.
(615), Nile Red (628), YO-PRO.RTM.-3 (631), YOYO.RTM.-3 (631),
R-phycocyanin (642), C-Phycocyanin (648), TO-PRO.RTM.-3 (660),
TOTO.RTM.-3 (660), DiD DilC(5) (665), Cy5.TM. (670),
Thiadicarbocyanine (671) and Cy5.5 (694).
[0156] In yet a further example, the anti-IL-6 antibodies may also
be used to determine the reduction in surface cell expression of
IL-6 on cells, for example, lymphocytes or monocytes.
[0157] Human anti-IL-6 antibodies or antigen-binding portions
thereof minimize the immunogenic and allergic responses intrinsic
to non-human or non-human-derivatized monoclonal antibodies (Mabs),
and thus increase the efficacy and safety of the administered
antibodies or antigen-binding portions thereof.
[0158] Another aspect provides human anti-IL-6 antibodies encoded
in part by a human germline sequence. The V.sub.H, V.sub.K,
V.sub..lamda. genes are classified into families on the basis of
sequence homology. Two V.sub.H, V.sub.K, or V.sub..lamda. genes
belong to the same family if they share the same nucleotide
sequence at more than 80% of the positions. An anti-IL-6 antibody
may comprise a human kappa light chain (V.sub.K) or a human lambda
light chain (V.sub..lamda.) or an amino acid sequence derived
therefrom. In some cases comprising a lambda light chain, the light
chain variable domain (V.sub.L) is encoded in part by a human
V.sub..lamda.1, V.sub..lamda.2, V.sub..lamda.3, V.sub..lamda.4,
V.sub..lamda.5, V.sub..lamda.6, V.sub..lamda.7, V.sub..lamda.8,
V.sub..lamda.9, or V.sub..lamda.10 family gene (Williams S. C. et
al. J. Mol. Bio. 264:220-232, 1996). In some cases comprising a
kappa light chain, the light chain variable domain (V.sub.L) is
encoded in part by a human V.sub.KI, V.sub.KII, V.sub.KIII,
V.sub.KIV, V.sub.KV, or V.sub.KVI family gene (Cox J. P. L., et al,
Eur. J. Immunol 24:827-836, 1994), preferably a V.sub.KI,
V.sub.KII, V.sub.KIII, or V.sub.KIV family gene, and preferably a
V.sub.KI or V.sub.KVI family gene. In some cases, the light chain
germline sequence is selected from human VK sequences including,
but not limited to, A1, A10, A11, A14, A17, A18, A19, A2, A20, A23,
A26, A27, A3, A30, A5, A7, B2, B3, L1, L10, L11, L12, L14, L15,
L16, L18, L19, L2, L20, L22, L23, L24, L25, L4/18a, L5, L6, L8, L9,
O1, O11, O12, O14, O18, O2, O4, and O8. In certain cases, this
light chain human germline framework is selected from V1-11, V1-13,
V1-16, V1-17, V1-18, V1-19, V1-2, V1-20, V1-22, V1-3, V1-4, V1-5,
V1-7, V1-9, V2-1, V2-11, V2-13, V2-14, V2-15, V2-17, V2-19, V2-6,
V2-7, V2-8, V3-2, V3-3, V3-4, V4-1, V4-2, V4-3, V4-4, V4-6, V5-1,
V5-2, V5-4, and V5-6. An anti-IL-6 antibody may comprise a heavy
chain variable domain (V.sub.H) encoded by a human V.sub.H1,
V.sub.H2, V.sub.H3, V.sub.H4, V.sub.HS, V.sub.H6 or V.sub.H7 family
gene. In particular examples, this heavy chain human germline
framework is selected from VH1-18, VH1-2, VH1-24, VH1-3, VH1-45,
VH1-46, VH1-58, VH1-69, VH1-8, VH2-26, VH2-5, VH2-70, VH3-11,
VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33,
VH3-35, VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66,
VH3-7, VH3-72, VH3-73, VH3-74, VH3-9, VH4-28, VH4-31, VH4-34,
VH4-39, VH4-4, VH4-59, VH4-61, VH5-51, VH6-1, and VH7-81. In
particular cases, the light chain variable region and/or heavy
chain variable region comprises a framework region or at least a
portion of a framework region (e.g., containing 2 or 3 subregions,
such as FR2 and FR3). In certain cases, at least FRL1, FRL2, FRL3,
or FRL4 is fully human. In other examples, at least FRH1, FRH2,
FRH3, or FRH4 is fully human. In some cases, at least FRL1, FRL2,
FRL3, or FRL4 is a germline sequence (e.g., human germline) or
comprises human consensus sequences for the particular framework
(readily available at the sources of known human Ig sequences
described herein). In other examples, at least FRH1, FRH2, FRH3, or
FRH4 is a germline sequence (e.g., human germline) or comprises
human consensus sequences for the particular framework.
[0159] The V.sub.L of the IL-6 antibody may comprise one or more
amino acid substitutions relative to the germline amino acid
sequence of the human gene. In some cases, the V.sub.L of the IL-6
antibody comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid
substitutions relative to the germline amino acid sequence. In an
example, one or more of those substitutions from germline is in the
CDR regions of the light chain. In one example, the amino acid
substitutions relative to germline are at one or more of the same
positions as the substitutions relative to germline in any one or
more of the V.sub.L of antibodies 9C8 IgG1, 9C8 IgG2, 9C8 N68T T83S
IgG1, 9C8 N68T T83S IgG2, 9C8 E31G N68T T83S IgG1, 9C8 E31G N68T
T83S IgG2, 9C8 I24V N68T T83S IgG1, 9C8 I24V N68T T83S IgG2 and
22B5 IgG1. For example, the V.sub.L of an IL-6 antibody may contain
one or more amino acid substitutions compared to germline found in
the V.sub.L of antibody 9C8 IgG1. In some cases, the amino acid
changes are at one or more of the same positions, but involve a
different substitution than in the reference antibody.
[0160] In some cases, amino acid changes relative to germline occur
at one or more of the same positions as in any of the V.sub.L of
antibodies 9C8 IgG1, 9C8 IgG2, 9C8 N68T T83S IgG1, 9C8 N68T T83S
IgG2, 9C8 E31G N68T T83S IgG1, 9C8 E31G N68T T83S IgG2, 9C8 I24V
N68T T83S IgG1, 9C8 I24V N68T T83S IgG2 and 22B5 IgG1, but the
changes may represent conservative amino acid substitutions at such
position(s) relative to the amino acid in the reference antibody.
For example, if a particular position in one of these antibodies is
changed relative to germline and is glutamate, one may substitute
aspartate at that position. Similarly, if an amino acid
substitution compared to germline is serine, one may conservatively
substitute threonine for serine at that position.
[0161] In some cases, the light chain of the human anti-IL-6
antibody comprises the V.sub.L amino acid sequence of antibody 9C8
IgG1, 9C8 IgG2, 9C8 N68T T83S IgG1, 9C8 N68T T83S IgG2, 9C8 E31G
N68T T83S IgG1, 9C8 E31G N68T T83S IgG2, 9C8 I24V N68T T83S IgG1,
9C8 I24V N68T T83S IgG2 and 22B5 IgG1 or the amino acid sequence
having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino
acid substitutions and/or a total of up to 3 non-conservative amino
acid substitutions. In some cases, the light chain comprises the
amino acid sequence from the beginning of the CDR1 to the end of
the CDR3 of any one of the foregoing antibodies.
[0162] In some cases, the light chain may comprise CDR1, CDR2 and
CDR3 regions independently selected from the light chain CDR1, CDR2
and CDR3, respectively, of the light chain of antibodies 9C8 IgG1,
9C8 IgG2, 9C8 N68T T83S IgG1, 9C8 N68T T83S IgG2, 9C8 E31G N68T
T83S IgG1, 9C8 E31G N68T T83S IgG2, 9C8 I24V N68T T83S IgG1, 9C8
I24V N68T T83S IgG2 and 22B5 IgG1, or CDR regions each having less
than 4 or less than 3 conservative amino acid substitutions and/or
a total of three or fewer non-conservative amino acid
substitutions. In some cases, the light chain of the anti-IL-6
antibody comprises a light chain CDR1, CDR2, and CDR3, each of
which are independently selected from the light chain CDR1, CDR2
and CDR3 regions of monoclonal antibody 9C8 IgG1, 9C8 IgG2, 9C8
N68T T83S IgG1, 9C8 N68T T83S IgG2, 9C8 E31G N68T T83S IgG1, 9C8
E31G N68T T83S IgG2, 9C8 I24V N68T T83S IgG1, 9C8 I24V N68T T83S
IgG2 and 22B5 IgG1. In certain cases, the light chain of the
anti-IL-6 antibody comprises the light chain CDR1, CDR2 and CDR3
regions of an antibody comprising the amino acid sequence of the
V.sub.L region of an antibody selected from 9C8 IgG1, 9C8 IgG2, 9C8
N68T T83S IgG1, 9C8 N68T T83S IgG2, 9C8 E31G N68T T83S IgG1, 9C8
E31G N68T T83S IgG2, 9C8 I24V N68T T83S IgG1, 9C8 I24V N68T T83S
IgG2 and 22B5 IgG1 or the CDR regions each having less than 4 or
less than 3 conservative amino acid substitutions and/or a total of
three or fewer non-conservative amino acid substitutions.
[0163] In some cases, the variable domain (V.sub.H) is encoded, at
least in part, by a human gene. In some cases, the V.sub.H sequence
of the IL-6 antibody contains one or more amino acid substitutions,
deletions or insertions (additions) relative to the germline amino
acid sequence. In some cases, the variable domain of the heavy
chain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, or 17 mutations from the germline amino acid sequence. In some
cases, the mutation(s) are non-conservative substitutions,
deletions or insertions, compared to the germline amino acid
sequence. In some examples, the mutations are in the CDR regions of
the heavy chain. In some examples, the amino acid changes are made
at one or more of the same positions as the mutations from germline
in any one or more of the V.sub.H of antibodies 9C8 IgG1, 9C8 IgG2,
9C8 N68T T83S IgG1, 9C8 N68T T83S IgG2, 9C8 E31G N68T T83S IgG1,
9C8 E31G N68T T83S IgG2, 9C8 I24V N68T T83S IgG1, 9C8 I24V N68T
T83S IgG2 and 22B5 IgG1. In other examples, the amino acid changes
are at one or more of the same positions but involve a different
mutation than in the reference antibody.
[0164] In some cases, the heavy chain comprises the V.sub.H amino
acid sequence of antibody 9C8 IgG1, 9C8 IgG2, 9C8 N68T T83S IgG1,
9C8 N68T T83S IgG2, 9C8 E31G N68T T83S IgG1, 9C8 E31G N68T T83S
IgG2, 9C8 I24V N68T T83S IgG1, 9C8 I24V N68T T83S IgG2 and 22B5
IgG1 the V.sub.H amino acid sequence having up to 1, 2, 3, 4, 6, 8,
or 10 conservative amino acid substitutions and/or a total of up to
3 non-conservative amino acid substitutions. In some examples, the
heavy chain comprises the amino acid sequence from the beginning of
the CDR1 to the end of the CDR3 of any one of the foregoing
antibodies.
[0165] In some cases, the heavy chain comprises the heavy chain
CDR1, CDR2 and CDR3 regions of antibody 9C8 IgG1, 9C8 IgG2, 9C8
N68T T83S IgG1, 9C8 N68T T83S IgG2, 9C8 E31G N68T T83S IgG1, 9C8
E31G N68T T83S IgG2, 9C8 I24V N68T T83S IgG1, 9C8 I24V N68T T83S
IgG2 and 22B5 IgG1 or the CDR regions each having less than 8, less
than 6, less than 4, or less than 3 conservative amino acid
substitutions and/or a total of three or fewer non-conservative
amino acid substitutions.
[0166] In some cases, the heavy chain CDR regions are independently
selected from the CDR regions of antibodies 9C8 IgG1, 9C8 IgG2, 9C8
N68T T83S IgG1, 9C8 N68T T83S IgG2, 9C8 E31G N68T T83S IgG1, 9C8
E31G N68T T83S IgG2, 9C8 I24V N68T T83S IgG1, 9C8 I24V N68T T83S
IgG2 and 22B5 IgG1. In other cases, the heavy chain comprises CDR
regions independently selected from two or more V.sub.H regions
selected from 9C8 IgG1, 9C8 IgG2, 9C8 N68T T83S IgG1, 9C8 N68T T83S
IgG2, 9C8 E31G N68T T83S IgG1, 9C8 E31G N68T T83S IgG2, 9C8 I24V
N68T T83S IgG1, 9C8 I24V N68T T83S IgG2 and 22B5 IgG1.
[0167] In other cases, the antibody comprises a light chain and a
heavy chain. In a further example, the light chain CDRs and the
heavy chain CDRs are from the same antibody.
[0168] One type of amino acid substitution that may be made is to
change one or more cysteines in the antibody, which may be
chemically reactive, to another residue, such as, without
limitation, alanine or serine. In one example, there is a
substitution of a non-canonical cysteine. The substitution can be
made in a CDR or framework region of a variable domain or in the
constant domain of an antibody. In some cases, the cysteine is
canonical.
[0169] Another type of amino acid substitution that may be made is
to change any potential proteolytic sites in the antibody. Such
sites may occur in a CDR or framework region of a variable domain
or in the constant domain of an antibody. Substitution of cysteine
residues and removal of proteolytic sites may decrease the risk of
any heterogeneity in the antibody product and thus increase its
homogeneity. Another type of amino acid substitution is to
eliminate asparagine-glycine pairs, which form potential
deamidation sites, by altering one or both of the residues.
[0170] In certain cases, the heavy and light chains of the IL-6
antibodies may optionally include a signal sequence.
