U.S. patent application number 11/563928 was filed with the patent office on 2007-05-31 for il-21 antagonists.
Invention is credited to Stephen R. Jaspers, Pallavur V. Sivakumar.
Application Number | 20070122413 11/563928 |
Document ID | / |
Family ID | 38541590 |
Filed Date | 2007-05-31 |
United States Patent
Application |
20070122413 |
Kind Code |
A1 |
Sivakumar; Pallavur V. ; et
al. |
May 31, 2007 |
IL-21 ANTAGONISTS
Abstract
Monoclonal antibodies are identified that bind the IL-21
protein. These antibodies are used to identify regions of the IL-21
protein to where binding neutralizes IL-21 activity. Hybridomas and
methods of producing anti-IL-21 monoclonal antibodies are
described. The monoclonal antibodies are useful in treating
IL-21-mediated diseases, which may include autoimmune and
inflammatory diseases such as pancreatitis, type I diabetes (IDDM),
Graves Disease, inflammatory bowel disease (IBD), Crohn's Disease,
ulcerative colitis, irritable bowel syndrome, multiple sclerosis,
rheumatoid arthritis, diverticulosis, systemic lupus erythematosus,
psoriasis, ankylosing spondylitis, scleroderma, systemic sclerosis,
psoriatic arthritis, osteoarthritis, atopic dermatitis, vitiligo,
graft vs. host disease (GVHD), cutaneous T cell lymphoma (CTCL),
Sjogren's syndrome, glomerulonephritis, IgA nephropathy, graft
versous host disease, transplant rejection, atopic dermatitis,
anti-phospholipid syndrome, and asthma, and other autoimmune
diseases.
Inventors: |
Sivakumar; Pallavur V.;
(Seattle, WA) ; Jaspers; Stephen R.; (Edmonds,
WA) |
Correspondence
Address: |
ZYMOGENETICS, INC.;INTELLECTUAL PROPERTY DEPARTMENT
1201 EASTLAKE AVENUE EAST
SEATTLE
WA
98102-3702
US
|
Family ID: |
38541590 |
Appl. No.: |
11/563928 |
Filed: |
November 28, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60740154 |
Nov 28, 2005 |
|
|
|
Current U.S.
Class: |
424/145.1 ;
435/335; 435/70.21; 530/388.23 |
Current CPC
Class: |
A61P 21/02 20180101;
A61P 17/00 20180101; A61P 5/14 20180101; C07K 2317/34 20130101;
C07K 2317/33 20130101; A61P 37/08 20180101; A61P 1/04 20180101;
A61P 17/04 20180101; Y10S 530/809 20130101; A61P 19/08 20180101;
C07K 16/244 20130101; A61K 39/3955 20130101; A61P 19/02 20180101;
C07K 2317/76 20130101; A61P 35/00 20180101; A61P 3/10 20180101;
A61K 2039/505 20130101; A61P 35/02 20180101; A61P 11/06 20180101;
A61P 17/06 20180101; A61P 37/06 20180101; A61P 43/00 20180101; A61P
25/00 20180101; A61P 1/00 20180101; A61P 29/00 20180101; C07K
2319/30 20130101; A61P 13/12 20180101; A61P 1/18 20180101; A61P
37/02 20180101 |
Class at
Publication: |
424/145.1 ;
530/388.23; 435/335; 435/070.21 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/24 20060101 C07K016/24; C12P 21/08 20060101
C12P021/08; C12N 5/06 20060101 C12N005/06 |
Claims
1. An anti-IL-21 monoclonal antibody that binds to an antigenic
region of human IL-21.
2. The anti-IL-21 monoclonal antibody of claim 1, wherein the
antigenic region of human IL-21 is shown in SEQ ID NO: 6 from amino
acid residues 97-122.
3. The anti-IL-21 monoclonal antibody of claim 2, wherein the
antibody neutralizes a human IL-21 protein activity, binds a human
IL-21-Fc protein, binds a human IL-21 mutein-Fc protein, and binds
a mouse IL-21-mouse Fc protein.
4. The anti-IL-21 monoclonal antibody of claim 1, wherein the
antigenic region of human IL-21 is shown in SEQ ID NO: 6 from amino
acid residues 145 to 148.
5. The anti-IL-21 monoclonal antibody of claim 4, wherein the
antibody also binds antigenic region as shown in SEQ ID NO: 6 from
amino acid residue 154 to 162.
6. The anti-IL-21 monoclonal antibody of claim 4, wherein the
antibody neutralizes a human IL-21 protein activity, binds a human
IL-21-Fc protein, does not bind a human IL-21 mutein-Fc protein,
and binds a mouse IL-21-mouse Fc protein.
7. The anti-IL-21 monoclonal antibody of claim 1, wherein the
antigenic region of human IL-21 is shown in SEQ ID NO: 6 from amino
acid residues 30 to 50.
8. The anti-IL-21 monoclonal antibody of claim 7, wherein the
antibody neutralizes a human IL-21 protein activity, binds a human
IL-21-Fc protein, binds a human IL-21 mutein-Fc protein, and binds
a mouse IL-21-mouse Fc protein.
9. The anti-IL-21 monoclonal antibody of claim 1, wherein the
antigenic region of human IL-21 is shown in SEQ ID NO: 6 from amino
acid residues 40 to 50.
10. The anti-IL-21 monoclonal antibody of claim 9, wherein the
antibody neutralizes a human IL-21 protein activity, binds a human
IL-21-Fc protein, binds a human IL-21 mutein-Fc protein, and binds
a mouse IL-21-mouse Fc protein.
11. A bin that is capable of competition with monoclonal antibody
272.21.1.13.4.2 (ATCC Accession No. PTA-7142) for binding a human
IL-21 antigen.
12. A bin that is capable of competition with monoclonal antibody
268.5.1.11.42.1.4.3.9 (ATCC Accession No. PTA-7143) for binding a
human IL-21 antigen.
13. The monoclonal antibody of claim 11, wherein the monoclonal
antibody specifically binds to the epitope to which monoclonal
antibody 272.21.1.13.4.2 (ATCC Accession No. PTA-7142) binds.
14. The monoclonal antibody of claim 11, wherein the monoclonal
antibody specifically binds to the epitope to which monoclonal
antibody 268.5.1.11.42.1.4.3.9 (ATCC Accession No. PTA-7143)
binds.
15. The monoclonal antibody of according to claims 1, 2, 4, 7, 9,
13 or 14 labeled with a detectable marker.
16. The monoclonal antibody of according to claims 1, 2, 4, 7, 9,
13 or 14 labeled with a detectable marker selected from the group
consisting of a radioactive isotope, enzyme, dye and biotin.
17. A hybridoma cell producing the monoclonal antibody according to
claims 1, 2, 4, 7, 9, 13 or 14.
18. The hybridoma cell producing the monoclonal antibody of claim
17.
19. A method of producing the monoclonal antibody according to
claims 1, 2, 4, 7, 9, 13 or 14 comprising: (a) providing a
hybridoma capable of producing the monoclonal antibody; and (b)
culturing the hybridoma under conditions that provide for
production of the monoclonal antibody by the hybridoma.
20. A method of treating an autoimmune disease comprising
administering a therapeutically effective amount of an anti-IL-21
monoclonal antibody according to claims 1, 2, 4, 7, 9, 13 or 14 to
a patient.
21. The method of claim 20, wherein the autoimmune disease is
selected from the group consisting of pancreatitis, type I diabetes
(IDDM), Graves Disease, inflammatory bowel disease (IBD), Crohn's
Disease, ulcerative colitis, irritable bowel syndrome, multiple
sclerosis, rheumatoid arthritis, diverticulosis, systemic lupus
erythematosus, psoriasis, ankylosing spondylitis, scleroderma,
systemic sclerosis, psoriatic arthritis, osteoarthritis, atopic
dermatitis, vitiligo, graft vs. host disease (GVHD), cutaneous T
cell lymphoma (CTCL), Sjogren's syndrome, glomerulonephritis, IgA
nephropathy, graft versous host disease, transplant rejection,
atopic dermatitis, anti-phospholipid syndrome, and asthma, and
other autoimmune diseases.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 60/740,154, filed Nov. 28, 2005, which is
herein incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] The immune system is the body's primary defense against
diseases caused by pathogens, namely bacteria, viruses, fungi etc,
as well as against diseases caused by abnormal growth of the body's
own cells and tissues (i.e. cancerous tumors). Normally, the immune
system is able to distinguish between the body's normal cells or
"self" and foreign pathogens or abnormal cells or "non-self". The
processes by which the immune system refrains from reacting to
one's own body is called tolerance. Sometimes, the immune system
loses the ability to recognize "self" as normal and the subsequent
response directed against the tissue or cells, results in loss of
tolerance, a state of autoimmunity. The pathologies resulting from
autoimmunity often have serious clinical consequences and are one
of the major health problems in the world, especially in developed
nations.
[0003] Cytokines generally stimulate proliferation or
differentiation of cells of the hematopoietic lineage or
participate in the immune and inflammatory response mechanisms of
the body. The interleukins are a family of cytokines that mediate
immunological responses. Receptors that bind cytokines are
typically composed of one or more integral membrane proteins that
bind the cytokine with high affinity and transduce this binding
event to the cell through the cytoplasmic portions of the certain
receptor subunits. Cytokine receptors have been grouped into
several classes on the basis of similarities in their extracellular
ligand binding domains. For example, the receptor chains
responsible for binding and/or transducing the effect of
interferons are members of the class II cytokine receptor family,
based upon a characteristic 200 residue extracellular domain.
[0004] The present invention provides anti-IL-21 monoclonal
antibodies and methods of using those antibodies that inhibit the
symptoms and biological activities that manifest as autoimmune and
inflammatory disorders and are associated with IL-21/IL-21 receptor
interaction.
BRIEF DESCRIPTION OF THE INVENTION
[0005] In one aspect, the present invention provides an anti-IL-21
monoclonal antibody that binds to an antigen region of human IL-21.
In certain embodiments, the monoclonal antibody binds to an
antigenic region of IL-21 that is shown in SEQ ID NO: 6 from amino
acid residues 97-122. In another embodiment, the monoclonal
antibody binds an antigenic region as shown in SEQ ID NO: 6 from
amino acid residues 145 to 148. In another embodiment, the
monoclonal antibody binds an antigenic region as shown in SEQ ID
NO: 6 from amino acid residues 154 to 162. In another embodiment,
the monoclonal antibody binds an antigen region as shown in SEQ ID
NO: 6 from amino acid residues 30 to 50. In another embodiment, the
monoclonal antibody binds to an antigen region as shown in SEQ ID
NO: 6 from amino acid residues 40 to 50. Additional embodiments
include monoclonal antibodies as described herein that can be shown
to neutralize a human IL-21 protein activity, binds a human
IL-21-Fc protein, bind a human mutein Fc protein, where the
mutations are at Gln 145 and/or Ile148 of SEQ ID NO: 6, or bind a
mouse IL-21-mouse Fc fusion protein. Generally, the monoclonal
antibodies of the present invention bind two or more IL-21
proteins.
[0006] In other aspects, the monoclonal antibody specifically binds
to the epitope to which monoclonal antibody 272.21.1.13.4.2 (ATCC
Accession No. PTA-7142) binds. In other embodiments, the monoclonal
antibody specifically binds to the epitope to which monoclonal
antibody 268.5.1.11.42.1.4.3.9 (ATCC Accession No. PTA-7143) binds.
The monoclonal antibodies of the present invention may also be
labeled with a detectable marker and the detectable marker can be
selected from, but is not limited to, radioactive isotopes,
enzymes, dyes and biotins.
[0007] In another aspect, the present invention provides a bin (or
group of antibodies) that is capable of competition with monoclonal
antibody 272.21.1.13.4.2 (ATCC Accession No. PTA-7142) for binding
a human IL-21 antigen.
[0008] Another aspect of the present invention provides a bin that
is capable of competition with monoclonal antibody
268.5.1.11.42.1.4.3.9 (ATCC Accession No. PTA-7143) for binding a
human IL-21 antigen.
[0009] Also included in the present invention are hybridomas
producing the claimed monoclonal antibodies.
[0010] The present invention provides a method of producing the
claimed monoclonal antibodies comprising: (a) providing a hybridoma
capable of producing the monoclonal antibody; and (b) culturing the
hybridoma under conditions that provide for production of the
monoclonal antibody by the hybridoma.
[0011] In another aspect, the present invention provides a method
of treating an autoimmune disease comprising administering a
therapeutically effective amount of the claimed anti-IL-21
monoclonal antibodies to a patient. In certain embodiments, the
autoimmune disease is selected from the group consisting of
pancreatitis, type I diabetes (IDDM), Graves Disease, inflammatory
bowel disease (IBD), Crohn's Disease, ulcerative colitis, irritable
bowel syndrome, multiple sclerosis, rheumatoid arthritis,
diverticulosis, systemic lupus erythematosus, psoriasis, ankylosing
spondylitis, scleroderma, systemic sclerosis, psoriatic arthritis,
osteoarthritis, atopic dermatitis, vitiligo, graft vs. host disease
(GVHD), cutaneous T cell lymphoma (CTCL), Sjogren's syndrome,
glomerulonephritis, IgA nephropathy, graft versous host disease,
transplant rejection, atopic dermatitis, anti-phospholipid
syndrome, and asthma, and other autoimmune diseases.
[0012] The present invention also provides a method of inhibiting
or reducing an IL-21-mediated disorder comprising administering an
anti-IL-21 monoclonal antibody in an amount sufficient to inhibit
or reduce IL-21 mediated biological activity in the subject.
DESCRIPTION OF THE INVENTION
[0013] The following definitions are provided to facilitate
understanding of the inventions described herein.
[0014] The term "antibody" or "antibody peptide(s)" refers to an
intact antibody, or a binding fragment thereof that competes with
the intact antibody for specific binding and includes chimeric,
humanized, fully human, and bispecific antibodies. In certain
embodiments, binding fragments are produced by recombinant DNA
techniques. In additional embodiments, binding fragments are
produced by enzymatic or chemical cleavage of intact antibodies.
Binding fragments include, but are not limited to, Fab, Fab',
F(ab').sub.2, Fv, and single-chain antibodies.
[0015] The term "isolated antibody" refers to an antibody that has
been identified and separated and/or recovered from a component of
its natural environment. Contaminant components of its natural
environment are materials which would interfere with diagnostic or
therapeutic uses for the antibody, and may include enzymes,
hormones, and other proteinaceous or nonproteinaceous solutes. In
preferred embodiments, the antibody will be purified (1) to greater
than 95% by weight of antibody as determined by the Lowry method,
and most preferably more than 99% by weight, (2) to a degree
sufficient to obtain at least 15 residues of N-terminal or internal
amino acid sequence by use of a spinning cup sequenator, or (3) to
homogeneity by SDS-PAGE under reducing or nonreducing conditions
using Coomassie blue or, preferably, silver stain. Isolated
antibody includes the antibody in situ within recombinant cells
since at least one component of the antibody's natural environment
will not be present. Ordinarily, however, isolated antibody will be
prepared by at least one purification step.
[0016] A "variant" anti-IL-21 antibody, refers herein to a molecule
which differs in amino acid sequence from a "parent" anti-IL-21
antibody amino acid sequence by virtue of addition, deletion and/or
substitution of one or more amino acid residue(s) in the parent
antibody sequence. In the preferred embodiment, the variant
comprises one or more amino acid substitution(s) in one or more
hypervariable region(s) of the parent antibody. For example, the
variant may comprise at least one, e.g. from about one to about
ten, and preferably from about two to about five, substitutions in
one or more hypervariable regions of the parent antibody.
Ordinarily, the variant will have an amino acid sequence having at
least 75% amino acid sequence identity with the parent antibody
heavy or light chain variable domain sequences, more preferably at
least 80%, more preferably at least 85%, more preferably at least
90%, and most preferably at least 95%. Identity or homology with
respect to this sequence is defined herein as the percentage of
amino acid residues in the candidate sequence that are identical to
the parent antibody residues, after aligning the sequences and
introducing gaps, if necessary, to achieve the maximum percent
sequence identity. None of N-terminal, C-terminal, or internal
extensions, deletions, or insertions into the antibody sequence
shall be construed as affecting sequence identity or homology. The
variant retains the ability to bind human IL-21 and preferably has
properties which are superior to those of the parent antibody. For
example, the variant may have a stronger binding affinity, enhanced
ability to inhibit IL-21-induced stimulation of immune cells. To
analyze such properties, one should compare a Fab form of the
variant to a Fab form of the parent antibody or a full length form
of the variant to a full length form of the parent antibody, for
example, since it has been found that the format of the anti-IL-21
antibody impacts its activity in the biological activity assays
disclosed herein. The variant antibody of particular interest
herein is one which displays at least about 10 fold, preferably at
least about 20 fold, and most preferably at least about 50 fold,
enhancement in biological activity when compared to the parent
antibody.
[0017] The term "parent antibody" as used herein refers to an
antibody which is encoded by an amino acid sequence used for the
preparation of the variant. Preferably, the parent antibody has a
human framework region and, if present, has human antibody constant
region(s). For example, the parent antibody may be a humanized or
human antibody.
[0018] The term "agonist" refers to any compound including a
protein, polypeptide, peptide, antibody, antibody fragment, large
molecule, or small molecule (less than 10 kD), that increases the
activity, activation or function of another molecule. IL-21
agonists cause, for example: stimulation of NK cells, T cell
subsets and B cell subsets and dendritic cells.
[0019] The term "antagonist" refers to any compound including a
protein, polypeptide, peptide, antibody, antibody fragment, large
molecule, or small molecule (less than 10 kD), that decreases the
activity, activation or function of another molecule. IL-21
antagonists cause: decreased immune function of NK cells, T cell
subsets and B cell subsets and dendritic cells; bind IL-21 such
that the interaction of IL-21 protein is blocked, inhibited,
reduced, antagonized or neutralized.
[0020] A "bivalent antibody" other than a "multispecific" or
"multifunctional" antibody, in certain embodiments, is understood
to comprise binding sites having identical antigenic
specificity.
[0021] A "bispecific" or "bifunctional" antibody is a hybrid
antibody having two different heavy/light chain pairs and two
different binding sites. Bispecific antibodies may be produced by a
variety of methods including, but not limited to, 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).
[0022] The term "chimeric antibody" or "chimeric antibodies" refers
to antibodies whose light and heavy chain genes have been
constructed, typically by genetic engineering, from immunoglobulin
variable and constant region genes belonging to different species.
For example, the variable segments of the genes from a mouse
monoclonal antibody may be joined to human constant segments, such
as gamma 1 and gamma 3. A typical therapeutic chimeric antibody is
thus a hybrid protein composed of the variable or antigen-binding
domain from a mouse antibody and the constant domain from a human
antibody, although other mammalian species may be used.
[0023] The term "effective neutralizing titer" as used herein
refers to the amount of antibody which corresponds to the amount
present in the serum of animals (human or cotton rat) that has been
shown to be either clinically efficacious (in humans) or to reduce
virus by 99% in, for example, cotton rats. The 99% reduction is
defined by a specific challenge of, e.g., 10.sup.3 pfu, 10.sup.4
pfu, 10.sup.5 pfu, 10.sup.6 pfu, 10.sup.7 pfu, 10.sup.8 pfu, or
10.sup.9 pfu) of RSV.
