Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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ANTIBODIES AGAINST INTERLEUKIN-10-LIKE CYTOKINES
AND USES THEREFOR
CROSS-REFERENCE TO RELATED APPLICATIONS
[001] This application claims the benefit of, and relies on the filing date
of,
U.S. provisional patent application number 61/064,718, filed 21 March 2008,
the entire
disclosure of which is hereby incorporated herein by reference.
FIELD
[002] Specific binding fragments, antibodies, and antigen-binding fragments
thereof that bind interleukin-22 (IL-22) are provided. The specific binding
fragments,
antibodies, and antigen-binding fragments disclosed herein are useful in
diagnosing,
preventing, and/or treating IL-22-associated disorders, including, e.g.,
autoimmune
disorders. Methods for determining which amino acids of the human IL-22
protein
sequence are important in the binding of IL-22 to its cell surface receptor
complex and to
its binding protein are also provided, along with the resulting epitope
defined by the
method.
BACKGROUND
[003] Interleukin 22 (IL-22) is a member of the Interleukin 10 (IL-10) -like
subgroup of type II cytokines.(Renauld, J.-C. Nature Reviews Immunology 3, 667-
76
(2003)). The members of this subgroup (i.e., IL-10, IL-19, IL-20, IL-22, IL-
24, and IL-
26) are proposed to have a conserved six a-helical structural and functional
unit that is
also shared with the interferons (Renauld et al. Nature Reviews Immunology 3,
667-76
(2003) and Langer et al. Cytokine & Growth Factor Reviews 15, 33-48 (2004)).
IL-22 is
produced by activated T helper (Th) 17 CD4+ lymphocytes, as well as monocytes,
and its
expression is highly dependent on IL-23 (Liang, S.C. et al. Journal of
Experimental
Medicine 203, 2271-9 (2006) and Zheng, Y. et al. Nature 445, 648-51 (2007)).
IL-22 is
known to regulate local tissue inflammation while acting only on non-immune
cells, and
plays a critical role in mucosal immunity as well as dysregulated inflammation
observed
in autoimmune disease. (Wolk, K. et al. Immunity 21, 241-54 (2004); Wolk et
al.,
Cytokine & Growth Factor Reviews 17, 367-80 (2006); Wolk et al. Journal of
Immunology 168, 5397-402 (2002); Pan et al. Cellular & Molecular Immunology 1,
43-9
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(2004); Zenewicz et al. 27, 647-59 (2007); Aujla, S.J. et al. Nature Medicine
in
press(2008); and Zheng, Y. et al. Nature Medicine in press(2008).). Recent
clinical and
pre-clinical studies strongly implicate Th17 cell and IL-22 activities in the
progression of
psoriasis, a human autoimmune disease of the skin (Zheng et al. Nature 445,
648-51
(2007); Nickoloff et al. Nature Medicine 13, 242-244 (2007); Zaba et al.
Journal of
Experimental Medicine 204, 3183-94 (2007); Ma et al. Journal of Clinical
Investigation
in press(2008); Lowes et al. Nature 445, 866-73 (2007); and Wolk et al.
European
Journal of Immunology 36, 1309-23 (2006). Adminstration of IL-22 has been
shown to
induce the hyperproliferation of skin keratinocytes and resultant thickening
of the
epidermis, both characteristics of psoriatic lesions (Boniface et al. Journal
of
Immunology 174, 3695-702 (2005)). In addition, the administration of IL-22has
been
shown to induce gene expression from keratinocytes that appear to be involved
in the
recruitment of immune cells and the maintenance of psoriatic tissue
inflammation (Wolk
et al. European Journal of Immunology 36, 1309-23 (2006); Boniface et al.
Journal of
Immunology 174, 3695-702 (2005); and Sa et al. Journal of Immunology 178, 2229-
40
(2007)[erratum appears in J Immunol. 2007 Jun 1;178(11):7487]).
[004] Expression of IL-22 is up-regulated in T cells by IL-9 or ConA
(Dumoutier L. et al. (2000) Proc Nail Acad Sci USA 97(18):10144-9). Further
studies
have shown that expression of IL-22 mRNA is induced in vivo in response to LPS
administration, and that IL-22 modulates parameters indicative of an acute
phase
response (Dumoutier L. et al. (2000) supra; Pittman D. et al. (2001) Genes and
Immunity
2:172). Taken together, these observations indicate that IL-22 plays a
critical role in
inflammation (Kotenko S.V. (2002) Cytokine & Growth Factor Reviews 13(3):223-
40).
[005] At low concentrations, IL-22 exits as a monomer in solution, sharing a
six a-helical structural and functional monomeric unit with the intercalated
IL- 10 dimer,
as well as other IL-10-like cytokines and IFN-y (Nagem et al. Vitamins &
Hormones 74,
77-103 (2006); Logsdon et al. Journal of Interferon & Cytokine Research 22,
1099-112
(2002); Nagem et al. Structure 10, 1051-62 (2002); and Chang et al. Journal of
Biological Chemistry 278, 3308-13 (2003)). The six defined helices of IL-22
are known
as helices A, B, C, D, E and F. The portion of the IL-22 protein that connects
helix A
with helix B is known as loop AB. Glycosylated IL-22, expressed from insect
cells, can
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exist and is proposed to function as a monomer (Logsdon, N.J., Jones, B.C.,
Josephson,
K., Cook, J. & Walter, M.R. Journal of Interferon & Cytokine Research 22, 1099-
112
(2002). A recent report indicates that E coli-derived IL-22 dimerizes at high
concentrations, via a proposed interaction between its DE loops, and is able
to associate
with the IL-22R receptor subunit (de Oliveira Neto, M. et al Biophys J. 94,
1754-65
(2008)). The resultant low resolution quaternary structure is similar to that
observed
between intercalated IL- 10 dimers and it's high affinity receptor, IL-IOR1
(Josephson,
K., Logsdon, N.J. & Walter, M.R. Immunity 15, 3 5-46 (2001)).
[006] The cell surface receptor for IL-22 is believed to be a receptor complex
consisting of an IL-22 receptor (IL-22R) and an IL-22 receptor 2 (IL-IOR2)
subunit, each
of which is a member of the type II cytokine receptor family (CRF2) (Xie M.H.
et al.
(2000) JBiol Chem 275(40):31335-9; Kotenko S.V. et al. (2001) JBiol Chem
276(4):2725-32). . CRF2 members are receptors for IFNa/(3, IFNy, coagulation
factor
VIIa, IL-10 and the IL-10 related proteins IL-19, IL-20, IL-22, IL-24, IL-26,
as well as
the recently identified IFN-like cytokines, IL-28 and IL-29 (Kotenko S.V.
(2002)
Cytokine & Growth Factor Reviews 13(3):223-40; Kotenko, S.V. et al. (2000)
Oncogene
19(21):2557-65; Sheppard, P. et al. (2003) Nature Immunology 4(1):63-8;
Kotenko, S.V.
et al. (2003) Nature Immunology 4(1):69-77). Each of the subunits, or chains,
of the IL-
22 receptor complex presents on epithelial cells and some fibroblasts within
various
tissues (Wolk et al. Journal of Immunology 168, 5397-402 (2002); Me et al.
Journal of
Biological Chemistry 275, 31335-9 (2000); Kotenko et al. Journal of Biological
Chemistry 276, 2725-32 (2001); Ikeuchi et al. Arthritis & Rheumatism 52, 1037-
46
(2005); Andoh et al. Gastroenterology 129, 969-84 (2005)). Both chains of the
IL-22
receptor complex are also expressed constitutively in a number of organs, and
epithelial
cell lines derived from these organs have been shown to be responsive to IL-22
in vitro
(Kotenko S.V. (2002) Gytokine & Growth Factor Reviews 13(3):223-40.
[007] While the IL-22R and IL- I OR2 subunits individually contribute to the
formation of different receptor complexes for other type II cytokines ,
together the
subunits form a single receptor complex that is specific for IL-22. IL-22 is
believed to
first bind to the extracellular domain (ECD) of IL-22R (Logsdon et al. Journal
of
Interferon & Cytokine Research 22, 1099-112 (2002) and Li et al. International
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Immunopharmacology 4, 693-708 (2004)). Due to a proposed IL-22R-induced
conformational change in IL-22, IL-1 OR2 is able to bind to the IL-22/IL-22R
surface (Li
et al. International Immunopharmacology 4, 693-708 (2004) and Logsdon et al.
Journal
of Molecular Biology 342, 503-14 (2004)). The resulting IL-22/IL-22R/IL-1OR2
complex, as either a heterotrimer or multimer thereof, transmits a signal into
the cell via
the JAK/STAT and MAPK (for example, ERK) signaling pathways (Dumoutier et al.
Journal of Immunology 164, 1814-9 (2000). Dumoutier et al. Proceedings of the
National
Academy of Sciences of the United States of America 97, 10144-9 (2000); and
Lej eune et
al. Journal of Biological Chemistry 277, 33676-82 (2002)). IL-22 induces
activation of
the JAK/STAT3 and MAPK (for example, ERK) pathways, as well as intermediates
of
other MAPK pathways (Dumoutier L. et al. (2000) supra; Xie M.H. et al. (2000)
supra;
Dumoutier L. et al. (2000) J Immunol 164(4): 1814-9; Kotenko S.V. et al.
(2001) JBiol
Chem 276(4):2725-32; Lejeune, D. et al. (2002) JBiol Chem 277(37):33676-82).
[008] The interaction between IL-22R and IL-1 OR2 has been characterized in
an ELISA based format using biotinylated cytokine and receptor extracellular
domain
(ECD) Fc fusion dimers. See, e.g., U.S. Published Patent Application No. 2005-
0042220. IL-22 was shown to have measurable affinity for the ECD of IL-22R and
no
detectable affinity for IL-IOR2 alone. IL-22 was also shown to have a
substantially
greater affinity for IL-22R/IL-1 OR2 ECD presented as Fc heterodimers. IL-10R2
appears
to bind to a surface created by the association between IL-22 and IL-22R,
suggesting that
IL-10R2 ECD further stabilizes the association of IL-22 within its cytokine
receptor
complex. See, e.g., U.S. Published Patent Application No. 2005-0042220.
[009] In addition to binding to the IL-22 receptor complex, IL-22 also binds
to
an IL-22 binding protein (IL-22BP), which is a secreted `receptor' specific
for IL-22 and
has 33 % primary sequence identity to the extracellular domain (ECD) of IL-22R
(Dumoutier, L., Lejeune, D., Colau, D. & Renauld, J.C. Cloning and
characterization of
IL-22 binding protein, a natural antagonist of IL-10-related T cell-derived
inducible
factor/IL-22. Journal of Immunology 166, 7090-5 (2001)). While a cell surface
form of
IL-22BP has not been specifically identified, in vitro, IL-22BP has been shown
to act as a
decoy receptor and block IL-22 signaling into the cell (Dumoutier et al.
Journal of
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Immunology 166, 7090-5 (2001) and Xu et al. Proceedings of the National
Academy of
Sciences of the United States of America 98, 9511-6 (2001)).
[010] Neutralizing anti-IL-22 antibodies have been generated and
characterized in terms of their binding specificity, affinity and IL-22
neutralizing activity
See, e.g., U.S. Published Patent Application No. 2005-0042220. Administration
of IL-22
in vivo has been shown to induce parameters of an acute phase response, and
the
administration of a neutralizing anti-IL-22 antibody has been shown to reduce
IL-22
activity and ameliorates inflammatory symptoms in a mouse collagen-induced
arthritis
(CIA) model See, e.g., U.S. Published Patent Application No. 2005-0042220. In
addition, the expression of IL-22 mRNA has been shown to be upregulated within
inflamed areas. Accordingly, IL-22 antagonists, such as, e.g., neutralizing
anti-IL-22
antibodies and fragments thereof, can be used to induce immune suppression in
vivo and
they provide a promising approach to the treatment of various inflammatory
and/or
autoimmune disorders. Thus, to aid in generating and evaluating neutralizing
anti-IL-22
antibodies, it would be beneficial to understand which IL-22 amino acids play
an
important role in the interaction between the IL-22 protein and its receptor
complex
and/or between the IL-22 protein and its binding protein.
[011] One focused mutagenesis study performed by Logsdon et al.
demonstrated that six amino acids in IL-22 helices A and D, including a
glycosylated
asparagine, are involved in binding to IL-1 OR2 (Logsdon et al. Journal of
Molecular
Biology 342, 503-14 (2004)). The relevance of helices A and D peptides for IL-
1OR2
binding was subsequently substantiated (Wolk et al. Genes & Immunity 6, 8-18
(2005)).
Residues within helices A and F, and loop AB of IL-22 have been proposed to be
important for binding to a receptor based on superimposition of IL-22
structure to
cytokine within IL-10/IL- I ORI ECG and IFN-y/IFN-7R1 ECG co-crystal
structures, as well as
inferences from an IL-22/IL-22RECD model (Logsdon et al. Journal of Interferon
&
Cytokine Research 22, 1099-112 (2002) and Nagem et al. Structure 10, 1051-62
(2002)).
However prior to the current invention, the IL-22 side chains that are
important for
binding to IL-22R and IL-22BP had not been determined. The present invention
provides
for systematic mutagenesis of human IL-22 to reveal specific amino acids that
are
involved in binding of IL-22 to IL-22R, IL-IOR2, and IL-22BP, and provides
methods for
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designing and making inhibitors of IL-22 activity, as well as inhibitors of IL-
19, IL-20,
IL-24 and IL-26 activity.
SUMMARY
[012] In certain embodiments, an IL-22 specific binding agent that binds to
the
wild-type human IL-22 but fails to bind to a mutant IL-22 is provided. In
certain
embodiments, the mutant IL-22 comprises one or more of the following changes
relative
to wild-type human IL-22: a) the amino acid at position 34 of the mutant IL-22
is alanine;
b) the amino acid at position 52 of the mutant IL-22 is alanine; c) the amino
acid at
position 56 of the mutant IL-22 is alanine; d) the amino acid at position 61
of the mutant
IL-22 is alanine; e) the amino acid at position 66 of the mutant IL-22 is
alanine; f) the
amino acid at position 83 of the mutant IL-22 is alanine; g) the amino acid at
position 88
of the mutant IL-22 is alanine; h) the amino acid at position 113 of the
mutant IL-22 is
alanine; i) the amino acid at position 121 of the mutant IL-22 is alanine; j)
the amino acid
at position 122 of the mutant IL-22 is alanine; k) the amino acid at position
125 of the
mutant IL-22 is alanine; and 1) the amino acid at position 172 of the mutant
IL-22 is
alanine. In certain embodiments, the mutant IL-22 further comprises one or
more of the
following changes relative to wild-type human IL-22: a) the amino acid at
position 51 of
the mutant IL-22 is alanine; b) the amino acid at position 54 of the mutant IL-
22 is
alanine; c) the amino acid at position 55 of the mutant IL-22 is alanine; d)
the amino acid
at position 114 of the mutant IL-22 is alanine; or e) the amino acid at
position 117 of the
mutant IL-22 is alanine. In certain embodiments, the mutant IL-22 further
comprises
one or more of the following changes relative to wild-type human IL-22: a) the
amino
acid at position 57 of the mutant IL-22 is alanine; b) the amino acid at
position 59 of the
mutant IL-22 is alanine; c) the amino acid at position 67 of the mutant IL-22
is alanine;
d) the amino acid at position 72 of the mutant IL-22 is alanine; e) the amino
acid at
position 159 of the mutant IL-22 is alanine; f) the amino acid at position 161
of the
mutant IL-22 is alanine; g) the amino acid at position 162 of the mutant IL-22
is alanine;
or h) the amino acid at position 169 of the mutant IL-22 is alanine. In
certain
embodiments, the mutant IL-22 further comprises one or more of the following
changes
relative to wild-type human IL-22: a) the amino acid at position 70 of the
mutant IL-22 is
alanine; b) the amino acid at position 71 of the mutant IL-22 is alanine; c)
the amino acid
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at position 73 of the mutant IL-22 is alanine; or d) the amino acid at
position 165 of the
mutant IL-22 is alanine. In certain embodiments, the IL-22 specific binding
agent is an
antibody.
[013] In certain embodiments, an IL-22 specific binding agent that binds to
the
wild-type human IL-22 but fails to bind to a mutant IL-22 is provided. In
certain
embodiments, the mutant IL-22 comprises one or more of the following changes
relative
to wild-type human IL-22: a) the amino acid at position 57 of the mutant IL-22
is alanine;
b) the amino acid at position 59 of the mutant IL-22 is alanine; c) the amino
acid at
position 67 of the mutant IL-22 is alanine; d) the amino acid at position 72
of the mutant
IL-22 is alanine; e) the amino acid at position 159 of the mutant IL-22 is
alanine; f) the
amino acid at position 161 of the mutant IL-22 is alanine; g) the amino acid
at position
162 of the mutant IL-22 is alanine; or h) the amino acid at position 169 of
the mutant IL-
22 is alanine. In certain embodiments, the mutant IL-22 further comprises one
or more of
the following changes relative to wild-type human IL-22: a) the amino acid at
position 70
of the mutant IL-22 is alanine; b) the amino acid at position 71 of the mutant
IL-22 is
alanine; c) the amino acid at position 73 of the mutant IL-22 is alanine; or
d) the amino
acid at position 165 of the mutant IL-22 is alanine. In certain embodiments,
the IL-22
specific binding agent is an antibody.
[014] In certain embodiments, an IL-22 specific binding agent that binds to
the
wild-type human IL-22 but fails to bind to a mutant IL-22 is provided. In
certain
embodiments, the mutant IL-22 comprises one or more of the following changes
relative
to wild-type human IL-22: a) the amino acid at position 67 of the mutant IL-22
is alanine;
b) the amino acid at position 73 of the mutant IL-22 is alanine; c) the amino
acid at
position 83 of the mutant IL-22 is alanine; or d) the amino acid at position
162 of the
mutant IL-22 is alanine. In certain embodiments, the IL-22 specific binding
agent is an
antibody.
[015] In certain embodiments, a method of selecting a specific binding agent
to
an IL-22 polypeptide is provided. In certain embodiments, the specific binding
agent
binds to at least a portion of an epitope on an IL-22 polypeptide. In certain
embodiments,
an IL-22 polypeptide is contacted with an agent. In certain embodiments, the
affinity of
the agent for the IL-22 polypeptide is determined. In certain embodiments, a
mutant IL-
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22 polypeptide is contacted with the agent, wherein the mutant IL-22
polypeptide
comprises at least one point mutation at at least one amino acid position
selected from
A34, I52, T56, K61, A66, V83, R88, P113, F121, L122, L125, or M172. In certain
embodiments, the affinity of the agent for the mutant IL-22 polypeptide is
determined. In
certain embodiments, the agent is selected if the affinity for the IL-22
polypeptide is
greater than the affinity for the mutant IL-22 polypeptide. In certain
embodiments, the
mutant IL-22 polypeptide is the same as the IL-22 polypeptide except for the
at least one
mutation. In certain embodiments, the agent is selected from a protein,
peptide, nucleic
acid, carbohydrate, lipid, and small molecule compound. In certain
embodiments, the
agent is an antibody. In certain embodiments, the agent is a small molecule
compound.
In certain embodiments, the amount of binding to the IL-22 polypeptide is at
least 2-fold
greater than the amount of binding to the mutant IL-22 polypeptide. In certain
embodiments, the affinity for the IL-22 polypeptide is at least 5-fold greater
than the
affinity for the mutant IL-22 polypeptide. In certain embodiments, the
affinity for the IL-
22 polypeptide is at least 10-fold greater than the affinity for the mutant IL-
22
polypeptide. In certain embodiments, the mutant IL-22 polypeptide comprises at
least
one point mutation selected from Y51, N54, R55, Y114, or E117. In certain
embodiments, the mutant IL-22 polypeptide comprises at least one point
mutation
selected from F57, L59, D67, T70, D71, V72, R73, G159,1161, K162, G165, or
L169.
In certain embodiments, the mutant IL-22 polypeptide comprises at least one
point
mutation selected from D67, R73, and K162. In certain embodiments, the IL-22
specific
binding agent is an antibody.
[016] In certain embodiments, a method of selecting a specific binding agent
to
an IL-22 polypeptide is provided. In certain embodiments, the specific binding
agent
binds to at least a portion of an epitope on an IL-22 polypeptide. In certain
embodiments,
an IL-22 polypeptide is contacted with an agent. In certain embodiments, the
affinity of
the agent for the IL-22 polypeptide is determined. In certain embodiments, a
mutant IL-
22 polypeptide is contacted with the agent, wherein the mutant IL-22
polypeptide
comprises at least one point mutation at at least one amino acid position
selected from
F57, L59, D67, V72, G159, I161, K162, or L169. In certain embodiments, the
affinity of
the agent for the mutant IL-22 polypeptide is determined. In certain
embodiments, the
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agent is selected if the affinity for the IL-22 polypeptide is greater than
the affinity for the
mutant IL-22 polypeptide. In certain embodiments, the mutant IL-22 polypeptide
is the
same as the IL-22 polypeptide except for the at least one mutation. In certain
embodiments, the agent is selected from a protein, peptide, nucleic acid,
carbohydrate,
lipid, and small molecule compound. In certain embodiments, the agent is an
antibody.
In certain embodiments, the agent is a small molecule compound. In certain
embodiments, the amount of binding to the IL-22 polypeptide is at least 2-fold
greater
than the amount of binding to the mutant IL-22 polypeptide. In certain
embodiments, the
affinity for the IL-22 polypeptide is at least 5-fold greater than the
affinity for the mutant
IL-22 polypeptide. In certain embodiments, the affinity for the IL-22
polypeptide is at
least 10-fold greater than the affinity for the mutant IL-22 polypeptide. In
certain
embodiments, the mutant IL-22 polypeptide comprises at least one point
mutation
selected from T70, D71, R73, or G165. In certain embodiments, the mutant IL-22
polypeptide comprises at least one point mutation selected from A34, Y51, 152,
N54,
R55,T56,K61,A66,V83,R88,P113,Y114,El17,F121,L122,L125,orM172.In
certain embodiments, the mutant IL-22 polypeptide comprises at least one point
mutation
selected from R73 or V83.
[017] In certain embodiments, a method of selecting a specific binding agent
to
an IL-22 polypeptide is provided. In certain embodiments, the specific binding
agent
binds to at least a portion of an epitope on an IL-22 polypeptide. In certain
embodiments,
an IL-22 polypeptide is contacted with an agent. In certain embodiments, the
affinity of
the agent for the IL-22 polypeptide is determined. In certain embodiments, a
mutant IL-
22 polypeptide is contacted with the agent, wherein the mutant IL-22
polypeptide
comprises at least one point mutation at at least one amino acid position
selected from
D67, R73, V83, or K162. In certain embodiments, the affinity of the agent for
the mutant
IL-22 polypeptide is determined. In certain embodiments, the agent is selected
if the
affinity for the IL-22 polypeptide is greater than the affinity for the mutant
IL-22
polypeptide. In certain embodiments, the mutant IL-22 polypeptide is the same
as the IL-
22 polypeptide except for the at least one mutation. In certain embodiments,
the agent is
selected from a protein, peptide, nucleic acid, carbohydrate, lipid, and small
molecule
compound. In certain embodiments, the agent is an antibody. In certain
embodiments,
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the agent is a small molecule compound. In certain embodiments, the amount of
binding
to the IL-22 polypeptide is at least 2-fold greater than the amount of binding
to the
mutant IL-22 polypeptide. In certain embodiments, the affinity for the IL-22
polypeptide
is at least 5-fold greater than the affinity for the mutant IL-22 polypeptide.
In certain
embodiments, the affinity for the IL-22 polypeptide is at least 10-fold
greater than the
affinity for the mutant IL-22 polypeptide. In certain embodiments, the mutant
IL-22
polypeptide comprises at least one point mutation selected from A34, Y51, 152,
N54,
R55, T56, K61, A66, R88, P113, Y114, El17, F121, L122, L125, or M172. In
certain
embodiments, the mutant IL-22 polypeptide comprises at least one point
mutation
selected from F57, L59, T70, D71, V72, G159,1161, G165, or L169.
[018] In certain embodiments, a pharmaceutical composition comprising an
IL-22 specific binding agent is provided. In certain embodiments, the
pharmaceutical
composition further comprising a cytokine inhibitor, a growth factor
inhibitor, an
immunosuppressant, an anti-inflammatory agent, a metabolic inhibitor, an
enzyme
inhibitor, a cytotoxic agent, or a cytostatic agent. In certain embodiments,
the
pharmaceutical composition further comprises at least one of a TNF antagonist,
an IL- 12
antagonist, an IL-15 antagonist, an IL-17 antagonist, an IL-18 antagonist, an
IL-21R
antagonist, a T cell depleting agent, a B cell depleting agent, methotrexate,
leflunomide,
sirolimus (rapamycin) or an analog thereof, a Cox-2 inhibitor, a cPLA2
inhibitor, an
NSAID, or a p3 8 inhibitor.
[019] In certain embodiments, a method of treating or preventing an IL22-
associated disorder, in a subject is provided, comprising, administering to
the subject an
IL-22 specific binding agent, in an amount sufficient to treat or prevent the
IL22-
associated disorder. In certain embodiments, the 11,22-associated disorder is
an
autoimmune disorder, a respiratory disorder, or an inflammatory condition. In
certain
embodiments, the 11,22-associated disorder is rheumatoid arthritis,
osteoarthritis, multiple
sclerosis, myasthenia gravis, Crohn's disease, inflammatory bowel disease,
lupus,
diabetes, psoriasis, asthma, chronic obstructive pulmonary disease (COPD),
cardiovascular inflammation, pancreatitis, hepatitis or nephritis. In certain
embodiments,
the method of treating or preventing an IL-22 associated disorder further
comprises
administering to the subject a cytokine inhibitor, a growth factor inhibitor,
an
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immunosuppressant, an anti-inflammatory agent, a metabolic inhibitor, an
enzyme
inhibitor, a cytotoxic agent, or a cytostatic agent. In certain embodiments,
the method of
treating or preventing an IL-22 associated disorder further comprises
administering to the
subject at least one of a TNF antagonist, an IL- 12 antagonist, an IL- 15
antagonist, an IL-
17 antagonist, an IL- 18 antagonist, an IL-21R antagonist, a T cell depleting
agent, a B
cell depleting agent, methotrexate, leflunomide, sirolimus (rapamycin) or an
analog
thereof, a Cox-2 inhibitor, a cPLA2 inhibitor, an NSAID, or a p38 inhibitor.
[020] In certain embodiments, an IL- 19 specific binding agent that binds to
the
wild-type human IL- 19 but fails to bind to a mutant IL- 19 is provided. In
certain
embodiments, the mutant IL- 19 comprises one or more of the following changes
relative
to wild-type human IL- 19: a) the amino acid at position 36 of the mutant IL-
19 is alanine;
b) the amino acid at position 37 of the mutant IL- 19 is alanine; c) the amino
acid at
position 39 of the mutant IL- 19 is alanine; d) the amino acid at position 40
of the mutant
IL-19 is alanine; e) the amino acid at position 41 of the mutant IL-19 is
alanine; f) the
amino acid at position 42 of the mutant IL- 19 is alanine; g) the amino acid
at position 44
of the mutant IL-19 is alanine; h) the amino acid at position 46 of the mutant
IL-19 is
alanine; i) the amino acid at position 51 of the mutant IL-19 is alanine; j)
the amino acid
at position 52 of the mutant IL-19 is alanine; k) the amino acid at position
55 of the
mutant IL-19 is alanine; 1) the amino acid at position 56 of the mutant IL-19
is alanine m)
the amino acid at position 57 of the mutant IL-19 is alanine; n) the amino
acid at position
58 of the mutant IL-19 is alanine; o) the amino acid at position 68 of the
mutant IL-19 is
alanine; p) the amino acid at position 74 of the mutant IL- 19 is alanine; q)
the amino acid
at position 102 of the mutant IL-19 is alanine; r) the amino acid at position
103 of the
mutant IL- 19 is alanine; s) the amino acid at position 106 of the mutant IL-
19 is alanine;
t) the amino acid at position 110 of the mutant IL- 19 is alanine; u) the
amino acid at
position 111 of the mutant IL-19 is alanine; v) the amino acid at position 114
of the
mutant IL- 19 is alanine; w) the amino acid at position 152 of the mutant IL-
19 is alanine;
x) the amino acid at position 154 of the mutant IL- 19 is alanine; y) the
amino acid at
position 155 of the mutant IL-19 is alanine; z) the amino acid at position 158
of the
mutant IL- 19 is alanine; aa) the amino acid at position 162 of the mutant IL-
19 is alanine;
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or bb) the amino acid at position 165 of the mutant IL- 19 is alanine. In
certain
embodiments, the IL- 19 specific binding agent is an antibody.
[021] In certain embodiments, a method of selecting a specific binding agent
to
an IL-19 polypeptide is provided. In certain embodiments, the specific binding
agent
binds to at least a portion of an epitope on an IL- 19 polypeptide. In certain
embodiments,
an IL- 19 polypeptide is contacted with an agent. In certain embodiments,
determining
the affinity of the agent for the IL- 19 polypeptide is determined. In certain
embodiments,
a mutant IL- 19 polypeptide is contacted with the agent. In certain
embodiments, the
mutant IL- 19 polypeptide comprises at least one point mutation at at least
one amino acid
position selected from H36,137, E39, S40, F41, Q42,144, R46, K51, D52, P55,
N56,
V57, T58, 168, V74, R102, K103, 5106, SI10, F111, M114, A152, 1541, 155K,
G158,
V162, or A165. In certain embodiments, the affinity of the agent for the
mutant IL-19
polypeptide is determined. In certain embodiments, the agent is selected if
the affinity
for the IL- 19 polypeptide is greater than the affinity for the mutant IL- 19
polypeptide. In
certain embodiments, the mutant IL- 19 polypeptide is the same as the IL- 19
polypeptide
except for the at least one mutation. In certain embodiments, the agent is
selected from a
protein, peptide, nucleic acid, carbohydrate, lipid, and small molecule
compound. In
certain embodiments, the agent is an antibody. In certain embodiments, the
agent is a
small molecule compound. In certain embodiments, the amount of binding to the
IL- 19
polypeptide is at least 2-fold greater than the amount of binding to the
mutant IL- 19
polypeptide. In certain embodiments, the affinity for the IL-19 polypeptide is
at least 5-
fold greater than the affinity for the mutant IL- 19 polypeptide. In certain
embodiments,
the affinity for the IL- 19 polypeptide is at least 10-fold greater than the
affinity for the
mutant IL- 19 polypeptide.
[022] In certain embodiments, a pharmaceutical composition comprising an
IL- 19 specific binding agent is provided. In certain embodiments, a method of
treating or
preventing an IL-19-associated disorder, in a subject is provided, comprising,
administering to the subject an IL- 19 specific binding agent, in an amount
sufficient to
treat or prevent the IL- 19-associated disorder.
[023] In certain embodiments, an IL-20 specific binding agent that binds to
the
wild-type human IL-20 but fails to bind to a mutant IL-20 is provided. In
certain
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embodiments, the mutant IL-20 comprises one or more of the following changes
relative
to wild-type human IL-20: a) the amino acid at position 41 of the mutant IL-20
is alanine;
b) the amino acid at position 42 of the mutant IL-20 is alanine; c) the amino
acid at
position 44 of the mutant IL-20 is alanine; d) the amino acid at position 45
of the mutant
IL-20 is alanine; e) the amino acid at position 46 of the mutant IL-20 is
alanine; f) the
amino acid at position 47 of the mutant IL-20 is alanine; g) the amino acid at
position 49
of the mutant IL-20 is alanine; h) the amino acid at position 51 of the mutant
IL-20 is
alanine; i) the amino acid at position 56 of the mutant IL-20 is alanine; j)
the amino acid
at position 57 of the mutant IL-20 is alanine; k) the amino acid at position
60 of the
mutant IL-20 is alanine; 1) the amino acid at position 61 of the mutant IL-20
is alanine m)
the amino acid at position 62 of the mutant IL-20 is alanine; n) the amino
acid at position
63 of the mutant IL-20 is alanine; o) the amino acid at position 73 of the
mutant IL-20 is
alanine; p) the amino acid at position 79 of the mutant IL-20 is alanine; q)
the amino acid
at position 107 of the mutant IL-20 is alanine; r) the amino acid at position
108 of the
mutant IL-20 is alanine; s) the amino acid at position 111 of the mutant IL-20
is alanine;
t) the amino acid at position 115 of the mutant IL-20 is alanine; u) the amino
acid at
position 116 of the mutant IL-20 is alanine; v) the amino acid at position 119
of the
mutant IL-20 is alanine; w) the amino acid at position 157 of the mutant IL-20
is alanine;
x) the amino acid at position 159 of the mutant IL-20 is alanine; y) the amino
acid at
position 160 of the mutant IL-20 is alanine; z) the amino acid at position 163
of the
mutant IL-20 is alanine; aa) the amino acid at position 170 of the mutant IL-
20 is alanine;
or bb) the amino acid at position 173 of the mutant IL-20 is alanine. In
certain
embodiments, the IL-20 specific binding agent is an antibody.
