NZ751498B2 - Il-22 polypeptides and il-22 fc fusion proteins and methods of use - Google Patents
Il-22 polypeptides and il-22 fc fusion proteins and methods of use Download PDFInfo
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- NZ751498B2 NZ751498B2 NZ751498A NZ75149814A NZ751498B2 NZ 751498 B2 NZ751498 B2 NZ 751498B2 NZ 751498 A NZ751498 A NZ 751498A NZ 75149814 A NZ75149814 A NZ 75149814A NZ 751498 B2 NZ751498 B2 NZ 751498B2
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Abstract
The invention relates to IL-22 Fc fusion proteins, wherein the Fc domain is an IgG1 Fc domain that lacks glycosylation, composition comprising the same, methods of making and methods of using the composition for the treatment of diseases. The invention also relates to IL-22 receptor associated reagents and methods of use thereof. nts and methods of use thereof.
Description
IL-22 POLYPEPTIDES AND IL-22 Fc FUSION PROTEINS AND
METHODS OF USE
This application is a divisional of New Zealand patent application 711095, which is the
national phase entry in New Zealand of PCT international application
(published as ). The instant application claims the benefit of priority to U.S.
provisional applications serial numbers 61/800,148, 61/800,795 and 61/801,144, all of which
were filed on March 15, 2013, U.S. provisional application serial number 61/821,062, filed on
May 8, 2013, and U.S. provisional application serial number 61/860,176, filed on July 30, 2013,
the contents of all of which are herein incorporated by reference in their entirety.
SEQUENCE LISTING
The instant application contains a Sequence Listing submitted via EFS-Web and hereby
incorporated by reference in its entirety. Said ASCII copy, created on March 14, 2014, is
named P5580R1-WO_Seq_Listing.txt, and is 106,763 bytes in size.
FIELD
The present invention generally relates to IL-22 and IL-22 Fc fusion proteins, IL-22
agonists, compositions comprising the same, and methods of making and method of using the
same.
BACKGROUND
Interleukin-22 (IL-22) is a member of the IL-10 family of cytokine that is produced by
Th22 cells, NK cells, lymphoid tissue inducer (LTi) cells, dendritic cells and Th17 cells. IL-22
binds to the IL-22R1/IL-10R2 receptor complex, which is expressed in innate cells such as
epithelial cells, hepatocytes, and keratinocytes and in barrier epithelial tissues of several organs
including dermis, pancreas, intestine and the respiratory system.
IL-22 plays an important role in mucosal immunity, mediating early host defense
against attaching and effacing bacterial pathogens. See Zheng et al., 2008, Nat. Med. 14:282-
89. IL-22 promotes the production of anti-microbial peptides and proinflammatory cytokines
from epithelial cells and stimulates proliferation and migration of colonic epithelial cells in the
gut. See Kumar et al., 2013, J. Cancer, 4:57-65. Upon bacterial infection, IL-22 knock-out
mice displayed impaired gut epithelial regeneration, high bacterial load and increased mortality.
Kumar et al., supra. Similarly, infection of IL-22 knock-out mice with influenza virus resulted
in severe weight loss and impaired regeneration of tracheal and bronchial epithelial cells. Thus,
IL-22 plays a pro-inflammatory role in suppressing microbial infection as well as an anti-
inflammatory protective role in epithelial regeneration in inflammatory responses. Much of IL-
22’s biological action promoting pathological inflammation and tissue repair remains to be
determined. The seemingly conflicting reports on the effects of IL-22 on epithelial cells are not
yet thoroughly understood. Kumar et al., supra.
The regulation of antimicrobial defensins, which limits bacterial replication and
dissemination, would help to stabilize intestinal microbiota by reducing subsequent LPS
production, and preserving mucosal integrity. IL-22 up-regulates expression of acute phase
proteins, including SAA, and contributes to the expression of a range of genes associated with
acute inflammatory responses, including IL-6, G-CSF, and IL-1a. Systemic administration of
IL-22 to healthy mice also up regulates LPS binding proteins to physiologically relevant
concentrations for neutralizing LPS in response to bacterial infection.
Increased expression of IL-22 is detected in inflammatory bowel disorder (IBD)
patients. See e.g., Wolk et al., 2007, J. Immunology, 178:5973; Andoh et al., 2005,
Gastroenterology, 129:969. IBDs such as Crohn’s disease (CD) and ulcerative colitis (UC) are
thought to result from a dysregulated immune response to the commensal microflora present in
the gut. Cox et al., 2012, Mucosal Immunol. 5:99-109. Both UC and CD are complex diseases
that occur in genetically susceptible individuals who are exposed to as yet poorly-defined
environmental stimuli. CD and UC are mediated by both common and distinct mechanisms and
exhibit distinct clinical features. See Sugimoto et al. 2008, J. Clinical Investigation, 118:534-
544.
In UC, inflammation occurs primarily in the mucosa of the colon and the rectum,
leading to debilitating conditions including diarrhea, rectal bleeding, and weight loss. It is
thought that UC is largely caused by an inappropriate inflammatory response by the host to
intestinal microbes penetrating through a damaged epithelial barrier (Xavier and Podolsky,
2007, Nature 448:427-434). Crohn’s disease is characterized by intestinal infilatratoin of
activated immune cells and distortion of the intestinal architechture. See Wolk et al., supra.
In recent years, a number of drugs based on various strategies to regulate the immune
response have been tested to treat IBD, including steroids, immunomodulators, and antibodies
against inflammatory cytokines, with variable success (Pastorelli et al., Expert opinion on
emerging drugs, 2009, 14:505-521). The complex variety of gut flora contributes to the
heterogeneity of the disease. Thus, there is a need for a better therapeutics for IBD.
Cardiovascular disease (CVD) is a leading cause of mortality that results, in part, from
atherosclerotic disease of large blood vessels. Atherosclerosis is the major culprit in CVD
events and is a slow and progressive disease that results from hypercholesterolemia and
chronically inflamed blood vessels. Atherosclerotic lesions are characterized as lipid laden
with infiltration of immunocytes, especially macrophages and T cells. It is now acknowledged
that both the innate and adaptive immune mechanisms contribute to the progression and
eventual thrombosis of the atherogenic plaque (Ross, Am Heart J. 1999 Nov;138 (5 Pt 2):S419-
; Hansson 2005 N Engl J Med 352(16): 1685-95; Hansson and Hermansson 2011 Nature
Immunology 12(3): 204-12).
Acute pancreatitis (AP) is an acute inflammatory process of the pancreas. Acute kidney
injury (AKI) is an abrupt loss of kidney function, resulting in the retention of urea and other
nitrogenous waste products and in the dysregulation of extracellular volume and electrolytes.
AKI was previously known as acute kidney failure. The change reflects recent recognition that
even smaller decreases in kidney function that do not result in overt organ failure are of
substantial clinical relevance and are associated with increased morbidity and mortality. There
remains a need for better treatment for AP and AKI.
Metabolic syndrome is a complex state characterized by a series of risk factors that
contribute to thrombosis, hypertension, dyslipidemia, and inflammation. Insulin resistance and
obesity are major pathogenic mechanisms underlying the metabolic syndrome.
Insulin resistance increases CVD risk because it induces endothelial dysfunction which,
in combination with atherogenic dyslipidemia, inflammation, and hypertension, contributes to
the mortality from coronary artery disease (CAD). Persistent insulin resistance also increases
the chance of developing diabetes mellitus type 2 (T2DM) although the atherogenic state occurs
many years before the onset of T2DM. It is likely therefore that the natural history of CAD lies
in the same pathway as T2DM but begins much earlier in life in a subclinical form, taking
longer to manifest clinically, with or without the presence of diabetes.
The term metabolic endotoxemia was coined to describe the condition of increased
plasma LPS induced by, for example, high-fat high-calorie diet (HFD) (Cani et al. 2007.
Diabetes 56(7): 1761-72). Mice fed with HFD have increased plasma levels of bacterial
lipopolysaccharide (LPS) and this elevation appears to be a direct consequence of the increased
dietary fat (Cani et al. 2007 supra; Cani et al. 2008 Diabetes 57(6): 1470-81; Ghoshal et al.
2009, J Lipid Res 50(1): 90-7). There is compelling evidence that gut microbiota play an
integral part in the host's energy balance and harvest of dietary nutrients and carbohydrate
metabolism, through modulation of gut mucosal epithelial cell function (Turnbaugh et al. 2009,
J Physiol (Lond) 587(Pt 17): 4153-8; Manco et al. 2010, Endocr Rev 31(6): 817-44). Alteration
in gut microbiota that occurs through disproportionate dietary fat composition or excess dietary
caloric consumption is a recognized initiator of obesity and insulin resistance, the established
sequela of cardiovascular disease. Lipopolysaccharides are found in outer membrane of gram-
negative bacteria and act as a source of endotoxin that can elicit a strong immune response
(Barcia et al. Clin Infect Dis 41 Suppl 7: S498-503). Alterations in the population, species and
regional distribution of intestinal microbiota can lead to changes in catabolism of LPS and a
high fat diet will facilities adsorption of LPS across the intestinal barrier. Under these
conditions, increased LPS in systemic circulation will induce low grade chronic inflammation,
activating the endogenous protective host response to elevate plasma lipids that, in the chronic
condition, contributes to diet induced obesity, insulin resistance and atherosclerosis, and
eventual CVD events.
Diabetes mellitus is a serious metabolic disease that is defined by the presence of
chronically elevated levels of blood glucose (hyperglycemia). This state of hyperglycemia is
the result of a relative or absolute lack of activity of the peptide hormone, insulin. Insulin is
produced and secreted by the β cells of the pancreas. Insulin is reported to promote glucose
utilization, protein synthesis, and the formation and storage of carbohydrate energy as
glycogen. Glucose is stored in the body as glycogen, a form of polymerized glucose, which can
be converted back into glucose to meet metabolism requirements. Under normal conditions,
insulin is secreted at both a basal rate and at enhanced rates following glucose stimulation, all to
maintain metabolic homeostasis by the conversion of glucose into glycogen. There remains a
need for new treatment paradigms for atherosclerosis and prevention of CVD events, metabolic
syndrome, acute endotoxemia and sepsis, and insulin-related disorders.
Wound healing is a complex process, involving an inflammation phase, a granulation
tissue formation phase, and a tissue remodeling phase (see, e.g., Singer and Clark, Cutaneous
Wound Healing, N. Engl. J. Med. 341:738-46 (1999)). These events are triggered by cytokines
and growth factors that are released at the site of injury. Many factors can complicate or
interfere with normal adequate wound healing. For example, such factors include age,
infection, poor nutrition, immunosuppression, medications, radiation, diabetes, peripheral
vascular disease, systemic illness, smoking, and stress.
For subjects with diabetes, a chronic, debilitating disease, development of a diabetic
foot ulcer (also referred to as a wound) is a common complication. A chronic ulcer is defined
as a wound that does not proceed through an orderly and timely repair process to produce
anatomic and functional integrity (see, e.g., Lazarus et al., Definitions and guidelines for
assessment of wounds and evaluation of healing, Arch. Dermatol. 130:489-93 (1994)). By its
nature, the diabetic foot ulcer is a chronic wound (American Diabetes Association, Consensus
development conference on diabetic foot wound care, Diabetes Care, 22(8):1354-60 (1999)).
Because the skin serves as the primary barrier again the environment, an open refractory wound
can be catastrophic; a major disability (including limb loss) and even death can result. Foot
ulceration is the precursor to about 85% of lower extremity amputations in persons with
diabetes (see, e.g., Apelqvist, et al., What is the most effective way to reduce incidence of
amputation in the diabetic foot? Diabetes Metab Res. Rev., 16(1 Suppl.): S75-S83 (2000)).
Thus, there is a need for accelerating or improving wound healing, including diabetic wound
healing. It is an object of the invention to go at least some way towards meeting this need; and/
or to at least provide the public with a useful choice.
SUMMARY
In a first aspect, the invention relates to an interleukin (IL)-22 Fc fusion protein
comprising an IL-22 polypeptide linked to an IgG1 Fc region by a linker, wherein the Fc region
is not glycosylated.
In a second aspect, the invention relates to an IL-22 Fc fusion protein according to the
first aspect produced by a method comprising the step of culturing a host cell capable of
expressing the IL-22 Fc fusion protein under conditions suitable for expression of the IL-22 Fc
fusion protein.
In a third aspect, the invention relates to an isolated nucleic acid encoding the IL-22 Fc
fusion protein according to the first or second aspect.
In a fourth aspect, the invention relates to a vector comprising the nucleic acid
according to the third aspect.
In a fifth aspect, the invention relates to an in vitro host cell comprising the vector
according to the fourth aspect.
In a sixth aspect, the invention relates to a method of making an IL-22 Fc fusion protein
comprising the step of culturing the host cell according to the fifth aspect under conditions
suitable for expression of the IL-22 Fc fusion protein.
In a seventh aspect, the invention relates to a pharmaceutical composition comprising an
IL-22 Fc fusion protein according to the first or second aspect and at least one pharmaceutically
acceptable carrier.
In an eighth aspect, the invention relates to use of the IL-22 Fc fusion protein according
to the first or second aspect in the manufacture of a medicament for treating inflammatory
bowel disease (IBD) in a subject in need thereof.
In an ninth aspect, the invention relates to use of the pharmaceutical composition
according to the seventh aspect in the manufacture of a medicament for treating inflammatory
bowel disease (IBD) in a subject in need thereof.
In a tenth aspect, the invention relates to use of the IL-22 Fc fusion protein according to
the first or second aspect in the manufacture of a medicament for inhibiting microbial infection
in the intestine, preserving goblet cells in the intestine during a microbial infection, enhancing
epithelial cell integrity, epithelial cell proliferation, epithelial cell differentiation, epithelial cell
migration or epithelial wound healing in the intestine, of a subject in need thereof.
In a eleventh aspect, the invention relates to use of the pharmaceutical composition
according to the seventh aspect in the manufacture of a medicament for inhibiting microbial
infection in the intestine, preserving goblet cells in the intestine during a microbial infection,
enhancing epithelial cell integrity, epithelial cell proliferation, epithelial cell differentiation,
epithelial cell migration or epithelial wound healing in the intestine, of a subject in need
thereof.
In an twelfth aspect, the invention relates to use of the IL-22 Fc fusion protein according
to the first or second aspect in the manufacture of a medicament for treating acute kidney injury
or acute pancreatitis in a subject in need thereof.
In a thirteenth aspect, the invention relates to use of the pharmaceutical composition
according to the seventh aspect in the manufacture of a medicament for treating acute kidney
injury or acute pancreatitis in a subject in need thereof.
In a fourteenth aspect, the invention relates to use of the IL-22 Fc fusion protein
according to the first or second aspect in the manufacture of a medicament for accelerating or
improving wound healing in a subject in need thereof.
In a fifteenth aspect, the invention relates to use of the pharmaceutical composition
according to the seventh aspect in the manufacture of a medicament for accelerating or
improving wound healing in a subject in need thereof.
In a sixteenth aspect, the invention relates to use of the IL-22 Fc fusion protein
according to the first or second aspect in the manufacture of a medicament for preventing or
treating a cardiovascular condition in a subject in need thereof, which condition includes a
pathology of atherosclerotic plaque formation.
In a seventeenth aspect, the invention relates to use of the pharmaceutical composition
according to the sixth aspect in the manufacture of a medicament for preventing or treating a
cardiovascular condition in a subject in need thereof, which condition includes a pathology of
atherosclerotic plaque formation.
In a eighteenth aspect, the invention relates to use of the IL-22 Fc fusion protein
according to the first or second aspect in the manufacture of a medicament for treating
metabolic syndrome in a subject in need thereof.
In an ninteenth aspect, the invention relates to use of the pharmaceutical composition
according to the seventh aspect in the manufacture of a medicament for treating metabolic
syndrome in a subject in need thereof.
In a twentieth aspect, the invention relates to use of the IL-22 Fc fusion protein
according to the first or second aspect in the manufacture of a medicament for treating acute
endotoxemia, sepsis, or both, in a subject in need thereof.
In a twenty-first aspect, the invention relates to use of the pharmaceutical composition
according to the seventh aspect in the manufacture of a medicament for treating acute
endotoxemia, sepsis, or both, in a subject in need thereof.
BRIEF DESCRIPTION
The inventionbroadly relates to IL-22 Fc fusion proteins, compositions comprising the
same, and methods of using the same.
In one embodiment, the description includes an IL-22 Fc fusion protein that binds to IL-
22 receptor, said IL-22 Fc fusion protein comprising an IL-22 polypeptide linked to an Fc
region by a linker, wherein the Fc region comprises a hinge region, an IgG CH2 domain and an
IgG CH3 domain, wherein the IL-22 Fc fusion protein comprises an amino acid sequence
having at least 95%, at least 96%, at least 97%, at least 98%, preferably at least 99% sequence
identity to the amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ
ID NO:10, SEQ ID NO:12 and SEQ ID NO:14, and wherein the Fc region is not glycosylated.
In certain embodiments, the N297 residue of the CH2 domain is changed to glycine or alanine.
In certain other embodiments, the N297 residue is changed to Gly; while in other embodiments,
the N297 residue is changed to Ala. In certain embodiments, the binding to IL-22 receptor
triggers IL-22 receptor downstream signaling, including activating STAT3.
In certain embodiments, the IL-22 Fc fusion protein comprises an amino acid sequence
having at least 98% sequence identity to the amino acid sequence of SEQ ID NO:8 or SEQ ID
NO:12. In certain other embodiments, the IL-22 Fc fusion protein comprises an amino acid
sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO:8 or
SEQ ID NO:12. In certain other embodiments, the IL-22 Fc fusion protein comprises an amino
acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID
NO:8. In certain other embodiments, the IL-22 Fc fusion protein comprises an amino acid
sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO:12.
In certain embodiments, the functions and/or activities of the IL-22 Fc fusion protein can be
assayed by in vitro or in vivo methods, for example, IL-22 receptor binding assay, Stat3
luciferase reporter activity assay, etc. In certain embodiments, the IL-22 Fc fusion protein
comprises the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:12. In certain particular
embodiments, the IL-22 Fc fusion protein comprises the amino acid sequence of SEQ ID NO:8.
In certain embodiments, the description includes the IL-22 Fc fusion protein produced by the
method comprising the step of culturing a host cell capable of expressing the IL-22 Fc fusion
protein under conditions suitable for expression of the IL-22 Fc fusion protein. In certain
embodiments, the method further comprises the step of obtaining the IL-22 Fc fusion protein
from the cell culture or culture medium. In certain embodiments, the host cell is a Chinese
hamster ovary (CHO) cell; while in other embodiments, the host cell is an E. coli cell.
In another embodiment, the description includes an IL-22 Fc fusion protein comprising
an IL-22 polypeptide linked to an IgG Fc region by a linker, wherein the Fc region comprises a
hinge region, an IgG CH2 domain and an IgG CH3 domain, and wherein the Fc region is not
glycosylated. In certain embodiments, the hinge region comprises the amino acid sequence of
CPPCP (SEQ ID NO:31). In certain other embodiments, the N297 residue in the Fc region is
changed and/or the T299 residue in the Fc region is changed. In certain embodiments, the
N297 residue in the CH2 domain is changed, preferably to glycine or alanine. In certain
particular embodiments, the N297 residue is changed to glycine. In certain other embodiments,
the N297 residue is changed to alanine. In yet other embodiments, the T299 residue is changed
to Ala, Gly or Val. In certain other embodiments, the linker is 8-20 amino acids long, 8-16
amino acids long, or 10-16 amino acids long.
In certain embodiments, the Fc region comprises the CH2 and CH3 domain of IgG1. In
certain particular embodiments, the linker comprises the amino acid sequence DKTHT (SEQ
ID NO:32). In certain embodiments, the linker comprises the amino acid sequence
GGGDKTHT (SEQ ID NO:41). In certain embodiments, the linker is at least 11 amino acids
long and comprises the amino acid sequence EPKSCDKTHT (SEQ ID NO:33). In certain other
embodiments, the linker comprises the amino acid sequence VEPKSCDKTHT (SEQ ID
NO:34), KVEPKSCDKTHT (SEQ ID NO:35), KKVEPKSCDKTHT (SEQ ID NO:36),
DKKVEPKSCDKTHT (SEQ ID NO:37), VDKKVEPKSCDKTHT (SEQ ID NO:38), or
KVDKKVEPKSCDKTHT (SEQ ID NO:39). In certain particular embodiments, the linker
comprises the amino acid sequence EPKSSDKTHT (SEQ ID NO:40). In certain embodiments,
the linker comprises the amino acid sequence VEPKSSDKTHT (SEQ ID NO:67),
KVEPKSSDKTHT (SEQ ID NO:68), KKVEPKSSDKTHT (SEQ ID NO:66),
DKKVEPKSSDKTHT (SEQ ID NO:64), VDKKVEPKSSDKTHT (SEQ ID NO:69), or
KVDKKVEPKSSDKTHT (SEQ ID NO:65). In certain particular embodiments, the linker does
not comprise the amino acid sequence of GGS (SEQ ID NO: 45), GGGS (SEQ ID NO:46) or
GGGGS (SEQ ID NO:47). In separate embodiments, the IL-22 IgG1 Fc fusion protein
comprises a linker sequence of GGGSTHT (SEQ ID NO:63). In other particular embodiments,
the IL-22 Fc fusion protein comprises the amino acid sequence of SEQ ID NO:12 or SE ID
NO:14. In certain other particular embodiments, the IL-22 Fc fusion protein comprises the
amino acid sequence of SEQ ID NO:12.
In certain embodiments, the IL-22 Fc fusion protein comprises the CH2 and CH3
domain of IgG4. In certain other embodiments, the linker comprises the amino acid sequence
SKYGPP (SEQ ID NO:43). In certain particular embodiments, the linker comprises the amino
acid sequence RVESKYGPP (SEQ ID NO:44). In certain embodiments, none of the linkers
comprise the amino acid sequence GGS (SEQ ID NO:45), GGGS (SEQ ID NO:46) or GGGGS
(SEQ ID NO:47). In other particular embodiments, the IL-22 Fc fusion protein comprises the
amino acid sequence of SEQ ID NO:8 or SE ID NO:10. In particular embodiments, the IL-22
Fc fusion protein comprises the amino acid sequence of SEQ ID NO:8. In another
embodiment, the IL-22 Fc fusion protein is produced by the method comprising the step of
culturing a host cell capable of expressing the IL-22 Fc fusion protein under conditions suitable
for expression of the IL-22 Fc fusion protein. In certain embodiments, the IL-22 Fc fusion
protein is produced by the method that further comprises the step of obtaining the IL-22 Fc
fusion protein from the cell culture or culture medium. In certain embodiments, the host cell is
a Chinese hamster ovary (CHO) cell. In certain other embodiments, the host cell is an E. coli
cell.
In yet another embodiment, the description includes a composition comprising an IL-22
Fc fusion protein, said IL-22 Fc fusion protein comprising an IL-22 polypeptide linked to an Fc
region by a linker, wherein the Fc region comprises a hinge region, an IgG CH2 domain and an
IgG CH3 domain, and wherein the composition has an afucosylation level in the CH2 domain
of no more than 5%. In certain embodiments, the afucosylation level is no more than 2%, more
preferably less than 1%. In certain embodiments, the afucosylation level is measured by mass
spectrometry. In certain embodiments, the Fc region comprises the CH2 and CH3 domain of
IgG4. In certain embodiments, the Fc region comprises a CH2 and CH3 domain of IgG1. In
certain other embodiments, the hinge region comprises the amino acid sequence of CPPCP
(SEQ ID NO:31). In certain embodiments, the IL-22 Fc fusion protein comprises the amino
acid sequence of SEQ ID NO:24 or SEQ ID NO:26. In certain embodiments, the IL-22 Fc
fusion protein comprises the amino acid sequence of SEQ ID NO:24. In certain embodiments,
the composition is produced by the process comprising the steps of culturing a host cell capable
of expressing the IL-22 Fc fusion protein under conditions suitable for expression of the IL-22
Fc fusion protein, and obtaining the IL-22 Fc fusion protein from the cell culture or culture
medium, wherein the composition has an afucosylation level in the CH2 domain of the Fc
region of no more than 5%. In certain embodiments, the afucosylation level is no more than
2%, more preferably less than 1%. In certain embodiments, the IL-22 Fc fusion protein is
obtained by purification, preferably purifying fucosylated species away from afucosylated
species. In certain embodiments, the IL-22 Fc fusion protein is purified by affinity
chromatography. In certain embodiments, the host cell is a CHO cell.
In a further embodiment, the description includes an IL-22 Fc fusion protein, or a
composition comprising IL-22 Fc fusion proteins, said IL-22 Fc fusion protein is produced by
the process comprising the step of culturing a host cell capable of expressing the IL-22 Fc
fusion protein under conditions suitable for expression of the IL-22 Fc fusion protein. In
certain embodiments, the process further comprises the step of obtaining the IL-22 Fc fusion
protein from the cell culture or culture medium. In certain embodiments, the host cell is a CHO
cell; while in other embodiments, the host cell is an E. coli cell.
In a further embodiment, the description includes a composition comprising an IL-22 Fc
fusion protein described herein. In yet another embodiment, the description includes a
pharmaceutical composition comprising an IL-22 Fc fusion protein described herein, and at
least one pharmaceutically acceptable carrier. In certain embodiments, the composition or
pharmaceutical composition comprises an IL-22 Fc fusion protein comprising an amino acid
sequence of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:24 or
SEQ ID NO:26. In certain particular embodiments, the composition or pharmaceutical
composition comprises an IL-22 Fc fusion protein comprising the amino acid sequence of SEQ
ID NO:8. In certain particular embodiments, the IL-22 Fc fusion protein is produced by E. coli.
In certain other embodiments, the Fc region of the IL-22 Fc fusion protein is not glycosylated.
In certain further embodiments, the IL-22 Fc fusion protein does not induce antibody dependent
cellular cytotoxicity (ADCC). In certain embodiments, the pharmaceutical composition further
comprises a suboptimal amount of a therapeutic agent such as dexamethasone. In certain
embodiments, the IL-22 polypeptide comprises the amino acid sequence of SEQ ID NO:4.
Further, according to each and every embodiment of the description, the IL-22 Fc fusion
protein can be a dimeric IL-22 Fc fusion protein (with respect to IL-22); while in other
embodiments, the IL-22 Fc fusion protein can be a monomeric Fc fusion protein (with respect
to IL22).
In a further embodiment, the description includes a monomeric IL-22 Fc fusion protein.