[0024] The term "epitope" includes any protein determinant capable
of specific binding to an immunoglobulin or T-cell receptor.
Epitopic determinants usually consist of chemically active surface
groupings of molecules such as amino acids or sugar side chains and
usually have specific three dimensional structural characteristics,
as well as specific charge characteristics. More specifically, the
term "IL-21 epitope" as used herein refers to a portion of a IL-21
polypeptide having antigenic or immunogenic activity in an animal,
preferably in a mammal, and most preferably in a mouse or a human.
An epitope having immunogenic activity is a portion of a IL-21
polypeptide that elicits an antibody response in an animal. An
epitope having antigenic activity is a portion of a IL-21
polypeptide to which an antibody immunospecifically binds as
determined by any method well known in the art, for example, by
immunoassays. Antigenic epitopes need not necessarily be
immunogenic.
[0025] The term "epitope tagged" when used herein refers to the
anti-IL-21 antibody fused to an "epitope tag". The epitope tag
polypeptide has enough residues to provide an epitope against which
an antibody can be made, yet is short enough such that it does not
interfere with activity of the IL-21 antibody. The epitope tag
preferably is sufficiently unique so that the antibody does not
substantially cross-react with other epitopes. Suitable tag
polypeptides generally have at least 6 amino acid residues and
usually between about 8-50 amino acid residues (preferably between
about 9-30 residues). Examples include the flu HA tag polypeptide
and its antibody 12CA5 (Field et al. Mol. Cell. Biol. 8:2159-2165
(1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10
antibodies thereto (Evan et al., Mol. Cell. Biol. 5(12):3610-3616
(1985)); and the Herpes Simplex virus glycoprotein D (gD) tag and
its antibody (Paborsky et al., Protein Engineering 3(6):547-553
(1990)). In certain embodiments, the epitope tag is a "salvage
receptor binding epitope". As used herein, the term "salvage
receptor binding epitope" refers to an epitope of the Fc region of
an IgG molecule (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, or
IgG.sub.4) that is responsible for increasing the in vivo serum
half-life of the IgG molecule.
[0026] The term "fragment" as used herein refers to a peptide or
polypeptide comprising an amino acid sequence of at least 5
contiguous amino acid residues, at least 10 contiguous amino acid
residues, at least 15 contiguous amino acid residues, at least 20
contiguous amino acid residues, at least 25 contiguous amino acid
residues, at least 40 contiguous amino acid residues, at least 50
contiguous amino acid residues, at least 60 contiguous amino
residues, at least 70 contiguous amino acid residues, at least
contiguous 80 amino acid residues, at least contiguous 90 amino
acid residues, at least contiguous 100 amino acid residues, at
least contiguous 125 amino acid residues, at least 150 contiguous
amino acid residues of the amino acid sequence of a IL-21
polypeptide or an antibody that immunospecifically binds to a IL-21
polypeptide.
[0027] As used herein, the term "immunoglobulin" refers to a
protein consisting of one or more polypeptides substantially
encoded by immunoglobulin genes. One form of immunoglobulin
constitutes the basic structural unit of an antibody. This form is
a tetramer and consists of two identical pairs of immunoglobulin
chains, each pair having one light and one heavy chain. In each
pair, the light and heavy chain variable regions are together
responsible for binding to an antigen, and the constant regions are
responsible for the antibody effector functions.
[0028] Full-length immunoglobulin "light chains" (about 25 Kd or
214 amino acids) are encoded by a variable region gene at the
NH.sub.2-terminus (about 110 amino acids) and a kappa or lambda
constant region gene at the COOH--terminus. Full-length
immunoglobulin "heavy chains" (about 50 Kd or 446 amino acids), are
similarly encoded by a variable region gene (about 116 amino acids)
and one of the other aforementioned constant region genes (about
330 amino acids). Heavy chains are classified as gamma, mu, alpha,
delta, or epsilon, and define the antibody's isotype as IgG, IgM,
IgA, IgD and IgE, respectively. 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 10 more amino acids. (See generally, Fundamental
Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989), Ch.
7.
[0029] An immunoglobulin light or heavy chain variable region
consists of a "framework" region interrupted by three hypervariable
regions. Thus, the term "hypervariable region" refers to the amino
acid residues of an antibody which are responsible for antigen
binding. The hypervariable region comprises amino acid residues
from a "Complementarity Determining Region" or "CDR" (i.e.,
residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain
variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the
heavy chain variable domain (Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)) and/or those residues
from a "hypervariable loop" (i.e., residues 26-32 (L1), 50-52 (L2)
and 91-96 (L3) in the light chain variable domain and 26-32 (H1),
53-55 (H2) and 96-101 (H3) in the heavy chain variable domain;
Chothia and Lesk, J. Mol. Biol. 196: 901-917, 1987) (both of which
are incorporated herein by reference). "Framework Region" or "FR"
residues are those variable domain residues other than the
hypervariable region residues as herein defined. The sequences of
the framework regions of different light or heavy chains are
relatively conserved within a species. Thus, a "human framework
region" is a framework region that is substantially identical
(about 85% or more, usually 90-95% or more) to the framework region
of a naturally occurring human immunoglobulin. The framework region
of an antibody, that is the combined framework regions of the
constituent light and heavy chains, serves to position and align
the CDR's. The CDR's are primarily responsible for binding to an
epitope of an antigen.
[0030] Accordingly, the term "humanized" immunoglobulin refers to
an immunoglobulin comprising a human framework region and one or
more CDR's from a non-human (usually a mouse or rat)
immunoglobulin. The non-human immunoglobulin providing the CDR's is
called the "donor" and the human immunoglobulin providing the
framework is called the "acceptor". Constant regions need not be
present, but if they are, they must be substantially identical to
human immunoglobulin constant regions, i.e., at least about 85-90%,
preferably about 95% or more identical. Hence, all parts of a
humanized immunoglobulin, except possibly the CDR's, are
substantially identical to corresponding parts of natural human
immunoglobulin sequences. A "humanized antibody" is an antibody
comprising a humanized light chain and a humanized heavy chain
immunoglobulin. For example, a humanized antibody would not
encompass a typical chimeric antibody as defined above, e.g.,
because the entire variable region of a chimeric antibody is
non-human.
[0031] As used herein, the term "human antibody" includes and
antibody that has an amino acid sequence of a human immunoglobulin
and includes antibodies isolated from human immunoglobulin
libraries or from animals transgenic for one or more human
immunoglobulin and that do not express endogenous immunoglobulins,
as described, for example, by Kucherlapati et al. in U.S. Pat. No.
5,939,598.
[0032] The term "genetically altered antibodies" means antibodies
wherein the amino acid sequence has been varied from that of a
native antibody. Because of the relevance of recombinant DNA
techniques in the generation of antibodies, one need not be
confined to the sequences of amino acids found in natural
antibodies; antibodies can be redesigned to obtain desired
characteristics. The possible variations are many and range from
the changing of just one or a few amino acids to the complete
redesign of, for example, the variable or constant region. Changes
in the constant region will, in general, be made in order to
improve or alter characteristics, such as complement fixation,
interaction with membranes and other effector functions. Changes in
the variable region will be made in order to improve the antigen
binding characteristics.
[0033] In addition to antibodies, immunoglobulins may exist in a
variety of other forms including, for example, single-chain or Fv,
Fab, and (Fab').sub.2, as well as diabodies, linear antibodies,
multivalent or multispecific hybrid antibodies (as described above
and in detail in: Lanzavecchia et al., Eur. J. Immunol. 17, 105
(1987)) and in single chains (e.g., Huston et al., Proc. Natl.
Acad. Sci. U.S.A., 85 5879-5883 (1988) and Bird et al., Science,
242:423-426 (1988), which are incorporated herein by reference).
(See, generally, Hood et al., "Immunology", Benjamin, N.Y., 2nd ed.
(1984), and Hunkapiller and Hood, Nature, 323:15-16 (1986), which
are incorporated herein by reference).
[0034] As used herein, the terms "single-chain Fv," "single-chain
antibodies," "Fv" or "scFv" refer to antibody fragments that
comprises the variable regions from both the heavy and light
chains, but lacks the constant regions, but within a single
polypeptide chain. Generally, a single-chain antibody further
comprises a polypeptide linker between the VH and VL domains which
enables it to form the desired structure which would allow for
antigen binding. Single chain antibodies are discussed in detail by
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315
(1994); see also International Patent Application Publication No.
WO 88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203, the
disclosures of which are incorporated by reference for any purpose.
In specific embodiments, single-chain antibodies can also be
bi-specific and/or humanized.
[0035] A "Fab fragment" is comprised of one light chain and the
C.sub.H1 and variable regions of one heavy chain. The heavy chain
of a Fab molecule cannot form a disulfide bond with another heavy
chain molecule.
[0036] A "Fab' fragment" contains one light chain and one heavy
chain that contains more of the constant region, between the
C.sub.H1 and C.sub.H2 domains, such that an interchain disulfide
bond can be formed between two heavy chains to form a F(ab').sub.2
molecule.
[0037] A "F(ab').sub.2 fragment" contains two light chains and two
heavy chains containing a portion of the constant region between
the C.sub.H1 and C.sub.H2 domains, such that an interchain
disulfide bond is formed between two heavy chains.
[0038] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy chain
variable domain (V.sub.H) connected to a light chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L).
By using a linker that is too short to allow pairing between the
two domains on the same chain, the domains are forced to pair with
the complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for
example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.
Acad. Sci. USA 90:6444-6448 (1993).
[0039] The term "linear antibodies" refers to the antibodies
described in Zapata et al. Protein Eng. 8 (10):1057-1062 (1995).
Briefly, these antibodies comprise a pair of tandem Fd segments
(V.sub.H-C.sub.H1-V.sub.H-C.sub.H1) which form a pair of antigen
binding regions. Linear antibodies can be bispecific or
monospecific.
[0040] The term "immunologically functional immunoglobulin
fragment" as used herein refers to a polypeptide fragment that
contains at least the variable domains of the immunoglobulin heavy
and light chains. An immunologically functional immunoglobulin
fragment of the invention is capable of binding to a ligand,
preventing binding of the ligand to its receptor, interrupting the
biological response resulting from ligand binding to the receptor,
or any combination thereof. Preferably, an immunologically
functional immunoglobulin fragment of the invention binds
specifically to IL-21.
[0041] The term "monoclonal antibody" as used herein is not limited
to antibodies produced through hybridoma technology. The term
"monoclonal antibody" refers to an antibody that is derived from a
single clone, including any eukaryotic, prokaryotic, or phage
clone, and not the method by which it is produced.
[0042] The present invention provides monoclonal antibodies and
antibody fragments that specifically bind with IL-21 proteins and
polypeptides. Human and mouse IL-21 polypeptides, proteins and
polynucleotides encoding the polypeptides are disclosed in
Parrish-Novak et al., Nature 408:57-63, 2003, U.S. Pat. Nos.
6,307,024 and 6,686,178 and WO 04/055168. Exemplary antibodies
include neutralizing antibodies, and may be murine monoclonal
antibodies, humanized antibodies derived from murine monoclonal
antibodies, and human monoclonal antibodies. Illustrative antibody
fragments include F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, and minimal
recognition units. Neutralizing antibodies preferably bind IL-21
such that the interaction of IL-21 protein is blocked, inhibited,
reduced, antagonized or neutralized. Described herein are epitopes
and structural and functional characteristics defining regions of
the human IL-21 protein that have been identified as targets for a
therapeutic monoclonal antibody. Exemplary mouse anti-human IL-21
monoclonal antibodies and rat anti-human monoclonal antibodies and
pools of these monoclonal antibodies with the ability to bind
wildtype human IL-21, a mutant IL-21 protein and/or peptide regions
of human IL-21 are presented. The present invention further
includes compositions comprising a carrier and a peptide,
polypeptide, or antibody described herein.
[0043] Thus, the present invention provides that antagonists to
IL-21 activity, such as anti-IL-21 antibodies, which are useful in
therapeutic treatment of inflammatory diseases. For example,
anti-IL-21 antibodies are useful in the treatment of pancreatitis,
type I diabetes (IDDM), Graves Disease, inflammatory bowel disease
(IBD), Crohn's Disease, ulcerative colitis, irritable bowel
syndrome, multiple sclerosis, rheumatoid arthritis, diverticulosis,
systemic lupus erythematosus, psoriasis, ankylosing spondylitis,
scleroderma, systemic sclerosis, psoriatic arthritis,
osteoarthritis, atopic dermatitis, vitiligo, graft vs. host disease
(GVHD), cutaneous T cell lymphoma (CTCL), Sjogren's syndrome,
glomerulonephritis, IgA nephropathy, graft versous host disease,
transplant rejection, atopic dermatitis, anti-phospholipid
syndrome, and asthma, and other autoimmune diseases, or other
diseases mediated by IL-21 and IL-21 receptor agonists.
[0044] The present invention also includes genetically altered
antibodies that are functionally equivalent to the above-described
antibodies. Modified antibodies providing improved stability and/or
therapeutic efficacy are preferred. Examples of modified antibodies
include those with conservative substitutions of amino acid
residues, and one or more deletions or additions of amino acids
which do not significantly deleteriously alter the antigen binding
utility. Substitutions can range from changing or modifying one or
more amino acid residues to complete redesign of a region as long
as the therapeutic utility is maintained. Antibodies of the present
invention can be can be modified post-tranlationally (e.g.,
acetylation, and phosphorylation) or can be modified synthetically
(e.g., the attachment of a labeling group).
[0045] The genetically altered antibodies also include chimeric
antibodies that derived from the anti-IL-21 antibodies. Preferably,
the chimeric antibodies comprise a variable region derived from a
mouse or rat and a constant region derived from a human so that the
chimeric antibody has a longer half-life and is less immunogenic
when administered to a human subject. The method of making chimeric
antibodies is known in the art. The variable regions of these
antibodies can be connected with a constant region of a human IgG
to form the desired chimeric antibody.
[0046] Preferably, the genetically altered anti-IL-21 antibodies
used in the present invention include humanized version of the
antibodies described herein. In certain embodiments, the humanized
antibody comprising CDRs of a mouse donor immunoglobulin and heavy
chain and light chain frameworks of a human acceptor
immunoglobulin. The method of making humanized antibody is
disclosed in U.S. Pat. Nos. 5,301,101; 5,585,089; 5,693,762; and
6,180,370 (each of which is incorporated by reference in its
entirety). The CDRs of these antibodies can then be grafted to any
selected human frameworks, which are known in the art, to generate
the desired humanized antibody.
[0047] Antibodies of the present invention may be described or
specified in terms of the epitope(s) or portion(s) of a polypeptide
of the present invention that they recognize or specifically bind.
The epitope(s) or polypeptide portion(s) may be specified as
described herein, e.g., by N-terminal and C-terminal positions, or
by size in contiguous amino acid residues. Antibodies of the
present invention may also be described or specified in terms of
their cross-reactivity. Antibodies that do not bind any other
analog, ortholog, or homolog of a polypeptide of the present
invention are included.
[0048] Epitope binning refers to the use of competitive binding
assays to identify pairs of antibodies that are, or are not,
capable of binding IL-21 protein simultaneously thereby identifying
antibodies that bind to the same, or overlapping epitopes on
protein. Families of antibodies (or bins) having the same binding
specificity can then be used to define specific epitopes on IL-21.
Epitope binning experiments provide evidence that antigenically
distinct epitopes are present. However, by themselves, they do not
identify, or "map" the epitope to a specific amino acid sequence or
location on the IL-21 protein molecule.
[0049] Competition for binding can be evaluated for any pair of
antibodies or fragments. For example, using the appropriate
detection reagents, the binding specificity of antibodies or
binding fragments from any species/source can be compared to the
binding specificity of the monoclonal antibodies disclosed herein.
Epitope binning can be performed with "isolated antibodies" or with
cell culture supernatants. Frequently binning is performed with
first round clonal supernatants to guide the choice of clones to be
developed further. The antibodies to be compared should have
substantially homogeneous antigen binding domains. In the case of
"bispecific" or "bifunctional" antibodies the binding specificity
of the two different binding sites need to be evaluated or binned
independently.
[0050] The present invention features both receptor-specific
antibodies and ligand-specific antibodies. In addition to
competitive binding of antibodies, epitope binning can also be used
to identify antibodies to either a receptor or a ligand that
competitively interfere with the binning of a ligand and its
receptor. Frequently, favorable properties, of a family (or bin) of
antibodies can correlated with a binding to a specific epitope
defined by the epitope bin.
[0051] Competitive binding experiments do not directly measure the
binding affinity, however the antibodies to be tested must bind
sufficiently strongly to act as competitors. Generally experimental
conditions are designed to minimize the effects of differences in
binding affinity.
[0052] Anti-Antigen IL-21 antibodies may also be useful in
diagnostic assays for IL-21 protein, e.g., detecting its expression
in specific cells, tissues, or serum. Antibodies assigned to
different bins and capable of binding to different immunogenic
portions, or epitopes, of IL-21 may be used as the reagents for
sandwich assays. In a sandwich assay, the test sample analyte is
captured by a first antibody which is immobilized on a solid
support, and thereafter detected by a second antibody that also
binds to the analyte, thus forming an insoluble three-part complex.
See, e.g., U.S. Pat. No. 4,376,110. The second antibody may itself
be labeled with a detectable moiety (direct sandwich assays) or may
be measured using an anti-immunoglobulin antibody that is labeled
with a detectable moiety (indirect sandwich assay). For example,
one type of sandwich assay is an ELISA assay, in which case the
detectable moiety is an enzyme.
[0053] The antibodies of the present invention may be assayed for
specific binding by any method known in the art. Many different
competitive binding assay format(s) can be used for epitope
binning. The immunoassays which can be used include, but are not
limited to, competitive and non-competitive assay systems using
techniques such as western blots, radioimmunoassays, ELISA (enzyme
linked immunosorbent assay), "sandwich" immunoassays,
immunoprecipitation assays, precipitin reactions, gel diffusion
precipitin reactions, immunodiffusion assays, agglutination assays,
complement-fixation assays, immunoradiometric assays, fluorescent
immunoassays, protein A immunoassays, to name but a few. Such
assays are routine and well known in the art (see, e.g., Ausubel et
al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John
Wiley & Sons, Inc., New York). Exemplary immunoassays are
described briefly below (but are not intended by way of
limitation). Additionally, a routine cross-blocking assay such as
that described in Antibodies, A Laboratory Manual, Cold Spring
Harbor Laboratory, Ed Harlow and David Lane (1988), can be
performed.
[0054] The Biacore is only one of a variety of assay formats that
are routinely used to epitope bin panels of monoclonal antibodies.
Many references (e.g. The Epitope Mapping Protocols, Methods in
Molecular Biology, Volume 6.6 Glenn E. Morris ed.) describe
alternative methods that could be used to bin antibodies and would
be expected to provide identical information regarding the binding
specificity of the antibodies to IL-21 protein. When using the
Biacore system, epitope binning experiments are performed with
soluble, native antigen. Epitope binning studies can be performed
on a Biacore1000.RTM. system (Biacore, Uppsalla Sweden).