[0171] In some cases, the antibody comprises the heavy and light
chain variants of monoclonal antibodies 9C8 IgG1, 9C8 IgG2, 9C8
N68T T83S IgG1, 9C8 N68T T83S IgG2, 9C8 E31G N68T T83S IgG1, 9C8
E31G N68T T83S IgG2, 9C8 I24V N68T T83S IgG1, 9C8 I24V N68T T83S
IgG2 and 22B5 IgG1. As discussed in greater detail in EXAMPLE 3,
numerous heavy and light chain variant mutations were made to match
those in the germline CDR regions. The specific amino acids that
were mutated to arrive at the germline versions are apparent to
those of skill in the art by comparing the sequences of the
germline vs. a non-germline antibody. For example, one amino acid
substitution is provided in the heavy chain of antibody 9C8,
wherein an isoleucine at residue 24 is changed to a valine and is
referred to as 9C8 I24V. A second exemplary amino acid substitution
is in the light chain of antibody 9C8, and substitutes the lysine
at residue 92 with an asparagine and is referred to as 9C8
K92N.
[0172] As will be appreciated, gene utilization analysis provides
only a limited overview of antibody structure. As human B-cells
stochastically generate V-D-J heavy or V-J kappa light chain
transcripts, there are a number of secondary processes that occur,
including, without limitation, somatic hypermutation, additions,
and CDR3 extensions. Accordingly, to further examine antibody
structures, predicted amino acid sequences of the antibodies were
generated from the cDNAs obtained from the clones. In addition,
N-terminal amino acid sequences were obtained through protein
sequencing.
Class and Subclass of Anti-IL-6 Antibodies
[0173] The class (e.g., IgG, IgM, IgE, IgA, or IgD) and subclass
(e.g. IgG1, IgG2, IgG3, or IgG4) of IL-6 antibodies may be
determined by any suitable method. In general, the class and
subclass of an antibody may be determined using antibodies that are
specific for a particular class and subclass of antibody. Such
antibodies are commercially available. The class and subclass can
be determined by ELISA, or Western Blot, as well as other
techniques.
[0174] Alternatively, the class and subclass may be determined by
sequencing all or a portion of the constant domains of the heavy
and/or light chains of the antibodies, comparing their amino acid
sequences to the known amino acid sequences of various class and
subclasses of immunoglobulins, and determining the class and
subclass of the antibodies. The IL-6 antibodies can be an IgG, an
IgM, an IgE, an IgA, or an IgD molecule. For example, the IL-6
antibodies can be an IgG that is an IgG1, IgG2, IgG3, or an IgG4
subclass. In one example, the IL-6 antibodies are IgG2 subclass. In
another example, the IL-6 antibodies are IgG1 subclass.
[0175] In one aspect methods are provided for converting the class
or subclass of an IL-6 antibody to another class or subclass. In
some cases, a nucleic acid molecule encoding a V.sub.L or V.sub.H
that does not include sequences encoding C.sub.L or C.sub.H is
isolated using any suitable methods. The nucleic acid molecule then
is operatively linked to a nucleic acid sequence encoding a C.sub.L
or C.sub.H from a desired immunoglobulin class or subclass. This
can be achieved using a vector or nucleic acid molecule that
comprises a C.sub.L or C.sub.H chain, as described above. For
example, an IL-6 antibody that was originally IgM can be class
switched to an IgG. Further, the class switching may be used to
convert one IgG subclass to another, e.g., from IgG1 to IgG2.
Another method for producing an antibody comprising a desired
isotype comprises the steps of isolating a nucleic acid encoding a
heavy chain of an IL-6 antibody and a nucleic acid encoding a light
chain of an IL-6 antibody, isolating the sequence encoding the
V.sub.H region, ligating the V.sub.H sequence to a sequence
encoding a heavy chain constant domain of the desired isotype,
expressing the light chain gene and the heavy chain construct in a
cell, and collecting the IL-6 antibody with the desired
isotype.
Binding Affinity of IL-6 Antibodies to IL-6
[0176] The binding affinity and dissociation rate of an anti-IL-6
antibody to IL-6 can be determined by any suitable method. The
binding affinity can be measured by ELISAs, RIAs, flow cytometry,
and surface plasmon resonance, such as BIACORE.TM.. The dissociate
rate can be measured by surface plasmon resonance. One can
determine whether an antibody has substantially the same K.sub.D as
an anti-IL-6 antibody by using any suitable method. Example 7
exemplifies a method for determining affinity constants of
anti-IL-6 monoclonal antibodies.
Identification of IL-6 Epitopes Recognized by Anti-IL-6
Antibodies
[0177] One can determine whether an antibody binds to the same
epitope or cross-competes for binding with an IL-6 antibody by
using any suitable method. In one example, one allows an IL-6
antibody to bind to IL-6 under saturating conditions and then
measures the ability of the test antibody to bind to IL-6. If the
test antibody is able to bind to IL-6 at the same time as the IL-6
antibody, then the test antibody binds to a different epitope as
the IL-6 antibody. However, if the test antibody is not able to
bind to IL-6 at the same time, then the test antibody binds to the
same epitope, an overlapping epitope, or an epitope that is in
close proximity to the epitope bound by the human IL-6 antibody.
This experiment can be performed using an ELISA, a RIA,
BIACORE.TM., or flow cytometry (FACS).
[0178] To test whether an IL-6 antibody cross-competes with another
IL-6 antibody, one may use the competition method described herein
in two directions, i.e., determining if the reference antibody
blocks the test antibody and vice versa. In one example, the
experiment is performed using an ELISA.
Methods of Producing Antibodies
[0179] IL-6 antibodies or antigen-binding portions thereof can be
produced by a variety of techniques, including conventional
monoclonal antibody methodology, for example the standard somatic
cell hybridization technique of Kohler and Milstein (1975) Nature
256: 495. Other techniques for producing monoclonal antibodies can
also be employed such as viral or oncogenic transformation of B
lymphocytes.
[0180] An exemplary animal system for preparing hybridomas is a
murine system. Immunization protocols and techniques for isolation
of immunized splenocytes for fusion are known in the art. Fusion
partners (e.g., murine myeloma cells) and fusion procedures are
also known.
[0181] Chimeric or humanized antibodies can be prepared based on
the sequence of a murine monoclonal antibody prepared as described
above. DNA encoding the heavy and light chain immunoglobulins can
be obtained from the murine hybridoma of interest and engineered to
contain non-murine (e.g., human) immunoglobulin sequences using
standard molecular biology techniques. For example, to create a
chimeric antibody, the murine variable regions can be linked to
human constant regions using methods known in the art (see e.g.,
U.S. Pat. No. 4,816,567 to Cabilly et al.). To create a humanized
antibody, the murine CDR regions can be inserted into a human
framework using methods known in the art (see e.g., U.S. Pat. No.
5,225,539, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762; and
6,180,370).
[0182] In one case, the anti-IL-6 antibodies are human monoclonal
antibodies. Such human monoclonal antibodies directed against IL-6
can be generated using transgenic or transchromosomic mice carrying
parts of the human immune system rather than the mouse system.
These transgenic and transchromosomic mice include mice referred to
herein as the HuMAb Mouse.RTM. and KM Mouse.RTM., respectively, and
are collectively referred to herein as "human Ig mice."
[0183] The HuMAb Mouse.RTM. (Medarex.RTM., Inc.) contains human
immunoglobulin gene miniloci that encode unrearranged human heavy
(.mu. and .gamma.) and .kappa. light chain immunoglobulin
sequences, together with targeted mutations that inactivate the
endogenous .mu. and .kappa. chain loci (see e.g., Lonberg, et al.
(1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit
reduced expression of mouse IgM or .kappa., and in response to
immunization, the introduced human heavy and light chain transgenes
undergo class switching and somatic mutation to generate high
affinity human IgG.kappa. monoclonal (Lonberg, N. et al. (1994);
reviewed in Lonberg, N. (1994) Handbook of Experimental
Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern.
Rev. Immunol. 13: 65-93, and Harding, F. and Lonberg, N. (1995)
Ann. N.Y. Acad. Sci. 764:536-546). Preparation and use of the HuMAb
Mouse.RTM., and the genomic modifications carried by such mice, is
further described in Taylor, L. et al. (1992) Nucleic Acids
Research 20:6287-6295; Chen, J. et al. (1993) International
Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad.
Sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics
4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et
al. (1994) J. Immunol. 152:2912-2920; Taylor, L. et al. (1994)
International Immunology 6: 579-591; and Fishwild, D. et al. (1996)
Nature Biotechnology 14: 845-851, the contents of all of which are
hereby specifically incorporated by reference in their entirety.
See further, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126;
5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299;
and 5,770,429; U.S. Pat. No. 5,545,807; PCT Publication Nos.: WO
92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO
99/45962; and PCT Publication No. WO 01/14424.
[0184] In another aspect, human anti-IL-6 antibodies can be raised
using a mouse that carries human immunoglobulin sequences on
transgenes and transchomosomes, such as a mouse that carries a
human heavy chain transgene and a human light chain
transchromosome. Such mice, referred to herein as "KM Mice.TM.",
are described in detail in PCT Publication No. WO 02/43478.
[0185] Still further, alternative transgenic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to raise IL-6 antibodies. For example, an alternative
transgenic system referred to as the Xenomouse.TM. (Abgenix, Inc.)
can be used; such mice are described in, for example, U.S. Pat.
Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584; and 6,162,963.
[0186] Moreover, alternative transchromosomic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to raise IL-6 antibodies. For example, mice carrying
both a human heavy chain transchromosome and a human light chain
tranchromosome, referred to as "TC mice" can be used; such mice are
described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA
97:722-727. Furthermore, cows carrying human heavy and light chain
transchromosomes have been described in the art (Kuroiwa et al.
(2002) Nature Biotechnology 20:889-894) and can be used to raise
IL-6 antibodies.
[0187] Human monoclonal antibodies can also be prepared using SCID
mice into which human immune cells have been reconstituted such
that a human antibody response can be generated upon immunization.
Such mice are described in, for example, U.S. Pat. Nos. 5,476,996;
and 5,698,767.
Immunization of Human Ig Mice
[0188] Production of Antibodies and Antibody-Producing Cell
Lines
[0189] After immunization of an animal with an IL-6 antigen,
antibodies and/or antibody-producing cells can be obtained from the
animal. In some cases, IL-6 antibody-containing serum is obtained
from the animal by bleeding or sacrificing the animal. The serum
may be used as it is obtained from the animal, an immunoglobulin
fraction may be obtained from the serum, or the IL-6 antibodies may
be purified from the serum.
[0190] In some cases, antibody-producing immortalized cell lines
are prepared from cells isolated from the immunized animal. After
immunization, the animal is sacrificed and lymph node and/or
splenic B cells are immortalized by any means known in the art.
Methods of immortalizing cells include, but are not limited to,
transfecting them with oncogenes, infecting them with an oncogenic
virus and cultivating them under conditions that select for
immortalized cells, subjecting them to carcinogenic or mutating
compounds, fusing them with an immortalized cell, e.g., a myeloma
cell, and inactivating a tumor suppressor gene. If fusion with
myeloma cells is used, the myeloma cells preferably do not secrete
immunoglobulin polypeptides (a non-secretory cell line).
Immortalized cells are screened using IL-6, a portion thereof, or a
cell expressing IL-6. In some cases, the initial screening is
performed using an enzyme-linked immunoassay (ELISA) or a
radioimmunoassay. An example of ELISA screening is provided in PCT
Publication No. WO 00/37504, incorporated herein by reference.
[0191] IL-6 antibody-producing cells, e.g., hybridomas, are
selected, cloned and further screened for desirable
characteristics, including robust growth, high antibody production
and desirable antibody characteristics. Hybridomas can be expanded
in vivo in syngeneic animals, in animals that lack an immune
system, e.g., nude mice, or in cell culture in vitro. Methods of
selecting, cloning and expanding hybridomas are well known to those
of ordinary skill in the art.
[0192] In one example, the immunized animal is a non-human animal
that expresses human immunoglobulin genes and the splenic B cells
are fused to a myeloma cell line from the same species as the
non-human animal. One such immunized animal is a Kirin TC Mouse.TM.
mouse and the myeloma cell line is a non-secretory mouse myeloma.
In a further example the myeloma cell line is Sp2/0-Ag14 (American
Type Culture Collection (ATCC) CRL-1581).
[0193] Also provided are methods for producing a cell line that
produces a human monoclonal antibody or a antigen-binding portion
thereof directed to IL-6 comprising: (a) immunizing a non-human
transgenic animal described herein with IL-6, a portion of IL-6 or
a cell or tissue expressing Il-6; (b) allowing the transgenic
animal to mount an immune response to IL-6; (c) isolating
antibody-producing cells from the transgenic animal; (d)
immortalizing the antibody-producing cells; (e) creating individual
monoclonal populations of the immortalized antibody-producing
cells; and (f) screening the immortalized antibody-producing cells
to identify an antibody directed to IL-6.
[0194] In another aspect, hybridomas are provided that produce a
human IL-6 antibody. The human IL-6 antibody produced by the
hybridoma may be an antagonist of IL-6. The hybridomas may be
produced in a non-human, non-mouse species such as, for example,
rats, sheep, pigs, goats, cattle or horses.
[0195] In one case, antibody-producing cells are isolated and
expressed in a host cell, for example myeloma cells. In still
another example, a transgenic animal is immunized with IL-6,
primary cells (e.g., spleen or peripheral blood cells) are isolated
from an immunized transgenic animal and individual cells producing
antibodies specific for the desired antigen are identified.
Polyadenylated mRNA from each individual cell is isolated and
reverse transcription polymerase chain reaction (RT-PCR) is
performed using sense primers that anneal to variable region
sequences, e.g., degenerate primers that recognize most or all of
the FR1 regions of human heavy and light chain variable region
genes and anti-sense primers that anneal to constant or joining
region sequences. cDNAs of the heavy and light chain variable
domains are then cloned and expressed in any suitable host cell,
e.g., a myeloma cell, as chimeric antibodies with respective
immunoglobulin constant regions, such as the heavy chain and
.kappa. or .lamda. constant domains. See Babcook, J. S. et al.