In certain particular embodiments, the monomeric fusion protein comprises an IL-22 Fc fusion
arm and an Fc arm. In certain embodiments, the IL-22 Fc fusion arm and the Fc arm comprises
either a knob or a hole in the Fc region. In certain embodiments, the Fc region of the IL-22 Fc
fusion arm (the monomer IL-22 Fc fusion) comprises a knob and the Fc region of the Fc arm
(the monomer Fc without linking to IL-22) comprises a hole. In certain embodiments, the Fc
region of the IL-22 Fc fusion arm (the monomer IL-22 Fc fusion) comprises a hole and the Fc
region of the Fc arm (the monomer Fc without linking to IL-22) comprises a knob. In certain
other embodiments, the monomeric IL-22 Fc fusion protein comprises the amino acid sequence
of SEQ ID NO:61 and SEQ ID NO:62. In certain other embodiments, the Fc region of both
arms further comprises an N297G mutation. In certain embodiments, the monomeric IL-22 Fc
is produced by the process comprising the step of culturing one or more host cells comprising
one or more nucleic acid molecules capable of expressing the first polypeptide comprising the
amino acid sequence of SEQ ID NO:61 and the second polypeptide comprising the amino acid
sequence of SEQ ID NO:62. In certain other embodiments, the method further comprises the
step of obtaining the monomeric IL-22 Fc fusion protein from the cell culture or culture
medium. In certain embodiments, the host cell is an E. coli cell. In a related embodiment, the
description includes a composition or pharmaceutical composition comprising the monomeric
IL-22 Fc fusion protein.
In yet another embodiment, the description includesan isolated nucleic acid encoding
the IL-22 Fc fusion protein described herein. In certain embodiments, the nucleic acid encodes
the IL-22 Fc fusion protein comprising the amino acid sequence of SEQ ID NO:8, SEQ ID
NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:24 or SEQ ID NO:26, preferably SEQ ID
NO:8 or SEQ ID NO:12, more preferably SEQ ID NO:8. In certain other embodiments, the
nucleic acid comprises the polynucleotide sequence of SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:11, SEQ ID NO:13, SEQ ID NO:23 or SEQ ID NO:25. In certain particular embodiments,
the nucleic acid comprises the polynucleotide sequence of SEQ ID NO:7 or SEQ ID NO:11,
preferably SEQ ID NO:7. In certain embodiments, the isolated nucleic acid comprises a
polynucleotide sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the polynucleotide
sequence of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13; SEQ ID NO:23 or
SEQ ID NO:25. In certain embodiments, the isolated nucleic acid comprises a polynucleotide
sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID
NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13; SEQ ID NO:23 or SEQ ID NO:25,
wherein the isolated nucleic acid is capable of encoding an IL-22 Fc fusion protein that is
capable of binding to IL-22R and/or triggering IL-22R activity and wherein the Fc region of the
IL-22 Fc fusion protein is not glycosylated. In certain embodiments, the isolated nucleic acid
comprises a polynucleotide sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the
polynucleotide sequence of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13;
SEQ ID NO:23 or SEQ ID NO:25, wherein the isolated nucleic acid is capable of encoding an
IL-22 Fc fusion protein comprising the amino acid sequence of SEQ ID NO:8, 10, 12, or 14. In
related embodiments, the description includesvectors comprising the nucleic acid described
above, and a host cell comprising the vector. In certain embodiments, the host cell is a
prokaryotic cell or eukaryotic cell. In certain particular embodiments, the host cell is a
prokaryotic cell, including without limitation, an E. coli cell. In certain other embodiments, the
host cell is a eukaryotic cell, including without limitation, a CHO cell. In certain embodiments,
the host cell comprises a vector comprising a nucleic acid encoding the IL-22 Fc fusion protein
comprising the amino acid sequence of SEQ ID NO:8.
In a further related embodiment, the description includesmethods of making the IL-22
Fc fusion protein comprising the step of culturing the host cell under conditions suitable for
expression of the IL-22 Fc fusion protein. In certain embodiments, the method further
comprises the step of obtaining the IL-22 Fc fusion protein from the cell culture or culture
medium. The IL-22 Fc fusion protein can be obtained from the cell culture or culture medium
by any methods of protein isolation or purification known in the art, including without
limitation, collecting culture medium, freezing/thawing, centrifugation, cell lysis,
homogenization, ammonium sulfate precipitation, HPLC, and affinity, gel filtration, and ion
exchanger column chromatography. In certain embodiments, the method further comprises the
step of removing afucosylated IL-22 Fc fusion protein. In certain other embodiments, the
afucosylated IL-22 Fc fusion protein is removed by affinity column chromatography. In certain
embodiments, the host cell is an E. coli cell. In other embodiments, the host cell is a CHO cell.
In yet another aspect, the invention provides a composition or pharmaceutical
composition comprising an IL-22 Fc fusion protein of the invention and at least one
pharmaceutically acceptable carrier. In certain embodiments, the IL-22 Fc fusion protein
comprises the amino acid sequence of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID
NO:14, SEQ ID NO:24, or SEQ ID NO:26. In other embodiments, the Fc region of the IL-22
Fc fusion protein is not glycosylated. In certain embodiments, the Fc region of the IL-22 Fc
fusion protein is not glycosylated while the IL-22 polypeptide is glycosylated. In certan such
embodiments, the IL-22 Fc fusion protein is produced in CHO cells. In certain embodiments,
the IL-22 Fc fusion protein does not induce antibody dependent cellular cytotoxicity. In yet
other embodiments, the pharmaceutical composition further comprises dexamethasone or a
TNF antagonist. In certain particular embodiments, the dexamethasone or a TNF antagonist is
present at a suboptimal amount.
In certain other embodiments, the pharmaceutical composition comprising IL-22 Fc
fusion proteins has an afucosylation level in the CH2 domain of no more than 5%, preferably
no more than 2%, more preferably less than 1%. In certain particular embodiments, the IL-22
Fc fusion protein comprises the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:26,
preferably SEQ ID NO:24. In certain other embodiments, the IL-22 Fc fusion protein is
produced in CHO cells. In certain particular embodiments, the subject is a human. In certain
embodiments, the pharmaceutical composition is administered systematically or topically. In
certain other embodiments, the pharmaceutical composition is administered intravenously,
subcutaneously, intraperitoneally or topically.
In a further embodiment, the description includesa pharmaceutical composition
comprising an IL-22 polypeptide or IL-22 Fc fusion protein described herein and at least one
pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutically acceptable
carrier is a gelling agent. In certain embodiments, the gelling agent is a polysaccharide. In some
embodiments, the gelling agent is, without limitation, methylcellulose, hydroxyethyl cellulose,
carboxymethyl cellulose, hydroxypropyl cellulose, POE-POP block polymers, alginate,
hyaluronic acid, polyacrylic acid, hydroxyethyl methylcellulose or hydroxypropyl
methylcellulose. In some embodiments, the polysaccharide is a cellulosic agent such as, without
limitation, hydroxyethyl methylcellulose or hydroxypropyl methylcellulose. In certain
embodiments, the gelling agent is hydroxypropyl methylcellulose. In some embodiments, the
pharmaceutical composition is for topical administration. In certain embodiments, the
pharmaceutical composition for topical administration comprises an IL-22 polypeptide. In some
embodiments, the pharmaceutical composition for topical administration comprises an IL-22 Fc
fusion protein. In certain embodiments, the pharmaceutical composition for topical
administration comprises an IL-22 polypeptide without an Fc fusion.
Also described are methods of treating IBD in a subject in need thereof comprising
administering to the subject the pharmaceutical composition comprising an IL-22 Fc fusion
protein described. In certain embodiments, the IBD is ulcerative colitis. In certain other
embodiments, the IBD is Crohn’s disease. In certain particular embodiments, the Fc region of
the IL-22 Fc fusion protein is not glycosylated. In certain embodiments, the N297 residue
and/or the T299 residue of the Fc region is changed. In certain embodiments, the N297 residue
of the Fc region is changed. In certain other embodiments, the N297 residue is changed to Gly
or Ala, preferably Gly. In certain other embodiments, the T299 residue is changed, preferably to
Val, Gly or Ala. In certain particular embodiments, the IL-22 Fc fusion protein comprises the
amino acid sequence of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or EQ ID NO:14,
preferably SEQ ID NO:8. In certain embodiments, the IL-22 Fc fusion protein is produced in E.
coli or a CHO cell. In certain embodiments, the subject is a human. In certain other
embodiments, the pharmaceutical composition is administered intravenously, subcutaneously,
intraperitoneally or topically.
In another embodiment, the description includesmethods of treating any one or
combination of the following diseases using an IL-22 polypeptide or an IL-22 Fc fusion protein
of this invention: Type II diabetes, Type II diabetes with morbid obesity, wounds (including
diabetic wounds and diabetic ulcers), burns, ulcers (including pressure ulcer and venous ulcer),
graft versus host disease (GVHD), atherosclerosis, cardiovascular disease, metabolic syndrome,
endotoxemia (acute and mild), sepsis, acute coronary heart disease, hypertension, dyslipemia,
obesity, hyperglycemia, lipid metabolism disorders, hepatitis, acute hepatitis, renal failure,
acute renal failure, acute kidney injury, renal draft failure, post cadaveric renal transplant
delayed graft function, contrast induced nephropathy, pancreatitis, acute pancreatitis, liver
fibrosis and lung fibrosis. In certain embodiments, acute pancreatitis can be mild to moderate
to severe disease. In certain embodiments, acute pancreatitis includes disease post ERCP
(endoscopic retrograde cholangiopancreatography). In some further embodiments, the patient to
be treated for the above disease is in need of a change in his HDL/LDL lipid profile, which IL-
22 polypeptide or IL-22 Fc fusion proteins can alter in the patient to increase HDL and decrease
LDL. In a related embodiment, the description includesuses of an IL-22 polypeptide or an IL-
22 Fc fusion protein in the preparation of a medicament for the treatment of any one or
combinations of the above diseases.
Also described are methods of inhibiting microbial infection in the intestine, or
preserving goblet cells in the intestine during a microbial infection, of a subject in need thereof
comprising the step of administering to the subject the pharmaceutical composition comprising
the IL-22 Fc fusion protein described. Also described are methods of enhancing epithelial cell
integrity, mucosal healing, epithelial cell proliferation, epithelial cell differentiation, epithelial
cell migration or epithelial wound healing in the intestine in a subject in need thereof
comprising administering to the subject the pharmaceutical composition comprising the IL-22
Fc fusion protein described. In certain embodiments, the epithelial cell is intestinal epithelial
cell.
In another embodiment, a method for preventing or treating a cardiovascular condition,
which condition includes a pathology of atherosclerotic plaque formation, is described. The
method includes administering to a subject in need thereof a therapeutically effective amount of
an IL-22 polypeptide or an IL-22 Fc fusion protein. The cardiovascular condition includes, for
example, coronary artery disease, coronary microvascular disease, stroke, carotid artery disease,
peripheral arterial disease, and chronic kidney disease. The method can include further slowing
down the progression of atherosclerotic plaque formation. The method can further include
administering one or more additional therapeutic agent to the subject for the prevention or
treatment of the cardiovascular condition.
In another embodiment, a method for treating metabolic syndrome is described. The
method includes administering to a subject in need thereof a therapeutically effective amount of
an IL-22 polypeptide or an IL-22 Fc fusion protein. The method can further include reducing
one or more risk factors associated with metabolic syndrome, including one or more of
abdominal obesity, hyperglycemia, dyslipidemia, and hypertension. The method can further
include reducing the level of bacterial lipopolysaccharide (LPS) in the subject. The method can
further include administering one or more additional agent to the subject for the prevention or
treatment of metabolic syndrome.
In another embodiment, a method for delaying or slowing down the progression of
atherosclerosis is described. The method includes administering to a subject in need thereof a
therapeutically effective amount of an IL-22 polypeptide or an IL-22 Fc fusion protein. The
method can further include administering one or more additional agent to the subject for
delaying or slowing down the progression of atherosclerosis.
In another embodiment, a method of preventing indicia of atherosclerosis is described.
The method includes administering a therapeutically effective amount of an IL-22 polypeptide
or an IL-22 Fc fusion protein to a subject at risk of atherosclerosis, wherein the IL-22
polypeptide of IL-22 Fc fusion protein is effective against the development of indicia of
atherosclerosis. In certain embodiments, the subject has been identified to be at risk to develop
a cardiovascular condition. In certain embodiments, the subject is genetically at risk of
developing a cardiovascular condition. In one or more embodiments, the indicia of
atherosclerosis include plaque accumulation. In some embodiments, the indicia of
atherosclerosis include vascular inflammation. The method can further include administering
one or more additional agent to the subject for preventing indicia of atherosclerosis.
In yet another embodiment, a method of treating one or more of acute endotoxemia and
sepsis is described. The method includes administering to a subject in need thereof a
therapeutically effective amount of an IL-22 polypeptide or an IL-22 Fc fusion protein. The
method can further include administering one or more additional agent to the subject for
treating one or more of acute endotoxemia and sepsis.
In one other embodiment, a method is described for accelerating or improving wound
healing, or both, in a subject. The method includes administering to a subject in need thereof a
therapeutically effective amount of an IL-22 polypeptide, an IL-22 Fc fusion protein or an IL-
22 agonist. In certain embodiments, the wound is a chronic wound. In certain other
embodiments, the wound is an infected wound. In certain embodiments, the subject is diabetic,
including a subject with type II diabetes. In one or more embodiments, the wound is a diabetic
foot ulcer. In certain embodiments, the therapeutically effective amount of an IL-22
polypeptide, IL-22 Fc fusion protein or IL-22 agonist is administered until there is complete
wound closure. In some embodiments, the administration is systemic; and in other
embodiments, the administration is topical. In certain embodiments, the IL-22 polypeptide, IL-
22 Fc fusion protein or IL-22 agonist is in a formulation for topical administration. In certain
embodiments, the topical formulation comprises an IL-22 polypeptide without an Fc fusion. In
certain embodiments, the IL22 agonist is selected from the group consisting of an IL-22
polypeptide, an IL-22 Fc fusion protein, an IL-22 agonist, an IL-19 polypeptide, an IL-19 Fc
fusion protein, an IL-19 agonist, an IL-20 polypeptide, an IL-20 Fc fusion protein, an IL-20
agonist, an IL-24 polypeptide, an IL-24 Fc fusion protein, an IL-24 agonist, an IL-26
polypeptide, an IL-26 Fc fusion protein, an IL-26 agonist, and an IL-22R1 agonist. In certain
other embodiments, the IL-22 agonist is selected from the group consisting of an IL-22
polypeptide, an IL-22 Fc fusion protein, an IL-22 agonist, an IL-20 polypeptide, an IL-20 Fc
fusion protein, an IL-20 agonist, an IL-24 polypeptide, an IL-24 Fc fusion protein, an IL-24
agonist and an IL-22R1 agonist. In certain embodiments, the IL-22R1 agonist is an anti-IL22R1
agonistic antibody.
In a further embodiment, the description includesmethods of treating a metabolic
syndrome comprising the step of administering to a subject in need thereof a therapeutically
effective amount of one or more IL-22 agonists. In certain embodiments, the IL22 agonist is
selected from the group consisting of an IL-22 polypeptide, an IL-22 Fc fusion protein, an IL-
22 agonist, an IL-19 polypeptide, an IL-19 Fc fusion protein, an IL-19 agonist, an IL-20
polypeptide, an IL-20 Fc fusion protein, an IL-20 agonist, an IL-24 polypeptide, an IL-24 Fc
fusion protein, an IL-24 agonist, an IL-26 polypeptide, an IL-26 Fc fusion protein, an IL-26
agonist, and an IL-22R1 agonist. In certain other embodiments, the IL-22 agonist is selected
from the group consisting of an IL-22 polypeptide, an IL-22 Fc fusion protein, an IL-22 agonist,
an IL-20 polypeptide, an IL-20 Fc fusion protein, an IL-20 agonist, an IL-24 polypeptide, an
IL-24 Fc fusion protein, an IL-24 agonist and an IL-22R1 agonist. In certain embodiments, the
IL-22R1 agonist is an anti-IL22R1 agonistic antibody. In certain other embodiments, the
metabolic syndrome is diabetes. In certain particular embodiments, the metabolic syndrome is
type II diabetes.
According to another embodiment, the subject is administered an IL-22 Fc fusion
protein of the invention. In certain embodiments, the subject is a human. In certain
embodiments, the IL-22 polypeptide or IL22 Fc fusion protein is administered intravenously,
subcutaneously, intraperitoneally, systemically or topically.
In certain embodiments, the Fc region of the IL-22 Fc fusion protein is not glycosylated.
In certain embodiments, the N297 residue and/or the T299 residue of the Fc region is changed.
In certain embodiments, the N297 residue of the Fc region is changed. In certain other
embodiments, the N297 residue is changed to Gly or Ala, preferably Gly. In certain other
embodiments, the T299 residue is changed, preferably to Val, Gly or Ala. In certain particular
embodiments, the IL-22 Fc fusion protein comprises the amino acid sequence of SEQ ID NO:8,
SEQ ID NO:10, SEQ ID NO:12 or EQ ID NO:14, preferably SEQ ID NO:8. In certain
embodiments, the IL-22 Fc fusion protein is produced in E. coli. In certain embodiments, the
subject is a human. In certain other embodiments, the pharmaceutical composition is
administered intravenously, subcutaneously or topically.
In certain other embodiments, the pharmaceutical composition comprising IL-22 Fc
fusion proteins has an afucosylation level in the CH2 domain of no more than 5%, preferably
no more than 2%, more preferably less than 1%. In certain particular embodiments, the IL-22
Fc fusion protein comprises the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:26,
preferably SEQ ID NO:24. In certain other embodiments, the IL-22 Fc fusion protein is
produced in CHO cells. In certain particular embodiments, the subject is a human. In certain
other embodiments, the pharmaceutical composition is administered intravenously,
subcutaneously or topically.
In yet other embodiments, the N-glycan attached to the Fc region of the IL-22 Fc fusion
protein is enzymatically removed by a glycolytic enzyme. In certain embodiments, the
glycolytic enzyme is peptide-N-glycosidase (PNGase). In certain particular embodiments, the
subject is a human.
In yet a further embodiment, the description also includes uses of an IL-22 Fc fusion
protein described herein in the preparation of a medicament for the treatment of IBD, including
UC and CD, in a subject in need thereof. In a related embodiment, the description includes uses
of an IL-22 Fc fusion protein described herein in the preparation of a medicament for inhibiting
microbial infection in the intestine, or preserving goblet cells in the intestine during a microbial
infection in a subject in need thereof. In yet another embodiment, the description includesuses
of an IL-22 Fc fusion protein described herein in the preparation of a medicament for enhancing
epithelial cell integrity, epithelial cell proliferation, epithelial cell differentiation, epithelial cell
migration or epithelial wound healing in the intestine, in a subject in need thereof. In other
related embodiment, the description includesuses of an IL-22 polypeptide or IL-22 Fc fusion
protein in the preparation of a medicament for treating a cardiovascular condition, metabolic
syndrome, atherosclerosis, acute kidney injury, acute pancreatitis, accelerating, promoting or
improving wound healing, including without limitation, healing of a chronic wound, diabetic
wound, infected wound, pressure ulcer or diabetic foot ulcer, in a subject in need thereof.
Each and every embodiment can be combined unless the context clearly suggests
otherwise. Each and every embodiment can be applied to each and every aspect of the
invention unless the context clearly suggests otherwise.
Specific embodiments of the present invention will become evident from the following
more detailed description of certain preferred embodiments and the claims.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows amino acid sequence alignment of mature IL-22 from different
mammalian species: human (GenBank Accession No.Q9GZX6, SEQ ID NO:4, chimpanzee
(GenBank Accession No.XP_003313906, SEQ ID NO:48), orangutan (GenBank Accession No.
XP_002823544, SEQ ID NO:49), mouse (GenBank Accession No. Q9JJY9, SEQ ID NO:50)
and dog (GenBank Accession No. XP_538274, SEQ ID NO:51).
Figure 2 shows mass spectrometry results of the glycosylation status of the Fc region of
a typical human monoclonal IgG1 Fc (Panel A), IL-22 IgG1 Fc fusion containing the linker
sequence EPKSCDKTHT (SEQ ID NO:33, Panel B), EPKSSDKTHT (SEQ ID NO:40, Panel
C), and GGGDKTHT (SEQ ID NO:41, Panel D), and IL-22 IgG4 Fc fusion containing the
linker sequence RVESKYGPP without or with the N297G mutation (SEQ ID NO:44, Panels E
and F, respectively) and IL-22 IgG1 Fc fusion containing the linker sequence EPKSSDKTHT
(SEQ ID NO:40) with the N297G mutation (Panel G).
Figure 3 shows sequence alignment of human IL-22 IgG4 Fc fusion (N297G, full
length Fc sequence with the C-terminal Lys, SEQ ID NO:16, without Lys SEQ ID NO:8), IL-22
IgG1 Fc fusion (N297G, full length Fc sequence with the C-terminal Lys, SEQ ID NO:20,
without Lys SEQ ID NO:12) and IL-22 (SEQ ID NO:4). The IL-22 sequence shown is the
matured form without the leader sequence. The hinge sequence CPPCP (SEQ ID NO:31) is
shown in the box, followed by the CH2 and CH3 domains. The N297G substitution and the
optional C-terminus Lys residue are marked.
Figure 4 presents a graph showing the results of STAT3 luciferase assay. Luciferase
activity stimulated by IL-22 IgG4 Fc fusion or IL-22 IgG1 Fc fusion was measured in 293 cells
expressing human IL-22R. The results show that IL-22 IgG4 and IL-22 IgG1 Fc fusion
exhibited similar in vitro activity.
Figure 5 shows the therapeutic effects of mouse IL-22 Fc fusion protein in the dextran
sodium sulfate (DSS)-induced mouse IBD model. Mouse IL-22 Fc fusion protein improved
colon histology in the DSS-induced IBD mice (Figure 5B) and the improvement was translated
to reduced colon histology score (Figure 5C). IL-22 Fc fusion protein treatment resulted in
reduced weight loss of the mice during treatment as compared to dexamethasone, currently the
best standard of care in this model (Figure 5A).
Figure 6 shows the rate of serum clearance of human IL-22 IgG4 and IgG1 Fc fusion
proteins in cynomolgus monkeys dosed at 0.15 mg/kg and 1.5 mg/kg on day 0 and day 7.
Figure 7 shows the serum levels of three IL-22R downstream genes in cynomolgus
monkeys after dosing at 0.15 mg/kg and 1.5 mg/kg at day 1 and day 8 (same dosing regimen as
day 0 and day 7 in Figure 6. Figure 7A shows dose-dependent increases in serum amyloid A
(SAA), Figure 7B shows does-dependent increases in lipopolysaccharide binding protein (LPS-
BP), Figure 7C shows dose-dependent increases in RegIII/Pancreatitis Associated Protein (PAP
or PancrePAP), following hIL-22 Fc administration.
Figure 8 shows a high resolution MicroCT demonstrating the atherosclerotic plaque
burden in the aorta arch and brachiocephalic artery of an 8 month old Ldlr-/-Apobec1-/- mouse
on high fat diet.
Figure 9 shows that Ldlr-/-Apobec1-/- mice were sensitive to dietary challenges and
showed a substantially increased level of atherosclerosis as measured from microCT (A), but
with only modestly increased serum LDL levels (B).
Figure 10 shows the response of Ldlr-/-Apobec1-/- mice to an acute low grade
inflammation stimulus, demonstrating an increase in sera MCP-1 (A) and IL-6 (B) greater than
observations in wt C57 mice and accompanied by loss of vascular function as assessed by flow
mediated dilation and infusion of nitroglycerine (C).
Figure 11 shows that chronic endotoxin exposure results in dyslipidemia (A) and
greater plaque burden (B) and instability (C).
Figure 12 shows fasting blood glucose was reduced in the ILFc treated group
compared to controls (A) and glucose clearance was improved with ILFc treatment as seen
from the glucose tolerance test (B, C).
Figure 13 shows reduction in total cholesterol occurs after treatment with ILFc. In
Ldlr-/-Apobec1-/- mice total cholesterol was elevated, in both the fasting and fed conditions,
and was reduced in the ILFc group compared with the controls as measured at the end of
the treatment period (A). Plasma triglycerides levels were also reduced upon ILFc
treatment with a marked reduction in the fed state (B).
Figure 14 shows the hyperlipidemia seen in the Ldlr-/-Apobec1-/- mouse was reduced
following ILFc treatment. LDL was reduced in both the fasting and fed state (A), HDL was
raised (B), and LDL/HDL ratio were reduced in both fast and fed (C). vLDL was reduced
under fed conditions (D). Results of HDL (E), LDL (F) and LDL/HDL ratio (G) were depicted
after 5 days with mice given two doses.
Figure 15 shows that plasma LPS levels were reduced after ILFc treatment.
Figure 16 shows improved endothelial function measure by vascular reactivity after IL-
22-Fc treatment.
Figure 17 depicts the quantitative analysis of plaque burden performed using contrast-
enhanced microCT on postmortem samples of the dissected aortic arch, ascending and
descending aorta (A), the brachiocephalic artery (B) and aortic valve (C).
Figure 18 shows body weights (A) and food intake (B) following ILFc treatment.
Figure 19 depicts a schematic of diabetic mouse model treatment regimen.
Figures 20A-C show body weight and serum glucose levels in db/db mice
demonstrating that ILFc significantly reduced glucose in the obese mice.
Figure 21 shows IL-22Fc treatment improves glucose tolerance and insulin sensitivity
based on the Glucose Tolerance Test (GTT). p < 0.05
Figures 22A-B show that IL-22Fc treatment improved insulin sensitivity based on the
Insulin Tolerance Test (ITT) as measured through mg/dL glucose levels (A) and % glucose
reduction (B).
Figure 23 shows that IL-22Fc increased insulin expression in islets. (A) Green shows
glucagon, red shows insulin. The circled area surrounded by red line shows islet area. Bar, 50
μm. (B) Average insulin staining intensity. (C) Average glucagon staining intensity. (D) Fed
insulin levels in HFD-fed mice. (E) Fasted insulin levels in HFD-fed mice. (F) IL-22 Fc
reversed insulin insensitivity in HFD-fed mice. **P<0.01, ***P<0.001. Error bars, s.e.m.
Figures 24A-B depict quantitative analysis of insulin-signal intensity in ILFc
treated animals.
Figures 25A-B show that the insulin-positive area was increased in ILFc treated
animals compared to control.
Figure 26 shows histological sections demonstrating a decrease in hepatic periportal
steatosis with ILFc treatment (B) as compared to control (A).
Figures 27A-B show an assessment of IL-22R in HFD induced glucose tolerance. (A)
glucose levels (mg/dL) over time post glucose ip injection. (B) Calculation of the total area
under the curve (AUC).
Figure 28 shows mass of IL-22 receptor KO mice compared to littermate control.
Figures 29A-D Ldlr -/-, Apobec1 -/- (dko) mice were treated with 50 ug IL-22Fc or 50
ug anti-ragweed (n=6 per group) for 48 hours. Serum LPS was reduced by 50% (p=0.0052) and
serum LDL/HDL was reduced by 30% (p=0.049) in IL-22Fc treated mice.
Figure 30 shows a nucleotide sequence of a cDNA encoding a native human IL-22
(SEQ ID NO:70).
Figure 31 shows the amino acid sequence derived from the coding sequence shown in
(SEQ ID NO:71).
Figure 32A shows the amino acid sequence of a mouse ILmouse-IgG2a fusion
protein (SEQ ID NO:73). Figure 32B shows the nucleotide sequence encoding mouse IL
mouse IgG2a fusion protein (SEQ ID NO:72).