BIAlogue.RTM. v. 1.2 software can be used for programming run
methods. For the example of using the Biacore to bin mouse
monoclonal antibodies raised against IL-21, polyclonal goat
anti-Mouse IgG Fc antibody (Jackson ImmunoResearch Laboratories,
West Grove, Pa.) can be covalently immobilized to a Biacorei CM5
sensor chip and used to bind (capture) the primary monoclonal
antibody of test series to the chip. Unoccupied Fc binding sites on
the chip are then blocked using a polyclonal IgG Fc fragment
(Jackson ImmunoResearch Laboratories, West Grove, Pa.).
Subsequently, IL-21 protein is injected and allowed to specifically
bind to the captured primary monoclonal antibody. The Biacore
instrument measures the mass of protein bound to the sensor chip,
and the binding of both the primary antibody and IL-21 antigen can
be verified for each cycle. Following the binding of the primary
antibody and antigen to the chip, soluble secondary antibody is
injected and allowed to bind to the pre-bound antigen. If the
secondary monoclonal antibody is capable of binding the IL-21
antigen simultaneously with the primary monoclonal antibody, its
binding is detected by the Biacore. If, however, the secondary
monoclonal antibody is not capable of binding the IL-21 antigen
simultaneously with the primary monoclonal antibody, no additional
binding is detected. Each monoclonal antibody is tested against
itself as a negative control to establish the level of the
background (no-binding) signal.
[0055] A label-free competitive ELISA format (LFC-ELISA) can also
be used to bin antibodies. This method is described by Nagata et
al., J. Immuno Methods 292:141-155, 2004. This method for epitope
binning utilized biotinylated IL-21. For the example of binning
mouse monoclonal antibodies raised against IL-21, microtiter plates
are coated at 100 .mu.L/well with 1 .mu.g/mL of a goat anti-mouse
IgG Fc-.gamma. specific antibody (Jackson ImmunoResearch) diluted
in ELISA B (PBS, 0.1% Tween 20, 1% BSA). After binding of this
coating antibody for 3 hours at ambient temperature, each
mAb-containing conditioned media is diluted in ELISA B to yield an
approximate mAb concentration of 0.5 .mu.g/mL and allowed to bind
to the goat anti-mouse IgG coated plates overnight at 4.degree. C.
(mAb#1). In parallel, a second set of conditioned medias (mAb#2)
are diluted in polystyrene test tubes to approximately 0.5 .mu.g/mL
mAb in ELISA B, mixed with 50 ng/mL biotinylated IL-21 antigen, and
incubated overnight at 4.degree. C. After incubation of mAb#1 with
the coating antibody, the plates are blocked with an unrelated
antibody to saturate unoccupied binding sites on the plate. The
mAb#2-biotin-IL-21 mixtures are added to the plate and allowed to
bind. As a control for (non-competition) in the assay, 50 ng/mL
biotinylated IL-21 is added directly (without pre-incubation with
mAb#2) to wells containing immobilized mAb#1. After incubation with
the biotinylated-IL-21-mAb#2 complex, streptavidin-HRP (Pierce,
Rockford, Ill.) is added to the plate at 0.5 .mu.g/mL. The plates
are developed with TMB substrate (BioFX Laboratories, Owings Mills,
Md.), and the absorbance of the individual wells at 450 nm is
measured with a plate reader (Molecular Devices SpectraMax.RTM.
340, Sunnyvale, Calif.). If mAb#1 binds to a different epitope from
mAb#2, the biotin-IL-21-mAb#2 complex will bind to the plate
resulting in a high absorbance reading. If mAb#1 binds to the same
epitope as mAb#2, the biotin-IL-21 -MAb#2 complex will not bind to
the plate resulting in a low absorbance reading.
[0056] Antibodies of the present invention act as antagonists of
IL-21. For example, the present invention includes antibodies which
disrupt IL-21's receptor/ligand interactions either partially or
fully. The invention features ligand-specific antibodies that
prevent receptor activation. The invention includes neutralizing
antibodies which bind the ligand and prevent binding of the ligand
to the receptor, as well as antibodies which bind the ligand,
thereby preventing receptor activation, but do not prevent the
ligand from binding the receptor. Receptor activation (i.e.,
signaling) may be determined by techniques described herein or
otherwise known in the art. For example, receptor activation can be
determined by detecting the phosphorylation (e.g., tyrosine or
serine/threonine) of the receptor or its substrate by
immunoprecipitation followed by western blot or luminex based
analysis (for example, as described supra). In specific
embodiments, antibodies are provided that inhibit ligand or
receptor activity by at least 90%, at least 80%, at least 70%, at
least 60%, or at least 50% of the activity in absence of the
antibody.
Production of Anti-IL-21 Antibodies
[0057] Antibodies to IL-21 can be obtained, for example, using the
product of a IL-21 expression vector or IL-21 isolated from a
natural source as an antigen. Particularly useful anti-IL-21
antibodies "bind specifically" with IL-21. Antibodies are
considered to be specifically binding if the antibodies exhibit at
least one of the following two properties: (1) antibodies bind to
IL-21 with a threshold level of binding activity, and (2)
antibodies do not significantly cross-react with polypeptides
related to IL-21.
[0058] With regard to the first characteristic, antibodies
specifically bind if they bind to a IL-21 polypeptide, peptide or
epitope with a binding affinity (Ka) of 10.sup.6 M.sup.-1 or
greater, preferably 10.sup.7 M.sup.-1 or greater, more preferably
10.sup.8 M.sup.-1 or greater, and most preferably 10.sup.9 M.sup.-1
or greater. The binding affinity of an antibody can be readily
determined by one of ordinary skill in the art, for example, by
Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 51:660 1949) or
using a commercially available biosensor instrument (BIAcore,
Pharmacia Biosensor, Piscataway, N.J.). With regard to the second
characteristic, antibodies do not significantly cross-react with
related polypeptide molecules, for example, if they detect IL-21,
but not other known polypeptides using a standard Western blot
analysis or capture ELISA. Examples of known related polypeptides
include known members of the IL-2 family.
[0059] Anti-IL-21 antibodies can be produced using antigenic IL-21
epitope-bearing peptides and polypeptides. Antigenic
epitope-bearing peptides and polypeptides of the present invention
contain a sequence of at least nine, or between 15 to about 30
amino acids contained within SEQ ID NO:2 or another amino acid
sequence disclosed herein. However, peptides or polypeptides
comprising a larger portion of an amino acid sequence of the
invention, containing from 30 to 50 amino acids, or any length up
to and including the entire amino acid sequence of a polypeptide of
the invention, also are useful for inducing antibodies that bind
with IL-21. It is desirable that the amino acid sequence of the
epitope-bearing peptide is selected to provide substantial
solubility in aqueous solvents (i.e., the sequence includes
relatively hydrophilic residues, while hydrophobic residues are
typically avoided). Moreover, amino acid sequences containing
proline residues may be also be desirable for antibody
production.
[0060] Monoclonal anti-IL-21 antibodies can be generated by methods
known to those skilled in the art. Rodent monoclonal antibodies to
specific antigens may be obtained by known methods (see, for
example, Kohler et al., Nature 256:495 (1975), Coligan et al.
(eds.), Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7
(John Wiley & Sons 1991) ["Coligan"], Picksley et al.,
"Production of monoclonal antibodies against proteins expressed in
E. coli," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover
et al. (eds.), page 93 (Oxford University Press 1995)).
[0061] Selection of binders from the display of a library of
antibody fragments is an in vitro alternative to the development of
monoclonal antibodies. The principle of display technology is
establishment of a physical connection between a binding moiety and
the encoding genetic material. This concept has been used in a
number of modes from display of protein and peptide libraries on
surfaces of bacteriophage, bacteria, and yeast to the display of
proteins attached to ribosomes in vitro (see for example Rothe et
al., FASEB J. 20:1599 (2006)). The display of antibodies on the
surface on single-stranded bacteriophage is the most highly
developed of these technologies. The typical method used for
antibody display is to fuse either the single chain Fv fragment or
the heavy chain Fd (heavy chain portion of a Fab) with the gene III
protein of the phage. Antibody libraries can be naive, representing
the natural immune repertoire, or semi-synthetic, consisting of
frameworks taken from native human templates combined with
synthetic CDR sequence libraries to increase diversity. Phage with
specific binding activities can be isolated from random libraries
of antibody fragments (particularly Fab and scFv) or peptides after
repeated rounds of growth and selection (see, for example,
Hoogenboom, Nature Biotech. 23:1105 (2005.)
[0062] In a further embodiment, antibodies of the present invention
can also be generated using various phage display methods known in
the art. In phage display methods, functional antibody domains are
displayed on the surface of phage particles which carry the
polynucleotide sequences encoding them. In a particular, such phage
can be utilized to display antigen-binding domains expressed from a
repertoire or combinatorial antibody library (e.g., human or
murine). Phage expressing an antigen binding domain that binds the
antigen of interest can be selected or identified with antigen,
e.g., using labeled antigen or antigen bound or captured to a solid
surface or bead. Phage used in these methods are typically
filamentous phage including fd and M13 binding domains expressed
from phage with Fab, Fv or disulfide stabilized Fv antibody domains
recombinantly fused to either the phage gene III or gene VIII
protein. Examples of phage display methods that can be used to make
the antibodies of the present invention include those disclosed in
Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al.,
J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur.
J. Immunol. 24:952-958 (1994); Persic et al., Gene 187:9-18 (1997);
Burton et al., Advances in Immunology 57:191-280 (1994); PCT
application No. PCT/GB91/01134; PCT publications WO 90/02809; WO
91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO
95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484;
5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908;
5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of
which is incorporated herein by reference in its entirety. In yet a
further embodiment, antibodies or antibody fragments can be
isolated from antibody phage libraries generated using the
techniques described in McCafferty et al., Nature, 348: 552-554
(1990). Clackson et al., Nature, 352: 624-628 (1991) and Marks et
al., J. Mol. Biol., 222: 581-597 (1991) describe the isolation of
murine and human antibodies, respectively, using phage libraries.
Subsequent publications describe the production of high affinity
(nM range) human antibodies by chain shuffling (Marks et al.,
Bio/Technology, 10: 779-783 (1992)), as well as combinatorial
infection and in vivo recombination as a strategy for constructing
very large phage libraries (Waterhouse et al., Nuc. Acids. Res.,
21: 2265-2266 (1993)). Thus, these techniques are viable
alternatives to traditional monoclonal antibody hybridoma
techniques for isolation of monoclonal antibodies.
[0063] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described in detail below. For
example, techniques to recombinantly produce Fab, Fab' and F(ab')2
fragments can also be employed using methods known in the art such
as those disclosed in PCT publication WO 92/22324; Mullinax et al.,
BioTechniques 12 (6):864-869, 1992; and Sawai et al., AJRI
34:26-34, 1995; and Better et al., Science 240:1041-1043, 1988 (all
references incorporated by reference in their entireties).
[0064] Human antibodies can also be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For example, the human heavy and light chain immunoglobulin gene
complexes may be introduced randomly or by homologous recombination
into mouse embryonic stem cells. Alternatively, the human variable
region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and
light chain genes. The mouse heavy and light chain immunoglobulin
genes may be rendered non-functional separately or simultaneously
with the introduction of human immunoglobulin loci by homologous
recombination. In particular, homozygous deletion of the JH region
prevents endogenous antibody production. The modified embryonic
stem cells are expanded and microinjected into blastocysts to
produce chimeric mice. The chimeric mice are then bred to produce
homozygous offspring that express human antibodies. The transgenic
mice are immunized in the normal fashion with a selected antigen,
e.g., all or a portion of a polypeptide of the invention.
Monoclonal antibodies directed against the antigen can be obtained
from the immunized, transgenic mice using conventional hybridoma
technology. The human immunoglobulin transgenes harbored by the
transgenic mice rearrange during B cell differentiation, and
subsequently undergo class switching and somatic mutation. Thus,
using such a technique, it is possible to produce therapeutically
useful IgG, IgA, IgM and IgE antibodies. For an overview of this
technology for producing human antibodies, see Lonberg and Huszar
(Int. Rev. Immunol. 13:65-93, 1995).
[0065] For a detailed discussion of this technology for producing
human antibodies and human monoclonal antibodies and protocols for
producing such antibodies, see, e.g., Jakobovits et al., Proc.
Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature,
362: 255-258 (1993); Bruggermann et al., Year in Immuno., 7:33
(1993); PCT publications WO 98/24893; WO 96/34096; WO 96/33735;
U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825;
5,661,016; 5,545,806; 5,814,318; and 5,939,598, which are
incorporated by reference herein in their entirety. In addition,
companies such as Medarex, Inc. (Princeton, N.J.) and Genpharm (San
Jose, Calif.) can be engaged to provide human antibodies directed
against a selected antigen using technology similar to that
described above. See, e.g. U.S. Pat. 7,135,287.
[0066] The antibodies of the invention can be produced by any
method known in the art for the synthesis of antibodies, in
particular, by chemical synthesis or preferably, by recombinant
expression techniques. Recombinant expression of an antibody of the
invention, or fragment, derivative or analog thereof, e.g., a heavy
or light chain of an antibody of the invention, requires
construction of an expression vector containing a polynucleotide
that encodes the antibody. Once a polynucleotide encoding an
antibody molecule or a heavy or light chain of an antibody, or
portion thereof (preferably containing the heavy or light chain
variable domain), of the invention has been obtained, the vector
for the production of the antibody molecule may be produced by
recombinant DNA technology using techniques well known in the art.
Thus, methods for preparing a protein by expressing a
polynucleotide containing an antibody encoding nucleotide sequence
are described herein. Methods which are well known to those skilled
in the art can be used to construct expression vectors containing
antibody coding sequences and appropriate transcriptional and
translational control signals. These methods include, for example,
in vitro recombinant DNA techniques, synthetic techniques, and in
vivo genetic recombination. The invention, thus, provides
replicable vectors comprising a nucleotide sequence encoding an
antibody molecule of the invention, or a heavy or light chain
thereof, or a heavy or light chain variable domain, operably linked
to a promoter. Such vectors may include the nucleotide sequence
encoding the constant region of the antibody molecule (see, e.g.,
PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S.
Pat. No. 5,122,464) and the variable domain of the antibody may be
cloned into such a vector for expression of the entire heavy or
light chain.
[0067] The expression vector is transferred to a host cell by
conventional techniques and the transfected cells are then cultured
by conventional techniques to produce an antibody of the invention.
Thus, the invention includes host cells containing a polynucleotide
encoding an antibody of the invention, or a heavy or light chain
thereof, operably linked to a heterologous promoter. In preferred
embodiments for the expression of double-chained antibodies,
vectors encoding both the heavy and light chains may be
co-expressed in the host cell for expression of the entire
immunoglobulin molecule, as detailed below.
[0068] A variety of host-expression vector systems may be utilized
to express the antibody molecules of the invention. Such
host-expression systems represent vehicles by which the coding
sequences of interest may be produced and subsequently purified,
but also represent cells which may, when transformed or transfected
with the appropriate nucleotide coding sequences, express an
antibody molecule of the invention in situ. These include but are
not limited to microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors containing antibody coding
sequences; yeast (e.g., Saccharomyces, Pichia) transformed with
recombinant yeast expression vectors containing antibody coding
sequences; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing antibody coding
sequences; plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing antibody coding
sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3
cells) harboring recombinant expression constructs containing
promoters derived from the genome of mammalian cells (e.g.,
metallothionein promoter) or from mammalian viruses (e.g., MPSV,
CMV, the adenovirus late promoter; the vaccinia virus 7.5K
promoter). Preferably, bacterial cells such as Escherichia coli,
and more preferably, eukaryotic cells, especially for the
expression of whole recombinant antibody molecule, are used for the
expression of a recombinant antibody molecule. For example,
mammalian cells such as Chinese hamster ovary cells (CHO), in
conjunction with a vector such as the major intermediate early gene
promoter element from human cytomegalovirus, CMV enhancer or MPSV
promoter is an effective expression system for antibodies (Foecking
et al., 1986, Gene 45:101; Cockett et al., 1990, Bio/Technology
8:2).
[0069] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
antibody molecule being expressed. For example, when a large
quantity of such a protein is to be produced, for the generation of
pharmaceutical compositions of an antibody molecule, vectors which
direct the expression of high levels of fusion protein products
that are readily purified may be desirable. Such vectors include,
but are not limited, to the E. coli expression vector pUR278
(Ruther et al., 1983, EMBO J. 2:1791), in which the antibody coding
sequence may be ligated individually into the vector in frame with
the lac Z coding region so that a fusion protein is produced; pIN
vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109,
1985; Van Heeke & Schuster J. Biol. Chem. 24:5503-5509, 1989);
and the like. pGEX vectors may also be used to express foreign
polypeptides as fusion proteins with glutathione S-transferase
(GST). In general, such fusion proteins are soluble and can easily
be purified from lysed cells by adsorption and binding to a matrix
glutathione-agarose beads followed by elution in the presence of
free glutathione. The pGEX vectors are designed to include thrombin
or factor Xa protease cleavage sites so that the cloned target gene
product can be released from the GST moiety.
[0070] In an insect system, Autographa califomica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The antibody
coding sequence may be cloned individually into non-essential
regions (for example the polyhedrin gene) of the virus and placed
under control of an AcNPV promoter (for example the polyhedrin
promoter).
[0071] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the antibody coding sequence of interest may be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric gene may then be inserted in the adenovirus genome by in
vitro or in vivo recombination. Insertion in a non-essential region
of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing the
antibody molecule in infected hosts. (e.g., see Logan & Shenk,
Proc. Natl. Acad. Sci. USA 81:355-359, 1984). Specific initiation
signals may also be required for efficient translation of inserted
antibody coding sequences. These signals include the ATG initiation
codon and adjacent sequences. Furthermore, the initiation codon
must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see Bittner et al., Methods in Enzymol.
153:51-544, 1987).
[0072] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. Such mammalian
host cells include but are not limited to CHO, VERO, BHK, Hela,
COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell
lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and
normal mammary gland cell line such as, for example, CRL7030 and
Hs578Bst.
[0073] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the antibody molecule may be engineered.
Rather than using expression vectors which contain viral origins of
replication, host cells can be transformed with DNA controlled by
appropriate expression control elements (e.g., promoter, enhancer,
sequences, transcription terminators, polyadenylation sites, etc.),
and a selectable marker. Following the introduction of the foreign
DNA, engineered cells may be allowed to grow for 1-2 days in an
enriched media, and then are switched to a selective media. The
selectable marker in the recombinant plasmid confers resistance to
the selection and allows cells to stably integrate the plasmid into
their chromosomes and grow to form foci which in turn can be cloned
and expanded into cell lines. This method may advantageously be
used to engineer cell lines which express the antibody molecule.
Such engineered cell lines may be particularly useful in screening
and evaluation of compounds that interact directly or indirectly
with the antibody molecule.
[0074] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler et
al., Cell 11:223, 1977), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl.
Acad. Sci. USA 48:202, 1992), and adenine phosphoribosyltransferase
(Lowy et al., Cell 22:817, 1980) genes can be employed in tk-,
hgprt- or aprt-cells, respectively. Also, antimetabolite resistance
can be used as the basis of selection for the following genes:
dhfr, which confers resistance to methotrexate (Wigler et al.,
Proc. Natl. Acad. Sci. USA 77:357, 1980; O'Hare et al., Proc. Natl.