(1996) Proc. Natl. Acad. Sci. USA 93: 7843-48, incorporated herein
by reference. IL-6 antibodies may then be identified and
isolated.
Recombinant Methods of Producing Antibodies
[0196] IL-6 antibodies or antigen-binding portions thereof can be
prepared by recombinant expression of immunoglobulin light and
heavy chain genes in a host cell. For example, 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 and,
preferably, secreted into the medium in which the host cells are
cultured, from which medium the antibodies can be recovered.
Various recombinant DNA methodologies are used to obtain antibody
heavy and light chain genes, to incorporate these genes into
recombinant expression vectors and to introduce the vectors into
host cells, such as those described in Sambrook, Fritsch and
Maniatis (eds), Molecular Cloning; A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F. M. et al.
(eds.) Current Protocols in Molecular Biology, Greene Publishing
Associates, (1989) and in U.S. Pat. No. 4,816,397, the disclosures
of which are incorporated herein by reference.
[0197] Mutations and Modifications
[0198] To express the IL-6 antibodies, DNA fragments encoding
V.sub.H and V.sub.L regions can first be obtained using any of the
methods discussed herein. Various mutations, deletions, and/or
additions can also be introduced into the DNA sequences using
various suitable methods. For example, mutagenesis can be carried
out using standard methods, such as PCR-mediated mutagenesis, in
which the mutated nucleotides are incorporated into the PCR primers
such that the PCR product contains the desired mutations or
site-directed mutagenesis. One type of substitution, for example,
that may be made is to change one or more cysteines in the
antibody, which may be chemically reactive, to another residue,
such as, without limitation, alanine or serine. For example, there
can be a substitution of a non-canonical or canonical cysteine. The
substitution can be made in a CDR or framework region of a variable
domain or in the constant domain of an antibody. The antibodies may
also be mutated in the variable domains of the heavy and/or light
chains, e.g., to alter a binding property of the antibody. For
example, a mutation may be made in one or more of the CDR regions
to increase or decrease the K.sub.D of the antibody for IL-6, to
increase or decrease k.sub.off, or to alter the binding specificity
of the antibody. Techniques in site-directed mutagenesis include,
for example, Sambrook et al. and Ausubel et al., which are
incorporated herein by reference.
[0199] A mutation may also be made in a framework region or
constant domain to increase the half-life of an IL-6 antibody. See,
e.g., PCT Publication No. WO 00/09560, incorporated herein by
reference. A mutation in a framework region or constant domain can
also be made to alter the immunogenicity of the antibody, to
provide a site for covalent or non-covalent binding to another
molecule, or to alter such properties as complement fixation, FcR
binding and antibody-dependent cell-mediated cytotoxicity (ADCC). A
single antibody may have mutations in any one or more of the CDRs
or framework regions of the variable domain or in the constant
domain.
[0200] In a process known as "germlining", certain amino acids in
the V.sub.H and V.sub.L sequences can be mutated to match those
found naturally in germline V.sub.H and V.sub.L sequences. In
particular, the amino acid sequences of the framework regions in
the V.sub.H and V.sub.L sequences can be mutated to match the
germline sequences to reduce the risk of immunogenicity when the
antibody is administered. Germline DNA sequences for human V.sub.H
and V.sub.L genes are known in the art (see e.g., the "Vbase" human
germline sequence database; see also Kabat, E. A., et al. (1991)
Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No.
91-3242; Tomlinson et al. (1992) J. Mol. Biol. 227:776-798; and Cox
et al. Eur. J. Immunol. 24:827-836 (1994); the contents of each of
which are incorporated herein by reference).
[0201] Another type of amino acid substitution that may be made is
to remove potential proteolytic sites in the antibody. Such sites
may occur in a CDR or framework region of a variable domain or in
the constant domain of an antibody. Substitution of cysteine
residues and removal of proteolytic sites may decrease the risk of
heterogeneity in the antibody product and thus increase its
homogeneity. Another type of amino acid substitution is to
eliminate asparagine-glycine pairs, which form potential
deamidation sites, by altering one or both of the residues. In
another example, the C-terminal lysine of the heavy chain of an
IL-6 antibody can be cleaved. In various examples, the heavy and
light chains of the IL-6 antibodies may optionally include a signal
sequence. In some cases, the C-terminal lysine of the heavy chain
of the anti-IL-6 antibody may be proteolytically cleaved.
[0202] Once DNA fragments encoding the V.sub.H and V.sub.L segments
are obtained, these DNA fragments can be further manipulated by
standard recombinant DNA techniques, for example to convert the
variable region genes to full-length antibody chain genes, to Fab
fragment genes, or to a scFv gene. In these manipulations, a
V.sub.L- or V.sub.H-encoding DNA fragment is operatively linked to
another DNA fragment encoding another protein, such as an antibody
constant region or a flexible linker. The term "operatively
linked", as used in this context, is intended to mean that the two
DNA fragments are joined such that the amino acid sequences encoded
by the two DNA fragments remain in-frame.
[0203] The isolated DNA encoding the V.sub.H region can be
converted to a full-length heavy chain gene by operatively linking
the V.sub.H-encoding DNA to another DNA molecule encoding heavy
chain constant regions (CH1, CH2 and CH3). The sequences of human
heavy chain constant region genes are known in the art (see e.g.,
Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242) 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, but most
preferably is an IgG1 or IgG2 constant region. The IgG1 constant
region sequence can be any of the various alleles or allotypes
known to occur among different individuals, such as Gm(1), Gm(2),
Gm(3), and Gm(17). These allotypes represent naturally occurring
amino acid substitution in the IgG1 constant regions. For a Fab
fragment heavy chain gene, the V.sub.H-encoding DNA can be
operatively linked to another DNA molecule encoding only the heavy
chain CH1 constant region. The CH1 heavy chain constant region may
be derived from any of the heavy chain genes.
[0204] The isolated DNA encoding the V.sub.L region can be
converted to a full-length light chain gene (as well as a Fab light
chain gene) by operatively linking the V.sub.L-encoding DNA to
another DNA molecule encoding the light chain constant region,
C.sub.L. The sequences of human light chain constant region genes
are known in the art (see e.g., Kabat, E. A., et al. (1991)
Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No.
91-3242) 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. The kappa constant
region may be any of the various alleles known to occur among
different individuals, such as Inv(1), Inv(2), and Inv(3). The
lambda constant region may be derived from any of the three lambda
genes.
[0205] To create a scFv gene, the V.sub.H- and V.sub.L-encoding DNA
fragments are operatively linked to another fragment encoding a
flexible linker, e.g., encoding the amino acid sequence
(Gly.sub.4-Ser).sub.3, such that the V.sub.H and V.sub.L sequences
can be expressed as a contiguous single-chain protein, with the
V.sub.L and V.sub.H 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)). The single chain antibody may be monovalent,
if only a single V.sub.H and V.sub.L are used, bivalent, if two
V.sub.H and V.sub.L are used, or polyvalent, if more than two
V.sub.H and V.sub.L are used. Bispecific or polyvalent antibodies
may be generated that bind specifically to IL-6 and to another
molecule.
[0206] In another case, a fusion antibody may be made that
comprises all or a portion of an IL-6 antibody linked to another
polypeptide. In another case, only the variable domains of the IL-6
antibody are linked to the polypeptide. In another case, the
V.sub.H domain of an IL-6 antibody is linked to a first
polypeptide, while the V.sub.L domain of an IL-6 antibody is linked
to a second polypeptide that associates with the first polypeptide
in a manner such that the V.sub.H and V.sub.L domains can interact
with one another to form an antigen binding site. In another case,
the V.sub.H domain is separated from the V.sub.L domain by a linker
such that the V.sub.H and V.sub.L domains can interact with one
another. The V.sub.H-linker-V.sub.L antibody is then linked to the
polypeptide of interest. In addition, fusion antibodies can be
created in which two (or more) single-chain antibodies are linked
to one another. This is useful if one wants to create a divalent or
polyvalent antibody on a single polypeptide chain, or if one wants
to create a bispecific antibody.
[0207] In other cases, other modified antibodies may be prepared
using IL-6 antibody encoding nucleic acid molecules. For instance,
"Kappa bodies" (Ill et al., Protein Eng. 10: 949-57 (1997)),
"Minibodies" (Martin et al., EMBO J. 13: 5303-9 (1994)),
"Diabodies" (Holliger et al., Proc. Natl. Acad. Sci. USA 90:
6444-6448 (1993)), or "Janusins" (Traunecker et al., EMBO J.
10:3655-3659 (1991) and Traunecker et al., Int. J. Cancer (Suppl.)
7:51-52 (1992)) may be prepared using suitable molecular biological
techniques following the teachings discussed herein.
[0208] Bispecific antibodies or antigen-binding fragments can be
produced by a variety of methods including fusion of hybridomas or
linking of Fab' fragments. See, e.g., Songsivilai & Lachmann,
Clin. Exp. Immunol. 79:315-321 (1990), Kostelny et al., J. Immunol.
148:1547-1553 (1992). In addition, bispecific antibodies may be
formed as "diabodies" or "Janusins." In some cases, the bispecific
antibody binds to two different epitopes of IL-6. In some cases,
the modified antibodies described herein are prepared using one or
more of the variable domains or CDR regions from a human IL-6
antibody.
Vectors and Host Cells
[0209] To express IL-6 antibodies and antigen-binding portions
thereof, DNAs encoding partial or full-length light and heavy
chains, obtained as described herein, are inserted into expression
vectors such that the genes are operatively linked to
transcriptional and translational control sequences. In this
context, the term "operatively linked" is intended to mean that an
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. Expression vectors include, for example, plasmids,
retroviruses, adenoviruses, adeno-associated viruses (AAV), plant
viruses such as cauliflower mosaic virus, tobacco mosaic virus,
cosmids, YACs, and EBV derived episomes. 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 vectors or both genes are inserted into the
same expression vector. The antibody genes are inserted into the
expression vector by various 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).
[0210] A convenient vector is one that encodes a functionally
complete human C.sub.H or C.sub.L immunoglobulin sequence, with
appropriate restriction sites engineered so that any V.sub.H or
V.sub.L sequence can easily be inserted and expressed, as described
above. In such vectors, splicing usually occurs between the splice
donor site in the inserted J region and the splice acceptor site
preceding the human C domain, and also at the splice regions that
occur within the human C.sub.H exons. Polyadenylation and
transcription termination occur at native chromosomal sites
downstream of the coding regions. The recombinant expression vector
also can encode a signal peptide that facilitates secretion of the
antibody chain from a host cell. The antibody chain gene may be
cloned into the vector such that the signal peptide is linked
in-frame to the amino terminus of the immunoglobulin chain. The
signal peptide can be an immunoglobulin signal peptide or a
heterologous signal peptide (i.e., a signal peptide from a
non-immunoglobulin protein).
[0211] In addition to the antibody chain genes, the recombinant
expression vectors carry regulatory sequences that control the
expression of the antibody chain genes in a host cell. 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.
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
retroviral LTRs, 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)), polyoma and strong mammalian promoters such as
native immunoglobulin and actin promoters. For further description
of viral regulatory elements, and sequences thereof, see e.g., U.S.
Pat. No. 5,168,062, U.S. Pat. No. 4,510,245 and U.S. Pat. No.
4,968,615, incorporated herein by reference. Methods for expressing
antibodies in plants, including a description of promoters and
vectors, as well as transformation of plants is known in the art.
See, e.g., U.S. Pat. No. 6,517,529, incorporated herein by
reference.
[0212] In addition to the antibody chain genes and regulatory
sequences, the recombinant expression vectors 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 (see e.g.,
U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, incorporated
herein by reference). 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. Selectable marker genes include the dihydrofolate
reductase (DHFR) gene (for use in dhfr-host cells with methotrexate
selection/amplification), the neomycin phosphotransferase gene (for
G418 selection), and the glutamate synthetase gene.
[0213] Nucleic acid molecules encoding IL-6 antibodies and vectors
comprising these nucleic acid molecules can be used for
transfection of a suitable mammalian, plant, bacterial or yeast
host cell. Transformation can be any suitable method for
introducing polynucleotides into a host cell. Methods for
introduction of heterologous polynucleotides into mammalian cells
include dextran-mediated transfection, calcium phosphate
precipitation, polybrene-mediated transfection, protoplast fusion,
electroporation, encapsulation of the polynucleotide(s) in
liposomes, and direct microinjection of the DNA into nuclei. In
addition, nucleic acid molecules may be introduced into mammalian
cells by viral vectors. Methods of transforming cells are well
known in the art. See, e.g., U.S. Pat. Nos. 4,399,216, 4,912,040,
4,740,461, and 4,959,455, incorporated herein by reference).
Methods of transforming plant cells are well known in the art,
including, e.g., Agrobacterium-mediated transformation, biolistic
transformation, direct injection, electroporation and viral
transformation. Methods of transforming bacterial and yeast cells
are also well known in the art.
[0214] Mammalian cell lines available as hosts for expression are
well known in the art and include many immortalized cell lines
available from the American Type Culture Collection (ATCC). These
include, for example, Chinese hamster ovary (CHO) cells, NSO cells,
SP2 cells, HEK-293T cells, NIH-3T3 cells, HeLa cells, baby hamster
kidney (BHK) cells, African green monkey kidney cells (COS), human
hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a
number of other cell lines. Cell lines of particular preference are
selected through determining which cell lines have high expression
levels. Other cell lines that may be used are insect cell lines,
such as Sf9 or Sf21 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 various
protein purification methods. Bacterial host cells include E. coli
and Streptomyces species. Yeast host cells include
Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia
pastoris.
[0215] Further, expression of antibodies from production cell lines
can be enhanced using any suitable techniques. For example, the
glutamine synthetase (the GS system) and DHFR gene expression
systems are common approaches for enhancing expression under
certain conditions. High expressing cell clones can be identified
using conventional techniques, such as limited dilution cloning and
Microdrop technology. The GS system is discussed in European Patent
Nos. 0 216 846, 0 256 055, 0 323 997 and 0 338 841.