Figure 33 shows that lack of signaling through IL-22R results in delayed wound
healing. IL-22R KO mice wounds were significantly delayed (p=0.0018 on day 10 & p=0.005
on day 12) in healing compared to WT littermate control mice.
Figures 34A-C represent individual mice (n=10) wound gap at days 10, 12 and 15.
Representative photo images of the wounds for both IL-22R KO mice and WT at day 14 are
shown (D).
Figure 35 illustrates wound healing comparison between Control WT mice (BKS) and
Diabetic db/db mice. (A) Wound healing in the db/db mice was considerably delayed
throughout the period of study and did not heal fully even at day 28. The bar graph in (B) shows
the level of IL-22 expression as fold change in wild type or db/db mice days after wound
excision.
Figure 36 is a schematic representation of the study design for testing ILFc in db/db
mice in a total of 3 groups (n=7). Anti-ragweed was used for control Fc protein and anti-
FGFR1 antibody was used as positive control for glucose regulation.
Figure 37 shows IL-22 Fc normalized fed glucose level of treated mice as compared to
controls from days 4 until day 27. Glucose levels were recorded using an Onetouch®
glucometer.
Figure 38 shows graphically comparative wound gap measurement of ILFc
compared to 2 control antibodies: anti-ragweed and anti-FGFR1. Each data point represents an
average of 7 mice/group.
Figures 39A-D show individual wound gap measurements at days 15, 19, 21, and day
27. The photographs of representative mice at day 27 are shown (E).
Figure 40 is a schematic representation of the study design for testing topical vs.
systemic dosing of ILFc compared to control antibody treatment in db/db mice; Total 3
groups (n=7).
Figure 41A-B show graphically comparative wound gap measurement of ILFc
topical vs. systemic dosing with control Fc topical treatment. Anti-ragweed antibody was used
as an Fc control antibody. Each data point represents an average of 7 mice/group.
Figure 42 shows photographically surgically removed wound tissue from representative
mice showing both top as well as back view on day 22 from ILFc (B) and control antibody
(A).
Figure 43A shows the strategy for generation of IL-22R KO mice. Figure 43B shows
RT-PCR results of IL-22Ra1 mRNA expression in colon from IL-22R KO and WT mice.
Figure 43 C shows RT-PCR results of Reg3b mRNA expression in colon from IL-22R KO and
WT mice 2 days after a single dose injection of IL-22 Fc or control IgG. ***P<0.001. Error
bars, s.e.m.
Figure 44 shows results demonstrating that obese mice mounted defective IL-22
responses. (A-D) Lymphocytes in draining lymph nodes of db/db (A-B), DIO (C-D) and control
mice immunized with OVA/CFA were analyzed for IL-22 expression on day 7 by flow
cytometry. Numbers on the FACS plots in (A, C) are percentage of IL-22 cells within CD4 T
cells. (E-F) db/db, lean controls, HFD and chow diet-fed normal mice were injected with
flagellin or PBS. Serum was harvested after 2 h. ELISA of IL-22 from db/db and lean controls
(E), and HFD and chow diet-fed mice (F). Data shown are representative of three (A-B) or two
(C-F) independent experiments. N=4 in all experiments. * P<0.05, **P<0.01, ***P<0.001,
Error bars, s.e.m.
Figure 45 shows defects in IL-17 and IL-22 production in leptin signal-deficient mice.
(A-B) IL-17A and IL-22 expression were analyzed on day 7 as percentage within CD4+ cells in
db/db and ob/ob mice immunized with OVA/CFA. (C) IL-22 ELISA from culture supernatant
of purified naïve WT CD4+ T cells that were stimulated under IL-22 producing conditions with
or without recombinant mouse leptin (1 μg/ml). (D) IL-22 ELISA from culture supernatant of
Rag2 KO splenocytes stimulated with IL-23 with or without recombinant mouse leptin (1
μg/ml). (E) ELISA of serum IL-22 from ob/ob or lean controls 2 hours after flagellin
stimulation. * P<0.05, **P<0.01, ***P<0.001, Error bars, s.e.m.
Figure 46 shows results demonstrating that the susceptibility of db/db (ob/ob) mice to
C. rodentium infection was associated with defective IL-22 production and rescued by
exogenous IL-22–Fc. (A) IL-22 mRNA expression in colons from WT, db/db and ob/ob mice
(n=5) after C. rodentium infection. (B) Body weight and (C) survival of db/db and lean control
mice (n=10) infected with C. rodentium. (D-E) Colon histology of lean control (D) and db/db
(E) mice on day 10, showing epithelial hyperplasia, enterocyte shedding into the gut lumen,
bacterial colonies (arrows) and submucosal edema (vertical bar). Horizontal bar, 200 μm. (F)
Clinical score determined by colon histology (n=5). (G-H) Bacterial burden of db/db and lean
control mice (n=5) in liver (G) and spleen (H) on day 10. (I) ELISA of anti-C. rodentium IgG in
lean control and db/db mice (n=5) on day 10. (J). Survival of lean control or db/db mice (n=10)
treated with IL-22–Fc or control IgG after infection. Data shown are representative of three
independent experiments. * P<0.05, **P<0.01, ***P<0.001, Error bars, s.e.m.
Figure 47 shows results demonstrating that diabetic disorders were reduced by IL-22–
Fc treatment. (A-D) HFD-fed mice were treated with IL-22–Fc twice per week (n=10). (A)
Blood glucose on day 20 (fed) and day 21 (16-hour fasting). (B) Body weight on day 30. (C)
Glucose tolerance test on day 21. (D) Insulin tolerance test on day 28. Data shown are
representative of two independent experiments. * P<0.05, **P<0.01, ***P<0.001, Error bars,
s.e.m.
Figure 48 shows results demonstrating that IL-22 prevents the diabetic disorders of
mice fed with HFD. (A) body weight, (B) blood glucose, (C) glucose tolerance test on day 23,
(D) blood glucose on day 23 after 16 h fast, and (E) abdominal fat pad on day 25. * P<0.05,
**P<0.01, ***P<0.001, Error bars, s.e.m.
Figure 49 shows results demonstrating that IL-22 regulates metabolic syndrome
through multiple mechanisms. (A-C) Two groups of db/db mice (n=8) were fed with food ad
libitum and treated with control IgG or IL-22–Fc twice per week. One group of db/db mice
(n=8) was fed with restricted food that matched the food intake of IL-22–Fc treated group, and
treated with control IgG. Accumulative food intake of first eight days of ad lib fed mice is
shown in (A), blood glucose in (B), and glucose tolerance test on day 25 in (C). (D-E) show
PYY levels in db/db (D) and HFD (E) mice treated with IL-22–Fc or control IgG on day 0 and
day 2. Serum was collected on day 2 before the 2 treatment and on day 5, and analyzed for
PYY. (F) shows serum LPS of db/db mice treated with IL-22–Fc or control IgG for 3 weeks.
(G-I) IL-22R KO (n=9) and WT mice (n=6) were fed with HFD starting at 6 weeks of age. The
results of body weight are shown in (G), results of glucose tolerance test at 3 months with HFD
are shown in (H) and results of Insulin tolerance test at 4 months with HFD are shown in (I).
Data shown are representative of two (A-C) or three (D-I) independent experiments. * P<0.05,
**P<0.01, ***P<0.001, Error bars, s.e.m.
Figure 50 shows results of pair-feeding restricted food intake. Three groups of db/db
mice were fed and treated as in Figure 49A. Accumulative food intake was measured.
Figure 51 shows results demonstrating IL-22 improved liver function and reduced fat
pad. (A) db/db mice treated with IL-22 Fc or control IgG as in Figure 20A. Liver enzymes were
measured at one month. (B-C) HFD-fed mice were treated with IL-22 Fc or control IgG as in
Figure 47A. Liver enzymes (B) and abdominal fat pad (C) were measured at one month.
**P<0.01, ***P<0.001, Error bars, s.e.m. (D-H) mice were fed with HFD for 10 weeks, and
then treated with IL-22 Fc or control twice per week for 6 weeks. (D) Lipid metabolic gene
expression from white adipose tissue. (E) Serum triglyceride, glycerol and free fatty acid. (F)
Hepatic triglyceride. (G) Hepatic cholesterol. (H) White adipose tissue triglyceride. (I-J) db/db
mice treated with IL-22 Fc or control IgG for 4 weeks. (I) Hepatic triglyceride. (J) White
adipose tissue triglyceride. *P<0.05. Error bars, s.e.m.
Figure 52 shows results demonstrating that IL-22 increased insulin secretion of β cells.
db/db mice were treated with IL-22 Fc as in Figure 20A, Pancreases were harvested on day 30
and stained for insulin and glucagon. (A) Percentage of islet area within total pancreas area. (B)
Percentage of β cell area within total islet area. (C) Percentage of α cell area within total islet
area.
Figure 53 IL-22 KO mice did not develop glucose intolerance with HFD. IL-22 KO
mice were fed with HFD starting at 6 weeks of age. Glucose tolerance test was done 3 months
after HFD. Error bars, s.e.m.
Figure 54 shows results demonstrating susceptibility of ob/ob mice to C. rodentium
infection: (A) body weight and (B) survival of ob/ob and lean mice (n = 10) infected with C.
rodentium; (C-D) colon histology of lean control (C) and ob/ob mice (D) on day 8, showing
epithelial hyperplasia, enterocyte shedding into the gut lumen, bacterial colonies (arrows) and
submucosal edema (vertical bar) (horizontal bar, 200 μm); (E) clinical score determined by
colon histology (n = 5); and (F-G) bacterial burden of ob/ob and lean control mice (n = 5) in
liver (F) and spleen (G) on day 8. *P < 0.05, ** P<0.01, ***P<0.001. Error bards, s.e.m.
Figure 55 shows results of db/db mice treated with IL-22 Fc, IL-20 Fc or IL-24 Fc in
(A) body weight, (B) serum glucose and (C) glucose tolerance test on day 20 of treatment.
Figures 56A and B show results comparing wound healing efficacy in db/db mice
treated with VEGF or IL-22 Fc.
Figures 57A-E show cytokine or chemokine induction by IL-22 Fc in reconstituted
epidermis.
Figure 58 shows results comparing wound closure using a splinted wound model in
wild type mice and db/db mice with or without S. aureus infection.
Figure 59 A and B show results comparing wound healing efficacy between VEGF and
IL-22 Fc in a splinted infected wound model.
Figure 60 shows results comparing wound healing efficacy between VEGF and IL-22
Fc at different concentrations in a splinted infected wound model.
Figure 61 shows results comparing wound healing efficacy between VEGF, PDGF and
IL-22 Fc at different concentrations in a splinted infected wound model.
Figure 62 shows that IL-22 Fc accelerated wound healing in a solution as well as in a
gel formulation in a splinted wound model.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
All publications, patents and patent applications cited herein are hereby expressly
incorporated by reference for all purposes.
the invention broadly relates to IL-22 protein or IL-22 Fc fusion proteins, composition
comprising the same, and methods of using the same. In particular, the description concerns
using IL-22 Fc fusion proteins or IL-22 polypeptide in the prevention and treatment of IBD,
atherosclerosis, cardiovascular diseases and conditions characterized by atherosclerotic plaque
formation, metabolic syndrome , mild and acute endotoxemia and sepsis, acute kidney injury,
acute pancreatitis, moderate acute pancreatitis, and insulin-related disorders. Further, the
description concerns using IL-22 Fc fusion proteins or IL-22 polypeptides in the prevention and
treatment of diabetic foot ulcer, accelerating wound healing and in particular diabetic wound
healing.
In one embodiment, it is believed that this is the first disclosure showing IL-22
polypeptide treating cardiovascular disease per se. The data herein supports the notion that an
IL-22 polypeptide or IL-22 Fc fusion protein can reduce the growth of atherosclerotic plaques,
reduce the frequency of rupture of atherosclerotic plaques and reduce endotoxemia. The
described polypeptides are particularly useful in treating subjects suffering from metabolic
syndrome, mild or acute endotoxemia, sepsis and insulin-related disorders, such as insulin-
resistance (no responsive to insulin) who need a change to their HDL/LDL lipid profile, as can
be determined by a doctor or clinician. The application shows data that indicate that IL-22
polypeptide or IL-22 Fc fusion protein can increase high density lipoproteins (HDL) and
decrease low density lipoproteins (LDL) in those subjects suffering from metabolic syndrome.
The data, without being bound by theory, also indicate gut-derived LPS a driver behind
endotoxemia and atherosclerosis. Mice treated with mIL-22 Fc fusion protein had reduced
hyperlipidemia, improved glucose tolerance with restored vascular function and these changes
culminated in a reduction in atherosclerotic plaque. IL-22 polypeptide or IL-22 Fc fusion
protein can attenuate the progression of cardiovascular disease.
Further, diabetes is a chronic disorder affecting carbohydrate, fat and protein
metabolism in animals. Diabetes is the leading cause of blindness, renal failure, and lower limb
amputations in adults and is a major risk factor for cardiovascular disease and stroke. Type I
diabetes mellitus (or insulin-dependent diabetes mellitus ("IDDM") or juvenile-onset diabetes)
comprises approximately 10% of all diabetes cases. The disease is characterized by a
progressive loss of insulin secretory function by beta cells of the pancreas. This characteristic
is also shared by non-idiopathic, or "secondary", diabetes having its origins in pancreatic
disease. Type I diabetes mellitus is associated with the following clinical signs or symptoms,
e.g., persistently elevated plasma glucose concentration or hyperglycemia; polyuria; polydipsia
and/or hyperphagia; chronic microvascular complications such as retinopathy, nephropathy and
neuropathy; and macrovascular complications such as hyperlipidemia and hypertension which
can lead to blindness, end-stage renal disease, limb amputation and myocardial infarction.
Type II diabetes mellitus (non-insulin-dependent diabetes mellitus or NIDDM, also
referred to as type II diabetes) is a metabolic disorder (or metabolic syndrome) involving the
dysregulation of glucose metabolism and impaired insulin sensitivity. Type II diabetes mellitus
usually develops in adulthood and is associated with the body's inability to utilize or make
sufficient insulin. In addition to the insulin resistance observed in the target tissues, patients
suffering from type II diabetes mellitus have a relative insulin deficiency -- that is, patients have
lower than predicted insulin levels for a given plasma glucose concentration. Type II diabetes
mellitus is characterized by the following clinical signs or symptoms, e.g., persistently elevated
plasma glucose concentration or hyperglycemia; polyuria; polydipsia and/or hyperphagia;
chronic microvascular complications such as retinopathy, nephropathy and neuropathy; and
macrovascular complications such as hyperlipidemia and hypertension which can lead to
blindness, end-stage renal disease, limb amputation and myocardial infarction.
I. DEFINITIONS
Unless otherwise defined, all terms of art, notations and other scientific terminology
used herein are intended to have the meanings commonly understood by those of skill in the art
to which this invention pertains. In some cases, terms with commonly understood meanings are
defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein
should not necessarily be construed to represent a substantial difference over what is generally
understood in the art.
Within this application, unless otherwise stated, the techniques utilized may be found in
any of several well-known references such as: Molecular Cloning: A Laboratory Manual
(Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), PCR Protocols: A Guide to
Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), and Harlow and
Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold
Spring Harbor, NY).
As appropriate, procedures involving the use of commercially available kits and
reagents are generally carried out in accordance with manufacturer defined protocols and/or
parameters unless otherwise noted. Before the present methods and uses therefore are
described, it is to be understood that this invention is not limited to the particular methodology,
protocols, cell lines, animal species or genera, constructs, and reagents described as such can, of
course, vary. It is also to be understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to limit the scope of the present
invention which will be limited only by the appended claims.
As used herein, the singular forms “a”, “an” and “the” include plural referents unless the
context clearly dictates otherwise. For example, reference to “an isolated peptide” means one
or more isolated peptides.
Throughout this specification and claims, the word "comprise," or variations such as
"comprises" or "comprising," will be understood to imply the inclusion of a stated integer or
group of integers but not the exclusion of any other integer or group of integers.
The term “IL-22 Fc fusion protein” or “IL-22 fusion protein” or “IL-22 Ig fusion
protein” as used herein refers to a fusion protein in which IL-22 protein or polypeptide is
linked, directly or indirectly, to an IgG Fc region. In certain preferred embodiments, the IL-22
Fc fusion protein of the description comprises a human IL-22 protein or polypeptide linked to a
human IgG Fc region. In certain embodiments, the human IL-22 protein comprises the amino
acid sequence of SEQ ID NO:4. However, it is understood that minor sequence variations such
as insertions, deletions, substitutions, especially conservative amino acid substitutions of IL-22
or Fc that do not affect the function and/or activity of IL-22 or IL-22 Fc fusion protein are also
contemplated by the invention. The IL-22 Fc fusion protein of the invention can bind to IL-22
receptor, which can lead to IL-22 receptor downstream signaling. In certain embodiments, the
IL-22 Fc fusion protein is capable of binding to IL-22 receptor, and/or is capable of leading to
IL-22 receptor downstream signaling. The functions and/or activities of the IL-22 Fc fusion
protein can be assayed by methods known in the art, including without limitation, ELISA,
ligand-receptor binding assay and Stat3 luciferase assay. In certain embodiments, the
description includes an IL-22 Fc fusion protein that binds to IL-22 receptor, the binding can
lead to IL-22 receptor downstream signaling, said IL-22 Fc fusion protein comprising an amino
acid sequence having at least 95% sequence identity to the amino acid sequence selected from
the group consisting of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14, and
wherein the Fc region is not glycosylated. In certain particular embodiments, the Fc region of
the IL-22 fusion protein does not possess effector activities (e.g., does not bind to FcγIIIR) or
exhibits substantially lower effector activity than a whole (e.g., wild type) IgG antibody. In
certain other embodiments, the Fc region of the IL-22 Fc fusion protein does not trigger
cytotoxicity such as antibody-dependent cellular cytotoxicity (ADCC) or complement
dependent cytotoxicity (CDC). Unless otherwise specified, “IL-22 fusion protein,” “IL-22 Fc
fusion,” “IL-22 Ig fusion protein,” “IL-22 Fc fusion protein” or “IL-22 Fc” are used
interchangeably throughout this application.
The term “IL-22” or “IL-22 polypeptide” or “IL-22 protein” as used herein, broadly
refers to any native IL-22 from any mammalian source, including primates (e.g. humans) and
rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,”
unprocessed IL-22 as well as any forms of IL-22 that result from processing in the cell. For
example, both full-length IL-22 containing the N-terminal leader sequence and the mature form
IL-22 are encompassed by the current invention. The leader sequence (or signal peptide) can be
the endogenous IL-22 leader sequence or an exogenous leader sequence of another mammalian
secretary protein. In certain embodiments, the leader sequence can be from a eukaryotic or
prokaryotic secretary protein. The term also encompasses naturally occurring variants of IL-22,
e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human IL-22
is shown in SEQ ID NO:4 (mature form, without a signal peptide). In certain embodiments, the
amino acid sequence of full-length IL-22 protein with the endogenous leader sequence is
provided in SEQ ID NO:71; while in other embodiments, the amino acid sequence of mature
IL-22 protein with an exogenous leader sequence is provided in SEQ ID NO:2. Minor sequence
variations especially conservative amino acid substitutions of IL-22 that do not affect the IL-
22’s function and/or activity (e.g., binding to IL-22 receptor) are also contemplated by the
invention. Figure 1 shows an amino acid sequence alignment of mature IL-22 from several
exemplary mammalian species. The asterisks indicate highly conserved amino acid residues
across species that are likely important for the functions and/or activities of IL-22. Accordingly,
in certain embodiments, the IL-22 Fc fusion protein of the invention comprises an IL-22
polypeptide comprising an amino acid sequence having at least 95%, at least 96%, at least 97%,
at least 98% or at least 99% sequence identity to SEQ ID NO:4. In certain other embodiments,
the IL-22 protein has 95% or more sequence identity to SEQ ID NO:71, 96% or more sequence
identity to SEQ ID NO:71, 97% or more sequence identity to SEQ ID NO:71; 98% or more
sequence identity to SEQ ID NO:71; 99% or more sequence identity to SEQ ID NO:71. The
IL-22 polypeptides described herein can be isolated from a variety of sources, such as from
human tissue or from another source, or prepared by recombinant or synthetic methods.
The term “IL-22 receptor” or “IL-22R” refers to a heterodimer consisting of IL-22R1
and IL-10R2 or naturally occurring allelic variants thereof. See Ouyang et al., 2011, Annu.
Rev. Immunol. 29:159-63. IL-10R2 is ubiquitously expressed by many cell types, and IL-22R1
is expressed only in innate cells such as epithelial cells, hepatocytes and keratinocytes. IL-
22R1 is also known as IL-22Ra1 or IL-22Rα1. IL-22R1 may be paired with other polypeptides
to form heterodimeric receptors for other IL-10 family members, for example IL-20 or IL-24.
See e.g., Ouyang et al., 2011, supra.
A "native sequence IL-22 polypeptide" or a "native sequence IL-22R polypeptide"
refers to a polypeptide comprising the same amino acid sequence as a corresponding IL-22 or
IL-22R polypeptide derived from nature. Such native sequence IL-22 or IL-22R polypeptides
can be isolated from nature or can be produced by recombinant or synthetic means. The terms
specifically encompass naturally-occurring truncated or secreted forms of the specific IL-22 or
IL-22R polypeptide (e.g., an IL-22 lacking its associated signal peptide), naturally-occurring
variant forms (e.g., alternatively spliced forms), and naturally-occurring allelic variants of the
polypeptide. In various embodiments of the description, the native sequence IL-22 or IL-22R
polypeptides disclosed herein are mature or full-length native sequence polypeptides. An
exemplary full length native human IL-22 is shown in Figure 30 (DNA, SEQ ID NO:70) and
Figure 31 (protein, SEQ ID NO:71). The start and stop codons are shown in bold font and
underlined in Figure 30. While the IL-22 and IL-22R polypeptide sequences disclosed in the
accompanying figures are shown to begin with methionine residues designated herein as amino
acid position 1, it is conceivable and possible that other methionine residues located either
upstream or downstream from the amino acid position 1 in the figures can be employed as the
starting amino acid residue for the IL-22 or IL-22R polypeptides.
An "IL-22 variant," an "IL-22R variant," an "IL-22 variant polypeptide," or an "IL-22R
variant polypeptide" means an active IL-22 or IL-22R polypeptide as defined above having at
least about 80% amino acid sequence identity with a full-length native sequence IL-22 or IL-
22R polypeptide sequence. Ordinarily, an IL-22 or IL-22R polypeptide variant will have at least
about 80% amino acid sequence identity, alternatively at least about 81% amino acid sequence
identity, alternatively at least about 82% amino acid sequence identity, alternatively at least
about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence
identity, alternatively at least about 85% amino acid sequence identity, alternatively at least
about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence
identity, alternatively at least about 88% amino acid sequence identity, alternatively at least
about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence
identity, alternatively at least about 91% amino acid sequence identity, alternatively at least
about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence
identity, alternatively at least about 94% amino acid sequence identity, alternatively at least
about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence
identity, alternatively at least about 97% amino acid sequence identity, alternatively at least
about 98% amino acid sequence identity, and alternatively at least about 99% amino acid
sequence identity to a full-length or mature native sequence IL-22 or IL-22R polypeptide
sequence.
The term “Fc region,” “Fc domain” or “Fc” refers to a C-terminal non-antigen binding
region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
The term includes native Fc regions and variant Fc regions. In certain embodiments, a human
IgG heavy chain Fc region extends from Cys226 to the carboxyl-terminus of the heavy
chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present,
without affecting the structure or stability of the Fc region. Unless otherwise specified herein,
numbering of amino acid residues in the IgG or Fc region is according to the EU numbering
system for antibodies, also called the EU index, as described in Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health,
Bethesda, MD, 1991.
In certain embodiments, Fc region refers to an immunoglobulin IgG heavy chain
constant region comprising a hinge region (starting at Cys226), an IgG CH2 domain and CH3
domain. The term “hinge region” or “hinge sequence” as used herein refers to the amino acid
sequence located between the linker and the CH2 domain. In certain embodiments, the hinge
region comprises the amino acid sequence CPPCP (SEQ ID NO:31). In certain embodiments,
the hinge region for IL-22 IgG4 Fc fusion protein comprises the CPPCP sequence (SEQ ID
NO:31), a sequence found in the native IgG1 hinge region, to facilitate dimerization. In certain
other embodiments, the Fc region starts at the hinge region and extends to the C-terminus of the
IgG heavy chain. In certain particular embodiments, the Fc region comprises the Fc region of
human IgG1, IgG2, IgG3 or IgG4. In certain particular embodiments, the Fc region comprises
the CH2 and CH3 domain of IgG4. In certain other particular embodiments, the Fc region
comprises the CH2 and CH3 domain of IgG1. As described in the Example section, it was
unexpectedly discovered by the applicants that IL-22 IgG4 Fc fusion protein exhibited even
superior pharmacokinetic properties than IL-22 IgG1 Fc fusion protein.
In certain embodiments, the IgG CH2 domain starts at Ala 231. In certain other
embodiments, the CH3 domain starts at Gly 341. It is understood that the C-terminus Lys
residue of human IgG can be optionally absent. It is also understood that conservative amino
acid substitutions of the Fc region without affecting the desired structure and/or stability of Fc
is contemplated within the scope of the invention.
In certain embodiments, the IL-22 is linked to the Fc region via a linker. In certain
particular embodiments, the linker is a peptide that connects the C-terminus of IL-22 to the Fc
region as described herein. In certain embodiments, native IgG sequences are present in the
linker and/or hinge region to minimize and/or avoid the risk of immunogenicity. In other
embodiments, minor sequence variations can be introduced to the native sequences to facilitate
manufacturing. IL-22 Fc fusion constructs comprising exogenous linker or hinge sequences
that exhibit high activity (as measured, e.g., by a luciferase assay) are also within the scope of
the invention. In certain embodiments, the linker comprises an amino acid sequence that is 8-
amino acids, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 10-11, 10-12, 10-13, 10-14, 10-
, 10-16, 11-16, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids long. In certain other
embodiments, the linker comprises the amino acid sequence DKTHT (SEQ ID NO:32).
In certain particular embodiments, the linker does not comprise the sequence Gly-Gly-
Ser (SEQ ID NO:45), Gly-Gly-Gly-Ser (SEQ ID NO:46) or Gly-Gly-Gly-Gly-Ser (SEQ ID
NO:47).
In certain embodiments, the IL-22 Fc fusion protein comprises an IL-22 polypeptide
linked to an Fc region by a linker. The term “linked to” or “fused to” refers to a covalent bond,
e.g., a peptide bond, formed between two moieties.
The term “afucosylation,” “afucosylated,” “defucosylation,” or “defucosylated” refers to
the absence or removal of core-fucose from the N-glycan attached to the CH2 domain of Fc.