Acad. Sci. USA 78:1527, 1981); gpt, which confers resistance to
mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA
78:2072, 1981); neo, which confers resistance to the aminoglycoside
G-418 (Wu and Wu, Biotherapy 3:87-95, 1991; Tolstoshev, Ann. Rev.
Pharmacol. Toxicol. 32:573-596, 1993; Mulligan, Science
260:926-932, 1993; and Morgan and Anderson, Ann. Rev. Biochem.
62:191-217, 1993; TIB TECH 11 (5):155-215), May, 1993; and hygro,
which confers resistance to hygromycin (Santerre et al., Gene
30:147, 1984). Methods commonly known in the art of recombinant DNA
technology which can be used are described in Ausubel et al.
(eds.), 1993, Current Protocols in Molecular Biology, John Wiley
& Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A
Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13,
Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics,
John Wiley & Sons, NY.; Colberre-Garapin et al., J. Mol. Biol.
150:1, 1981; which are incorporated by reference herein in their
entireties.
[0075] The expression levels of an antibody molecule can be
increased by vector amplification (for a review, see Bebbington and
Hentschel, The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells in DNA cloning, Vol.
3. (Academic Press, New York, 1987)). When a marker in the vector
system expressing antibody is amplifiable, increase in the level of
inhibitor present in culture of host cell will increase the number
of copies of the marker gene. Since the amplified region is
associated with the antibody gene, production of the antibody will
also increase (Crouse et al., Mol. Cell. Biol. 3:257, 1983).
[0076] The host cell may be co-transfected with two expression
vectors of the invention, the first vector encoding a heavy chain
derived polypeptide and the second vector encoding a light chain
derived polypeptide. The two vectors may contain identical
selectable markers which enable equal expression of heavy and light
chain polypeptides. Alternatively, a single vector may be used
which encodes both heavy and light chain polypeptides. In such
situations, the light chain should be placed before the heavy chain
to avoid an excess of toxic free heavy chain (Proudfoot, Nature
322:52, 1986; Kohler, Proc. Natl. Acad. Sci. USA 77:2197, 1980).
The coding sequences for the heavy and light chains may comprise
cDNA or genomic DNA.
[0077] Once an antibody molecule of the invention has been
recombinantly expressed, it may be purified by any method known in
the art for purification of an immunoglobulin molecule, for
example, by chromatography (e.g., ion exchange, affinity,
particularly by affinity for the specific antigen after Protein A,
and sizing column chromatography), centrifugation, differential
solubility, or by any other standard technique for the purification
of proteins.
[0078] For particular uses, it may be desirable to prepare
fragments of anti-IL-21 antibodies. Such antibody fragments can be
obtained, for example, by proteolytic hydrolysis of the antibody.
Antibody fragments can be obtained by pepsin or papain digestion of
whole antibodies by conventional methods. As an illustration,
antibody fragments can be produced by enzymatic cleavage of
antibodies with pepsin to provide a 5S fragment denoted F(ab')2.
This fragment can be further cleaved using a thiol reducing agent
to produce 3.5S Fab' monovalent fragments. Optionally, the cleavage
reaction can be performed using a blocking group for the sulfhydryl
groups that result from cleavage of disulfide linkages. As an
alternative, an enzymatic cleavage using pepsin produces two
monovalent Fab fragments and an Fc fragment directly. These methods
are described, for example, by Goldenberg, U.S. Pat. No. 4,331,647,
Nisonoff et al., Arch Biochem. Biophys. 89:230, 1960; Porter,
Biochem. J. 73:119, 1959; Edelman et al., in Methods in Enzymology
Vol. 1, page 422 (Academic Press 1967), and by Coligan at pages
2.8.1-2.8.10 and 2.10.-2.10.4.
[0079] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody.
[0080] For example, Fv fragments comprise an association of VH and
VL chains. This association can be noncovalent, as described by
Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659, 1972.
Alternatively, the variable chains can be linked by an
intermolecular disulfide bond or cross-linked by chemicals such as
glutaraldehyde (see, for example, Sandhu, Crit. Rev. Biotech.
12:437, 1992).
[0081] The Fv fragments may comprise VH and VL chains which are
connected by a peptide linker. These single-chain antigen binding
proteins (scFv) are prepared by constructing a structural gene
comprising DNA sequences encoding the VH and VL domains which are
connected by an oligonucleotide. The structural gene is inserted
into an expression vector which is subsequently introduced into a
host cell, such as E. coli. The recombinant host cells synthesize a
single polypeptide chain with a linker peptide bridging the two V
domains. Methods for producing scFvs are described, for example, by
Whitlow et al., Methods: A Companion to Methods in Enzymology 2:97
(1991) (also see, Bird et al., Science 242:423, 1988, Ladner et
al., U.S. Pat. No. 4,946,778, Pack et al., Bio/Technology 11:1271,
1993, and Sandhu, supra).
[0082] As an illustration, a scFV can be obtained by exposing
lymphocytes to IL-21 polypeptide in vitro, and selecting antibody
display libraries in phage or similar vectors (for instance,
through use of immobilized or labeled IL-21 protein or peptide).
Genes encoding polypeptides having potential IL-21 polypeptide
binding domains can be obtained by screening random peptide
libraries displayed on phage (phage display) or on bacteria, such
as E. coli. Nucleotide sequences encoding the polypeptides can be
obtained in a number of ways, such as through random mutagenesis
and random polynucleotide synthesis. These random peptide display
libraries can be used to screen for peptides which interact with a
known target which can be a protein or polypeptide, such as a
ligand or receptor, a biological or synthetic macromolecule, or
organic or inorganic substances. Techniques for creating and
screening such random peptide display libraries are known in the
art (Ladner et al., U.S. Pat. No. 5,223,409, Ladner et al., U.S.
Pat. No. 4,946,778, Ladner et al., U.S. Pat. No. 5,403,484, Ladner
et al., U.S. Pat. No. 5,571,698, and Kay et al., Phage Display of
Peptides and Proteins (Academic Press, Inc. 1996)) and random
peptide display libraries and kits for screening such libraries are
available commercially, for instance from CLONTECH Laboratories,
Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New
England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKB
Biotechnology Inc. (Piscataway, N.J.). Random peptide display
libraries can be screened using the IL-21 sequences disclosed
herein to identify proteins which bind to IL-21.
[0083] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing cells
(see, for example, Larrick et al., Methods: A Companion to Methods
in Enzymology 2:106 (1991), Courtenay-Luck, "Genetic Manipulation
of Monoclonal Antibodies," in Monoclonal Antibodies: Production,
Engineering and Clinical Application, Ritter et al. (eds.), page
166 (Cambridge University Press 1995), and Ward et al., "Genetic
Manipulation and Expression of Antibodies," in Monoclonal
Antibodies: Principles and Applications, Birch et al., (eds.), page
137 (Wiley-Liss, Inc. 1995)).
[0084] Alternatively, an anti-IL-21 antibody may be derived from a
"humanized" monoclonal antibody. Humanized monoclonal antibodies
are produced by transferring mouse or rat complementary determining
regions from heavy and light variable chains of the mouse
immunoglobulin into a human variable domain. Typical residues of
human antibodies are then substituted in the framework regions of
the murine counterparts. The use of antibody components derived
from humanized monoclonal antibodies obviates potential problems
associated with the immunogenicity of murine constant regions.
General techniques for cloning murine immunoglobulin variable
domains are described, for example, by Orlandi et al., Proc. Nat'l
Acad. Sci. USA 86:3833, 1989. Techniques for producing humanized
monoclonal antibodies are described, for example, by Jones et al.,
Nature 321:522, 1986; Carter et al., Proc. Nat'l Acad. Sci. USA
89:4285, 1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; Singer et
al., J. Immun. 150:2844, 1993; Sudhir (ed.), Antibody Engineering
Protocols (Humana Press, Inc. 1995), Kelley, "Engineering
Therapeutic Antibodies," in Protein Engineering: Principles and
Practice, Cleland et al. (eds.), pages 399-434 (John Wiley &
Sons, Inc. 1996), and by Queen et al., U.S. Pat. No. 5,693,762.
[0085] It is also possible to construct alternative frameworks by
using a collection of monomeric proteins to form a monomer domain.
These monomer domains can be small enough to penetrate tissues. The
monomer domains can be naturally-occurring or non-natural variants
or combination thereof. Monomer domains can form multimers of two
more domains. The monomer domain binds a position, analogous to
epitopes described herein, on a target molecule. In some cases, the
multimer can be formed from variety of monomer domains. (See, e.g.
U. S. Patent Application 2004-0132028 and U.S. Patent Application
2006-0177831.)
[0086] The antibodies of the present invention include derivatives
that are modified, i.e, by the covalent attachment of any type of
molecule to the antibody such that covalent attachment does not
prevent the antibody from binding IL-21 or preventing receptor
activation. For example, but not by way of limitation, the antibody
derivatives include antibodies that have been modified, e.g., by
glycosylation, acetylation, pegylation, phosphylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, linkage to a cellular ligand or other protein, etc. Any
of numerous chemical modifications may be carried out by known
techniques, including, but not limited to specific chemical
cleavage, acetylation, formylation, metabolic synthesis of
tunicamycin, etc. Additionally, the derivative may contain one or
more non-classical amino acids.
[0087] An anti-IL-21 antibody can be conjugated with a detectable
label to form an anti-IL-21 immunoconjugate. Suitable detectable
labels include, for example, a radioisotope, a fluorescent label, a
chemiluminescent label, an enzyme label, a bioluminescent label or
colloidal gold. Methods of making and detecting such
detectably-labeled immunoconjugates are well-known to those of
ordinary skill in the art, and are described in more detail below.
The detectable label can be a radioisotope that is detected by
autoradiography. Isotopes that are particularly useful for the
purpose of the present invention are .sup.3H, .sup.125I, .sup.131I,
.sup.35S and .sup.14C.
[0088] Anti-IL-21 immunoconjugates can also be labeled with a
fluorescent compound. The presence of a fluorescently-labeled
antibody is determined by exposing the immunoconjugate to light of
the proper wavelength and detecting the resultant fluorescence.
Fluorescent labeling compounds include fluorescein isothiocyanate,
rhodamine, phycoerytherin, phycocyanin, allophycocyanin,
o-phthaldehyde and fluorescamine.
[0089] It is also possible that anti-IL-21 immunoconjugates can be
detectably labeled by coupling an antibody component to a
chemiluminescent compound. The presence of the
chemiluminescent-tagged immunoconjugate is determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of chemiluminescent labeling compounds
include luminol, isoluminol, an aromatic acridinium ester, an
imidazole, an acridinium salt and an oxalate ester.
[0090] Similarly, a bioluminescent compound can be used to label
anti-IL-21 immunoconjugates of the present invention.
Bioluminescence is a type of chemiluminescence found in biological
systems in which a catalytic protein increases the efficiency of
the chemiluminescent reaction. The presence of a bioluminescent
protein is determined by detecting the presence of luminescence.
Bioluminescent compounds that are useful for labeling include
luciferin, luciferase and aequorin.
[0091] Alternatively, anti-IL-21 immunoconjugates can be detectably
labeled by linking an anti-IL-21 antibody component to an enzyme.
When the anti-IL-21-enzyme conjugate is incubated in the presence
of the appropriate substrate, the enzyme moiety reacts with the
substrate to produce a chemical moiety which can be detected, for
example, by spectrophotometric, fluorometric or visual means.
Examples of enzymes that can be used to detectably label
polyspecific immunoconjugates include .beta.-galactosidase, glucose
oxidase, peroxidase and alkaline phosphatase.
[0092] Those of skill in the art will know of other suitable labels
which can be employed in accordance with the present invention. The
binding of marker moieties to anti-IL-21 antibodies can be
accomplished using standard techniques known to the art. Typical
methodology in this regard is described by Kennedy et al., Clin.
Chim. Acta 70:1, 1976;, Schurs et al., Clin. Chim. Acta 81:1,
1977;, Shih et al., Int'l J. Cancer 46:1101, 1990;, Stein et al.,
Cancer Res. 50:1330, 1990; and Coligan, supra.
[0093] Moreover, the convenience and versatility of immunochemical
detection can be enhanced by using anti-IL-21 antibodies that have
been conjugated with avidin, streptavidin, and biotin (see, for
example, Wilchek et al. (eds.), "Avidin-Biotin Technology," Methods
In Enzymology, Vol. 184 (Academic Press 1990), and Bayer et al.,
"Immunochemical Applications of Avidin-Biotin Technology," in
Methods In Molecular Biology, Vol. 10, Manson (ed.), pages 149-162
(The Humana Press, Inc. 1992).
[0094] Methods for performing immunoassays are well-established.
See, for example, Cook and Self, "Monoclonal Antibodies in
Diagnostic Immunoassays," in Monoclonal Antibodies: Production
Engineering and Clinical Application, Ritter and Ladyman (eds.),
pages 180-208, (Cambridge University Press, 1995), Perry, "The Role
of Monoclonal Antibodies in the Advancement of Immunoassay
Technology," in Monoclonal Antibodies: Principles and Applications,
Birch and Lennox (eds.), pages 107-120 (Wiley-Liss, Inc. 1995), and
Diamandis, Immunoassay (Academic Press, Inc. 1996).
[0095] Antibodies or fragments thereof having increased in vivo
half-lives can be generated by techniques known to those of skill
in the art. For example, antibodies or fragments thereof with
increased in vivo half-lives can be generated by modifying (e.g.,
substituting, deleting or adding) amino acid residues identified as
involved in the interaction between the Fc domain and the FcRn
receptor (see, e.g., International Publication Nos. WO 97/34631 and
WO 02/060919, which are incorporated herein by reference in their
entireties). Antibodies or fragments thereof with increased in vivo
half-lives can be generated by attaching to said antibodies or
antibody fragments polymer molecules such as high molecular weight
polyethyleneglycol (PEG). PEG can be attached to said antibodies or
antibody fragments with or without a multifunctional linker either
through site-specific conjugation of the PEG to the N- or
C-terminus of said antibodies or antibody fragments or via
epsilon-amino groups present on lysine residues. Linear or branched
polymer derivatization that results in minimal loss of biological
activity will be used. The degree of conjugation will be closely
monitored by SDS-PAGE and mass spectrometry to ensure proper
conjugation of PEG molecules to the antibodies. Unreacted PEG can
be separated from antibody-PEG conjugates by, e.g., size exclusion
or ion-exchange chromatography.
Pharmaceutical Compositions
[0096] The present invention further includes pharmaceutical
compositions, comprising a pharmaceutically acceptable carrier and
a polypeptide or antibody described herein. The pharmaceutical
composition can include additional therapeutic agents, including
but not limited to cytotoxic agents a cytotoxin, e.g., a cytostatic
or cytocidal agent, a therapeutic agent or a radioactive metal ion.
A cytotoxin or cytotoxic agent includes any agent that is
detrimental to cells. Examples include paclitaxol, cytochalasin B,
gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,
tenoposide, vincristine, vinblastine, colchicin, doxorubicin,
daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,
actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, and puromycin and analogs or
homologs thereof Therapeutic agents include, but are not limited
to, antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine). For example, the pharmaceutical
composition can comprise a protein or polypeptide possessing a
desired biological activity. Such proteins may include, for
example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor,
.alpha.-IFN, .beta.-IFN, nerve growth factor, platelet derived
growth factor, tissue plasminogen activator, a thrombotic agent or
an anti-angiogenic agent, e.g., angiostatin or endostatin; or,
biological response modifiers such as, for example, lymphokines,
interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6
("IL-6"), granulocyte macrophage colony stimulating factor
("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or
other growth factors.
[0097] For purposes of therapy, anti-IL-21 antibody molecules and a
pharmaceutically acceptable carrier are administered to a patient
in a therapeutically effective amount. A combination of a
therapeutic molecule of the present invention and a
pharmaceutically acceptable carrier is said to be administered in a
"therapeutically effective amount" if the amount administered is
physiologically significant. An agent is physiologically
significant if its presence results in a detectable change in the
physiology of a recipient patient. For example, an agent used to
treat inflammation is physiologically significant if its presence
alleviates the inflammatory response.
[0098] A pharmaceutical composition comprising anti-IL-21 antibody
can be furnished in liquid form, in an aerosol, or in solid form.
Liquid forms, are illustrated by injectable solutions and oral
suspensions. Exemplary solid forms include capsules, tablets, and
controlled-release forms. The latter form is illustrated by
miniosmotic pumps and implants (Bremer et al., Pharm. Biotechnol.
10:239 (1997); Ranade, "Implants in Drug Delivery," in Drug
Delivery Systems, Ranade and Hollinger (eds.), pages 95-123 (CRC
Press 1995); Bremer et al., "Protein Delivery with Infusion Pumps,"
in Protein Delivery: Physical Systems, Sanders and Hendren (eds.),
pages 239-254 (Plenum Press 1997); Yewey et al., "Delivery of
Proteins from a Controlled Release Injectable Implant," in Protein
Delivery: Physical Systems, Sanders and Hendren (eds.), pages
93-117 (Plenum Press 1997)).
[0099] Liposomes provide one means to deliver therapeutic
polypeptides to a subject intravenously, intraperitoneally,
intrathecally, intramuscularly, subcutaneously, or via oral
administration, inhalation, or intranasal administration. Liposomes
are microscopic vesicles that consist of one or more lipid bilayers
surrounding aqueous compartments (see, generally, Bakker-Woudenberg
et al., Eur. J. Clin. Microbiol. Infect. Dis. 12 (Suppl. 1):S61
(1993), Kim, Drugs 46:618 (1993), and Ranade, "Site-Specific Drug
Delivery Using Liposomes as Carriers," in Drug Delivery Systems,
Ranade and Hollinger (eds.), pages 3-24 (CRC Press 1995)).
Liposomes are similar in composition to cellular membranes and as a
result, liposomes can be administered safely and are biodegradable.
Depending on the method of preparation, liposomes may be
unilamellar or multilamellar, and liposomes can vary in size with
diameters ranging from 0.02 .mu.m to greater than 10 .mu.m. A
variety of agents can be encapsulated in liposomes: hydrophobic
agents partition in the bilayers and hydrophilic agents partition
within the inner aqueous space(s) (see, for example, Machy et al.,
Liposomes In Cell Biology And Pharmacology (John Libbey 1987), and
Ostro et al., American J. Hosp. Pharm. 46:1576 (1989)). Moreover,
it is possible to control the therapeutic availability of the
encapsulated agent by varying liposome size, the number of
bilayers, lipid composition, as well as the charge and surface
characteristics of the liposomes.
[0100] Alternatively, various targeting ligands can be bound to the
surface of the liposome, such as antibodies, antibody fragments,
carbohydrates, vitamins, and transport proteins. For example,
liposomes can be modified with branched type galactosyllipid
derivatives to target asialoglycoprotein (galactose) receptors,
which are exclusively expressed on the surface of liver cells (Kato
and Sugiyama, Crit. Rev. Ther. Drug Carrier Syst. 14:287, 1997;
Murahashi et al., Biol. Pharm. Bull. 20:259, 1997). Similarly, Wu
et al., Hepatology 27:772, 1998, have shown that labeling liposomes
with asialofetuin led to a shortened liposome plasma half-life and
greatly enhanced uptake of asialofetuin-labeled liposome by
hepatocytes. On the other hand, hepatic accumulation of liposomes
comprising branched type galactosyllipid derivatives can be
inhibited by preinjection of asialofetuin (Murahashi et al., Biol.