[0216] It is likely that antibodies expressed by different cell
lines or in transgenic animals will have different glycosylation
from each other. However, all antibodies encoded by the nucleic
acid molecules provided herein, or comprising the amino acid
sequences provided herein are part of the present disclosure,
regardless of the glycosylation of the antibodies.
Phage Display Libraries
[0217] Also provided are methods for producing an IL-6 antibody or
antigen-binding portion thereof comprising the steps of
synthesizing a library of human antibodies on phage, screening the
library with IL-6 or a portion thereof, isolating phage that bind
IL-6, and obtaining the antibody from the phage. By way of example,
one method for preparing the library of antibodies for use in phage
display techniques comprises the steps of immunizing a non-human
animal comprising human immunoglobulin loci with IL-6 or an
antigenic portion thereof to create an immune response, extracting
antibody-producing cells from the immunized animal; isolating RNA
encoding heavy and light chains of antibodies from the extracted
cells, reverse transcribing the RNA to produce cDNA, amplifying the
cDNA using primers, and inserting the cDNA into a phage display
vector such that antibodies are expressed on the phage. Recombinant
IL-6 antibodies may be obtained in this way.
[0218] Recombinant IL-6 human antibodies can be isolated by
screening a recombinant combinatorial antibody library. Preferably
the library is a scFv phage display library, generated using human
V.sub.L and V.sub.H cDNAs prepared from mRNA isolated from B cells.
Methods for preparing and screening such libraries are known in the
art. Kits for generating phage display libraries are commercially
available (e.g., the Pharmacia Recombinant Phage Antibody System,
catalog no. 27-9400-01; and the Stratagene SurfZAP.TM. phage
display kit, catalog no. 240612). There also are other methods and
reagents that can be used in generating and screening antibody
display libraries (see, e.g., U.S. Pat. No. 5,223,409; PCT
Publication Nos. WO 92/18619, WO 91/17271, WO 92/20791, WO
92/15679, WO 93/01288, WO 92/01047, WO 92/09690; Fuchs et al.,
Bio/Technology 9:1370-1372 (1991); Hay et al., Hum. Antibod.
Hybridomas 3:81-85 (1992); Huse et al., Science 246:1275-1281
(1989); McCafferty et al., Nature 348:552-554 (1990); Griffiths et
al., EMBO J. 12:725-734 (1993); Hawkins et al., J. Mol. Biol.
226:889-896 (1992); Clackson et al., Nature 352:624-628 (1991);
Gram et al., Proc. Natl. Acad. Sci. USA 89:3576-3580 (1992); Garrad
et al., Bio/Technology 9:1373-1377 (1991); Hoogenboom et al., Nuc.
Acid Res. 19:4133-4137 (1991); and Barbas et al., Proc. Natl. Acad.
Sci. USA 88:7978-7982 (1991), all incorporated herein by
reference.
[0219] To isolate and produce human IL-6 antibodies with the
desired characteristics, a human IL-6 antibody is first used to
select human heavy and light chain sequences having similar binding
activity toward IL-6, using the epitope imprinting methods
described in PCT Publication No. WO 93/06213, incorporated herein
by reference. The antibody libraries used in this method are
preferably scFv libraries prepared and screened as described in PCT
Publication No. WO 92/01047, McCafferty et al., Nature 348:552-554
(1990); and Griffiths et al., EMBO J. 12:725-734 (1993), all
incorporated herein by reference.
[0220] Once initial human V.sub.L and V.sub.H domains are selected,
"mix and match" experiments are performed, in which different pairs
of the initially selected V.sub.L and V.sub.H segments are screened
for IL-6 binding to select preferred V.sub.L/V.sub.H pair
combinations. Additionally, to further improve the quality of the
antibody, the V.sub.L and V.sub.H segments of the preferred
V.sub.L/V.sub.H pair(s) can be randomly mutated, preferably within
the CDR3 region of V.sub.H and/or V.sub.L, in a process analogous
to the in vivo somatic mutation process responsible for affinity
maturation of antibodies during a natural immune response. This in
vitro affinity maturation can be accomplished by amplifying V.sub.H
and V.sub.L domains using PCR primers complimentary to the V.sub.H
CDR3 or V.sub.L CDR3, respectively, which primers have been
"spiked" with a random mixture of the four nucleotide bases at
certain positions such that the resultant PCR products encode
V.sub.H and V.sub.L segments into which random mutations have been
introduced into the V.sub.H and/or V.sub.L CDR3 regions. These
randomly mutated V.sub.H and V.sub.L segments can be re-screened
for binding to IL-6.
[0221] Following screening and isolation of an IL-6 antibody from a
recombinant immunoglobulin display library, nucleic acids encoding
the selected antibody can be recovered from the display package
(e.g., from the phage genome) and subcloned into other expression
vectors by standard recombinant DNA techniques. If desired, the
nucleic acid can further be manipulated to create other antibody
forms, as described herein. To express a recombinant human antibody
isolated by screening of a combinatorial library, the DNA encoding
the antibody is cloned into a recombinant expression vector and
introduced into mammalian host cells, as described above.
Deimmunized Antibodies
[0222] In another aspect, the IL-6 antibodies or antigen binding
portions thereof may be deimmunized to reduce their immunogenicity
using the techniques described in, e.g., PCT Publication Nos.:
WO98/52976 and WO00/34317 (incorporated herein by reference).
Derivatized and Labeled Antibodies
[0223] An IL-6 antibody or antigen-binding portion can be
derivatized or linked to another molecule (e.g., another peptide or
protein). In general, the antibodies or antigen-binding portion
thereof are derivatized such that the IL-6 binding is not affected
adversely by the derivatization or labeling. Accordingly, the
antibodies and antigen-binding portions are intended to include
both intact and modified forms of the human IL-6 antibodies
described herein. For example, an antibody or antigen-binding
portion can be functionally linked (by chemical coupling, genetic
fusion, noncovalent association or otherwise) to one or more other
molecular entities, such as another antibody (e.g., a bispecific
antibody or a diabody), a detection agent, a label, a cytotoxic
agent, a pharmaceutical agent, and/or a protein or peptide that can
mediate association of the antibody or antigen-binding portion with
another molecule (such as a streptavidin core region or a
polyhistidine tag).
[0224] One type of derivatized antibody is produced by crosslinking
two or more antibodies (of the same type or of different types,
e.g., to create bispecific antibodies). Suitable crosslinkers
include those that are heterobifunctional, having two distinctly
reactive groups separated by an appropriate spacer (e.g.,
m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional
(e.g., disuccinimidyl suberate). Such linkers are available from
Pierce Chemical Company, Rockford, Ill.
[0225] Another type of derivatized antibody is a labeled antibody.
Useful detection agents with which an antibody or antigen-binding
portion may be derivatized include fluorescent compounds,
including, for example, fluorescein, fluorescein isothiocyanate,
rhodamine, 5-dimethylamine-1-naphthalene-sulfonyl chloride,
phycoerythrin, lanthanide phosphors. An antibody can also be
labeled with enzymes that are useful for detection, such as, for
example, horseradish peroxidase, .beta.-galactosidase, luciferase,
alkaline phosphatase, or glucose oxidase. When an antibody is
labeled with a detectable enzyme, it is detected by adding
additional reagents that the enzyme uses to produce a reaction
product that can be discerned. For example, when the agent
horseradish peroxidase is present the addition of hydrogen peroxide
and diaminobenzidine leads to a colored reaction product, which is
detectable. An antibody can also be labeled with biotin, and
detected through indirect measurement of avidin or streptavidin
binding. An antibody can also be labeled with a predetermined
polypeptide epitope recognized by a secondary reporter (e.g.,
leucine zipper pair sequences, binding sites for secondary
antibodies, metal binding domains, epitope tags). In some cases,
labels are attached by spacer arms of various lengths to reduce
potential steric hindrance. An IL-6 antibody can also be
derivatized with a chemical group such as polyethylene glycol
(PEG), a methyl or ethyl group, or a carbohydrate group. These
groups are useful to improve the biological characteristics of the
antibody, e.g., to increase serum half-life.
[0226] The specific Examples set forth herein are intended to
illustrate particular aspects of the disclosure and are not
intended to limit the scope of the claims.
Example 1
Generation of Hybridomas Producing Anti-IL-6 Antibody
[0227] Exemplary antibodies in accordance with the disclosure were
prepared, selected, and assayed as follows:
Mouse Strains
[0228] Fully human monoclonal antibodies to human IL-6 were
prepared using human Ig transgenic mouse strains HCo7 and HCo12, as
well as the human transchromosomal/transgenic strain, KM (Medarex,
Inc.). These strains all express fully human antibodies that are
indistinguishable from antibodies isolated from humans.
[0229] In all three strains, both the endogenous mouse kappa light
chain gene and the endogenous mouse heavy chain gene have been
homozygously disrupted as described in Chen et al. (1993) EMBO J.
12:811-820 and in Example 1 of PCT Publication WO 01/09187,
respectively. Moreover, all three carry a human kappa light chain
transgene, KCo5, as described in Fishwild et al. (1996) Nature
Biotechnology 14:845-851. In contrast, the three strains are
distinct with respect to their human heavy chain genes. The HCo7
strain carries the HCo7 human heavy chain transgene as described in
U.S. Pat. Nos. 5,545,806, 5,625,825, and 5,545,807; the HCo12
strain carries the HCo12 human heavy chain transgene as described
in Example 2 of PCT Publication WO 01/09187; and the KM strain
carries a human mini-chromosome as described in Ishida et al.,
(2002), Cloning and Stem Cells, 4: 91-102.
Immunization with IL-6 Antigen and Selection of HuMab Mice
Producing Anti-IL-6 Monoclonal Antibodies
[0230] General immunization schemes for HuMab mice are described in
Lonberg et al (1994) Nature 368(6474): 856-859; Fishwild et al.
(1996) Nature Biotechnology 14: 845-851 and PCT Publication WO
98/24884. In the present case, a total of 81 HuMab mice of the
HCo7, HCo12 and KM strains were immunized beginning at 6-16 weeks
of age with 5-25 .mu.gs of purified human IL-6 in Ribi adjuvant.
Human IL-6 was isolated from a human bone marrow-derived stromal
cell, HS-5 (ATCC CRL-11882, Roecklein B. A. & Torok-Storb B.,
Blood 85: 997-1005, 1995), which endogenously secretes IL-6 into
the media. Human IL-6 was purified from IL-6 expressing HS-5 media,
which was concentrated by ultrafiltration followed by a Q Sepharose
anion exchange chromatography step and an affinity chromatography
step using a mouse anti-hIL-6 MAb (R&D Systems, Catalog number
MAB2061, clone 1936). The purified human IL-6 had a purity of about
90% by SDS-PAGE. The major bands were excised from the SDS-PAGE
gel, digested with trypsin and the extracted gel purified tryptic
peptides were analyzed via MALDI/MS on the 4700 TOF/TOF Proteomics
Analyzer and were confirmed to be human IL-6. Alternatively, one
can use recombinant human IL-6 from commercial sources (for
example, Recombinant Human IL-6, catalog number 206-IL-6/CF.
R&D System Inc. 614 Mckinley Place NE, Minneapolis, Minn.
55413). Administration was via injection intra-peritoneally,
subcutaneously or into the footpad at 3-14 day intervals, up to a
total of 8 immunizations. Immune response was monitored via ELISA
screening, as described below.
Selection of HuMab Mice Producing Anti-IL-6 Antibodies
[0231] Blood from the transgenic mice described above was obtained
via retro-orbital bleeds and analyzed by ELISA for specific binding
to purified human IL-6 recombinant protein, as described by
Fishwild et al. (1996), Nature Biotechnology 14: 845-851.
[0232] Briefly, microtiter plates were coated using 50 .mu.l/well
of a purified recombinant IL-6 solution containing 1 .mu.g/ml in
PBS, and incubated overnight at 4.degree. C. The wells were then
blocked using 200 .mu.l/well of 5% chicken serum in PBS/Tween
(0.05%). Dilutions of plasma from IL-6-immunized mice were added to
each well and incubated for 1 hour at ambient temperature. The
plates were washed with PBS/Tween and then incubated with a
goat-anti-human IgG Fc polyclonal antibody conjugated with
horseradish peroxidase (HRP) for 1 hour at room temperature. After
washing, the plates were developed with ABTS substrate (Moss Inc,
product #: ABTS-1000 mg/ml) and analyzed by spectrophotometer at OD
415-495. Mice that developed the highest titers of anti-IL-6
antibodies (22 animals total) were used for fusions.
Generation of Hybridomas Producing Human Monoclonal Antibodies to
IL-6:
[0233] The mice selected above were boosted intravenously with IL-6
at 3 days and then again at 2 days prior to sacrifice and removal
of the spleen and/or lymph nodes. A total of 17 fusions were
performed.
[0234] The mouse splenocytes and/or lymph node lymphocytes isolated
from immunized HuMab or KM mice, were fused to SP2/0 non-secreting
mouse myeloma cells (ATCC, CRL-1581, ATCC American Type Culture
Collection, 1080 University Boulevard, Manassas, Va. 20110-2209
USA) using electrofusion (E-fusion, Cyto Pulse.TM. technology, Cyto
Pulse.TM. Sciences, Inc., Glen Burnie, Md.), according to standard
or manufacturer-recommended protocols.
[0235] Briefly, single cell suspensions of splenocytes and/or lymph
node lymphocytes from immunized mice were prepared and then
combined with an equal number of Sp2/0 non-secreting mouse myeloma
cells; E-fusion was then performed.
[0236] The cells were then plated at 2.times.10.sup.4 cells/well in
flat bottom microtiter plate, and incubated for 10-14 days in
selective medium containing 10% fetal bovine serum, 10% P388D1
(ATCC, CRL-TIB-63) conditioned medium, 3-5% (IGEN) in DMEM
(Mediatech, Herndon, Va., Cat. No. CRL 10013, with high glucose,
L-glutamine and sodium pyruvate), 5 mM HEPES, 0.055 mM
2-mercaptoethanol, 50 mg/ml gentamycin and 1.times.HAT (Sigma, Cat.