[024] In certain embodiments, a method of selecting a specific binding agent
to
an IL-20 polypeptide is provided. In certain embodiments, the specific binding
agent
binds to at least a portion of an epitope on an IL-20 polypeptide. In certain
embodiments,
an IL-20 polypeptide is contacted with an agent. In certain embodiments,
determining
the affinity of the agent for the IL-20 polypeptide is determined. In certain
embodiments,
a mutant IL-20 polypeptide is contacted with the agent. In certain
embodiments, the
mutant IL-20 polypeptide comprises at least one point mutation at at least one
amino acid
position selected from E41,142, N44, G45, F46, S47,149, G51, D57,160, D61,162,
R63,
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164, L65, D73, R79, R107, K108, 5111, S115, F116,1119, A157, V159, K160, G163,
1170, or Q173. In certain embodiments, the affinity of the agent for the
mutant IL-20
polypeptide is determined. In certain embodiments, the agent is selected if
the affinity
for the IL-20 polypeptide is greater than the affinity for the mutant IL-20
polypeptide. In
certain embodiments, the mutant IL-20 polypeptide is the same as the IL-20
polypeptide
except for the at least one mutation. In certain embodiments, the agent is
selected from a
protein, peptide, nucleic acid, carbohydrate, lipid, and small molecule
compound. In
certain embodiments, the agent is an antibody. In certain embodiments, the
agent is a
small molecule compound. In certain embodiments, the amount of binding to the
IL-20
polypeptide is at least 2-fold greater than the amount of binding to the
mutant IL-20
polypeptide. In certain embodiments, the affinity for the IL-20 polypeptide is
at least 5-
fold greater than the affinity for the mutant IL-20 polypeptide. In certain
embodiments,
the affinity for the IL-20 polypeptide is at least 10-fold greater than the
affinity for the
mutant IL-20 polypeptide.
[025] In certain embodiments, a pharmaceutical composition comprising an
IL-20 specific binding agent is provided. In certain embodiments, a method of
treating or
preventing an IL-20-associated disorder, in a subject is provided, comprising,
administering to the subject an IL-20 specific binding agent, in an amount
sufficient to
treat or prevent the IL-20-associated disorder.
[026] In certain embodiments, an IL-24 specific binding agent that binds to
the
wild-type human IL-24 but fails to bind to a mutant IL-24 is provided. In
certain
embodiments, the mutant IL-24 comprises one or more of the following changes
relative
to wild-type human IL-24: a) the amino acid at position 68 of the mutant IL-24
is alanine;
b) the amino acid at position 69 of the mutant IL-24 is alanine; c) the amino
acid at
position 71 of the mutant IL-24 is alanine; d) the amino acid at position 72
of the mutant
IL-24 is alanine; e) the amino acid at position 73 of the mutant IL-24 is
alanine; f) the
amino acid at position 74 of the mutant IL-24 is alanine; g) the amino acid at
position 76
of the mutant IL-24 is alanine; h) the amino acid at position 78 of the mutant
IL-24 is
alanine; i) the amino acid at position 83 of the mutant IL-24 is alanine; j)
the amino acid
at position 84 of the mutant IL-24 is alanine; k) the amino acid at position
87 of the
mutant IL-24 is alanine; 1) the amino acid at position 88 of the mutant IL-24
is alanine;
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m) the amino acid at position 89 of the mutant IL-24 is alanine; n) the amino
acid at
position 90 of the mutant IL-24 is alanine; o) the amino acid at position 100
of the mutant
IL-24 is alanine; p) the amino acid at position 105 of the mutant IL-24 is
alanine; q) the
amino acid at position 13 5 of the mutant IL-24 is alanine; r) the amino acid
at position
136 of the mutant IL-24 is alanine; s) the amino acid at position 139 of the
mutant IL-24
is alanine; t) the amino acid at position 143 of the mutant IL-24 is alanine;
u) the amino
acid at position 144 of the mutant IL-24 is alanine; v) the amino acid at
position 147 of
the mutant IL-24 is alanine; w) the amino acid at position 185 of the mutant
IL-24 is
alanine; x) the amino acid at position 187 of the mutant IL-24 is alanine; y)
the amino
acid at position 188 of the mutant IL-24 is alanine; z) the amino acid at
position 191 of
the mutant IL-24 is alanine; aa) the amino acid at position 195 of the mutant
IL-24 is
alanine; or bb) the amino acid at position 198 of the mutant IL-24 is alanine.
In certain
embodiments, the IL-24 specific binding agent is an antibody.
[027] In certain embodiments, a method of selecting a specific binding agent
to
an IL-24 polypeptide is provided. In certain embodiments, the specific binding
agent
binds to at least a portion of an epitope on an IL-24 polypeptide. In certain
embodiments,
an IL-24 polypeptide is contacted with an agent. In certain embodiments,
determining
the affinity of the agent for the IL-24 polypeptide is determined. In certain
embodiments,
a mutant IL-24 polypeptide is contacted with the agent. In certain
embodiments, the
mutant IL-24 polypeptide comprises at least one point mutation at at least one
amino acid
position selected from K68, L69, E71, A72, F73, W74, V76, D78, Q83, D84, T87,
S88,
A89, R90, V100, S105, K135, S136, T139, N143, F144,1147, A185, T187, K188,
G191,
1195, or T198. In certain embodiments, the affinity of the agent for the
mutant IL-24
polypeptide is determined. In certain embodiments, the agent is selected if
the affinity
for the IL-24 polypeptide is greater than the affinity for the mutant IL-24
polypeptide. In
certain embodiments, the mutant IL-24 polypeptide is the same as the IL-24
polypeptide
except for the at least one mutation. In certain embodiments, the agent is
selected from a
protein, peptide, nucleic acid, carbohydrate, lipid, and small molecule
compound. In
certain embodiments, the agent is an antibody. In certain embodiments, the
agent is a
small molecule compound. In certain embodiments, the amount of binding to the
IL-24
polypeptide is at least 2-fold greater than the amount of binding to the
mutant IL-24
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polypeptide. In certain embodiments, the affinity for the IL-24 polypeptide is
at least 5-
fold greater than the affinity for the mutant IL-24 polypeptide. In certain
embodiments,
the affinity for the IL-24 polypeptide is at least 10-fold greater than the
affinity for the
mutant IL-24 polypeptide.
[028] In certain embodiments, a pharmaceutical composition comprising an
IL-24 specific binding agent is provided. In certain embodiments, a method of
treating or
preventing an IL-24-associated disorder, in a subject is provided, comprising,
administering to the subject an IL-24 specific binding agent, in an amount
sufficient to
treat or prevent the IL-24-associated disorder.
[029] In certain embodiments, an IL-26 specific binding agent that binds to
the
wild-type human IL-26 but fails to bind to a mutant IL-26 is provided. In
certain
embodiments, the mutant IL-26 comprises one or more of the following changes
relative
to wild-type human IL-26: a) the amino acid at position 40 of the mutant IL-26
is alanine;
b) the amino acid at position 41 of the mutant IL-26 is alanine; c) the amino
acid at
position 43 of the mutant IL-26 is alanine; d) the amino acid at position 44
of the mutant
IL-26 is alanine; e) the amino acid at position 45 of the mutant IL-26 is
alanine; f) the
amino acid at position 46 of the mutant IL-26 is alanine; g) the amino acid at
position 48
of the mutant IL-26 is alanine; h) the amino acid at position 50 of the mutant
IL-26 is
alanine; i) the amino acid at position 55 of the mutant IL-26 is alanine; j)
the amino acid
at position 59 of the mutant IL-26 is alanine; k) the amino acid at position
61 of the
mutant IL-26 is alanine; 1) the amino acid at position 62 of the mutant IL-26
is alanine;
m) the amino acid at position 63 of the mutant IL-26 is alanine; n) the amino
acid at
position 64 of the mutant IL-26 is alanine; o) the amino acid at position 75
of the mutant
IL-26 is alanine; p) the amino acid at position 78 of the mutant IL-26 is
alanine; q) the
amino acid at position 106 of the mutant IL-26 is alanine; r) the amino acid
at position
107 of the mutant IL-26 is alanine; s) the amino acid at position 110 of the
mutant IL-26
is alanine; t) the amino acid at position 114 of the mutant IL-26 is alanine;
u) the amino
acid at position 115 of the mutant IL-26 is alanine; v) the amino acid at
position 118 of
the mutant IL-26 is alanine; w) the amino acid at position 148 of the mutant
IL-26 is
alanine; x) the amino acid at position 150 of the mutant IL-26 is alanine; y)
the amino
acid at position 151 of the mutant IL-26 is alanine; z) the amino acid at
position 154 of
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the mutant IL-26 is alanine; aa) the amino acid at position 158 of the mutant
IL-26 is
alanine; or bb) the amino acid at position 161 of the mutant IL-26 is alanine.
In certain
embodiments, the IL-26 specific binding agent is an antibody.
[030] In certain embodiments, a method of selecting a specific binding agent
to
an IL-26 polypeptide is provided. In certain embodiments, the specific binding
agent
binds to at least a portion of an epitope on an IL-26 polypeptide. In certain
embodiments,
an IL-26 polypeptide is contacted with an agent. In certain embodiments,
determining
the affinity of the agent for the IL-26 polypeptide is determined. In certain
embodiments,
a mutant IL-26 polypeptide is contacted with the agent. In certain
embodiments, the
mutant IL-26 polypeptide comprises at least one point mutation at at least one
amino acid
position selected from Q40, A41, D43, A44, L45, Y46, K48, A50, T55, D59,161,
K62,
N63,164, F75, N78, R106, F107, D110, L114, R115, L118, G148, Y150, K151, S154,
I158, or 5161. In certain embodiments, the affinity of the agent for the
mutant IL-26
polypeptide is determined. In certain embodiments, the agent is selected if
the affinity
for the IL-26 polypeptide is greater than the affinity for the mutant IL-26
polypeptide. In
certain embodiments, the mutant IL-26 polypeptide is the same as the IL-26
polypeptide
except for the at least one mutation. In certain embodiments, the agent is
selected from a
protein, peptide, nucleic acid, carbohydrate, lipid, and small molecule
compound. In
certain embodiments, the agent is an antibody. In certain embodiments, the
agent is a
small molecule compound. In certain embodiments, the amount of binding to the
IL-26
polypeptide is at least 2-fold greater than the amount of binding to the
mutant IL-26
polypeptide. In certain embodiments, the affinity for the IL-26 polypeptide is
at least 5-
fold greater than the affinity for the mutant IL-26 polypeptide. In certain
embodiments,
the affinity for the IL-26 polypeptide is at least 10-fold greater than the
affinity for the
mutant IL-26 polypeptide.
[031] In certain embodiments, a pharmaceutical composition comprising an
IL-26 specific binding agent is provided. In certain embodiments, a method of
treating or
preventing an IL-26-associated disorder, in a subject is provided, comprising,
administering to the subject an IL-26 specific binding agent, in an amount
sufficient to
treat or prevent the IL-26-associated disorder.
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[032] In certain embodiments, a method of systematic mutagenesis of a target
protein is provided. In certain embodiments, a series of ten or more different
nucleic acid
molecules is generated. In certain embodiments, each different nucleic acid
molecule
comprises a nucleic acid sequence that encodes a polypeptide comprising a
different
mutant of the target protein. In certain embodiments, each polypeptide
comprising a
different mutant further comprises a secretory sequence; a first tag, and a
second tag. In
certain embodiments, each of the ten or more different nucleic acid molecules
is
introduced into a different group of cells, wherein each different group of
cells is in a
separate well comprising liquid media. In certain embodiments, ten or more
different
mutants of the target protein are expressed in the separate wells; wherein the
ten or more
different mutants of the target protein are secreted into the liquid media of
the separate
wells. In certain embodiments, the ten or more different mutants of the target
protein are
quantitated in the liquid media in the separate wells using the first tag and
the second tag.
In certain embodiments, the ten or more different mutants of the target
protein are
subjected to at least one assay. In certain embodiments, the cells are
eukaryotic cells. In
certain embodiments, the introducing each of the ten or more different nucleic
acid
molecules into a different group of cells comprises transfecting each of the
ten or more
different nucleic acid molecules into a different group of cells. In certain
embodiments,
the cells are bacterial cells. In certain embodiments, each amino acid of the
target protein
is mutated in a different mutant target protein. In certain embodiments, each
amino acid
of the target protein that is not alanine is mutated to alanine in a different
mutant target
protein. In certain embodiments, the first tag is a six histidine tag and the
second tag is a
FLAG tag.
[033] In certain embodiments, a peptidomimetic that mimics an epitope
comprising two or more of the following amino acids of IL-22: A34,152, T56,
K61, A66,
V83, R88, P113, F121, L122, L125, or M172, is provided. In certain
embodiments, the
peptidomimetic mimics an epitope further comprising one or more of the
following
amino acids of IL-22: Y51, N54, R55, Y114, or E117. In certain embodiments,
the
peptidomimetic mimics an epitope further comprising one or more of the
following
amino acids of IL-22: F57, L59, D67, T70, D71, V72, R73, G159,1161, K162,
G165, or
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L169. In certain embodiments, the peptidomimetic mimics an epitope further
comprising
one or more of he following amino acids of IL-22: D67, R73, or K162.
[034] In certain embodiments, a peptidomimetic that mimics an epitope
comprising two or more of the following amino acids of IL-22: F57, L59, D67,
V72,
G159,1161, K162, or L169, is provided. In certain embodiments, the
peptidomimetic
mimics an epitope further comprising one or more of the following amino acids
of IL-22:
T70, D71, R73, or G165. In certain embodiments, the peptidomimetic mimics an
epitope
further comprising one or more of he following amino acids of IL-22: A34,
Y51,152,
N54, R55, T56, K61, A66, V83, R88, P113, Y114, El 17, F121, L122, L125, or
M172.
In certain embodiments, the peptidomimetic mimics an epitope further
comprising one or
more of the following amino acids of IL-22: R73 or V83.
[035] In certain embodiments, a peptidomimetic that mimics an epitope
comprising two or more of the following amino acids of IL-22: D67, R73, V83,
or K162,
is provided. In certain embodiments, the peptidomimetic mimics an epitope
further
comprising one or more of the following amino acids of IL-22: A34, Y51,152,
N54, R55,
T56, K61, A66, R88, P113, Y114, El17, F121, L122, L125, or M172. In certain
embodiments, the peptidomimetic mimics an epitope further comprising one or
more of
the following amino acids of IL-22: F57, L59, T70, D71, V72, G159,1161, G165,
or
L169.
[036] In certain embodiments, a peptidomimetic that mimics an epitope
comprising two or more of the following amino acids of IL-19: H36, I37, E39,
S40, F41,
Q42, 144, R46, K51, D52, P55, N56, V57, T58, 168, V74, R102, K103, 5106, SI10,
F111, M114, A152, 1541, 155K, G158, V162, or A165, is provided.
[037] In certain embodiments, peptidomimetic that mimics an epitope
comprising two or more of the following amino acids of IL-20: E41,142, N44,
G45, F46,
S47,149, G51, D57,160, D61,162, R63,164, L65, D73, R79, R107, K108, 5111,
S115,
F116,1119, 157A, V159, K160, G163,1170, or Q173 is provided.
[038] In certain embodiments, a peptidomimetic that mimics an epitope
comprising two or more of the following amino acids of IL-24: K68, L69, E71,
A72, F73,
W74, V76, D78, Q83, D84, T87, S88, A89, R90, V100, S105, K135, S136, T139,
N143,
F144,1147, A185, T187, K188, G191,1195, or T198, is provided.
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[039] In certain embodiments, a peptidomimetic that mimics an epitope
comprising two or more of the following amino acids of IL-26: Q40, A41, D43,
A44,
L45, Y46, K48, A50, T55, D59,16 1, K62, N63,164, F75, N78, R106, F107, Dl 10,
L114,
R115, L118, G148, Y150, K151, 5154, 1158, or 5161, is provided.
[040] In certain embodiments, a method for increasing antagonistic activity of
an antagonist of IL-22 comprising the step of altering a structure of the
antagonist such
that the antagonist binds to one or more of the following amino acids of IL-
22: F57, L59,
D67, T70, D71, V72, R73, G159,1161, K162, G165, or L169, is provided. In
certain
embodiments, the step of altering the structure is performed by computer
modeling. In
certain embodiments, the structure is an amino acid sequence. In certain
embodiments,
the antagonist is an antibody. In certain embodiments, the structure is a
crystal structure.
In certain embodiments, the antagonist is a small molecule.
[041] In certain embodiments, a method for increasing antagonistic activity of
an antagonist of IL-22 comprising the step of altering a structure of the
antagonist such
that the antagonist binds to one or more of the following amino acids of IL-
22: A34,152,
T56, K61, A66, V83, R88, P113, F121, L122, L125, or M172, is provided. In
certain
embodiments, the step of altering the structure is performed by computer
modeling. In
certain embodiments, the structure is an amino acid sequence. In certain
embodiments,
the antagonist is an antibody. In certain embodiments, the structure is a
crystal structure.
In certain embodiments, the antagonist is a small molecule.
[042] In certain embodiments, a method for increasing antagonistic activity of
an antagonist of IL-22 comprising the step of altering a structure of the
antagonist such
that the antagonist binds to one or more of the following amino acids of IL-
22: IL-22:
D67, R73, V83, or K162, is provided. In certain embodiments, the step of
altering the
structure is performed by computer modeling. In certain embodiments, the
structure is an
amino acid sequence. In certain embodiments, the antagonist is an antibody. In
certain
embodiments, the structure is a crystal structure. In certain embodiments, the
antagonist
is a small molecule.
[043] In certain embodiments, a method for increasing antagonistic activity of
an antagonist of IL- 19 comprising the step of altering a structure of the
antagonist such
that the antagonist binds to one or more of the following amino acids of IL-
19: H3 6, 13 7,
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E39, S40, F41, Q42,144, R46, K51, D52, P55, N56, V57, T58,168, V74, R102,
K103,
S106, 5110, Fill, M114, A152, 1541, 155K, G158, V162, or A165, is provided. In
certain embodiments, the step of altering the structure is performed by
computer
modeling. In certain embodiments, the structure is an amino acid sequence. In
certain
embodiments, the antagonist is an antibody. In certain embodiments, the
structure is a
crystal structure. In certain embodiments, the antagonist is a small molecule.
[044] In certain embodiments, a method for increasing antagonistic activity of
an antagonist of IL-20 comprising the step of altering a structure of the
antagonist such
that the antagonist binds to one or more of the following amino acids of IL-
20: E41, 142,
N44, G45, F46, 547,149, G51, D57,160, D61, 162, R63,164, L65, D73, R79, R107,
K108, 5111, S115, F116,1119, 157A, V159, K160, G163,1170, or Q173, is
provided. In
certain embodiments, the step of altering the structure is performed by
computer
modeling. In certain embodiments, the structure is an amino acid sequence. In
certain
embodiments, the antagonist is an antibody. In certain embodiments, the
structure is a
crystal structure. In certain embodiments, the antagonist is a small molecule.
[045] In certain embodiments, a method for increasing antagonistic activity of
an antagonist of IL-24 comprising the step of altering a structure of the
antagonist such
that the antagonist binds to one or more of the following amino acids of IL-
24: K68, L69,
E71, A72, F73, W74, V76, D78, Q83, D84, T87, S88, A89, R90, V100, S105, K135,
S136, T139, N143, F144,1147, A185, T187, K188, G191, 1195, or T198, is
provided. In
certain embodiments, the step of altering the structure is performed by
computer
modeling. In certain embodiments, the structure is an amino acid sequence. In
certain
embodiments, the antagonist is an antibody. In certain embodiments, the
structure is a
crystal structure. In certain embodiments, the antagonist is a small molecule.
[046] In certain embodiments, a method for increasing antagonistic activity of
an antagonist of IL-26 comprising the step of altering a structure of the
antagonist such
that the antagonist binds to one or more of the following amino acids of IL-
26: Q40, A4 1,
D43, A44, L45, Y46, K48, A50, T55, D59,161, K62, N63, 164, F75, N78, R106,
F107,
D110, L114, R115, L118, G148, Y150, K151, S154,1158, or 5161, is provided. In
certain embodiments, the step of altering the structure is performed by
computer
modeling. In certain embodiments, the structure is an amino acid sequence. In
certain
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embodiments, the antagonist is an antibody. In certain embodiments, the
structure is a
crystal structure. In certain embodiments, the antagonist is a small molecule.
BRIEF DESCRIPTION OF THE DRAWINGS
[047] Figure 1 shows the results of systematic mutagenesis of human IL-22.
Systematic mutagenesis of human IL-22 revealed specific amino acids that are
involved
in the binding of IL-22 to IL-22R, IL-IOR2, and IL-22BP. Figure 1(a) shows the
mature
primary sequence of IL-22 with its numbering beginning at the N terminus of
the
secretory leader. The dashed lines indicate intramolecular disulfide bonds
while the stick
models indicate glycosylation of asparagines at 54, 68, and 97. Figure 1(b)
shows a
ribbon rendering of the IL-22 tertiary backbone with helices A-F annotated.
Amino acids
defined as being involved in the binding of IL-22 to IL-22R (F57, L59, D67,
T70, D71,
V72, R73, G159,1161, K162, G165, and L169)), IL-1OR2 (A34, Y51,152, N54, R55,
T56, K61, A66, V83, R88, P113, Y114, E117, F121, L122, L125, and M172), and IL-
22BP (D67, R73, V83, and K162) are highlighted.
[048] Figure 2 shows certain IL-22 point substitutions in helices A, D, and F
and loop AB that bind more weakly than wild-type IL-22 to IL-22BP, IL-22R,
and/or a
complex of IL22R/IL- I OR2. Purified H/F-IL-22 mutants were evaluated for
binding to
plates coated indirectly with (a) IL-22R-Fc, (b) IL-22R-Fc/IL- I OR2-Fc, or
(c) IL-22BP-
Fc using His-Probe-HRP to detect the HIS-tag at the N terminus of the
cytokine. Nine
IL-22 substitutions that exhibited weak binding to IL-22BP (D67A, R73A, and
K162A),
IL-22R (D67A, V72A, R73A, 1161A, K162A, and L169A), or IL-1OR2 (Y51A, R55A,
and E117A) in the systematic high-throughput assays were evaluated as were two
substitutions that had an adverse effect in all five binding assays (L100A and
C132A) and
two substitutions that had normal binding characteristics in all five assays
(S86A and
Q94A). The binding of wild-type IL-22, with no mutations, is shown as a dashed
line.
Data are representative of at least two experiments.
[049] Figure 3 shows IL-22 point substitutions in helices A, D, and F, and
loop
AB that reduce the ability of IL-22 to induce the proliferation of cells. IL-
22-dependent
proliferation of BaF3 cells expressing both receptor subunits was evaluated
after 72 hours
by 3H-thymidine incorporation. Nine IL-22 substitutions were evaluated that
exhibited
relatively poor binding to IL-22R (D67A, V72A, R73A, 1161A, K162A, and L169A)
or
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IL-22R/IL- I OR2 (Y51A, R55A, and E117A) in the ELISA-based receptor binding
assays. In addition, two IL-22 mutants were evaluated that had an adverse
effect in all
five assays (L 100A and C 132A) as well as two IL-22 mutants that had normal
binding
characteristics in all five assays (S86A and Q94A). The binding of IL-22 with
no
mutations, is shown as a dashed line. Data are representative of at least two
experiments.
[050] Figure 4 shows that the IL-22 amino acids that are involved in binding
to
IL-22R, IL-IOR2, and IL-22BP contribute to adjacent and overlapping binding
sites on
the surface of IL-22. Figures (a)-(d) are sequential 90 rotations to the
right, around the
vertical axis, of a CPK rendering of IL-22. Shown are amino acid side chains
that were
defined as involved in binding to IL-22R (F57, D67, T70, D71, V72, R73,1161,
K162,
and L169), IL-1OR2 (Y51,152, N54, R55, K61, A66, V83, R88, P113, Y114, E117,
F121, L125, and M172), and IL-22BP-Fc (D67, R73, V83, and K162). Figures (e)-
(h)
are comparable sequential rotations to those in (a)-(d), respectively. The
shading of the
IL-22 CPK renderings in (e)-(h) delineates the various helices and loops in
the IL-22
structure. Renderings (a) and (e) can be compared, as can the other horizontal
pairs of
renderings, to visualize the contribution of a given amino acid (e.g., F57 in
(a) from a
given secondary structure (e.g., helix A in (e) to a given binding site (e.g.,
IL-22R in (a)).
[051] Figure 5 shows a structure-based alignment of IL-22 and IL- 10
sequences and receptor binding sites. The IL-22 sequence in Figure 5
corresponds to
amino acids 44-179 of SEQ ID NO:2. The IL-10 sequence in Figure 5 corresponds
to
SEQ ID NO:9. This alignment of IL-22 and IL-10 sequences was derived from the
superimposition of IL- 10, IL- 19, and IL-22 monomeric structures. The first
line of
sequence is IL-22 with amino acids highlighted that are involved in binding to
IL-22R
(F57, L59, D67, T70, D71, V72, R73, G159,1161, K162, G165, and L169), IL-1OR2
(Y51,152, N54, R55, T56, K61, A66, V83, R88, P113, Y114, E117, F121, L122,
L125,
and M172), and IL-22BP (D67, R73, V83, and K162). The second line of sequence
is
IL- 10 with the short bars underneath corresponding to certain amino acids
that have been
previously demonstrated to be important for binding to IL-IOR1 (upper bars)
and IL-
IOR2 (lower bars) (Josephson et al. Immunity 15, 35-46 (2001) and Yoon et al.
Journal of
Biological Chemistry 281, 35088-96 (2006)). The cylinders show the positions
of the IL-
22 and IL- 10 helices in relation to their respective above sequences. The
dashed bars
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below the helices indicate the regions that contribute to IL-22's IL-22R
binding site. , IL-
22's IL-10R2 binding site, IL- IO's IL-IOR1 binding site, and IL-22's IL-10R2
binding
site.
[052] Figure 6 shows side-chain atoms within helix A, loop AB and helix F of
IL-22 and IL- 10 that define the cytokine binding interfaces for the
respective high
affinity receptor subunits. Figure 6(a) shows a solvent accessible surface
(1.4 angstrom
probe radius) rendering of a portion of IL-22's tertiary structure. The
residues that
contribute to the IL-22R binding site are F57, L59, D67, T70, D71, V72, R73,
G159,
1161, K162, G165, and L169. K161's solvent accessible surface cannot be seen
from this
perspective; residues L59 and G159 are completely buried. Residues K61, S64,
N68,
E166, and D168 were previously proposed, based on modeling, to participate in
recognition of IL-22R (Logsdon et al. Journal of Interferon & Cytokine
Research 22,
1099-112 (2002)); however, point substitution to alanine had no impact on
binding to IL-
22R in our study. Figure 6(b) shows the same orientation as in (a) of helices
A, and F
and loop AB of IL-22 superimposed with the corresponding IL- 10 side chains,
as aligned
in Figure 5. The light ribbon represents the IL-22 backbone with the annotated
ball and
stick models representing side chains that are involved in binding to IL-22R
and includes
those that are also involved in binding to IL-22BP (corresponding to F57, L59,
D67, T70,
D71, V72, R73, G159,1161, K162, G165, and L169 residues in Figures 1 and 5).
The
orientation of the helices is comparable to those shown in Figures 1(b), 4(a)
and (e), and
8. G159 of IL-22 cannot be seen well from this perspective since it is
immediately
behind K162. The darker ribbon represents the superimposed IL-10 backbone with
the
remaining annotated models indicating side chains that are involved in IL-IORI
binding
(R42, R45, Q56, Q60, D62, K156, S159, E160, D162, and E169), based on the
analysis
of structure from IL-10/IL-10R1ECD co-crystals (Josephson et al. Immunity 15,
35-46
(2001) and Yoon et al. Journal of Biological Chemistry 281, 35088-96 (2006)).
The
alignment, numbering and annotation are as shown in Figure 5. Figure 5(c)
shows a
solvent accessible surface (1.4 angstrom probe radius) rendering of IL-22,
with residues
D67, R73, V83, and K162 that contribute to the IL-22BP binding site.
[053] Figure 7 shows side-chain atoms mostly within helix A and D of IL-22
and IL-10 that define the low affinity receptor binding sites for IL-IOR2.
Figure 7(a)
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shows a solvent accessible surface (1.4 angstrom probe radius) rendering of a
portion of
IL-22's structure. The residues that contribute to the IL-22/IL- I OR2 binding
interface are
Y51,152, N54, R55, T56, L59, K61, A66, R88, P113, Y114, E117, F121, L122,
L125,
G159, and M172. T56 and L122 have very low solvent accessible surface and
cannot be
seen from this perspective. Residues L59 and G159 are completely buried.
Figure 7(b)
shows the light ribbon that represents the IL-22 backbone for helix A and D
secondary
structure with the annotated ball and stick models indicating side chains that
are involved
in binding to IL-1OR2 in the presence of IL-22R (Y51,152, N54, R55, T56, L59,
K61,
A66, R88, P113, Y114, E117, F121, L122, L125, G159, and M172). The orientation
of
the helices is similar to those shown in Figures 4(d) and (h). The dark ribbon
represents
the superimposed IL- 10 backbone with the remaining annotated models
indicating side
chains that are proposed to contribute to IL-10R2 binding (N39, M40, R42, S49,
R50,
H108, and 5111) (Yoon et al. Journal of Biological Chemistry 281, 35088-96
(2006)).
The alignment, numbering, and annotation are the same as in Figure 5.
[054] Figure 8 shows putative receptor binding sites for IL- 19, IL-20, IL-24,
and IL-26 based on the elucidated IL-22 receptor binding sites and the
proposed
conservation of the structure-function relationship between the IL-10-like
cytokines. The
ribbon representations of IL- 19 and IL-22 backbones are from crystal
structures, while
those for IL-20, IL-24, and IL-26 are from models generated as described in
the
Examples. The IL-22 amino acid side chains that were defined as involved in
binding to
IL-22R (F57, L59, T70, D71, V72, G159,1161, G165, and L169), IL-1OR2 (A34,
Y51,
152, N54, R55, T56, K61, A66, R88, P113, Y114, E117, F121, L122, L125, and
M172),
and IL-22BP (D67, R73, V83, and K162) are highlighted as ball and stick
models, and
correspond to the same highlighted residues as in Figures 1 and 4(a)-(d).
Based on a
structural alignment of the IL-10-like cytokines, shown at the primary
sequence level in
Figure 10, the above IL-22 binding sites were transposed to the equivalent
positions in
the IL-19, IL-20, IL-24, and IL-26 sequences and to the tertiary structures
shown here.
These transpositions may be predictive of the cognate high affinity and low
affinity
receptor subunit binding sites for these cytokines.
[055] Figure 9 shows certain IL-22 point mutants that do not bind as well as
control cytokine to IL-22R, IL-22R/IL- I OR2 and IL-22BP. A systematic
collection of
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146 IL-22 point mutants were expressed in mammalian cells from linear
expression
cassettes and evaluated, relative to silent substitution mutants, at a fixed
and limiting
concentration for binding to receptors. Figure 9(a) shows data for the twenty-
nine IL-22
substitutions that bound weakly (shown in solid symbols) relative to control
IL-22 (silent
substitutions; shown in open symbols) in the high-throughput receptor binding
assays.
Data were normalized to binding of purified IL-22 with no mutations, the value
of this
sample was set to `1' in each assay. Figure 9(b) shows a solvent accessible
surface (1.4
angstrom probe radius) rendering of IL-22 structure with those amino acids
highlighted
that had statistically weaker than normal binding in the binding assays. Most
of these 30
residues have low solvent accessibility and are proposed to be required for
maintaining
IL-22 secondary or tertiary structure.
[056] Figure 10 shows a structure-based alignment of IL-10-like cytokines.
The first line of sequence is IL-22 with amino acids highlighted that are
involved in
binding to IL-22R (F57, L59, D67, T70, D71, V72, R73, G159, I161,K162, G165,
and
L169), IL-10R2 (Y51,152, N54, R55, T56, K61, A66, V83, R88, P113, Y114, E117,
F121, L122, L125, and M172), and IL-22BP (D67, R73, V83, and K162). The IL-22
sequence repeatd in the first and second lines in Figure 10 corresponds to
amino acids 44-
179 of SEQ ID NO:2. The IL-10-like cytokine primary sequence alignment is
derived
from the structural superimposition and modeling described in the Examples.
The IL- 10
sequence in Figure 10 corresponds to SEQ ID NO:9. The IL-19 sequence in Figure
10
corresponds to amino acid residues 31 to 177 in SEQ ID NO:5. The IL-20
sequence in
Figure 10 corresponds to amino acid residues 36 to 176 in SEQ ID NO:6. The IL-
24
sequence in Figure 10 corresponds to amino acid residues 63 to 206 in SEQ ID
NO:7.