Pharm. Bull. 20:259, 1997). Polyaconitylated human serum albumin
liposomes provide another approach for targeting liposomes to liver
cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681, 1997).
Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe a
hepatocyte-directed liposome vesicle delivery system, which has
specificity for hepatobiliary receptors associated with the
specialized metabolic cells of the liver.
[0101] In a more general approach to tissue targeting, target cells
are prelabeled with biotinylated antibodies specific for a ligand
expressed by the target cell (Harasym et al., Adv. Drug Deliv. Rev.
32:99, 1998). After plasma elimination of free antibody,
streptavidin-conjugated liposomes are administered. In another
approach, targeting antibodies are directly attached to liposomes
(Harasym et al., ibid. (1998)).
[0102] Polypeptides and antibodies can be encapsulated within
liposomes using standard techniques of protein microencapsulation
(see, for example, Anderson et al., Infect. Immun. 31:1099, 1981,
Anderson et al., Cancer Res. 50:1853, 1990, and Cohen et al.,
Biochim. Biophys. Acta 1063:95, 1991, Alving et al. "Preparation
and Use of Liposomes in Immunological Studies," in Liposome
Technology, 2nd Edition, Vol. III, Gregoriadis (ed.), page 317 (CRC
Press 1993), Wassef et al., Meth. Enzymol. 149:124, 1987). As noted
above, therapeutically useful liposomes may contain a variety of
components. For example, liposomes may comprise lipid derivatives
of poly(ethylene glycol) (Allen et al., Biochim. Biophys. Acta
1150:9, 1993).
[0103] Degradable polymer microspheres have been designed to
maintain high systemic levels of therapeutic proteins. Microspheres
are prepared from degradable polymers such as
poly(lactide-co-glycolide) (PLG), polyanhydrides, poly (ortho
esters), nonbiodegradable ethylvinyl acetate polymers, in which
proteins are entrapped in the polymer (Gombotz and Pettit,
Bioconjugate Chem. 6:332, 1995; Ranade, "Role of Polymers in Drug
Delivery," in Drug Delivery Systems, Ranade and Hollinger (eds.),
pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, "Degradable
Controlled Release Systems Useful for Protein Delivery," in Protein
Delivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92
(Plenum Press 1997); Bartus et al., Science 281:1161, 1998; Putney
and Burke, Nature Biotechnology 16:153, 1998; Putney, Curr. Opin.
Chem. Biol. 2:548, 1998). Polyethylene glycol (PEG)-coated
nanospheres can also provide carriers for intravenous
administration of therapeutic proteins (see, for example, Gref et
al., Pharm. Biotechnol. 10:167, 1997).
[0104] Other dosage forms can be devised by those skilled in the
art, as shown, for example, by Ansel and Popovich, Pharmaceutical
Dosage Forms and Drug Delivery Systems, 5th Edition (Lea &
Febiger 1990), Gennaro (ed.), Remington's Pharmaceutical Sciences,
19th Edition (Mack Publishing Company 1995), and by Ranade and
Hollinger, Drug Delivery Systems (CRC Press 1996).
[0105] Pharmaceutical compositions may be supplied as a kit
comprising a container that comprises a neutralizing anti-IL-21
antibody). Therapeutic polypeptides can be provided in the form of
an injectable solution for single or multiple doses, or as a
sterile powder that will be reconstituted before injection.
Alternatively, such a kit can include a dry-powder disperser,
liquid aerosol generator, or nebulizer for administration of a
therapeutic polypeptide. Such a kit may further comprise written
information on indications and usage of the pharmaceutical
composition.
[0106] A pharmaceutical composition comprising anti-IL-21
antibodies can be furnished in liquid form, in an aerosol, or in
solid form. Liquid forms, are illustrated by injectable solutions,
aerosols, droplets, topological solutions and oral suspensions.
Exemplary solid forms include capsules, tablets, and
controlled-release forms. The latter form is illustrated by
miniosmotic pumps and implants (Bremer et al., Pharm. Biotechnol.
10:239, 1997; Ranade, "Implants in Drug Delivery," in Drug Delivery
Systems, Ranade and Hollinger (eds.), pages 95-123 (CRC Press
1995); Bremer et al., "Protein Delivery with Infusion Pumps," in
Protein Delivery: Physical Systems, Sanders and Hendren (eds.),
pages 239-254 (Plenum Press 1997); Yewey et al., "Delivery of
Proteins from a Controlled Release Injectable Implant," in Protein
Delivery: Physical Systems, Sanders and Hendren (eds.), pages
93-117 (Plenum Press 1997)). Other solid forms include creams,
pastes, other topological applications, and the like.
Therapeutic Uses for Anti-IL-21 Antibodies
[0107] IL-21 is a CD4.sup.+ T cell-derived cytokine that is
important for optimal CD8.sup.+ T cell mediated immunity, NK cell
activation, and optimal humoral responses, such as antibody
production and B cell maturation. IL-21 has been shown to induce a
number of proinflammatory chemokines and cytokines, such as IL-18,
IL-15, IL-5, IL-6, TNFRII, sCD25, and RANTES. IL-21 also induces an
acute phase response in non-human primates and humans. Increased
expression of IL-21 receptor has been shown in epidermis in
patients with systemic sclerosis (Distler et al., Arthritis &
Rheumatism 52:865-864, 2004) and rheumatoid arthritis synovial
fibroblasts (Jungel et al., Arthritis & Rheumatism
50:1468-1476, 2004). Moreover, autoimmune, diabetic NOD mice have
increased IL-21 receptor expression (King et al., Cell 117:265-277,
2004.) It has been shown that IgG and IL-21 expression is increased
in the BXSB-Yaa mouse model which develop an autoimmune lupus
erythematosus-like disease (Ozaki et al., J. Immunol.
173:5361-5371, 2004); IL-21 expression is higher in lupus-prone
Sanroque mice (Vinuesa et al. Nature 435:452, 2005); IL-21
expression is higher in patients with Crohn's disease (Monteleone,
et al., Gastroenterology 128:687-694, 2005).
[0108] A therapeutically effective amount of an anti-IL-21 antibody
refers to an amount of antibody which when administered to a
subject is effective to prevent, delay, reduce or inhibit a symptom
or biological activity associated with a disease or disorder.
Administration may consist of a single dose or multiple doses and
may be given in combination with other pharmaceutical
compositions.
[0109] The present invention provides compositions and methods for
using IL-21 antagonists in inflammatory and immune diseases or
conditions such as pancreatitis, type I diabetes (IDDM), Graves
Disease, inflammatory bowel disease (IBD), Crohn's Disease,
ulcerative colitis, irritable bowel syndrome, multiple sclerosis,
rheumatoid arthritis, diverticulosis, systemic lupus erythematosus,
psoriasis, ankylosing spondylitis, scleroderma, systemic sclerosis,
psoriatic arthritis, osteoarthritis, atopic dermatitis, vitiligo,
graft vs. host disease (GVHD), cutaneous T cell lymphoma (CTCL),
Sjogren's syndrome, glomerulonephritis, IgA nephropathy, graft
versous host disease, transplant rejection, atopic dermatitis,
anti-phospholipid syndrome, and asthma, and other autoimmune
diseases.
[0110] Contact Dermatitis
[0111] Allergic contact dermatitis is defined as a T cell mediated
immune reaction to an antigen that comes into contact with the
skin. The CLA+ T cell population is believed to be involved in the
initiation of dermatitis since allergen dependent T cell responses
are largely confined to the CLA+ population of cells (See
Santamaria-Babi, L.F., et al. J Exp Med 181:1935, (1995)). Recent
data have found that only memory (CD45RO+) CD4+ CLA+ and not CD8+ T
cells proliferate and produce both type-1 (IFN-.gamma.) and type-2
(IL-5) cytokines in response to nickel, a common contact
hypersensitivity allergen. Furthermore, cells expressing CLA in
combination with CD4, CD45RO (memory) or CD69 are increased after
nickel-specific stimulation and express the chemokine receptors
CXCR3, CCR4, CCR10 but not CCR6. See Moed H., et al., Br J Dermatol
51:32, (2004).
[0112] In animal models, it has been demonstrated that allergic
contact dermatitis is T cell-dependent and that the
allergic-responsive T cells migrate to the site of allergen
application. See generally: Engeman T. M., et al., J Immunol
164:5207, (2000); Ferguson T. A. & Kupper T. S. J Immunol
150:1172, (1993); and Gorbachev A. V. & Fairchild R. L. Crit
Rev Immunol. 21:451 (2001).
[0113] Atopic Dermatitis
[0114] Atopic dermatitis (AD) is a chronically relapsing
inflammatory skin disease with a dramatically increasing incidence
over the last decades. Clinically AD is characterized by highly
pruritic, often excoriated, plaques and papules that show a chronic
relapsing course. The diagnosis of AD is mostly based on major and
minor clinical findings. See Hanifin J. M., Arch Dermatol 135:1551
(1999). Histopathology reveals spongiosis, hyperparakeratosis and
focal parakeratosis in acute lesions, whereas marked epidermal
hyperplasia with hyperparakeratosis and parakeratosis,
acanthosis/hypergranulosis and perivascular infiltration of the
dermis with lymphocytes and abundant mast cells are the hallmarks
of chromic lesions.
[0115] T cells play a central role in the initiation of local
immune responses in tissues and evidence suggests that
skin-infiltrating T cells in particular, may play a key role in the
initiation and maintenance of disregulated immune responses in the
skin. Approximately 90% of infiltrating T cells in cutaneous
inflammatory sites express the cutaneous lymphocyte-associated Ag
(CLA+) which binds E-selectin, an inducible adhesion molecule on
endothelium (reviewed in Santamaria-Babi L. F., et al., Eur J
Dermatol 14:13, (2004)). A significant increase in circulating CLA+
T cells among AD patients compared with control individuals has
been documented (See Teraki Y., et al., Br J Dermatol 143:373
(2000), while others have demonstrated that memory CLA+ T cells
from AD patients preferentially respond to allergen extract
compared to the CLA- population (See Santamaria-Babi, L. F., et
al., J Exp Med.181:1935, (1995)). In humans, the pathogenesis of
atopic disorders of the skin have been associated with increases in
CLA+ T cells that express increased levels of Th-2-type cytokines
like IL-5 and IL-13. See Akdis M., et al., Eur J Immunol 30:3533
(2000); and Hamid Q., et al., J Allergy Clin Immunol 98: 225
(1996).
[0116] NC/Nga mice spontaneously develop AD-like lesions that
parallel human AD in many aspects, including clinical course and
signs, histophathology and immunopathology when housed in
non-specified pathogen-free (non-SPF) conditions at around 6-8
weeks of age. In contrast, NC/Nga mice kept under SPF conditions do
not develop skin lesions. However, onset of spontaneous skin
lesions and scratching behaviour can be synchronized in NC/Nga mice
housed in a SPF facility by weekly intradermal injection of crude
dust mite antigen. See Matsuoka H., et al., Allergy 58:139 (2003).
Therefore, the development of AD in NC/Nga is a useful model for
the evaluation of novel therapeutics for the treatment of AD.
[0117] In addition to the NC/Nga model of spontaneous AD,
epicutaneous sensitization of mice using OVA can also be used as a
model to induce antigen-dependent epidermal and dermal thickening
with a mononuclear infiltrate in skin of sensitized mice. This
usually coincides with elevated serum levels of total and specific
IgE, however no skin barrier dysfunction or pruritus normally
occurs in this model. See Spergel J. M., et al., J Clin Invest,
101:1614, (1998). This protocol can be modified in order to induce
skin barrier disregulation and pruritis by sensitizing DO11.10 OVA
TCR transgenic mice with OVA. Increasing the number of
antigen-specific T cells that could recognize the sensitizing
antigen may increase the level of inflammation in the skin to
induce visible scratching behaviour and lichenification/scaling of
the skin.
[0118] Arthritis
[0119] Arthritis, including osteoarthritis, rheumatoid arthritis,
arthritic joints as a result of injury, and the like, are common
inflammatory conditions which would benefit from the therapeutic
use of anti-inflammatory antibodies and binding polypeptides. For
example, rheumatoid arthritis (RA) is a systemic disease that
affects the entire body and is one of the most common forms of
arthritis. It is characterized by the inflammation of the membrane
lining the joint, which causes pain, stiffness, warmth, redness and
swelling. Inflammatory cells release enzymes that may digest bone
and cartilage. As a result of rheumatoid arthritis, the inflamed
joint lining, the synovium, can invade and damage bone and
cartilage leading to joint deterioration and severe pain amongst
other physiologic effects. The involved joint can lose its shape
and alignment, resulting in pain and loss of movement.
[0120] Rheumatoid arthritis (RA) is an immune-mediated disease
particularly characterized by inflammation and subsequent tissue
damage leading to severe disability and increased mortality. A
variety of cytokines are produced locally in the rheumatoid joints.
Numerous studies have demonstrated that IL-1 and TNF-alpha, two
prototypic pro-inflammatory cytokines, play an important role in
the mechanisms involved in synovial inflammation and in progressive
joint destruction. Indeed, the administration of TNF-alpha and IL-1
inhibitors in patients with RA has led to a dramatic improvement of
clinical and biological signs of inflammation and a reduction of
radiological signs of bone erosion and cartilage destruction.
However, despite these encouraging results, a significant
percentage of patients do not respond to these agents, suggesting
that other mediators are also involved in the pathophysiology of
arthritis (Gabay, Expert. Opin. Biol. Ther. 2 (2):135-149,
2002).
[0121] There are several animal models for rheumatoid arthritis
known in the art. For example, in the collagen-induced arthritis
(CIA) model, mice develop chronic inflammatory arthritis that
closely resembles human rheumatoid arthritis. Since CIA shares
similar immunological and pathological features with RA, this makes
it an ideal model for screening potential human anti-inflammatory
compounds. The CIA model is a well-known model in mice that depends
on both an immune response, and an inflammatory response, in order
to occur. The immune response comprises the interaction of B-cells
and CD4+ T-cells in response to collagen, which is given as
antigen, and leads to the production of anti-collagen antibodies.
The inflammatory phase is the result of tissue responses from
mediators of inflammation, as a consequence of some of these
antibodies cross-reacting to the mouse's native collagen and
activating the complement cascade. An advantage in using the CIA
model is that the basic mechanisms of pathogenesis are known. The
relevant T-cell and B-cell epitopes on type II collagen have been
identified, and various immunological (e.g., delayed-type
hypersensitivity and anti-collagen antibody) and inflammatory
(e.g., cytokines, chemokines, and matrix-degrading enzymes)
parameters relating to immune-mediated arthritis have been
determined, and can thus be used to assess test compound efficacy
in the CIA model (Wooley, Curr. Opin. Rheum. 3:407-20, 1999;
Williams et al., Immunol. 89:9784-788, 1992; Myers et al., Life
Sci. 61:1861-78, 1997; and Wang et al., Immunol. 92:8955-959,
1995).
[0122] The administration of anti-IL-21 antibodies to these CIA
model mice are used to evaluate the use of anti-IL-21 antibodies to
ameliorate symptoms and alter the course of disease.
[0123] Inflammatory Bowel Disease (IBD)
[0124] In the United States approximately 500,000 people suffer
from inflammatory bowel disease (IBD) which can affect either colon
and rectum (ulcerative colitis) or both, small and large intestine
(Crohn's Disease). The pathogenesis of these diseases is unclear,
but they involve chronic inflammation of the affected tissues.
Ulcerative colitis (UC) is an inflammatory disease of the large
intestine, commonly called the colon, characterized by inflammation
and ulceration of the mucosa or innermost lining of the colon. This
inflammation causes the colon to empty frequently, resulting in
diarrhea. Symptoms include loosening of the stool and associated
abdominal cramping, fever and weight loss. Although the exact cause
of UC is unknown, recent research suggests that the body's natural
defenses are operating against proteins in the body which the body
thinks are foreign (an "autoimmune reaction"). Perhaps because they
resemble bacterial proteins in the gut, these proteins may either
instigate or stimulate the inflammatory process that begins to
destroy the lining of the colon. As the lining of the colon is
destroyed, ulcers form releasing mucus, pus and blood. The disease
usually begins in the rectal area and may eventually extend through
the entire large bowel. Repeated episodes of inflammation lead to
thickening of the wall of the intestine and rectum with scar
tissue. Death of colon tissue or sepsis may occur with severe
disease. The symptoms of ulcerative colitis vary in severity and
their onset may be gradual or sudden. Attacks may be provoked by
many factors, including respiratory infections or stress.
[0125] Although there is currently no cure for UC available,
treatments are focused on suppressing the abnormal inflammatory
process in the colon lining. Treatments including corticosteroids
immunosuppressives (eg. azathioprine, mercaptopurine, and
methotrexate) and aminosalicytates are available to treat the
disease. However, the long-term use of immunosuppressives such as
corticosteroids and azathioprine can result in serious side effects
including thinning of bones, cataracts, infection, and liver and
bone marrow effects. In the patients in whom current therapies are
not successful, surgery is an option. The surgery involves the
removal of the entire colon and the rectum.
[0126] There are several animal models that can partially mimic
chronic ulcerative colitis. The most widely used model is the
2,4,6-trinitrobenesulfonic acid/ethanol (TNBS) induced colitis
model, which induces chronic inflammation and ulceration in the
colon. When TNBS is introduced into the colon of susceptible mice
via intra-rectal instillation, it induces T-cell mediated immune
response in the colonic mucosa, in this case leading to a massive
mucosal inflammation characterized by the dense infiltration of
T-cells and macrophages throughout the entire wall of the large
bowel. Moreover, this histopathologic picture is accompanies by the
clinical picture of progressive weight loss (wasting), bloody
diarrhea, rectal prolapse, and large bowel wall thickening (Neurath
et al. Intem. Rev. Immunol. 19:51-62, 2000).
[0127] Another colitis model uses dextran sulfate sodium (DSS),
which induces an acute colitis manifested by bloody diarrhea,
weight loss, shortening of the colon and mucosal ulceration with
neutrophil infiltration. DSS-induced colitis is characterized
histologically by infiltration of inflammatory cells into the
lamina propria, with lymphoid hyperplasia, focal crypt damage, and
epithelial ulceration. These changes are thought to develop due to
a toxic effect of DSS on the epithelium and by phagocytosis of
lamina propria cells and production of TNF-alpha and IFN-gamma.
Despite its common use, several issues regarding the mechanisms of
DSS about the relevance to the human disease remain unresolved. DSS
is regarded as a T cell-independent model because it is observed in
T cell-deficient animals such as SCID mice.
[0128] The administration of anti-IL-21 antibodies to these TNBS,
DSS or CD4 transfer models can be used to evaluate the use of IL-21
antagonists to ameliorate symptoms and alter the course of
gastrointestinal disease. IL-21 may play a role in the inflammatory
response in colitis, and the neutralization of IL-21 activity by
administrating IL-21 antagonists is a potential therapeutic
approach for IBD.