No. CRL-P-7185).
[0237] After 1-2 weeks, cells were cultured in medium in which the
HAT was replaced with HT. Approximately 10-14 days after cell
plating supernatants from individual wells were screened for the
presence of human gamma, kappa antibodies. The supernatants which
scored positive for human gamma, kappa were then screened by ELISA
(using the protocol described above) for human anti-IL-6 monoclonal
IgG antibodies. The antibody-secreting hybridomas were transferred
to 24 well plates, screened again and, if confirmed positive for
human anti-IL-6 IgG monoclonal antibodies, were subcloned at least
twice by limiting dilution. The stable subclones were then cultured
in vitro to generate small amounts of antibody in tissue culture
medium for further characterization.
Example 2
Sequencing of IL-6 Antibodies
[0238] Full-length anti-IL-6 antibodies were cloned and sequence
verified from hybridomas as follows: Poly(A).sup.+ mRNA was
isolated using an RNeasy Mini Kit (Qiagen) and cDNA synthesized
from the mRNA with the Advantage RT-for-PCR kit (BD Biosciences)
using oligo(dT) priming. The oligo(dT) primed cDNA for clone 9C8
was amplified using degenerate primers listed in Table 1
respectively.
TABLE-US-00001 TABLE 1 Degenerate primers (5' to 3') for 9C8
VH4_5UTR_F CTTTCTGAGASTCMTGGAKCTCMTG SEQ ID NO: 49 G_3UTR_R
TACGTGCCAAGCATCCTCGC SEQ ID NO: 50 VK1a_5UTR_F
GSARTCAGWCYCWVYCAGGACACAGC SEQ ID NO: 51 K_3UTR_F
AGGCTGGAACTGAGGAGCAGGTG SEQ ID NO: 52
[0239] Amplification was achieved using the High Fidelity
Polymerase (Roche) and a PTC-200 DNA Engine (MJ Research) with
cycling as follows: 2'@95.degree. C.; 25.times.(20''@95.degree. C.,
30''@52.degree. C., 2'@72.degree. C.); 10'@72.degree. C. PCR
amplicons were cloned into the pCR2.1 TOPO and transformed into
TOP10 chemically competent cells (Invitrogen) using the standard
protocol. Clones were sequence verified using Grills 16.sup.th
BDTv3.1/dGTP chemistry (Applied Biosystems Inc) and a 3730xl DNA
Analyzer (Applied Biosystems Inc). All sequences were analyzed by
alignments to the `V BASE sequence directory` (Tomlinson, et al, J.
Mol. Biol., 227, 776-798 (1992); Hum. Mol. Genet., 3, 853-860
(1994); EMBO J., 14, 4628-4638 (1995). The germline gene segment
usages of exemplary anti IL-6 antibodies are listed in Table 2.
TABLE-US-00002 TABLE 2 Heavy chain Light chain Clone V.sub.H D
J.sub.H V.sub.K J.sub.K Subtype 9C8 4-34 -- 3b L15 JK1 IgG1 22B5
4-34 -- 3b L15 JK1 IgG1
Full-Length Sequences of the ANTI-IL-6 Antibody Derived from
Hybridomas 9C8
TABLE-US-00003 DNA sequence of 9C8 heavy chain from hybridoma cells
(variable domain in uppercase) SEQ ID NO: 1
CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCAC
CTGCGCTATCTATGGTGGGTCCTTCAGGGAGTACTACTGGAGCTGGATCCGCCAGCCCCCAG
GGAAGGGGCTGGAGTGGATTGGGGAAATCTTTCATAGTGGAAGCACCAACTACAACCCGTCC
CTCAAGAGTCGAGTCAACATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAC
CTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGAGGAATTAGATGATTTTG
ATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAgcctccaccaagggcccatcggtc
ttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggt
caaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcg
tgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgacc
gtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaa
caccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgt
gcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggac
accctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaaga
ccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagc
cgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccag
gactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccat
cgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccc
catcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctat
cccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccac
gcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaaga
gcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccac
tacacgcagaagagcctctccctgtctccgggtaaa Derived protein sequence (by
translation) of 9C8 heavy chain from hybridoma cells (variable
domain in uppercase) SEQ ID NO: 3
QVQLQQWGAGLLKPSETLSLTCAIYGGSFREYYWSWIRQPPGKGLEWIGEIFHSGSTNYNPS
LKSRVNISVDTSKNQFSLKLTSVTAADTAVYYCAREELDDFDIWGQGTMVTVSSastkgpsv
fplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvt
vpssslgtqtyicnvnhkpsntkvdkrvepkscdkthtcppcpapellggpsvflfppkpkd
tlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhq
dwingkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfy
psdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnh
ytqkslslspgk DNA sequence of 9C8 light chain from hybridoma cells
(variable domain in uppercase) SEQ ID NO: 12
GACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCAT
CACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGAGA
AAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTC
AGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGTAGCCTGCAGCCTGAAGATTT
TGCAACTTATTACTGCCAACAGTATAAAAGTTACCCTCGGACGTTCGGCCAAGGGACCAAGG
TGGAAATCAAAcgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcag
ttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaa
agtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagc
aggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactac
gagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaa
gagcttcaacaggggagagtgt Derived protein sequence (by translation) of
9C8 light chain from hybridoma cells (variable domain in uppercase)
SEQ ID NO: 14
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRF
SGSGSGTDFTLTISSLQPEDFATYYCQQYKSYPRTFGQGTKVEIKrtvaapsvfifppsdeq
lksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskady
ekhkvyacevthqglsspvtksfnrgec
ANTI-IL-6 Antibodies Variable Domains were Cloned into Expression
Vectors as Follows:
[0240] The variable domains were amplified from pCR2.1 cloned cDNA
using primers listed in Table 3. Amplification was achieved using
the Pfx Platinum polymerase (Invitrogen) and a PTC-200 DNA Engine
(MJ Research) with cycling as follows: 2'@94.degree. C.;
20.times.(30''@94.degree. C., 45''@55.degree. C., 1'@68.degree.
C.); 5'@68.degree. C. The variable domains were then cloned into
expression vectors containing constant domains of the appropriate
isotype. These clones were sequence verified using Grills 16.sup.th
BDTv3.1/dGTP chemistry (Applied Biosystems Inc) and a 3730xl DNA
Analyzer (Applied Biosystems Inc).
TABLE-US-00004 TABLE 3 Variable domain primers (5' to 3') for 9C8
H4_34 ttacagtGCGCGCACTCCCAGGTGCAGCTACAGCAGTGG SEQ ID NO: 53 K_O12
ttacagtGTGCACTCCGACATCCAGATGACCCAGTCTCC SEQ ID NO: 54 G1/2_ch1
GAAGACCGATGGGCCCTTGG (Apal)_R SEQ ID NO: 55 JK1_R
tatattccttaattaagttattctactcacGTTT GATTTCCACCTTGGTCCCT SEQ ID NO:
56
Full-Length Sequences of Recombinant ANTI-IL-6 Antibodies 9C8
TABLE-US-00005 [0241] DNA sequence of recombinant 9C8 lgG2 heavy
chain (variable domain in uppercase) SEQ ID NO: 23
CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCAC
CTGCGCTATCTATGGTGGGTCCTTCAGGGAGTACTACTGGAGCTGGATCCGCCAGCCCCCAG
GGAAGGGGCTGGAGTGGATTGGGGAAATCTTTCATAGTGGAAGCACCAACTACAACCCGTCC
CTCAAGAGTCGAGTCAACATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAC
CTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGAGGAATTAGATGATTTTG
ATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAgcctccaccaagggcccatcggtc
ttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggt
caaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcg
tgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtagtgacc
gtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaa
caccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcac
cacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatc
tcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtcca
gttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagc
agttcaacagcacgttccgtgtggtcagcgtcctcaccgtcgtgcaccaggactggctgaac
ggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccat
ctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggagg
agatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatc
gccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgct
ggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagc
aggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaag
agcctctccctgtctccgggtaaa Derived protein sequence (by translation)
of recombinant 9C8 lgG2 heavy chain (variable domain in uppercase)
SEQ ID NO: 24
QVQLQQWGAGLLKPSETLSLTCAIYGGSFREYYWSWIRQPPGKGLEWIGEIFHSGSTNYNPS
LKSRVNISVDTSKNQFSLKLTSVTAADTAVYYCAREELDDFDIWGQGTMVTVSSastkgpsv
fplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvt
vpssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappvagpsvflfppkpkdtlmi
srtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwln
gkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdi
avewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqk
slslspgk DNA sequence of recombinant 9C8 Kappa light chain
(variable domain in uppercase) SEQ ID NO: 25
GACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCAT
CACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGAGA
AAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTC
AGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGTAGCCTGCAGCCTGAAGATTT
TGCAACTTATTACTGCCAACAGTATAAAAGTTACCCTCGGACGTTCGGCCAAGGGACCAAGG
TGGAAATCAAAcgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcag
ttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaa
agtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagc
aggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactac
gagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaa
gagcttcaacaggggagagtgt Derived protein sequence (by translation) of
recombinant 9C8 Kappa light chain (variable domain in uppercase)
SEQ ID NO: 26
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRF
SGSGSGTDFTLTISSLQPEDFATYYCQQYKSYPRTFGQGTKVEIKrtvaapsvfifppsdeq
lksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskady
ekhkvyacevthqglsspvtksfnrgec
Example 3
Mutagenesis of Anti-IL-6 Antibody
[0242] Amino acid substitution variants of the anti-IL-6 antibody
9C8 in the heavy chain variable domain were made at positions 24
(124V), 30 (R30S), 31 (E31 G), 52 (F52N), 68 (N68T), 83 (T83S)
either singly or in combination both in the context of an IgG1 or
an IgG2 format. Amino acid substitution variants of the anti-IL-6
antibody 9C8 in the light chain variable domain were made at 92
(K92N) both as an IgG1 or an IgG2 format. Antibodies having both a
heavy chain variable domain and a light chain variable domain
variant were made. Some of the various mutation combinations are
shown in Table 6a and 6b. Mutagenesis, in the V.sub.H (124V),
V.sub.H (E31 G), V.sub.H (N68T), and V.sub.H (T83S) regions of
clone 9C8, was conducted with the primers listed in Table 4 (sense
strands shown; targeted residue shown in bold) and the QuickChange
kit (Stratagene) according to the manufacturer's instructions. The
mutated variants were sequence verified and cloned into expression
vectors by standard procedures.
TABLE-US-00006 TABLE 4 Mutagenic primers (5' to 3') for 9C8
9C8_H_I24V CCTCACCTGCGCTGTCTATGGTGGGTCC SEQ ID NO: 57 9C8_H_E31G
GGGTCCTTCAGGGGGTACTACTGGAGCTG SEQ ID NO: 58 9C8_H_N68T
CCTCAAGAGTCGAGTCACCATATCAGTAGACACG SEQ ID NO: 59 9C8_H_T83S
CTCCCTGAAGCTGAGCTCTGTGACCGCC SEQ ID NO: 60
TABLE-US-00007 DNA sequence of recombinant 9C8 lgG2 heavy chain
(N68T, T83S) heavy chain SEQ ID NO: 27
CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCAC
CTGCGCTATCTATGGTGGGTCCTTCAGGGAGTACTACTGGAGCTGGATCCGCCAGCCCCCAG
GGAAGGGGCTGGAGTGGATTGGGGAAATCTTTCATAGTGGAAGCACCAACTACAACCCGTCC
CTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAG
CTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGAGGAATTAGATGATTTTG
ATATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCAgcctccaccaagggcccatcggtc
ttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggt
caaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcg
tgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtagtgacc
gtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaa
caccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcac
cacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatc
tcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtcca
gttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagc
agttcaacagcacgttccgtgtggtcagcgtcctcaccgtcgtgcaccaggactggctgaac
ggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccat
ctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggagg
agatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatc
gccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgct
ggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagc
aggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaag
agcctctccctgtctccgggtaaa Derived protein sequence (by translation)
of recombinant 9C8 lgG2 heavy chain (N68T, T83S) heavy chain SEQ ID
NO: 28
QVQLQQWGAGLLKPSETLSLTCAIYGGSFREYYWSWIRQPPGKGLEWIGEIFHSGSTNYNPS
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREELDDFDIWGQGTMVTVSSastkgpsv
fplapcsrstsestaalgolvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvt
vpssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappvagpsvflfppkpkdtlmi
srtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwln
gkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdi
avewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqk
slslspgk DNA sequence of recombinant 9C8 lgG1 heavy chain (N68T,
T83S) heavy chain SEQ ID NO: 31
CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCAC
CTGCGCTATCTATGGTGGGTCCTTCAGGGAGTACTACTGGAGCTGGATCCGCCAGCCCCCAG
GGAAGGGGCTGGAGTGGATTGGGGAAATCTTTCATAGTGGAAGCACCAACTACAACCCGTCC
CTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAG
CTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGAGGAATTAGATGATTTTG
ATATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCAgcctccaccaagggcccatcggtc
ttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggt
caaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcg
tgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtagtgacc
gtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaa
caccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgt
gcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggac
accctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaaga
ccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagc
cgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccag
gactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccat
cgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccc
catcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctat
cccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccac
gcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaaga
gcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccac
tacacgcagaagagcctctccctgtctccgggtaaa Protein sequence of
recombinant 9C8 lgG1 heavy chain (N68T, T83S) heavy chain SEQ ID
NO: 32
QVQLQQWGAGLLKPSETLSLTCAIYGGSFREYYWSWIRQPPGKGLEWIGEIFHSGSTNYNPS
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREELDDFDIWGQGTMVTVSSastkgpsv
fplapsskstsggtaalgolvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvt
vpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkd
tlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhq
dwingkeykckvsnkalpapiektiskakgqprepqvytlppsreemtknqvsltclvkgfy
psdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnh
ytqkslslspgk Variable domain DNA sequence of recombinant 9C8 (E31G,
N68T, T83S) heavy chain SEQ ID NO: 33
CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCAC
CTGCGCTATCTATGGTGGGTCCTTCAGGGGGTACTACTGGAGCTGGATCCGCCAGCCCCCAG
GGAAGGGGCTGGAGTGGATTGGGGAAATCTTTCATAGTGGAAGCACCAACTACAACCCGTCC
CTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAG
CTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGAGGAATTAGATGATTTTG
ATATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCA Variable domain translated
protein sequence of recombinant 9C8 (E31G, N68T, T83S) heavy chain
SEQ ID NO: 34
QVQLQQWGAGLLKPSETLSLTCAIYGGSFRGYYWSWIRQPPGKGLEWIGEIFHSGSTNYNPS
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREELDDFDIWGQGTMVTVSS Variable
domain DNA sequence of recombinant 9C8 (124V, N68T, T83S) heavy
chain SEQ ID NO: 36
CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCAC
CTGCGCTGTCTATGGTGGGTCCTTCAGGGAGTACTACTGGAGCTGGATCCGCCAGCCCCCAG
GGAAGGGGCTGGAGTGGATTGGGGAAATCTTTCATAGTGGAAGCACCAACTACAACCCGTCC
CTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAG
CTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGAGGAATTAGATGATTTTG
ATATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCA Variable domain translated
protein sequence of recombinant 9C8 (124V, N68T, T83S) heavy chain
SEQ ID NO: 37
QVQLQQWGAGLLKPSETLSLTCAVYGGSFREYYWSWIRQPPGKGLEWIGEIFHSGSTNYNPS
LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREELDDFDIWGQGTMVTVSS
[0243] The CMV promoter containing expression vectors were
transfected in 293 Freestyle (Invitrogen) cells according to the
vendor's protocol. Supernatants from these cells were collected by
centrifugation and purified by standard Protein-A affinity
chromatography to isolate recombinant immunoglobulins. These
proteins were then characterized by SDS-PAGE, light scatter, and
spectrophotometrically.