The IL-26 sequence in Figure 10 corresponds to amino acid residues 36 to 171
in SEQ ID
NO: 8. Shared physicochemical properties of amino acid groups are indicated in
the
visual legend. The short bars underneath the IL- 10 sequence correspond to
certain amino
acids that have been previously demonstrated to be important for binding to IL-
1 OR1
(upper bars) and IL-10R2 (lower bars) (Josephson et al., Immunity 15, 35-46
(2001) and
Yoon et al. Journal of Biological Chemistry 281, 35088-96 (2006)). The
cylinders show
the positions of the IL-22, IL- 10 and IL- 19 helices in relation to the above
sequence, with
dashed bars below indicating the regions that contribute to IL-22's IL-22R
binding site
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and IL-22's IL-10R2 binding site and in relation to IL-10's IL-IOR1 binding
site and IL-
10's IL-10R2 binding site.
[057] Figure 11 shows IL-22 point substitutions that impact binding to at
least
one of IL-22BP, IL-22R, and IL-10R2 as described in the Examples.
[058] Figure 12 shows the effect of IL-22 point substitutions on binding
assays
as described in the Examples.
[059] Figure 13 shows the solvent accessibility of certain IL-22 amino acids
as
described in the Examples.
[060] Figure 14 shows the solvent accessibility of certain IL-22 amino acids
identified as being involved in binding at least one of IL-22BP, IL-22R, and
IL- I OR2 as
described in the Examples.
[061] Figure 15 shows the human IL-22 amino acid and nucleotide sequences.
[062] Figure 16 shows the mouse IL-22 amino acid and nucleotide sequences.
[063] Figure 17(a) shows the human IL-19 amino acid sequence. Figure 17(b)
shows the human IL-20 amino acid sequence.
[064] Figure 18(a) shows the human IL-24 amino acid sequence. Figure 18(b)
shows the human IL-26 amino acid sequence.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[065] Unless otherwise defined, all technical and scientific terms used herein
have the same meaning as commonly understood by one of ordinary skill in the
art.
Although methods and materials similar or equivalent to those described herein
can be
used in the practice or testing of the claims, suitable methods and materials
are described
below. All publications, patent applications, patents, and other references
mentioned
herein are incorporated by reference in their entirety. In addition, the
materials, methods,
and examples are illustrative only and not intended to be limiting.
[066] In order that the present invention may be more readily understood,
certain terms are first defined. Additional definitions are set forth
throughout the detailed
description. Unless specific definitions are provided, the nomenclatures
utilized in
connection with, and the laboratory procedures and techniques of, analytical
chemistry,
synthetic organic chemistry, and medicinal and pharmaceutical chemistry
described
herein are those well known and commonly used in the art. Standard techniques
may be
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used for chemical syntheses, chemical analyses, pharmaceutical preparation,
formulation,
delivery, and treatment of patients.
[067] In this application, the use of the singular includes the plural unless
specifically stated otherwise. In this application, the use of "or" means
"and/or" unless
stated otherwise. In the context of a multiple dependent claim, the use of
"or" refers back
to more than one preceding independent or dependent claim in the alternative
only.
Furthermore, the use of the term "including", as well as other forms, such as
"includes"
and "included", is not limiting. Also, terms such as "element" or "component"
encompass both elements and components comprising one unit and elements and
components that comprise more than one subunit unless specifically stated
otherwise.
[068] Other features and advantages will be apparent from the following
detailed description and claims.
[069] The present application provides for, at least in part, antibodies and
antigen-binding fragments thereof that bind to IL-22, in particular, human IL-
22, with
high affinity and specificity. In certain embodiments, the anti-IL-22 antibody
or
fragment thereof reduces, inhibits or antagonizes at least one IL-22-
associated activity.
For example, the anti-IL- 22 antibody or fragment thereof can bind to IL-22,
e.g., an
epitope of IL-22, and interfere with an interaction, e.g., binding, between IL-
22 and an
IL-22 receptor complex, e.g., a complex comprising IL-22 receptor ("IL-22R")
and
interelukin-10 receptor 2 ("IL-IOR2"), or a subunit thereof (e.g., IL-22R or
IL-IOR2,
individually). Thus, in certain embodiments, the antibodies and fragments
thereof can be
used to interfere with (e.g., inhibit, block or otherwise reduce) an
interaction, e.g.,
binding, between IL-22 and an IL-22 receptor complex, or a subunit thereof. In
certain
embodiments, the anti-IL-22 antibodies or fragments thereof can be used to
diagnose,
treat or prevent IL-22-associated disorders, .e.g., autoimmune disorders,
e.g., arthritis
(including rheumatoid arthritis, juvenile rheumatoid arthritis,
osteoarthritis, psoriatic
arthritis, lupus-associated arthritis or ankylosing spondylitis), scleroderma,
systemic
lupus erythematosis, HIV, Sjogren's syndrome, vasculitis, multiple sclerosis,
autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous
dermatitis), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's
disease,
colitis, diabetes mellitus (type I); inflammatory conditions of, e.g., the
skin (e.g.,
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psoriasis), cardiovascular system (e.g., atherosclerosis), nervous system
(e.g.,
Alzheimer's disease), liver (e.g., hepatitis), kidney (e.g., nephritis) and
pancreas (e.g.,
pancreatitis); cardiovascular disorders, e.g., cholesterol metabolic
disorders, oxygen free
radical injury, ischemia; disorders associated with wound healing; respiratory
disorders,
e.g., asthma and COPD (e.g., cystic fibrosis); acute inflammatory conditions
(e.g.,
endotoxemia, sepsis and septicaemia, toxic shock syndrome and infectious
disease);
transplant rejection and allergy. In one embodiment, the IL-22-associated
disorder is, an
arthritic disorder, e.g., a disorder chosen from one or more of rheumatoid
arthritis,
juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, or
ankylosing spondylitis; a
respiratory disorder (e.g., asthma, chronic obstructive pulmonary disease
(COPD); or an
inflammatory condition of, e.g., the skin (e.g., psoriasis), cardiovascular
system (e.g.,
atherosclerosis), nervous system (e.g., Alzheimer's disease), liver (e.g.,
hepatitis), kidney
(e.g., nephritis), pancreas (e.g., pancreatitis), and gastrointestinal organs,
e.g., colitis,
Crohn's disease and IBD.
[070] The term "interleukin-22" or "IL-22" refers to a class II cytokine
(which
may be mammalian) capable of binding to IL-22R and/or a receptor complex of IL-
22R
and IL-IOR2, and has at least one of the following features: (1) an amino acid
sequence
of a naturally occurring mammalian IL-22 polypeptide (full length or mature
form) or a
fragment thereof, e.g., an amino acid sequence shown as SEQ ID NO:1 (human) or
SEQ
ID NO:3 (murine) or a fragment thereof; (2) an amino acid sequence
substantially
identical to, e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
or
99% identical to, an amino acid sequence shown as SEQ ID NO:1 or amino acids
34-179
thereof (human) or SEQ ID NO:3 (murine) or a fragment thereof; (3) an amino
acid
sequence which is encoded by a naturally occurring mammalian IL-22 nucleotide
sequence or a fragment thereof (e.g., SEQ ID NO:2 or nucleotides 71 to 610
(human) or
SEQ ID NO:4 (murine) or a fragment thereof); (4) an amino acid sequence
encoded by a
nucleotide sequence which is substantially identical to, e.g., at least 85%,
90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, a nucleotide sequence
shown as SEQ ID NO:2 or nucleotides 71 to 610 thereof (human) or SEQ ID NO:4
(murine) or a fragment thereof, (5) an amino acid sequence encoded by a
nucleotide
sequence degenerate to a naturally occurring IL-22 nucleotide sequence or a
fragment
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thereof, e.g., SEQ ID NO:2 (human) or SEQ ID NO:4 (murine) or a fragment
thereof; or
(6) a nucleotide sequence that hybridizes to one of the foregoing nucleotide
sequences
under stringent conditions, e.g., highly stringent conditions. The IL-22 may
bind to IL-
22R and/or a receptor complex of IL-22R and IL- I OR2 of mammalian origin,
e.g., human
or mouse.
[071] The nucleotide sequence and the predicted amino acid sequence of
human IL-22 are shown in SEQ ID NO:2 and SEQ ID NO:1, respectively. The amino
acid sequence of mature human IL-22 corresponds to amino acids 34-179 of SEQ
ID
NO:1. Analysis of recombinant human IL-22 reveals many structural domains.
(Nagem
et al. (2002) Structure, 10:1051-62; U.S. Patent Application No. US
2002/0187512 Al).
[072] The human IL-22 cDNA was deposited with the American Type Culture
Collection (10801 University Boulevard, Manassas, Virginia, U.S.A. 20110-2209)
on
April 28, 1999 as an original deposit under the Budapest Treaty and was given
the
accession number ATCC 207231.
[073] The phrase "an IL-22 activity" or "IL-22 associated activity" refers to
one or more of the biological activities of an IL-22 polypeptide, e.g., a
mature IL-22
polypeptide (e.g., a mammalian, e.g., human or murine IL-22 having an amino
acid
sequence as shown in SEQ ID NO:2 and 4, respectively), including, but not
limited to,
(1) interacting with, e.g., binding to, an IL-22 receptor (e.g., an IL-22R or
IL-1OR2 or a
complex thereof, preferably of mammalian, e.g., murine or human origin); (2)
associating
with one or more signal transduction molecules; (3) stimulating
phosphorylation and/or
activation of a protein kinase, e.g., JAK/STAT3, ERK, and MAPK; (4)
modulating, e.g.,
stimulating or decreasing, proliferation, differentiation, effector cell
function, cytolytic
activity, cytokine or chemokine secretion, and/or survival of an IL-22
responsive cell,
e.g., an epithelial cell from, e.g., kidney, liver, colon, small intestine,
thyroid gland,
pancreas, skin); (5) modulating at least one parameter of an acute phase
response, e.g., a
metabolic, hepatic, hematopoietic (e.g., anemia, platelet increase) or
neuroendocrine
change, or a change (e.g., increase or decrease in an acute phase protein,
e.g., an increase
in fibrinogen and/or serum amyloid A, or a decrease in albumin); and/or (6)
modulating
at least one parameter of an inflammatory state, e.g., modulating cytokine-
mediated
proinflammatory actions (e.g., fever, and/or prostaglandin synthesis, for
example PGE2
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synthesis), modulating cellular immune responses, modulating cytokine,
chemokine (e.g.,
GRO1), or lymphokine production and/or secretion (e.g., production and/or
secretion of a
proinflammatory cytokine).
[074] As used herein, a "therapeutically effective amount" of an antagonist,
e.g., an antibody or a fragment thereof, refers to an amount of an agent which
is effective,
upon single or multiple dose administration to a subject, e.g., a human
patient, at treating,
curing, reducing the severity of, or ameliorating one or more symptoms of a
disorder, or
in prolonging the survival of the subject beyond that expected in the absence
of such
treatment.
[075] As used herein, "a prophylactically effective amount" of an antagonist,
e.g., antibody, refers to an amount of an agent which is effective, upon
single- or
multiple-dose administration to a subject, e.g., a human patient, in
preventing or delaying
the occurrence of the onset or recurrence of a disorder, e.g., a disorder as
described
herein.
[076] The term "induce", "reduce," "inhibit," "potentiate," "elevate,"
"increase," "decrease" or the like, e.g., which denote quantitative
differences between
two states, refer to at least statistically significant differences between
the two states.
[077] As used herein, an "IL-22 antagonist" refers to an agent which reduces,
inhibits or otherwise diminishes one or biological activities of an IL-22
polypeptide, e.g.,
a human IL-22 polypeptide, or fragment thereof. Preferably, the antagonist
interacts
with, e.g., binds to, an IL-22 polypeptide. Antagonism using an IL-22
antagonist does
not necessarily indicate a total elimination of the IL-22-associated
biological activity. IL-
22 antagonists include without limitation antibodies directed to human IL-22
proteins;
soluble forms of the receptor or other target to which human IL-22 is
directed; antibodies
directed to the receptor or other target to which human IL-22 is directed; and
peptide and
small molecule compounds that inhibit or interfere with the interaction of
human IL-22
with its receptor or other target.
[078] As used herein, an "IL-22 agonist" refers to an agent which potentiates,
induces or otherwise enhances one or biological activities of an IL-22
polypeptide.
[079] The terms "specific binding" or "specifically binds" refers to two
molecules forming a complex that is relatively stable under physiologic
conditions.
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Specific binding is characterized by a high affinity and a low to moderate
capacity as
distinguished from nonspecific binding which usually has a low affinity with a
moderate
to high capacity. Typically, binding is considered specific when the
association constant
KA is higher than 106 M-'. If necessary, nonspecific binding can be reduced
without
substantially affecting specific binding by varying the binding conditions.
The
appropriate binding conditions, such as concentration of antibodies, ionic
strength of the
solution, temperature, time allowed for binding, concentration of a blocking
agent (e.g.,
serum albumin, milk casein), etc., may be optimized by a skilled artisan using
routine
techniques.
[080] The term "specific binding agent" refers to a natural or non-natural
molecule that specifically binds to a target. Examples of specific binding
agents include,
but are not limited to, proteins, peptides, nucleic acids, carbohydrates,
lipids, and small
molecule compounds. In certain embodiments, a specific binding agent is an
antibody.
In certain embodiments, a specific binding agent is an antigen binding region.
[081] The term "structure" encompasses both structures of biologics (for
example and not limitation, antibodies and fragments thereof) and small
molecules.
[082] The term "antibody" refers to an immunoglobulin or fragment thereof,
such as Fab, Fab', F(ab')2, Fc, Fd, Fd', Fv, single chain antibodies (scFv for
example),
single variable domain antibodies (Dab), diabodies (bivalent and bispecific),
and
chimeric (e.g., humanized) antibodies, which may be produced by the
modification of
whole antibodies or those synthesized de novo using recombinant DNA
technologies.
These functional antibody fragments retain the ability to selectively bind
with their
respective antigen or receptor. Antibodies and antibody fragments can be from
any class
of antibodies including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and
from any
subclass (e.g., IgGI, IgG2, IgG3, and IgG4) of antibodies. The antibodies of
the present
invention can be monoclonal or polyclonal. The antibody can also be a
monospecific,
polyspecific, non-specific, humanized, human, single-chain, chimeric,
synthetic,
recombinant, hybrid, mutated, CDR-grafted, and/or in vitro generated antibody.
The
antibody can have a heavy chain constant region chosen from, e.g., IgGI, IgG2,
IgG3, or
IgG4. The antibody can also have a light chain chosen from, e.g., kappa or
lambda.
Constant regions of the antibodies can be altered, e.g., mutated, to modify
the properties
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of the antibody (e.g., to increase or decrease one or more of. Fc receptor
binding,
antibody glycosylation, the number of cysteine residues, effector cell
function, or
complement function). Typically, the antibody specifically binds to a
predetermined
antigen, e.g., an antigen associated with a disorder, e.g., a
neurodegenerative, metabolic,
inflammatory, autoimmune and/or a malignant disorder. Unless preceded by the
word
"intact", the term "antibody" includes, in addition to complete antibody
molecules,
antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody
fragments
that retain antigen-binding function. Typically, such fragments comprise an
antigen-binding domain.
[083] The antibody can further include a heavy and light chain constant
region,
to thereby form a heavy and light immunoglobulin chain, respectively. In
certain
embodiments, the antibody is a tetramer of two heavy immunoglobulin chains and
two
light immunoglobulin chains, wherein the heavy and light immunoglobulin chains
are
inter-connected by, e.g., disulfide bonds. The heavy chain constant region is
comprised
of three domains, CH1, CH2 and CH3. The light chain constant region is
comprised of
one domain, CL. The variable region of the heavy and light chains contains a
binding
domain that interacts with an antigen. The constant regions of the antibodies
typically
mediate the binding of the antibody to host tissues or factors, including
various cells of
the immune system (e.g., effector cells) and the first component (Clq) of the
classical
complement system.
[084] The terms "antigen-binding domain" and "antigen-binding fragment"
refer to a part of an antibody molecule that comprises amino acids responsible
for the
specific binding between antibody and antigen. The part of the antigen that is
specifically recognized and bound by the antibody is referred to as the
"epitope." An
antigen-binding domain may comprise an antibody light chain variable region
(VL) and
an antibody heavy chain variable region (VH); however, it does not have to
comprise
both. Fd fragments, for example, have two VH regions and often retain some
antigen-
binding function of the intact antigen-binding domain. Examples of antigen-
binding
fragments of an antibody include (1) a Fab fragment, a monovalent fragment
having the
VL, VH, CL and CHI domains; (2) a F(ab')2 fragment, a bivalent fragment having
two Fab
fragments linked by a disulfide bridge at the hinge region; (3) a I'd fragment
having the
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two VH and CH1 domains; (4) a Fv fragment having the VL and VH domains of a
single
arm of an antibody, (5) a dAb fragment (Ward et al., (1989) Nature 341:544-
546), which
has a VH domain; (6) an isolated complementarity determining region (CDR); and
(7) a
single chain Fv (scFv). Although the two domains of the Fv fragment, VL and
VH, are
coded for by separate genes, they can be joined, using recombinant methods, by
a
synthetic linker that enables them to be made as a single protein chain in
which the VL
and VH regions pair to form monovalent molecules (known as single chain Fv
(scFv); see
e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.
Natl. Acad.
Sci. USA 85:5879-5883). These antibody fragments are obtained using
conventional
techniques known to those with skill in the art, and the fragments are
evaluated for
function in the same manner as are intact antibodies.
[085] As used herein, the term "immunoglobulin" refers to a protein consisting
of one or more polypeptides substantially encoded by immunoglobulin genes. The
recognized human immunoglobulin genes include the kappa, lambda, alpha (IgAl
and
IgA2), gamma (IgGi, IgG2, IgG3, IgG4), delta, epsilon and mu constant region
genes, as
well as the myriad immunoglobulin variable region genes. Full-length
immunoglobulin
"light chains" (about 25 Kd or 214 amino acids) are encoded by a variable
region gene at
the NH2-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, e.g., gamma
(encoding about
330 amino acids).
[086] As used herein, "isotype" refers to the antibody class (e.g., 1gM or
IgGI)
that is encoded by heavy chain constant region genes.
[087] The term "human antibody" includes antibodies having variable and
constant regions corresponding substantially to human germline immunoglobulin
sequences known in the art , including, for example, those described by Kabat
et al. (See
Kabat, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S.
Department of Health and Human Services, NIH Publication No. 91-3242). In
certain
embodiments, human antibodies may include amino acid residues not encoded by
human
germline immunoglobulin sequences (e.g., mutations introduced by random or
site-
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specific mutagenesis in vitro or by somatic mutation in vivo), for example in
the CDRs,
and in particular, CDR3. The human antibody can have at least one, two, three,
four,
five, or more positions replaced with an amino acid residue that is not
encoded by the
human germline immunoglobulin sequence.
[088] The phrase "inhibit" or "antagonize" IL-22 activity and its cognates
refer
to a reduction, inhibition, or otherwise diminution of at least one activity
of IL-22 due to
binding an anti-IL-22 antibody, wherein the reduction is relative to the
activity of IL-22
in the absence of the same antibody. The activity can be measured using any
technique
known in the art. Inhibition or antagonism does not necessarily indicate a
total
elimination of the IL-22 polypeptide biological activity. A reduction in
activity may be
about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.
[089] The term "IL-22 activity" or "IL-22-associated activity" refers to at
least
one cellular process initiated or interrupted as a result of IL-22 binding to
a receptor
complex consisting of IL-22R and IL-1OR2 on the cell. IL-22 activities include
at least
one of, but are not limited to: (1) binding IL-22R or a receptor complex of IL-
22R and
IL-1OR2 (e.g., human IL-22R with or without human IL-1OR2); (2) associating
with
signal transduction molecules (e.g., JAK-1); (3) stimulating phosphorylation
of STAT
proteins (e.g., STATS, STAT3, or combination thereof); (4) activating STAT
proteins;
and (5) modulating (e.g., increasing or decreasing) proliferation,
differentiation, effector
cell function, cytolytic activity, cytokine secretion, survival, or
combinations thereof, of
epithelial cells, fibroblasts, or immune cells. Epithelial cells include, but
are not limited
to, cells of the skin, gut, liver, and kidney, as well as endothelial cells.
Fibroblasts
include, but are not limited to, synovial fibroblasts. Immune cells may
include CD8+ and
CD4+ T cells, NK cells, B cells, macrophages, and megakaryocytes. IL-22
activity can
be determined in vitro, for example, using an IL-22 receptor inhibition assay,
a GROa
secretion assay, or a BAF3 proliferation assay. IL-22 activity can also be
determined in
vivo, for example, by scoring progression of an immune response or disorder.
[090] The term "isolated" refers to a molecule that is substantially free of
its
natural environment. For instance, an isolated protein is substantially free
of cellular
material or other proteins from the cell or tissue source from which it was
derived. The
term also refers to preparations where the isolated protein is sufficiently
pure for
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pharmaceutical compositions; or at least 70-80% (w/w) pure; or at least 80-90%
(w/w)
pure; or at least 90-95% pure; or at least 95%, 96%, 97%, 98%, 99%, or 100%
(w/w)
pure.
[091] The term "therapeutic agent" is a substance that treats or assists in
treating a medical disorder. Therapeutic agents may include, but are not
limited to,
substances that modulate immune cells or immune responses in a manner that
complements the IL-22 activity of anti-IL-22 antibodies. Non-limiting examples
and
uses of therapeutic agents are described herein.
[092] The term "treatment" refers to a therapeutic or preventative measure.
The treatment may be administered to a subject having a medical disorder or
who
ultimately may acquire the disorder, in order to prevent, cure, delay, reduce
the severity
of, and/or ameliorate one or more symptoms of a disorder or recurring
disorder, or in
order to prolong the survival of a subject beyond that expected in the absence
of such
treatment.
[093] The term "effective amount" refers to a dosage or amount that is
sufficient to regulate an activity to ameliorate clinical symptoms or achieve
a desired
biological outcome, e.g., decreased T cell and/or B cell activity, suppression
of
autoimmunity, suppression of transplant rejection, etc.
[094] The term "in combination" in the context of treatment with two agents
means that the agents are given substantially contemporaneously, either
simultaneously
or sequentially. If given sequentially, at the onset of administration of the
second
compound, the first of the two compounds is preferably still detectable at
effective
concentrations at the site of treatment.
[095] The phrase "percent identical" or "percent identity" refers to the
similarity between at least two different sequences. This percent identity can
be
determined by standard alignment algorithms, for example, the Basic Local
Alignment
Tool (BLAST) described by Altshul et al. ((1990) J. Mol. Biol., 215: 403-410);
the
algorithm of Needleman et al. ((1970) J. Mol. Biol., 48: 444-453); or the
algorithm of
Meyers et al. ((1988) Comput. Appl. Biosci., 4: 11-17). A set of parameters
may be the
Blosum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4,
and a
frameshift gap penalty of 5. The percent identity between two amino acid or
nucleotide
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sequences can also be determined using the algorithm of E. Meyers and W.
Miller
((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program
(version 2.0), using a PAM 120 weight residue table, a gap length penalty of
12 and a gap
penalty of 4. The percent identity is usually calculated by comparing
sequences of
similar length.
[096] In certain embodiments, sequences similar or homologous (e.g., at least
about 85% sequence identity) to the sequences disclosed are provided. In
certain
embodiments, the sequence identity can be about 90%, 91%, 92%, 93%, 94%, 95%,
96%,
97%, 98%, 99% or higher. Alternatively, substantial identity exists when the
nucleic acid
segments will hybridize under selective hybridization conditions (e.g., highly
stringent
hybridization conditions), to the complement of the strand. The nucleic acids
may be
present in whole cells, in a cell lysate, or in a partially purified or
substantially pure form.
[097] Isolated polynucleotides may be used as hybridization probes and
primers to identify and isolate nucleic acids having sequences identical to or
similar to
those encoding the disclosed polynucleotides. Polynucleotides isolated in this
fashion
may be used, for example and not limitation, to produce antibodies against IL-
22 or other
IL-10-like cytokines or to identify cells expressing such antibodies.
Hybridization
methods for identifying and isolating nucleic acids include Southern
hybridizations, in
situ hybridization and Northern hybridization, and are well known to those
skilled in the
art.
[098] Hybridization reactions can be performed under conditions of different
stringencies. Preferably, each hybridizing polynucleotide hybridizes to its
corresponding
polynucleotide under reduced stringency conditions, more preferably stringent
conditions, and most preferably highly stringent conditions. Examples of
stringency
conditions are shown in Table 1 below: highly stringent conditions are those
that are at
least as stringent as, for example, conditions A-F; stringent conditions are
at least as
stringent as, for example, conditions G-L; and reduced stringency conditions
are at least
as stringent as, for example, conditions M-R.
Table 1
Condition Hybrid Hybrid Length Hybridization Wash Temperature
(bp)' Temperature and and Buffer2
Buffer2
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Condition Hybrid Hybrid Length Hybridization Wash Temperature
(bp)' Temperature and and Buffer2
Buffer2
A DNA:DNA > 50 65 C; 1X SSC -or- 65 C; 0.3X SSC
42 C; 1X SSC,
50% formamide
B DNA:DNA <50 TB*; 1X SSC TB*; 1X SSC
C DNA:RNA > 50 67 C; 1X SSC -or- 67 C; 0.3X SSC
45 C; 1X SSC,
50% formamide
D DNA:RNA <50 TD*; 1X SSC TD*; 1X SSC
E RNA:RNA >50 70 C; 1X SSC -or- 70 C; 0.3X SSC
50 C; 1X SSC,
50% formamide
F RNA:RNA <50 TF*; 1X SSC TF*; 1X SSC
G DNA:DNA >50 65 C; 4X SSC -or- 65 C; 1X SSC
42 C; 4X SSC,
50% formamide
H DNA:DNA <50 TH*; 4X SSC TH*; 4X SSC
I DNA:RNA >50 67 C; 4X SSC -or- 67 C; 1X SSC
45 C; 4X SSC,
50% formamide
J DNA:RNA <50 Tj*; 4X SSC Tj*; 4X SSC
K RNA:RNA >50 70 C; 4X SSC -or- 67 C; 1X SSC
50 C; 4X SSC,
50% formamide
L RNA:RNA <50 TL*; 2X SSC TL*; 2X SSC
M DNA:DNA >50 50 C; 4X SSC -or- 50 C; 2X SSC
40 C; 6X SSC,
50% formamide
N DNA:DNA <50 TN*; 6X SSC TN*; 6X SSC
0 DNA:RNA >50 55 C; 4X SSC -or- 55 C; 2X SSC
42 C; 6X SSC,
50% formamide
P DNA:RNA <50 Tp*; 6X SSC Tp*; 6X SSC
Q RNA:RNA >50 60 C; 4X SSC -or- 60 C; 2X SSC
45 C; 6X SSC,
50% formamide
R RNA:RNA <50 TR*; 4X SSC TR*; 4X SSC
' The hybrid length is that anticipated for the hybridized region(s) of the
hybridizing
polynucleotides. When hybridizing a polynucleotide to a target polynucleotide
of
unknown sequence, the hybrid length is assumed to be that of the hybridizing
polynucleotide. When polynucleotides of known sequence are hybridized, the
hybrid
length can be determined by aligning the sequences of the polynucleotides and
identifying the region or regions of optimal sequence complementarity.
2 SSPE (1xSSPE is 0.15M NaCl, 10mM NaH2PO4, and 1.25mM EDTA, pH 7.4) can be
substituted for SSC (1xSSC is 0.15M NaCl and 15mM sodium citrate) in the
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hybridization and wash buffers; washes are performed for 15 minutes after
hybridization
is complete.
TB* - TR*: The hybridization temperature for hybrids anticipated to be less
than 50 base
pairs in length should be 5-10 C less than the melting temperature (T.) of the
hybrid,
where T. is determined according to the following equations. For hybrids less
than 18
base pairs in length, Tm( C) = 2(# of A + T bases) + 4(# of G + C bases). For
hybrids
between 18 and 49 base pairs in length, Tm( C) = 81.5 + 16.6(logioNa+) +
0.41(%G + C)
- (600/N), where N is the number of bases in the hybrid, and Na+ is the
concentration of
sodium ions in the hybridization buffer (Na+ for 1X SSC = 0.165 M).
Additional examples of stringency conditions for polynucleotide hybridization
are
provided in Sambrook et al., Molecular Cloning: A Laboratory Manual, Chs. 9 &
11,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), and
Ausubel et
al., eds., Current Protocols in Molecular Biology, Sects. 2.10 & 6.3-6.4, John
Wiley &
Sons, Inc. (1995), herein incorporated by reference.
[099] Isolated polynucleotides may be used as hybridization probes and
primers to identify and isolate DNAs having sequences encoding allelic
variants of the
disclosed polynucleotides. Allelic variants are naturally occurring
alternative forms of
the disclosed polynucleotides that encode polypeptides that are identical to
or have
significant similarity to the polypeptides encoded by the disclosed
polynucleotides.
Preferably, allelic variants have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%,
98%, or 99% sequence identity with the disclosed polynucleotides.
[0100] Isolated polynucleotides may also be used as hybridization probes and
primers to identify and isolate DNAs having sequences encoding polypeptides
homologous to the disclosed polynucleotides. These homologs are
polynucleotides and
polypeptides isolated from a different species than that of the disclosed
polypeptides and
polynucleotides, or within the same species, but with significant sequence
similarity to
the disclosed polynucleotides and polypeptides. In certain embodiments,
polynucleotide
homologs have at least 50%, 75%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
or
99% identity with the disclosed polynucleotides, whereas polypeptide homologs
have at
least 30%, 45%, or 60% identity with the disclosed antibodies/polypeptides. In
certain
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embodiments, homologs of the disclosed polynucleotides and polypeptides are
those
isolated from mammalian species.
[0101] Isolated polynucleotides may also be used as hybridization probes and
primers to identify cells and tissues that express proteins, including
antibodies, and the
conditions under which they are expressed.
[0102] It is understood that the IL-22 polypeptides and antagonists, e.g.,
antibodies, may have additional conservative or non-essential amino acid
substitutions,
which do not have a substantial effect on their functions. A "conservative
amino acid
substitution" is one in which the amino acid residue is replaced with an amino
acid
residue having a similar side chain. Families of amino acid residues having
similar side
chains have been defined in the art. These families include amino acids with
basic side
chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic
acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine,
serine, threonine,
tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.,
threonine,
valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,
tryptophan,
histidine).
IL-22 Proteins, Fragments and Polynucleotides Encoding the Same
[0103] IL-22 nucleotide and amino acid sequences are described in US Patent
7,307,161 and provided below. The nucleotide sequence of each clone can also
be
determined by sequencing of the deposited clone in accordance with known
methods. As
used herein, a "secreted" protein is one which, when expressed in a suitable
host cell, is
transported across or through a membrane, including transport as a result of
signal
sequences in its amino acid sequence. "Secreted" proteins include without
limitation
proteins secreted wholly (e.g., soluble proteins) or partially (e.g.,
receptors) from the cell
in which they are expressed. "Secreted" proteins also include without
limitation proteins
that are transported across the membrane of the endoplasmic reticulum.
[0104] The nucleotide sequence of human IL-22 is provided in SEQ ID NO:1,
and includes a poly(A) tail. The disclosed nucleotide sequence includes an
open reading
frame and the amino acid sequence of full-length IL-22 protein corresponding
to the
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foregoing nucleotide sequence is reported in SEQ ID NO:2. The amino acid
sequence of
mature IL-22 corresponds to about amino acids 34-179 of SEQ ID NO:2.
[0105] Nucleotide sequences encoding murine IL-22, and the sequence of the
encoded polypeptide, are provided in SEQ. ID. NO. 3 and 4, respectively.
[0106] Any form of IL-22 proteins less than full length can be used in the
methods and compositions of the present claims. IL-22 fragments, e.g., IL-22
proteins of
less than full length, can be produced by expressing a corresponding fragment
of the
polynucleotide encoding the full-length IL-22 protein in a host cell. Modified
polynucleotides as described above may be made by standard molecular biology
techniques, including construction of appropriate desired deletion mutants,
site-directed
mutagenesis methods, or by the polymerase chain reaction using appropriate
oligonucleotide primers.
[0107] Fragments of the protein can be in linear form, or they can be cyclized
using known methods, for example, as described in H.U. Saragovi, et al.,
Bio/Technology 10, 773-778 (1992) and in R.S. McDowell, et al., J. Amer. Chem.
Soc.
114, 9245-9253 (1992), both of which are incorporated herein by reference.
Such
fragments can be fused to carrier molecules such as immunoglobulins for many
purposes,
including increasing the valency of protein binding sites. For example,
fragments of the
protein can be fused through "linker" sequences to the Fc portion of an
immunoglobulin.
For a bivalent form of the protein, the fusion can be to the Fc portion of an
IgG molecule.
Other immunoglobulin isotypes may be used to generate such fusions. For
example, a
protein- 1 gM fusion can be used to generate a decavalent form of the protein.