It was unexpectedly discovered by the applicants that IL-22 IgG1 Fc fusion proteins,
unlike other Fc fusion proteins or antibodies comprising Fc, exhibited high levels (e.g., 30%) of
afucosylation in the N-glycans attached to the Fc region. The N-glycans attached to the CH2
domain of Fc is heterogeneous. Antibodies or Fc fusion proteins generated in CHO cells are
fucosylated by fucosyltransferase activity. See Shoji-Hosaka et al., J. Biochem. 2006, 140:777-
83. Normally, a small percentage of naturally occurring afucosylated IgGs may be detected in
human serum. N-glycosylation of the Fc is important for binding to FcγR; and afucosylation of
the N-glycan increases Fc’s binding capacity to FcγRIIIa. Increased FcγRIIIa binding can
enhance antibody-dependent cellular cytotoxicity (ADCC), which can be advantageous in
certain antibody therapeutic applications in which cytotoxicity is desirable. See Shoji-Hosaka et
al., supra. Such an enhanced effector function, however, can be detrimental when Fc-mediated
cytotoxicity is undesirable such as in the case of IL-22 Fc fusion.
IgG4 Fc is known to exhibit less effector activity than IgG1 Fc. Applicants
unexpectedly discovered that IL-22 IgG4 Fc fusion protein also showed high levels of
afucosylation in the Fc region. The high-level of afucosylated N-glycan attached to the Fc of
IgG4 can increase the undesirable effector activity.
Thus, in one embodiment, the description includes an IL-22 Fc fusion protein in which
the Fc region or CH2 domain is not glycosylated. In certain embodiments, the N-glycosylation
site in the CH2 domain is mutated to prevent from glycosylation.
In certain other embodiments, the glycosylation in the CH2 domain of the Fc region can
be eliminated by altering the glycosylation consensus site, i.e., Asn at position 297 followed by
any amino acid residue (in the case of human IgG, Ser) and Thr (see Figure 3). The
glycosylation site can be altered by amino acid insertions, deletions and/or substitutions. For
example, one or more amino acid residues can be inserted between Asn and Ser or between Ser
and Thr to alter the original glycosylation site, wherein the insertions do not regenerate an N-
glycosylation site. In certain particular embodiments, the N297 residue (e.g., the N-
glycosylated site in Fc, see Figure 3) within the CH2 domain of human IgG Fc is mutated to
abolish the glycosylation site. In certain particular embodiments, the N297 residue is changed
to Gly, Ala, Gln, Asp or Glu. In some particular embodiments, the N297 residue is changed to
Gly or Ala. In other particular embodiments, the N297 residue is changed to Gly. In certain
other embodiments, the T299 residue can be substituted with another amino acid, for example
Ala, Val or Gly. In certain particular embodiments, the mutations that result in an
aglycosylated Fc do not affect the structure and/or stability of the IL-22 Fc fusion protein.
In a related embodiment, the description includes a method of treating IBD, including
UC and CD, methods of inhibiting bacterial infection in the intestine, and methods of
improving epithelial integrity, epithelial proliferation, differentiation and/or migration in the
intestine, and methods of treating metabolic disorders or metabolic syndrome, type II diabetes,
atherosclerosis and diabetic wound healing in a patient in need thereof comprising
administering to the patient a pharmaceutical composition comprising an IL-22 Fc fusion
protein wherein the Fc region is not glycosylated.
In a further embodiment, the description includes a composition comprising IL-22 Fc
fusion proteins having low level of or no afucosylation in the Fc region. Specifically, the
description includes a composition comprising IL-22 Fc fusion proteins having an overall
afucosylation level in the Fc region of no more than 10%, preferably no more than 5%, more
preferably no more than 2%, and most preferably less than 1%. In another embodiment, the
description includes methods of treating IBD, including UC and CD, methods of inhibiting
bacterial infection in the intestine, and methods of improving epithelial integrity, epithelial
proliferation, differentiation and/or migration in the intestine, and methods of treating metabolic
disorders, type II diabetes, type II diabetes with morbid obesity, graft versus host disease
(GVHD), atherosclerosis, cardiovascular disease, metabolic syndrome, endotoxemia (acute and
mild), sepsis, acute coronary heart disease, hypertension, dyslipemia, obesity, hyperglycemia,
lipid metabolism disorders, hepatitis, acute hepatitis, renal failure, acute renal failure, acute
kidney injury, rental draft failure, pancreatitis, acute pancreatitis, liver fibrosis and lung
fibrosis, wound, infected wound, accelerating wound healing, including diabetic wound
healing, in a patient in need thereof comprising administering to the patient a pharmaceutical
composition comprising IL-22 Fc fusion proteins having an afucosylation level in the Fc region
of no more than 10 %, preferably no more than 5%, more preferably no more than 2%, and
most preferably less than 1%.
The term “% afucosylation” refers to the level of afucosylation in the Fc region in a
composition of IL-22 Fc fusion proteins. The % afucosylation can be measured by mass
spectrometry (MS) and presented as the percentage of afucosylated glycan species (species
without the fucose on one Fc domain (minus 1) and on both Fc domains (minus 2) combined)
over the entire population of IL-22 Fc fusion proteins. For example, % afucosylation can be
calculated as the percentage of the combined area under the minus 1 fucose peak and minus 2
fucose peak over the total area of all glycan species analyzed by MS, such as determined by an
Agilent 6520B TOF Mass Spectrometer as described in Figure 2 and in the examples shown
below. The level of afucosylation can be measured by any other suitable methods known in the
art, including without limitation HPLC-Chip Cube MS (Agilent) and reverse phase-HPLC. The
% afucosylation of IL-22 Fc composition can be used as an indication for determining whether
the composition will likely trigger unacceptable level of ADCC, unsuitable for the intended
purposes. Accordingly, in certain particular embodiments, the composition comprises IL-22 Fc
fusion proteins having an afucosylation level of no more than 10%, preferably no more than
%, more preferably no more than 3%, and most preferably no more than 1%. In certain
embodiments, the composition comprises IL-22 Fc fusion proteins having an afucosylation
level of no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than
6%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, or no more than
In certain embodiments, the desired level of afucosylation of an IL-22 Fc composition
can be achieved by methods known in the art, including without limitation, by purification. For
example, the fucosylated species in a composition can be enriched by affinity chromatography
having resins conjugated with a fucose binding moiety, such as an antibody or lectin specific
for fucose, especially fucose present in the 1-6 linkage. See e.g., Kobayashi et al, 2012, J. Biol.
Chem. 287:33973-82. In certain other embodiments, the fucosylated species can be enriched
and separated from afucosylated species using an anti-fucose specific antibody in an affinity
column. Alternatively or additionally, afucosylated species can be separated from fucosylated
species based on the differential binding affinity to FcγRIIIa using affinity chromatography.
In certain other embodiments, the IL-22 Fc fusion protein comprises an Fc region in
which the N297 residue in the CH2 domain is mutated. In certain embodiments, the N297
residue is changed to Gly or Ala, preferably to Gly. In certain other embodiments, the N297
residue is deleted. In certain embodiments, the IL-22 Fc fusion protein comprising an Fc
having an amino acid substitution at N297 is aglycosylated or not glycosylated. The term
“aglycosylated” as used herein refers to a protein or a portion of a protein of interest that is not
glycosylated. For example, an IL-22 Fc fusion protein with an aglycosylated Fc region can be
made by mutagenizing the N297 residue in the CH2 domain of the Fc region.
In other embodiments, the N-glycan attached to the wild type N297 residue can be
removed enzymatically, e.g., by deglycosylation. Suitable glycolytic enzymes include without
limitation, peptide-N-glycosidase (PNGase).
The term “dimeric IL-22 Fc fusion protein” refers to a dimer in which each monomer
comprises an IL-22 Fc fusion protein. The term “monomeric IL-22 Fc fusion protein” refers to
a dimer in which one monomer comprises an IL-22 Fc fusion protein (the IL-22 Fc arm), while
the other monomer comprises an Fc region without the IL-22 polypeptide (the Fc arm).
Accordingly, the dimeric IL-22 Fc fusion protein is bivalent with respect to IL-22R binding,
whereas the monomeric IL-22 Fc fusion protein is monovalent with respect to IL-22R binding.
The heterodimerization of the monomeric IL-22 Fc fusion protein can be facilitated by methods
known in the art, including without limitation, heterodimerization by the knob-into-hole
technology. The structure and assembly method of the knob-into-hole technology can be found
in, e.g., US5,821,333, US7,642,228, US 2011/0287009 and , hereby
incorporated by reference in their entireties. This technology was developed by introducing a
“knob” (or a protuberance) by replacing a small amino acid residue with a large one in the CH3
domain of one Fc, and introducing a “hole” (or a cavity) in the CH3 domain of the other Fc by
replacing one or more large amino acid residues with smaller ones. In certain embodiments, the
IL-22 Fc fusion arm comprises a knob, and the Fc only arm comprises a hole.
The preferred residues for the formation of a knob are generally naturally occurring
amino acid residues and are preferably selected from arginine (R), phenylalanine (F), tyrosine
(Y) and tryptophan (W). Most preferred are tryptophan and tyrosine. In one embodiment, the
original residue for the formation of the knob has a small side chain volume, such as alanine,
asparagine, aspartic acid, glycine, serine, threonine or valine. Exemplary amino acid
substitutions in the CH3 domain for forming the knob include without limitation the T366W,
T366Y or F405W substitution.
The preferred residues for the formation of a hole are usually naturally occurring amino
acid residues and are preferably selected from alanine (A), serine (S), threonine (T) and valine
(V). In one embodiment, the original residue for the formation of the hole has a large side
chain volume, such as tyrosine, arginine, phenylalanine or tryptophan. Exemplary amino acid
substitutions in the CH3 domain for generating the hole include without limitation the T366S,
L368A, F405A, Y407A, Y407T and Y407V substitutions. In certain embodiments, the knob
comprises T366W substitution, and the hole comprises the T366S/L368A/Y407V substitutions.
In certain particular embodiments, the Fc region of the monomeric IL-22 Fc fusion protein
comprises an IgG1 Fc region. In certain particular embodiments, the monomeric IL-22 IgG1 Fc
fusion comprises an IL-22 Fc knob arm and an Fc hole arm. In certain embodiments, the IL-22
Fc knob arm comprises a T366W substitution (SEQ ID NO:61), and the Fc hole arm comprises
T366S, L368A and Y407V (SEQ ID NO:62). In certain other embodiments, the Fc region of
both arms further comprises an N297G or N297A mutation. In certain embodiments, the
monomeric IL-22 Fc fusion protein is expressed in E. coli cells. It is understood that other
modifications to the Fc region known in the art that facilitate heterodimerization are also
contemplated and encompassed by the instant application.
The term “wound” refers to an injury, especially one in which the skin or another
external surface is torn, pierced, cut, or otherwise broken.
The term “ulcer” is a site of damage to the skin or mucous membrane that is often
characterized by the formation of pus, death of tissue, and is frequently accompanied by an
inflammatory reaction.
The term “intestine” or “gut” as used herein broadly encompasses the small intestine
and large intestine.
The term “accelerating wound healing” or “acceleration of wound healing” refers to the
increase in the rate of healing, e.g., a reduction in time until complete wound closure occurs or
a reduction in time until a % reduction in wound area occurs.
A “diabetic wound” is a wound that associated with diabetes.
A “diabetic ulcer” is an ulcer that is associated with diabetes.
A “chronic wound” refers to a wound that does not heal. See, e.g., Lazarus et al.,
Definitions and guidelines for assessment of wounds and evaluation of healing, Arch.
Dermatol. 130:489-93 (1994). Chronic wounds include, but are not limited to, e.g., arterial
ulcers, diabetic ulcers, pressure ulcers or bed sores, venous ulcers, etc. An acute wound can
develop into a chronic wound. Acute wounds include, but are not limited to, wounds caused
by, e.g., thermal injury (e.g., burn), trauma, surgery, excision of extensive skin cancer, deep
fungal and bacterial infections, vasculitis, scleroderma, pemphigus, toxic epidermal necrolysis,
etc. See, e.g., Buford, Wound Healing and Pressure Sores, HealingWell.com, published on:
October 24, 2001. Thus, in certain embodiments, a chronic wound is an infected wound. A
“normal wound” refers to a wound that undergoes normal wound healing repair.
An “acceptor human framework” for the purposes herein is a framework comprising the
amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable
domain (VH) framework derived from a human immunoglobulin framework or a human
consensus framework, as defined below. An acceptor human framework “derived from” a
human immunoglobulin framework or a human consensus framework may comprise the same
amino acid sequence thereof, or it may contain amino acid sequence changes. In some
embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6
or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human
framework is identical in sequence to the VL human immunoglobulin framework sequence or
human consensus framework sequence.
“Affinity” refers to the strength of the sum total of non-covalent interactions between a
single binding site of a molecule (e.g., a ligand or an antibody) and its binding partner (e.g., a
receptor or an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to
intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair
(e.g., IL-22 Fc fusion protein and IL-22 receptor). The affinity of a molecule X for its partner
Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by
common methods known in the art, including those described herein. Specific illustrative and
exemplary embodiments for measuring binding affinity are described in the following.
The term "antibody" herein is used in the broadest sense and encompasses various
antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies,
multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they
exhibit the desired antigen-binding activity.
An "antibody fragment" refers to a molecule other than an intact antibody that
comprises a portion of an intact antibody that binds the antigen to which the intact antibody
binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab’-SH,
F(ab') ; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and
multispecific antibodies formed from antibody fragments.
An “antibody that binds to the same epitope” as a reference antibody refers to an
antibody that blocks binding of the reference antibody to its antigen in a competition assay by
50% or more, and conversely, the reference antibody blocks binding of the antibody to its
antigen in a competition assay by 50% or more. An exemplary competition assay is provided
herein.
The term "chimeric" antibody refers to an antibody in which a portion of the heavy
and/or light chain is derived from a particular source or species, while the remainder of the
heavy and/or light chain is derived from a different source or species.
The “class” of an antibody refers to the type of constant domain or constant region
possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG,
and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG ,
IgG , IgG , IgG , IgA , and IgA . The heavy chain constant domains that correspond to the
2 3 4 1 2
different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.
The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents
a cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not
211 131 125 90 186 188 153 212 32 212
limited to, radioactive isotopes (e.g., At , I , I , Y , Re , Re , Sm , Bi , P , Pb
and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate,
adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan,
mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory
agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as
small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin,
including fragments and/or variants thereof; and the various antitumor or anticancer agents
disclosed below.
“Effector functions” or “effector activities” refer to those biological activities
attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of
antibody effector functions include: C1q binding and complement dependent cytotoxicity
(CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC);
phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell
activation. In certain embodiments, the IL-22 Fc fusion protein does not exhibit any effector
function or any detectable effector function. In certain other embodiments, the IL-22 Fc fusion
protein exhibits substantially reduced effector function, e.g., about 50%, 60%, 70% 80%, or
90% reduced effector function.
An “effective amount” or “therapeutically effective amount” of an agent, e.g., a
pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time
necessary, to achieve the desired therapeutic or prophylactic result.
For example, in the case of a cardiovascular disease or condition, the therapeutically
effective amount of the IL-22 polypeptide, fusion protein or agonist can reduce the degree of
atherosclerotic plaque formation; reduce the size of the atherosclerotic plaque(s); inhibit (i.e.,
slow to some extent and preferably stop) atherosclerotic plaque; inhibit (i.e., slow to some
extent and preferably stop) thrombosis or rupture of an atherosclerotic plaque; and/or relieve to
some extent one or more of the symptoms associated with the disease or condition.
By "reduce or inhibit" is meant the ability to cause an overall decrease preferably of
% or greater, more preferably of 50% or greater, and most preferably of 75%, 85%, 90%,
95%, or greater. Reduce or inhibit can refer to the symptoms of the disorder being treated, the
presence or size of atherosclerotic plaques, or the number of atherosclerotic plaque(s).
A “suboptimal amount” refers to the amount less than the optimal amount of a
therapeutic agent typically used for a certain treatment. When two therapeutic agents are given
to a subject, either concurrently or sequentially, each therapeutic agent can be given at a
suboptimal amount as compared to the treatment when each therapeutic agent is given alone.
For example, in certain embodiments, the subject in need of IBD treatment is administered with
the pharmaceutical composition comprising the IL-22 Fc fusion protein of the invention and a
dexamethasone at a suboptimal amount.
"Framework" or "FR" refers to variable domain residues other than hypervariable region
(HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1,
FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the
following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
The terms “full length antibody,” “intact antibody,” and “whole antibody” are used
herein interchangeably to refer to an antibody having a structure substantially similar to a native
antibody structure or having heavy chains that contain an Fc region as defined herein.
The terms "host cell," "host cell line," and "host cell culture" are used interchangeably
and refer to cells into which exogenous nucleic acid has been introduced, including the progeny
of such cells. Host cells include "transformants" and "transformed cells," which include the
primary transformed cell and progeny derived therefrom without regard to the number of
passages. The transformed cell includes transiently or stably transformed cell. Progeny may not
be completely identical in nucleic acid content to a parent cell, but may contain mutations.
Mutant progeny that have the same function or biological activity as screened or selected for in
the originally transformed cell are included herein. In certain embodiments, the host cell is
transiently transfected with the exogenous nucleic acid. In certain other embodiments, the host
cell is stably transfected with the exogenous nucleic acid.
A “human antibody” is one which possesses an amino acid sequence which corresponds
to that of an antibody produced by a human or a human cell or derived from a non-human
source that utilizes human antibody repertoires or other human antibody-encoding sequences.
This definition of a human antibody specifically excludes a humanized antibody comprising
non-human antigen-binding residues.
A “human consensus framework” is a framework which represents the most commonly
occurring amino acid residues in a selection of human immunoglobulin VL or VH framework
sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a
subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as
in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication
91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for the VL, the subgroup is
subgroup kappa I as in Kabat et al., supra. In one embodiment, for the VH, the subgroup is
subgroup III as in Kabat et al., supra.
A “humanized” antibody refers to a chimeric antibody comprising amino acid residues
from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a
humanized antibody will comprise substantially all of at least one, and typically two, variable
domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a
non-human antibody, and all or substantially all of the FRs correspond to those of a human
antibody. A humanized antibody optionally may comprise at least a portion of an antibody
constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a
non-human antibody, refers to an antibody that has undergone humanization.
The term “hypervariable region” or “HVR” as used herein refers to each of the regions
of an antibody variable domain which are hypervariable in sequence (“complementarity
determining regions” or “CDRs”) and/or form structurally defined loops (“hypervariable
loops”) and/or contain the antigen-contacting residues (“antigen contacts”). Generally,
antibodies comprise six HVRs: three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3).
Exemplary HVRs herein include:
(a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96
(L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917
(1987));
(b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b
(H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));
(c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96
(L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745
(1996)); and
(d) combinations of (a), (b), and/or (c), including HVR amino acid residues 46-56 (L2),
47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-
102 (H3).
Unless otherwise indicated, HVR residues and other residues in the variable domain
(e.g., FR residues) are numbered herein according to Kabat et al., supra.
An “immunoconjugate” is an antibody or a fragment of an antibody conjugated to one
or more heterologous molecule(s), including but not limited to a cytotoxic agent.
An “individual,” “subject” or “patient” is a mammal. Mammals include, but are not
limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g.,
humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
In certain embodiments, the individual, subject or patient is a human.
An “isolated” IL-22 fusion protein is one which has been separated from the
environment of a host cell that recombinantly produces the fusion protein. In some
embodiments, an IL-22 fusion protein is purified to greater than 95% or 99% purity as
determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF),
capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC).
An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from
a component of its natural environment. An isolated nucleic acid includes a nucleic acid
molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic
acid molecule is present extrachromosomally or at a chromosomal location that is different
from its natural chromosomal location.
“Isolated nucleic acid encoding IL-22 Fc fusion protein” refers to one or more nucleic
acid molecules encoding the IL-22 Fc fusion protein, including such nucleic acid molecule(s) in
a single vector or separate vectors, such nucleic acid molecule(s) transiently or stably
transfected into a host cell and such nucleic acid molecule(s) present at one or more locations in
a host cell.
The term "control sequences" refers to DNA sequences necessary for the expression of
an operably linked coding sequence in a particular host organism. The control sequences that
are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence,
and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
Nucleic acid is "operably linked" when it is placed into a functional relationship with
another nucleic acid sequence. For example, DNA for a presequence or secretory leader is
operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in
the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence
if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a
coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked"
means that the DNA sequences being linked are contiguous, and, in the case of a secretory
leader, contiguous and in reading phase. However, enhancers do not have to be contiguous.
Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the
synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
The term “monoclonal antibody” as used herein refers to an antibody obtained from a
population of substantially homogeneous antibodies, i.e., the individual antibodies comprising
the population are identical and/or bind the same epitope, except for possible variant antibodies,
e.g., containing naturally occurring mutations or arising during production of a monoclonal
antibody preparation, such variants generally being present in minor amounts. In contrast to
polyclonal antibody preparations, which typically include different antibodies directed against
different determinants (epitopes), each monoclonal antibody of a monoclonal antibody
preparation is directed against a single determinant on an antigen. Thus, the modifier
“monoclonal” indicates the character of the antibody as being obtained from a substantially
homogeneous population of antibodies, and is not to be construed as requiring production of the
antibody by any particular method. For example, the monoclonal antibodies to be used in
accordance with the present invention may be made by a variety of techniques, including but
not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and
methods utilizing transgenic animals containing all or part of the human immunoglobulin loci,
such methods and other exemplary methods for making monoclonal antibodies being described
herein.
A “naked antibody” refers to an antibody that is not conjugated to a heterologous moiety
(e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical
formulation.
“Native antibodies” refer to naturally occurring immunoglobulin molecules with
varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of
about 150,000 daltons, composed of two identical light chains and two identical heavy chains
that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region
(VH), also called a variable heavy domain or a heavy chain variable domain, followed by three
constant domains (CH1, CH2, and CH3). Similarly, from N- to C-terminus, each light chain
has a variable region (VL), also called a variable light domain or a light chain variable domain,
followed by a constant light (CL) domain. The light chain of an antibody may be assigned to
one of two types, called kappa ( κ) and lambda ( λ), based on the amino acid sequence of its
constant domain.
A “native sequence Fc region” comprises an amino acid sequence identical to the amino
acid sequence of an Fc region found in nature. Native sequence human Fc regions include,
without limitation, a native sequence human IgG1 Fc region (non-A and A allotypes); native
sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence
human IgG4 Fc region, as well as naturally occurring variants thereof.
A “variant Fc region” comprises an amino acid sequence which differs from that of a
native sequence Fc region by virtue of at least one amino acid modification, preferably one or
more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid
substitution compared to a native sequence Fc region or to the Fc region of a parent
polypeptide, e.g. from about one to about ten amino acid substitutions, and preferably from
about one to about five amino acid substitutions in a native sequence Fc region or in the Fc
region of the parent polypeptide. The variant Fc region herein will preferably possess at least
about 80% homology with a native sequence Fc region and/or with an Fc region of a parent
polypeptide, and most preferably at least about 90% homology therewith, more preferably at
least about 95% homology therewith. In certain embodiments, the variant Fc region is not
glycosylated.
The term “inflammatory bowel disorder,” “inflammatory bowel disease” or IBD is used
herein in the broadest sense and includes all diseases and pathological conditions the
pathogenesis of which involves recurrent inflammation in the intestine, including small
intestine and colon. Commonly seen IBD includes ulcerative colitis and Crohn’s disease. IBD is
not limited to UC and CD. The manifestations of the disease include but not limited to
inflammation and a decrease in epithelial integrity in the intestine.
The term "cardiovascular disease" or “cardiovascular disorder” is used herein in the
broadest sense and includes all diseases and pathological conditions the pathogenesis of which
involves abnormalities of the blood vessels, such as, for example, atherosclerotic plaque
formation (including stable or unstable/vulnerable plaques), atherosclerosis, arteriosclerosis,
arteriolosclerosis, and elevated systemic lipopolysaccharide (LPS) exposure. The term
additionally includes diseases and pathological conditions that benefit from the inhibition of the
formation of atherosclerotic plaques. Cardiovascular diseases include, without limitation,
coronary artery atherosclerosis, coronary microvascular disease, stroke, carotid artery disease,
peripheral arterial disease, ischemia, coronary artery disease (CAD), acute coronary syndrome
(ACS), coronary heart disease (CHD), conditions associated with CAD and CHD,
cerebrovascular disease, peripheral vascular disease, aneurysm, vasculitis, venous thrombosis,
diabetes mellitus, and metabolic syndromechronic kidney disease, remote tissue injury after
ischemia and reperfusion, cardiopulmonary bypass. Specifically included within this group are
all cardiovascular diseases associated with the occurrence, development, or progression of
which can be controlled by the inhibition of the atherosclerotic plaque formation.
The term "cardiovascular condition" is used herein in the broadest sense and includes all
cardiovascular conditions and diseases the pathology of which involves atherosclerotic plaque
formation (including stable or unstable/vulnerable plaques), atherosclerosis, arteriosclerosis,
arteriolosclerosis, and elevated systemic lipopolysaccharide (LPS) exposure. Specifically
included within this group are all cardiovascular conditions and diseases associated with the
atherosclerotic plaque formation, the occurrence, development, or progression of which can be
controlled by the inhibition of the atherosclerotic plaque formation. The term specifically
includes diseases and pathological conditions that benefit from the inhibition of the formation
of atherosclerotic plaques. Cardiovascular conditions include, without limitation, coronary
artery atherosclerosis, coronary microvascular disease, stroke, carotid artery disease, peripheral
arterial disease, ischemia, coronary artery disease (CAD), coronary heart disease (CHD),
conditions associated with CAD and CHD, cerebrovascular disease and conditions associated
with cerebrovascular disease, peripheral vascular disease and conditions associated with
peripheral vascular disease, aneurysm, vasculitis, venous thrombosis, diabetes mellitus, and
metabolic syndromechronic kidney disease, remote tissue injury after ischemia and reperfusion,
and cardiopulmonary bypass. "Conditions associated with cerebrovascular disease" as used
herein include, for example, transient ischemic attack (TIA) and stroke. "Conditions associated
with peripheral vascular disease" as used herein include, for example, claudication. Specifically
included within this group are all cardiovascular diseases and conditions associated with the
occurrence, development, or progression of which can be controlled by the inhibition of the
atherosclerostic plaque formation.
Atherosclerotic plaque formation can occur as a result of an innate immune response to
metabolic endotoxemia, which is characterized by elevated levels of systemic
lipopolysaccharides (LPS) that originate from gut microbiota and a loss of functional integrity
in the gut mucosal barrier. The innate immune response to endotoxemia results in the low-
grade chronic inflammation that is responsible for plaque formation.