[0244] The heavy and light chains of the anti-IL-6 antibodies
indicated in Table 5 were deposited under terms in accordance with
the Budapest Treaty with the American Type Culture Collection
(ATCC), 10801 University Blvd., Manassas, Va. 20110-2209. The heavy
& light chains have been assigned the following accession
numbers:
TABLE-US-00008 TABLE 5 Clone ATCC Patent Deposit Deposit Antibody
Designation Designation Designation Date 9C8 heavy E. coli; pCR2.1
UC25510 PTA-8013 Nov. 21, chain IgG1 TOPO 2006 9C8H(WT) 9C8 light
chain E. coli; pCR2.1 UC25511 PTA-8014 Nov. 21, IgG1 TOPO 2006
9C8L(WT) 9C8 N68T, E. coli; pCR2.1 UC25512 PTA-8015 Nov. 21, T83S
IgG2 TOPO 2006 heavy chain 9C8H(NT)hG2 9C8 N68T, E. coli; pCR2.1
UC25513 PTA-8016 Nov. 21, T83S IgG2 TOPO 2006 light chain
9C8L(wt)hk 9C8 E31G, E. coli; pCR2.1 UC25514 PTA-8017 Nov. 21,
N68T, T83S TOPO 2006 variable region 9C8H(ENT) 9C8 I24V, E. coli;
pCR2.1 UC25515 PTA-8018 Nov. 21, N68T, T83S TOPO 2006 variable
region 9C8H(INT) 9C8 N68T, E. coli; pCR2.1 UC25526 PTA-8019 Nov.
21, T83S IgG1 TOPO 2006 heavy chain 9C8H(NT)hG1
Example 4
TF-1 Proliferation
[0245] Human cells, TF-1 cells, from American Type Culture
Collection (ATCC) (Manassas, Va.), were obtained and maintained in
RPMI-1640 medium containing 10% heat inactivated fetal bovine serum
(FBS) (Invitrogen, Carlsbad, Ca.), with 2 ng/ml recombinant human
GM-CSF. TF-1 cells were split to 1-2.times.10.sup.5 for next day
use. Prior to plating, the cells were washed three times with
RPMI-1640, counted, and the volume adjusted with assay medium to
yield 2.times.10.sup.5 cells/ml. To each well, 50 .mu.l of the
washed cells was added and incubated overnight at 37.degree. C.
with 5% CO.sub.2. All conditions were conducted in triplicate in
96-well tissue culture treated plates (Corning, Corning, N.Y.). To
each well, either 25 ng/ml or 2.5 ng/ml IL-6 in a volume of 25
.mu.l and test or control antibodies at various concentrations in a
volume of 25 .mu.l in sodium phosphate buffer (10 mM sodium
phosphate and 150 mM sodium chloride, pH 7.4) to a final volume of
100 .mu.l was added. Antibodies were tested alone and with human
IL-6. The plates were incubated for 48 hours (hrs) at 37.degree. C.
with 5% CO.sub.2. After 48 hours, 10 .mu.l/well of 0.5 .mu.Ci
.sup.3H-thymidine (Amersham Biosciences, Piscataway, N.J.) was
added and pulsed with the cells for 3 hrs. To detect the amount of
incorporated thymidine, the cells were harvested onto pre-wet
unifilter GF/C filterplates (Packard, Meriden, Ct.) and washed 10
times with water. The plates were allowed to dry overnight. Bottom
seals were added to the filterplates. Next, 45 .mu.l Microscint 20
(Packard, Meriden, Ct.) per well was added. After a top seal was
added, the plates were counted in a Trilux microbeta counter
(Wallac, Norton, Ohio) and data is reported as CPM (counts per
minute). Tables 6a and 6b show the IC50s in the TF-1 proliferation
assay of the 9C8 antibody and the various antibodies having amino
acid substitutions. The results shown in Table 6a and Table 6b are
from assays done on two separate occasions.
TABLE-US-00009 TABLE 6a Average IC50 Fold Hc Lc (.mu.g/ml)
Difference 9C8 9C8 0.0022 1.0 9C8 I24V 9C8 0.0071 3.3 9C8 R30S 9C8
0.0260 12.1 9C8 N68T 9C8 0.0028 1.3 9C8 T83S 9C8 0.0026 1.2 9C8
I24V, N68T, T83S 9C8 0.0145 6.7 9C8 I24V, R30S, N68T, T83S 9C8
0.2040 94.9 9C8 E31G 9C8 0.0051 2.4 9C8 F52N 9C8 1.3850 644.2 9C8
9C8 K92N 0.0120 5.6
TABLE-US-00010 TABLE 6b Average IC50 Fold Hc Lc (.mu.g/ml)
Difference 9C8 9C8 0.0031 1.4 9C8 N68T, T83S 9C8 0.0030 1.4 9C8
E31G, N68T, T83S 9C8 0.0083 3.9 9C8 I24V, E31G, N68T, T83S 9C8
0.0528 24.5 9C8 N68T, T83S 9C8 9C8 0.0186 8.6 K92N 9C8 E31G, N68T
T83S 9C8 K92N 0.1015 47.2 9C8 I24V, E31G N68T, T83S 9C8 K92N 0.9670
449.9 Hc = heavy chain, Lc = light chain
Example 5
C-Reactive Protein from LPS-Monkey Study
[0246] The in-vivo portion of this study was conducted by Charles
River Laboratories Preclinical Services at their Worcester, Mass.
Test Facility. Briefly, the study consisted of fifteen male
cynomolgus monkeys (five groups; 3 monkeys/group). On Day 1 animals
in group 1 received vehicle; animals in Groups 2 and 3 received the
antibody 9C8 N68T T83S IgG.sub.1 at doses of 0.5 and 5 mg/kg,
respectively; and animals in Groups 4 and 5 received 9C8 N68T T83S
IgG.sub.2 at doses of 0.5 and 5 mg/kg, respectively. All treatments
were administered by IV bolus injection at dose volumes of 1 mL/kg
each. Approximately 2 hours after treatment, all animals were
challenged with 10 .mu.g/kg bacterial lipopolysaccharide (LPS) at a
volume of 1 mL/kg by slow IV bolus injection. On Day 1 blood was
collected from a peripheral vessel at baseline (prior to
treatment); immediately following treatment; 2 hours post-treatment
(immediately prior to LPS administration); and at 30 minutes and 1,
2, 3, 4, 6, 8, and 22 hours after LPS challenge. Blood samples were
also collected on Days 3, 4, 5, 6 and 7. Whole blood samples were
processed for serum and serum samples were stored frozen.
C-Reactive Protein (CRP) was measured using a human Vascular Injury
Panel II Multi-Spot.RTM. Assay Kit from Meso Scale Discovery
(MSD.RTM.), Gaithersburg, Md. Assays were performed as outlined in
published kit instructions from MSD. FIG. 2 shows the total serum
CRP for the 9C8 N68T T83S IgG.sub.2 antibody.
Example 6
pSTAT3 Assay by Flow Cytometry
[0247] In vitro assays were conducted in human whole blood and
human peripheral blood mononuclear cells (PBMCs) stimulated with
recombinant human IL-6 (rhIL-6) to measure phosphorylated STAT3
(pSTAT3) levels in the presence of anti-IL-6 antibodies.
Whole Blood Assay
[0248] Freshly collected heparinized human whole blood was
incubated with anti-IL-6 antibody or vehicle control (final volume
300 .mu.L in 15 mL polypropylene tube) for 15 minutes at 37.degree.
C. Samples were stimulated with recombinant human (rh) IL-6 for a
final concentration of 25 ng/mL and incubated for 10 minutes at
37.degree. C. Red blood cells (RBCs) were then lysed with 4 mL RBC
Lysing Buffer (Sigma), and samples were mixed gently for 10 minutes
at 37.degree. C. Samples were spun at 400.times.g for 5 min to
pellet cells. Supernatants were removed. Two milliliters of wash
buffer (4% BSA in PBS, Gibco) were added to each tube, and then
samples were spun at 400.times.g for 5 min to pellet cells.
Supernatants were removed, and cell pellets were resuspended in 200
.mu.L of preheated (37.degree. C.) permeabilization buffer (2%
formaldehyde in PBS, Polysciences, Inc.) and incubated at
37.degree. C. for 10 minutes. Three milliliters of ice cold MeOH
was added to each sample to fix the cells. Samples were mixed and
placed on ice for at least 30 min. Samples were spun (400.times.g
for 5 min.) and supernatants were removed. Pellets were washed once
with wash buffer. Cell pellets were then stained with
anti-phosphorylated STAT3 (Y705) antibody conjugated to Alexa Fluor
488 (BD Pharmingen) diluted in wash buffer, final volume 100 .mu.L.
Samples were incubated on ice for 30 minutes then washed 2.times.
in wash buffer. Final cell pellets were resuspended in 400 .mu.L IF
Buffer (Hank's Buffered Saline Solution, 2% Fetal Calf Serum, 10 mM
Hepes, 0.2% Sodium Azide, Gibco). A FACSCalibur instrument using
CellQuest software (BD Biosciences) was used to collect and analyze
data. Table 7 shows the pSTAT3 levels in the presence of anti-IL-6
antibodies as measured in human whole blood.
TABLE-US-00011 TABLE 7 IL-6 Conc WB IC50 Anti IL-6 Antibody ng/ml
.mu.g/ml Assay # 9C8 25 0.487 1 25 0.432 2 25 0.482 3 I24V, N68T,
T83S 25 0.420 1 25 0.527 2 25 0.749 3 E31G, N68T, T83S 25 0.626 1
25 0.714 2 25 0.968 3 I24V, E31G, N68T, T83S 25 0.364 1 25 0.603 2
25 0.821 3
PBMC Assay
[0249] Peripheral blood mononuclear cells (PBMCs) were isolated
from freshly collected heparinized human whole blood using Accuspin
System-Histopaque 1077 columns according to manufacture's protocol
(Sigma-Aldrich A7054) and placed in (0.1% penicillin streptomycin
in Macrophage SFM Media, Gibco). Approximately 2.times.10.sup.6
PBMCs were incubated with anti-IL-6 antibody or vehicle control
(final vol. 300 .mu.L) for 15 min. at 37.degree. C. PBMCs were
stimulated with rhIL-6 for a final concentration of 25 ng/mL and
incubated for 10 min. at 37.degree. C. Samples were spun at
400.times.g for 5 min to pellet cells. Supernatants were removed.
Two mL wash buffer (4% BSA in PBS) was added to each sample.
Samples were spun at 400.times.g 5 min to pellet cells.
Supernatants were removed and cell pellets were resuspended in 200
uL of preheated (37.degree. C.) permeabilization buffer (2%
formaldehyde in PBS, Polysciences, Inc.) and incubated at
37.degree. C. for 10 min. Three milliliters of ice cold MeOH was
added to each sample to fix the cells. Samples were mixed and kept
on ice for at least 30 min. Samples were spun at 400.times.g for 5
min. and supernatants were removed. Cell pellets were washed once
with wash buffer then stained and analyzed as described in the
whole blood assay. Table 8 shows the pSTAT3 levels in the presence
of anti-IL-6 antibodies as measured in human peripheral blood
mononuclear cells.
TABLE-US-00012 TABLE 8 IL-6 conc. PBMC IC50 Anti IL-6 Antibody
ng/ml .mu.g/ml 9C8 25 0.210 R&D MAB2061 25 0.580
Example 7
Binding Affinity
Preparation of BIAcore Chips
[0250] The anti-IL-6 antibody 9C8 N68T T83S IgG2 was immobilized
onto BIAcore CM5 chips (GE Biosciences--formerly BIAcore Inc,
Piscataway, N.J.) by amine coupling to carboxymethylcellulose (CM)
attached to the dextran matrix of the chip as described by Lofas
& Johnsson (J. Chem. Soc. Chem. Commun. (1990); 21:pp
1526-1528). The chip was pretreated with 1 M NaCl and 50 mM NaOH
prior to coupling. Amine coupling was accomplished by combining EDC
(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and NHS
(N-hydroxysuccinimide) and passing across the chip to activate the
CM groups. The ligand in 10 mM acetate buffer pH 5 was passed
across the chip surface and covalently bound to the activated CM
group. The remaining active CM groups on the chip surface were
quenched by passing 1M ethanolamine pH 8.5 across the chip. The
EDC, NHS, and ethanolamine were obtained as part of the Amine
Coupling kit obtained from GE Biosciences. Coupling was performed
using the automated Surface Preparation Wizard included with the
BiaControl Software V3.2.