[0108] IL-22 proteins and fragments thereof include proteins with amino acid
sequence lengths that are at least 25%(more preferably at least 50%, and most
preferably
at least 75%) of the length of a disclosed protein and have at least 60%
sequence identity
(more preferably, at least 75% identity; most preferably at least 90%, 91%,
92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identity along the length of the
fragment)
with that disclosed protein, where sequence identity is determined by
comparing the
amino acid sequences of the proteins when aligned so as to maximize overlap
and
identity while minimizing sequence gaps. In certain embodiments, proteins and
protein
fragments contain a segment comprising 8 or more (more preferably 20 or more,
most
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preferably 30 or more) contiguous amino acids that shares at least 75%
sequence identity
(more preferably, at least 85% identity; most preferably at least 91%, 92%,
93%, 94%,
95%, 96%, 97%, 98%, or 99% identity) with any such segment of any of the
disclosed
proteins.
[0109] In certain embodiments, proteins, protein fragments, and recombinant
proteins include those that can be identified based on the presence of at
least one "IL-22
receptor-binding motif." As used herein, the term "IL-22 receptor-binding
motif
includes amino acid sequences or residues that are important for binding of IL-
22 to its
receptor. In certain embodiments, an IL-22 protein contains an IL-22 receptor-
binding
motif comprising amino acids 50-60 of SEQ ID NO:2. In one embodiment, an IL-22
protein contains an IL-22 receptor-binding motif comprising amino acids 63-81
of SEQ
ID NO:2. In certain embodiments, an IL-22 protein contains a IL-22 receptor-
binding
motif comprising amino acids 168-177 of SEQ II) NO:2. In another embodiment,
an IL-
22 protein contains an IL-22 receptor-binding motif comprising at least one of
amino
acids 50-60, amino acids 63-81, and/or amino acids 168-177 of SEQ ID NO:2. In
certain
embodiments, an IL-22 protein contains an IL-22 receptor-binding motif
comprising
amino acids 20-90, 30-80, 40-70, and/or 50-60 of SEQ ID NO: 2. In certain
embodiments, the IL-22 protein comprises an IL-22 receptor binding motif
comprising
amino acids 33-111, 43-101, 53-91, and/or 63-81 of SEQ ID NO: 2. In certain
embodiments, the IL-22 protein comprises an IL-22 receptor binding motif
comprising
amino acids 138-179,148-179,158-179, and/or 168-177 of SEQ ID NO: 2.
[0110] In certain embodiments, an IL-22 receptor-binding motif is bound by an
antibody or binding fragment that binds to IL-22. In certain embodiments, an
IL-22
antibody or binding fragment binds to at least part of a receptor-binding
motif comprising
amino acids 20-90, 30-80, 40-70, and/or 50-60 of SEQ ID NO: 2. In certain
embodiments, an IL-22 antibody or binding fragment binds to at least part of a
receptor-
binding motif comprising amino acids 33-111, 43-101, 53-91, and/or 63-81 of
Seq Id 2.
In certain embodiments, an IL-22 antibody or binding fragment binds to at
least part of a
receptor-binding motif comprising amino acids 138-179, 148-179, 158-179,
and/or 168-
177 of SEQ ID NO: 2. In certain embodiments, binding of an IL-22 antibody or
binding
fragment to an IL-22 receptor-binding motif interferes with IL-22 binding to
IL-22R, IL-
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l OR, and/or IL-2213P. In certain such embodiments, an IL-22 antibody or
binding
fragment reduces an IL-22 associated activity and/or treats or prevents an IL-
22
associated disorder.
[0111] In certain embodiments, an IL-22 receptor binding motif has an amino
acid sequence at least 95%, 96%, 97%, 98%, 99%, or more identical to an amino
acid
sequence selected from the group consisting of amino acids 50-60 of SEQ ID
NO:2,
amino acids 63-81 of SEQ ID NO:2, and amino acids 168-177 of SEQ ID NO:2. In
other
embodiments, proteins, protein fragments, and/or recombinant proteins include
those
which can be identified based on the presence of at least one, two, three,
four or more
sites for N-linked glycosylation. Length can be determined by aligning the
sequences of
the polynucleotides and identifying the region or regions of optimal sequence
complementarity.
Vectors and Host Cells
[0112] Recombinant polynucleotides can be operably linked to an expression
control sequence such as, for example and not limitation, the pMT2 or pED
expression
vectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991),
to produce
the protein recombinantly. Many suitable expression control sequences are
known in the
art. General methods of expressing recombinant proteins are also known and are
exemplified in R. Kaufman (1990) Methods in Enzymology 185, 537-566. As
defined
herein "operably linked" means that the isolated polynucleotide and an
expression control
sequence are situated within a vector or cell in such a way that the protein
is expressed by
a host cell which has been transformed (transfected) with the ligated
polynucleotide/expression control sequence.
[0113] The term "vector", as used herein, is intended to refer to a nucleic
acid
molecule capable of transporting another nucleic acid to which it has been
linked. One
type of vector is a "plasmid", which refers to a circular double stranded DNA
loop into
which additional DNA segments may be ligated. Another type of vector is a
viral vector,
wherein additional DNA segments may be ligated into the viral genome. Certain
vectors
are capable of autonomous replication in a host cell into which they are
introduced (e.g.,
bacterial vectors having a bacterial origin of replication and episomal
mammalian
vectors). Other vectors (e.g., non-episomal mammalian vectors) can be
integrated into
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44
the genome of a host cell upon introduction into the host cell, and thereby
are replicated
along with the host genome. Moreover, certain vectors are capable of directing
the
expression of genes to which they are operatively linked. Such vectors are
referred to
herein as "recombinant expression vectors" (or simply, "expression vectors").
In general,
expression vectors of utility in recombinant DNA techniques are often in the
form of
plasmids.
[0114] The term "regulatory sequence" as used herein includes promoters,
enhancers and other expression control elements (e.g., polyadenylation
signals) that
control the transcription or translation of the antibody chain genes. Such
regulatory
sequences are described, for example, in Goeddel; Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, CA (1990). It will be
appreciated by those skilled in the art that the design of the expression
vector, including
the selection of regulatory sequences may depend on such factors as the choice
of the
host cell to be transformed, the level of expression of protein desired, etc.
Regulatory
sequences for mammalian host cell expression include, but are not limited to,
viral
elements that direct high levels of protein expression in mammalian cells,
such as
promoters and/or enhancers derived from FF-la promoter and BGH poly A,
cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40
(SV40)
(such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major
late
promoter (AdMLP)) and polyoma. For further description of viral regulatory
elements,
and sequences thereof, see e.g., U.S. Patent No. 5,168,062 by Stinski, U.S.
Patent No.
4,510,245 by Bell et al. and U.S. Patent No. 4,968,615 by Schaffner et al.
[0115] In certain embodiments, recombinant expression vectors may carry
additional sequences, such as sequences that regulate replication of the
vector in host
cells (e.g., origins of replication) and selectable marker genes. The
selectable marker
gene facilitates selection of host cells into which the vector has been
introduced (see e.g.,
U.S. Patents Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For
example,
typically the selectable marker gene confers resistance to drugs, such as
G418,
hygromycin or methotrexate, on a host cell into which the vector has been
introduced.
Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for
use in
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dhfr host cells with methotrexate selection/amplification) and the neo gene
(for G418
selection).
[0116] A number of types of cells may act as suitable host cells for
expression
of the IL-22 protein or fusion protein thereof. Any cell type capable of
expressing
functional IL-22 protein may be used. Suitable mammalian host cells include,
for
example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293
cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells,
other
transformed primate cell lines, normal diploid cells, cell strains derived
from in vitro
culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK,
HL-60,
U937, HaK, Rat2, BaF3, 32D, FDCP-1, PC12, M I x or C2C12 cells.
[0117] In certain embodiments, an IL-22 protein or fusion protein thereof may
also be produced by operably linking an isolated polynucleotide to suitable
control
sequences in one or more insect expression vectors, and employing an insect
expression
system. Materials and methods for baculovirus/insect cell expression systems
are
commercially available in kit form from, e.g., Invitrogen, San Diego, Calif.
U.S.A. (the
MaxBac kit), and such methods are well known in the art, as described in
Summers and
Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987),
incorporated
herein by reference. Soluble forms of the IL-22 protein may also be produced
in insect
cells using appropriate isolated polynucleotides as described above.
[0118] Alternatively, the IL-22 protein or fusion protein thereof maybe
produced in lower eukaryotes such as yeast or in prokaryotes such as bacteria.
Suitable
yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe,
Kluyveroimyces strains, Candida, or any yeast strain capable of expressing
heterologous
proteins. Suitable bacterial strains include Escherichia coli, Bacillus
subtilis, Salmonella
typhimurium, or any bacterial strain capable of expressing heterologous
proteins.
[0119] Expression in bacteria may result in formation of inclusion bodies
incorporating the recombinant protein. Thus, refolding of the recombinant
protein may
be required in order to produce active or more active material. Several
methods for
obtaining correctly folded heterologous proteins from bacterial inclusion
bodies are
known in the art. These methods generally involve solubilizing the protein
from the
inclusion bodies, then denaturing the protein completely using a chaotropic
agent. When
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46
cysteine residues are present in the primary amino acid sequence of the
protein, it is often
necessary to accomplish the refolding in an environment which allows correct
formation
of disulfide bonds (a redox system). General methods of refolding are
disclosed in
Kohno, Meth. Enzym., 185:187-195 (1990). EP 0433225 and copending application
U.S.
Ser. No. 08/163,877 describe other appropriate methods.
[0120] An IL-22 protein or fusion protein thereof may also be expressed as a
product of transgenic animals, e.g., as a component of the milk of transgenic
cows, goats,
pigs, or sheep which are characterized by somatic or germ cells containing a
polynucleotide sequence encoding the IL-22 protein or fusion protein thereof.
[0121] The IL-22 protein or fusion protein thereof maybe prepared by growing
a culture transformed host cells under culture conditions necessary to express
the desired
protein. The resulting expressed protein may then be purified from the culture
medium
or cell extracts. Soluble forms of the IL-22 protein or fusion protein thereof
can be
purified from conditioned media. In certain embodiments, membrane-bound forms
of IL-
22 protein can be purified by preparing a total membrane fraction from the
expressing
cell and extracting the membranes with a non-ionic detergent such as Triton X-
100.
[0122] In certain embodiments, the IL-22 protein can be purified using methods
known to those skilled in the art. For example, and not limitation, the IL-22
protein can
be concentrated using a commercially available protein concentration filter,
including,
but not limited to, an Amicon or Millipore Pellicon ultrafiltration unit.
Following the
concentration step, the concentrate can be applied to a purification matrix
such as a gel
filtration medium. Alternatively, an anion exchange resin can be employed, for
example,
a matrix or substrate having pendant diethylaminoethyl (DEAB) or
polyetheyleneimine
(PEI) groups. The matrices can be acrylamide, agarose, dextran, cellulose or
other types
commonly employed in protein purification. Alternatively, a cation exchange
step can be
employed. Suitable cation exchangers include various insoluble matrices
comprising
sulfopropyl or carboxymethyl groups. In certain embodiments, sulfopropyl
groups are
preferred (e.g., S-Sepharose columns). The purification of the IL-22 protein
or fusion
protein from culture supernatant may also include one or more column steps
over such
affinity resins as concanavalin A-agarose, heparin-toyopearl or Cibacrom blue
3GA
Sepharose ; or by hydrophobic interaction chromatography using such resins as
phenyl
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ether, butyl ether, or propyl ether; or by immunoaffinity chromatography.
Finally, one or
more reverse-phase high performance liquid chromatography (RP-HPLC) steps
employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or
other
aliphatic groups, can be employed to further purify the IL-22 protein.
Affinity columns
including antibodies to the IL-22 protein can also be used in purification in
accordance
with known methods. Some or all of the foregoing purification steps, in
various
combinations or with other known methods, can also be employed to provide a
substantially purified isolated recombinant protein. Preferably, the isolated
IL-22 protein
is purified so that it is substantially free of other mammalian proteins.
[0123] In certain embodiments, IL-22 proteins or fusion proteins may also be
used to screen for agents (e.g., IL-22 antagonists, e.g., anti-IL-22
antibodies or fragments
thereof) that are capable of binding to IL-22 and/or to various portions of IL-
22. Binding
assays using a desired binding protein, immobilized or not, are well known in
the art and
may be used for this purpose using the IL-22 protein. Purified cell based or
protein based
(cell free) screening assays may be used to identify such agents. For example,
IL-22
protein may be immobilized in purified form on a carrier and binding or
potential ligands
to purified IL-22 protein may be measured.
[0124] IL-22 polypeptides may also be produced by known conventional
chemical synthesis. Methods for constructing proteins by synthetic means are
known to
those skilled in the art. The synthetically-constructed protein sequences, by
virtue of
sharing primary, secondary or tertiary structural and/or conformational
characteristics
with proteins may possess biological properties in common therewith, including
protein
activity. Thus, they can be employed as biologically active or immunological
substitutes
for natural, purified proteins in screening of therapeutic compounds and in
immunological processes for the development of antibodies.
Antibodies and Antigen-Binding Fragments Thereof
[0125] Antibodies, also known as immunoglobulins, are typically tetrameric
glycosylated proteins composed of two light (L) chains of approximately 25 kDa
each
and two heavy (H) chains of approximately 50 kDa each. Two types of light
chain,
termed lambda and kappa, may be found in antibodies. Depending on the amino
acid
sequence of the constant domain of heavy chains, immunoglobulins can be
assigned to
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48
five major classes: A, D, E, G, and M, and several of these may be further
divided into
subclasses (isotypes), e.g., IgGi, IgG2, IgG3, IgG4, IgAi, and IgA2. Each
light chain
includes an N-terminal variable (V) domain (VL) and a constant (C) domain
(CL). Each
heavy chain includes an N-terminal V domain (VH), three or four C domains
(CHs), and
a hinge region. The CH domain most proximal to VH is designated as CHI. The VH
and VL domains consist of four regions of relatively conserved sequences
called
framework regions (FRI, FR2, FR3, and FR4), which form a scaffold for three
regions of
hypervariable sequences (complementarity determining regions, CDR5). The CDRs
contain most of the residues responsible for specific interactions of the
antibody with the
antigen. CDRs are referred to as CDRI, CDR2, and CDR3. Accordingly, CDR
constituents on the heavy chain are referred to as H1, H2, and H3, while CDR
constituents on the light chain are referred to as L 1, L2, and U.
[0126] CDR3 is typically the greatest source of molecular diversity within the
antibody-binding site. H3, for example, can be as short as two amino acid
residues or
greater than 26 amino acids. The subunit structures and three-dimensional
configurations
of different classes of immunoglobulins are well known in the art. For a
review of the
antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, eds. Harlow et al., 1988. One of skill in the art will recognize
that each
subunit structure, e.g., a CH, VH, CL, VL, CDR, FR structure, comprises active
fragments, e.g., the portion of the VH, VL, or CDR subunit the binds to the
antigen, i.e.,
the antigen-binding fragment, or, e.g., the portion of the CH subunit that
binds to and/or
activates, e.g., an Fc receptor and/or complement. The CDRs typically refer to
the Kabat
CDRs, as described in Sequences of Proteins of Immunological Interest, US
Department
of Health and Human Services (1991), eds. Kabat et al. Another standard for
characterizing the antigen binding site is to refer to the hypervariable loops
as described
by Chothia. See, e.g., Chothia, D. et al. (1992) J. Mol. Biol. 227:799-817;
and Tomlinson
et al. (1995) EMBO J. 14:4628-4638. Still another standard is the AbM
definition used
by Oxford Molecular's AbM antibody modeling software. See, generally, e.g.,
Protein
Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody
Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag,
Heidelberg). Embodiments described with respect to Kabat CDRs can
alternatively be
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49
implemented using similar described relationships with respect to Chothia
hypervariable
loops or to the AbM-defined loops.
[0127] The Fab fragment (Fragment antigen-binding) consists of VH-CH1 and
VL-CL domains covalently linked by a disulfide bond between the constant
regions. The
Fv fragment is smaller and consists of VH and VL domains non-covalently
linked. To
overcome the tendency of non-covalently linked domains to dissociate, a single
chain Fv
fragment (scFv) can be constructed. The scFv contains a flexible polypeptide
that links
(1) the C-terminus of VH to the N-terminus of VL, or (2) the C-terminus of VL
to the
N-terminus of VH. A 15-mer (G1y4Ser)3 peptide may be used as a linker, but
other linkers
are known in the art.
[0128] The sequence of antibody genes after assembly and somatic mutation is
highly varied, and these varied genes are estimated to encode 1010 different
antibody
molecules (Immunoglobulin Genes, 2nd ed., eds. Jonio et al., Academic Press,
San
Diego, CA, 1995).
[0129] Numerous methods known to those skilled in the art are available for
obtaining antibodies or antigen-binding fragments thereof. For example,
antibodies can
be produced using recombinant DNA methods (U.S. Patent 4,816,567). Monoclonal
antibodies may also be produced by generation of hybridomas (see e.g., Kohler
and
Milstein (1975) Nature, 256: 495-499) in accordance with known methods.
Hybridomas
formed in this manner are then screened using standard methods, such as enzyme-
linked
immunosorbent assay (ELISA) and surface plasmon resonance (BIACORETM)
analysis,
to identify one or more hybridomas that produce an antibody that specifically
binds with
a specified antigen. Any form of the specified antigen may be used as the
immunogen,
e.g., recombinant antigen, naturally occurring forms, any variants or
fragments thereof, as
well as antigenic peptide thereof.
[0130] One exemplary method of making antibodies includes screening protein
expression libraries, e.g., phage or ribosome display libraries. Phage display
is described,
for example, in Ladner et al., U.S. Patent No. 5,223,409; Smith (1985) Science
228:1315-
1317; Clackson et al. (1991) Nature, 352: 624-628; Marks et al. (1991) J. Mol.
Biol., 222:
581-597WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288;
WO 92/01047; WO 92/09690; and WO 90/02809.
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[0131] In addition to the use of display libraries, the specified antigen can
be
used to immunize a non-human animal, e.g., including, but not limited to,
mouse,
hamster, rat, monkey, camel, llama, fish, shark, goat, rabbit, and bovine. In
certain
embodiments, the non-human animal includes at least a part of a human
immunoglobulin
gene. For example, it is possible to engineer mouse strains deficient in mouse
antibody
production with large fragments of the human Ig loci. Using the hybridoma
technology,
antigen-specific monoclonal antibodies derived from the genes with the desired
specificity may be produced and selected. See, e. g., XENOMOUSETM, Green et
al.
(1994) Nature Genetics 7:13-21, US 2003-0070185, WO 96/34096, published Oct.
31,
1996, and PCT Application No. PCT/US96/05928, filed Apr. 29, 1996.
[0132] In certain embodiments, a monoclonal antibody is obtained from a non-
human animal, e.g., including, but not limited to, mouse, hamster, rat,
monkey, camel,
llama, fish, shark, goat, rabbit, and bovine and then modified, e.g.,
humanized or
deimmunized. In certain embodiments chimeric antibodies may be produced using
recombinant DNA techniques known in the art. A variety of approaches for
making
chimeric antibodies have been described. See e.g., Morrison et al., Proc.
Natl. Acad. Sci.
U.S.A. 81:6851, 1985; Takeda et al., Nature 314:452, 1985, Cabilly et al.,
U.S. Patent
No. 4,816,567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi et al.,
European Patent
Publication EP171496; European Patent Publication 0173494, United Kingdom
Patent
GB 2177096B. Humanized antibodies may also be produced, for example, using
transgenic mice that express human heavy and light chain genes, but are
incapable of
expressing the endogenous mouse immunoglobulin heavy and light chain genes.
Winter
describes an exemplary CDR-grafting method that may be used to prepare the
humanized
antibodies described herein (U.S. Patent No. 5,225,539). All of the CDRs of a
particular
human antibody may be replaced with at least a portion of a non-human CDR, or
only
some of the CDRs may be replaced with non-human CDRs. It is only necessary to
replace the number of CDRs required for binding of the humanized antibody to a
predetermined antigen.
[0133] Humanized antibodies or fragments thereof can be generated by
replacing sequences of the Fv variable domain that are not directly involved
in antigen
binding with equivalent sequences from human Fv variable domains. Exemplary
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51
methods for generating humanized antibodies or fragments thereof are provided
by
Morrison (1985) Science 229:1202-1207; by Oi et al. (1986) BioTechniques
4:214; and
by US 5,585,089; US 5,693,761; US 5,693,762; US 5,859,205; and US 6,407,213.
Those
methods include isolating, manipulating, and expressing the nucleic acid
sequences that
encode all or part of immunoglobulin Fv variable domains from at least one of
a heavy or
light chain. Such nucleic acids may be obtained from a hybridoma producing an
antibody against a predetermined target, as described above, as well as from
other
sources. The recombinant DNA encoding the humanized antibody molecule can then
be
cloned into an appropriate expression vector.
[0134] In certain embodiments, a humanized antibody is optimized by the
introduction of conservative substitutions, consensus sequence substitutions,
germline
substitutions and/or back mutations. Such altered immunoglobulin molecules can
be
made by any of several techniques known in the art, (e.g., Teng et al., Proc.
Natl. Acad.
Sci. USA., 80: 7308-7312, 1983; Kozbor et al., Immunology Today, 4: 7279,
1983;
Olsson et al., Meth. Enzymol., 92: 3-16, 1982), and may be made according to
the
teachings of PCT Publication W092/06193 or EP 0239400).
[0135] An antibody or fragment thereof may also be modified by specific
deletion of human T cell epitopes or "deimmunization" by the methods disclosed
in WO
98/52976 and WO 00/34317. Briefly, the heavy and light chain variable domains
of an
antibody can be analyzed for peptides that bind to MHC Class II; these
peptides represent
potential T-cell epitopes (as defined in WO 98/52976 and WO 00/34317). For
detection
of potential T-cell epitopes, a computer modeling approach termed "peptide
threading"
can be applied, and in addition a database of human MHC class II binding
peptides can
be searched for motifs present in the VH and VL sequences, as described in WO
98/52976
and WO 00/34317. These motifs bind to any of the 18 major MHC class II DR
allotypes,
and thus constitute potential T cell epitopes. Potential T-cell epitopes
detected can be
eliminated by substituting small numbers of amino acid residues in the
variable domains,
or preferably, by single amino acid substitutions. Typically, conservative
substitutions
are made. Often, but not exclusively, an amino acid common to a position in
human
germline antibody sequences may be used. Human germline sequences, e.g., are
disclosed in Tomlinson, et al. (1992) J. Mol. Biol. 227:776-798; Cook, G. P.
et al. (1995)
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Immunol. Today Vol. 16 (5): 237-242; Chothia, D. et al. (1992) J. Mol. Biol.
227:799-
817; and Tomlinson et al. (1995) EMBO J. 14:4628-4638. The V BASE directory
provides a comprehensive directory of human immunoglobulin variable region
sequences
(compiled by Tomlinson, I.A. et al. MRC Centre for Protein Engineering,
Cambridge,
UK). These sequences can be used as a source of human sequence, e.g., for
framework
regions and CDRs. Consensus human framework regions can also be used, e.g., as
described in U.S. Patent No. 6,300,064.
[0136] In certain embodiments, an antibody can contain an altered
immunoglobulin constant or Fc region. For example, an antibody produced in
accordance with the teachings herein may bind more strongly or with more
specificity to
effector molecules such as complement and/or Fc receptors, which can control
several
immune functions of the antibody such as effector cell activity, lysis,
complement-
mediated activity, antibody clearance, and antibody half-life. Typical Fc
receptors that
bind to an Fc region of an antibody (e.g., an IgG antibody) include, but are
not limited to,
receptors of the FcyRI, FcyRII, and FcyRIII and FcRn subclasses, including
allelic
variants and alternatively spliced forms of these receptors. Fc receptors are
reviewed in
Ravetch and Kinet, Annu. Rev. Immunol 9:457-92, 1991; Capel et al.,
Imimunoimethods
4:25-34,1994; and de Haas et al., J. Lab. Clin. Med. 126:330-41, 1995).
[0137] For additional antibody production techniques, see Antibodies: A
Laboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988.
[0138] A bispecific or bifunctional antibody is an artificial hybrid antibody
having two different heavy/light chain pairs and two different binding sites.
Bispecific
antibodies can be produced by a variety of methods including fusion of
hybridomas or
linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp.
Immunol.
79:315-321 (1990); Kostelny et al., J. Imimunol. 148, 1547-1553 (1992). In
certain
embodiments, the bispecific antibody comprises a first binding domain
polypeptide, such
as a Fab' fragment, linked via an immunoglobulin constant region to a second
binding
domain polypeptide.
[0139] Antibodies of the present invention can also be single domain
antibodies.
Single domain antibodies can include antibodies whose complementary
determining
regions are part of a single domain polypeptide. Examples include, but are not
limited to,
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heavy chain antibodies, antibodies naturally devoid of light chains, single
domain
antibodies derived from conventional 4-chain antibodies, engineered antibodies
and
single domain scaffolds other than those derived from antibodies. Single
domain
antibodies may be any of the art, or any future single domain antibodies.
Single domain
antibodies may be derived from any species including, but not limited to
mouse, human,
camel, llama, fish, shark, goat, rabbit, and bovine. In one aspect of the
invention, a single
domain antibody can be derived from a variable region of the immunoglobulin
found in
fish, such as, for example, that which is derived from the immunoglobulin
isotype known
as Novel Antigen Receptor (NAR) found in the serum of shark. Methods of
producing
single domain antibodies derived from a variable region of NAR ("IgNAR5")
are described in WO 03/014161 and Streltsov (2005) Protein Sci. 14:2901-2909.
[0140] According to another aspect of the invention, a single domain antibody
is
a naturally occurring single domain antibody known as heavy chain antibody
devoid of
light chains. Such single domain antibodies are disclosed in WO 9404678, for
example.
For clarity reasons, this variable domain derived from a heavy chain antibody
naturally
devoid of light chain is known herein as a VHH or nanobody to distinguish it
from the
conventional VH of four chain immunoglobulins. Such a VHH molecule can be
derived
from antibodies raised in Camelidae species, for example in camel, llama,
dromedary,
alpaca and guanaco. Other species besides Camelidae may produce heavy chain
antibodies naturally devoid of light chain; such VHHs are within the scope of
the
invention.
[0141] The invention also contemplates the use of Small Modular
ImmunoPharmaceuticals ("SMIPsTM) which typically refers to binding domain-
immunoglobulin fusion proteins including a binding domain polypeptide that is
fused or
otherwise connected to an immunoglobulin hinge or hinge-acting region
polypeptide,
which in turn is fused or otherwise connected to a region comprising one or
more native
or engineered constant regions from an immunoglobulin heavy chain, other than
CHI, for
example, the CH2 and CH3 regions of IgG and IgA, or the CH3 and CH4 regions of
IgE
(see e.g., U.S. 2005/0136049 by Ledbetter, J. et al. for a more complete
description). The
binding domain-immunoglobulin fusion protein can further include a region that
includes
a native or engineered immunoglobulin heavy chain CH2 constant region
polypeptide (or
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54
CH3 in the case of a construct derived in whole or in part from IgE) that is
fused or
otherwise connected to the hinge region polypeptide and a native or engineered
immunoglobulin heavy chain CH3 constant region polypeptide (or CH4 in the case
of a
construct derived in whole or in part from IgE) that is fused or otherwise
connected to the
CH2 constant region polypeptide (or CH3 in the case of a construct derived in
whole or
in part from IgE). Typically, such binding domain-immunoglobulin fusion
proteins are
capable of at least one immunological activity selected from the group
consisting of
antibody dependent cell-mediated cytotoxicity, complement fixation, and/or
binding to a
target, for example, a target antigen.
[0142] In certain embodiments, therapeutic proteins, i.e., a protein or
peptide
that has a biological effect on a region in the body on which it acts or on a
region of the
body on which it remotely acts via intermediates, and method of designing and
making
these therapeutic proteins, are provided. Therapeutic proteins of the current
invention
can include peptide mimetics. Mimetics are peptide-containing molecules that
mimic
elements of protein secondary structure. See, for example, Johnson et al.,
"Peptide Turn
Mimetics" in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds., Chapman
and Hall, New York (1993), incorporated herein by reference. The underlying
rationale
behind the use of peptide mimetics is that the peptide backbone of proteins
exists chiefly
to orient amino acid side chains in such a way as to facilitate molecular
interactions, such
as those of antibody and antigen. A peptide mimetic is expected to permit
molecular
interactions similar to the natural molecule. In conjunction with the
information provided
by the current invention, these principles may be used to engineer second
generation
molecules having many of the natural properties of the targeting peptides
disclosed
herein. These second generation molecules can also be altered and provide
potentially
improved characteristics. Using the current invention, both protein and small
molecule
therapeutics can be designed to interrupt the desired cytokine activity by,
for example,
being specifically designed to bind at the desired positions, i.e., at the
amino acid
positions demonstrated as being important for a binding complex, and therefore
effectively reducing or inhibiting the activity associated with the cytokine
and its receptor
or receptor complex.
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[0143] Other embodiments of therapeutic proteins include fusion proteins.
These molecules generally have all or a substantial portion of a targeting
peptide, for
example, IL-22 or an anti-IL-22 antibody, linked at the N- or C-terminus, to
all or a
portion of a second polypeptide or protein. For example, fusions may employ
leader
sequences from other species to permit the recombinant expression of a protein
in a
heterologous host. Another useful fusion includes the addition of an
immunologically
active domain, such as an antibody epitope, to facilitate purification of the
fusion protein.
Inclusion of a cleavage site at or near the fusion junction will facilitate
removal of the
extraneous polypeptide after purification. Other useful fusions include
linking of
functional domains, such as active sites from enzymes, glycosylation domains,
cellular
targeting signals or transmembrane regions. Examples of proteins or peptides
that may
be incorporated into a fusion protein include cytostatic proteins, cytocidal
proteins, pro-
apoptosis agents, anti-angiogenic agents, hormones, cytokines, growth factors,
peptide
drugs, antibodies, Fab fragments of antibodies, antigens, receptor proteins,
enzymes,
lectins, MHC proteins, cell adhesion proteins and binding proteins. Methods of
generating fusion proteins are well known to those of skill in the art. Such
proteins can
be produced, for example, by chemical attachment using bifunctional cross-
linking
reagents, by de novo synthesis of the complete fusion protein, or by
attachment of a DNA
sequence encoding the targeting peptide to a DNA sequence encoding the second
peptide
or protein, followed by expression of the intact fusion protein.
[0144] In certain embodiments, the targeting peptide, for example, IL-22 or an
anti-IL-22 antibody, is fused with an immunoglobulin heavy chain constant
region, such
as an Fc fragment, which contains two constant region domains and a hinge
region but
lacks the variable region (See, U.S. Pat. Nos. 6,018,026 and 5,750,375,
incorporated
herein by reference). The Fc region may be a naturally occurring Fc region, or
may be
altered to improve certain qualities, such as therapeutic qualities,
circulation time,
reduced aggregation, etc. Peptides and proteins fused to an Fc region
typically exhibit a
greater half-life in vivo than the unfused counterpart. Also, a fusion to an
Fc region
permits dimerization/multimerization of the fusion polypeptide.
[0145] In certain embodiments, mutagenesis is used to make an antibody more
similar to one or more germline sequences. This may be desirable when
mutations are
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introduced into the framework region of an antibody through somatic
mutagenesis or
through error prone PCR. Germline sequences for the VH and VL domains can be
identified by performing amino acid and nucleic acid sequence alignments
against the
VBASE database (MRC Center for Protein Engineering, UK). VBASE is a
comprehensive directory of all human germline variable region sequences
compiled from
over a thousand published sequences, including those in the current releases
of the
Genbank and EMBL data libraries. In some embodiments, the FR regions of the
scFvs
are mutated in conformity with the closest matches in the VBASE database and
the CDR
portions are kept intact.
[0146] Using recombinant DNA methodology, a disclosed CDR sequence may
be introduced into a repertoire of VH or VL domains lacking the respective CDR
(Marks
et al. (BioTechnology (1992) 10: 779-783). For example, a primer adjacent to
the 5' end
of the variable domain and a primer to the third FR can be used to generate a
repertoire of
variable domain sequences lacking CDR3. This repertoire can be combined with a
CDR3
of a disclosed antibody. Using analogous techniques, portions of a disclosed
CDR
sequence may be shuffled with portions of CDR sequences from other antibodies
to
provide a repertoire of antigen-binding fragments that bind IL-22. Either
repertoire can
be expressed in a host system such as phage display (described in WO 92/01047
and its
corresponding U.S. Patent No. 5,969,108) so suitable antigen-binding fragments
that bind
to IL-22 can be selected.
[0147] A further alternative uses random mutagenesis of the disclosed VH or VL
sequences to generate variant VH or VL domains still capable of binding IL-22.
A
technique using error-prone PCR is described by Gram et al. (Proc. Nat. Acad.
Sci.
U.S.A. (1992) 89: 3576-3580).
[0148] Another method uses direct mutagenesis of the disclosed VH or VL
sequences. Such techniques are disclosed by Barbas et al. (Proc. Nat. Acad.
Sci. U.S.A.
(1994) 91: 3809-3813) and Schier et al. (J. Mol. Biol. (1996) 263: 551-567).