The term “metabolic syndrome” is used herein in the broadest sense. Metabolic
syndrome includes the co-occurrence in an adult subject of several metabolic risk factors,
including at least three of the following five traits: abdominal obesity, which can be, for
example, a waist circumference in men of greater than or equal to 90 cm and in women greater
than or equal to 80 cm; elevated serum triglycerides, which can be, for example, greater than or
equal to 150 mg/dL, or drug treatment for elevated triglycerides; reduced serum HDL
cholesterol level, which can be, for example, below 40 mg/dL in men and below 50 mg/dL in
women, or drug treatment for low HDL cholesterol; hypertension, which can be, for example,
systolic blood pressure greater than 130 mmHg and diastolic blood pressure greater than 85
mmHg, or drug treatment for hypertension; and elevated fasting plasma glucose, which can be,
for example, greater than or equal to 100 mg/dL, drug treatment for elevated glucose, or
previously diagnosed type 2 diabetes. See also Meigs, the Metabolic Syndrome (Insulin
Resistance Syndrome or Syndrome X), http://www.uptodate.com/contents/the-metabolic-
syndrome-insulin-resistance-syndrome-or-syndrome-x, the disclosure of which is hereby
incorporated by reference herein.
For children over 16 years old, the above criteria for adults can be used. For children
between 10-16 year old, metabolic syndrome includes the co-occurrence in a subject of several
metabolic risk factors, including at least three of the following five traits: abdominal obesity,
which can be, for example, a waist circumference greater than 90 percentile; elevated serum
triglycerides, which can be, for example, greater than or equal to 110 mg/dL, greater than 95
percentile, or drug treatment for elevated triglycerides; reduced serum HDL cholesterol level,
which can be, for example, below 40 mg/dL, less than 5 percentile, or drug treatment for low
HDL cholesterol; hypertension, which can be, for example, systolic blood pressure greater than
130 mmHg and diastolic blood pressure greater than 85 mmHg, greater than 90 percentile, or
drug treatment for hypertension; and elevated fasting plasma glucose, which can be, for
example, greater than or equal to 100 mg/dL, impaired glucose tolerance, drug treatment for
elevated glucose, or previously diagnosed type 2 diabetes.
Generally speaking, the risk factors that co-occur in metabolic syndrome include obesity
(such as abdominal obesity), hyperglycemia, dyslipidemia, insulin resistance, and/or
hypertension. All these risk factors promote the development of atherosclerotic cardiovascular
disease, diabetes, or both. Metabolic syndrome can also feature chronic adipose tissue
inflammation.
Metabolic syndrome can be recognized as a proinflammatory, prothrombic state, and
can be associated with elevated levels of one or more of C-reactive protein, IL-6, LPS, and
plasminogen activator inhibitor 1; such markers can be associated with an increased risk for
subsequent development of atherosclerotic cardiovascular disease, diabetes, or both.
Metabolic syndrome can be associated with several obesity-related disorders, including
one or more of fatty liver disease with steatosis, fibrosis, and cirrhosis, hepatocellular and
intrahepatic cholangiocarcinoma, chronic kidney disease, polycystic ovary syndrome, sleep
disordered breathing, including obstructive sleep apnea, and hyperuricemia and gout.
The term “insulin-related disorder” encompasses diseases or conditions characterized by
impaired glucose tolerance. In one embodiment, the insulin-related disorder is diabetes mellitus
including, without limitation, Type I (insulin-dependent diabetes mellitus or IDDM), Type II
(non-insulin dependent diabetes mellitus or NIDDM) diabetes, gestational diabetes, and any
other disorder that would be benefited by agents that stimulate insulin secretion. In another
embodiment, the insulin-related disorder is characterized by insulin resistance.
The term “sepsis” is used in its broadest sense and can encompass a systemic
inflammatory state caused by severe infection. Sepsis can caused by the immune system's
response to a serious infection, most commonly bacteria, but also fungi, viruses, and parasites
in the blood, urinary tract, lungs, skin, or other tissues.
The term “acute endotoxemia” is used in its broadest sense and can encompass the
condition of increased plasma bacterial lipopolysaccharide (LPS). Acute endotoxemia in turn
could result in sepsis. Increased LPS in systemic circulation will induce low grade chronic
inflammation, activating the endogenous protective host response to elevate plasma lipids that,
in the chronic condition contributes to diet induced obesity, insulin resistance and
atherosclerosis, and eventual CVD events.
As used herein, “treatment” (and grammatical variations thereof such as “treat” or
“treating”) refers to clinical intervention in an attempt to alter the natural course of the
individual being treated, and can be performed either for prophylaxis or during the course of
clinical pathology. Desirable effects of treatment include, but are not limited to, preventing
occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or
indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of
disease progression, amelioration or palliation of the disease state, and remission or improved
prognosis.
For example, with regard to IBD, “treatment” can refer to a decrease in the likelihood of
developing IBD, a decrease in the rate of developing IBD and a decrease in the severity of the
disease. As another example, with regard to atherosclerotic plaque formation, "treatment" can
refer to a decrease in the likelihood of developing atherosclerotic plaque deposits, a decrease in
the rate of development of deposits, a decrease in the number or size of existing deposits, or
improved plaque stability. Those in need of treatment include those already with the disorder
as well as those in which the disorder is to be prevented. Desirable effects of treatment include,
but are not limited to, preventing occurrence or recurrence of disease, alleviating symptoms,
diminishing any direct or indirect pathological consequences of the disease, preventing the
disease, decreasing the rate of disease progression, ameliorating or palliating the disease state,
and causing remission or improved prognosis. In some embodiments, an IL-22 polypeptide or
IL-22 Fc fusion protein of the description are used to delay development of a disease or to slow
the progression of a disease.
In certain embodiments, a "subject in need thereof" in the context of preventing or
treating a cardiovascular condition refers to a subject diagnosed with a cardiovascular disease
or cardiovascular condition (CVD) or metabolic syndrome or exhibiting one or more
conditions associated with CVD or metabolic syndrome , a subject who has been diagnosed
with or exhibited one or more conditions associated with CVD or metabolic syndrome in the
past, or a subject who has been deemed at risk of developing CVD or metabolic syndrome or
one or more conditions associated with CVD or metabolic syndrome in the future due to
hereditary or environmental factors. Therefore, in certain embodiments, a subject in need
thereof can be a subject exhibiting a CVD or metabolic syndrome or a condition associated with
a CVD or metabolic syndrome or a subject that has exhibited a CVD or metabolic syndrome or
a condition associated with a CVD or metabolic syndrome in the past or has been deemed at
risk for developing a CVD or metabolic syndrome or a condition associated with a CVD or
metabolic syndrome in the future.
In treatment of a cardiovascular disease or condition, a therapeutic agent can directly
alter the magnitude of response of a component of the immune response, or render the disease
more susceptible to treatment by other therapeutic agents, e.g., antibiotics, antifungals, anti-
inflammatory agents, chemotherapeutics, etc. In treatment of an arterial disease, treatment
might, for example, prevent or slow down the progression of a disease. Thus, treatment of an
arterial disease specifically includes the prevention, inhibition, or slowing down of the
development of the condition, or of the progression from one stage of the condition to another,
more advanced stage, or into a more severe, related condition.
The "pathology" of a disease or condition includes all phenomena that compromise the
well-being of the subject. In the case of a cardiovascular disease or condition, this includes,
without limitation, atherosclerotic plaque formation (including stable or unstable/vulnerable
plaques), atherosclerosis, arteriosclerosis, arteriolosclerosis, and elevated systemic
lipopolysaccharide (LPS) exposure.
"Alleviation", “alleviating” or equivalents thereof, refers to both therapeutic treatment
and prophylactic or preventative measures, wherein the object is to ameliorate, prevent, slow
down (lessen), decrease or inhibit a disease or condition, e.g., the formation of atherosclerotic
plaques. Those in need of treatment include those already with the disease or condition as well
as those prone to having the disease or condition or those in whom the disease or condition is to
be prevented.
"Chronic" administration refers to administration of an agent(s) in a continuous mode as
opposed to an acute mode, so as to maintain the initial therapeutic effect for an extended period
of time.
“Intermittent” administration is treatment that is not consecutively done without
interruption, but rather is cyclic in nature.
The term “package insert” is used to refer to instructions customarily included in
commercial packages of therapeutic products, that contain information about the indications,
usage, dosage, administration, combination therapy, contraindications and/or warnings
concerning the use of such therapeutic products.
“Percent (%) amino acid sequence identity" with respect to a reference polypeptide
sequence is defined as the percentage of amino acid residues in a candidate sequence that are
identical with the amino acid residues in the reference polypeptide sequence, after aligning the
sequences and introducing gaps, if necessary, to achieve the maximum percent sequence
identity, and not considering any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence identity can be achieved in
various ways that are within the skill in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those
skilled in the art can determine appropriate parameters for aligning sequences, including any
algorithms needed to achieve maximal alignment over the full length of the sequences being
compared. For purposes herein, however, % amino acid sequence identity values are generated
using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence
comparison computer program was authored by Genentech, Inc., and the source code has been
filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it
is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is
publicly available from Genentech, Inc., South San Francisco, California, or may be compiled
from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating
system, including digital UNIX V4.0D. All sequence comparison parameters are set by the
ALIGN-2 program and do not vary.
In situations where ALIGN-2 is employed for amino acid sequence comparisons, the %
amino acid sequence identity of a given amino acid sequence A to, with, or against a given
amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A
that has or comprises a certain % amino acid sequence identity to, with, or against a given
amino acid sequence B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by the sequence
alignment program ALIGN-2 in that program’s alignment of A and B, and where Y is the total
number of amino acid residues in B. It will be appreciated that where the length of amino acid
sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence
identity of A to B will not equal the % amino acid sequence identity of B to A. Unless
specifically stated otherwise, all % amino acid sequence identity values used herein are
obtained as described in the immediately preceding paragraph using the ALIGN-2 computer
program.
As further examples of % amino acid sequence identity calculations using this method, below
demonstrate how to calculate the % amino acid sequence identity of the amino acid sequence
designated "Comparison Protein" or “Reference Protein” to the amino acid sequence designated
"IL-22", wherein "IL-22" represents the amino acid sequence of an IL-22 polypeptide of
interest, "Comparison Protein" represents the amino acid sequence of a polypeptide against
which the "IL-22 " polypeptide of interest is being compared, and "X, "Y" and "Z" each
represent different amino acid residues.
As examples of % amino acid sequence identity calculations using this method, Tables 1
and 2 demonstrate how to calculate the % amino acid sequence identity of the amino acid
sequence designated "Comparison Protein" to the amino acid sequence designated "IL-22 ",
wherein "IL-22 " represents the amino acid sequence of an IL-22 polypeptide of interest,
"Comparison Protein" represents the amino acid sequence of a polypeptide against which the
"IL-22 " polypeptide of interest is being compared, and "X, "Y" and "Z" each represent
different amino acid residues.
"Stringency" of hybridization reactions is readily determinable by one of ordinary skill
in the art, and generally is an empirical calculation dependent upon probe length, washing
temperature, and salt concentration. In general, longer probes require higher temperatures for
proper annealing, while shorter probes need lower temperatures. Hybridization generally
depends on the ability of denatured DNA to re-anneal when complementary strands are present
in an environment below their melting temperature. The higher the degree of desired homology
between the probe and hybridizable sequence, the higher the relative temperature which can be
used. As a result, it follows that higher relative temperatures would tend to make the reaction
conditions more stringent, while lower temperatures less so. For additional details and
explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in
Molecular Biology, Wiley Interscience Publishers, (1995).
"Stringent conditions" or "high stringency conditions", as defined herein, can be
identified by those that: (1) employ low ionic strength and high temperature for washing, for
example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at
5OC; (2) employ during hybridization a denaturing agent, such as formamide, for example,
50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%
polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride,
75 mM sodium citrate at 42°C; or (3) overnight hybridization in a solution that employs 50%
formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH
6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50
μg/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with a 10 minute wash at 42°C in 0.2 x
SSC (sodium chloride/sodium citrate) followed by a 10 minute high-stringency wash consisting
of 0.1 x SSC containing EDTA at 55°C.
"Moderately stringent conditions" can be identified as described by Sambrook et al.,
Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Press, 1989, and
include the use of washing solution and hybridization conditions (e.g., temperature, ionic
strength, and %SDS) less stringent that those described above. An example of moderately
stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide,
x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x
Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA,
followed by washing the filters in 1 x SSC at about 37-500C. The skilled artisan will recognize
how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as
probe length and the like.
The term "agonist" is used in the broadest sense and includes any molecule that partially
or fully mimics a biological activity of an IL-22 polypeptide. Also encompassed by “agonist”
are molecules that stimulate the transcription or translation of mRNA encoding the polypeptide.
Suitable agonist molecules include, e.g., agonist antibodies or antibody fragments; a
native polypeptide; fragments or amino acid sequence variants of a native polypeptide;
peptides; antisense oligonucleotides; small organic molecules; and nucleic acids that encode
polypeptides agonists or antibodies. Reference to “an” agonist encompasses a single agonist or
a combination of two or more different agonists.
The term “IL-22 agonist" is used in the broadest sense, and includes any molecule that
mimics a qualitative biological activity (as hereinabove defined) of a native sequence IL-22
polypeptide. IL-22 agonists specifically include ILFc or IL-22 Ig polypeptides
(immunoadhesins), but also small molecules mimicking at least one IL-22 biological activity.
Preferably, the biological activity is binding of the IL-22 receptor, interacting with IL-22BP,
facilitating an innate immune response pathway, or in the case of a cardiovascular disease or
condition, to affect the formation of atherosclerotic plaques, in particular to inhibit formation of
atherosclerotic plaque formation. Inhibition of plaque formation can be assessed by any suitable
imaging method known to those of ordinary skill in the art.
IL-22R1 pairs with other proteins to form heterodimers as the receptors for certain IL-
family members. See Quyang et al., 2011, supra. Thus, in certain embodiments, IL-22
agonists may include an IL-22 receptor agonist, including a cytokine (or a fusion protein or
agonist thereof) that binds to and triggers downstream signaling of the IL-22 R1. In certain
embodiments, the IL-22 agonists include an IL-22R1 agonist, including without limitation an
anti-IL-22R1 agonist antibody; an IL-20 agonist, including without limitation IL-20
polypeptide or IL-20 Fc fusion protein; and an IL-24 agonist, including without limitation IL-24
polypeptide or IL-24 fusion protein. In certain other embodiments, the IL-22R1 agonists
include an IL-19 agonist, including without limitation IL-19 polypeptide or IL-19 Fc fusion
protein; and an IL-26 agonist, including without limitation IL-26 polypeptide or IL-26 Fc fusion
protein. Exemplary sequences for IL-19 (GenBank Accession No. AAG16755.1, SEQ ID
NO:77), IL-20 (GenBank Accession No. AAH69311.1, SEQ ID NO:78), IL-24 (GenBank
Accession No. AAH09681.1, SEQ ID NO:79) and IL-26 (GenBank Accession No.
NP_060872.1, SEQ ID NO:80) are provided herein. In certain embodiments, an IL-19
polypeptide comprises the amino acid sequence of SEQ ID NO:77 or the mature protein
without the signal peptide. In certain other embodiments, an IL-20 polypeptide comprises the
amino acid sequence of SEQ ID NO:78 or the mature protein without the signal peptide. In yet
other embodiments, an IL-24 polypeptide comprises the amino acid sequence of SEQ ID NO:79
or the mature protein without the signal peptide. In certain other embodiments, an IL-26
polypeptide comprises the amino acid sequence of SEQ ID NO:80 or the mature protein
without the signal peptide.
A "small molecule" is defined herein to have a molecular weight below about 600,
preferably below about 1000 daltons.
An “agonist antibody,” as used herein, is an antibody which partially or fully mimics a
biological activity of an IL-22 polypeptide.
The term "pharmaceutical formulation" or “pharmaceutical composition” refers to a
preparation which is in such form as to permit the biological activity of an active ingredient
contained therein to be effective, and which contains no additional components which are
unacceptably toxic to a subject to which the formulation would be administered.
A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical
formulation, other than an active ingredient, which is nontoxic to a subject., A
pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, diluent,
stabilizer, or preservative.
The term “variable region” or “variable domain” refers to the domain of an antibody
heavy or light chain that is involved in binding the antibody to antigen. The variable domains
of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally
have similar structures, with each domain comprising four conserved framework regions (FRs)
and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6 ed.,
W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to
confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may
be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library
of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol.
150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
The term "vector," as used herein, refers to a nucleic acid molecule capable of
propagating another nucleic acid to which it is linked. The term includes the vector as a self-
replicating nucleic acid structure as well as the vector incorporated into the genome of a host
cell into which it has been introduced. Certain vectors are capable of directing the expression
of nucleic acids to which they are operatively linked. Such vectors are referred to herein as
"expression vectors."
II. COMPOSITIONS AND METHODS
In one embodiment, the description is based, in part, on compositions comprising
therapeutics that ameliorate IL-22 associated diseases or disorders by increasing IL-22 activities
or signaling. In certain embodiments, IL-22 polypeptide and IL-22 Fc fusion proteins that bind
to and activate IL-22 receptor are described. IL-22 Fc fusion proteins of the description are
useful, e.g., for the diagnosis or treatment of IL-22 associated diseases such as inflammatory
bowel disease and accelerating wound healing. In addition, IL-22 polypeptide and IL-22 Fc
fusion proteins for the treatment of other IL-22 associated diseases for example cardiovascular
conditions, metabolic syndrome and accelerating diabetic wound healing are also described.
A. Exemplary IL-22 Polypeptide
IL-22 polypeptide as used herein includes a polypeptide comprising an amino acid
sequence comprising SEQ ID NO:71 (human IL-22 with the endogenous IL-22 leader
sequence) (see Figure 31), or a polypeptide comprising an amino acid sequence that has at least
95% sequence identity with SEQ ID NO:71. In certain embodiments, the IL-22 polypeptide
comprises an amino acid sequence comprising SEQ ID NO:4 (human IL-22 without a leader
sequence) or a polypeptide comprising an amino acid sequence that has at least 95% sequence
identity. In certain embodiments, the IL-22 polypeptide comprises an amino acid sequence
comprising SEQ ID NO:4. In certain embodiments, the IL-22 polypeptide does not comprise an
Fc fusion.
The preparation of native IL-22 molecules, along with their nucleic acid and
polypeptide sequences, can be achieved through methods known to those of ordinary skill in the
art. For example, IL-22 polypeptides can be produced by culturing cells transformed or
transfected with a vector containing IL-22 nucleic acid. It is, of course, contemplated that
alternative methods, which are well known in the art, can be employed to prepare IL-22. For
instance, the IL-22 sequence, or portions thereof, can be produced by direct peptide synthesis
using solid-phase techniques (see, e.g., Stewart et al., 1969, Solid-Phase Peptide Synthesis,
W.H. Freeman Co., San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 1963,
85:2149-2154). In vitro protein synthesis can be performed using manual techniques or by
automation. Automated synthesis can be accomplished, for instance, using an Applied
Biosystems Peptide Synthesizer (Foster City, Calif.) using manufacturer's instructions. Various
portions of IL-22 can be chemically synthesized separately and combined using chemical or
enzymatic methods to produce the full-length IL-22.
IL-22 variants can be prepared by introducing appropriate nucleotide changes into the
DNA encoding a native sequence IL-22 polypeptide, or by synthesis of the desired IL-22
polypeptide. Those skilled in the art will appreciate that amino acid changes can alter post-
translational processes of IL-22, such as changing the number or position of glycosylation sites
or altering the membrane anchoring characteristics.
Variations in the native sequence IL-22 polypeptides described herein can be made, for
example, using any of the techniques and guidelines for conservative and non-conservative
mutations set forth, for instance, in U.S. Pat. No. 5,364,934. Variations can be a substitution,
deletion or insertion of one or more codons encoding a native sequence or variant IL-22 that
results in a change in its amino acid sequence as compared with a corresponding native
sequence or variant IL-22. Optionally the variation is by substitution of at least one amino acid
with any other amino acid in one or more of the domains of a native sequence IL-22
polypeptide. Guidance in determining which amino acid residue can be inserted, substituted or
deleted without adversely affecting the desired activity can be found by comparing the
sequence of the IL-22 with that of homologous known protein molecules and minimizing the
number of amino acid sequence changes made in regions of high homology. Amino acid
substitutions can be the result of replacing one amino acid with another amino acid having
similar structural and/or chemical properties, such as the replacement of a leucine with a serine,
i.e., conservative amino acid replacements. Insertions or deletions can optionally be in the range
of 1 to 5 amino acids. The variation allowed can be determined by systematically making
insertions, deletions or substitutions of amino acids in the sequence and testing the resulting
variants for activity in the in vitro assay described in the Examples below.
In particular embodiments, conservative substitutions of interest are shown in Table 1
under the heading of preferred substitutions. If such substitutions result in a change in
biological activity, then more substantial changes, denominated exemplary substitutions in
Table 1, or as further described below in reference to amino acid classes, are introduced and the
products screened.
The variations can be made using methods known in the art such as oligonucleotide-
mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed
mutagenesis (Carter et al., 1986, Nucl. Acids Res, 13:4331; Zoller et al., 1987, Nucl. Acids
Res., 10:6487), cassette mutagenesis (Wells et al., 1985, Gene, 34:315), restriction selection
mutagenesis (Wells et al., 1986, Philos. Trans. R. Soc. London SerA, 317:415) or other known
techniques can be performed on the cloned DNA to produce the IL-22 variant DNA.
Fragments of an IL-22 polypeptide of the present description are also included herein.
Such fragments can be truncated at the N-terminus or C-terminus, or can lack internal residues,
for example, when compared with a full length native protein. Certain fragments lack amino
acid residues that are not essential for a desired biological activity of an IL-22 polypeptide of
the present description. Accordingly, in certain embodiments, a fragment of an IL-22
polypeptide is biologically active. In certain embodiments, a fragment of full length IL-22 lacks
the N-terminal signal peptide sequence.
Covalent modifications of native sequence and variant IL-22 polypeptides are included
within the scope of this description. One type of covalent modification includes reacting
targeted amino acid residues of IL-22 with an organic derivatizing agent that is capable of
reacting with selected side chains or the N- or C-terminal residues of the IL-22 polypeptide.
Derivatization with bifunctional agents is useful, for instance, for crosslinking IL-22 to a water-
insoluble support matrix or surface, for example, for use in the method for purifying anti-IL-22
antibodies. Commonly used crosslinking agents include, e.g., 1,1-bis(diazo-acetyl)
phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-
azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-
dithiobis(succinimidyl-propionate), bifunctional maleimides such as bis-N-maleimido-1,8-
octane and agents such as methyl[(p-azidophenyl)dithio]propioimidate.
Other modifications include deamidation of glutaminyl and asparaginyl residues to the
corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the .alpha.-
amino groups of lysine, arginine, and histidine side chains (T. E. Creighton, 1983, Proteins:
Structure and Molecular Properties, W. H. Freeman & Co., San Francisco, pp. 79-86i),
acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.
Another type of covalent modification of the IL-22 polypeptides included within the
scope of this description comprises altering the native glycosylation pattern of the polypeptides.
"Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one
or more carbohydrate moieties found in native sequence IL-22, and/or adding one or more
glycosylation sites that are not present in the native sequence IL-22, and/or alteration of the
ratio and/or composition of the sugar residues attached to the glycosylation site(s).
Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked
glycosylation refers to the attachment of the carbohydrate moiety to the side-chain of an
asparagine residue. The tripeptide sequences, asparagine-X-serine and asparagine-X-threonine,
wherein X is any amino acid except proline, are recognition sequences for enzymatic
attachment of the carbohydrate moiety to the asparagine side chain. O-linked glycosylation
refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a
hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-
hydroxylysine can also be involved in O-linked glycosylation. Addition of glycosylation sites
to the IL-22 polypeptide can be accomplished by altering the amino acid sequence. The
alteration can be made, for example, by the addition of, or substitution by, one or more serine or
threonine residues to the native sequence IL-22 (for N-linked glycosylation sites), or the
addition of a recognition sequence for O-linked glycosylation. The IL-22 amino acid sequence
can optionally be altered through changes at the DNA level, particularly by mutating the DNA
encoding the IL-22 polypeptide at preselected bases such that codons are generated that will
translate into the desired amino acids.
Another means of increasing the number of carbohydrate moieties on the IL-22
polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such
methods are described in the art, e.g., in WO 87/05330 published 11 Sep. 1987, and in Aplin
and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).
Removal of carbohydrate moieties present on an IL-22 polypeptide can be accomplished
chemically or enzymatically or by mutational substitution of codons encoding for amino acid
residues that serve as targets for glycosylation. Chemical deglycosylation techniques are
known in the art and described, for instance, by Hakimuddin, et al., Arch. Biochem. Biophys.,
259:52 (1987) and by Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of
carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and
exo-glycosidases as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).
Another type of covalent modification of IL-22 comprises linking the IL-22 polypeptide
to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene
glycol, or polyoxyalkylenes, for example in the manner set forth in U.S. Pat. Nos. 4,640,835;
4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. The native sequence and variant IL-
22 can also be modified in a way to form a chimeric molecule comprising IL-22, including
fragments of IL-22, fused to another, heterologous polypeptide or amino acid sequence.
In one embodiment, such a chimeric molecule comprises a fusion of IL-22 with a tag
polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The
epitope tag is generally placed at the amino- or carboxyl-terminus of the IL-22 polypeptide.
The presence of such epitope-tagged forms of the IL-22 polypeptide can be detected using an
antibody against the tag polypeptide. Also, provision of the epitope tag enables the IL-22
polypeptide to be readily purified by affinity purification using an anti-tag antibody or another
type of affinity matrix that binds to the epitope tag. Various tag polypeptides and their
respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or
poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5
(Field et al., 1988, Mol. Cell. Biol., 8:2159-2165); the c-myc tag and the 8F9, 3C7, 6E10, G4,
and 9E10 antibodies thereto (Evan et al., 1985, Molecular and Cellular Biology, 5:3610-3616);
and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al., 1990,
Protein Engineering, 3(6):547-553). Other tag polypeptides include the Flag-peptide (Hopp et
al., 1988, BioTechnology, 6:1204-1210); the KT3 epitope peptide (Martin et al., 1992, Science,
255:192-194); an .quadrature.-tubulin epitope peptide (Skinner et al., 1991, J. Biol. Chem.,
266:15163-15166); and the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al., 1990, Proc.
Natl. Acad. Sci. USA, 87:6393-6397).
In another embodiment, the chimeric molecule can comprise a fusion of the IL-22
polypeptide or a fragment thereof with an immunoglobulin or a particular region of an
immunoglobulin. For a bivalent form of the chimeric molecule, such a fusion can be to the Fc
region of an IgG molecule. These fusion polypeptides are antibody-like molecules which
combine the binding specificity of a heterologous protein (an "adhesin") with the effector
functions of immunoglobulin constant domains, and are often referred to as immunoadhesins.
Structurally, the immunoadhesins comprise a fusion of an amino acid sequence of IL-22, or a
variant thereof, and an immunoglobulin constant domain sequence. The adhesin part of an
immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the
binding site of a receptor or a ligand. The immunoglobulin constant domain sequence in the
immunoadhesin can be obtained from any immunoglobulin, such as IgG1, IgG2, IgG3, or IgG4
subtypes, IgA (including IgA1 and IgA2), IgE, IgD or IgM. In certain embodiments, the IL-22
Fc fusion protein exhibits modified effector activities.