Determination of Binding Affinity
[0251] All SPR measurements were performed on a BIAcore 3000
instrument (GE Biosciences, Piscataway, N.J.). BIAcore
Software-BIAcore 3000 Control Software V3.2 was used for the
operation and control of the BIAcore 3000 instrument. BiaEvaluation
Software V4.1 was used for the analysis of SPR data from the
BIAcore 3000 instrument and data was plotted using Graph Pad Prism
Software Version 5. The binding affinity of IL-6 to monoclonal
antibodies (mAb) was measured in HBS-EP buffer (10 mM HEPES, 150 mM
NaCl, 3.4 mM EDTA, 0.005% P20) at 25.degree. C. The flow rate for
the affinity study was 40 uL/minute to minimize mass transport
effects (Myszka, D. G., et al., Biophysical chemistry. 64, 127-137,
1997). Human IgG.sub.K was used as the ligand for the construction
of the reference channel of the chip. Analyte (rhIL-6; R&D
Systems, Minneapolis, Minn., 206-IL) binding to the immobilized
ligand (9C8 N68T T83S IgG.sub.2) was measured in duplicate and the
concentration of the IL-6 ranged from 0 to 25 nM. Injection time
for IL-6 was 6 minutes and dissociation time was 25 minutes. The
surface was regenerated between cycles by 10 mM glycine pH 1.7 for
30 seconds at a flow rate of 30 uL/min. The regeneration conditions
were established to be optimal after a regeneration study (data not
shown). Data was analyzed by using the Kinetics Wizard and the
manual fitting programs that are both included with the
BiaEvaluation Software V4.1 using a 1:1 Langmuir model (Karlson R
& Falt A., J. Immunol. Methods. 200: pp 121-133, 1997). The
anti IL-6 antibody 9C8 N68T T83S IgG.sub.2 was shown to have
k.sub.a=9.95E+05 (Ms).sup.-1, a k.sub.d=1.34E-04 s.sup.-1, and a
K.sub.D.sup.=1.35 E-10 M or 135 pM.
TABLE-US-00013 SUMMARY OF SEQUENCE LISTING (sequences are amino
acid sequences except those indicated by `n.a.` for nucleic acid)
SEQ ID NO: SEQUENCE 1 heavy 9C8 IgG1 n.a. 2 V.sub.H 9C8 n.a. 3
heavy 9C8 IgG1 4 V.sub.H 9C8 5 V.sub.H CDR1 9C8 6 V.sub.H CDR2 9C8
7 V.sub.H CDR3 9C8 8 V.sub.H FR1 9C8 9 V.sub.H FR2 9C8 10 V.sub.H
FR3 9C8 11 V.sub.H FR4 9C8 12 light 9C8 IgG1 n.a. 13 V.sub.L 9C8
n.a. 14 light 9C8 IgG1 15 V.sub.L 9C8 16 V.sub.L CDR1 9C8 17
V.sub.L CDR2 9C8 18 V.sub.L CDR3 9C8 19 V.sub.L FR1 9C8 20 V.sub.L
FR2 9C8 21 V.sub.L FR3 9C8 22 V.sub.L FR4 9C8 23 heavy 9C8 IgG2
n.a. 24 heavy 9C8 IgG2 25 light 9C8 IgG2 n.a. 26 light 9C8 IgG2 27
heavy 9C8 N68T T83S IgG2 n.a. 28 heavy 9C8 N68T T83S IgG2 29
V.sub.H 9C8 N68T T83S 30 V.sub.H FR3 9C8 N68T T83S 31 heavy 9C8
N68T T83S IgG1 n.a. 32 heavy 9C8 N68T T83S IgG1 33 V.sub.H 9C8 E31G
N68T T83S n.a. 34 V.sub.H 9C8 E31G N68T T83S 35 V.sub.H CDR1 9C8
E31G N68T T83S 36 V.sub.H 9C8 I24V N68T T83S n.a. 37 V.sub.H 9C8
I24V N68T T83S 38 V.sub.H FR1 9C8 I24V N68T T83S 39 V.sub.H 22B5
n.a. 40 V.sub.H 22B5 41 V.sub.H CDR3 22B5 42 V.sub.L 22B5 n.a. 43
V.sub.L 22B5 44 Consensus 1 CDR3 45 Consensus 2 CDR3
Sequence CWU 1
1
6011338DNAhomo sapien 1caggtgcagc tacagcagtg gggcgcagga ctgttgaagc
cttcggagac cctgtccctc 60acctgcgcta tctatggtgg gtccttcagg gagtactact
ggagctggat ccgccagccc 120ccagggaagg ggctggagtg gattggggaa
atctttcata gtggaagcac caactacaac 180ccgtccctca agagtcgagt
caacatatca gtagacacgt ccaagaacca gttctccctg 240aagctgacct
ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag agaggaatta
300gatgattttg atatctgggg ccaagggaca atggtcaccg tctcttcagc
ctccaccaag 360ggcccatcgg tcttccccct ggcaccctcc tccaagagca
cctctggggg cacagcggcc 420ctgggctgcc tggtcaagga ctacttcccc
gaaccggtga cggtgtcgtg gaactcaggc 480gccctgacca gcggcgtgca
caccttcccg gctgtcctac agtcctcagg actctactcc 540ctcagcagcg
tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac
600gtgaatcaca agcccagcaa caccaaggtg gacaagagag ttgagcccaa
atcttgtgac 660aaaactcaca catgcccacc gtgcccagca cctgaactcc
tggggggacc gtcagtcttc 720ctcttccccc caaaacccaa ggacaccctc
atgatctccc ggacccctga ggtcacatgc 780gtggtggtgg acgtgagcca
cgaagaccct gaggtcaagt tcaactggta cgtggacggc 840gtggaggtgc
ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt
900gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga
gtacaagtgc 960aaggtctcca acaaagccct cccagccccc atcgagaaaa
ccatctccaa agccaaaggg 1020cagccccgag aaccacaggt gtacaccctg
cccccatccc gggatgagct gaccaagaac 1080caggtcagcc tgacctgcct
ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1140gagagcaatg
ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac
1200ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca
gcaggggaac 1260gtcttctcat gctccgtgat gcatgaggct ctgcacaacc
actacacgca gaagagcctc 1320tccctgtctc cgggtaaa 13382348DNAhomo
sapien 2caggtgcagc tacagcagtg gggcgcagga ctgttgaagc cttcggagac
cctgtccctc 60acctgcgcta tctatggtgg gtccttcagg gagtactact ggagctggat
ccgccagccc 120ccagggaagg ggctggagtg gattggggaa atctttcata
gtggaagcac caactacaac 180ccgtccctca agagtcgagt caacatatca
gtagacacgt ccaagaacca gttctccctg 240aagctgacct ctgtgaccgc
cgcggacacg gctgtgtatt actgtgcgag agaggaatta 300gatgattttg
atatctgggg ccaagggaca atggtcaccg tctcttca 3483446PRThomo sapien
3Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1
5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Tyr Gly Gly Ser Phe Arg Glu
Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu
Glu Trp Ile 35 40 45 Gly Glu Ile Phe His Ser Gly Ser Thr Asn Tyr
Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Asn Ile Ser Val Asp Thr
Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Thr Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Glu Leu Asp
Asp Phe Asp Ile Trp Gly Gln Gly Thr Met Val 100 105 110 Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135
140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Arg Val Glu
Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260
265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385
390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 435 440 445 4116PRThomo sapien 4Gln Val Gln Leu Gln
Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser
Leu Thr Cys Ala Ile Tyr Gly Gly Ser Phe Arg Glu Tyr 20 25 30 Tyr
Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45 Gly Glu Ile Phe His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys
50 55 60 Ser Arg Val Asn Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
Ser Leu 65 70 75 80 Lys Leu Thr Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95 Arg Glu Glu Leu Asp Asp Phe Asp Ile Trp
Gly Gln Gly Thr Met Val 100 105 110 Thr Val Ser Ser 115 55PRThomo
sapien 5Glu Tyr Tyr Trp Ser 1 5 616PRThomo sapien 6Glu Ile Phe His
Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 78PRThomo
sapien 7Glu Glu Leu Asp Asp Phe Asp Ile 1 5 830PRThomo sapien 8Gln
Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10
15 Thr Leu Ser Leu Thr Cys Ala Ile Tyr Gly Gly Ser Phe Arg 20 25 30
914PRThomo sapien 9Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
Ile Gly 1 5 10 1031PRThomo sapien 10Arg Val Asn Ile Ser Val Asp Thr
Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu Thr Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys Ala 20 25 30 1111PRThomo sapien
11Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 1 5 10 12642DNAhomo
sapien 12gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga
cagagtcacc 60atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca
gcagaaacca 120gagaaagccc ctaagtccct gatctatgct gcatccagtt
tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagat
ttcactctca ccatcagtag cctgcagcct 240gaagattttg caacttatta
ctgccaacag tataaaagtt accctcggac gttcggccaa 300gggaccaagg
tggaaatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca
360tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa
taacttctat 420cccagagagg ccaaagtaca gtggaaggtg gataacgccc
tccaatcggg taactcccag 480gagagtgtca cagagcagga cagcaaggac
agcacctaca gcctcagcag caccctgacg 540ctgagcaaag cagactacga
gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcgc
ccgtcacaaa gagcttcaac aggggagagt gt 64213321DNAhomo sapien
13gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc
60atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca
120gagaaagccc ctaagtccct gatctatgct gcatccagtt tgcaaagtgg
ggtcccatca 180aggttcagcg gcagtggatc tgggacagat ttcactctca
ccatcagtag cctgcagcct 240gaagattttg caacttatta ctgccaacag
tataaaagtt accctcggac gttcggccaa 300gggaccaagg tggaaatcaa a
32114214PRThomo sapien 14Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln
Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Lys Ser Tyr Pro Arg 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205
Phe Asn Arg Gly Glu Cys 210 15107PRThomo sapien 15Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35
40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Lys Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 1611PRThomo sapien 16Arg Ala Ser Gln Gly Ile Ser
Ser Trp Leu Ala 1 5 10 177PRThomo sapien 17Ala Ala Ser Ser Leu Gln
Ser 1 5 189PRThomo sapien 18Gln Gln Tyr Lys Ser Tyr Pro Arg Thr 1 5
1923PRThomo sapien 19Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20
2015PRThomo sapien 20Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys
Ser Leu Ile Tyr 1 5 10 15 2132PRThomo sapien 21Gly Val Pro Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 30
2210PRThomo sapien 22Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 1 5 10
231326DNAhomo sapien 23caggtgcagc tacagcagtg gggcgcagga ctgttgaagc
cttcggagac cctgtccctc 60acctgcgcta tctatggtgg gtccttcagg gagtactact
ggagctggat ccgccagccc 120ccagggaagg ggctggagtg gattggggaa
atctttcata gtggaagcac caactacaac 180ccgtccctca agagtcgagt
caacatatca gtagacacgt ccaagaacca gttctccctg 240aagctgacct
ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag agaggaatta
300gatgattttg atatctgggg ccaagggaca atggtcaccg tctcttcagc
ctccaccaag 360ggcccatcgg tcttccccct ggcgccctgc tccaggagca
cctccgagag cacagcggcc 420ctgggctgcc tggtcaagga ctacttcccc
gaaccggtga cggtgtcgtg gaactcaggc 480gctctgacca gcggcgtgca
caccttcccg gctgtcctac agtcctcagg actctactcc 540ctcagcagcg
tagtgaccgt gccctccagc aacttcggca cccagaccta cacctgcaac
600gtagatcaca agcccagcaa caccaaggtg gacaagacag ttgagcgcaa
atgttgtgtc 660gagtgcccac cgtgcccagc accacctgtg gcaggaccgt
cagtcttcct cttcccccca 720aaacccaagg acaccctcat gatctcccgg
acccctgagg tcacgtgcgt ggtggtggac 780gtgagccacg aagaccccga
ggtccagttc aactggtacg tggacggcgt ggaggtgcat 840aatgccaaga
caaagccacg ggaggagcag ttcaacagca cgttccgtgt ggtcagcgtc
900ctcaccgtcg tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa
ggtctccaac 960aaaggcctcc cagcccccat cgagaaaacc atctccaaaa
ccaaagggca gccccgagaa 1020ccacaggtgt acaccctgcc cccatcccgg
gaggagatga ccaagaacca ggtcagcctg 1080acctgcctgg tcaaaggctt
ctaccccagc gacatcgccg tggagtggga gagcaatggg 1140cagccggaga
acaactacaa gaccacacct cccatgctgg actccgacgg ctccttcttc
1200ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt
cttctcatgc 1260tccgtgatgc atgaggctct gcacaaccac tacacacaga
agagcctctc cctgtctccg 1320ggtaaa 132624442PRThomo sapien 24Gln Val
Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Ile Tyr Gly Gly Ser Phe Arg Glu Tyr 20
25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
Ile 35 40 45 Gly Glu Ile Phe His Ser Gly Ser Thr Asn Tyr Asn Pro
Ser Leu Lys 50 55 60 Ser Arg Val Asn Ile Ser Val Asp Thr Ser Lys
Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Thr Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Glu Leu Asp Asp Phe
Asp Ile Trp Gly Gln Gly Thr Met Val 100 105 110 Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Cys Ser
Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150
155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Asn Phe 180 185 190 Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His
Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Thr Val Glu Arg Lys
Cys Cys Val Glu Cys Pro Pro 210 215 220 Cys Pro Ala Pro Pro Val Ala
Gly Pro Ser Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp 260 265 270
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275
280 285 Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val
Val 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn 305 310 315 320 Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Thr Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu 340 345 350 Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr
Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395
400 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
405 410 415 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 420 425 430 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
25642DNAhomo sapien 25gacatccaga tgacccagtc tccatcctca ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca gggtattagc agctggttag
cctggtatca gcagaaacca 120gagaaagccc ctaagtccct gatctatgct
gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc
tgggacagat ttcactctca ccatcagtag cctgcagcct 240gaagattttg
caacttatta ctgccaacag tataaaagtt accctcggac gttcggccaa
300gggaccaagg tggaaatcaa acgaactgtg gctgcaccat ctgtcttcat
cttcccgcca
360tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa
taacttctat 420cccagagagg ccaaagtaca gtggaaggtg gataacgccc
tccaatcggg taactcccag 480gagagtgtca cagagcagga cagcaaggac
agcacctaca gcctcagcag caccctgacg 540ctgagcaaag cagactacga
gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcgc
ccgtcacaaa gagcttcaac aggggagagt gt 64226214PRThomo sapien 26Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp
20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser
Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Lys Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145
150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
271326DNAhomo sapien 27caggtgcagc tacagcagtg gggcgcagga ctgttgaagc
cttcggagac cctgtccctc 60acctgcgcta tctatggtgg gtccttcagg gagtactact
ggagctggat ccgccagccc 120ccagggaagg ggctggagtg gattggggaa
atctttcata gtggaagcac caactacaac 180ccgtccctca agagtcgagt
caccatatca gtagacacgt ccaagaacca gttctccctg 240aagctgagct
ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag agaggaatta
300gatgattttg atatctgggg ccaagggaca atggtcaccg tctcctcagc
ctccaccaag 360ggcccatcgg tcttccccct ggcgccctgc tccaggagca
cctccgagag cacagcggcc 420ctgggctgcc tggtcaagga ctacttcccc
gaaccggtga cggtgtcgtg gaactcaggc 480gctctgacca gcggcgtgca
caccttcccg gctgtcctac agtcctcagg actctactcc 540ctcagcagcg
tagtgaccgt gccctccagc aacttcggca cccagaccta cacctgcaac
600gtagatcaca agcccagcaa caccaaggtg gacaagacag ttgagcgcaa
atgttgtgtc 660gagtgcccac cgtgcccagc accacctgtg gcaggaccgt
cagtcttcct cttcccccca 720aaacccaagg acaccctcat gatctcccgg
acccctgagg tcacgtgcgt ggtggtggac 780gtgagccacg aagaccccga
ggtccagttc aactggtacg tggacggcgt ggaggtgcat 840aatgccaaga
caaagccacg ggaggagcag ttcaacagca cgttccgtgt ggtcagcgtc
900ctcaccgtcg tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa
ggtctccaac 960aaaggcctcc cagcccccat cgagaaaacc atctccaaaa
ccaaagggca gccccgagaa 1020ccacaggtgt acaccctgcc cccatcccgg
gaggagatga ccaagaacca ggtcagcctg 1080acctgcctgg tcaaaggctt
ctaccccagc gacatcgccg tggagtggga gagcaatggg 1140cagccggaga
acaactacaa gaccacacct cccatgctgg actccgacgg ctccttcttc
1200ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt
cttctcatgc 1260tccgtgatgc atgaggctct gcacaaccac tacacgcaga
agagcctctc cctgtctccg 1320ggtaaa 132628442PRThomo sapien 28Gln Val
Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Ile Tyr Gly Gly Ser Phe Arg Glu Tyr 20
25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
Ile 35 40 45 Gly Glu Ile Phe His Ser Gly Ser Thr Asn Tyr Asn Pro
Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys
Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Glu Leu Asp Asp Phe
Asp Ile Trp Gly Gln Gly Thr Met Val 100 105 110 Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Cys Ser
Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150
155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Asn Phe 180 185 190 Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His
Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Thr Val Glu Arg Lys
Cys Cys Val Glu Cys Pro Pro 210 215 220 Cys Pro Ala Pro Pro Val Ala
Gly Pro Ser Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp 260 265 270
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275
280 285 Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val
Val 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn 305 310 315 320 Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Thr Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu 340 345 350 Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr
Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395
400 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
405 410 415 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 420 425 430 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
29116PRThomo sapien 29Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu
Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Tyr
Gly Gly Ser Phe Arg Glu Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Phe His
Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val
Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90
95 Arg Glu Glu Leu Asp Asp Phe Asp Ile Trp Gly Gln Gly Thr Met Val
100 105 110 Thr Val Ser Ser 115 3032PRThomo sapien 30Arg Val Thr
Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu
Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25
30 311338DNAhomo sapien 31caggtgcagc tacagcagtg gggcgcagga
ctgttgaagc cttcggagac cctgtccctc 60acctgcgcta tctatggtgg gtccttcagg
gagtactact ggagctggat ccgccagccc 120ccagggaagg ggctggagtg
gattggggaa atctttcata gtggaagcac caactacaac 180ccgtccctca
agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg
240aagctgagct ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag
agaggaatta 300gatgattttg atatctgggg ccaagggaca atggtcaccg
tctcctcagc ctccaccaag 360ggcccatcgg tcttccccct ggcaccctcc
tccaagagca cctctggggg cacagcggcc 420ctgggctgcc tggtcaagga
ctacttcccc gaaccggtga cggtgtcgtg gaactcaggc 480gccctgacca
gcggcgtgca caccttcccg gctgtcctac agtcctcagg actctactcc
540ctcagcagcg tagtgaccgt gccctccagc agcttgggca cccagaccta
catctgcaac 600gtgaatcaca agcccagcaa caccaaggtg gacaagaaag
ttgagcccaa atcttgtgac 660aaaactcaca catgcccacc gtgcccagca
cctgaactcc tggggggacc gtcagtcttc 720ctcttccccc caaaacccaa
ggacaccctc atgatctccc ggacccctga ggtcacatgc 780gtggtggtgg
acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc
840gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag
cacgtaccgt 900gtggtcagcg tcctcaccgt cctgcaccag gactggctga
atggcaagga gtacaagtgc 960aaggtctcca acaaagccct cccagccccc
atcgagaaaa ccatctccaa agccaaaggg 1020cagccccgag aaccacaggt
gtacaccctg cccccatccc gggaggagat gaccaagaac 1080caggtcagcc
tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg
1140gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct
ggactccgac 1200ggctccttct tcctctatag caagctcacc gtggacaaga
gcaggtggca gcaggggaac 1260gtcttctcat gctccgtgat gcatgaggct
ctgcacaacc actacacgca gaagagcctc 1320tccctgtctc cgggtaaa
133832446PRThomo sapien 32Gln Val Gln Leu Gln Gln Trp Gly Ala Gly
Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile
Tyr Gly Gly Ser Phe Arg Glu Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Phe
His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg
Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85
90 95 Arg Glu Glu Leu Asp Asp Phe Asp Ile Trp Gly Gln Gly Thr Met
Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210
215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330
335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445
33348DNAhomo sapien 33caggtgcagc tacagcagtg gggcgcagga ctgttgaagc
cttcggagac cctgtccctc 60acctgcgcta tctatggtgg gtccttcagg gggtactact
ggagctggat ccgccagccc 120ccagggaagg ggctggagtg gattggggaa
atctttcata gtggaagcac caactacaac 180ccgtccctca agagtcgagt
caccatatca gtagacacgt ccaagaacca gttctccctg 240aagctgagct
ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag agaggaatta
300gatgattttg atatctgggg ccaagggaca atggtcaccg tctcctca
34834116PRThomo sapien 34Gln Val Gln Leu Gln Gln Trp Gly Ala Gly
Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile
Tyr Gly Gly Ser Phe Arg Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Phe
His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg
Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85
90 95 Arg Glu Glu Leu Asp Asp Phe Asp Ile Trp Gly Gln Gly Thr Met
Val 100 105 110 Thr Val Ser Ser 115 355PRThomo sapien 35Gly Tyr Tyr
Trp Ser 1 5 36348DNAhomo sapien 36caggtgcagc tacagcagtg gggcgcagga
ctgttgaagc cttcggagac cctgtccctc 60acctgcgctg tctatggtgg gtccttcagg
gagtactact ggagctggat ccgccagccc 120ccagggaagg ggctggagtg
gattggggaa atctttcata gtggaagcac caactacaac 180ccgtccctca
agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg
240aagctgagct ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag
agaggaatta 300gatgattttg atatctgggg ccaagggaca atggtcaccg tctcctca
34837116PRThomo sapien 37Gln Val Gln Leu Gln Gln Trp Gly Ala Gly
Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val
Tyr Gly Gly Ser Phe Arg Glu Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Phe
His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg
Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85
90 95 Arg Glu Glu Leu Asp Asp Phe Asp Ile Trp Gly Gln Gly Thr Met
Val 100 105 110 Thr Val Ser Ser 115 3830PRThomo sapien 38Gln Val
Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Arg 20 25 30
39348DNAhomo sapien 39caggtgcagc tacagcagtg gggcgcagga ctgttgaagc
cttcggagac cctgtccctc 60acctgcgctg tctatggtgg gtcattcaga ggttactact
ggagctggat ccgccagccc 120ccaggaaagg ggctggagtg gattggggaa
atctttcata gtggaagcac caactacaac 180ccgtccctca agagtcgagt
caccatatca gtagacacgt ccaagaacca gttctccctg 240aaactgaggt
ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag agaagatatt
300gatgattttg atatctgggg ccaagggaca atggtcaccg tctcttca
34840116PRThomo sapien 40Gln Val Gln Leu Gln Gln Trp Gly Ala Gly
Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val
Tyr Gly Gly Ser Phe Arg Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Phe
His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg
Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80
Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85
90 95 Arg Glu Asp Ile Asp Asp Phe Asp Ile Trp Gly Gln Gly Thr Met
Val 100 105
110 Thr Val Ser Ser 115 418PRThomo sapien 41Glu Asp Ile Asp Asp Phe
Asp Ile 1 5 42321DNAhomo sapien 42gacatccaga tgacccagtc tccatcctca
ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca gggtattagc
agctggttag cctggtatca gcagaaacca 120gagaaagccc ctaagtccct
gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg
gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct
240gaagactttg caacttatta ttgccaacag tataagagtt accctcggac
gttcggccaa 300gggaccaagg tggaaatcaa a 32143107PRThomo sapien 43Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp
20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser
Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Lys Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 105 449PRThomo sapienMISC_FEATURE(2)..()X =
D, E, N, or Q 44Glu Xaa Xaa Leu Asp Asp Phe Asp Ile 1 5 459PRThomo
sapienMISC_FEATURE(2)..(2)X = D or E 45Glu Xaa Xaa Leu Asp Asp Phe
Asp Ile 1 5 46112PRThomo sapien 46Gln Val Gln Leu Gln Gln Trp Gly
Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys
Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu
Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65
70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95 Arg Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val
Thr Val Ser Ser 100 105 110 47107PRThomo sapien 47Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35
40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Asn Ser Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 48212PRThomo sapien 48Met Asn Ser Phe Ser Thr Ser
Ala Phe Gly Pro Val Ala Phe Ser Leu 1 5 10 15 Gly Leu Leu Leu Val
Leu Pro Ala Ala Phe Pro Ala Pro Val Pro Pro 20 25 30 Gly Glu Asp
Ser Lys Asp Val Ala Ala Pro His Arg Gln Pro Leu Thr 35 40 45 Ser
Ser Glu Arg Ile Asp Lys Gln Ile Arg Tyr Ile Leu Asp Gly Ile 50 55
60 Ser Ala Leu Arg Lys Glu Thr Cys Asn Lys Ser Asn Met Cys Glu Ser
65 70 75 80 Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro Lys
Met Ala 85 90 95 Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe Asn Glu
Glu Thr Cys Leu 100 105 110 Val Lys Ile Ile Thr Gly Leu Leu Glu Phe
Glu Val Tyr Leu Glu Tyr 115 120 125 Leu Gln Asn Arg Phe Glu Ser Ser
Glu Glu Gln Ala Arg Ala Val Gln 130 135 140 Met Ser Thr Lys Val Leu
Ile Gln Phe Leu Gln Lys Lys Ala Lys Asn 145 150 155 160 Leu Asp Ala
Ile Thr Thr Pro Asp Pro Thr Thr Asn Ala Ser Leu Leu 165 170 175 Thr
Lys Leu Gln Ala Gln Asn Gln Trp Leu Gln Asp Met Thr Thr His 180 185
190 Leu Ile Leu Arg Ser Phe Lys Glu Phe Leu Gln Ser Ser Leu Arg Ala
195 200 205 Leu Arg Gln Met 210 4925DNAArtificial
Sequencesequencing primer 49ctttctgaga stcmtggakc tcmtg
255020DNAArtificial Sequencesequencing primer 50tacgtgccaa
gcatcctcgc 205126DNAArtificial Sequencesequencing primer
51gsartcagwc ycwvycagga cacagc 265223DNAArtificial
Sequencesequencing primer 52aggctggaac tgaggagcag gtg
235339DNAArtificial Sequencecloning primer 53ttacagtgcg cgcactccca
ggtgcagcta cagcagtgg 395439DNAArtificial Sequencecloning primer
54ttacagtgtg cactccgaca tccagatgac ccagtctcc 395520DNAArtificial
Sequencecloning primer 55gaagaccgat gggcccttgg 205651DNAArtificial
Sequencecloning primer 56tatattcctt aattaagtta ttctactcac
gtttgatttc caccttggtc c 515728DNAArtificial Sequencemutagenesis
primer 57cctcacctgc gctgtctatg gtgggtcc 285829DNAArtificial
Sequencemutagenesis primer 58gggtccttca gggggtacta ctggagctg
295934DNAArtificial Sequencemutagensis primer 59cctcaagagt
cgagtcacca tatcagtaga cacg 346028DNAArtificial Sequencemutagenesis
primer 60ctccctgaag ctgagctctg tgaccgcc 28
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