[0149] A portion of a variable domain will comprise at least one CDR region
substantially as set out herein and, optionally, intervening framework regions
from the
VH or VL domains as set out herein. The portion may include the C-terminal
half of FR1
and/or the N-terminal half of FR4. Additional residues at the N-terminal or C-
terminal
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end of the variable domain may not be the same residues found in naturally
occurring
antibodies. For example, construction of antibodies by recombinant DNA
techniques
often introduces N- or C-terminal residues from its use of linkers. Some
linkers may be
used to join variable domains to other variable domains (e.g., diabodies),
constant
domains, or proteinaceous labels.
[0150] The disclosed antibodies can be modified to alter their glycosylation;
that
is, at least one carbohydrate moiety can be deleted or added to the antibody.
Deletion or
addition of glycosylation sites can be accomplished by changing amino acid
sequence to
delete or create glycosylation consensus sites, which are well known in the
art. Another
means of adding carbohydrate moieties is the chemical or enzymatic coupling of
glycosides to amino acid residues of the antibody (see WO 87/05330 and Aplin
et al.
(1981) CRC Crit. Rev. Biochem., 22: 259-306). Removal of carbohydrate moieties
can
also be accomplished chemically or enzymatically (see Hakimuddin et al. (1987)
Arch.
Biochem. Biophys., 259: 52; Edge et al. (1981) Anal. Biochem., 118: 131;
Thotakura et al.
(1987) Meth. Enzymol., 138: 350).
[0151] Methods for altering an antibody constant region are known in the art.
Antibodies with altered function (e.g., altered affinity for an effector
ligand such as FcR
on a cell or the Cl component of complement) can be produced by replacing at
least one
amino acid residue in the constant portion of the antibody with a different
residue (see
e.g., EP 388,151 Al, US 5,624,821 and US 5,648,260). Similar types of
alterations could
be described which if applied to a murine or other species antibody would
reduce or
eliminate similar functions.
[0152] For example, it is possible to alter the affinity of an Fc region of an
antibody (e.g., an IgG, such as a human IgG) for FcR (e.g., Fc gamma Rl) or
Clq. The
affinity may be altered by replacing at least one specified residue with at
least one residue
having an appropriate functionality on its side chain, or by introducing a
charged
functional group, such as glutamate or aspartate, or perhaps an aromatic non-
polar
residue such as phenylalanine, tyrosine, tryptophan or alanine (see e.g., US
5,624,821).
[0153] In another example, replacing residue 297 (asparagine) with alanine in
the IgG constant region significantly inhibits recruitment of effector cells,
while only
slightly reducing (about three fold weaker) affinity for Clq (see e.g., US
5,624,821). The
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numbering of the residues in the heavy chain is that of the EU index (see
Kabat et al.,
1991 supra). This alteration destroys the glycosylation site and it is
believed that the
presence of carbohydrate is required for Fc receptor binding. Any other
substitution at
this site that destroys the glycosylation site is believed to cause a similar
decrease in lytic
activity. Other amino acid substitutions, e.g., changing any one of residues
318 (Glu),
320 (Lys) and 322 (Lys), to Ala, are also known to abolish Clq binding to the
Fc region
of IgG antibodies (see e.g., US 5,624,821).
[0154] Modified antibodies can be produced which have a reduced interaction
with an Fc receptor. For example, it has been shown that in human IgG3, which
binds to
the human Fc gamma RI receptor, changing Leu 235 to Glu destroys its
interaction with
the receptor. Mutations on adjacent or close sites in the hinge link region of
an antibody
(e.g., replacing residues 234, 236 or 237 with Ala) can also be used to affect
antibody
affinity for the Fc gamma R1 receptor. The numbering of the residues in the
heavy chain
is based in the EU index (see Kabat et al., 1991 supra).
[0155] Additional methods for altering the lytic activity of an antibody, for
example, by altering at least one amino acid in the N-terminal region of the
CH2 domain,
are described in WO 94/29351 by Morgan et al. and US 5,624,821.
[0156] In certain embodiments, antibodies maybe tagged with a detectable or
functional label. These labels include radiolabels (e.g., 1311 or 99Tc),
enzymatic labels
(e.g., horseradish peroxidase or alkaline phosphatase), and other chemical
moieties (e.g.,
biotin).
[0157] In certain embodiments, the IL-22 antagonists are antibodies, or
fragments thereof (e.g., antigen-binding fragments thereof), that bind to
mammalian (e.g.,
human or murine) IL-22. In certain embodiments, the anti-IL22 antibody or
fragment
thereof (e.g., an Fab, F(ab')2, Fv or a single chain Fv fragment) is a
monoclonal or single
specificity antibody. The antibody or fragment thereof can also be a human,
humanized,
chimeric, or in vitro generated antibody against human IL-22.
[0158] The production of anti-IL-22 antibodies is described in more detail in
U.S. Published Patent Application Nos. 2005-0042220 and 2007-0243589. One non-
limiting example of an anti-IL22 antibody that interferes with IL-22 binding
to IL-22R is
referred to as "Ab-04" or "IL22-04" in US Published Patent Application No.
2005-
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0042220. Ab-04 (also referred to herein as rat monoclonal antibody "P3/2")
binds to
human IL-22 and neutralizes human IL-22 activity. A hybridoma cell line
producing Ab-
04 has been deposited with the ATCC on June 5, 2003 and has been assigned ATCC
accession number PTA-5255. Another non-limiting example of an anti-11,22
antibody
that interferes with IL-22 binding to IL-1OR2 is "Ab-02" or "IL22-02." Ab-02
(also
referred to herein as rat monoclonal antibody "P3/3") binds to mouse and human
IL-22
and neutralizes the activity of mouse and human IL-22. A hybridoma cell line
producing
Ab-02 has been deposited on June 5, 2003 with the ATCC and has been assigned
ATCC
accession number PTA-5254. Additional examples of IL-22 antibodies that
reduce,
inhibit or antagonize IL-22 activity are found in U.S. Published Patent
Application No.
2007-0243589, which describes germlined antibodies identified as GILO1, GIL
16,
GIL45, GIL60, GIL68, GIL92, 062A09, 087B03, 166B06, 166G05, 354A08, 355B06,
355E04, 356A11, and 368D04.
[0159] In certain embodiments, an antibody is provided that binds to the wild-
type human IL-22 but fails to bind to a mutant IL-22 comprising one or more
point
mutations.
In certain embodiments, an antibody is provided that binds to the wild-type
human IL-22
but fails to bind to a mutant IL-22 wherein the mutant IL-22 comprises one or
more of
the following changes relative to wild-type human IL-22(a) the amino acid at
position 34
of the mutant IL-22 is alanine; (b) the amino acid at position 52 of the
mutant IL-22 is
alanine; (c) the amino acid at position 56 of the mutant IL-22 is alanine; (d)
the amino
acid at position 61 of the mutant IL-22 is alanine; e) the amino acid at
position 66 of the
mutant IL-22 is alanine; f) the amino acid at position 83 of the mutant IL-22
is alanine; g)
the amino acid at position 88 of the mutant IL-22 is alanine; h) the amino
acid at position
113 of the mutant IL-22 is alanine; i) the amino acid at position 121 of the
mutant IL-22
is alanine; j) the amino acid at position 122 of the mutant IL-22 is alanine;
k) the amino
acid at position 125 of the mutant IL-22 is alanine; or 1) the amino acid at
position 172 of
the mutant IL-22 is alanine.
[0160] In certain embodiments, an antibody is provided that binds to the wild-
type human IL-22 but fails to bind to a mutant IL-22 wherein the mutant IL-22
comprises
one or more of the following changes relative to wild-type human IL-22: a) the
amino
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acid at position 57 of the mutant IL-22 is alanine; b) the amino acid at
position 59 of the
mutant IL-22 is alanine; c) the amino acid at position 67 of the mutant IL-22
is alanine;
d) the amino acid at position 72 of the mutant IL-22 is alanine; e) the amino
acid at
position 159 of the mutant IL-22 is alanine; f) the amino acid at position 161
of the
mutant IL-22 is alanine; g) the amino acid at position 162 of the mutant IL-22
is alanine;
or h) the amino acid at position 169 of the mutant IL-22 is alanine.
[0161] In certain embodiments, an antibody is provided that binds to the wild-
type human IL-22 but fails to bind to a mutant IL-22 wherein the mutant IL-22
comprises
one or more of the following changes relative to wild-type human IL-22: a) the
amino
acid at position 67 of the mutant IL-22 is alanine; b) the amino acid at
position 73 of the
mutant IL-22 is alanine; c) the amino acid at position 83 of the mutant IL-22
is alanine;
or d) the amino acid at position 162 of the mutant IL-22 is alanine.
Methods of Systematic Mutagenesis of Target Proteins
[0162] In certain embodiments, methods of systematic mutagenesis of a target
protein are provided.
[0163] The term "systematic mutagenesis" refers to the creation of multiple
different mutants of the same target protein. Examples of systematic
mutagenesis
include, but are not limited to, alanine scanning. In certain embodiments,
each amino
acid of a target protein is individually mutagenized. For example, and not
limitation, if a
target protein consists of 400 amino acids, in certain embodiments, 400
different mutants
are created. In certain embodiments, less than all of the amino acids in a
target protein
are mutagenized. For example, and not limitation, in certain embodiments, 90%,
80%,
70%, 60%, 50%, or fewer of the amino acids of a target protein are mutagenized
during
systematic mutagenesis.
[0164] In certain embodiments, a method of systematic mutagenesis of a target
protein comprising generating a series of ten or more different nucleic acid
molecules is
provided. In certain embodiments, each different nucleic acid molecule
comprises a
nucleic acid sequence that encodes a polypeptide comprising a different mutant
of the
target protein. In certain embodiments, each different polypeptide comprising
a different
mutant of the target protein further comprises a secretory sequence, a first
tag, and a
second tag;. Secretory sequences are known by those of skill in the art and
are
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commonly used in molecular biology. Tags are also known by those of skill in
the art
and are commonly used in molecular biology. Examples of tags include, but are
not
limited to, FLAG tag, His tag, c-myc-tag, Maltose Binding Protein tag,
Thioredoxin tag,
GFP tag, and Glutothione-S-transferase tag.
[0165] In certain embodiments, ten or more different mutants of the target
protein are expressed in separate wells. In certain such embodiments, the ten
or more
different mutants of the target protein are secreted into the liquid media of
the separate
wells. In certain embodiments, the ten or more different mutants of the target
protein in
the liquid media in the separate wells are quantitated using a first tag and a
second tag.
For example, and not limitation, in certain embodiments, a small sample of the
media
may be removed and subjected to a sandwich assay using the first tag and the
second tag.
[0166] In certain embodiments, the use of two tags added to a polypeptide
allows for a more accurate detection of a target protein than using a single
tag and an
epitope on the target protein. For example, and not limitation, if a target
protein has been
mutated, one or more of the epitopes that would have been present in the wild-
type
protein may be disrupted in the mutant protein. If an antibody used to
quantitate that
mutant protein in an assay happens to overlap with a disrupted epitope, the
quantitation
of the assay may be inaccurate due to the disrupted epitope. In comparison, if
the same
mutant were detected with two antibodies, each specific for a separate
artificial tag added
to the mutant protein, the chances of an inaccurate quantitation of the mutant
protein are
decreased.
[0167] In certain embodiments, the use of two artificial tags allows for the
rapid
systematic mutagenesis of a protein. In certain such embodiments, the presence
of two
artificial tags on each different mutant protein allows for the use of assays
to quantitate
the different proteins without having to be concerned about the effect of
mutations to the
target protein on the quantitation assay.
[0168] In certain embodiments, systematic mutagenesis comprises generating a
series of 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or
more, 70 or
more, 80 or more, 90 or more, or 100 or more different nucleic acid molecules
wherein
each different nucleic acid molecule comprises a nucleic acid sequence that
encodes a
polypeptide comprising a different mutant of the target protein.
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[0169] In certain embodiments, systematic mutagenesis provides information
concerning which residues in a protein are important for a particular
function. In certain
embodiments, systematic mutagenesis includes testing the mutated target
proteins in an
assay for a particular function. For example, and not limitation, systematic
mutagenesis
can be performed on a receptor to determine which amino acids are important
for binding
of the receptor to the receptor's ligand by testing the mutated target
proteins in a ligand
binding assay. Examples of systematic mutagenesis include, but are not limited
to,
analysis of amino acids of a receptor involved in binding to a ligand;
analysis of the
amino acids of a ligand involved in binding to a receptor; analysis of the
amino acids of a
first protein involved in binding to a second protein; and analysis of the
amino acids of an
enzyme that are involved in that enzyme's activity, e.g., the amino acids of a
kinase
involved in kinase activity.
[0170] In certain embodiments, identification of amino acids involved in a
particular function aids in rational design and evaluation of antagonists
and/or agonists of
the target protein. For example, and not limitation, identification of amino
acids of a first
protein involved in binding to a second protein provides insight into which
portions of the
first protein are involved in binding to the second protein. That information
can be used
in the rational design and evaluation of antagonists and/or agonists of the
first protein.
[0171] In certain embodiments, identification of amino acids involved in a
particular function aids in identifying amino acids involved in a similar
function in
related proteins. In certain embodiments, the information from systematic
mutagenesis
can be combined with other information concerning related proteins to identify
amino
acids involved in a similar function in those related proteins. Examples of
information
that can be used to help identify amino acids involved in a similar function
in related
proteins includes, but is not limited to, sequence alignments, crystal
structures of the
target protein and/or related proteins, structural modeling based on amino
acid sequences,
and predicted structure and/or function based on analysis of amino acid
sequences.
[0172] In certain embodiments, systematic mutagenesis is used to define which
amino acids of the IL-22 protein sequence are important for the binding of IL-
22 to IL-
22R, IL-IOR2, and/or IL-2213P. In certain embodiments, systematic mutagenesis
of IL-
22 allows for the design of binding fragments, antibodies, and fragments
thereof that
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specifically block one or more of IL-22 binding to IL-22R, IL-22 binding to IL-
IOR2,
and IL-22 binding to IL-2213P.
[0173] The study of IL-22 as described herein is the first to explore by
systematic mutagenesis a structure/ function relationship for an IL-10-like
cytokine. The
members of this group (i.e., IL-10, IL-19, IL-20, IL-22, IL-24, and IL-26) are
proposed to
have a conserved six a-helical structural and functional unit that is also
shared with the
interferons. The receptors for these cytokines and the interferons belong to
the cytokine
receptor family 2 (CRF 2) and studies have suggested that these receptors
share a
conserved structure. As described in detail below, single substitutions of
amino acids in
the DE loop of IL-22 derived from mammalian cells did not reduce binding to
either IL-
22R or IL-22R/IL- I OR2 in the assays performed, indicating that none of the
DE loop
residues are singularly involved in IL-22 receptor binding. However,
dimerization via
the DE loop would be quite removed from the receptor binding sites described
in this
application and therefore dimerization via the DE loop would be compatible
with the
receptor binding sites described in this application.
Protein Structure and Crystallography
[0174] Structural data describing a crystal can be obtained, for example, by X-
ray diffraction. X-ray diffraction data can be collected by a variety of
sources, X-ray
wavelengths and detectors. In some embodiments, rotating anodes and
synchrotron
sources (e.g., Advanced Light Source (ALS), Berkeley, California; or Advanced
Photon
Source (APS), Argonne, Illinois) can be used as the source(s) of X-rays. In
certain
embodiments, X-rays for generating diffraction data can have a wavelength of
from about
0.5 A to about 1.6 A (e.g., about 0.7 A, about 0.9 A, about 1.0 A, about 1.1
A, about 1.3
A, about 1.4 A, about 1.5 A, or about 1.6 A). In some embodiments, area
detectors
and/or charge-couple devices (CCDs) can be used as the detector(s).
[0175] X-ray diffraction data of a crystal of a polypeptide can be used to
obtain
the structural coordinates of the atoms in the complex. The structural
coordinates are
Cartesian coordinates that describe the location of atoms in three-dimensional
space in
relation to other atoms in the complex. The structural coordinates can be
modified by
mathematical manipulation, such as by inversion or integer additions or
subtractions. As
such, structural coordinates are relative coordinates.
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[0176] The structural coordinates of a polypeptide can be used to derive a
representation (e.g., a two dimensional representation or three dimensional
representation) of the polypeptide or a fragment of the polypeptide. Such
representations
can be useful for a number of applications, including, for example, the
visualization,
identification and characterization of an active site of the polypeptide. In
certain
embodiments, a three-dimensional representation can include the structural
coordinates of
a polypeptide according to certain coordinates a root mean square (rms)
deviation from
the alpha carbon atoms of amino acids of not more than about 1.5 A (e.g., not
more than
about 1.0 A, not more than about 0.5 A).
[0177] RMS deviation is the square root of the arithmetic mean of the squares
of
the deviations from the mean, and is a way of expressing deviation or
variation from
structural coordinates. Conservative substitutions (see discussion below) of
amino acids
can result in a molecular representation having structural coordinates within
the stated
rms deviation. For example, two molecular models of polypeptides that differ
from one
another by conservative amino acid substitutions can have coordinates of
backbone atoms
within a stated rms deviation, such as less than about 1.5 A (e.g., less than
about 1.0 A,
less than about 0.5 A). Backbone atoms of a polypeptide include the alpha
carbon (Ca or
CA) atoms, carbonyl carbon (C) atoms, and amide nitrogen (N) atoms.
[0178] Various software programs allow for the graphical representation of a
set
of structural coordinates to obtain a representation of a polypeptide or a
fragment of the
polypeptide. In general, such a representation should accurately reflect
(relatively and/or
absolutely) structural coordinates, or information derived from structural
coordinates,
such as distances or angles between features. In some embodiments, the
representation is
a two-dimensional figure, such as a stereoscopic two-dimensional figure. In
certain
embodiments, the representation is an interactive two-dimensional display,
such as an
interactive stereoscopic two-dimensional display. An interactive two-
dimensional
display can be, for example, a computer display that can be rotated to show
different
faces of a polypeptide or a fragment of a polypeptide. In some embodiments,
the
representation is a three-dimensional representation. As an example, a three-
dimensional
model can be a physical model of a molecular structure (e. g., a ball-and-
stick model). As
another example, a three dimensional representation can be a graphical
representation of
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a molecular structure (e.g., a drawing or a figure presented on a computer
display). A
two-dimensional graphical representation (e.g., a drawing) can correspond to a
three-
dimensional representation when the two-dimensional representation reflects
three-
dimensional information, for example, through the use of perspective, shading,
or the
obstruction of features more distant from the viewer by features closer to the
viewer. In
some embodiments, a representation can be modeled at more than one level. As
an
example, when the three-dimensional representation includes a polypeptide, the
polypeptide can be represented at one or more different levels of structure,
such as
primary (amino acid sequence), secondary (e.g., a-helices and (3-sheets),
tertiary (overall
fold), and quaternary (oligomerization state) structure. A representation can
include
different levels of detail. For example, the representation can include the
relative
locations of secondary structural features of a protein without specifying the
positions of
atoms. A more detailed representation could, for example, include the
positions of
atoms.
[0179] In some embodiments, a representation can include information in
addition to the structural coordinates of the atoms in a polypeptide. For
example, a
representation can provide information regarding the shape of a solvent
accessible
surface, the van der Waals radii of the atoms of the model, and the van der
Waals radius
of a solvent (e.g., water). Other features that can be derived from a
representation
include, for example, electrostatic potential, the location of voids or
pockets within a
macromolecular structure, and the location of hydrogen bonds and salt bridges.
[0180] An agent that interacts with (e. g., binds) a polypeptide can be
identified
or designed by a method that includes using a representation of the
polypeptide or a
fragment of the polypeptide. Exemplary types of representations include the
representations discussed above. In some embodiments, the representation can
be of an
analog polypeptide or polypeptide fragment. A candidate agent that interacts
with the
representation can be designed or identified by performing computer fitting
analysis of
the candidate agent with the representation. In general, an agent is a
molecule. Examples
of agents include polypeptides, nucleic acids (including DNA or RNA), steroids
and non-
steroidal organic compounds. An agent that interacts with a polypeptide can
interact
transiently or stably with the polypeptide. The interaction can be mediated by
any of the
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forces noted herein, including, for example, hydrogen bonding, electrostatic
forces,
hydrophobic interactions, and van der Waals interactions.
[0181] As noted above, X-ray crystallography can be used to obtain structural
coordinates of a polypeptide. However, such structural coordinates can be
obtained using
other techniques including NMR techniques. Additional structural information
can be
obtained from spectral techniques (e.g., optical rotary dispersion (ORD),
circular
dichroism (CD)), homology modeling, and computational methods (e.g.,
computational
methods that can include data from molecular mechanics, computational methods
that
include data from dynamics assays).
[0182] In some embodiments, the X-ray diffraction data can be used to
construct
an electron density map of a polypeptide or a fragment of the polypeptide, and
the
electron density map can be used to derive a representation (e. g., a two
dimensional
representation, a three dimensional representation) of the polypeptide.
Creation of an
electron density map typically involves using information regarding the phase
of the X-
ray scatter. Phase information can be extracted, for example, either from the
diffraction
data or from supplementing diffraction experiments to complete the
construction of the
electron density map. Methods for calculating phase from X-ray diffraction
data include,
for example, multiwavelength anomalous dispersion (MAD), multiple isomorphous
replacement (MIR), multiple isomorphous replacement with anomalous scattering
(MIRAS), single isomorphous replacement with anomalous scattering (SIRAS),
reciprocal space solvent flattening, molecular replacement, or any combination
thereof.
These methods generate phase information by making isomorphous structural
modifications to the native protein, such as by including a heavy atom or
changing the
scattering strength of a heavy atom already present, and then measuring the
diffraction
amplitudes for the native protein and each of the modified cases. If the
position of the
additional heavy atom or the change in its scattering strength is known, then
the phase of
each diffracted X-ray can be determined by solving a set of simultaneous phase
equations. The location of heavy atom sites can be identified using a computer
program,
such as SHELXD (Bruker-AXS, Madison, WI) or SHELXS (Sheldrick, Institut Anorg.
Chemie, Gottingen, Germany), and XPREP (Bruker-AXS, Madison, WI); and
diffraction
data can be processed using computer programs such as MOSFLM, SCALA,
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SOLOMON, and SHARP ("The CCP4 Suite: Programs for Protein Crystallography,"
Acta Crystallogr. Sect. D, 54:905-921, 1997; deLa Fortelle and Brigogne, Meth.
Enzym.
276:472-494, 1997). The phase of X-ray scatter for a crystalline polypeptide,
for
example, can be determined by SIRAS using crystals of a platinum derivative of
the
polypeptide. To create a platinum derivative of a crystalline polypeptide, the
crystalline
polypeptide can be soaked in a solution containing platinum. Phases obtained
by SIRAS
from the platinum derivative can then be refined using, for example, non-
crystallographic
symmetry (NCS) averaging and phase extension in a computer program such as DM
(Cowtan and Main, Acta Cryst. D49:148-157, 1993). The resulting model can be
further
derived by molecular replacement with a second data set. For example, a model
derived
from a crystalline polypeptide having space group 1222 can be refined using
molecule
replacement with a data set from a crystalline polypeptide having space group
P21212.
Phases obtained by SIRAS from crystals of the native crystalline polypeptide
and the
platinum derivative can then be used to create an electron density map of the
polypeptide.
[0183] The electron density map can be used to derive a representation of a
polypeptide or a fragment of a polypeptide by aligning a three-dimensional
model of a
polypeptide with the electron density map. For example, the electron density
map
corresponding to the native crystalline polypeptide can be aligned with the
electron
density map corresponding to the platinum derivative of the crystalline
polypeptide
complex derived by an isomorphous replacement method.
[0184] The alignment process results in a comparative model that shows the
degree to which the calculated electron density map varies from the model of
the
previously known polypeptide or the previously known complex. The comparative
model is then refined over one or more cycles (e.g., two cycles, three cycles,
four cycles,
five cycles, six cycles, seven cycles, eight cycles, nine cycles, ten cycles)
to generate a
better fit with the electron density map. Software programs such as CNS
(Brunger et al,
Acta Crystallogr. D54:905-921, 1998) and REFMAC (Collaborative Computational
Project, Number 4. The CCP4 suite: Programs for Protein Crystallography, Acta
Crystallogr. D50:760-776, 1994) can be used to refine the model. The quality
of fit in
the comparative model can be measured by, for example, an Rfactor or Rfree
value. A
smaller value of Rfactor or Rfree generally indicates a better fit.
Misalignments in the
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comparative model can be adjusted to provide a modified comparative model and
a lower
Rfactor or Rfree value. The adjustments can be based on information relating
to variations
of the polypeptide (e.g., sequence variation information, alternative
structure information,
heavy atom derivative information) as appropriate. As an example, in
embodiments in
which a model of a heavy atom derivative of a crystalline polypeptide is used,
an
adjustment can include fitting an approximate model of the native polypeptide
over the
model of the heavy atom derivative. As another example, in certain
embodiments, an
adjustment can include replacing an amino acid in the previously known
polypeptide with
an amino acid having a similar structure (a conservative amino acid change).
When
adjustments to the modified comparative model satisfy a best fit to the
electron density
map, the resulting model is that which is determined to describe the
polypeptide or
complex from which the X-ray data was derived. Methods of such processes are
disclosed, for example, in Carter and Sweet, eds., "Macromolecular
Crystallography" in
Methods in Enzymology, Vol. 277, Part B, New York: Academic Press, 1997, and
articles therein, e.g., Jones and Kjeldgaard, "Electron-Density Map
Interpretation," p.
173, and Kleywegt and Jones, "Model Building and Refinement Practice," p. 208.
[0185] A machine, such as a computer, can be programmed in memory with the
structural coordinates of a polypeptide together with a program capable of
generating a
graphical representation of the structural coordinates on a display connected
to the
machine. A software system can also be designed and/or utilized to accept and
store the
structural coordinates. The software system can be capable of generating a
graphical
representation of the structural coordinates. The software system can also be
capable of
accessing external databases to identify one or more candidate agents likely
to interact
with the polypeptide.
[0186] A machine having a memory containing structure data or a software
system containing such data can aid in the rational design or selection of a
polypeptide
agonist or antagonist of a polypeptide. For example, such a machine or
software system
can aid in the evaluation of the ability of an agent to associate with the
polypeptide, or
can aid in the modeling of compounds or proteins related by structural or
sequence
homology to the polypeptide.
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[0187] The machine can produce a representation (e. g., a two dimensional
representation, a three dimensional representation) of the polypeptide or a
fragment of the
polypeptide. A software system, for example, can cause the machine to produce
such
information. The machine can include a machine-readable data storage medium
including a data storage material encoded with machine-readable data. The
machine-
readable data can include structural coordinates of atoms of the polypeptide.
Machine-
readable storage media (e.g., data storage material) include, for example,
conventional
computer hard drives, floppy disks, DAT tape, CD-ROM, DVD, and other magnetic,
magneto-optical, optical, and other media which may be adapted for use with a
machine
(e.g., a computer). The machine can also have a working memory for storing
instructions
for processing the machine-readable data, as well as a central processing unit
(CPU)
coupled to the working memory and to the machine-readable data storage medium
for the
purpose of processing the machine-readable data into the desired three-
dimensional
representation. A display can be connected to the CPU so that the three-
dimensional
representation can be visualized by the user. Accordingly, when used with a
machine
programmed with instructions for using the data (e.g., a computer loaded with
one or
more programs of the sort described herein) the machine is capable of
displaying a
graphical representation (e.g., a two dimensional graphical representation, a
three-
dimensional graphical representation) of any of the polypeptides, polypeptide
fragments,
complexes, or complex fragments described herein.
[0188] A display (e.g., a computer display) can show a representation of a
polypeptide or a fragment of a polypeptide. The user can inspect the
representation and,
using information gained from the representation, generate a model of the
polypeptide or
polypeptide fragment bound to a ligand. The model can be generated, for
example, by
altering a previously existing representation of the polypeptide. Optionally,
the user can
superimpose a three-dimensional model of an agent on the representation of the
polypeptide. The agent can be an agonist or antagonist of the polypeptide. In
some
embodiments, the agent can be a known compound or a fragment of a known
compound.
In certain embodiments, the agent can be a previously unknown compound, or a
fragment
of a previously unknown compound.
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[0189] It can be desirable for the agent to have a shape that complements the
shape of the active site. There can be a preferred distance, or range of
distances, between
atoms of the agent and atoms of a polypeptide. Distances longer than a
preferred distance
may be associated with a weak interaction between the agent and active site.
Distances
shorter than a preferred distance may be associated with repulsive forces that
can weaken
the interaction between the agent and the polypeptide. A steric clash can
occur when
distances between atoms are too short. A steric clash occurs when the
locations of two
atoms are unreasonably close together, for example, when two atoms are
separated by a
distance less than the sum of their van der Waals radii. If a steric clash
exists, the user
can adjust the position of the agent relative to the polypeptide (e.g., a
rigid body
translation or rotation of the agent) until the steric clash is relieved. The
user can adjust
the conformation of the agent or of the polypeptide in the vicinity of the
agent in order to
relieve a steric clash. Steric clashes can also be removed by altering the
structure of the
agent, for example, by changing a "bulky group," such as an aromatic ring, to
a smaller
group, such as to a methyl or hydroxyl group, or by changing a rigid group to
a flexible
group that can accommodate a conformation that does not produce a steric
clash.
Electrostatic forces can also influence an interaction between an agent and a
ligand-
binding domain. For example, electrostatic properties can be associated with
repulsive
forces that can weaken the interaction between the agent and the polypeptide.
Electrostatic repulsion can be relieved by altering the charge of the agent,
e.g., by
replacing a positively charged group with a neutral group.
[0190] Forces that influence binding strength between a candidate agent and a
polypeptide, respectively, can be evaluated in the polypeptide/agent model.
These can
include, for example, hydrogen bonding, electrostatic forces, hydrophobic
interactions,
van der Waals interactions, dipole-dipole interactions, t-stacking forces, and
cation-t
interactions. The user can evaluate these forces visually, for example by
noting a
hydrogen bond donor/acceptor pair arranged with a distance and angle suitable
for a
hydrogen bond. Based on the evaluation, the user can alter the model to find a
more
favorable interaction between the polypeptide and the agent. Altering the
model can
include changing the three-dimensional structure of the polypeptide without
altering its
chemical structure, for example by altering the conformation of amino acid
side chains or
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backbone dihedral angles. Altering the model can include altering the position
or
conformation of the agent, as described above. Altering the model can also
include
altering the chemical structure of the agent, for example by substituting,
adding, or
removing groups. For example, if a hydrogen bond donor on the polypeptide is
located
near a hydrogen bond donor on the agent, the user can replace the hydrogen
bond donor
on the agent with a hydrogen bond acceptor.
[0191] The relative locations of an agent and a polypeptide, or their
conformations, can be adjusted to find an optimized binding geometry for a
particular
agent to the polypeptide. An optimized binding geometry is characterized by,
for
example, favorable hydrogen bond distances and angles, maximal electrostatic
attractions, minimal electrostatic repulsions, the sequestration of
hydrophobic moieties
away from an aqueous environment, and the absence of steric clashes. The
optimized
geometry can have the lowest calculated energy of a family of possible
geometries for the
polypeptide/agent complex. An optimized geometry can be determined, for
example,
through molecular mechanics or molecular dynamics calculations.
[0192] A series of representations of a polypeptide having different bound
agents can be generated. A score can be calculated for each representation.
The score
can describe, for example, an expected strength of interaction between the
polypeptide
and the agent. The score can reflect one of the factors described above that
influence
binding strength. The score can be an aggregate score that reflects more than
one of the
factors. The different agents can be ranked according to their scores.
[0193] Steps in the design of the agent can be carried out in an automated
fashion by a machine. For example, a representation of the polypeptide can be
programmed in the machine, along with representations of candidate agents. The
machine can find an optimized binding geometry for each of the candidate
agents to an
active site, and calculate a score to determine which of the agents in the
series is likely to
interact most strongly with the polypeptide.
[0194] A software system can be designed and/or implemented to facilitate
these steps. Software systems (e.g., computer programs) used to generate
representations
or perform the fitting analyses include, for example: MCSS, Ludi, QUANTA
(macromolecular X-ray crystallography software), Insight II (biological
compound
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modeling and simulation software), Cerius2 (modeling and simulation
software),
CHARMm (software for simulation of biological macromolecules) CHARMm
(software for simulation of biological macromolecules), and Modeler from
Accelrys, Inc.
(San Diego, CA); SYBYL (molecular modeling software), Unity, F1eXX, and
LEAPFROG from TRIPOS, Inc. (St. Louis, MO); AUTODOCK (Scripps Research
Institute, La Jolla, CA); GRID (Oxford University, Oxford, UK); DOCK
(University of
California, San Francisco, CA); and Flo+ and F1o99 (Thistlesoft, Morris
Township, NJ).