The IL-22 polypeptide, or a fragment thereof, can be fused, for example, to an
immunoglobulin heavy chain constant region sequence to produce an ILIg fusion protein
(e.g., IL-22 Fc fusion protein). The IL-22 polypeptide can be human or murine IL-22. The
immunoglobulin heavy chain constant region sequence can be human or murine
immunoglobulin heavy chain constant region sequence.
B. Exemplary IL-22 Fc Fusion Protein
In one embodiment, the description includes isolated IL-22 fusion protein. In certain
embodiments, the IL-22 fusion protein binds to and induces IL-22 receptor activity or signaling
and/or is an agonist of IL-22 receptor activity.
In another embodiment, an IL-22 Fc fusion protein comprises a polypeptide having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to
the amino acid sequence of SEQ ID NO:4. In other embodiments, the IL-22 Fc fusion protein
comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% sequence identity contains substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but an IL-22 Fc fusion protein comprising that
sequence retains the ability to bind to IL-22 receptor. In certain embodiments, a total of 1 to 10
amino acids have been substituted, inserted and/or deleted in SEQ ID NOs:8, 10, 12, 14, 24 or
26. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the
IL22 (i.e., in the Fc). In certain particular embodiments, the C-terminus Lys residue of Fc is
deleted. In certain other embodiments, the C-terminus Gly and Lys residues of Fc are both
deleted.
In certain embodiments, IL-22 Fc fusion proteins variants having one or more amino
acid substitutions are described. Conservative substitutions are shown in Table 1 under the
heading of "preferred substitutions." More substantial changes are provided in Table 1 under
the heading of "exemplary substitutions," and as further described below in reference to amino
acid side chain classes. Amino acid substitutions may be introduced into the IL-22 Fc fusion
protein and the products screened for a desired activity, e.g., retained/improved IL-22 receptor
binding, decreased immunogenicity, or improved IL-22 receptor signaling.
TABLE 1
Original Exemplary Preferred
Residue Substitutions Substitutions
Ala (A) Val; Leu; Ile Val
Arg (R) Lys; Gln; Asn Lys
Asn (N) Gln; His; Asp, Lys; Arg Gln
Asp (D) Glu; Asn Glu
Cys (C) Ser; Ala Ser
Gln (Q) Asn; Glu Asn
Glu (E) Asp; Gln Asp
Gly (G) Ala Ala
His (H) Asn; Gln; Lys; Arg Arg
Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu
Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile
Lys (K) Arg; Gln; Asn Arg
Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr
Pro (P) Ala Ala
Ser (S) Thr Thr
Thr (T) Val; Ser Ser
Trp (W) Tyr; Phe Tyr
Tyr (Y) Trp; Phe; Thr; Ser Phe
Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu
Amino acids may be grouped according to common side-chain properties:
(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
(3) acidic: Asp, Glu;
(4) basic: His, Lys, Arg;
(5) residues that influence chain orientation: Gly, Pro;
(6) aromatic: Trp, Tyr, Phe.
Non-conservative substitutions will entail exchanging a member of one of these classes
for another class.
A useful method for identification of residues or regions of a protein that may be
targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham
and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues
(e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced by a neutral
or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the
interaction of the protein with its binding partner is affected. Further substitutions may be
introduced at the amino acid locations demonstrating functional sensitivity to the initial
substitutions. Alternatively, or additionally, a crystal structure of a protein complex (e.g., a
cytokine-receptor complex) can be used to identify contact points between a protein and its
binding partner. Such contact residues and neighboring residues may be targeted or eliminated
as candidates for substitution. Variants may be screened to determine whether they contain the
desired properties.
Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions
ranging in length from one residue to polypeptides containing a hundred or more residues, as
well as intrasequence insertions of single or multiple amino acid residues.
a) Glycosylation variants
In certain embodiments, an Fc fusion protein described herein is altered to increase or
decrease the extent to which the fusion protein, especially the Fc portion of the fusion protein,
is glycosylated. Addition or deletion of glycosylation sites to a protein may be conveniently
accomplished by altering the amino acid sequence such that one or more glycosylation sites is
created or removed.
Where the fusion protein comprises an Fc region, the carbohydrate attached thereto may
be altered. Native antibodies produced by mammalian cells typically comprise a branched,
biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2
domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The
oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine
(GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of
the biantennary oligosaccharide structure. In some embodiments, modifications of the
oligosaccharide in an antibody or the Fc region of an antibody may be made in order to create
Fc variants with certain improved properties.
The amount of fucose attached to the CH2 domain of the Fc region can be determined
by calculating the average amount of fucose within the sugar chain at Asn297, relative to the
sum of all glycostructures attached to Asn 297 or N297 (e. g. complex, hybrid and high
mannose structures) as measured by MALDI-TOF mass spectrometry, as described in
, for example. Asn297 refers to the asparagine residue located at about
position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also
be located about ± 3 amino acids upstream or downstream of position 297, i.e., between
positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation
variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US
2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of
publications related to “defucosylated” or “fucose-deficient” antibody variants include: US
2003/0157108; ; ; US 2003/0115614; US 2002/0164328; US
2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865;
; ; ; ; WO2005/053742;
WO2002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al.
Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated
antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch.
Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 A1, Presta, L; and
A1, Adams et al., especially at Example 11), and knockout cell lines, such as
alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al.
Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006);
and WO2003/085107).
Antibodies variants are further described with bisected oligosaccharides, e.g., in which a
biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc.
Such antibody variants may have reduced fucosylation and/or improved ADCC function.
Examples of such antibody variants are described, e.g., in (Jean-Mairet et
al.); US Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Antibody
variants with at least one galactose residue in the oligosaccharide attached to the Fc region are
also described. Such antibody variants may have improved CDC function. Such antibody
variants are described, e.g., in (Patel et al.); (Raju, S.); and
(Raju, S.).
b) Fc region variants
In certain embodiments, one or more amino acid modifications may be introduced into
the Fc region of an Fc fusion protein described herein, thereby generating an Fc region variant.
The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2,
IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or
more amino acid positions.
In certain embodiments, the description contemplates an Fc variant that possesses some
but not all effector functions, which make it a desirable candidate for applications in which the
half life of the antibody or a fusion protein comprising an Fc region in vivo is important yet
certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In
vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of
CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted
to ensure that the antibody or Fc lacks FcγR binding (hence likely lacking ADCC activity), but
retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express
FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on
hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.
Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity
of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g. Hellstrom, I. et al.
Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat’l Acad. Sci.
USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361
(1987)). Alternatively, non-radioactive assays methods may be employed (see, for example,
ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain
View, CA; and CytoTox 96 non-radioactive cytotoxicity assay (Promega, Madison, WI).
Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and
Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al.
Proc. Nat’l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be carried out to
confirm that the antibody or Fc is unable to bind C1q and hence lacks CDC activity. See, e.g.,
C1q and C3c binding ELISA in and . To assess
complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et
al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and
Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo
clearance/half-life determinations can also be performed using methods known in the art (see,
e.g., Petkova, S.B. et al., Int’l. Immunol. 18(12):1759-1769 (2006)).
Antibodies with reduced effector function include those with substitution of one or more
of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Such
Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265,
269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues
265 and 297 to alanine (US Patent No. 7,332,581).
Certain antibody or Fc variants with improved or diminished binding to FcRs are
described. (See, e.g., U.S. Patent No. 6,737,056; , and Shields et al., J. Biol.
Chem. 9(2): 6591-6604 (2001).)
In certain embodiments, an IL-22 Fc fusion protein comprises an Fc variant with one or
more amino acid substitutions which reduce ADCC, e.g., substitution at position 297 of the Fc
region to remove the N-glycosylation site and yet retain FcRn binding activity (EU numbering
of residues).
In some embodiments, alterations are made in the Fc region that result in diminished
C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US
Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
Antibodies with increased half lives and improved binding to the neonatal Fc receptor
(FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J.
Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in
US2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more
substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include
those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305,
307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution
of Fc region residue 434 (US Patent No. 7,371,826).
See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260;
U.S. Patent No. 5,624,821; and WO 94/29351 concerning other examples of Fc region variants.
c) Cysteine engineered variants
In certain embodiments, it may be desirable to create cysteine engineered Fc fusion
protein, in which one or more residues of the Fc region of an antibody are substituted with
cysteine residues. In particular embodiments, the substituted residues occur at accessible sites
of the Fc. By substituting those residues with cysteine, reactive thiol groups are thereby
positioned at accessible sites of the Fc and may be used to conjugate the Fc to other moieties,
such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described
further herein. For example, S400 (EU numbering) of the heavy chain Fc region can be
substituted with Cysteine. See e.g., U.S. Patent No. 7,521,541.
C. Recombinant Methods and Compositions
The IL-22 polypeptides can be prepared by routine recombinant methods, e.g., culturing
cells transformed or transfected with a vector containing a nucleic acid encoding an IL-22
polypeptide, a fragment or variant thereof, or fusion protein comprising the same. Host cells
comprising any such vector are also described. By way of example, host cells can be CHO
cells, E. coli, or yeast. A process for producing any of the herein described polypeptides is
further described and comprises culturing host cells under conditions suitable for expression of
the desired polypeptide and recovering the desired polypeptide from the cell culture.
Host cells are transfected or transformed with expression or cloning vectors described
herein for IL-22 polypeptide production and cultured in conventional nutrient media modified
as appropriate for inducing promoters, selecting transformants, or amplifying the genes
encoding the desired sequences. The culture conditions, such as media, temperature, pH and
the like, can be selected by the skilled artisan without undue experimentation. In general,
principles, protocols, and practical techniques for maximizing the productivity of cell cultures
can be found in Mammalian Cell Biotechnology: A Practical Approach, M. Butler, ed. (IRL
Press, 1991) and Sambrook et al., supra.
Methods of transfection are known to the ordinarily skilled artisan, for example, by
CaPO and electroporation. Depending on the host cell used, transformation is performed using
standard techniques appropriate to such cells. The calcium treatment employing calcium
chloride, as described in Sambrook et al., supra, or electroporation is generally used for
prokaryotes or other cells that contain substantial cell-wall barriers. Infection with
Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by
Shaw et al., Gene, 23:315 (1983) and WO 89/05859 published 29 June 1989. For mammalian
cells without such cell walls, the calcium phosphate precipitation method of Graham and van
der Eb, Virology, 52:456-457 (1978) can be employed. General aspects of mammalian cell
host system transformations have been described in U.S. Pat. No. 4,399,216. Transformations
into yeast are typically carried out according to the method of Van Solingen et al., J. Bact,
130:946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, other
methods for introducing DNA into cells, such as by nuclear microinjection, electroporation,
bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, can
also be used. For various techniques for transforming mammalian cells, see Keown et al.,
Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature, 336:348-352 (1988).
Recombinantly expressed polypeptides of the present invention can be recovered from
culture medium or from host cell lysates. The following procedures are exemplary of suitable
purification procedures: by fractionation on an ion-exchange column; ethanol precipitation;
reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE;
chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for
example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG;
and metal chelating columns to bind epitope-tagged forms of a polypeptide of the present
invention. Various methods of protein purification can be employed and such methods are
known in the art and described for example in Deutscher, Methods in Enzymology, 182 (1990);
Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The
purification step(s) selected will depend, for example, on the nature of the production process
used and the particular polypeptide produced.
Alternative methods, which are well known in the art, can be employed to prepare a
polypeptide of the present invention. For example, a sequence encoding a polypeptide or
portion thereof, can be produced by direct peptide synthesis using solid-phase techniques (see,
e.g., Stewart et al., 1969, Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco,
CA; Merrifield, J. 1963,Am. Chem. Soc., 85:2149-2154. In vitro protein synthesis can be
performed using manual techniques or by automation. Automated synthesis can be
accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, CA)
using manufacturer's instructions. Various portions of a polypeptide of the present invention or
portion thereof can be chemically synthesized separately and combined using chemical or
enzymatic methods to produce the full-length polypeptide or portion thereof.
In other embodiments, the description includes chimeric molecules comprising any of
the herein described polypeptides fused to a heterologous polypeptide or amino acid sequence.
Examples of such chimeric molecules include, but are not limited to, any of the herein
described polypeptides fused to an epitope tag sequence or an Fc region of an immunoglobulin.
Suitable host cells for cloning or expressing the DNA in the vectors herein include
prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes include but are not limited to
eubacteria, such as Gram-negative or Gram-positive organisms, for example,
Enterobacteriaceae such as E. coli. Various E. coli strains are publicly available, such as E. coli
K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110
(ATCC 27,325) and K5 772 (ATCC 53,635).
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are
suitable cloning or expression hosts for ILencoding vectors. Saccharomyces cerevisiae is a
commonly used lower eukaryotic host microorganism.
Suitable host cells for the expression of glycosylated -IL-22 are derived from
multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila
S2 and Spodoptera Sf9, as well as plant cells. Examples of useful mammalian host cell lines
include Chinese hamster ovary (CHO) and COS cells. More specific examples include monkey
kidney CV1 cells transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney
cells (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol.,
36:59 (1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl.
Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251
(1980)); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and
mouse mammary tumor cells (MMT 060562, ATCC CCL51). The selection of the appropriate
host cell is deemed to be within the skill in the art.
The nucleic acid (e.g., cDNA or genomic DNA) encoding IL-22 can be inserted into a
replicable vector for cloning (amplification of the DNA) or for expression. Various vectors are
publicly available. The vector can, for example, be in the form of a plasmid, cosmid, viral
particle, or phage. The appropriate nucleic acid sequence can be inserted into the vector by a
variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease
site(s) using techniques known in the art. Vector components generally include, but are not
limited to, one or more of a signal sequence, an origin of replication, one or more marker genes,
an enhancer element, a promoter, and a transcription termination sequence. Construction of
suitable vectors containing one or more of these components employs standard ligation
techniques which are known to the skilled artisan.
The IL-22 polypeptides can be produced recombinantly not only directly, but also as a
fusion polypeptide with a heterologous polypeptide, which can be a signal sequence or other
polypeptide having a specific cleavage site at the N-terminus of the mature protein or
polypeptide, as well as an IL-22 Fc fusion protein. In general, the signal sequence can be a
component of the vector, or it can be a part of the IL-22 DNA that is inserted into the vector.
The signal sequence can be a prokaryotic signal sequence selected, for example, from the group
of the alkaline phosphatase, penicillinase, 1 pp, or heat-stable enterotoxin II leaders. For yeast
secretion the signal sequence can be, e.g., the yeast invertase leader, alpha factor leader
(including Saccharomyces and Kluyveromyces "--factor leaders, the latter described in U.S.
Pat. No. 5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP
362,179 published 4 Apr. 1990), or the signal described in WO 90/13646 published 15 Nov.
1990. In mammalian cell expression, mammalian signal sequences can be used to direct
secretion of the protein, such as signal sequences from secreted polypeptides of the same or
related species, as well as viral secretory leaders.
Both expression and cloning vectors contain a nucleic acid sequence that enables the
vector to replicate in one or more selected host cells. Such sequences are well known for a
variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is
suitable for most Gram-negative bacteria, the 2: plasmid origin is suitable for yeast, and various
viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in
mammalian cells.
Expression and cloning vectors will typically contain a selection gene, also termed a
selectable marker. Typical selection genes encode proteins that (a) confer resistance to
antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic deficiencies, or (c) supply critical nutrients not available from
complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
An example of suitable selectable markers for mammalian cells is one that enables the
identification of cells competent to take up the IL-22 nucleic acid, such as DHFR or thymidine
kinase. An appropriate host cell when wild-type DHFR is employed is the CHO cell line
deficient in DHFR activity, prepared and propagated as described by Urlaub et al., Proc. Natl.
Acad. Sci. USA, 77:4216 (1980). A suitable selection gene for use in yeast is the trp1 gene
present in the yeast plasmid YRp7 [see, e.g., Stinchcomb et al., Nature, 282:39(1979);
Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1 gene
provides a selection marker for a mutant strain of yeast lacking the ability to grow in
tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].
Expression and cloning vectors usually contain a promoter operably linked to the IL-22
nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of
potential host cells are well known. Promoters suitable for use with prokaryotic hosts include
the quadrature-lactamase and lactose promoter systems [see, e.g., Chang et al., Nature, 275:615
(1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, a tryptophan (trp)
promoter system [see, e.g., Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and
hybrid promoters such as the tac promoter [see, e.g., deBoer et al., Proc. Natl. Acad. Sci. USA,
80:21-25 (1983)]. Promoters for use in bacterial systems also will contain a Shine-Dalgarno
(S.D.) sequence operably linked to the DNA encoding IL-22.
Examples of suitable promoter sequences for use with yeast hosts include the promoters
for 3-phosphoglycerate kinase [see, e.g., Hitzeman et al., J. Biol. Chem, 255:2073 (1980)] or
other glycolytic enzymes [see, e.g., Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland,
Biochemistry, 17:4900 (1978)], such as enolase, glyceraldehydephosphate dehydrogenase,
hexokinase, pyruvate decarboxylase, phosphofructokinase, glucosephosphate isomerase, 3-
phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose
isomerase, and glucokinase.
Other yeast promoters, which are inducible promoters having the additional advantage
of transcription controlled by growth conditions, are the promoter regions for alcohol
dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with
nitrogen metabolism, metallothionein, glyceraldehydephosphate dehydrogenase, and
enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for
use in yeast expression are further described in EP 73,657.
IL-22 transcription from vectors in mammalian host cells is controlled, for example, by
promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK
2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus,
avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40
(SV40), from heterologous mammalian promoters, e.g., the actin promoter or an
immunoglobulin promoter, and from heat-shock promoters, provided such promoters are
compatible with the host cell systems.
Transcription of a DNA encoding the IL-22 polypeptides by higher eukaryotes can be
increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements
of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription.
Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-
fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell
virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-
270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of
the replication origin, and adenovirus enhancers. The enhancer can be spliced into the vector at
a position 5' or 3' to the IL-22 coding sequence, but is preferably located at a site 5' from the
promoter.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal,
human, or nucleated cells from other multicellular organisms) will also contain sequences
necessary for the termination of transcription and for stabilizing the mRNA. Such sequences
are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or
viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as
polyadenylated fragments in the untranslated portion of the mRNA encoding IL-22.
Still other methods, vectors, and host cells suitable for adaptation to the synthesis of IL-
22 in recombinant vertebrate cell culture are described in Gething et al., Nature, 293:620-625
(1981); Mantei et al., Nature, 281:4046 (1979); EP 117,060; and EP 117,058.
Gene amplification and/or expression can be measured in a sample directly, for
example, by conventional Southern blotting, Northern blotting to quantitate the transcription of
mRNA [see, e.g., Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting
(DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the
sequences described herein. Alternatively, antibodies can be employed that can recognize
specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or
DNA-protein duplexes. The antibodies in turn can be labeled and the assay can be carried out
where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the
presence of antibody bound to the duplex can be detected.
Gene expression, alternatively, can be measured by immunological methods, such as
immunohistochemical staining of cells or tissue sections and assay of cell culture or body
fluids, to quantitate directly the expression of gene product. Antibodies useful for
immunohistochemical staining and/or assay of sample fluids can be either monoclonal or
polyclonal, and can be prepared in any mammal. Conveniently, the antibodies can be prepared
against a native sequence IL-22 polypeptide or against a synthetic peptide based on the DNA
sequences described herein or against exogenous sequence fused to IL-22 DNA and encoding a
specific antibody epitope.
Forms of IL-22 can be recovered from culture medium or from host cell lysates. If
membrane-bound, it can be released from the membrane using a suitable detergent solution
(e.g. Triton-X 100) or by enzymatic cleavage. Cells employed in expression of IL-22 can be
disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication,
mechanical disruption, or cell lysing agents.
It may be desired to purify IL-22 from recombinant cell proteins or polypeptides. The
following procedures are exemplary of suitable purification procedures: by fractionation on an
ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or
on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate
precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to
remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms of
the IL-22 polypeptide. Various methods of protein purification may be employed and such
methods are known in the art and described for example in Deutscher, Methods in Enzymology,
182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York
(1982). The purification step(s) selected will depend, for example, on the nature of the
production process used and the particular IL-22 produced. The above-described general
methods can be applied to the preparation of IL-2 Fc fusion protein as well.
Similarly, IL-22 Fc fusion proteins may be produced using recombinant methods and
compositions, as described in, e.g., Molecular Cloning: A Laboratory Manual (Sambrook, et
al., 1989, Cold Spring Harbor Laboratory Press) and PCR Protocols: A Guide to Methods and
Applications (Innis, et al. 1990. Academic Press, San Diego, CA). In one embodiment, an
isolated nucleic acid encoding IL-22 Fc fusion proteins described herein is included. In a
further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic
acid are described. In a further embodiment, a host cell comprising such nucleic acid is
described. In one such embodiment, a host cell comprises (e.g., has been transformed with) a
vector comprising a nucleic acid that encodes an amino acid sequence comprising the IL-22 Fc
fusion protein. In certain embodiment, the vector is an expression vector. In one embodiment,
the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0,
NS0, Sp20 cell). In one embodiment, a method of making an IL-22 Fc fusion protein is
described, wherein the method comprises culturing a host cell comprising a nucleic acid
encoding the IL-22 Fc fusion protein, as described above, under conditions suitable for
expression of the Fc fusion protein, and optionally recovering the Fc fusion protein from the
host cell (or host cell culture medium).
For recombinant production of an IL-22 Fc fusion protein, nucleic acid encoding an Fc
fusion protein, e.g., as described herein, is isolated and inserted into one or more vectors for
further cloning and/or expression in a host cell. Such nucleic acid may be readily isolated and
sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable
of binding specifically to genes encoding the fusion protein). In certain embodiments, when
preparing the IL-22 Fc fusion proteins, nucleic acid encoding the IL-22 polypeptide or a
fragment thereof can be ligated to nucleic acid encoding an immunoglobulin constant domain
sequence at specified location on the constant domain to result in an Fc fusion at the C-terminus
of IL-22; however N-terminal fusions are also possible.
As an example of constructing an IL-22 Fc fusion protein, the DNA encoding IL-22 is
cleaved by a restriction enzyme at or proximal to the 3' end of the DNA encoding IL-22 and at a
point at or near the DNA encoding the N-terminal end of the mature polypeptide (where use of
a different leader is contemplated) or at or proximal to the N-terminal coding region for IL-22
full-length protein (where a native signal is employed). This DNA fragment then is readily
inserted into DNA encoding an immunoglobulin light or heavy chain constant region and, if
necessary, tailored by deletional mutagenesis. Preferably, this is a human immunoglobulin
when the fusion protein is intended for in vivo therapy for humans.
In some embodiments, the ILimmunoglobulin chimeras are assembled as
monomers, hetero- or homo-multimer, or as dimers or tetramers. Generally, these assembled
immunoglobulins will have known unit structures as represented by the following diagrams. A
basic four chain structural unit is the form in which IgG, IgD, and IgE exist. A four chain unit
is repeated in the higher molecular weight immunoglobulins; IgM generally exists as a
pentamer of, basic four-chain units held together by disulfide bonds. IgA globulin, and
occasionally IgG globulin, may also exist in a multimeric form in serum. In the case of
multimers, each four chain unit may be the same or different. See also Capon et al. U.S. Patent
No. 5,116,964, incorporated herein by reference in its entirety.
In the diagrams herein, "A" means at least a portion of a binding partner (such as IL-22)
containing a binding site which is capable of binding its ligand or receptor (such as IL-22 R); X
is an additional agent, which may be another functional binding partner (same as A or
different), a multiple subunit (chain) polypeptide as defined above (e.g., an integrin), a portion
of an immunoglobulin superfamily member such as a variable region or a variable region-like
domain, including a native or chimeric immunoglobulin variable region, a toxin such as
pseudomonas exotoxin or ricin, or a polypeptide therapeutic agent not otherwise normally
associated with a constant domain; and V , V , C and C represent light or heavy chain
L H L H
variable or constant domains of an immunoglobulin. These diagrams are understood to be
merely exemplary of general assembled immunoglobulin structures, and do not encompass all
possibilities. It will be understood, for example, that there might desirably be several different
"A"s or "X"s in any of these constructs.
It will be understood that these diagrams are merely illustrative, and that the chains of
the multimers are believed to be disulfide bonded in the same fashion as native
immunoglobulins. According to this description, hybrid immunoglobulins are readily secreted
from mammalian cells transformed with the appropriate nucleic acid. The secreted forms
include those wherein the binding partner epitope is present in heavy chain dimers, light chain
monomers or dimers, and heavy and light chain heterotetramers wherein the binding partner
epitope is present fused to one or more light or heavy chains, including heterotetramers wherein
up to and including all four variable region analogues are substituted. Where a light-heavy
chain non-binding partner variable-like domain is present, a heterofunctional antibody thus is
described.
Chains or basic units of varying structure may be utilized to assemble the monomers and
hetero- and homo-multimers and immunoglobulins of this description. Specific examples of
these basic units are diagrammed below and their equivalents (for purposes of the attenuated
formulae infra) are indicated.
Various exemplary assembled novel immunoglobulins produced in accordance with this
description are schematically diagrammed below. In addition to the symbols defined above, n
is an integer, and Y designates a covalent cross-linking moiety.
A, X, V or C may be modified with a covalent cross-linking moiety (Y) so to be (A-Y) ,
(X-Y) etc.
The binding partner A (such as IL-22) may also be a multi-chain molecule, e.g. having
chains arbitrarily denoted as A and A . These chains as a unit are located at the sites noted for
the single chain "A" above. One of the multiple chains is fused to one immunoglobulin chain
(with the remaining chains covalently or noncovalently associated with the fused chain in the
normal fashion) or, when the ligand binding partner contains two chains, one chain is separately
fused to an immunoglobulin light chain and the other chain to an immunoglobulin heavy chain.
Basic units having the structures as diagrammed below are examples of those used to
create monomers, and hetero- and homo-multimers, particularly dimers and trimers with multi-
chain ligand binding partners:
Various exemplary novel assembled antibodies having a two-chain ligand binding
partner ("A and A ") utilized in unit structures as above are schematically diagrammed below.
The structures shown in the above tables show only key features, e.g. they do not show
joining (J) or other domains of the immunoglobulins, nor are disulfide bonds shown. These are
omitted in the interests of brevity. However, where such domains are required for binding
activity they shall be constructed as being present in the ordinary locations which they occupy
in the binding partner or immunoglobulin molecules as the case may be.
DNA encoding immunoglobulin light or heavy chain constant regions is known or
readily available from cDNA libraries or is synthesized. See for example, Adams et al.,
Biochemistry 19:2711-2719 (1980); Gough et al., Biochemistry 19:2702-2710 (1980); Dolby et
al; P.N.A.S. USA, 77:6027-6031 (1980); Rice et al P.N.A.S USA 79:7862-7865 (1982);
Falkner et al; Nature 298:286-288 (1982); and Morrison et al; Ann. Rev. Immunol. 2:239-256
(1984). DNA sequence encoding human IL-22 with the endogenous leader sequence is
included herein (SEQ ID NO:70). DNA sequences encoding other desired binding partners
which are known or readily available from cDNA libraries are suitable in the practice of this
invention.