[0129] Psoriasis
[0130] Psoriasis is a chronic skin condition that affects more than
seven million Americans. Psoriasis occurs when new skin cells grow
abnormally, resulting in inflamed, swollen, and scaly patches of
skin where the old skin has not shed quickly enough. Plaque
psoriasis, the most common form, is characterized by inflamed
patches of skin ("lesions") topped with silvery white scales.
Psoriasis may be limited to a few plaques or involve moderate to
extensive areas of skin, appearing most commonly on the scalp,
knees, elbows and trunk. Although it is highly visible, psoriasis
is not a contagious disease. The pathogenesis of the diseases
involves chronic inflammation of the affected tissues. Anti-IL-21
antibodies of the present invention, could serve as a valuable
therapeutic to reduce inflammation and pathological effects in
psoriasis, other inflammatory skin diseases, skin and mucosal
allergies, and related diseases.
[0131] Psoriasis is a T-cell mediated inflammatory disorder of the
skin that can cause considerable discomfort. It is a disease for
which there is no cure and affects people of all ages. Psoriasis
affects approximately two percent of the populations of European
and North America. Although individuals with mild psoriasis can
often control their disease with topical agents, more than one
million patients worldwide require ultraviolet or systemic
immunosuppressive therapy. Unfortunately, the inconvenience and
risks of ultraviolet radiation and the toxicities of many therapies
limit their long-term use. Moreover, patients usually have
recurrence of psoriasis, and in some cases rebound, shortly after
stopping immunosuppressive therapy. Anti-IL-21 antibodies can be
tested using a recently developed a model of psoriasis based on the
CD4+CD45RB transfer model (Davenport et al., Internat.
Immunopharmacol., 2:653-672, 2002).
[0132] In addition to other disease models described herein, the
activity of anti-IL-21 antibodies on inflammatory tissue derived
from human psoriatic lesions can be measured in vivo using a severe
combined immune deficient (SCID) mouse model. Several mouse models
have been developed in which human cells are implanted into
immunodeficient mice (collectively referred to as xenograft
models); see, for example, Cattan A R, Douglas E, Leuk. Res.
18:513-22, 1994 and Flavell, D J, Hematological Oncology 14:67-82,
1996. As an in vivo xenograft model for psoriasis, human psoriatic
skin tissue is implanted into the SCID mouse model, and challenged
with an appropriate antagonist. Moreover, other psoriasis animal
models in ther art may be used to evaluate IL-21 antagonists, such
as human psoriatic skin grafts implanted into AGR129 mouse model,
and challenged with an appropriate antagonist (e.g., see, Boyman,
O. et al., J. Exp. Med. Online publication #20031482, 2004,
incorporated herein by reference). Similarly, tissues or cells
derived from human colitis, IBD, arthritis, or other inflammatory
lestions can be used in the SCID model to assess the
anti-inflammatory properties of the anti-IL-21 antibodies described
herein.
[0133] Efficacy of treatment is measured and statistically
evaluated as increased anti-inflammatory effect within the treated
population over time using methods well known in the art. Some
exemplary methods include, but are not limited to measuring for
example, in a psoriasis model, epidermal thickness, the number of
inflammatory cells in the upper dermis, and the grades of
parakeratosis. Such methods are known in the art and described
herein. For example, see Zeigler, M. et al. Lab Invest 81:1253,
2001; Zollner, T. M. et al. J. Clin. Invest. 109:671, 2002;
Yamanaka, N. et al. Microbio.l Immunol. 45:507, 2001; Raychaudhuri,
S. P. et al. Br. J. Dermatol. 144:931, 2001; Boehncke, W. H et al.
Arch. Dermatol. Res. 291:104, 1999; Boehncke, W. H et al. J.
Invest. Dermatol. 116:596, 2001; Nickoloff, B. J. et al. Am. J.
Pathol. 146:580, 1995; Boehncke, W. H et al. J. Cutan. Pathol.
24:1, 1997; Sugai, J., M. et al. J. Dermatol. Sci. 17:85, 1998; and
Villadsen L. S. et al. J. Clin. Invest. 112:1571, 2003.
Inflammation may also be monitored over time using well-known
methods such as flow cytometry (or PCR) to quantitate the number of
inflammatory or lesional cells present in a sample, score (weight
loss, diarrhea, rectal bleeding, colon length) for IBD, paw disease
score and inflammation score for CIA RA model.
[0134] Systemic Lupus Erythematosus
[0135] Systemic lupus erythematosus (SLE) is an immune-complex
related disorder characterized by chronic IgG antibody production
directed at ubiquitous self antigens (e.g. anti-dsDNA). The effects
of SLE are systemic, rather than localized to a specific organ.
Multiple chromosomal loci have been associated with the disease and
may contribute towards different aspects of the disease, such as
anti-dsDNA antibodies and glomerulonephritis. CD4+ T cells have
been shown to play an active part in mouse models of SLE (Horwitz,
Lupus 10:319-320, 2001; Yellin and Thienel, Curr. Rheumatol. Rep.,
2:24-37, 2000). The role for CD8+ T cells is not clearly defined,
but there is evidence to suggest that "suppressor" CD8+ T cell
function is impaired in lupus patients (Filaci et al., J. Immunol.,
166:6452-6457, 2001; Sakane et al, J. Immunol., 137:3809-3813,
1986).
[0136] IL-21 has been shown to modulate antibody responses by
directly acting on B cells. (Mehta et al., J. Immunol.,
170:4111-4118, 2003; Ozaki et al., Science, 298:1630-1634, 2002;
Suto et al., Blood, 100:4565-4573, 2002). For example, Ozaki et
al., (J. Immunol. 173:5361, 2004) demonstrated that in BXSB-Yaa
mice, a model for SLE, there is an elevated serum IL-21 level.
Moreover, because IL-21 enhances CD8.sup.+ T cell activity,
administration of anti-IL-21 antibodies would provide a more robust
T cell suppressor function in lupus patients where that function is
compromised.
[0137] Anti-IL-21 antibodies can be administered in combination
with other agents already in use in autoimmunity including immune
modulators such as IFN.gamma., NOVANTRONE.RTM., ENBREL.RTM.,
REMICADE.RTM., LEUKINE.RTM. and IL-2. Establishing the optimal dose
level and scheduling for anti-IL-21 antibodies is done by a variety
of means, including study of the pharmacokinetics and
pharmacodynamics of anti-IL-21 antibodies; determination of
effective doses in animal models, and evaluation of the toxicity of
anti-IL-21 antibodies. Direct pharmacokinetic measurements done in
primates and clinical trials can then be used to predict
theoretical doses in patients that achieve plasma anti-IL-21
antibody levels that are of sufficient magnitude and duration to
achieve a biological response in patients.
[0138] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
Preparation of IL-21 Proteins
[0139] IL-21 protein was produced as described in U.S. Patent
Application No. 2006-0134754 and WO 04/055168, incorporated in its
entirety herein. Briefly, a IL-21 nucleotide sequence was optimized
and inserted in an E. coli expression vector which was deposited as
ATCC Accession No. PTA-4853. The expression vector was introduced
into E. coli strain W3110 (ATCC Accession No. 27325).
[0140] Host cells were fermented by growing E. coli strains
expressing IL-21 in a suitable medium in shake flask culture to in
a suitable medium and may be supplemented with carbohydrates, such
as fructose, glucose, galactose, lactose, and glycerol. Isopropyl
thiogalactopyranoside (IPTG) is may be added to the culture to a
concentration 0.1 to 2.0 mM.
[0141] Following fermentation the cells were harvested by
centrifugation, re-suspended in homogenization buffer and
homogenized. After the homogenate was collected, it was resuspended
a guanidine containing solution and the supernatant containing
solubilized IL-21 was decanted and retained. The concentration of
the IL-21 in the solubilized fraction was determined by reversed
phase HPLC. Once the inclusion bodies were solubilized and
denatured in guanidine solution containing a reducing agent, the
reduced IL-21 was then oxidized in a controlled renaturation step.
This step involved dilution in a refold buffer containing arginine
hydrochloride, salts, and an oxido-shuffling system.
[0142] Purification of IL-21 protein may include purification of
the IL-21 using hydrophobic interaction chromatography. The IL-21
may be further purified by high performance cation exchange
chromatography. The methods for purifying IL-21 can comprise
concentrating and carrying out a buffer exchange of the protein.
This step is designed to concentrate the high performance cation
exchange column eluate and exchange it into formulation buffer. The
final column eluate pool is concentrated to increase the
concentration of IL-21. Further purification of IL-21 to remove the
remaining impurities and contaminants may be desirable. For
example, an anion exchange column can be used to reduce the
endotoxin level.
Example 2
Preparation of IL-21 Receptor Proteins
[0143] The IL-21 receptor (also designated as zalpha11 or IL-21r)
heterodimer protein can be produced as described in U.S. Patent
Application No. 2002-0137677, incorporated in its entirety herein.
Briefly, a vector expressing a secreted human hzalpha11/hIL2Rgamma
heterodimer is constructed. In this construct, the extracellular
domain of hzalpha11 is fused to the CH1 domain of IgG .gamma.1. The
CH1 domain is cloned into a mammalian expression vector. The CL1
domain of the human .kappa. light chain is cloned in a mammalian
expression vector.
[0144] A construct having human zalpha11 fused to CH1 is made, and
the vector is sequenced to confirm that the fusion is correct. A
separate construct having hIL2Rgamma fused to CL1 can be also
constructed. The resulting vector is sequenced to confirm that the
human IL-2Rgamma/CL1 fusion is correct.
[0145] The human zalpha11 (IL-21r) and human IL-2Rgamma receptor
fusions are co-expressed. Each expression vector is co-transfected
into mammalian host cells by methods known to those skilled in the
art. The transfected cells are selected for 10 days in methotrexate
(MTX) and G418 (Gibco/BRL) for 10 days. The resulting pool of
transfectants is selected again in MTX and G418 for 10 days.
[0146] The resulting pool of doubly-selected cells is used to
generate protein. Factories (Nunc, Denmark) of this pool are used
to generate conditioned medium. This serum free, conditioned media
is passed over a protein-A column and eluted in fractions.
Fractions found to have the highest concentration are pooled and
dialyzed (10 kD MW cutoff) against PBS. Finally the dialyzed
material is submitted for amino acid analysis (AAA). The purified
soluble human zalpha11 receptor/IL-2Rgamma receptor can be used to
assess its ability to compete for binding of the human zalpha11
Ligand a BaF3 proliferation assay.
[0147] B. The extracellular domain of human zalpha11 fused to Fc9
(Fc region of human gamma1 (Kabat numbering 221-447; Kabat et al,
Sequences of Proteins of Immunological Interest, U.S. Dept. Health
and Human Serv., Bethesa, Md., 1991)) with an GluGlu tag
(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 198))
at the carboxyl terminus was generated by overlap PCR. The cDNA was
inserted into pZMP31 (described in US Patent application,
US2003/023414; a hybrid vector having a cytomegalovirus enhancer
and myeloproliferative sarcoma virus promoter) by recombination in
yeast. The extracellular domain of the human IL2 receptor common
gamma chain was fused to Fc9 with a 6.times.His tag at the carboxyl
terminus of Fc9. This construct was inserted into pZMP21z by yeast
recombination using the same method as described for
zalpha11Fc9CEE. The resulting constructs were sequenced to verify
that the inserts were correct. Both plasmids were transfected into
suspension, serum-free-adapted CHO cells by electroporation and
selected in protein-free PFCHO media (BioWhittaker) without
hypoxanthine and thymidine with 200 ng/mL zeomycin added. These
cells were then selected in the same medium plus increasing
concentrations of methotrexate until the cells were resistant to
both 1 uM methotrexate and 200 ng/mL zeomycin. The cells were
tested for production of heterodimeric IL21 receptor by western
blot analysis for the presence of both EE and his tags.
[0148] The design of zcytor26f2 (extracellular domain of the human
IL2 receptor common gamma chain was fused to Fc9 with a 6.times.His
tag) is such that three tags are available for purification
(GluGlu, His, and Fc), of which two are utilized to best
discriminate heterodimer from the two homodimer contaminants. All
molecules containing an Fc domain (homodimer contaminants and
heterodimer target) were captured and purified from host cell
components and related media products. The pool containing all
species was concentrated and injected over an appropriate size
exclusion column (Superdex 200) in order to remove aggregates. The
SEC pool containing all three species (two homodimers and one
heterodimer) was subjected to Immobilized Metal Affinity
Chromatography (IMAC) using the Ni counter ion under highly
discriminating load and elution conditions. The IMAC elution pool
contained highly pure heterodimer, with only residual homodimer
contamination. IMAC pool buffer was exchanged into formulation
buffer using size exclusion chromatography (Superdex 200), which
also removes any residual aggregation products. This IL-21
heterodimeric protein was used as a comparator when testing an
antibody's neutralizing activity.
Example 3
Preparation of IL-21 Monoclonal Antibodies
[0149] Rat monoclonal antibodies are prepared by immunizing 4
female Sprague-Dawley Rats (Charles River Laboratories, Wilmington,
Mass.), with the purified recombinant IL-21 protein. The rats are
each given an initial intraperitoneal (IP) injection of 25 .mu.g of
the purified recombinant protein in Complete Freund's Adjuvant
(Pierce, Rockford, Ill.) followed by booster IP injections of 10
.mu.g of the purified recombinant protein in Incomplete Freund's
Adjuvant every two weeks. Seven days after the administration of
the second booster injection, the animals are bled and serum is
collected.
[0150] The IL-21-specific rat sera samples are characterized by
ELISA using 1 ug/ml of the purified recombinant IL-21 receptor
protein as the specific antibody target. ELISAs comprise preparing
IL-21 antigen, coating the wells of a 96-well microtiter plate with
the antigen, adding the rat sera of interest to the wells and
incubating for a period of time to allow the antibodies in the rat
sera to bind to the antigen. A second detection antibody (which
recognizes the antibodies of interest contained within the rate
sera) conjugated to a detectable compound conjugated to a
detectable compound such as an enzymatic substrate (e.g.,
horseradish peroxidase or alkaline phosphatase) is added to the
wells. One of skill in the art would be knowledgeable as to the
parameters that can be modified to increase the signal detected as
well as other variations of ELISAs known in the art. For further
discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994,
Current Protocols in Molecular Biology, Vol. 1, John Wiley &
Sons, Inc., New York at 11.2.1.
[0151] Splenocytes are harvested from a single high-titer rat and
fused to SP2/0 (mouse) myeloma cells using PEG 1500 in a single
fusion procedure (4:1 fusion ratio, splenocytes to myeloma cells,
"Antibodies: A Laboratory Manual", E. Harlow and D. Lane, Cold
Spring Harbor Press). Following 9 days growth post-fusion, specific
antibody-producing hybridoma pools are identified by ELISA using
500 ng/ml of the recombinant IL-21 protein as specific antibody
target. Positive hybridoma pools are analyzed further for their
ability to block the cell-proliferative activity ("neutralization
assay") of purified recombinant IL-21 protein on BaF3 cells
expressing the IL-21 receptor sequence.
[0152] Hybridoma pools yielding positive results by the
"neutralization assay" are cloned at least two times by limiting
dilution.
[0153] The monoclonal antibodies produced by clones are
characterized in a number of ways including binning (i.e,
determining if each antibody could inhibit the binding of any other
binding), relative affinity, and neutralization. Monoclonal
antibodies purified from tissue culture media are characterized for
their ability to block the cell-proliferative activity
("neutralization assay") of purified recombinant IL-21 on Baf3
cells expressing the receptor sequences. "Neutralizing" monoclonal
antibodies are identified in this manner.
[0154] Samples were taken from the hybridoma pools and assayed
using both the neutralization assay and a direct titration ELISA.
In this assay a sample was titrated out using four-fold serial
dilutions to see which clone could maintain the highest OD reading.
Using the results from both the neutralization and titration
assays, specific clones from each initial master well were chosen
to go forward with. Another neutralization screen was performed
that ran all these samples in the same assay and at this point the
number of cell lines was narrowed down to four top picks. These
were subjected to an additional round of cloning to ensure culture
homogeneity and screened using the direct ELISA. After one more
titration assay, two final IL-21 clones were chosen and designated
268.5.1.11.42.1.4.3.9 (rat anti-mouse IL-21, ATCC Accession no.
PTA-7143) and 272.21.1.3.4.2 (rat anti-human IL-21, ATCC Accession
no. PTA-7142). The monoclonal antibodies produced by these
hybridoma clones can be cultured in a growth medium of 90% Iscove's
Modified Dulbecco's medium with 2 mM L-glutamine, 100 .mu.g/mL
penicillin, and 100 .mu.g/mL streptomycin sulfate, and 10% Fetal
Clone I Serum (Hyclone Laboratories). The clones can be propagated
by starting cultures at 2.times.10.sup.5 cells/ml and maintaining
between 1.times.10.sup.5 and 5.times.10.sup.5 cell ml at 37.degree.
C. and 5-6% CO. Cells can be adapted to serum free conditions upon
subsequent transfers. Cells that are frozen are stored in 90%
serum, 10% DMSO and stored in vapor phase of liquid nitrogen
freezer.
Example 4
Serum Screening of Monoclonal Antibodies
[0155] The activity of anti-IL-21 antibodies is measured using a
cell-based potency bioassay. The bioassay utilizes a BaF3 reporter
cell line that was engineered to express the IL-21 receptor
(IL-21R) through stable transfection with IL-21R cDNA. The
IL-21R/BaF3 transfected cells are highly dependent upon rIL-21 or
IL-3 for growth and, in their absence, are unable to proliferate
and undergo apoptosis within 24 hours. In the cell-based bioassay,
the IL-21R/BaF3 transfected cells are incubated with varying
concentrations of serum containing anti-IL-21 antibodies and
subsequent cellular proliferation is measured.
Example 5
Characterization of Antibodies
[0156] Epitope Binning
[0157] Epitope binning studies are performed on a Biacore1000.TM.
system (Biacore, Uppsalla Sweden). Methods are programmed using
Method Definition Language (MDL) and run using Biacore Control
Software, v 1.2. Polyclonal goat anti-Mouse IgG Fc antibody
(Jackson ImmunoResearch Laboratories, West Grove, Pa.) is
covalently immobilized to a Biacore CM5 sensor chip and is used to
bind (capture) the primary monoclonal antibody of test series to
the chip. Unoccupied Fc binding sites on the chip are then blocked
using a polyclonal IgG Fc fragment (Jackson ImmunoResearch
Laboratories, West Grove, Pa.). Subsequently, IL-21 is injected and
allowed to specifically bind to the captured primary monoclonal
antibody. The Biacore instrument measures the mass of protein bound
to the sensor chip surface, and thus, binding of both the primary
antibody and IL-21 antigen are verified for each cycle. Following
the binding of the primary antibody and antigen to the chip, a
monoclonal antibody of the test series is injected as the secondary
antibody, and allowed to bind to the pre-bound antigen. If the
secondary monoclonal antibody is capable of binding the IL-21
antigen simultaneously with the primary monoclonal antibody, an
increase in mass on the surface of the chip, or binding, is
detected. If, however, the secondary monoclonal antibody is not
capable of binding the IL-21 antigen simultaneously with the
primary monoclonal antibody, no additional mass, or binding, is
detected. Each monoclonal antibody tested against itself is used as
the negative control to establish the level of the background
(no-binding) signal. Data are compiled using BioEvaluation 3.2 RCI
software, then loaded into Excel.TM. for data processing.