Other useful programs include ROCS, ZAP, FRED, Vida, and Szybki from Openeye
Scientific Software (Santa Fe, NM); Maestro, Macromodel, and Glide from
Schrodinger,
LLC (Portland, OR); MOE (Chemical Computing Group, Montreal, Quebec), Allegrow
(Boston De Novo, Boston, MA), CNS (Brunger, et al., Acta Crystall. Sect. D
54:905-921,
1997) and GOLD (Jones et al., J. Mol. Biol. 245:43-53, 1995). The structural
coordinates
can also be used to visualize the three-dimensional structure of the human
LINGO-1
polypeptide using MOLSCRIPT, RASTER3D, or PYMOL (Kraulis, J. Appl.
Crystallogr.
24: 946-950, 1991; Bacon and Anderson, J. Mol. Graph. 6: 219-220, 1998;
DeLano, The
PYMOL Molecular Graphics System (2002) DeLano Scientific, San Carlos, CA).
[0195] The agent can, for example, be selected by screening an appropriate
database, can be designed de novo by analyzing the steric configurations and
charge
potentials of a polypeptide in conjunction with the appropriate software
systems, and/or
can be designed using characteristics of known ligands. The method can be used
to
design or select agonists or antagonists of the polypeptide. A software system
can be
designed and/or implemented to facilitate database searching, and/or agent
selection and
design.
[0196] Once an agent has been designed or identified, it can be obtained or
synthesized and further evaluated for its effect on the activity of the
polypeptide. For
example, the agent can be evaluated by contacting it with the polypeptide and
measuring
the effect of the agent on an activity of the polypeptide. A method for
evaluating the
agent can include an activity assay performed in vitro or in vivo.
[0197] An activity assay can be a cell-based assay, for example. A cell based
assay can include monitoring the effect of a candidate agent on myelin
production. Such
assays for polypeptide inhibitors may involve contacting a candidate inhibitor
with cells
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expressing the polypeptide and assaying for an effect on cell morphology.
Protein levels
can be assayed by standard protein detection techniques, such as
immunohistochemistry
by Western blot analysis or in situ hybridization in cultured cells or whole
tissue sections.
[0198] Depending upon the action of the agent on the polypeptide, the agent
can
act either as an agonist or antagonist of an activity of the polypeptide. A
crystal
containing the polypeptide bound to the identified agent can be grown and the
structure
determined by X-ray crystallography. A second agent can be designed or
identified
based on the interaction of the first agent with the polypeptide.
[0199] Various molecular analysis and rational drug design techniques are
further disclosed in, for example, U.S. Patent Nos. 5,834,228, 5,939,528 and
5,856,116,
as well as in PCT Application No. PCT/US98/16879, published as WO 99/09148.
Small Molecules
[0200] Peptide analogs are commonly used in the pharmaceutical industry as
non-peptide drugs with properties analogous to those of the template peptide.
These
types of non-peptide compounds are termed "peptide mimetics" or
"peptidomimetics".
Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p.392
(1985); and
Evans et al. J. Med. Chem. 30:1229 (1987). Such compounds are often developed
with
the aid of computerized molecular modeling. Peptide mimetics that are
structurally
similar to therapeutically useful peptides may be used to produce a similar
therapeutic or
prophylactic effect. In certain embodiments, peptidomimetics are structurally
similar to a
paradigm polypeptide (i.e., a polypeptide that has a biochemical property or
pharmacological activity), such as human antibody, but have one or more
peptide
linkages optionally replaced by a linkage selected from: --CH2 NH--, --CH2 5--
, --CH2 -
CH2 --, --CH=CH-(cis and trans), --COCH2 --, --CH(OH)CH2 --, and --CH2 SO--,
by
methods well known in the art. Systematic substitution of one or more amino
acids of a
consensus sequence with a D-amino acid of the same type (e.g., D-lysine in
place of L-
lysine) may be used in certain embodiments to generate more stable peptides.
In
addition, constrained peptides comprising a consensus sequence or a
substantially
identical consensus sequence variation may be generated by methods known in
the art
(Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992)); for example, by adding
internal
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cysteine residues capable of forming intramolecular disulfide bridges which
cyclize the
peptide.
[0201] Methods of designing peptidomimetics can be combined with other
methods useful in elucidating the structure of epitopes and/or the
identification of small
molecules that bind to such epitopes, including, but not limited to, alanine
scanning, high
throughput screening, Structural Activity Relationship ("SAR") analysis, X-ray
crystallography, medicinal chemistry, NMR spectroscopy, computer analysis,
sequence
alignments, and predictions of three-dimensional protein structure. Examples
of methods
to screen for small molecules that disrupt protein-protein interactions are
known in the
art, e.g., Wells et al., Nature 450:1001-9 (2007) and Voronkov et al., Journal
of
Molecular Graphics and Modeling 26:1179-87 (2008).
[0202] In certain embodiments, an epitope defined on a target protein
comprises
amino acid residues that are not contiguous in the polymer chain. In certain
such
embodiments, small molecules, such as peptidomimetics can be designed to mimic
the
structure of the defined epitope. Computer programs are known in the art to
aid in
designing small molecules, including, but not limited to, peptidomimetics. In
certain
embodiments, computer programs known in the art can aid in designing
peptidomimetics
that mimic at least a portion of an epitope based on a crystal structure or
predicted
structure of a protein. Examples of computer programs that can used to design
a
peptidomimetic include, but are not limited to, Charmm, Insightll Glide,
Maestro and
Macromodel. In certain embodiments, a peptidomimetic designed to mimic a
defined
epitope binds to the same molecule that is naturally bound by the defined
epitope.
[0203] In certain embodiments, a peptidomimetic is provided that mimics an
epitope comprising two or more of the following amino acids of IL-22: A34,152,
T56,
K61, A66, V83, R88, P113, F121, L122, L125, or M172. In certain such
embodiments,
the peptidomimetic binds to IL-1 OR2. In certain embodiments, the
peptidomimetic that
mimics an epitope is a peptidomimetic that mimics an epitope further
comprising one or
more of the following amino acids of IL-22: Y51, N54, R55, Y114, or E117. In
certain
embodiments, the peptidomimetic that mimics an epitope is a peptidomimetic
that
mimics an epitope further comprising one or more of the following amino acids
of IL-22:
F57, L59, D67, T70, D71, V72, R73, G159,1161, K162, G165, or L169. In certain
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embodiments, the peptidomimetic that mimics an epitope is a peptidomimetic
that
mimics an epitope further comprising one or more of he following amino acids
of IL-22:
D67, R73, or K162. In certain embodiments, a peptidomimetic that mimics an
epitope
comprising two or more amino acids of IL-22 binds to one or more of IL-IOR2,
IL-22R,
or IL-2213P.
[0204] In certain embodiments, a method of making a peptidomimetic that
mimics an epitope comprising two or more of the following amino acids of IL-
22: A34,
152, T56, K61, A66, V83, R88, P113, F121, L122, L125, or M172, is provided. In
certain embodiments, the peptidomimetic that mimics an epitope is a
peptidomimetic that
mimics an epitope further comprising one or more of the following amino acids
of IL-22:
Y5 1, N54, R55, Y114, or El 17. In certain embodiments, the peptidomimetic
that mimics
an epitope is a peptidomimetic that mimics an epitope further comprising one
or more of
the following amino acids of IL-22: F57, L59, D67, T70, D71, V72, R73,
G159,1161,
K162, G165, or L169. In certain embodiments, the peptidomimetic that mimics an
epitope is a peptidomimetic that mimics an epitope further comprising one or
more of he
following amino acids of IL-22: D67, R73, or K162. In certain embodiments, the
method
of making a peptidomimetic comprises structural modeling of the epitope
through the use
of a computer.
[0205] In certain embodiments, a peptidomimetic is provided that mimics an
epitope comprising two or more of the following amino acids of IL-22: F57,
L59, D67,
V72, G159, I161, K162, or L169. In certain such embodiments, the
peptidomimetic
binds to IL-22R. In certain embodiments, the peptidomimetic that mimics an
epitope is a
peptidomimetic that mimics an epitope further comprising one or more of the
following
amino acids of IL-22: T70, D71, R73, or G165. In certain embodiments, the
peptidomimetic that mimics an epitope is a peptidomimetic that mimics an
epitope
further comprising one or more of the following amino acids of IL-22: A34,
Y51,152,
N54, R55, T56, K61, A66, V83, R88, P113, Y114, El 17, F121, L122, L125, or
M172.
In certain embodiments, the peptidomimetic that mimics an epitope is a
peptidomimetic
that mimics an epitope further comprising one or more of the following amino
acids of
IL-22: R73 or V83. In certain embodiments, a peptidomimetic that mimics an
epitope
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comprising two or more amino acids of IL-22 binds to one or more of IL-IOR2,
IL-22R,
or IL-22BP.
[0206] In certain embodiments, a method of making a peptidomimetic that
mimics an epitope comprising two or more of the following amino acids of IL-
22: F57,
L59, D67, V72, G159,1161, K162, or L169. In certain embodiments, the
peptidomimetic
that mimics an epitope is a peptidomimetic that mimics an epitope further
comprising one
or more of the following amino acids of IL-22: T70, D71, R73, or G165. In
certain
embodiments, the peptidomimetic that mimics an epitope is a peptidomimetic
that
mimics an epitope further comprising one or more of the following amino acids
of IL-22:
A34, Y51,152, N54, R55, T56, K61, A66, V83, R88, P113, Y114, E117, F121, L122,
L125, or M172. In certain embodiments, the peptidomimetic that mimics an
epitope is a
peptidomimetic that mimics an epitope further comprising one or more of the
following
amino acids of IL-22: R73 or V83. In certain embodiments, the method of making
a
peptidomimetic comprises structural modeling of the epitope through the use of
a
computer.
[0207] In certain embodiments, a peptidomimetic is provided that mimics an
epitope comprising two or more of the following amino acids of IL-22: D67,
R73, V83,
and K162. In certain such embodiments, the peptidomimetic binds to IL-22BP. In
certain embodiments, the peptidomimetic that mimics an epitope is a
peptidomimetic that
mimics an epitope further comprising one or more of the following amino acids
of IL-22:
A34, Y51,152, N54, R55, T56, K61, A66, R88, P113, Y114, E117, F121, L122,
L125, or
M172. In certain embodiments, the peptidomimetic that mimics an epitope is a
peptidomimetic that mimics an epitope further comprising one or more of the
following
amino acids of IL-22: F57, L59, T70, D71, V72, G159,1161, G165, or L169. In
certain
embodiments, a peptidomimetic that mimics an epitope comprising two or more
amino
acids of IL-22 binds to one or more of IL-IOR2, IL-22R, or IL-22BP.
[0208] In certain embodiments, a method of making a peptidomimetic that
mimics an epitope comprising two or more of the following amino acids of IL-
22: D67,
R73, V83, or K162. In certain embodiments, the peptidomimetic that mimics an
epitope
is a peptidomimetic that mimics an epitope further comprising one or more of
the
following amino acids of IL-22: A34, Y51,152, N54, R55, T56, K61, A66, R88,
P113,
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Y114, E117, F121, L122, L125, or M172. In certain embodiments, the
peptidomimetic
that mimics an epitope is a peptidomimetic that mimics an epitope further
comprising one
or more of the following amino acids of IL-22: F57, L59, T70, D71, V72, G159,
I161,
G165, or L169. In certain embodiments, the method of making a peptidomimetic
comprises structural modeling of the epitope through the use of a computer.
Pharmaceutical Compositions
[0209] IL-22 binding agents, e.g., IL-22 antagonists, (e.g., anti-IL-22
antibodies
and fragments thereof (e.g., antigen-binding fragments thereof)) can be used
as a
pharmaceutical composition when combined with a pharmaceutically acceptable
carrier.
Such a composition may contain, in addition to the IL-22-agonists or
antagonists and
carrier, various diluents, fillers, salts, buffers, stabilizers, solubilizers,
and other materials
well known in the art. The term "pharmaceutically acceptable" means a non-
toxic
material that does not interfere with the effectiveness of the biological
activity of the
active ingredient(s). The characteristics of the carrier will depend on the
route of
administration.
[0210] In certain embodiments, a pharmaceutical composition may be in the
form of a liposome in which IL-22- antagonists are combined, in addition to
other
pharmaceutically acceptable carriers, with amphipathic agents such as lipids
which exist
in aggregated form as micelles, insoluble monolayers, liquid crystals, or
lamellar layers
which in aqueous solution. Suitable lipids for liposomal formulation include,
without
limitation, monoglycerides, diglycerides, sulfatides, lysolecithin,
phospholipids, saponin,
bile acids, and the like. Preparation of such liposomal formulations is within
the level of
skill in the art, as disclosed, for example, in U.S. Pat. No. 4,235,871; U.S.
Pat. No.
4,501,728; U.S. Pat. No. 4,837,028; and U.S. Pat. No. 4,737,323, all of which
are
incorporated herein by reference.
[0211] In certain embodiments, a therapeutically effective amount of an IL-22
antagonist is administered to a subject, e.g., mammal (e.g., a human). An IL-
22
antagonist may be administered either alone or in combination with other
therapies, e.g.,
anti-inflammatory agents described in more detail below. When co-administered
with
one or more agents, an IL-22 antagonist may be administered either
simultaneously with
the second agent, or sequentially. If administered sequentially, the attending
physician
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will decide on the appropriate sequence of administering an IL-22 antagonist
in
combination with other agents.
[0212] Administration of an IL-22 antagonist used in a pharmaceutical
composition can be carried out in a variety of conventional ways, such as oral
ingestion,
inhalation, or cutaneous, subcutaneous, or intravenous injection. In certain
embodiments,
intravenous administration to the patient is preferred.
[0213] When a therapeutically effective amount of an IL-22 antagonist is
administered orally, the binding agent will be in the form of a tablet,
capsule, powder,
solution or elixir. When administered in tablet form, a pharmaceutical
composition may
additionally contain a solid carrier such as a gelatin. The tablet, capsule,
and powder
contain from about 5 to 95% binding agent, and preferably from about 25 to 90%
binding
agent. When administered in liquid form, a liquid carrier such as water,
petroleum, oils
of animal or plant origin such as peanut oil, mineral oil, soybean oil, or
sesame oil, or
synthetic oils may be added. The liquid form of the pharmaceutical composition
may
further contain physiological saline solution, dextrose or other saccharide
solution, or
glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When
administered in liquid form, the pharmaceutical composition contains from
about 0.5 to
90% by weight of the binding agent, and preferably from about 1 to 50% the
binding
agent.
[0214] When a therapeutically effective amount of an IL-22 antagonist is
administered by intravenous, cutaneous or subcutaneous injection, binding
agent will be
in the form of a pyrogen-free, parenterally acceptable aqueous solution. The
preparation
of such parenterally acceptable protein solutions, having due regard to pH,
isotonicity,
stability, and the like, is within the skill in the art. In certain
embodiments, a
pharmaceutical composition for intravenous, cutaneous, or subcutaneous
injection should
contain, in addition to binding agent an isotonic vehicle such as Sodium
Chloride
Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium
Chloride
Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
In certain
embodiments, a pharmaceutical composition may also contain stabilizers,
preservatives,
buffers, antioxidants, or other additives known to those of skill in the art.
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[0215] In certain embodiments, the amount of an IL-22 binding agent in a
pharmaceutical composition will depend upon the nature and severity of the
condition
being treated, and on the nature of prior treatments that the patient has
undergone. In
certain embodiments, the attending physician will decide the amount of binding
agent
with which to treat each individual patient. In certain embodiments, the
attending
physician will initially administer low doses of binding agent and observe the
patient's
response. In certain embodiments, larger doses of binding agent may be
administered
until the optimal therapeutic effect is obtained for the patient, and at that
point the dosage
is not generally increased further. In certain embodiments, the various
pharmaceutical
compositions should contain about 0.1 g to about 100 mg IL-22 binding agent
per kg
body weight.
[0216] In certain embodiments, the duration of intravenous therapy will vary,
depending on the severity of the disease being treated and the condition and
potential
idiosyncratic response of each individual patient. In certain embodiments, the
duration of
each application of the IL-22 binding agent will be in the range of 12 to 24
hours of
continuous intravenous administration. In certain embodiments, the attending
physician
will decide on the appropriate duration of intravenous therapy using a
pharmaceutical
composition.
Uses of IL-22 Agonists and IL-22 Antagonists
[0217] IL-22 is a cytokine involved in pro-inflammatory actions, e.g.,
inducing
an acute phase response. As described in U.S. Published Patent Application No.
2005-
0042220, IL-22 induces changes associated with those caused by inflammatory
cytokines
(such as IL-1 and TNFa), and inhibitors of IL-22 ameliorate symptoms of
rheumatoid
arthritis. Therefore, IL-22, and/or agents that increase levels of IL-22 or
mimic the
actions of IL-22 are useful as agonists in certain clinical indications, and
antagonists of
this molecule are useful in other clinical situations, particularly in those
in which
modulation of an inflammatory state is desired. Whether the agonist or
antagonist is
preferred depends on the particular aspects of the disease pathology, such as
the cell
types involved, the nature of the stimulus and the cellular microenvironment.
[0218] Human IL-22 agonists include without limitation human IL-22 proteins
and fragments, deletion mutants and addition mutants thereof; and peptide and
small
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molecule compounds that interact with the receptor or other target to which
human IL-22
is directed. Human IL-22 antagonists include without limitation antibodies
directed to
human IL-22 proteins; soluble forms of the receptor or other target to which
human IL-22
is directed; antibodies directed to the receptor or other target to which
human IL-22 is
directed; and peptide and small molecule compounds that inhibit or interfere
with the
interaction of human IL-22 with its receptor or other target.
[0219] In certain embodiments, a method is provided of inhibiting at least one
IL-22-associated activity, by contacting a cell, e.g., an epithelial cell,
with an IL-22
antagonist (e.g., an anti-IL-22 antibody or an antigen-binding fragment
thereof), in an
amount sufficient to inhibit the activity. Antagonists of IL-22 (e.g., a
neutralizing
antibody, as described herein) can also be administered to subjects for which
inhibition of
an immune IL-22-associated activity is desired. For example, inhibition of IL-
22-
associated activities would be desired in order to prevent, treat, ameliorate
or reduce at
least one symptom of an IL-22-associated disorder, such as, for example,
autoimmune
disorders, e.g., arthritis (including rheumatoid arthritis, juvenile
rheumatoid arthritis,
osteoarthritis, psoriatic arthritis, lupus-associated arthritis or ankylosing
spondylitis),
scleroderma, systemic lupus erythematosis, HIV, Sjogren's syndrome,
vasculitis,
multiple sclerosis, autoimmune thyroiditis, dermatitis (including atopic
dermatitis and
eczematous dermatitis), myasthenia gravis, inflammatory bowel disease (IBD),
Crohn's
disease, colitis, diabetes mellitus (type I); inflammatory conditions of,
e.g., the skin (e.g.,
psoriasis), cardiovascular system (e.g., atherosclerosis), nervous system
(e.g.,
Alzheimer's disease), liver (e.g., hepatitis), kidney (e.g., nephritis) and
pancreas (e.g.,
pancreatitis); cardiovascular disorders, e.g., cholesterol metabolic
disorders, oxygen free
radical injury, ischemia; disorders associated with wound healing; respiratory
disorders,
e.g., asthma and COPD (e.g., cystic fibrosis); acute inflammatory conditions
(e.g.,
endotoxemia, sepsis and septicaemia, toxic shock syndrome and infectious
disease);
transplant rejection and allergy. In one embodiment, the IL-22-associated
disorder is, an
arthritic disorder, e.g., a disorder chosen from one or more of rheumatoid
arthritis,
juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, or
ankylosing spondylitis; a
respiratory disorder (e.g., asthma, chronic obstructive pulmonary disease
(COPD); or an
inflammatory condition of, e.g., the skin (e.g., psoriasis), cardiovascular
system (e.g.,
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atherosclerosis), nervous system (e.g., Alzheimer's disease), liver (e.g.,
hepatitis), kidney
(e.g., nephritis), pancreas (e.g., pancreatitis), and gastrointestinal organs,
e.g., colitis,
Crohn's disease and IBD.
[0220] Reduction of IL-22 activity by using a neutralizing anti-IL-22 antibody
has been shown to ameliorate inflammatory symptoms in mouse collagen-induced
arthritis (CIA) animal models. In addition, it has been demonstrated that
expression of
IL-22 mRNA is upregulated in the paws of CIA mice (See, e.g., U.S. Published
Patent
Application No. 2005-0042220, Examples 9 and 10). ). Accordingly, IL-22
antagonists
can be used to induce immune suppression in vivo, e.g., for treating or
preventing IL-22-
associated disorders, in a subject.
[0221] As used herein, the term "subject" is intended to include human and non-
human animals. Human animals include a human patient having a disorder
characterized
by abnormal IL-22 activity. The term "non-human animals" includes all
vertebrates, e.g.,
mammals and non-mammals, such as non-human primates, rodents, sheep, llamas,
camels, dogs, cows, sharks, fish, chickens, amphibians, reptiles, etc.
[0222] Non-limiting examples of IL-22-associated disorders that can be treated
or prevented include, but are not limited to, transplant rejection, autoimmune
diseases
(including, for example, diabetes mellitus, arthritis (including rheumatoid
arthritis,
juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple
sclerosis,
encephalomyelitis, myasthenia gravis, systemic lupus erythematosis, autoimmune
thyroiditis, dermatitis (including atopic dermatitis and eczematous
dermatitis), Sjogren's
Syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis,
keratoconjunctivitis,
ulcerative colitis, spondyoarthropathy, ankylosing spondylitis, intrinsic
asthma, allergic
asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug
eruptions,
leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis,
allergic
encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic
bilateral
progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia,
idiopathic
thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active
hepatitis,
Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease,
sarcoidosis,
primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis);
respiratory
disorders, e.g., asthma or COPD; inflammatory conditions of the skin (e.g.,
psoriasis),
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liver (e.g., hepatitis), kidney (e.g., nephritis) and pancreas (e.g.,
pancreatitis); graft-
versus-host disease, and allergy such as, atopic allergy; cancers (e.g., solid
or soft tissue
tumors), arthritic disorders (e.g., rheumatoid arthritis, juvenile rheumatoid
arthritis,
osteoarthritis, psoriatic arthritis, and ankylosing spondylitis), multiple
sclerosis, type I
diabetes, lupus (SLE), IBD, ulcerative colitis, Crohn's disease, COPD, asthma,
vasculitis,
allergy, scleroderma and inflammatory conditions of the skin (e.g.,
psoriasis), liver (e.g.,
hepatitis), kidney (e.g., nephritis) and pancreas (e.g., pancreatitis).
[0223] In certain embodiments, IL-22 antagonists, alone or in combination
with,
other therapeutic agents as described herein (e.g., TNF antagonists) can be
used to treat
multiple myeloma and related B lymphocytic malignancies (Brenne, A. et al.
(2002)
Blood Vol. 99(10):3756-3762).
[0224] In certain embodiments, the IL-22 antagonists, e.g., pharmaceutical
compositions thereof, are administered in combination therapy, i.e., combined
with other
agents, e.g., therapeutic agents that are useful for treating pathological
conditions or
disorders, such as immune and inflammatory disorders.
[0225] For example, the combination therapy can include one or more IL-22
antagonists, e.g., an antibody or an antigen-binding fragment thereof as
described herein
(e.g., a chimeric, humanized, human, or in vitro generated antibody or antigen-
binding
fragment thereof against IL-22) co-formulated with, and/or co-administered
with, one or
more additional therapeutic agents, e.g., one or more cytokine and growth
factor
inhibitors, immunosuppressants, anti-inflammatory agents, metabolic
inhibitors, enzyme
inhibitors, and/or cytotoxic or cytostatic agents, as described in more detail
below.
Furthermore, one or more IL-22 antagonists described herein may be used in
combination
with two or more of the therapeutic agents described herein.
[0226] Such combination therapies may advantageously utilize lower dosages of
the administered therapeutic agents, thus avoiding possible toxicities or
complications
associated with the various monotherapies.
[0227] Accordingly, inhibition of IL-22 activity using, e.g., an anti-11,22
antibody or fragment thereof described herein, may provide a more effective
tissue-
specific, anti-inflammatory activity than systemic anti-inflammatory
modalities as
described herein. Furthermore, inhibition of local IL-22 activity using, e.g.,
an anti-11,22
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antibody or fragment thereof described herein, may provide a useful candidate
for
combination with systemic anti-inflammatory modalities described herein.
[0228] In certain embodiments, one or more IL-22 antagonist described herein
may be co-formulated with, and/or co-administered with, one or more additional
agents
such as other cytokine or growth factor antagonists (e.g., soluble receptors,
peptide
inhibitors, small molecules, ligand fusions); or antibodies or antigen-binding
fragments
thereof that bind to other targets (e.g., antibodies that bind to other
cytokines or growth
factors, their receptors, or other cell surface molecules); and anti-
inflammatory cytokines
or agonists thereof. Non-limiting examples of the agents that can be used in
combination
with the IL-22 antagonists described herein, include, but are not limited to,
antagonists of
one or more interleukins (ILs) or their receptors, e.g., antagonists of IL-1,
IL-2, IL-6, IL-
7, IL8, IL-12, IL-13, IL-15, IL-16, IL-18, and IL-21/IL-21R; antagonists of
cytokines or
growth factors or their receptors, such as tumor necrosis factor (TNF), LT,
EMAP-II,
GM-CSF, FGF and PDGF. IL-22 antagonists can also be combined with inhibitors
of,
e.g., antibodies to, cell surface molecules such as CD2, CD3, CD4, CD8, CD25,
CD28,
CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, or their ligands,
including CD154 (gp39 or CD40L), or LFA-1/ICAM-1 and VLA-4/VCAM-1(Yusuf-
Makagiansar H. et al. (2002) Med Res Rev 22(2):146-67). In certain
embodiments,
antagonists that can be used in combination with IL-22 antagonists described
herein
include antagonists of IL-1, IL-12, TNFa, IL-15, IL-17, IL-18, and IL-21/IL-
21R.
[0229] Examples of those agents include, but are not limited to, IL-12
antagonists, such as chimeric, humanized, human or in vitro generated
antibodies (or
antigen-binding fragments thereof) that bind to IL- 12 (preferably human IL-
12), e.g., the
antibody disclosed in WO 00/56772, Genetics Institute/BASF); IL-12 receptor
inhibitors,
e.g., antibodies to human IL-12 receptor; and soluble fragments of the IL-12
receptor,
e.g., human IL-12 receptor. Examples of IL-15 antagonists include antibodies
(or
antigen-binding fragments thereof) against IL-15 or its receptor, e.g.,
chimeric,
humanized, human or in vitro generated antibodies to human IL-15 or its
receptor,
soluble fragments of the IL-15 receptor, and IL-15-binding proteins. Examples
of IL-18
antagonists include antibodies, e.g., chimeric, humanized, human or in vitro
generated
antibodies (or antigen-binding fragments thereof), to human IL- 18, soluble
fragments of
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the IL-18 receptor, and IL-18 binding proteins (IL-18BP, Mallet et al. (2001)
Circ. Res.
28). Examples of IL-1 antagonists include Interleukin-1-converting enzyme
(ICE)
inhibitors, such as Vx740, IL-1 antagonists, e.g., IL- IRA (ANIKINRA, AMGEN),
sIL 1 RII (Immunex), and anti-IL-1 receptor antibodies (or antigen-binding
fragments
thereof).
[0230] Examples of TNF antagonists include, but are not limited to, chimeric,
humanized, human or in vitro generated antibodies (or antigen-binding
fragments
thereof) to TNF (e.g., human TNF a), such as D2E7, (human TNFa antibody, US.
6,258,562; BASF), CDP-571/CDP-870/BAY-10-3356 (humanized anti-TNFa antibody;
Celltech/Pharmacia), cA2 (chimeric anti-TNFa antibody; RemicadeTM, Centocor);
anti-
TNF antibody fragments (e.g., CPD870); soluble fragments of the TNF receptors,
e.g.,
p55 or p75 human TNF receptors or derivatives thereof, e.g., 75 kdTNFR-IgG (75
kD
TNF receptor-IgG fusion protein, EnbrelTM; Immunex; see e.g., Arthritis &
Rheumatism
(1994) Vol. 37, S295; J. Invest. Med. (1996) Vol. 44, 235A), p55 kdTNFR-IgG
(55 kD
TNF receptor-IgG fusion protein (Lenercept)); enzyme antagonists, e.g., TNFa
converting enzyme (TACE) inhibitors (e.g., an alpha-sulfonyl hydroxamic acid
derivative, WO 01/55112, and N-hydroxyformamide TACE inhibitor GW 3333, -005,
or
-022); and TNF-bp/s-TNFR (soluble TNF binding protein; see e.g., Arthritis &
Rheumatism (1996) Vol. 39, No. 9 (supplement), S284; Amer. J. Physiol. - Heart
and
Circulatory Physiology (1995) Vol. 268, pp. 37-42). In certain embodiments,
TNF
antagonists are soluble fragments of the TNF receptors, e.g., p55 or p75 human
TNF
receptors or derivatives thereof, e.g., 75 kdTNFR-IgG, and TNFa converting
enzyme
(TACE) inhibitors.
[0231] In certain embodiments, the IL-22 antagonists described herein can be
administered in combination with one or more of the following: IL-13
antagonists, e.g.,
soluble IL-13 receptors (sIL-13) and/or antibodies against IL-13; IL-2
antagonists, e.g.,
DAB 486-IL-2 and/or DAB 389-IL-2 (IL-2 fusion proteins; Seragen; see e.g.,
Arthritis &
Rheumatism (1993) Vol. 36, 1223), and/or antibodies to IL-2R, e.g., anti-Tac
(humanized
anti-IL-2R; Protein Design Labs, Cancer Res. 1990 Mar 1;50(5):1495-502). Yet
another
combination includes IL-21 antagonists in combination with non-depleting anti-
CD4
inhibitors (IDEC-CE9.1/SB 210396 (non-depleting primatized anti-CD4 antibody;
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IDEC/SmithKline). In certain embodiments, combinations include antagonists of
the co-
stimulatory pathway CD80 (B7.1) or CD86 (B7.2) including antibodies, soluble
receptors
or antagonistic ligands; as well as p-selectin glycoprotein ligand (PSGL),
anti-
inflammatory cytokines, e.g., IL-4 (DNAX/Schering); IL-10 (SCH 52000;
recombinant
IL-10 DNAX/Schering); IL-13 and TGF(3, and agonists thereof (e.g., agonist
antibodies).
[0232] In certain embodiments, one or more IL-22 antagonists can be co-
formulated with, and/or co-administered with, one or more anti-inflammatory
drugs,
immunosuppressants, or metabolic or enzymatic inhibitors. Non-limiting
examples of the
drugs or inhibitors that can be used in combination with the IL-22 antagonists
described
herein, include, but are not limited to, one or more of. non-steroidal anti-
inflammatory
drug(s) (NSAID5), e.g., ibuprofen, Tenidap (see e.g., Arthritis & Rheumatism
(1996)
Vol. 39, No. 9 (supplement), S280)), Naproxen (see e.g., Neuro Report (1996)
Vol. 7, pp.
1209-1213), Meloxicam, Piroxicam, Diclofenac, and Indomethacin; Sulfasalazine
(see
e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S281);
corticosteroids
such as prednisolone; cytokine suppressive anti-inflammatory drug(s) (CSAID5);
inhibitors of nucleotide biosynthesis, e.g., inhibitors of purine
biosynthesis, folate
antagonists (e.g., methotrexate (N-[4-[[(2,4-diamino-6-
pteridinyl)methyl]methylamino]benzoyl]-L-glutamic acid); and inhibitors of
pyrimidine
biosynthesis, e.g., dihydroorotate dehydrogenase (DHODH) inhibitors (e.g.,
leflunomide
(see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), 5131;
Inflammation Research (1996) Vol. 45, pp. 103-107). In certain embodiments,
therapeutic agents for use in combination with IL-22 antagonists include
NSAIDs,
CSAIDs, (DHODH) inhibitors (e.g., leflunomide), and folate antagonists (e.g.,
methotrexate).
[0233] Examples of additional inhibitors include, but are not limited to, one
or
more of: corticosteroids (oral, inhaled and local injection);
immunosuppresants, e.g.,
cyclosporin, tacrolimus (FK-506); and mTOR inhibitors, e.g., sirolimus
(rapamycin) or
rapamycin derivatives, e.g., soluble rapamycin derivatives (e.g., ester
rapamycin
derivatives, e.g., CCI-779 (Elit. L. (2002) Current Opinion Investig. Drugs
3(8): 1249-
53; Huang, S. et al. (2002) Current Opinion investig. Drugs 3(2):295-304);
agents which
interfere with signaling by proinflammatory cytokines such as TNFa or IL-1
(e.g. IRAK,
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NIX, IKK, p38 or MAP kinase inhibitors); COX2 inhibitors, e.g., celecoxib and
variants
thereof, MK-966, see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9
(supplement),
S81); phosphodiesterase inhibitors, e.g., 8973401 (phosphodiesterase Type IV
inhibitor;
see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S282));
phospholipase inhibitors, e.g., inhibitors of cytosolic phospholipase 2
(cPLA2) (e.g.,
trifluoromethyl ketone analogs (U.S. 6,350,892)); inhibitors of vascular
endothelial cell
growth factor or growth factor receptor, e.g., VEGF inhibitor and/or VEGF-R
inhibitor;
and inhibitors of angiogenesis. In certain embodiments, therapeutic agents for
use in
combination with IL-22 antagonists immunosuppresants, e.g., cyclosporin,
tacrolimus
(FK-506); and mTOR inhibitors, e.g., sirolimus (rapamycin) or rapamycin
derivatives,
e.g., soluble rapamycin derivatives (e.g., ester rapamycin derivatives, e.g.,
CCI-779;
COX2 inhibitors, e.g., celecoxib and variants thereof; and phospholipase
inhibitors, e.g.,
inhibitors of cytosolic phospholipase 2 (cPLA2) (e.g., trifluoromethyl ketone
analogs).