DNA encoding an IL-22 Fc fusion protein of this invention is transfected into a host cell
for expression. If multimers are desired then the host cell is transformed with DNA encoding
each chain that will make up the multimer, with the host cell optimally being selected to be
capable of assembling the chains of the multimers in the desired fashion. If the host cell is
producing an immunoglobulin prior to transfection then one needs only transfect with the
binding partner fused to light or to heavy chain to produce a heteroantibody. The
aforementioned immunoglobulins having one or more arms bearing the binding partner domain
and one or more arms bearing companion variable regions result in dual specificity for the
binding partner ligand and for an antigen or therapeutic moiety. Multiply cotransformed cells
are used with the above-described recombinant methods to produce polypeptides having
multiple specificities such as the heterotetrameric immunoglobulins discussed above.
Although the presence of an immunoglobulin light chain is not required in the
immunoadhesins of the present description, an immunoglobulin light chain might be present
either covalently associated to an ILimmunoglobulin heavy chain fusion polypeptide. In
this case, DNA encoding an immunoglobulin light chain is typically co-expressed with the
DNA encoding the ILimmunoglobulin heavy chain fusion protein. Upon secretion, the
hybrid heavy chain and the light chain will be covalently associated to provide an
immunoglobulin-like structure comprising two disulfide-linked immunoglobulin heavy chain-
light chain pairs. Methods suitable for the preparation of such structures are, for example,
disclosed in U.S. Pat. No. 4,816,567 issued Mar. 28, 1989.Suitable host cells for cloning or
expression of target protein-encoding vectors include prokaryotic or eukaryotic cells described
herein. For example, IL-22 fusion protein may be produced in bacteria, in particular when
glycosylation and Fc effector function are not needed or are detrimental. For expression of
polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See
also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa,
NJ, 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After
expression, the Fc fusion protein may be isolated from the bacterial cell paste in a soluble
fraction and can be further purified. As exemplified in the example section, further purification
methods include without limitation purification using a Protein A column.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are
suitable cloning or expression hosts, including fungi and yeast strains whose glycosylation
pathways have been “humanized,” resulting in the production of an antibody with a partially or
fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li
et al., Nat. Biotech. 24:210-215 (2006).
Suitable host cells for the expression of glycosylated proteins are also derived from
multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include
plant and insect cells. Numerous baculoviral strains have been identified which may be used in
conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.
Plant cell cultures can also be utilized as hosts. See, e.g., US Patent Nos. 5,959,177,
6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES technology for
producing antibodies in transgenic plants).
Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are
adapted to grow in suspension may be useful. Other examples of useful mammalian host cell
lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney
line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby
hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol.
Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells
(VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat
liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary
tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci.
383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include
Chinese hamster ovary (CHO) cells, including DHFR CHO cells (Urlaub et al., Proc. Natl.
Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp2/0. For a
review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki
and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ),
pp. 255-268 (2003).
D. IL-22 Agonists
In one embodiment, the present description includes IL-22 agonists for method
embodiments. The IL-22 agonists have IL-22 biological activity as defined herein. In one
embodiment, the IL-22 agonist is an antibody. In certain embodiments, an anti-IL-22 antibody
is an agonistic antibody that promotes the interaction of IL-22 with IL-22R. In a particular
embodiment, an IL-22 agonist is an antibody that binds IL-22BP and blocks or inhibits binding
of IL-22BP to IL-22, and thereby induces or increases an IL-22 activity (e.g., binding to IL-
22R). In another embodiment, an IL-22 agonist is an oligopeptide that binds to IL-22.
Oligopeptides can be chemically synthesized using known oligopeptide synthesis methodology
or can be prepared and purified using recombinant technology. Such oligopeptides are usually
at least about 5 amino acids in length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14,
, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,
65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in length. Such oligopeptides can be
identified without undue experimentation using well known techniques. In this regard, it is
noted that techniques for screening oligopeptide libraries for oligopeptides that are capable of
specifically binding to a polypeptide target are well known in the art (see, e.g., U.S. Patent Nos.
5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT
Publication Nos. WO 84/03506 and WO84/03564; Geysen et al., Proc. Natl. Acad. Sci. U.S.A.,
81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. USA, 82:178-182 (1985); Geysen et
al., in Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J. Immunol. Meth.,
102:259-274 (1987); Schoofs et al., J. Immunol., 140:611-616 (1988), Cwirla, S. E. et al.
(1990) Proc. Natl. Acad. Sci. USA, 87:6378; Lowman, H.B. et al. (1991) Biochemistry,
:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol.,
222:581; Kang, A.S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991)
Current Opin. Biotechnol., 2:668).
In yet another embodiment, an IL-22 agonist of the present description is an organic
molecule that binds to IL-22, other than an oligopeptide or antibody as described herein. An
organic molecule can be, for example, a small molecule. An organic molecule that binds to IL-
22 can be identified and chemically synthesized using known methodology (see, e.g., PCT
Publication Nos. WO00/00823 and WO00/39585). Such organic molecules are usually less
than about 2000 daltons in size, alternatively less than about 1500, 750, 500, 250 or 200 daltons
in size, wherein such organic molecules that are capable of binding to IL-22 of the present
description can be identified without undue experimentation using well known techniques. In
this regard, it is noted that techniques for screening organic molecule libraries for molecules
that are capable of binding to a polypeptide target are well known in the art (see, e.g., PCT
Publication Nos. WO00/00823 and WO00/39585). In a particular embodiment, an IL-22
agonist is an organic molecule that binds IL-22BP and blocks or inhibits binding of IL-22BP to
IL-22, and thereby induces or increases an IL-22 activity (e.g., binding to IL-22R). In yet
another embodiment, agonists of IL-22 are described. Exemplary agonists include, but are not
limited to, native IL-22 or IL-22R; fragments, variants, or modified forms of IL-22 or IL-22R
that retain at least one activity of the native polypeptide; agents that are able to bind to and
activate IL-22R; and agents that induce over-expression of IL-22 or IL-22R or nucleic acids
encoding IL-22 or IL-22R.
E. Assays
IL-22 Fc fusion protein described herein may be identified, screened for, or
characterized for their physical/chemical properties and/or biological activities by various
assays known in the art.
1. Binding assays and other assays
In one aspect, an IL-22 Fc fusion protein of the invention is tested for its receptor
binding activity, e.g., by known methods such as ELISA, western blotting analysis, cell surface
binding by Scatchard, surface plasmon resonance. In another embodiment, competition assays
may be used to identify an antibody that competes with the IL-22 Fc fusion protein for binding
to the IL-22 receptor. In a further aspect, an IL-22 Fc fusion protein of the invention can be
used for detecting the presence or amount of IL-22 receptor or IL22-Binding Protein (soluble
receptor) present in a biological sample. In a further aspect, an IL-22 Fc fusion protein of the
invention can be used for detecting the presence or amount of IL-22 receptor present in a
biological sample. In certain embodiments, the biological sample is first blocked with a non-
specific isotype control antibody to saturate any Fc receptors in the sample.
2. Activity assays
In one embodiment, assays are described for identifying biological activity of IL-22 Fc
fusion protein. Biological activity of an IL-22 polypeptide or IL-22 Fc fusion protein may
include, e.g., binding to IL-22 receptor, stimulating IL-22 signaling, and inducing STAT3,
RegIII and/or PancrePAP expression. Further, in the case of a cardiovascular disease or
condition, the biological activity may include affecting the formation of atherosclerotic plaques,
in particular to inhibit formation of atherosclerotic plaque formation. Inhibition of plaque
formation can be assessed by any suitable imaging method known to those of ordinary skill in
the art.
F. Conjugates
The description also describes conjugates comprising an IL-22 Fc fusion protein
described herein conjugated to one or more agents for detection, formulation, half-life
extension, mitigating immunogenicity or tissue penetration. Exemplary conjugation includes
without limitation PEGylation and attaching to radioactive isotopes.
In another embodiment, a conjugate comprises an IL-22 Fc fusion protein as described
herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive
211 131 125
isotopes are available for the production of radioconjugates. Examples include At , I , I ,
90 186 188 153 212 32 212
Y , Re , Re , Sm , Bi , P , Pb and radioactive isotopes of Lu. When the
radioconjugate is used for detection, it may comprise a radioactive atom for scintigraphic
studies, for example tc99m or I123, or a spin label for nuclear magnetic resonance (NMR)
imaging (also known as magnetic resonance imaging, mri), such as iodine-123 again, iodine-
131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or
iron.
G. Methods and Compositions for Detection
In certain embodiments, any of the IL-22 Fc fusion described herein is useful for
detecting the presence of IL-22 receptor in a biological sample. In certain embodiments, the
method further comprises the step of blocking any Fc receptors in the sample with a non-
specific isotype control antibody. The term “detecting” as used herein encompasses
quantitative or qualitative detection. In certain embodiments, a biological sample comprises a
cell or tissue, such as epithelial tissues.
In one embodiment, an IL-22 Fc fusion protein for use in a method of detection is
described. In a further embodiment, a method of detecting the presence of IL-22 receptor in a
biological sample is included. In certain embodiments, the method comprises contacting the
biological sample with an IL-22 Fc fusion protein as described herein under conditions
permissive for binding of the IL-22 Fc fusion protein to IL-22 receptor, and detecting whether a
complex is formed between the IL-22 Fc fusion protein and IL-22 receptor. In certain
embodiments, the method further comprises the step of blocking any Fc receptors in the sample
with a non-specific isotype control antibody. Such method may be an in vitro or in vivo
method. In one embodiment, an IL-22 Fc fusion protein is used to select subjects eligible for
therapy with IL-22 Fc fusion protein, e.g. where IL-22 receptor is a biomarker for selection of
patients.
In certain embodiments, labeled IL-22 Fc fusion proteins are described. Labels include,
but are not limited to, labels or moieties that are detected directly (such as fluorescent,
chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties,
such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or
molecular interaction. Exemplary labels include, but are not limited to, the radioisotopes P,
14 125 3 131
C, I, H, and I, fluorophores such as rare earth chelates or fluorescein and its derivatives,
rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and
bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,
horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme,
saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucosephosphate
dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an
enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP,
lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free
radicals, and the like.
H. Pharmaceutical Formulations
The ILbased compositions (which in certain embodiments, include IL-22 Fc fusion
proteins, and IL-22 polypeptide or agonists) herein will be formulated, dosed, and administered
in a fashion consistent with good medical practice. Factors for consideration in this context
include the particular disorder being treated, the particular mammal being treated, the clinical
condition of the individual subject, the cause of the disorder, the site of delivery of the agent,
the method of administration, the scheduling of administration, and other factors known to
medical practitioners. In one embodiment, the composition can be used for increasing the
duration of survival of a human subject susceptible to or diagnosed with the disease or
condition disease. Duration of survival is defined as the time from first administration of the
drug to death.
Pharmaceutical formulations are prepared using standard methods known in the art by
mixing the active ingredient having the desired degree of purity with one or more optional
pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol,
A. Ed. (1980) and Remington's Pharmaceutical Sciences 20 edition, ed. A. FGennaro, 2000,
Lippincott, Williams & Wilkins, Philadelphia, Pa), in the form of lyophilized formulations or
aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at
the dosages and concentrations employed, and include, but are not limited to: buffers such as
phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and
methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol;
cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic
polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine,
histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as
sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal
complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene
glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include
insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins
(sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as
rHuPH20 (HYLENEX , Baxter International, Inc.). Certain exemplary sHASEGPs and
methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186
and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional
glycosaminoglycanases such as chondroitinases.
Optionally, but preferably, the formulation contains a pharmaceutically acceptable salt,
preferably sodium chloride, and preferably at about physiological concentrations.
Optionally, the formulations of the description can contain a pharmaceutically
acceptable preservative. In some embodiments the preservative concentration ranges from 0.1
to 2.0%, typically v/v. Suitable preservatives include those known in the pharmaceutical arts.
Benzyl alcohol, phenol, m-cresol, methylparaben, benzalkonium chloride and propylparaben
are preferred preservatives. Optionally, the formulations of the description can include a
pharmaceutically acceptable surfactant at a concentration of 0.005 to 0.02%.
The formulation herein can also contain more than one active compound as necessary
for the particular indication being treated, preferably those with complementary activities that
do not adversely affect each other. Such molecules are suitably present in combination in
amounts that are effective for the purpose intended.
Exemplary lyophilized formulations are described in US Patent No. 6,267,958.
Aqueous formulations include those described in US Patent No. 6,171,586 and
WO2006/044908, the latter formulations including a histidine-acetate buffer.
The formulation herein may also contain more than one active ingredients as necessary
for the particular indication being treated, preferably those with complementary activities that
do not adversely affect each other. For example, it may be desirable to further include a steroid,
TNF antagonist or other anti-inflammatory therapeutics Such active ingredients are suitably
present in combination in amounts that are effective for the purpose intended.
Active ingredients may be entrapped in microcapsules prepared, for example, by
coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in
colloidal drug delivery systems (for example, liposomes, albumin microspheres,
microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are
disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
Sustained-release preparations may be prepared. Suitable examples of sustained-release
preparations include semipermeable matrices of solid hydrophobic polymers containing the IL-
22 Fc fusion protein, which matrices are in the form of shaped articles, e.g. films, or
microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for
example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat.
No. 3,773,919), copolymers of L-glutamic acid and .gamma. ethyl-L-glutamate, non-degradable
ethylene -vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON
DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and
leuprolide acetate), and poly-D-(-)hydroxybutyric acid. While polymers such as ethylene-
vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain
hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the
body for a long time, they may denature or aggregate as a result of exposure to moisture at 37
C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational
strategies can be devised for stabilization depending on the mechanism involved. For example,
if the aggregation mechanism is discovered to be intermolecular S-S bond formation through
thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues,
lyophilizing from acidic solutions, controlling moisture content, using appropriate additives,
and developing specific polymer matrix compositions.
A pharmaceutical composition for topical administration can be formulated, for
example, in the form of a topical gel. See e.g., US 4,717,717, US 5,130,298, US 5,427,778, US
,457,093, US 5,705,485, US 6,331,309 and WO2006/138,468. In certain embodiments, the
composition can be formulated in the presence of cellulose derivatives. In certain other
embodiments, the topical formulation can be reconstituted from lyophilized formulation with
sufficient buffer or diluent before administration. In certain embodiments, IL-22 polypeptide or
IL-22 Fc fusion protein is formulated for topical administration to a subject having a defect in
epithelial wound healing. In certain particular embodiments, the epithelial wound healing
occurs in the skin. In certain other particular embodiments, the subject is a human having a
defect in wound healing. In certain other embodiments, the topical formulation comprising an
IL-22 Fc fusion protein of the invention can be used to improve wound healing after internal or
external surgical incisions.
In one embodiment of the description, an IL-22 polypeptide or IL-22 Fc fusion protein
for use in accelerating, promoting or improving wound healing is in a formulation of a topical
gel, e.g., in a pre-filled syringe or container, or alternatively, the compound of the description
can be mixed with a gel matrix right before topical administration to a patient. In certain
embodiments, an additional therapeutic agent is also administered topically, either concurrently
or sequentially. Other routes of administration can also be optionally used, e.g., administered by
any suitable means, including but not limited to, parenteral, subcutaneous, intraperitoneal,
intrapulmonary, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal,
oral, and intranasal administration. Parenteral infusions include intramuscular, intravenous,
intraarterial, intraperitoneal, or subcutaneous administration.
Typically for wound healing, an IL-22 polypeptide or IL-22 Fc fusion protein is
formulated for site-specific delivery. When applied topically, the IL-22 polypeptide or IL-22 Fc
fusion is suitably combined with other ingredients, such as carriers and/or adjuvants. There are
no limitations on the nature of such other ingredients, except that they must be
pharmaceutically acceptable and efficacious for their intended administration, and cannot
degrade the activity of the active ingredients of the composition. Examples of suitable vehicles
include ointments, creams, gels, sprays, or suspensions, with or without purified collagen. The
compositions also may be impregnated into sterile dressings, transdermal patches, plasters, and
bandages, optionally in liquid or semi-liquid form. An oxidized regenerated cellulose/collagen
matrices can also be used, e.g., PROMOGRAN Matrix Wound Dressing or PROMOGRAN
PRISMA MATRIX.
Sustained-release preparations may be prepared. Suitable examples of sustained-release
preparations include semipermeable matrices of solid hydrophobic polymers containing a
polypeptide of the invention, which matrices are in the form of shaped articles, e.g. films, or
microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for
example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol), polylactides (U.S. Pat. No.
3,773,919), copolymers of L-glutamic acid and gamma ethyl-L-glutamate, non-degradable
ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON
DEPOT (injectable microspheres composed of lactic acid-glycolic acid copolymer and
leuprolide acetate), poly-lactic-coglycolic acid (PLGA) polymer, and poly-D-(-)
hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic
acid enable release of molecules for over 100 days, certain hydrogels release proteins for
shorter time periods. When encapsulated polypeptides remain in the body for a long time, they
may denature or aggregate as a result of exposure to moisture at 37 C., resulting in a loss of
biological activity and possible changes in immunogenicity. Rational strategies can be devised
for stabilization depending on the mechanism involved. For example, if the aggregation
mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide
interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from
acidic solutions, controlling moisture content, using appropriate additives, and developing
specific polymer matrix compositions.
For obtaining a gel formulation, the IL-22 polypeptide or IL-22 Fc fusion protein
formulated in a liquid composition may be mixed with an effective amount of a water-soluble
polysaccharide or synthetic polymer to form a gel (e.g., a gelling agent) such as polyethylene
glycol to form a formulation of the proper viscosity to be applied topically. The polysaccharide
or gelling agent that may be used includes, for example, cellulose derivatives such as etherified
cellulose derivatives, including alkyl celluloses, hydroxyalkyl celluloses, and alkylhydroxyalkyl
celluloses, for example, methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose,
hydroxypropyl methylcellulose, and hydroxypropyl cellulose; Sodium carboxymethyl cellulose;
POE-POP block polymers: poloxamer USP in various grades; Hyaluronic acid; Polyacrylic acid
such as carbopol 940; starch and fractionated starch; agar; alginic acid and alginates; gum
Arabic; pullullan; agarose; carrageenan; dextrans; dextrin; fructans; inulin; mannans; xylans;
arabinans; chitosans; glycogens; glucans; and synthetic biopolymers; as well as gums such as
xanthan gum; guar gum; locust bean gum; gum Arabic; tragacanth gum; and karaya gum; and
derivatives, combinations and mixtures thereof. In one embodiment, the gelling agent herein is
one that is, e.g., inert to biological systems, nontoxic, simple to prepare, and/or not too runny or
viscous, and will not destabilize the IL-22 polypeptide or IL-22 Fc fusion held within it.
In certain embodiments, the polysaccharide is an etherified cellulose derivative, in
another embodiment one that is well defined, purified, and listed in USP, e.g., methylcellulose
and the hydroxyalkyl cellulose derivatives, such as hydroxypropyl cellulose, hydroxyethyl
cellulose, and hydroxypropyl methylcellulose (all referred to as cellulosic agents). In some
embodiments, the polysaccharide is hydroxyethyl methylcellulose or hydroxypropyl
methylcellulose.
The polyethylene glycol useful for gelling is typically a mixture of low and high
molecular weight polyethylene glycols to obtain the proper viscosity. For example, a mixture of
a polyethylene glycol of molecular weight 400-600 with one of molecular weight 1500 would
be effective for this purpose when mixed in the proper ratio to obtain a paste.
The term "water soluble" as applied to the polysaccharides and polyethylene glycols is
meant to include colloidal solutions and dispersions. In general, the solubility of the cellulose
derivatives is determined by the degree of substitution of ether groups, and the stabilizing
derivatives useful herein should have a sufficient quantity of such ether groups per
anhydroglucose unit in the cellulose chain to render the derivatives water soluble. A degree of
ether substitution of at least 0.35 ether groups per anhydroglucose unit is generally sufficient.
Additionally, the cellulose derivatives may be in the form of alkali metal salts, for example, the
Li, Na, K, or Cs salts.
In certain embodiments, methylcellulose is employed in the gel, for example, it
comprises about 1-5%, or about 1%, about 2%, about 3%, about 4% or about 5%, of the gel and
the IL-22 polypeptide or IL-22 Fc fusion protein is present in an amount of about 50-2000 μg,
100-2000 μg, or 100-1000 μg per ml of gel. In certain embodiments, the effective amount of IL-
22 polypeptide or IL-22 Fc fusion protein for wound healing by topical administration can be
about 25 μg to about 500 μg, about 50 μg to about 300 μg, about 100 μg to about 250 μg, about
50 μg to about 250 μg, about 50 μg to about 150 μg, about 75 μg, about 100 μg, about 125 μg,
about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 300 μg, or about
350 μg, per cm wound area.
The formulations to be used for in vivo administration are generally sterile. Sterility
may be readily accomplished, e.g., by filtration through sterile filtration membranes.
The present description includes dosages for the ILbased therapeutics. For
example, depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g.
0.1-20 mg/kg) of polypeptide is an initial candidate dosage for administration to the subject,
whether, for example, by one or more separate administrations, or by continuous infusion. A
typical daily dosage might range from about 1 μ.g/kg to 100 mg/kg or more, depending on the
factors mentioned above. For repeated administrations over several days or longer, depending
on the condition, the treatment is sustained until a desired suppression of disease symptoms
occurs. However, other dosage regimens can be useful. The progress of this therapy is easily
monitored by conventional techniques and assays.
For the prevention or treatment of disease, the appropriate dosage of a polypeptide of
the invention (when used alone or in combination with one or more other additional therapeutic
agents) will depend on the type of disease to be treated, the type of polypeptide, the severity
and course of the disease, whether the polypeptide is administered for preventive or therapeutic
purposes, previous therapy, the subject's clinical history and response to the polypeptide, and
the discretion of the attending physician. The polypeptide is suitably administered to the subject
at one time or over a series of treatments. Depending on the type and severity of the disease,
about 1 μg/kg to 20 mg/kg (e.g. 0.1mg/kg-15mg/kg) of the polypeptide can be an initial
candidate dosage for administration to the subject, whether, for example, by one or more
separate administrations, or by continuous infusion. One typical daily dosage might range from
about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the condition, the treatment would
generally be sustained until a desired suppression of disease symptoms occurs. One exemplary
dosage of the polypeptide would be in the range from about 0.05 mg/kg to about 20 mg/kg.
Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, 10 mg/kg, 12 mg/kg, 15
mg/kg, or 20 mg/kg (or any combination thereof) may be administered to the subject. In certain
embodiments, about 0.5 mg/kg, 1.0 mg.kg, 2.0 mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6.0
mg/kg, 7.0 mg/kg, 8.0 mg/kg, 9.0 mg/kg, 10 mg/kg, 12 mg/kg, 15 mg/kg, or 20 mg/kg (or any
combination thereof) may be administered to the subject. Such doses may be administered
intermittently, e.g. every week, every two weeks, or every three weeks (e.g. such that the
subject receives from about two to about twenty, or e.g. about six doses of the polypeptide). An
initial higher loading dose, followed by one or more lower doses may be administered. An
exemplary dosing regimen comprises administering an initial loading dose of about 4 mg/kg,
followed by a weekly maintenance dose of about 2 mg/kg of the antibody. However, other
dosage regimens may be useful. The progress of this therapy is easily monitored by
conventional techniques and assays.
The compounds of the description for prevention or treatment of a cardiovascular
disease or condition, metabolic syndrome, acute endotoxemia or sepsis, or diabetes are typically
administered by intravenous injection.
Other methods of administration can also be used, which includes but is not limited to,
topical, parenteral, as intravenous, subcutaneous, intraperitoneal, intrapulmonary, intranasal,
ocular, intraocular, intravitreal, intralesional, intracerobrospinal, intra-articular, intrasynovial,
intrathecal, oral, or inhalation administration. Parenteral infusions include intramuscular,
intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the
compounds described herein are administered to a human subject, in accord with known
methods, such as intravenous administration as a bolus or by continuous infusion over a period
of time.
I. Therapeutic Methods and Compositions
Any of the IL-22 Fc fusion proteins or IL-22 polypeptides or IL-22 agonists described
herein may be used in therapeutic methods.
a) Inflammatory Bowel Disease
In one embodiment, an IL-22 Fc fusion protein for use as a medicament is described. In
further embodiments, an IL-22 Fc fusion protein for use in treating IBD, including UC and CD,
is described. In certain embodiments, an IL-22 Fc fusion protein for use in a method of
treatment is described. In certain embodiments, the description includes an IL-22 Fc fusion
protein for use in a method of treating an individual having UC or CD comprising
administering to the individual an effective amount of the IL-22 Fc fusion protein. In one such
embodiment, the method further comprises administering to the individual an effective amount
of at least one additional therapeutic agent, e.g., as described below. In further embodiments,
the description includes an IL-22 Fc fusion protein for use in enhancing epithelial proliferation,
differentiation and/or migration. In certain particular embodiments, the epithelial tissue is
intestinal epithelial tissue. In certain embodiments, the description includes an IL-22 Fc fusion
protein for use in a method of enhancing epithelial proliferation, differentiation and/or
migration in an individual comprising administering to the individual an effective amount of the
IL-22 Fc fusion protein to enhance epithelial proliferation, differentiation and/or migration. In
yet other embodiments, the description includes an IL-22 Fc fusion protein for use in treating
diabetes, especially type II diabetes, diabetic wound healing, metabolic syndromes and
atherosclerosis. In certain embodiments, the description includes an IL-22 Fc fusion protein
for use in a method of treating diabetes, especially type II diabetes, diabetic wound healing,
metabolic syndromes and atherosclerosis in an individual comprising administering to the
individual an effective amount of the IL-22 Fc fusion protein. See Genentech applications
Docket numbers PR5586, application serial number 61/800795, entitled “Using an IL-22
polypeptide for wound healing,” and PR5590, application serial number 61/801144, entitled
“Methods of treating cardiovascular conditions and metabolic syndrome using an IL-22
polypeptide,” both filed on March 15, 2013. The disclosures of both of the applications are
incorporated herein by reference in their entireties. An “individual” or “subject” or “patient”
according to any of the above embodiments is preferably a human.
In a further embodiment, the description includes the use of an IL-22 polypeptide or IL-
22 Fc fusion protein in the manufacture or preparation of a medicament. In one embodiment,
the medicament is for treatment of IBD and wound healing. In a further embodiment, the
medicament is for use in a method of treating IBD and wound healing comprising administering
to an individual having IBD an effective amount of the medicament. In one such embodiment,
the method further comprises administering to the individual an effective amount of at least one
additional therapeutic agent, e.g., as described below. In a further embodiment, the
medicament is for suppressing inflammatory response in the gut epithelial cells. In a further
embodiment, the medicament is for use in a method of enhancing epithelial proliferation,
differentiation and/or migration in an individual comprising administering to the individual an
amount effective of the medicament to enhance epithelial proliferation, differentiation and/or
migration. An “individual” according to any of the above embodiments may be a human.