[0158] Western Blotting
[0159] The ability of the neutralizing monoclonal antibodies from
clones to detect denatured and reduced/denatured IL-21 from two
sources is assessed using a Western blot format. A rabbit
polyclonal antibody known to detect IL-21 in a Western blot format
is used as a positive control.
[0160] IL-21 protein is loaded onto 4-12% NuPAGE Bis-Tris gels
(Invitrogen, Carlsbad, Calif.) in either non-reducing or reducing
sample buffer (Invitrogen) along with molecular weight standards
(SeeBlue; Invitrogen), and electrophoresis is performed. Following
electrophoresis, protein is transferred from the gel, the
nitrocellulose blots are blocked overnight and exposed to each
antibody. The blots are then probed with a secondary antibody
conjugated to horseradish peroxidase; sheep anti-mouse IgG-HRP
(Amersham: Piscataway, N.J.) for the monoclonal antibodies and
donkey anti-rabbit Ig-HRP (Amersham) for the polyclonal antibodies.
Bound antibody is detected using a chemiluminescent reagent
(Lumi-Light Plus Reagent: Roche, Mannheim, Germany) and images of
the blots were recorded on a Lumi-Imager (Mannheim-Boehringer).
Example 6
DTH Mouse Model
[0161] DTH responses are classic immune responses that are
initiated by CD4+ T cells and mediated by T cells, neutrophils and
macrophages. A DTH response is a good indicator of a CD4+ T cell
mediated response. Mice are immunized sub-cutaneously with chicken
ovalbumin protein (OVA) in either of 2 adjuvants, RIBI or CFA. This
phase is called the sensitization phase (days 0-6). Ear
measurements are taken seven days later. Mice are then injected in
the ear with control PBS (left ear) or OVA (right ear). This phase
is called the challenge phase (days 7-8). Immune responses
generated to OVA induce inflammation in the ear resulting an
increase in ear thickness in 24 hours in the OVA-treated, but not
in the PBS-treated ear. This is measured using calipers.
[0162] C57BL/6 mice (n=8/group) are immunized in the back with 100
.mu.g chicken ovalbumin (OVA) emulsified in RIBI adjuvant (Corixa,
Seattle, Wash.) in a total volume of 200 .mu.l. A 0.5 mg/ml of
ovalbumin is added to a single vial of RIBI and vortexed vigorously
for 2 minutes to form an emulsion that is used to inject mice.
Seven days after the immunization, mice are injected with 10 .mu.l
PBS in the left ear (control) and with 10 .mu.g OVA in PBS in the
right ear in a volume of 10 .mu.l. Ear thickness of all mice is
measured before injecting mice in the ear (0 measurement). Ear
thickness is measured 24 hours after challenge. The difference in
ear thickness between the 0 measurement and the 24 hour measurement
is calculated and is reflective of the inflammation in the ear.
Groups of mice are injected with PBS or different concentration of
anti-IL-21 antibody intra-peritoneally from either days 0-6
(sensitization phase) or from days 7-8 (challenge phase). The
injection on day 7 and 8 is given 2 hours before measuring ear
thickness at the 0 and 24 hour time points. At the end of the 24
hour period, once ear thickness was measured, the ears were cut and
placed in formalin for histological analysis.
Example 7
Mouse Model for Multiple Sclerosis
[0163] To test if anti-IL-21 has any effects on multiple sclerosis,
the ablility of anti-IL-21 antibodies to inhibit experimental
autoimmune encephalomyelitis (EAE), a mouse model for MS is tested.
The well characterized myelin oligodendrocyte glycoprotein (MOG)
35-55 peptide immunization model in C57BL/6 mice is used. The
experiment is run to determine that anti-IL-21 antibody could delay
and/or inhibit disease scores in EAE either by inhibiting DC
mediated antigen presentation or by enhancing CD8 T cell responses.
Absence of efficient CD8 T cell responses in this model exacerbates
EAE (Malipiero et. al., Eur. J. Immunol., 27:3151-3160, 1997).
Delayed onset of disease in the EAE model in a dose dependent
manner suggests that use of anti-IL-21 antibody may be beneficial
in MS.
[0164] Experimental autoimmune encephalomyelitis (EAE) is a mouse
model for MS. In one such model, C57BL/6 mice are immunized with
100 .mu.g MOG peptide (MOG35-55) or 100 .mu.g recombinant MOG
protein emulsified in RIBI adjuvant. Two milliliters of a 0.5 mg/ml
preparation of the MOG35-55 in PBS is added to a vial of RIBI and
vortexed vigorously to emulsify the solution or a 1:1 ratio of
recombinant MOG in DFA is prepared. The backs of mice are shaved
and 100 .mu.g MOG/RIBI is injected s.c in the backs of mice.
Weights of mice are taken 2 days before and every day after the
immunization. Mice are then injected on day 2 i.v. with 200 .mu.l
pertussis toxin (PT), a final concentration of 200 ng/mouse. Mice
are monitored daily for clinical scores. Groups of mice are
injected i.p. with 200 .mu.l PBS, 100 .mu.g BSA, 10 .mu.g-200 .mu.g
anti-IL-21 antibody in a 200 .mu.l volume from days 0-20, or
3.times. a week for 3 weeks. The weights of mice, clinical scores
and incidence are evaluated and plotted for analysis.
Example 8
CD4+CD45RBhi (CD25-) Colitis and Psoriasis Mouse Model
[0165] Transfer of CD4+CD45RBhi or CD4+CD25- T cells into syngenic
SCID mice results in colitis in the mice. Co-transfer of regulatory
T cells (CD4+CD25+or CD4+CD45RBlo) inhibits this colitis. After
transfer of CD4+CD25- T cells into mice, if mice are additionally
injected with staphylococcal enterotoxin B (SEB), mice not only
develop colitis, but also psoriasis. Anti-IL-21 antibody is
administered from days 0-21 after cell transfer and symptoms for
colitis and psoriasis are monitored. Inhibition of psoriatic score
or colitis (histology) indicates that anti-IL-21 antibody can
inhibit these autoimmune diseases.
[0166] Spleens and inguinal lymph nodes are isolated from B10.D2
mice. Single cell suspensions are formed and counted. Using the
Miltenyi Bead system, CD25+ cells are sorted out by positive
selection. Cells are stained with CD25-PE (BD Pharmingen) at 1:100
dilution and incubated for 15 minutes. Excess antibody is washed
out and the cells are incubated with 10 ul anti-PE beads/106 cells
for 20 minutes. The cells are washed with PBS and passed over an LS
column (Miltenyi Biotech). Cells that pass through the column
(CD25-) are retained for further analysis. A CD4 enrichment
cocktail (Stem Cell technologies) is added (1:100) to these CD25-
cells and incubated for 15 minutes. Cells are washed with PBS. A 1:
10 dilution of anti-biotin tetramer is added to the cells for 15
minutes followed by a magnetic colloid (60 ul/106 cells) for 15
minutes (all from Stem Cell Technologies). Cells are passed through
a negative selection column (0.5'', Stem cell Technologies). Cells
that pass through are the CD4+CD25- cells. Purity is analyzed using
flow cytometry. 0.4.times.106 cells are injected i.v into naive
CB-17 SCID mice in a total volume of 200 .mu.l. Mice are injected
i.p with 10 .mu.g SEB the following day (d1). Symptoms for
psoriasis and colitis are followed from 2-5 weeks. Groups of mice
are injected i.p. with PBS, 100 .mu.g BSA or 10-200 .mu.g IL-21
from days 1-20, or 3.times. a week for 3 weeks.
[0167] Inhibition of psoriatic and colitis symptoms in anti-IL-21
antibody treated mice indicates that anti-IL-21 antibodies can
inhibit autoimmune symptoms in this model for psoriasis and
colitis.
Example 9
Contact Hypersensitivity Mouse Model
[0168] Contact hypersensitivity can be induced in mice using a
variety of contact allergens including dinitrofluorobenzene (DNFB)
and oxazolone. Mice are sensitized topically with the allergen in a
vehicle of acetone and olive oil and then challenged in the ear
with the allergen in olive oil alone. Change in ear thickness is a
measure of the immune response against the allergen. Anti-IL-21
antibodies are administered either at the sensitization phase
(dO-5) or during the challenge phase (d5-6). Inhibition of ear
thickness by IL-21 indicates a role for IL-21 in inhibiting contact
hypersensitivity.
[0169] C57B1/6 mice are painted in the back with 0.5% DNFB in
acetone:olive oil (4:1) or acetone:olive oil alone on d0. On d5,
ear thickness of mice is measured using calipers and mice are
challenged in the ears with olive oil alone (control) or 0.25% DNFB
in olive oil by dropping a 25 .mu.l solution onto the ear. Change
in ear thickness is measured on d6 and the inflammation calculated
as a difference in ear thickness between d5 and d6. Groups of mice
are injected i.p. with PBS or 10-100 .mu.g anti-IL-21 antibodies on
either days 0-5 or days 5-6.
[0170] Inhibition of ear thickness by anti-IL-21 antibodies
demonstrate that anti-IL-21 antibodies can be useful in inhibiting
contact hypersensitivity.
[0171] Splenocytes are harvested and pooled from two high-titer
Balb/c mice and fused to P3-X63-Ag8.653 mouse myeloma cells using
PEG 1450 in a single fusion procedure (2:1 fusion ratio,
splenocytes to myeloma cells, "Antibodies: A Laboratory Manual", E.
Harlow and D. Lane, Cold Spring Harbor Press). Following 9 days
growth post-fusion, specific antibody-producing hybridoma pools are
identified by Direct and Capture ELISA using recombinant IL-21
protein, untagged and human IgG Fc tagged, as specific antibody
target. Positive hybridoma pools are analyzed further for their
ability to block the cell-proliferative activity ("neutralization
assay") of purified recombinant IL-21 protein on BaF3 cells
expressing the IL-21 receptor sequence. Monoclonal antibodies
purified from tissue culture media are characterized for their
ability to block the cell-proliferative activity ("neutralization
assay") of purified recombinant IL-21 on Baf3 cells expressing the
receptor sequences. "Neutralizing" monoclonal antibodies are
identified in this manner.
[0172] Hybridoma pools yielding positive results by the
"neutralization assay" and ELISA formats are cloned at least two
times by limiting dilution. In these assays, samples are titrated
using four-fold serial dilutions to see which clone will maintain
the highest OD reading. Using the results from both the
neutralization and titration assays, two specific clones from each
initial master well are selected for further analysis. These are
subjected to an additional round of cloning to ensure culture
homogeneity and screened using the Direct ELISA. After one
additional titration assay, two final IL-21 clones are selected.
Hybridoma clones are cultured in a growth medium of 90% Iscove's
Modified Dulbecco's medium with 2 mM L-glutamine, 100 .mu.g/mL
penicillin, and 100 .mu.g/mL streptomycin sulfate, and 10% Fetal
Clone I Serum (Hyclone Laboratories). The clones are propagated by
seeding cultures at 2.times.10.sup.5 cells/ml and maintaining
between 1.times.10.sup.5 and 5.times.10.sup.5 cell/ml at 37.degree.
C. and 5-6% CO. Cells are adapted to serum free conditions upon
subsequent transfers. Cells are frozen in 90% serum, 10% DMSO and
stored in vapor phase of a liquid nitrogen freezer.
[0173] The purified monoclonal antibodies produced by the hybridoma
clones are characterized in a number of ways including binning
(i.e, determining if each antibody could inhibit the binding of any
other binding), epitope mapping using peptides, relative affinity,
and neutralization.
Example 11
Selection of Peptide Sequences For Use in the Evaluation of
Monoclonal Antibodies Directed against Human IL-21
[0174] An assessment of the binding of murine anti-human IL21
antibodies to the various domains of IL-21 was conducted in part
through the ability of the monoclonal antibodies to bind synthetic
peptides derived from the native human IL-21 sequence. Peptides of
18-29 amino acids were selected to provide significant coverage of
the cytokine polypeptide while focusing on domains predicted from
cytokine mutein studies and the structure of IL-21 related
cytokines to be important in receptor binding or activation.
Peptides in this size range are also efficient to manufacture and
are of a size that may provide limited secondary structure for
antibody recognition. Peptides 1, 3, and 4 were synthesized with an
amidated carboxyl terminus to better mimic the electrostatic charge
found in the native peptide bonds.
[0175] Peptide #1
[0176] The N-terminus of human IL-21 following the mammalian
processing of the signal peptide sequence, along with the adjacent
amino acid sequence of IL-21 (SEQ ID NO: 6) was chosen for one
peptide. Mammalian expression of human IL-21 and N-terminal
sequencing of the cytokine had previously demonstrated that
following cleavage of the signal peptide, the resulting N-terminal
amino acid was the pyroglutamate derivative of glutamine-30. This
derivative was chosen for the N-terminal amino acid for the peptide
and along with the subsequent 20 amino acids found at the amino
terminus of human IL-21. To permit efficient and specific coupling
of the peptide to carrier proteins or solid phase matrix necessary
for the analysis with this peptide, an additional cysteine residue
was added to the carboxyl terminus of the peptide. The complete
peptide sequence is pyroGQDRHMIRMRQLIDIVDQLKCamide (SEQ ID NO:
1).
[0177] Peptide #2
[0178] The second peptide was chosen due to its hydrophilic
character, the presence of proline residues (predicted non-helical
segment), and its location in the IL-21 sequence between the
predicted A- and B-helical regions. The carboxy terminal end of the
peptide was selected due to the presence of a cysteine residue in
the human IL-21 polypeptide sequence. The peptide sequence is
NDLVPEFLPAPEDVETNC (SEQ ID NO: 2).
[0179] Peptide #3
[0180] This peptide sequence was selected for its predicted
location comprising a significant portion of the hydrophilic
C-helix of the IL-21 structure. This very hydrophilic region is
predicted to be important in ligand-receptor interaction, and the
peptide span was also selected to allow for the inclusion of a
native cysteine residue (Cys-122) to enable efficient conjugations
when appropriate as noted above. The peptide sequence is
NVSIKKLKRKPPSTNAGRRQKHRLTCamide (SEQ ID NO: 3).
[0181] Peptide #4
[0182] This peptide was selected due to its location near the
carboxyl terminus of the cytokine and because studies utilizing
IL-21 muteins have demonstrated this peptide region to be important
for ligand-receptor activation but not ligand binding. Mutation of
Gln-145 and/or Ile-148 contained within the sequence of this
peptide, have been shown to affect human IL-21 receptor activation.
The 29 amino acid peptide was initiated at the native Cys-125 to
enable its use in chemical coupling of the peptide as noted above,
and to end at Ser-153. This serine is the final amino acid in
murine IL-21 so the peptide sequence amino terminal of this residue
is predicted to contain the elements of the human IL-21 sequence
that are important for ligand activity. The peptide sequence is
CDSYEKKPPKEFLERFKSLLQKMIHQHLSamide (SEQ ID NO: 4).
Example 12
Phosiphorylated-STAT3 Assay for Detection of IL-21
Neutralization
[0183] Previously derived Baf3/human IL21 receptor (hIL-21R)
transfectants were used (see, U.S. Pat. Nos. 6,307,024 and
6,686,178, incorporated herein by reference). The cells were washed
three times in Baf3 bioassay media which consists of: RPMI,
1.times. Glutamax, 10% Fetal Bovine Serum, 50 uM
Beta-mercaptoethanol, 200 ug/mL Zeocin, 1 mg/mL G418 (all from
Invitrogen Corporation, Carlsbad, Calif.). After third wash, cells
were counted using standard methods (hemacytometer) and resuspended
to 6.times.10.sup.5 cells per mL in bioassay media. Cells were then
plated in a 96-well round bottom tissue culture plate at 30,000
cells per well. The plate was then transferred to a 37.degree. C.
tissue culture incubator while the other assay plates were set
up.
[0184] The samples plate was then set up with 30 uL of 2.0 ng/mL
human IL-21 plus 30 uL of one of the following: diluted mouse serum
(1:10, 1:50 or 1:100 final concentrations), media, anti-IL-21
neutralizing antibody (various lots and concentrations), soluble
hIL-21R (example 2) or irrelevant controls. The plate was then
transferred to a 37.degree. C. incubator. After 30-40 minutes, both
the cell plate and the sample plates were removed from the
incubator and 50 uL of each well in the sample plate was
transferred to the cell plate and mixed. The plates were then
placed back in the 37.degree. C. incubator for exactly 8 minutes.
At this point, the reaction was stopped by placing the plate on ice
and adding 150 uL of ice cold BioPlex Cell Wash Buffer (BioRad
Laboratories, Hercules, Calif.). The plate was centrifuged for 5
minutes at 1500 RPM and 4.degree. C. Following centrifugation, the
supernatant was disgarded into the sink and cells were lysed in 60
uL BioPlex Cell Lysis Buffer containing Factor 1, Factor 2 and PMSF
(all from BioRad). Lysed cells were pipetted to break up clumps and
then shaken at 600 RPM on at 4.degree. C. for 20 minutes. The plate
was then centrifuged again for 20 minutes at 3000 RPM at 4.degree.
C. After centrifugation, 55 uL of lysate was removed and mixed with
55 uL of Phosphoprotein Testing Assay Buffer (BioRad).
[0185] At this point a filter plate was pre-wetted with 50 uL
Phosphoprotein Wash Buffer (PWB), aspirated and 50 uL of
PhosphoSTAT3 Coupled Beads (BioRad) plated. These beads were then
aspirated and the plate was washed three times with 75 uL of PWB.
Following final aspiration, 50 uL of diluted lysate was transferred
to the plate which was then covered and shaken overnight at room
temperature. The following morning, the plate was washed three
times with PWB, and biotinylated-PhosphoSTAT3 Detection Antibodies
(BioRad) were then added for 20 minutes at room temperature. The
plate was washed three more times in PWB and then Streptavidin-PE
was added for 10 minutes. Finally, the plate was washed three times
with Phosphoprotein Resuspension Buffer (PRB) and the beads were
resuspended in 125 uL of PRB.
[0186] Total phosphorylated-STAT3 was measured in each well by
following the standard Luminex 100 data collection protocol as
recommended by the manufacturer (Luminex Inc., Austin, Tex.). Data
were then analyzed and expressed as fold-induction of
phosphorylated-STAT3 as compared to media alone.
[0187] Serum from mice immunized with various IL-21 peptides (see
table 1) was tested for neutralization of IL21-induced STAT3
phosphorylation. IL-21 was premixed with a dilution of serum for 30
minutes at 37.degree. C. This solution was then added to
BaB3/hIL21R transfectants for 8 minutes. The reaction was then
stopped, cells lysed and phosphorylated-STAT3 measured. The fold
increase in phosphoylated-STAT3 is calculated using the "media
alone" control as a baseline. Lower numbers correlate to stronger
IL-21 neutralization. The data is summarized in Table 2 below.