[0234] Additional examples of therapeutic agents that can be combined with an
IL-22 antagonist include, but are not limited to, one or more of: 6-
mercaptopurines (6-
MP); azathioprine sulphasalazine; mesalazine; olsalazine
chloroquinine/hydroxychloroquine; pencillamine; aurothiomalate (intramuscular
and
oral); azathioprine; cochicine; beta-2 adrenoreceptor agonists (salbutamol,
terbutaline,
salmeteral); xanthines (theophylline, aminophylline); cromoglycate;
nedocromil;
ketotifen; ipratropium and oxitropium; mycophenolate mofetil; adenosine
agonists;
antithrombotic agents; complement inhibitors; and adrenergic agents.
[0235] The use of the IL-22 antagonists disclosed herein in combination with
other therapeutic agents to treat or prevent specific immune disorders is
discussed in
further detail below.
[0236] Non-limiting examples of agents for treating or preventing arthritic
disorders (e.g., rheumatoid arthritis, inflammatory arthritis, rheumatoid
arthritis, juvenile
rheumatoid arthritis, osteoarthritis and psoriatic arthritis), with which an
IL-22
antagonists can be combined include one or more of the following: IL- 12
antagonists as
described herein, NSAIDs; CSAIDs; TNF's, e.g., TNFa, antagonists as described
herein;
non-depleting anti-CD4 antibodies as described herein; IL-2 antagonists as
described
herein; anti-inflammatory cytokines, e.g., IL-4, IL-10, IL- 13 and TGFa, or
agonists
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thereof; IL-1 or IL-1 receptor antagonists as described herein);
phosphodiesterase
inhibitors as described herein; COX-2 inhibitors as described herein; Iloprost
(see e.g.,
Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S82); methotrexate;
thalidomide (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9
(supplement), S282)
and thalidomide-related drugs (e.g., Celgen); leflunomide; inhibitor of
plasminogen
activation, e.g., tranexamic acid; see e.g., Arthritis & Rheumatism (1996)
Vol. 39, No. 9
(supplement), S284); cytokine inhibitor, e.g., T-614; see e.g., Arthritis &
Rheumatism
(1996) Vol. 39, No. 9 (supplement), S282); prostaglandin El (see e.g.,
Arthritis &
Rheumatism (1996) Vol. 39, No. 9 (supplement), S282); azathioprine (see e.g.,
Arthritis
& Rheumatism (1996) Vol. 39, No. 9 (supplement), S281); an inhibitor of
interleukin-1
converting enzyme (ICE); zap-70 and/or lck inhibitor (inhibitor of the
tyrosine kinase
zap-70 or lck); an inhibitor of vascular endothelial cell growth factor or
vascular
endothelial cell growth factor receptor as described herein; an inhibitor of
angiogenesis as
described herein; corticosteroid anti-inflammatory drugs (e.g., SB203580); TNF-
convertase inhibitors; interleukin-i i (see e.g., Arthritis & Rheumatism
(1996) Vol. 39,
No. 9 (supplement), S296); interleukin-13 (see e.g., Arthritis & Rheumatism
(1996) Vol.
39, No. 9 (supplement), S308); interleukin-17 inhibitors (see e.g., Arthritis
&
Rheumatism (1996) Vol. 39, No. 9 (supplement), S120); gold; penicillamine;
chloroquine; hydroxychloroquine; chlorambucil; cyclophosphamide; cyclosporine;
total
lymphoid irradiation; anti-thymocyte globulin; CD5-toxins; orally-administered
peptides
and collagen; lobenzarit disodium; Cytokine Regulating Agents (CRAs) HP228 and
HP466 (Houghten Pharmaceuticals, Inc.); ICAM-1 antisense phosphorothioate
oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble
complement
receptor 1 (TP10; T Cell Sciences, Inc.); prednisone; orgotein;
glycosaminoglycan
polysulphate; minocycline; anti-IL2R antibodies; marine and botanical lipids
(fish and
plant seed fatty acids; see e.g., DeLuca et al. (1995) Rheum. Dis. Clin. North
Am.
21:759-777); auranofin; phenylbutazone; meclofenamic acid; flufenamic acid;
intravenous immune globulin; zileuton; mycophenolic acid (RS61443); tacrolimus
(FK-
506); sirolimus (rapamycin); amiprilose (therafectin); cladribine (2-
chlorodeoxyadenosine); and azaribine. In certain embodiments, combinations
include
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one or more IL-21 antagonists in combination with methotrexate or leflunomide,
and in
moderate or severe rheumatoid arthritis cases, cyclosporine.
[0237] In certain embodiments, examples of inhibitors to use in combination
with IL-22 antagonists to treat arthritic disorders include TNF antagonists
(e.g., chimeric,
humanized, human or in vitro generated antibodies, or antigen-binding
fragments thereof,
that bind to TNF; soluble fragments of a TNF receptor, e.g., p55 or p75 human
TNF
receptor or derivatives thereof, e.g., 75 kdTNFR-IgG (75 kD TNF receptor-IgG
fusion
protein, EnbrelTM), p55 kD TNF receptor-IgG fusion protein; TNF enzyme
antagonists,
e.g., TNFa converting enzyme (TACE) inhibitors); antagonists of IL- 12, IL-
15, IL- 17,
IL-18, IL-21/IL-21R; T cell and B cell depleting agents (e.g., anti-CD4 or
anti-CD22
antibodies); small molecule inhibitors, e.g., methotrexate and leflunomide;
sirolimus
(rapamycin) and analogs thereof, e.g., CCI-779; Cox-2 and cPLA2 inhibitors;
NSAIDs;
p38 inhibitors, TPL-2, Mk-2 and NFkb inhibitors; RAGE or soluble RAGE; P-
selectin or
PSGL-1 inhibitors (e.g., small molecule inhibitors, antibodies thereto, e.g.,
antibodies to
P-selectin); estrogen receptor beta (ERB) agonists or ERB-NFkI3 antagonists.
Most
additional therapeutic agents that can be co-administered and/or co-formulated
with one
or more IL-22 antagonists include one or more of: a soluble fragment of a TNF
receptor,
e.g., p55 or p75 human TNF receptor or derivatives thereof, e.g., 75 kd
TNFRIgG (75 kD
TNF receptor-IgG fusion protein, EnbrelTM) methotrexate, leflunomide, or a
sirolimus
(rapamycin) or an analog thereof, e.g., CCI-779.
[0238] Non-limiting examples of agents for treating or preventing multiple
sclerosis with which an IL-22 antagonists can be combined include the
following:
interferons, e.g., interferon-alphala (e.g., AvonexTM; Biogen) and interferon-
1(3
(BetaseronTM Chiron/Berlex); Copolymer 1 (Cop-1; CopaxoneTM Teva
Pharmaceutical
Industries, Inc.); hyperbaric oxygen; intravenous immunoglobulin; clabribine;
TNF
antagonists as described herein; corticosteroids; prednisolone;
methylprednisolone;
azathiopnne; cyclophosphamide; cyclosporine; methotrexate; 4-aminopyridine;
and
tizanidine. Additional antagonists that can be used in combination with IL-22
antagonists
include antibodies to or antagonists of other human cytokines or growth
factors, for
example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-12 IL-15, IL-16, IL-18,
EMAP-11,
GMCSF, FGF, and PDGF. IL-21 antagonists as described herein can be combined
with
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antibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28,
CD30,
CD40, CD45, CD69, CD80, CD86, CD90 or their ligands. The IL-22 antagonists may
also be combined with agents, such as methotrexate, cyclosporine, FK506,
rapamycin,
mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen,
corticosteroids
such as prednisolone, phosphodiesterase inhibitors, adenosine agonists,
antithrombotic
agents, complement inhibitors, adrenergic agents, agents which interfere with
signaling
by proinflammatory cytokines as described herein, IL-I(3 converting enzyme
inhibitors
(e.g., Vx740), anti-P7s, PSGL, TACE inhibitors, T-cell signaling inhibitors
such as
kinase inhibitors, metal loproteinase inhibitors, sulfasalazine, azathloprine,
6-
mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine
receptors
and derivatives thereof, as described herein, and anti-inflammatory cytokines
(e.g. IL-4,
IL-10, IL-13 and TGF).
[0239] Examples of therapeutic agents for multiple sclerosis with which the IL-
22 antagonists can be combined include, but are not limited to, interferon-(3,
for example,
IFNb-1 a and 1 FNb-1(3; copaxone, corticosteroids, IL-I inhibitors, TNF
inhibitors,
antibodies to CD40 ligand and CD80, IL-12 antagonists.
[0240] Non-limiting examples of agents for treating or preventing inflammatory
bowel disease or Crohn's disease with which an IL-22 antagonist can be
combined
include the following: budenoside; epidermal growth factor; corticosteroids;
cyclosporin,
sulfasalazine; aminosalicylates; 6-mercaptopurine; azathioprine;
metronidazole;
lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide; antioxidants;
thromboxane
inhibitors; IL-1 receptor antagonists; anti-IL-1 monoclonal antibodies; anti-
IL-6
monoclonal antibodies; growth factors; elastase inhibitors; pyridinyl-
imidazole
compounds; TNF antagonists as described herein; IL-4, IL-10, IL-13 and/or
TGFI3
cytokines or agonists thereof (e.g., agonist antibodies); interleukin-11;
glucuronide- or
dextran-conjugated prodrugs of prednisolone, dexamethasone or budesonide; ICAM-
1
antisense phosphorothioate oligodeoxynucleotides (ISIS 2302; Isis
Pharmaceuticals,
Inc.); soluble complement receptor 1 JP 10; T Cell Sciences, Inc.); slow-
release
mesalazine; methotrexate; antagonists of Platelet Activating Factor (PAF);
ciprofloxacin;
and lignocaine.
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[0241] In certain embodiments, an IL-22 antagonists can be used in combination
with one or more antibodies directed at other targets involved in regulating
immune
responses, e.g., transplant rejection or graft-v-host disease. Non-limiting
examples of
agents for treating or preventing immune responses with which an IL-22
antagonist can
be combined include the following: antibodies against cell surface molecules,
including
but not limited to CD25 (interleukin-2 receptor-a), CDI la (LFA-1), CD54 (ICAM-
1),
CD4, CD45, CD28/CTLA4, CD80 (B7-2) and/or CD86 (B7-2). In yet another
embodiment, an IL-22 antagonist is used in combination with one or more
general
immunosuppressive agents, such as cyclosporin A or FK506.
[0242] In certain embodiments, kits for carrying out the combined
administration of the IL-22 antagonists with other therapeutic compounds are
provided.
In certain embodiments, the kit comprises one or more binding agents
formulated in a
pharmaceutical carrier, and at least one agent, e.g., therapeutic agent,
formulated as
appropriate, in one or more separate pharmaceutical preparations.
Diagnostic Assays
[0243] Antibodies may also be used to detect the presence of IL-22 in
biological
samples. By correlating the presence or level of these proteins with a medical
condition,
one of skill in the art can diagnose the associated medical condition. For
example, IL-22
induces changes associated with those caused by inflammatory cytokines (such
as IL-1
and TNFa), and inhibitors of IL-22 ameliorate symptoms of rheumatoid arthritis
(WO
2005/000897 A2). Exemplary medical conditions that may be diagnosed by the
antibodies include, but are not limited to, multiple sclerosis, rheumatoid
arthritis,
psoriasis, inflammatory bowel disease, pancreatitis, and transplant rejection.
[0244] Antibody-based detection methods are well known in the art, and include
ELISA, radioimmunoassays, immunoblots, Western blots, flow cytometry,
immunofluorescence, immunoprecipitation, and other related techniques. The
antibodies
may be provided in a diagnostic kit that incorporates at least one of these
procedures to
detect IL-22. The kit may contain other components, packaging, instructions,
or other
material to aid the detection of the protein and use of the kit.
[0245] Antibodies may be modified with detectable markers, including ligand
groups (e.g., biotin), fluorophores and chromophores, radioisotopes, electron-
dense
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reagents, or enzymes. Enzymes are detected by their activity. For example,
horseradish
peroxidase is detected by its ability to convert tetramethylbenzidine (TMB) to
a blue
pigment, quantifiable with a spectrophotometer. Other suitable binding
partners include
biotin and avidin, IgG and protein A, and other receptor-ligand pairs known in
the art.
[0246] Antibodies can also be functionally linked (e.g., by chemical coupling,
genetic fusion, non-covalent association or otherwise) to at least one other
molecular
entity, such as another antibody (e. g., a bispecific or a multispecific
antibody), toxins,
radioisotopes, cytotoxic or cytostatic agents, among others. Other
permutations and
possibilities are apparent to those of ordinary skill in the art.
Assaying Effects of IL-22 Agonists or Antagonists
[0247] The activity of an IL-22 agonist or antagonist can be measure by the
following methods:
[0248] Assays for T-cell or thymocyte proliferation include without limitation
those described in: Current Protocols in Immunology, Ed by J. B. Coligan, A.M.
Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing
Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse
Lymphocyte
Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J.
Immunol.
137:3494-3500, 1986; Bertagnolli et al., J. Immunol. 145:1706-1712, 1990;
Bertagnolli et
al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., J. Immunol.
149:3778-
3783, 1992; Bowman et al., J. Immunol. 152: 1756-1761, 1994.
[0249] Assays for cytokine production and/or proliferation of spleen cells,
lymph node cells or thymocytes include, without limitation, those described
in:
Polyclonal T cell stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current
Protocols
in Immunology. J.E. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and
Sons,
Toronto. 1994; and Measurement of mouse and human Interferon y, Schreiber,
R.D. In
Current Protocols in Immunology. J.E. Coligan eds. Vol i pp. 6.8.1-6.8.8, John
Wiley and
Sons, Toronto. 1994.
[0250] Assays for proliferation and differentiation of hematopoietic and
lymphopoietic cells include, without limitation, those described in:
Measurement of
Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S.
and
Lipsky, P.E. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1
pp. 6.3.1-
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6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med.
173:1205-1211,
1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl.
Acad. Sci.
U.S.A. 80:2931-2938, 1983; Measurement of mouse and human interleukin 6 -
Nordan,
R. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1-
6.6.5, John
Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. Acad. Sci. U.S.A.
83:1857-1861,
1986; Measurement of human Interleukin 11 - Bennett, F., Giannotti, J., Clark,
S.C. and
Turner, K. J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1
pp. 6.15.1
John Wiley and Sons, Toronto. 1991; Measurement of mouse and human Interleukin
9 -
Ciarletta, A., Giannotti, J., Clark, S.C. and Turner, K.J. In Current
Protocols in
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons,
Toronto.
1991.
[0251] Assays for T-cell clone responses to antigens (which will identify,
among others, proteins that affect APC-T cell interactions as well as direct T-
cell effects
by measuring proliferation and cytokine production) include, without
limitation, those
described in: Current Protocols in Immunology, Ed by J. B. Coligan, A.M.
Kruisbeek,
D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and
Wiley
Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function;
Chapter 6,
Cytokines and their cellular receptors; Chapter 7, Immunologic studies in
Humans);
Weinberger et al., Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger
et al.,
Eur. J. Immun. 11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500,
1986; Takai
et al., J. Immunol. 140:508-512, 1988.
Antibodies and Specific Binding Fragments against IL-19, IL-20, IL-24 and IL-
26
[0252] In certain embodiments, a specific binding fragment or antibody against
IL- 19 is provided. IL- 19 is a cytokine that belongs to the IL- 10 cytokine
subfamily. IL-
19 is preferentially expressed in monocytes. It can bind the IL20 receptor
complex and
lead to the activation of the signal transducer and activator of transcription
3 (STAT3). A
similar cytokine in mouse is reported to up-regulate the expression of IL-6
and TNF-
alpha and induce apoptosis, which suggests a role of this cytokine in
inflammatory
responses. Alternatively spliced transcript variants encoding the distinct
isoforms have
been described.
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[0253] Non-limiting examples of IL-19-associated disorders that can be treated
or prevented include, but are not limited to, transplant rejection, autoimmune
diseases
(including, for example, diabetes mellitus, arthritis (including rheumatoid
arthritis,
juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple
sclerosis,
encephalomyelitis, myasthenia gravis, systemic lupus erythematosis, autoimmune
thyroiditis, dermatitis (including atopic dermatitis and eczematous
dermatitis), Sjogren's
Syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis,
keratoconjunctivitis,
ulcerative colitis, spondyoarthropathy, ankylosing spondylitis, intrinsic
asthma, allergic
asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug
eruptions,
leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis,
allergic
encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic
bilateral
progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia,
idiopathic
thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active
hepatitis,
Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease,
sarcoidosis,
primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis);
respiratory
disorders, e.g., asthma or COPD; inflammatory conditions of the skin (e.g.,
psoriasis),
liver (e.g., hepatitis), kidney (e.g., nephritis) and pancreas (e.g.,
pancreatitis); graft-
versus-host disease, and allergy such as, atopic allergy; cancers (e.g., solid
or soft tissue
tumors), arthritic disorders (e.g., rheumatoid arthritis, juvenile rheumatoid
arthritis,
osteoarthritis, psoriatic arthritis, and ankylosing spondylitis), multiple
sclerosis, type I
diabetes, lupus (SLE), IBD, ulcerative colitis, Crohn's disease, COPD, asthma,
vasculitis,
allergy, scleroderma and inflammatory conditions of the skin (e.g.,
psoriasis), liver (e.g.,
hepatitis), kidney (e.g., nephritis) and pancreas (e.g., pancreatitis).
[0254] The amino acid sequence of the human IL-19 polypeptide is provided in
Figure 17(a) and is identified as SEQ ID NO. 5. Where particular amino acids
of IL-19
are identified by position, SEQ ID NO. 5 should be used as the reference IL-19
amino
acid sequence.
[0255] In certain embodiments, an IL-19 specific binding agent that binds to
the
wild-type human IL- 19 but fails to bind to a mutant IL- 19 wherein the mutant
IL- 19
comprises one or more of the following changes relative to wild-type human IL-
19: H36,
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I37, E39, S40, F41, Q42,144, R46, K51, D52, P55, N56, V57, T58,168, V74, R102,
K103, S106, 5110, F111, M114, A152,1541, 155K, G158, V162, or A165.
[0256] In certain embodiments, a peptidomimetic is provided that mimics an
epitope comprising two or more of the following amino acids of IL- 19: H3 6,
I37, E3 9,
S40, F41, Q42,144, R46, K51, D52, P55, N56, V57, T58,168, V74, R102, K103,
5106,
5110, F111, M114, A152, 154I, 155K, G158, V162, or A165.
[0257] In certain embodiments, a method of making a peptidomimetic that
mimics an epitope comprising two or more of the following amino acids of IL-
19: H3 6,
I37, E39, S40, F41, Q42,144, R46, K51, D52, P55, N56, V57, T58,168, V74, R102,
K103, S106, 5110, F111, M114, A152,1541, 155K, G158, V162, or A165, is
provided.
In certain embodiments, the method of making a peptidomimetic comprises
structural
modeling of the epitope through the use of a computer.
[0258] IL-19 antibodies and specific binding fragments can be generated and
used according to the techniques described above for IL-22. Pharmaceutical
compositions comprising IL- 19 antibodies and specific binding fragments can
be
generated and used according to the techniques described above for IL-22.
[0259] In certain embodiments, a method of selecting a specific binding agent
to
an IL-19 polypeptide is provided. In certain such embodiments, the specific
binding
agent binds to at least a portion of an epitope on an IL- 19 polypeptide. In
certain
embodiments, an IL- 19 polypeptide is contacted with an agent. In certain
embodiments,
the affinity of the agent for the IL- 19 polypeptide is determined. In certain
embodiments,
a mutant IL- 19 polypeptide is contacted with the agent, wherein the mutant IL-
19
polypeptide comprises at least one point mutation at at least one amino acid
position
selected from H36,137, E39, S40, F41, Q42,144, R46, K51, D52, P55, N56, V57,
T58,
I68, V74, R102, K103, 5106, SI10, F111, M114, A152, 154I, 155K, G158, V162, or
A165. In certain embodiments, the affinity of the agent for the mutant
polypeptide is
determined. In certain embodiments, the agent is selected if the affinity for
the IL- 19
polypeptide is greater than the affinity for the mutant IL- 19 polypeptide.
[0260] In certain embodiments, a specific binding fragment or antibody against
IL-20 is provided. IL-20 is a cytokine structurally related to IL-10. IL-20
has been
shown to transduce its signal through signal transducer and activator of
transcription 3
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(STAT3) in keratinocytes. A specific receptor for this cytokine is found to be
expressed
in skin and upregulated dramatically in psoriatic skin, suggesting a role for
this protein in
epidermal function and psoriasis.
[0261] Non-limiting examples of IL-20-associated disorders that can be treated
or prevented include, but are not limited to, transplant rejection, autoimmune
diseases
(including, for example, diabetes mellitus, arthritis (including rheumatoid
arthritis,
juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple
sclerosis,
encephalomyelitis, myasthenia gravis, systemic lupus erythematosis, autoimmune
thyroiditis, dermatitis (including atopic dermatitis and eczematous
dermatitis), Sjogren's
Syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis,
keratoconjunctivitis,
ulcerative colitis, spondyoarthropathy, ankylosing spondylitis, intrinsic
asthma, allergic
asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug
eruptions,
leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis,
allergic
encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic
bilateral
progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia,
idiopathic
thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active
hepatitis,
Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease,
sarcoidosis,
primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis);
respiratory
disorders, e.g., asthma or COPD; inflammatory conditions of the skin (e.g.,
psoriasis),
liver (e.g., hepatitis), kidney (e.g., nephritis) and pancreas (e.g.,
pancreatitis); graft-
versus-host disease, and allergy such as, atopic allergy; cancers (e.g., solid
or soft tissue
tumors), arthritic disorders (e.g., rheumatoid arthritis, juvenile rheumatoid
arthritis,
osteoarthritis, psoriatic arthritis, and ankylosing spondylitis), multiple
sclerosis, type I
diabetes, lupus (SLE), IBD, ulcerative colitis, Crohn's disease, COPD, asthma,
vasculitis,
allergy, scleroderma and inflammatory conditions of the skin (e.g.,
psoriasis), liver (e.g.,
hepatitis), kidney (e.g., nephritis) and pancreas (e.g., pancreatitis).
[0262] The amino acid sequence of the human IL-20 polypeptide is provided in
Figure 17(b) and is identified as SEQ ID NO. 6. Where particular amino acids
of IL-20
are identified by position, SEQ ID NO. 6 should be used as the reference IL-20
amino
acid sequence.
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[0263] In certain embodiments, an IL-20 specific binding agent that binds to
the
wild-type human IL-20 but fails to bind to a mutant IL-20 wherein the mutant
IL-20
comprises one or more of the following changes relative to wild-type human IL-
20: E4 1,
142, N44, G45, F46, S47,149, G51, D57,160, D61,162, R63,164, L65, D73, R79,
R107,
K108, 5111, S115, F116,1119, 157A, V159, K160, G163,1170, or Q173.
[0264] In certain embodiments, a peptidomimetic is provided that mimics an
epitope comprising two or more of the following amino acids of IL-20: E41,142,
N44,
G45, F46, S47,149, G51, D57,160, D61,162, R63,164, L65, D73, R79, R107, K108,
5111, S115, F116,1119, 157A, V159, K160, G163,1170, or Q173.
[0265] In certain embodiments, a method of making a peptidomimetic that
mimics an epitope comprising two or more of the following amino acids of IL-
20: E41,
142, N44, G45, F46, S47,149, G51, D57,160, D61,162, R63,164, L65, D73, R79,
R107,
K108, 5111, S115, F116, I119, 157A, V159, K160, G163,1170, or Q173, is
provided. In
certain embodiments, the method of making a peptidomimetic comprises
structural
modeling of the epitope through the use of a computer.
[0266] IL-20 antibodies and specific binding fragments can be generated and
used according to the techniques described above for IL-22. Pharmaceutical
compositions comprising IL-20 antibodies and specific binding fragments can be
generated and used according to the techniques described above for IL-22.
[0267] In certain embodiments, a method of selecting a specific binding agent
to
an IL-20 polypeptide is provided. In certain such embodiments, the specific
binding
agent binds to at least a portion of an epitope on an IL-20 polypeptide. In
certain
embodiments, an IL-20 polypeptide is contacted with an agent. In certain
embodiments,
the affinity of the agent for the IL-20 polypeptide is determined. In certain
embodiments,
a mutant IL-20 polypeptide is contacted with the agent, wherein the mutant IL-
20
polypeptide comprises at least one point mutation at at least one amino acid
position
selected from E41,142, N44, G45, F46, S47,149, G51, D57,160, D61,162, R63,164,
L65, D73, R79, R107, K108, 5111, 5115, F116, 1119, 157A, V159, K160,
G163,1170, or
Q173. In certain embodiments, the affinity of the agent for the mutant
polypeptide is
determined. In certain embodiments, the agent is selected if the affinity for
the IL-20
polypeptide is greater than the affinity for the mutant IL-20 polypeptide.
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[0268] In certain embodiments, a specific binding fragment or antibody against
IL-24 is provided. IL-24 was identified as a gene induced during terminal
differentiation
in melanoma cells. The protein encoded by this gene can induce apoptosis
selectively in
various cancer cells. Overexpression of this gene leads to elevated expression
of several
GADD family genes, which correlates with the induction of apoptosis. The
phosphorylation of mitogen-activated protein kinase 14 (MAPK7/P38), and heat
shock
27kDa protein 1 (HSPB2/HSP27) are found to be induced by this gene in melanoma
cells, but not in normal immortal melanocytes. Alternatively spliced
transcript variants
encoding distinct isoforms have been reported. IL-24 signals through two
distinct
receptor complexes - an IL-22R1 and IL-20R2 receptor complex and an IL-20R1
and IL-
22R2 receptor complex (U.S. Published Application Nos. 20030078381,
20030023033,
and 20060013815).
[0269] Non-limiting examples of IL-24-associated disorders that can be treated
or prevented include, but are not limited to, transplant rejection, autoimmune
diseases
(including, for example, diabetes mellitus, arthritis (including rheumatoid
arthritis,
juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple
sclerosis,
encephalomyelitis, myasthenia gravis, systemic lupus erythematosis, autoimmune
thyroiditis, dermatitis (including atopic dermatitis and eczematous
dermatitis), Sjogren's
Syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis,
keratoconjunctivitis,
ulcerative colitis, spondyoarthropathy, ankylosing spondylitis, intrinsic
asthma, allergic
asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug
eruptions,
leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis,
allergic
encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic
bilateral
progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia,
idiopathic
thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active
hepatitis,
Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease,
sarcoidosis,
primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis);
respiratory
disorders, e.g., asthma or COPD; inflammatory conditions of the skin (e.g.,
psoriasis),
liver (e.g., hepatitis), kidney (e.g., nephritis) and pancreas (e.g.,
pancreatitis); graft-
versus-host disease, and allergy such as, atopic allergy; cancers (e.g., solid
or soft tissue
tumors), arthritic disorders (e.g., rheumatoid arthritis, juvenile rheumatoid
arthritis,
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osteoarthritis, psoriatic arthritis, and ankylosing spondylitis), multiple
sclerosis, type I
diabetes, lupus (SLE), IBD, ulcerative colitis, Crohn's disease, COPD, asthma,
vasculitis,
allergy, scleroderma and inflammatory conditions of the skin (e.g.,
psoriasis), liver (e.g.,
hepatitis), kidney (e.g., nephritis) and pancreas (e.g., pancreatitis).
[0270] The amino acid sequence of the human IL-24 polypeptide is provided in
Figure 18(a) and is identified as SEQ ID NO. 7. Where particular amino acids
of IL-24
are identified by position, SEQ ID NO. 7 should be used as the reference IL-24
amino
acid sequence.
[0271] In certain embodiments, an IL-24 specific binding agent that binds to
the
wild-type human IL-24 but fails to bind to a mutant IL-24 wherein the mutant
IL-24
comprises one or more of the following changes relative to wild-type human IL-
24: K68,
L69, E71, A72, F73, W74, V76, D78, Q83, D84, T87, S88, A89, R90, V100, S105,
K135, S136, T139, N143, F144,1147, A185, T187, K188, G191,1195, or T198.
[0272] In certain embodiments, a peptidomimetic is provided that mimics an
epitope comprising two or more of the following amino acids of IL-24: K68,
L69, E71,
A72, F73, W74, V76, D78, Q83, D84, T87, S88, A89, R90, V100, S105, K135, S136,
T139, N143, F144,1147, A185, T187, K188, G191,1195, or T198.
[0273] In certain embodiments, a method of making a peptidomimetic that
mimics an epitope comprising two or more of the following amino acids of IL-
24: K68,
L69, E71, A72, F73, W74, V76, D78, Q83, D84, T87, S88, A89, R90, V100, S105,
K135, S136, T139, N143, F144,1147, A185, T187, K188, G191,1195, or T198, is
provided. In certain embodiments, the method of making a peptidomimetic
comprises
structural modeling of the epitope through the use of a computer.
[0274] IL-24 antibodies and specific binding fragments can be generated and
used according to the techniques described above for IL-22. Pharmaceutical
compositions comprising IL-24 antibodies and specific binding fragments can be
generated and used according to the techniques described above for IL-22.
[0275] In certain embodiments, a method of selecting a specific binding agent
to
an IL-24 polypeptide is provided. In certain such embodiments, the specific
binding
agent binds to at least a portion of an epitope on an IL-24 polypeptide. In
certain
embodiments, an IL-24 polypeptide is contacted with an agent. In certain
embodiments,
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the affinity of the agent for the IL-24 polypeptide is determined. In certain
embodiments,
a mutant IL-24 polypeptide is contacted with the agent, wherein the mutant IL-
24
polypeptide comprises at least one point mutation at at least one amino acid
position
selected from K68, L69, E71, A72, F73, W74, V76, D78, Q83, D84, T87, S88, A89,
R90, V100, S105, K135, S136, T139, N143, F144,1147, A185, T187, K188,
G191,1195,
or T198. In certain embodiments, the affinity of the agent for the mutant
polypeptide is
determined. In certain embodiments, the agent is selected if the affinity for
the IL-24
polypeptide is greater than the affinity for the mutant IL-24 polypeptide.
[0276] In certain embodiments, a specific binding fragment or antibody against
IL-26 is provided. IL-26 was identified by its overexpression specifically in
herpesvirus
samimiri-transformed T cells (Knappe et al. Journal of Virolology, 74:3881-
3887
(2000)). The encoded protein is a member of the IL-10 family of cytokines. IL-
26 is a
secreted protein and may function as a homodimer. IL-26 may contribute to the
transformed phenotype of T cells after infection by herpesvirus samimiri. IL-
26 signals
through a receptor complex of IL-20R1 and IL-IOR2 (Sheikh et al. Journal of
Immunology, 172:2006-2010 (2004) and Hor et al. J Biol. Chem. 279 (32): 33343-
51
(2004)).
[0277] Non-limiting examples of IL-26-associated disorders that can be treated
or prevented include, but are not limited to, transplant rejection, autoimmune
diseases
(including, for example, diabetes mellitus, arthritis (including rheumatoid
arthritis,
juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple
sclerosis,
encephalomyelitis, myasthenia gravis, systemic lupus erythematosis, autoimmune
thyroiditis, dermatitis (including atopic dermatitis and eczematous
dermatitis), Sjogren's
Syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis,
keratoconjunctivitis,
ulcerative colitis, spondyoarthropathy, ankylosing spondylitis, intrinsic
asthma, allergic
asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug
eruptions,
leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis,
allergic
encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic
bilateral
progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia,
idiopathic
thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active
hepatitis,
Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease,
sarcoidosis,
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primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis);
respiratory
disorders, e.g., asthma or COPD; inflammatory conditions of the skin (e.g.,
psoriasis),
liver (e.g., hepatitis), kidney (e.g., nephritis) and pancreas (e.g.,
pancreatitis); graft-
versus-host disease, and allergy such as, atopic allergy; cancers (e.g., solid
or soft tissue
tumors), arthritic disorders (e.g., rheumatoid arthritis, juvenile rheumatoid
arthritis,
osteoarthritis, psoriatic arthritis, and ankylosing spondylitis), multiple
sclerosis, type I
diabetes, lupus (SLE), IBD, ulcerative colitis, Crohn's disease, COPD, asthma,
vasculitis,
allergy, scleroderma and inflammatory conditions of the skin (e.g.,
psoriasis), liver (e.g.,
hepatitis), kidney (e.g., nephritis) and pancreas (e.g., pancreatitis).