[0188] Briefly: one of three mice (#1976) immunized with peptide #1
and two of three mice (#1979, 1980) immunized with peptide #2,
generated IL-21 neutralizing antibodies. No mice immunized with
peptides #3 and #4 generated neutralizing antibodies. The
neutralization seen in the 1:10 dilution column may be due to a
serum effect, not specific anti-IL-21 activity (refer to
"irrelevant serum" data). TABLE-US-00001 TABLE 1 IL21 peptides and
corresponding serum sample numbers Sample # Peptide # 1976-1978
Peptide #1 (see example 11) 1979-1981 Peptide #2 (see example 11)
1982-1984 Peptide #4 (see example 11) 1985-1987 Peptide #3 (see
example 11) 1988-1990 Peptide #3 (see example 11)
[0189] TABLE-US-00002 TABLE 2 Average fold increase in
phosphorylated-STAT3 levels as compared to media Serum Dilution
Sample# 1:10 1:50 1:100 1976 1.89 5.07 5.15 1977 4.98 10.14 8.63
1978 6.72 8.33 12.99 1979 1.04 1.81 2.17 1980 0.65 1.4 1.81 1981
6.91 9.8 17.06 1982 4.04 10.8 12.73 1983 9.68 12.05 12.42 1984 5.84
10.84 11.78 1985 6.94 12.64 11.5 1986 9.01 13.41 16.61 1987 8.48
14.11 17.36 1988 4.56 8.66 9.38 1989 4.88 11.63 12.66 1990 9.56
10.29 14.58 Controls Media Alone 1 IL-21 12.4 NMS 11.28 11.72 10.31
Irrelevant Serum 3.52 11.09 9.49
Example 13
[0190] Assay Description of Direct EIA
[0191] The ability of human IL-21 peptides (example 11) to bind
anti-human IL-21 antibodies was assessed using a "direct" style
ELISA assay. In this assay, wells of 96 well polystyrene ELISA
plates were first coated with 100 .mu.L /well of human IL-21
protein at a concentration of 1 .mu.g/mL in Coating Buffer (0.1M
Na.sub.2CO.sub.3, pH 9.6). Plates were incubated overnight at
4.degree. C. after which unbound peptides were aspirated and the
plates washed twice with 300 .mu.L/well of Wash Buffer (PBS-Tween
defined as 0.137M NaCl, 0.0022M KCl, 0.0067M Na.sub.2HPO.sub.4,
0.0020M KH.sub.2PO.sub.4, 0.05% v/w polysorbate 20, pH 7.2). Wells
were blocked with 200 .mu.L/well of Blocking Buffer (PBS-Tween plus
1% v/w bovine serum albumin (BSA)) for 1 hour, after which the
plates were washed twice with Wash Buffer. Antibody dilutions were
prepared in 5% FBS/IMDM medium and adjusted to 1 ug/ml. Duplicate
samples of each antibody dilution were then transferred to the
assay plates, 100 .mu.L/well, in order to bind anti-human IL-21
peptides. Following 1 hour incubation at RT, the wells were
aspirated and the plates washed twice as described above.
Horseradish peroxidase labeled Goat anti Mouse IgG, Fe specific
(Jackson ImmunoResearch Laboratories, West Grove, Pa.) at a
dilution of 1:5000 with 5%/IMDM medium was then added to each well,
100 .mu.L/well, and the plates incubated at RT for 1 hour. After
removal of unbound HRP conjugated antibody, the plates were washed
five times, 100 .mu.L/well of tetra methyl benzidine (TMB) (BioFX
Laboratories, Owings Mills, Md.) added to each well and the plates
incubated for 3 minutes at RT. Color development was stopped by the
addition of 100 .mu.L/well of 450 nm TMB Stop Reagent (BioFX
Laboratories, Owings Mills, Md.) and the absorbance values of the
wells read on a Molecular Devices Spectra MAX 340 instrument at 450
nm.
Example 14
[0192] Characterization of Neutralizing Antibodies
[0193] Characterization of samples derived from hybridoma culture
supernatant included binding assays using a human IL-21 Fc fusion
protein, mouse IL-21 Fc fusion protein, a human IL-21 protein
mutated at Gln 145 and Ile148 (SEQ ID NO: 6) and peptides described
above. IL-21 Fc fusions are disclosed in U.S. Pat. Nos. 6,307,024
and 6,686,178, and methods for generating Fc fusions are disclosed
in U.S. Pat. Nos. 5,155,027 and 5,567,584, all incorporated herein
by reference. The IL-21 mutant protein is described in U.S. Pat.
6,929,932 and U.S. Patent Application No. 2005-0244930, all
incorporated herein by reference. TABLE-US-00003 TABLE 3 Binds
hIL-21-Fc Binds hIL-21 Binds mIL-21-mFc Binds peptide Designation
Isotype Neutralization protein mutein protein number Mouse Ab
338.17 IgG1 Yes Yes Yes Yes No. 3 Mouse Ab 338.24 IgG1 Yes Yes Yes
Yes No. 3 Mouse Ab 338.25 IgG1 Yes Yes No Yes None Mouse Ab 338.29
IgG1 Yes Yes Yes Yes No. 1 Rat Ab IgG2a Yes No Yes Yes No. 1
297.21.1.3.4.2
[0194] Mouse Ab 338.17 This sample bound the human IL-21 protein
structure in an area predicted to be in the region of the C-Helix
based on its ability to bind Peptide #3.
[0195] Mouse Ab 338.24 This sample bound the human IL-21 protein
structure in an area predicted to be in the region of the C-Helix-C
based on its ability to bind Peptide #3.
[0196] Mouse Ab 338.25 This sample bound the D-helix side of the
molecule near Q145 and/or I148 (SEQ ID NO: 6) based on its ability
to bind the native hIL-21 but not the hIL-21 mutein. The binding is
likely to be to a discontinuous epitope based on its inability to
bind peptide #4, which contains the native peptide sequence mutated
in the IL-21 mutein, or the epitope may require the presence of
Ser154-Ser162 of the human IL-21 sequence (SEQ ID NO: 6).
[0197] Mouse Ab 338.29 This sample bound the human IL-21 protein
structure in an area predicted to be in the region of the A-Helix
based on its ability to bind Peptide #1. Based on its ability to
react moderately with a C-terminally conjugated Peptide #1 but
react very strongly to the non-conjugated peptide, one would
predict the epitope for this antibody to include the middle or
C-terminal domain of Helix A as represented in Peptide #1.
[0198] Rat Ab 297.21.1.3.4.2 Based on its ability to react weakly
with both the C-terminally conjugated and non-conjugated Peptide
#1, its ability to neutralize hIL-21 but its inability to capture
hIL-21-Fc from solution., this antibody binds to a largely
discontinuous epitope on the human IL-21 protein structure
comprising an area predicted to be in the region of the N-terminus
of IL-21 and the A-Helix and require space near the adjacent
C-terminus of hIL-21 where it is linked to the Fc fusion protein.
Sequence CWU 1
1
8 1 21 PRT Artificial Sequence synthesized peptide VARIANT
(1)...(1) Xaa = pyroGlu AMIDATION (21)...(21) 1 Xaa Gln Asp Arg His
Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val 1 5 10 15 Asp Gln Leu
Lys Cys 20 2 19 PRT Artificial Sequence synthesized peptide 2 Asn
Asp Leu Val Pro Glu Phe Leu Pro Ala Pro Glu Asp Val Glu Asn 1 5 10
15 Thr Asn Cys 3 26 PRT Artificial Sequence synthesized peptide
AMIDATION (26)...(26) 3 Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro
Pro Ser Thr Asn Ala 1 5 10 15 Gly Arg Arg Gln Lys His Arg Leu Thr
Cys 20 25 4 29 PRT Artificial Sequence synthesized peptide
AMIDATION (29)...(29) 4 Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu
Phe Leu Glu Arg Phe 1 5 10 15 Lys Ser Leu Leu Gln Lys Met Ile His
Gln His Leu Ser 20 25 5 642 DNA Homo sapiens CDS (47)...(532) 5
gctgaagtga aaacgagacc aaggtctagc tctactgttg gtactt atg aga tcc 55
Met Arg Ser 1 agt cct ggc aac atg gag agg att gtc atc tgt ctg atg
gtc atc ttc 103 Ser Pro Gly Asn Met Glu Arg Ile Val Ile Cys Leu Met
Val Ile Phe 5 10 15 ttg ggg aca ctg gtc cac aaa tca agc tcc caa ggt
caa gat cgc cac 151 Leu Gly Thr Leu Val His Lys Ser Ser Ser Gln Gly
Gln Asp Arg His 20 25 30 35 atg att aga atg cgt caa ctt ata gat att
gtt gat cag ctg aaa aat 199 Met Ile Arg Met Arg Gln Leu Ile Asp Ile
Val Asp Gln Leu Lys Asn 40 45 50 tat gtg aat gac ttg gtc cct gaa
ttt ctg cca gct cca gaa gat gta 247 Tyr Val Asn Asp Leu Val Pro Glu
Phe Leu Pro Ala Pro Glu Asp Val 55 60 65 gag aca aac tgt gag tgg
tca gct ttt tcc tgt ttt cag aag gcc caa 295 Glu Thr Asn Cys Glu Trp
Ser Ala Phe Ser Cys Phe Gln Lys Ala Gln 70 75 80 cta aag tca gca
aat aca gga aac aat gaa agg ata atc aat gta tca 343 Leu Lys Ser Ala
Asn Thr Gly Asn Asn Glu Arg Ile Ile Asn Val Ser 85 90 95 att aaa
aag ctg aag agg aaa cca cct tcc aca aat gca ggg aga aga 391 Ile Lys
Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg Arg 100 105 110
115 cag aaa cac aga cta aca tgc cct tca tgt gat tct tat gag aaa aaa
439 Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys Lys
120 125 130 cca ccc aaa gaa ttc cta gaa aga ttc aaa tca ctt ctc caa
aag atg 487 Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu Gln
Lys Met 135 140 145 att cat cag cat ctg tcc tct aga aca cac gga agt
gaa gat tcc 532 Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu
Asp Ser 150 155 160 tgaggatcta acttgcagtt ggacactatg ttacatactc
taatatagta gtgaaagtca 592 tttctttgta ttccaagtgg aggagcccta
ttaaattata taaagaaata 642 6 162 PRT Homo sapiens 6 Met Arg Ser Ser
Pro Gly Asn Met Glu Arg Ile Val Ile Cys Leu Met 1 5 10 15 Val Ile
Phe Leu Gly Thr Leu Val His Lys Ser Ser Ser Gln Gly Gln 20 25 30
Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp Gln 35
40 45 Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala
Pro 50 55 60 Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser
Cys Phe Gln 65 70 75 80 Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn
Asn Glu Arg Ile Ile 85 90 95 Asn Val Ser Ile Lys Lys Leu Lys Arg
Lys Pro Pro Ser Thr Asn Ala 100 105 110 Gly Arg Arg Gln Lys His Arg
Leu Thr Cys Pro Ser Cys Asp Ser Tyr 115 120 125 Glu Lys Lys Pro Pro
Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu 130 135 140 Gln Lys Met
Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu 145 150 155 160
Asp Ser 7 3072 DNA Mus musculus CDS (54)...(491) 7 gagaaccaga
ccaaggccct gtcatcagct cctggagact cagttctggt ggc atg 56 Met 1 gag
agg acc ctt gtc tgt ctg gta gtc atc ttc ttg ggg aca gtg gcc 104 Glu
Arg Thr Leu Val Cys Leu Val Val Ile Phe Leu Gly Thr Val Ala 5 10 15
cat aaa tca agc ccc caa ggg cca gat cgc ctc ctg att aga ctt cgt 152
His Lys Ser Ser Pro Gln Gly Pro Asp Arg Leu Leu Ile Arg Leu Arg 20
25 30 cac ctt att gac att gtt gaa cag ctg aaa atc tat gaa aat gac
ttg 200 His Leu Ile Asp Ile Val Glu Gln Leu Lys Ile Tyr Glu Asn Asp
Leu 35 40 45 gat cct gaa ctt cta tca gct cca caa gat gta aag ggg
cac tgt gag 248 Asp Pro Glu Leu Leu Ser Ala Pro Gln Asp Val Lys Gly
His Cys Glu 50 55 60 65 cat gca gct ttt gcc tgt ttt cag aag gcc aaa
ctc aag cca tca aac 296 His Ala Ala Phe Ala Cys Phe Gln Lys Ala Lys
Leu Lys Pro Ser Asn 70 75 80 cct gga aac aat aag aca ttc atc att
gac ctc gtg gcc cag ctc agg 344 Pro Gly Asn Asn Lys Thr Phe Ile Ile
Asp Leu Val Ala Gln Leu Arg 85 90 95 agg agg ctg cct gcc agg agg
gga gga aag aaa cag aag cac ata gct 392 Arg Arg Leu Pro Ala Arg Arg
Gly Gly Lys Lys Gln Lys His Ile Ala 100 105 110 aaa tgc cct tcc tgt
gat tcg tat gag aaa agg aca ccc aaa gaa ttc 440 Lys Cys Pro Ser Cys
Asp Ser Tyr Glu Lys Arg Thr Pro Lys Glu Phe 115 120 125 cta gaa aga
cta aaa tgg ctc ctt caa aag atg att cat cag cat ctc 488 Leu Glu Arg
Leu Lys Trp Leu Leu Gln Lys Met Ile His Gln His Leu 130 135 140 145
tcc tagaacacat aggacccgaa gattcctgag gatccgagaa gattcccgag 541 Ser
gactgaggag acgccggaca ctatagacgc tcacgaatgc aggagtacat cttgcctctt
601 gggattgcaa gtggagaagt acgatacgtt atgataagaa caactcagaa
aagctatagg 661 ttaagatcct ttcgcccatt aactaagcag acattgtggt
tccctgcaca gactccatgc 721 tgtcaacatg gaaaatctca actcaacaag
agcccagctt cccgtgtcag ggatttctgg 781 tgcttctcaa gctgtggctt
catcttattg cccaactgtg acattctttg attggaaggg 841 gaaaactaaa
gcttttagca aaaatacagc tagggaattt gtcgatctgc gagagtaaga 901
cctcttatga tcctaacgga atgatgtaag ctggaaataa taagcataag atgaaattga
961 aaattgaagt ctttattctt taagaaaaac tttgtacttg aaagcatgtc
tgaagagttt 1021 actcattacc acaaacatct agcatattga taactaacat
ctttatactc tacaagagag 1081 gctttccaga taggtacagt ttttcttctc
tattaggtct atcaaaattt aacctattat 1141 gagggtcacc cctggctttc
actgtttttc taaagaggca agggtgtagt aagaagcagg 1201 cttaagttgc
cttcctccca atgtcaagtt cctttataag ctaatagttt aatcttgtga 1261
agatggcaat gaaagcctgt ggaagtgcaa acctcactat cttctggagc caagtagaat
1321 tttcaagttt gtagctctca cctcaagtgg ttatgggtgt cctgtgatga
atctgctagc 1381 tccagcctca gtctcctctc ccacatcctt tcctttcttt
cctctttgaa acttctaaga 1441 aaaagcaatc caaacaagtt cagcacttaa
gacacattgc atgcacactt ttgataagtt 1501 aaatccaacc atctatttaa
aatcaaaatc aggagatgag ccaagagacc agaggttctg 1561 ttccagtttt
aaacagactt ttactgaaca tcccaatctt ttaaccacag aggctaaatt 1621
gagcaaatag ttttgccatt tgatataatt tccaacagta tgtttcaatg tcaagttaaa
1681 aagtctacaa agctattttc cctggagtgg tatcatcgct ttgagaattt
cttatggtta 1741 aaatggatct gagatccaag catggcctgg gggatggttt
tgatctaagg aaaaaggtgt 1801 ctgtacctca cagtgccttt aaaacaagca
gagatcccgt gtaccgccct aagatagcac 1861 agactagtgt taactgattc
ccagaaaagt gtcacaatca gaaccaacgc attctcttaa 1921 actttaaaaa
tatgtattgc aaagaacttg tgtaactgta aatgtgtgac tgttgatgac 1981
attatacaca catagcccac gtaagtgtcc aatggtgcta gcattggttg ctgagtttgc
2041 tgctcgaaag ctgaagcaga gatgcagtcc ttcacaaagc aatgatggac
agagagggga 2101 gtctccatgt tttattcttt tgttgtttct ggctgtgtaa
ctgttgactt cttgacattg 2161 tgatttttat atttaagaca atgtatttat
tttggtgtgt ttattgttct agccttttaa 2221 atcactgaca atttctaatc
aagaagtaca aataattcaa tgcagcacag gctaagagct 2281 tgtatcgttt
ggaaaagcca gtgaaggctt ctccactagc catgggaaag ctacgcttta 2341
gagtaaacta gacaaaattg cacagcagtc ttgaacctct ctgtgctcaa gactcagcca
2401 gtcctttgac attattgttc actgtgggtg ggaacacatt ggacctgaca
cactgttgtg 2461 tgtccatgaa ggttgccact ggtgtaagct ttttttggtt
ttcattctct tatctgtaga 2521 acaagaatgt ggggctttcc taagtctatt
ctgtatttta ttctgaactt cgtatgtctg 2581 agttttaatg ttttgagtac
tcttacagga acacctgacc acacttttga gttaaatttt 2641 atcccaagtg
tgatatttag ttgttcaaaa agggaaggga tatacataca tacatacata 2701
catacataca tatatatata tatatataca tatatatata tatatatatg tatatatata
2761 tatatataga gagagagaga gagagagaga gagaaagaga gagaggttgt
tgtaggtcat 2821 aggagttcag aggaaatcag ttatggccgt taatactgta
gctgaaagtg ttttctttgt 2881 gaataaattc atagcattat tgatctatgt
tattgctctg ttttatttac agtcacacct 2941 gagaatttag ttttaatatg
aatgatgtac tttataactt aatgattatt tattatgtat 3001 ttggttttga
atgtttgtgt tcatggcttc ttatttaaga cctgatcata ttaaatgcta 3061
cccagtccgg a 3072 8 146 PRT Mus musculus 8 Met Glu Arg Thr Leu Val
Cys Leu Val Val Ile Phe Leu Gly Thr Val 1 5 10 15 Ala His Lys Ser
Ser Pro Gln Gly Pro Asp Arg Leu Leu Ile Arg Leu 20 25 30 Arg His
Leu Ile Asp Ile Val Glu Gln Leu Lys Ile Tyr Glu Asn Asp 35 40 45
Leu Asp Pro Glu Leu Leu Ser Ala Pro Gln Asp Val Lys Gly His Cys 50
55 60 Glu His Ala Ala Phe Ala Cys Phe Gln Lys Ala Lys Leu Lys Pro
Ser 65 70 75 80 Asn Pro Gly Asn Asn Lys Thr Phe Ile Ile Asp Leu Val
Ala Gln Leu 85 90 95 Arg Arg Arg Leu Pro Ala Arg Arg Gly Gly Lys
Lys Gln Lys His Ile 100 105 110 Ala Lys Cys Pro Ser Cys Asp Ser Tyr
Glu Lys Arg Thr Pro Lys Glu 115 120 125 Phe Leu Glu Arg Leu Lys Trp
Leu Leu Gln Lys Met Ile His Gln His 130 135 140 Leu Ser 145
* * * * *