[0278] The amino acid sequence of the human IL-26 polypeptide is provided in
Figure 18(b) and is identified as SEQ ID NO. 8. Where particular amino acids
of IL-26
are identified by position, SEQ ID NO. 8 should be used as the reference IL-26
amino
acid sequence.
[0279] In certain embodiments, an IL-26 specific binding agent that binds to
the
wild-type human IL-26 but fails to bind to a mutant IL-26 wherein the mutant
IL-26
comprises one or more of the following changes relative to wild-type human IL-
26: Q40,
A41, D43, A44, L45, Y46, K48, A50, T55, D59,161, K62, N63,164, F75, N78, R106,
F107,D110,L114,R115,L118,G148,Y150,K151,S154,1158,orS161.
[0280] In certain embodiments, a peptidomimetic is provided that mimics an
epitope comprising two or more of the following amino acids of IL-26: Q40,
A41, D43,
A44, L45, Y46, K48, A50, T55, D59,161, K62, N63,164, F75, N78, R106, F107, Dl
10,
L114,R115,L118,G148,Y150,K151,S154,1158,orS161.
[0281] In certain embodiments, a method of making a peptidomimetic that
mimics an epitope comprising two or more of the following amino acids of IL-
26: Q40,
A41, D43, A44, L45, Y46, K48, A50, T55, D59,161, K62, N63,164, F75, N78, R106,
F107, D110, L114, R115, L118, G148, Y150, K151, S154,1158, or 5161, is
provided. In
certain embodiments, the method of making a peptidomimetic comprises
structural
modeling of the epitope through the use of a computer.
[0282] IL-26 antibodies and specific binding fragments can be generated and
used according to the techniques described above for IL-22. Pharmaceutical
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compositions comprising IL-26 antibodies and specific binding fragments can be
generated and used according to the techniques described above for IL-22.
[0283] In certain embodiments, a method of selecting a specific binding agent
to
an IL-26 polypeptide is provided. In certain such embodiments, the specific
binding
agent binds to at least a portion of an epitope on an IL-26 polypeptide. In
certain
embodiments, an IL-26 polypeptide is contacted with an agent. In certain
embodiments,
the affinity of the agent for the IL-26 polypeptide is determined. In certain
embodiments,
a mutant IL-26 polypeptide is contacted with the agent, wherein the mutant IL-
26
polypeptide comprises at least one point mutation at at least one amino acid
position
selected from Q40, A41, D43, A44, L45, Y46, K48, A50, T55, D59,161, K62,
N63,164,
F75, N78, R106, F107, D110, L114, R115, L118, G148, Y150, K151, S154,1158, or
S 161. In certain embodiments, the affinity of the agent for the mutant
polypeptide is
determined. In certain embodiments, the agent is selected if the affinity for
the IL-26
polypeptide is greater than the affinity for the mutant IL-26 polypeptide.
EXAMPLES
Evaluation of 146 distinct point substitutions in IL-22
[0284] A panel of IL-22 mutants was generated by a method employing three
polymerase chain reactions (PCR) for each point mutation (Ho et al. Gene 77,
51-9
(1989)). A mammalian expression plasmid that encoded IL-22 with an N-terminal
hexahistidine/FLAG octapeptide (H/F) tag was used as template for the first
two of
three PCR steps (Li et al. International Imnunopharmacology 4, 693-708
(2004)). The
third PCR generated a linear DNA expression cassette that encoded a CMV
promoter,
H/F-IL-22 protein with a single point substitution, and an SV40
polyadenylation
sequence. This 1816 bp linear DNA encoded a mammalian promoter, translation
start
and secretory leader, the latter fused in-frame to an N-terminal H/F-tagged IL-
22, and
then followed by a polyadenylation sequence. Using this PCR-based method, the
seven
alanines of the mature IL-22 ORF were individually mutated, either to glycine
or a
different alanine codon. The remaining amino acids of IL-22 were individually
changed
to alanines.
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[0285] The panel of mutated IL-22 linear DNA expression cassettes was used
directly, without purification, for transfection of mammalian cells (Liang et
al. Journal of
Biological Chemistry 277, 3593-8 (2002)). Transfection of suspension HEK293FT
cells
(Invitrogen, Carlsbad, CA) was performed in 48 deep-well plates using
293fectinTM
(Invitrogen, Carlsbad, CA) and conditioned media was collected after five
days. For
purification of select IL-22 mutants, point-substituted cDNA was subcloned
into a
mammalian expression plasmid and the resulting subcloning products were used
for
transient transfection of adherent HEK293. H/F-tagged and mutated IL-22
protein was
purified using Ni-NTA resin (Qiagen, Valencia, CA). All together, 146 distinct
point
mutants encompassing the mature IL-22 ORF, as well as seven silent alanine
substitution
controls, were expressed in mammalian cells.
[0286] IL-22 in the conditioned media was quantitated by sandwich ELISA,
exploiting both entities of the H/F tag. Using standard methods with very
gentle wash
steps, H/F-IL-22 was captured with anti-FLAG HS M2 antibody covalently coated
to
plates (P2983; Sigma, St. Louis, MO) and detected with HRP-conjugated HisProbe
(Pierce, Rockford, IL).
[0287] To determine which IL-22 amino acids are important for binding to IL-
22 cell surface and soluble receptors, we evaluated our panel of control IL-22
and point
mutants in five distinct ELISAs. The five ELISAs used were 1) an IL-22BP-Fc
homodimer binding assay; 2) an IL-22R-Fc homodimer binding assay; 3) an IL-22R-
Fc/IL-IOR2-Fc heterodimer binding assay; 4) an IL-22 antibody (IL22-02)
binding assay;
and a second IL-22 antibody (IL22-04) binding assay (Li et al. International
Immunopharmacology 4, 693-708 (2004)). The two antibody assays, which detect
distinct IL-22 epitopes (Li et al. International Immunopharmacology 4, 693-708
(2004)),
were used in concert with the three IL-22 receptor assays to identify those IL-
22
mutations that affect the overall stability of IL-22 secondary and/or tertiary
structure and
only indirectly affect receptor or antibody binding. As described previously
by Li et al.,
ELISAs for HF-IL-22 binding to receptors used plates coated indirectly with
either 25
ng/ml IL-22BP-Fc or 50 ng/ml IL-22R-Fc homodimers or 50 ng/ml IL-22R-Fc/IL-
lOR2-
Fc heterodimers, the latter two containing only the extracellular domains of
the
corresponding cell surface receptor subunits (Li et al. International
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Immunopharmacology 4, 693-708 (2004)). Mutant and control IL-22 cytokines in
conditioned media were tested in duplicate for binding at either 200 ng/ml (IL-
22R) or 20
ng/ml (IL-22R-Fc/IL-1OR2-Fc and IL-22BP-Fc). Rat IL22-02 and IL22-04
antibodies
were coated directly at 3 g/ml and 1 ug/ml, respectively, and IL-22 mutants
were tested
in duplicate for binding at 200 ng/ml and 20 ng/ml, respectively. Bound H/F-IL-
22 was
detected by conventional methods using HRP- conjugated HisProbe. Serial
dilutions of
purified IL-22 mutants were evaluated in at least two independent experiments.
Approximately 800 independent data points were collected in duplicate that
described the
binding characteristics of single amino acid substitutions spanning the mature
primary
sequence of IL-22.
[0288] The binding of silent alanine substitutions, derived from thirteen
separate
transfections, served as the control IL-22 cytokine for the high-throughput
method (open
squares in Figure 9(a)). The signal threshold for the control IL-22 binding
was set at 1.8
standard deviations below the average signal in a given assay and is a one-
sided 95%
confidence interval for individual observations. IL-22 mutants that gave a
binding signal
below the threshold were defined as being weaker than normal for binding to a
given
receptor or antibody.
[0289] Sixty-five of the 146 (45%) substitutions bound comparably to control
IL-22 in the five IL-22 receptor and antibody assays (see Figure 12). The IL-
22 amino
acid side chains defined by this group of substitutions range from entirely
surface
exposed to buried, and their individual integrity was not essential for normal
binding to
IL-22R, IL-IOR2, or IL-22BP.
[0290] Thirty of the 146 (20 %) substitutions had statistically significant
weaker
than normal binding compared to control IL-22, in four or five assays (Figure
12).
Twenty alanine substitutions had weaker than normal binding in all five
binding assays
(Figure 12). Alanine substitutions at 175, L100, C132, E166, and C178 showed
the
strongest inhibition of binding in the five assays. The ten additional
substitutions
classified as statistically significant in only four assays (Figure 12) may
also have had a
subtle impact in the IL-22R binding assay, since those mutants had a binding
value that
was just above the statistical threshold for the IL-22R assay. Considering
that relatively
few of the 30 corresponding IL-22 side chains are exposed to the surface (see
solvent
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accessibility values in Supplementary Table II and surface visualization of
these in
Supplementary Fig. lb), these IL-22 side chains may not contribute
specifically to the
receptor binding sites. Rather, these 30 amino acids may be important for
maintaining
IL-22 secondary and/or tertiary structures.
29 side chains in IL-22 are involved in binding to IL-22BP, IL-22R, and/or IL-
I OR2
[0291] Twelve of the 146 (8%) amino acid substitutions in the IL-22 protein
were determined to be involved in the binding of IL-22 to IL-22R due to the
fact that
these mutants did not bind as well to the IL-22R as the control IL-22 protein
did in the
IL-22R binding assay (F57A, L59A, D67A, T70A, D71A, V72A, R73A, G159A, 116 1A,
K162A, G165A, and L169A substitutions in Figure 11 and corresponding residues
in
Figure 1(a); IL-22R in Figure 9(a)). Alanine substitution of 1161, V72, G165,
D71, and
L169 had the most deleterious effect in the IL-22R-Fc binding assay. Three of
the twelve
individual substitutions (D67A, R73A, and K162A) were also shown to not bind
as well
as the control IL-22 to IL-22BP (Figure 11 and Figure 1(a); IL-22BP in Figure
9(a)),
indicating that the corresponding IL-22 side chains are involved in binding to
IL-22BP.
Collectively, these data suggest that the IL-22R and IL-22BP binding sites on
IL-22 are
overlapping.
[0292] IL-1 OR2 binds to a pre-formed IL-22/IL-22R complex (Logsdon et al.
Journal of Interferon & Cytokine Research 22, 1099-112 (2002) and Li et al.
International Immunopharmacology 4, 693-708 (2004)). Seventeen of the 146
(12%)
substitutions in IL-22 were determined to be involved in binding to IL-1 OR2.
Twenty-
nine amino acid substitutions did not bind as well as control IL-22 in the IL-
22R-Fc/IL-
l OR2-Fc binding assay. As twelve of these 29 mutants were also less effective
in the IL-
22R-Fc binding assay, we inferred that the remaining seventeen mutants
correspond to
IL-22 side chains that are involved in binding to IL-10R2 (A34G, Y51A, 152A,
N54A,
R55A, T56A, K61A, A66G, V83A, R88A, P113A, Y114A, E117A, F121A, L122A,
L125A, and M172A substitutions in Figure 11 and corresponding residues in
Figure 1(a);
IL-1OR2 in Figure 9(a)). Alanine substitution of T56, Y51, R55, N54, F121, and
E117
had the most deleterious and specific effect in the IL-22R-Fc/IL-1 OR2 binding
assay.
The alanine substitution of V83 bound less well in both the IL-22R-Fc/IL- I
OR2-Fc and
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IL-22BP-Fc assays (Figure 11 and Figure 1; Figure 9(a)), indicating that the
corresponding IL-22 side chain may be involved in both IL-1 OR2 and IL-22BP
binding.
[0293] None of the 146 point substitutions in IL-22 had a uniquely deleterious
effect in the IL-22BP binding assay. Rather, the four IL-22 side chains that
were
involved in binding to IL-22BP were also involved in binding to IL-22R or IL-
I OR2
(D67, R73, V83, and K162 residues/solid symbols, respectively, in Figure 11,
Figure
1(a), and Figure 9(a)).
[0294] To study the activity of certain IL-22 substitutions in more detail,
thirteen IL-22 point mutants were purified and evaluated over a three-log
range of
concentrations in the five receptor and antibody binding assays. The binding
data
collected with purified IL-22 point mutants (Figure 2) confirmed the weaker
than normal
effects on binding observed in the high throughput evaluation (summarized in
Figure 11
and Figure 9(a)). IL-22 mutants that contained alanine substitution of D67,
V72, R73,
I161, K162, or L169 were approximately 50-fold less effective than control
cytokine for
binding to IL-22R-Fc (Figure 2(a), D67A, V72A, R73A, 1161A, K162A, and L169A
substitutions). Those mutants that blocked binding to IL-22R were also less
effective, to
varying degrees, in the IL-22R-Fc/IL- I OR2-Fc binding assay (Figure 2(b),
D67A, V72A,
R73A, 1161A, K162A, and L169A substitutions). As expected, three of these
purified
IL-22 substitutions (D67A, R73A, and K162A) were also deleterious for binding
to IL-
22BP-Fc (Figure 2(c)). Mutants that were specifically defective for binding to
IL-IOR2
in the high-throughput screen were also weaker than normal for binding to IL-
22R-Fc/IL-
IOR2-Fc when purified and evaluated at different concentrations (Figure 2(b),
Y5 IA,
R55A, and E117A substitutions) and bound normally to IL-22R-Fc or IL-22BP-Fc
(Figures 2(a) and (c)). Accordingly, the data collected with the high-
throughput methods
were predictive of observations that were subsequently obtained with purified
mutants.
IL-22 side chains involved in binding to IL-22R or IL-IOR2 are also involved
in
signaling into a cell
[0295] The purified IL-22 substitutions were evaluated for the ability to
induce
proliferation of an IL-22-dependent BaF3 cell line that over-expressed both
human IL-
22R and IL-10R2 (Figure 3). A BaF3 cell line that expressed IL-1082/YFP and IL-
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22R/GFP was generated by sequential retroviral transductions and proliferated
in
response to IL-22. Serial dilutions of IL-22, control or mutated, were added
to 5 x 103
GFP+YFP+ cells in RPMI with 10 % FCS and standard concentrations of
penicillin,
streptomycin, and glutamine. Cells were incubated at 37 C and 5% CO2 for 72
hours,
and then evaluated for proliferation by conventional methods using 3H-
thymidine
incorporation.
[0296] The eleven mutants that did not bind as well as control IL-22 to IL-22R
or IL-IOR2 in the ELISA binding assays (Figure 2A and B) were, in general,
similarly
weaker than normal for inducing IL-22-dependent cell proliferation (Figure 3).
One
exception was the R73A substitution that bound poorly to IL-22R-Fc/IL-1 OR2-Fc
(R73A,
Figure 2b). The R73A substitution mutant effected a signal into a cell when a
40-fold
higher concentration was added relative to control IL-22 (R73A, Figure 3). The
two
other exceptions were the alanine substitutions of 1161 and L169. In the IL-
22R-Fc/IL-
l OR2-Fc binding assay, the I161A mutant IL-22 was a stronger binder than the
V72A and
L169A mutants (Figure 2b). In contrast, in the IL-22-dependent cell-based
assay, the
V72A, I161A, and L169A substitutions were similarly less effective at lower
concentrations with the L169A substitution inducing more proliferation at
higher
concentrations (Figure 3 and data not depicted). Overall, however, the above
studies in a
cell-based assay indicate that how effectively an IL-22 mutant interacts with
IL-22R and
IL-10R2 by ELISA is predictive of its effectiveness for signaling into a cell.
Side chains mostly in IL-22 helices A, D, and F, and loop AB are involved in
binding IL-
22BP, IL-22R, and/or IL- I OR2 and define the cell surface receptor binding
sites
[0297] To explore the structural implications of mutagenesis results, the IL-
22
residues that have been previously reported as being involved in cell
signaling and/or
receptor binding were considered in the context of existing IL-22 crystal
structure
(Nagem et al. Structure 10, 1051-62 (2002) and Xu et al. Acta
Crystallographica Section
D-Biological Crystallography 61, 942-50 (2005)). Based on the new data
described in
this application, the proposed IL-22R binding site is defined by IL-22 side
chains within
helices A and F, and loop AB (Figure lb and CPK (Figure 4) renderings of
structure).
The IL-IOR2 binding site is adjacent and defined by IL-22 side chains in
helices A and D
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with contributions from loops BC and CD, and helices C and F. The binding site
for IL-
22BP involves four IL-22 side chains, three of which are in loop AB and helix
F, and
transect the proposed IL-22R binding site. Sequential 90' rotations of IL-22,
counterclockwise around the vertical axis, are shown in the CPK structures of
Figure 4
(a-d) and (e-h), and emphasize the restriction of the IL-22 cell surface and
soluble
receptor binding sites to certain surface regions of the IL-22 structure.
IL-22R binding is dependent on the integrity of IL-22 side chains in helix A,
and F and
loop AB
[0298] IL-22 binds first to IL-22R, its high affinity receptor subunit
(Logsdon et
al. Journal of Interferon & Cytokine Research 22, 1099-112 (2002) and Li, et
al.
International bmimunopharimacology 4, 693-708 (2004)). The twelve IL-22 amino
acids
identified as being involved in its binding to IL-22R are located in helix A
(F57, L59),
loop AB (D67, T70, D71, V72, R73), and helix F (G159,1161, K162, G165, L169)
(Figure 1). Eight of these amino acids (F57, D67, T70, D71, V72, R73, G165,
and L169)
have 12-86% solvent accessibility (see Figure 14) suggesting that each may
contribute
atoms to the IL-22R receptor interface. The remaining four amino acids (i.e.,
L59, G159,
I161, K162) are almost or completely buried, suggesting that these contribute
indirectly
by facilitating a local surface structure. The proposed IL-22R binding site on
IL-22 is
illustrated in CPK (F57, D67, T70, D71, V72, R73,1161, K162, and L169
residues,
Figure 4(a-d)) and solvent accessibility (F57, L59, D67, T70, D71, V72, R73,
G159,
K161, K162, G165, and L169 residues, Figure 6(a)) renderings.
[0299] Nagem et al. superimposed their IL-22 crystal structure to the cytokine
within IL-10/IL-IORIECD and IFN-y/IFN-7R1ECD co-crystal structures, and
proposed that
T70 and D7 1, as well as four other side chains of IL-22, are important for
the recognition
of IL-22's corresponding high affinity receptor subunit (Nagem et al.
Structure 10, 1051-
62 (2002)). Logsdon et al., using a model of IL-22/IL-22R based on their
solved IL-
10/IL-IORIECD co-crystal structure, subsequently proposed that D71, R73, and
G165, as
well as seven other side chains of IL-22, may contribute to IL-22R binding
(Logsdon et
al. Journal of Interferon & Cytokine Research 22, 1099-112 (2002) and
Josephson et al.
Immunity 15, 35-46 (2001)). The studies in this application prove
experimentally that
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four of those previously proposed side chains (i.e., T70, D71, R73, and G165),
as well as
eight additional side-chains identified in the present application, are part
of the generation
of the IL-22R binding interface. Five amino acids proposed by Logsdon et al.
to
contribute to IL-22R binding are surface neighbors (K61, S64, N68, E166, and
D168
residues in Figure 6(a)) to the residues identified in the present application
as
contributory (F57, L59, D67, T70, D71, V72, R73, G159, K161, K162, G165, and
L169
residues; Figure 6(a)) (Logsdon et al. Journal of Interferon & Cytokine
Research 22,
1099-112 (2002)). Further, it is possible that K61, S64, N68, E166, and D168
may also
contribute to the IL-22R binding interface.
[0300] The high affinity receptor binding interface for IL-10 was elucidated
from IL-10/IL-IORIECD co-crystal structure (Josephson et al. Immunity 15, 35-
46 (2001)
and Yoon et al. Journal of Biological Chemistry 281, 35088-96 (2006)). The IL-
IOR1
binding site encompasses much of IL-10's helix A, loop AB and helix F (see IL-
LORI-a
and -b dashed bars in alignment of Figure 5, and the renderings in Figure 6b).
Nine of
the IL-22R amino acids identified as contributing to the IL-22R binding
interface (i.e.,
L59, D67, T70, D71, V72, R73, K162, G165, and L169) align with the IL-LORI-a
region.
The three remaining residues (i.e., F57, G159, and 1161) are immediately to
the N
terminal side of the IL-LORI-a sites in helix A and F (Figure 5). Accordingly,
the IL-22R
binding site may be slightly expanded in helix A and translated upstream in
helix F of IL-
22 (see IL-22R dashed bars in Figure 5) relative to the IL-IORl-a binding site
in these
same helices of IL-10.
[0301] None of the IL-22 amino acids that align with the IL-IORl-b regions of
IL-10 (Figure 5 and Figure 6b) appear to be involved in IL-22's interaction
with IL-22R.
This absence may be due to the use of different methods, as described above,
to explore
the structure and function of IL- 10 and IL-22. Or, the identified amino acids
may reflect
a more compact IL-22R binding site than the corresponding IL-1 OR1 binding
site on IL-
10, as shown by comparing the IL-22 and IL-10 side chains in the superimposed
ribbon
structures of Figure 6b. In consideration of the distinct methods used to
study the
interaction between IL- 10 or IL-22 and their respective high affinity
receptors, a gross
conservation of function as dictated by structure appears to exist between
these two
cytokines (see alignments and structures shown in Figure 5 and 6b,
respectively).
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IL-22 surface structure that is involved in IL-22R binding is also involved in
binding
22BP
[0302] IL-22BP specifically blocks an interaction between IL-22 and IL-22R,
suggesting that IL-22BP has at least a partially overlapping binding site with
IL-22R (Li
et al. International Immunopharmacology 4, 693-708 (2004)). The mutagenesis
analysis
described above demonstrated that four IL-22 side chains in loops AB (D67 and
R73),
and BC (V83) and helix F (K162), are involved in binding to IL-22BP as well as
IL-22R.
The side chains D67, R73, and K162 may contribute directly to those binding
sites
(Figure 6a and c). V83 may contribute to those binding sites indirectly since
it has only
3% solvent accessibility. Amino acids corresponding to D67, R73, V83, and K162
are
conserved between IL-22 and IL-24. IL-22BP, however, is specific for IL-22.
Thus,
those four amino acids of IL-22 may not be sufficient for binding to IL-22BP.
In
consideration of IL-22BP's high affinity for IL-22, other IL-22 side chains in
the vicinity
of D67, R73, and K162 (see Figure 6c) may also contribute to the IL-22BP
binding site
but may contribute to a lesser extent than the four identified amino acids,
and thus were
not detected in the mutagenesis screens described above. The existence of IL-
22BP, its
specificity and high affinity for IL-22, and the fact that it interferes with
binding to IL-
22R, by a mechanism proposed above, indicates that it has a physiologic role,
yet to be
explored, in vivo.
IL-IOR2 binding to IL-22 requires the integrity of side chains mostly in helix
A and D
[0303] IL-1OR2 binds to a surface created by the interaction of IL-22 and IL-
22R (Logsdon et al. Journal of Interferon & Cytokine Research 22, 1099-112
(2002) and
Li et al. International Immunopharmacology 4, 693-708 (2004)). This surface is
proposed to include a conformational change in IL-22 that is induced by
binding to IL-
22R (Li et al. International Immunopharmacology 4, 693-708 (2004)). The
mutagenesis
analysis described above determined that IL-22 binding to IL-IOR2 involves at
least
seventeen side chains, located in pre-helix A (A34); helix A (Y51,152, N54,
R55, T56,
K61); loop AB (A66); loop BC (V83); helix C (R88); loop CD (P113); helix D
(Y114,
El 17, F121, L122, L125); and helix F (M172) (Figure lb). Solvent
accessibility values
(see Figure 14) indicate that most of these IL-22 side chains could contribute
atoms to the
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interface with IL-IOR2, as is shown in the solvent accessible rendering of IL-
22 in Figure
7a. A conformational change in IL- 10 has already been determined to occur
with binding
to its high affinity receptor, IL-I ORI (Moon et al. Journal of Biological
Chemistry 281,
35088-96 (2006)). With this conformational change, the solvent accessibility
of the five
IL-10 side chains of helix A that contribute to the IL- I OR2 interface - N39,
M40, R42,
S49, R50 - are altered by 9 to 21%, in one direction or the other. Determining
how
significantly the putative conformational change in IL-22 will modify the
surface
displayed in Figure 7a may involve the determination of an IL-22/IL-22R
structure
[0304] If a conformational change in IL-22 does occur with binding to IL-22R,
then certain side chain atoms on the surface of IL-22 may contribute to both
the IL-22R
and IL-1 OR2 binding sites. Yoon et al. demonstrated, using surface plasmon
resonance
methods, that R42 of IL- 10 is important for both IL-IORi and IL-IOR2 binding
(Yoon et
al. Journal of Biological Chemistry 281, 35088-96 (2006)). To interpret the
mutagenesis
data, an amino acid substitution of IL-22 that was weaker than normal for
binding to IL-
22R was assumed to bind similarly less effectively to a complex of IL-22R/IL-
I OR2.
Substitutions that had a deleterious impact on binding to IL-22R/IL-1 OR2, and
not IL-
22R, were assumed to affect only IL-IOR2 binding. This interpretation does
not,
however, consider those IL-22 amino acids that might be involved in the
binding of both
the high and low affinity receptor subunits.
[0305] D67, R73, and K162 of IL-22, which are centrally located within the IL-
22R binding site and are involved in IL-22BP recognition, may also contribute
to the IL-
l OR2 binding site. To validate the high-throughput screen, the binding of
nine purified
IL-22 substitutions was evaluated over a three-log range of concentrations.
Six of these
substitutions bind very poorly to IL-22R at relatively low concentrations of
cytokine (i.e.,
10-100 ng/ml; D67A, V72A, R73A, I161A, K162A, and L169A substitutions in
Figure
2A). However, these six substitutions do not behave comparably in the presence
of both
IL-22R and IL-IOR2, in either a receptor binding (i.e., 10-100 ng/ml, Figure
2b) or cell
signaling (0.1-1 ng/ml, Figure 3) assay. Three of the substitutions (V72A,
I161A,
L169A) bind or signal relatively well, suggesting that the presence of IL-IOR2
shifts an
equilibrium, compensating for an initial IL-22 defect on IL-22R binding. The
other three
substitutions (D67A, R73A, and K162A) still bind poorly in the presence of IL-
1 OR2,
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indicating that the presence of IL-1 OR2 cannot compensate for the poor
binding of these
substitutions. These observations suggest that D67, R73, and K162 also
contribute,
directly or indirectly, to the interaction between IL-22/IL-22R and IL-1 OR2.
[0306] To validate their structure-function model for IL-22, Logsdon et al.
evaluated the impact of fifteen alanine or cysteine point substitutions,
demonstrating that
Q48, Y51, N54, R55 in helix A (i.e. bold residues in QQPYITNR) and Y114, El 17
in
helix D (i.e., YMQE) are important for the interaction between IL-22/IL-22R
and IL-
lOR2 (Logsdon et al. Journal of Molecular Biology 342, 503-14 (2004)).
Subsequently,
Wolk et al. determined that soluble IL-10R2 binds to surface coupled IL-22
peptides that
include either QQPYITNRT of helix A or LARLS of helix D (Wolk et al. Genes &
Immunity 6, 8-18 (2005)). While the mutagenesis analysis described above did
not detect
an impact of the Q48A substitution, the mutagenesis analysis supports these
prior
observations and demonstrates that twelve additional side chains of IL-22
contribute to
the recognition of IL-22/IL-22R by IL-1 OR2.
Comparison of surface regions of IL- 10 and IL-22 that are recognized by IL-
IOR2
[0307] All cells express the IL-1 OR2 receptor subunit (Kotenko et al.
Cytokine
& Growth Factor Reviews 13, 223-40 (2002)). The expression of IL-10R2 suggests
that
IL-10R2 is a receptor subunit for several type II cytokines (i.e., IL-10, IL-
22, IL-26 and
IFN-2) (Kotenko et al. International Immunopharmacology 4, 593-608 (2004)).
Yoon et
al. determined that that M40, R42, and R50 in helix A are critical for IL-10R2
binding to
IL-10/IL10R1, with additional side chains (i.e., N39 and S49 in helix A and
H108 and
S 111 in helix D; see dashed bars under IL- 10 sequence in Figure 5) having
subtler
impacts in some assays (Yoon et al. Journal of Biological Chemistry 281, 3
5088-96
(2006)). In Figure 7(b), helix A and D structures from IL-22 and IL-10 are
superimposed. Figure 7(b) shows the IL-22 (Y51,152, N54, R55, T56, L59, K61,
A66,
R88, P113, Y114, E117, F121, L122, L125, G159, and M172) and IL-10 (N39, M40,
R42, S49, R50, H108, and 5111) side chains that may contribute to the binding
site for
IL-1 OR2. While the surface regions that are recognized by IL-1 OR2 are
grossly
overlapping, the systematic evaluation of IL-22 mutants suggests that IL-22's
binding site
for IL-10R2 covers a broader surface than that elucidated to date for IL-10.
The study of
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a more comprehensive panel of IL- 10 substitutions, similar to the systematic
evaluation
of IL-22 mutants described above, may elucidate a similar aspect of IL-10
surface
contributing to the IL- I OR2 binding site.
IL-22 structure-function model suggests the high and low affinity binding
sites for other
IL-10-like cytokines
[0308] A monomeric structure of IL-10 (PDB:ILK3) (Josephson et al. Structure
10, 981-7 (2002)) was used for superimposition with the monomeric crystal
structures of
IL-22 (PDB:IM4R) (Nagem et al. Structure 10, 1051-62 (2002)) and IL-19
(PDB:INIF)
(Chang et al. Journal of Biological Chemistry 278, 3308-13 (2003)), using the
least
squares method based on Ca positions as implemented by the Malign3D routine of
Modeler (Marti-Renom et al. Annual Review of Biophysics & Biomolecular
Structure 29,
291-325 (2000)). Structural models for IL-20 (NP_061194.2) (Blumberg et al.
Cell 104,
9-19 (2001)) and IL-24 (NP_006841.1) (Jiang et al. Oncogene 11, 2477-86
(1995)) were
generated using helices B through G of IL-19 (PDB:INIF) as the template.
Helical
secondary structure for these three cytokines, as shown in Figures 1(a), 5 and
10, were
derived from Discovery Studio Visualizer 2.0 evaluation of the above structure
files.
Structural models for IL-26 (NP_060872.1) (Knappe et al. Journal of Virology
74, 3881-
7 (2000)) were generated using the monomeric IL-10 structure (PDB:ILK3) as the
template. A virtual construct of IL-26 containing a six-residue flexible
linker
(GGGSGG) inserted in the predicted DE loop between residues E130 and M131 was
used in model building to mimic the engineered monomeric IL-10 (Josephson et
al.
Structure 10, 981-7 (2002)). From the 100 initial models for IL-20, IL-24 and
IL-26, the
model with the lowest restraint violations, as defined by the molecular
probability density
function, was chosen for further optimization. For model optimization, an
energy
minimization cascade consisting of Steepest Descent, Conjugate Gradient and
Adopted
Basis Newton Raphson methods was performed until an RMS gradient of 0.01 was
satisfied using CHARMm force field (Accelrys Software Inc.) and Generalized
Born
implicit solvation as implemented in Discovery Studio 1.7 (Accelrys Software
Inc.).
During energy minimization, backbone atom movements were restrained using a
harmonic constraint of 10 mass force.
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[0309] The systematic mutagenesis analysis of IL-22 described above defines at
the molecular level those localized regions of IL-22 that are involved in
binding to IL-
22R and IL-IOR2. Side chains, mostly from helices A, D, and F, and loop AB,
are
individually involved in recognition by IL-22's high affinity and low affinity
receptor
subunits. In consideration of the actual (i.e., IL-10, IL-19, IL-22) and
modeled (IL-20,
IL-24, IL-26) structural homology between the IL-10-like cytokines (summarized
in
Figure 10) and the present demonstration that there is functional conservation
between
the IL-22 and IL-10 structures (Figure 6b, 7b), positional transposition of
the
experimentally-defined IL-22 receptor binding sites is a first step towards
the elucidation
of other IL-10-like cytokines' receptor binding sites. The IL- 19, IL-20, IL-
24 and IL-26
residues that align (see Figure 10) with the IL-22R and IL-10R2 binding sites
are shown
as ball and stick models on the ribbon structures in Figure 8. Accordingly,
based on
information known in the art and that provided by the present invention,
protein and
small molecule therapeutics can also be designed for targeting and/or
interrupting the IL-
19, IL-20, IL-24 and IL-26 cytokine signaling pathways, therefore providing a
method
for treating and/or preventing disorders associated with these cytokines.
Equivalents
[0310] Those skilled in the art will recognize, or be able to ascertain using
no
more than routine experimentation, many equivalents to the specific
embodiments
described herein. Such equivalents are encompassed by the following claims.