In a further embodiments, the description includes a method for treating IBD, including
UC and CD. In one embodiment, the method comprises administering to an individual having
IBD an effective amount of an IL-22 polypeptide or an IL-22 Fc fusion protein. In one such
embodiment, the method further comprises administering to the individual an effective amount
of at least one additional therapeutic agent, as described below. An “individual” according to
any of the above embodiments may be a human.
In a further embodiments, the description includes a method for enhancing epithelial
proliferation, differentiation and/or migration in an individual. In one embodiment, the method
comprises administering to the individual an effective amount of an IL-22 polypeptide or IL-22
Fc fusion protein to enhance epithelial proliferation, differentiation and/or migration. In one
embodiment, an “individual” is a human.
b) Other Therapeutic Indications
The present description includes ILbased therapeutic agents for cardiovascular
diseases and conditions, metabolic syndrome, acute endotoxemia and sepsis, and diabetes. For
the prevention, treatment or reduction in the severity of a given disease or condition, the
appropriate dosage of a compound of the invention will depend on the type of disease or
condition to be treated, as defined above, the severity and course of the disease or condition,
whether the agent is administered for preventive or therapeutic purposes, previous therapy, the
subject's clinical history and response to the compound, and the discretion of the attending
physician. The compound is suitably administered to the subject at one time or over a series of
treatments. Preferably, it is desirable to determine the dose-response curve and the
pharmaceutical composition of the invention first in vitro, and then in useful animal models
prior to testing in humans.
In one embodiment, the description includes methods of treatment for a cardiovascular
disease or disorder, metabolic syndrome, acute endotoxemia and sepsis, and an insulin-related
disorder. In one embodiment, the method comprises administering to a subject in need a
therapeutically effective amount of an IL-22 polypeptide, an IL-22 Fc fusion protein, or an IL-
22 agonist. In another embodiment, the description includes a method for the delaying or
slowing down of the progression of a cardiovascular disease or disorder, metabolic syndrome,
and an insulin-related disorder. In one embodiment, the method comprises administering to
subject diagnosed with the disease, condition, or disorder, an effective amount of an IL-22
polypeptide, IL-22 Fc fusion protein, or IL-22 agonist. In another embodiment, the description
includes a method for preventing indicia of a cardiovascular disease or disorder, and an insulin-
related disorder. In one embodiment, the method comprises administering an effective amount
of an IL-22 polypeptide, IL-22 Fc fusion protein, or IL-22 agonist to a subject at risk of the
disease, condition, or disorder, wherein the IL-22 polypeptide, IL-22 Fc fusion protein, or IL-22
agonist is effective against the development of indicia of the disease, condition, or disorder.
Cardiovascular diseases and conditions
In one embodiment, the IL-22 polypeptides, IL-22 Fc fusion proteins and IL-22 agonists
include a preventative or prophylactic effect against the development of, or the progression of,
clinical and/or histological and/or biochemical and/or pathological indicia (including both
symptoms and signs) of cardiovascular diseases or conditions in a subject. In one embodiment,
the disease or condition is atherosclerosis. In one embodiment, the indicia include
atherosclerotic plaque formation and/or vascular inflammation. In another embodiment, the
subject is at risk for cardiovascular disease. In general, a subject at risk will previously have
had a cardiovascular disease or condition as described herein, or will have a genetic
predisposition for a cardiovascular disease or condition.
The efficacy of the treatment of cardiovascular diseases and conditions can be measured
by various assessments commonly used in evaluating cardiovascular diseases. For example,
cardiovascular health can be assessed. Cardiovascular health can be evaluated by, but not
limited to, e.g., blood tests (e.g., total cholesterol, LDL-C, HDL-C, triglyceride, C-reactive
protein, fibrinogen, homocysteine, fasting insulin, ferritin, lipoprotein, LPS), blood pressure,
auscultation, electrocardiogram, cardiac stress testing, cardiac imaging (e.g., coronary
catheterization, echocardiogram, intravascular ultrasound, positron emission tomography,
computed tomography angiography, and magnetic resonance imaging).
Metabolic syndrome
In one embodiment, the IL-22 polypeptides, IL-22 Fc fusion proteins and IL-22 agonists
include a therapeutic, preventative or prophylactic effect against the development of, or the
progression of, clinical and/or histological and/or biochemical and/or pathological indicia
(including both symptoms and signs) of metabolic syndrome (or metabolic disorder or disease)
in a subject. In one or more embodiment, the subject is at risk for metabolic syndrome.
The efficacy of the treatment of metabolic syndrome can be measured by various
assessments commonly used in evaluating metabolic syndrome. For example, obesity can be
measured. As a further example, hyperglycemia, dyslipidemia, insulin resistance, chronic
adipose tissue inflammation, and/or hypertension can be measured. Reduction in in levels of
one or more of C-reactive protein, IL-6, LPS, and plasminogen activator inhibitor 1 can be
measured. These measurements can be performed by any methods well known in the art.
Insulin-related disorders
For insulin-related disorders, the term “treatment” refers to both therapeutic treatment
and prophylactic or preventative measures for the disorder, wherein the object is to prevent or
slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment
include those already with an insulin-related disorder as well as those prone to have such a
disorder or those in whom the disorder is to be prevented.
In one embodiment, the IL-22 polypeptides, IL-22 Fc fusion proteins and IL-22 agonists
include a preventative or prophylactic effect against the development of, or the progression of,
clinical and/or histological and/or biochemical and/or pathological indicia (including both
symptoms and signs) of an insulin-related disorder in a subject. In one embodiment, the
disorder is Type I diabetes, Type II diabetes, or gestational diabetes. In one embodiment, the
pathology or pathological indicia include one or more of: little or no insulin production by the
pancreas (e.g., islet cells), insulin resistance, and hyperglycemia. In another embodiment, the
subject is at risk for an insulin-related disorder. In general, a subject at risk has a genetic
predisposition for an insulin-related disorder, has been exposed to a virus that triggers
autoimmune destruction of islet cells (e.g., Epstein-Barr virus, coxsackievirus, mumps virus or
cytomegalovirus), is obese, is pre-diabetic (higher than normal blood sugar levels), or has
gestational diabetes.
The efficacy of the treatment of an insulin-related disorder can be measured by various
assessments commonly used in evaluating such disorders. For example, both Type I and Type II
diabetes can be evaluated with one or more of the following: a glycated hemoglobin test (A1C),
a regular blood sugar test, and a fasting blood sugar test. Type I can also be evaluated by
testing for autoantibodies in the blood and/or ketones in the urine. Type II can also be
evaluated by testing for oral glucose tolerance.
Acute endotoxemia and sepsis
In one embodiment, the IL-22 polypeptides, IL-22 Fc fusion proteins and IL-22 agonists
include a therapeutic, preventative or prophylactic effect against the development of, or the
progression of, clinical and/or histological and/or biochemical and/or pathological indicia
(including both symptoms and signs) of acute endotoxemia, sepsis, or both, in a subject. In
one or more embodiment, the subject is at risk for acute endotoxemia, sepsis, or both.
The efficacy of the treatment of acute endotoxemia, sepsis, or both can be measured by
various assessments commonly used in evaluating acute endotoxemia, sepsis, or both. For
example, reduction in in levels of LPS or inflammatory markers can be measured. These
measurements can be performed by any methods well known in the art.
Wound healing
There are a variety of ways to measure wound healing. Often images are taken to
calculate linear dimensions, perimeter and area. The NIH has a free program, Image J, that
allows measurement of wound areas from an image. The final healing prognosis can be
extrapolated from initial healing rates based on the migration of the periphery towards the
center. This is done using a number of mathematical equations, the most common of which is a
modified Gilman's equation. In addition to visual inspection, wound healing measurement can
also be aided by spectroscopic methods or MRI. See e.g.,Dargaville et al., Biosensors
Bioelectronics, 2013, 41:30-42, Tan et al., 2007, British J. Radiol. 80:939-48. If healing is
slow/inadequate, biopsies of the wound edges may be taken to rule out or determine infection
and malignancy. In certain embodiments, the acceleration or improvement of wound healing
can be assessed by comparing wound closure in ILtreated and control wounds. In certain
embodiments, the acceleration or improvement of wound healing is at least 20%, 30%, 40%,
50%, 60%, 70%, 80% or 90% faster or better than the control.
In certain embodiment, the description includes methods for
promoting/accelerating/improving healing of a wound with or without active infection,
microbial contamination or colonization in the wound. The IL-22 polypeptides, IL-22 Fc fusion
proteins or IL-22 agonists can be used for treating infected wounds or
promoting/accelerating/improving infected wound healing. In certain embodiments, the IL-22
polypeptides, IL-22 Fc fusion proteins or IL-22 agonists can be used for treating wounds, or
promoting/accelerating/improving wound healing, in the presence of infection. In some
embodiments, the IL-22 polypeptides, IL-22 Fc fusion proteins or IL-22 agonists can be used
for treating wounds or promoting/accelerating/improving wound healing in the presence of
microbial contamination or colonization with risk for infection. In further embodiments, the
patient in need of wound healing treatment can be a diabetic patient. Accordingly, in some
embodiments, the wound is a diabetic wound, for example, diabetic foot ulcer. In some further
embodiments, the wound is an infected diabetic wound, for example, infected diabetic foot
ulcer.
In a further embodiment, the description includes pharmaceutical formulations
comprising an IL-22 polypeptide, IL-22 Fc fusion protein or IL-22 agonist described herein,
e.g., for use in any of the above therapeutic methods. In one embodiment, a pharmaceutical
formulation comprises an IL-22 polypeptide, IL-22 Fc fusion protein or IL-22 agonist described
herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical
formulation comprises an IL-22 polypeptide, IL-22 Fc fusion protein or IL-22 agonist described
herein and at least one additional therapeutic agent, e.g., as described below.
IL-22 Fc fusion protein of the invention can be used either alone or in combination with
other agents in a therapy. For instance, an IL-22 polypeptide, IL-22 Fc fusion protein or IL-22
agonist of the description may be co-administered with at least one additional therapeutic agent.
In certain embodiments, an additional therapeutic agent is an immune suppressant that reduces
the inflammatory response including without limitation methotrexate, TNF inhibitor, TNF
antagonist, mesalazine, steroid, dexamethasone, and azathioprine, and combination thereof.
Suitable additional therapeutic agents that reduce an inflammatory response include without
limitation 5-aminosalicylic acid (5-ASA), mercaptopurine (also called 6-mercaptopurine or 6-
MP) or combination thereof. In certain embodiments, the IL22 polypeptide or IL-22 Fc fusion
may be co-administered with one or more additional therapeutic agents that reduce an
inflammatory response (for example, 5-ASA, 6-MP, or an TNF antagonist) for the treatment of
IBD. In certain other embodiments, the IL22 polypeptide or IL-22 Fc fusion may be co-
administered with an integrin antagonist such as etrolizumab for the treatment of IBD. In one
embodiment, the IL-22 polypeptide or IL-22 Fc fusion protein is used in combination with an
IL-22 agonist.
For accelerating chronic wound healing, such as for the treatment of diabetic foot ulcer,
the administration of an IL-22 polypeptide or fragments or variants thereof, IL-22 Fc fusion
proteins or IL-22 agonists can be combined with one or more additional wound healing agents.
Suitable additional wound healing agents include without limitation growth factors (e.g., EGF,
FGF, IGF, PDGF, TGF, and VEGF), nerve growth factor (NGF), angiogenesis factors (e.g.,
HGF, TNF-α, angiogenin, IL-8, angiopoietins 1 and 2, Tie-2, integrin α5, matrix
metalloproteinases, nitric oxide, COX-2), members of the platelet derived growth factor
(PDGF) family (e.g., PDGF-A, PDGF-B, PDGF-C, and PDGF-D), members of the insulin
growth factor (IGF) family (e.g., IGF-I, IGF-II), members of the transforming growth factor
(TGF) family (e.g., TGF-α TGF-β) and anabolic oxygen (vacuum therapy). In certain
embodiments, the IL-22 polypeptide or IL-22 Fc fusion can be co-administered with one or
more additional wound healing agents described herein and/or one or more antibacterial agents
or antibiotics suitable for use in topical administration. See WO2006/138468, incorporated
herein by reference in its entirety. In such embodiments, the antibiotic can be sulfur antibiotic
including without limitation silver sulfadiazine, i.e., silvadeen. The co-administered one or
more additional agents can be administered concurrently, alternatively or sequentially with IL-
22 polypeptide, IL-22 fusion protein or IL22 agonist.
In further exemplary embodiments, if the target is prevention or treatment of
cardiovascular diseases or conditions or metabolic syndrome, the administration of an IL-22
polypeptide or fragments or variants thereof, IL-22 Fc fusion proteins or IL-22 agonists can be
combined with or supplement the administration of the cholesterol-lowering agents such as
statins (e.g., lovastatin, rosuvastatin, fluvastatin, atorvastatin, pravastatin, and simvastatin), bile
acid binding resins (colestipol, cholestyramine sucrose, and colesevelam), ezetimibe, or a
ezetimibe-simvastatin combination; anti-platelet agents such as cyclooxygenase inhibitors
(aspirin), adenosine diphosphate (ADP) receptor inhibitors (clopidogrel, prasugrel, ticagrelor,
ticlopidine), phosphodiesterase inhibitors (cilostazol), glycoprotein IIB/IIIA inhibitors
(abciximab, eptifibatide, tirofiban), adenosine reuptake inhibitors (dipyridamole), thromboxane
inhibitors (thromboxane synthase inhibitors, thromboxane receptor antagonists, terutroban);
beta blockers such as alprenolol, bucindolol, carteolol, carvedilol, labetalol, nadolol,
oxprenolol, penbutolol, pindolol, propranolol, sotalol, timolol, eucommia bark, acebutolol,
atenolol, betaxolol, bisoprolol, celiprolol, esmolol, metoprolol, nebivolol, butaxamine, ICI-
118,551, and SR 59230A; angiotensin-converting enzyme (ACE) inhibitors such as captopril,
zofenopril, dicarboxylate-containing agents (enalapril, ramipril, quinapril, perindopril,
lisinopril, benazepril, imidapril, zofenopril), phosphonate-containing agents (fosinopril),
casokinins, lactokinins, lactotripeptides (Val-Pro-Pro, and Ile-Pro-Pro produced by the probiotic
Lactobacillus helveticus or derived from casein); calcium channel blockers such as
dihydropyridines (e.g., amlodipine, aranidipine, azelnidipine, barnidipine, benidipine,
cilnidipine, clevidipine, isradipine, efonidipine, felodipine, lacidipine, lercanidipine,
manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine, and
pranidipine), phenylalkylamine (e.g., verapamil), benzothiazepines (e.g., diltiazem), mibefradil,
bepridil, fluspirilene, and fendiline; diuretics such as high ceiling loop diuretics (e.g.,
furosemide, ethacrynic acid, torsemide and bumetanide), thiazides (e.g., hydrochlorothiazide
acid), carbonic anhydrase inhibitors (e.g., acetazolamide and methazolamide), potassium-
sparing diuretics (e.g., aldosterone antagonists: spironolactone, and epithelial sodium channel
blockers: amiloride and triamterene), and calcium-sparing diuretics, and pharmaceutically
acceptable salts, acids or derivatives of any of the above.
For insulin-related disorders or metabolic syndrome, the administration of an IL-22
polypeptide or fragments or variants thereof or IL-22 Fc fusion protein or IL-22 agonists can be
combined with or supplement the administration of various therapeutic agents. In the case of
Type I diabetes (insulin-dependent diabetes mellitus or IDDM), the IL-22 polypeptide, Fc
fusion protein or agonist described herein are combined with one or more of regular insulin
replacement therapy (including rapid-acting and long-acting insulin), immunosuppression
treatment, islet transplantation and stem cell therapy. In one embodiment, the regular insulin
replacement therapy includes, without limitation, regular insulin (e.g., Humulin R, Novolin R),
insulin isophane (e.g., Humulin N, Novolin N), insulin lispro (e.g., Humalog), insulin aspart
(e.g., NovoLog), insulin glargine (e.g., Lantus) and insulin detemir (e.g., Levemir). In other
embodiments, the insulin replacement therapy further includes pramlintide (Symlin).
In the case of Type II diabetes (non-insulin dependent diabetes mellitus or NIDDM) or
metabolic syndrome, the IL-22 polypeptide, Fc fusion protein and agonist described herein can
be combined with one or more of insulin replacement therapy (as discussed above), an agent to
lower glucose production by the liver, an agent to stimulate pancreatic production and release
of insulin, an agent that blocks enzymatic break down of carbohydrates or increases insulin
sensitivity. In one embodiment, the agent to lower glucose production is metformin (e.g.,
Glucophage, Glumetza). In another embodiment, the agent to stimulate pancreatic production
and release of insulin is glipizide (e.g., Glucotrol, Glucotrol XL), glyburide (e.g., DiaBeta,
Glynase) and glimepiride (e.g., Amaryl). In one other embodiment, the agent that blocks
enzymatic break down of carbohydrates or increases insulin sensitivity is pioglitazone (e.g.,
Actos). In another embodiment, the IL-22 polypeptide, Fc fusion protein and agonist can be
combined with one of the following replacements for metformin: sitagliptin (e.g., Januvia),
saxagliptin (e.g., Onglyza), repaglinide (e.g., Prandin) and nateglinide (e.g., Starlix). Exenatide
(e.g., Byetta) and liraglutide (e.g., Victoza). In another embodiment, the IL-22 polypeptide, Fc
fusion protein and agonist are combined with an oral hypoglycemic agent, e.g., sulfonylureas.
In the case of gestational diabetes or metabolic syndrome, the IL-22 polypeptide, Fc
fusion and agonist described herein are combined with an oral blood sugar control medication.
In one embodiment, the medication is glyburide.
The combination therapy can provide "synergy" and prove "synergistic", i.e. the effect
achieved when the active ingredients used together is greater than the sum of the effects that
results from using the compounds separately. A synergistic effect can be attained when the
active ingredients are: (1) co-formulated and administered or delivered simultaneously in a
combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate
formulations; or (3) by some other regimen. When delivered in alternation therapy, a
synergistic effect can be attained when the compounds are administered or delivered
sequentially, e.g. by different injections in separate syringes. In general, during alternation
therapy, an effective dosage of each active ingredient is administered sequentially, i.e. serially,
whereas in combination therapy, effective dosages of two or more active ingredients are
administered together.
Such combination therapies noted above encompass combined administration (where
two or more therapeutic agents are included in the same or separate formulations), and separate
administration, in which case, administration of the IL-22 polypeptide or IL-22 Fc fusion
protein of the description can occur prior to, simultaneously, and/or following, administration
of the additional therapeutic agent or agents. In one embodiment, administration of the IL-22
Fc fusion protein and administration of an additional therapeutic agent occur within about one
month, or within about one, two or three weeks, or within about one, two, three, four, five, or
six days, of each other.
An IL-22 polypeptide or IL-22 Fc fusion protein of the description (and any additional
therapeutic agent) can be administered by any suitable means, including parenteral,
intrapulmonary, topical and intranasal, and, if desired for local treatment, intralesional
administration. Parenteral infusions include intramuscular, intravenous, intraarterial,
intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by
injections, such as intravenous or subcutaneous injections, depending in part on whether the
administration is brief or chronic. Various dosing schedules including but not limited to single
or multiple administrations over various time-points, bolus administration, and pulse infusion
are contemplated herein.
IL-22 polypeptide or IL-22 Fc fusion protein of the description would be formulated,
dosed, and administered in a fashion consistent with good medical practice. Factors for
consideration in this context include the particular disorder being treated, the particular
mammal being treated, the clinical condition of the individual patient, the cause of the disorder,
the site of delivery of the agent, the method of administration, the scheduling of administration,
and other factors known to medical practitioners. The IL-22 polypeptide or IL-22 Fc fusion
protein need not be, but is optionally formulated with one or more agents currently used to
prevent or treat the disorder in question. The effective amount of such other agents depends on
the amount of the fusion protein present in the formulation, the type of disorder or treatment,
and other factors discussed above. These are generally used in the same dosages and with
administration routes as described herein, or about from 1 to 99% of the dosages described
herein, or in any dosage and by any route that is empirically/clinically determined to be
appropriate.
For the prevention or treatment of disease, the appropriate dosage of an IL-22 Fc fusion
protein of the invention (when used alone or in combination with one or more other additional
therapeutic agents) will depend on the type of disease to be treated, the type of Fc region, the
severity and course of the disease, whether the fusion protein is administered for preventive or
therapeutic purposes, previous therapy, the patient's clinical history and response to the IL-22
Fc fusion protein, and the discretion of the attending physician. The IL-22 Fc fusion protein is
suitably administered to the patient at one time or over a series of treatments. Depending on the
type and severity of the disease, about 1 µg/kg to 15 mg/kg (e.g. 0.1mg/kg-10mg/kg) or about
0.1 μg/kg to 1.5 mg/kg (e.g., 0.01 mg/kg – 1 mg/kg) of the IL-22 Fc fusion protein can be an
initial candidate dosage for administration to the patient, whether, for example, by one or more
separate administrations, or by continuous infusion. One typical daily dosage might range from
about 1 µg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the condition, the treatment would
generally be sustained until a desired suppression of disease symptoms occurs. One exemplary
dosage of the IL-22 Fc fusion protein would be in the range from about 0.05 mg/kg to about 10
mg/kg. Certain other dosages include the range from about 0.01 mg/kg to about 10 mg/kg,
about 0.02mg/kg to about 10 mg/kg, and about 0.05 mg/kg to about 10 mg/kg. Thus, one or
more doses of about 0.01 mg/kg, 0.02mg/kg, 0.03mg/kg, 0.04mg/kg, 0.05mg/kg, 0.06 mg/kg,
0.07mg/kg, 0.08mg/kg, 0.09mg/kg, 0.1mg/kg, 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, 0.5mg/kg ,
0.6mg/kg, 0.7mg/kg, 0.8mg/kg , 0.9mg/kg , 1.0 mg/kg, 2.0 mg/kg, 3.0 mg/kg, 4.0 mg/kg,
5mg/kg, 6mg/kg, 7mg/kg, 8mg/kg, 9mg/kg or 10 mg/kg (or any combination thereof) may be
administered to the patient. For topical wound healing, one or more doses of about
2 2 2 2
0.001mg/cm - about 10mg/cm wound area, about 0.05mg/cm - about 5mg/cm wound area,
2 2 2 2
about 0.01mg/cm - about 1mg/cm wound area, about 0.05 mg/cm - about 0.5 mg/cm wound
2 2 2 2
area, about 0.01 mg/cm - about 0.5 mg/cm wound area, about 0.05 mg/cm - about 0.2 mg/cm
wound area, or about 0.1mg/cm - about 0.5mg/cm wound area (or any combination thereof)
may be administered to the patient. In certain embodiments, one or more doses of about
2 2 2 2 2 2 2
0.01mg/cm , 0.02mg/cm , 0.03mg/cm , 0.04mg/cm , 0.05mg/cm , 0.06mg/cm , 0.07mg/cm ,
2 2 2 2 2 2 2
0.08mg/cm , 0.09mg/cm , 0.1mg/cm , 0.15mg/cm , 0.2mg/cm , 0.25mg/cm , 0.3mg/cm ,
0.4mg/cm , or 0.5mg/cm wound area may be administered to the patient. Such doses may be
administered intermittently, e.g. every week or every three weeks (e.g. such that the patient
receives from about two to about twenty, or e.g. about six doses of the IL-22 Fc fusion protein).
An initial higher loading dose, followed by one or more lower doses may be administered.
However, other dosage regimens may be useful. The progress of this therapy is easily
monitored by conventional techniques and assays. Similar dosage ranges can be applied to an
IL-22 polypeptide.
It is understood that any of the above formulations or therapeutic methods may be
carried out using conjugate of the description in place of or in addition to an IL-22 Fc fusion
protein.
J. Articles of Manufacture
In another embodiment of the description, an article of manufacture containing materials
useful for the treatment, prevention and/or diagnosis of the disorders described above is
described. The article of manufacture comprises a container and a label or package insert on or
associated with the container. Suitable containers include, for example, bottles, vials, syringes,
IV solution bags, etc. The containers may be formed from a variety of materials such as glass
or plastic. The container holds a composition which is by itself or combined with another
composition effective for treating, preventing and/or diagnosing the condition and may have a
sterile access port (for example the container may be an intravenous solution bag or a vial
having a stopper pierceable by a hypodermic injection needle). At least one active agent in the
composition is an IL-22 Fc fusion protein of the invention. The label or package insert
indicates that the composition is used for treating the condition of choice. Moreover, the article
of manufacture may comprise (a) a first container with a composition contained therein,
wherein the composition comprises an IL-22 Fc fusion protein of the invention; and (b) a
second container with a composition contained therein, wherein the composition comprises a
further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment
of the description may further comprise a package insert indicating that the compositions can be
used to treat a particular condition. Alternatively, or additionally, the article of manufacture
may further comprise a second (or third) container comprising a pharmaceutically-acceptable
buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's
solution and dextrose solution. It may further include other materials desirable from
a commercial and user standpoint, including other buffers, diluents, filters, needles, and
syringes.
It is understood that any of the above articles of manufacture may include a conjugate of
the description in place of or in addition to an IL-22 Fc fusion protein.
K. Screening Assays and Animal Models
As exemplified in the Example sections, IL-22, IL-22 Fc fusion protein and IL-22
agonists can be evaluated in a variety of cell-based assays and animal models of IBD,
cardiovascular diseases or conditions and metabolic syndrome.
Recombinant (transgenic) animal models can be engineered by introducing the coding
portion of the genes of interest into the genome of animals of interest, using standard techniques
for producing transgenic animals. Animals that can serve as a target for transgenic
manipulation include, without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and
non-human primates, e.g. baboons, chimpanzees and other monkeys. Techniques known in the
art to introduce a transgene into such animals include pronucleic microinjection (Hoppe and
Wanger, U.S. Pat. No. 4,873,191); retrovirus-mediated gene transfer into germ lines (e.g., Van
der Putten et al., Proc. Natl. Acad. Sci. USA 82, 6148-615 [1985]); gene targeting in embryonic
stem cells (Thompson et al., Cell 56, 313-321 [1989]); electroporation of embryos (Lo, Mol.
Cell. Biol. 3, 1803-1814 [1983]); sperm-mediated gene transfer (Lavitrano et al., Cell 57, 717-
73 [1989]). For review, see, for example, U.S. Pat. No. 4,736,866.
For the purpose of the present description, transgenic animals include those that carry
the transgene only in part of their cells ("mosaic animals"). The transgene can be integrated
either as a single transgene, or in concatamers, e.g., head-to-head or head-to-tail tandems.
Selective introduction of a transgene into a particular cell type is also possible by following, for
example, the technique of Lasko et al., Proc. Natl. Acad. Sci. USA 89, 623-636 (1992).
The expression of the transgene in transgenic animals can be monitored by standard
techniques. For example, Southern blot analysis or PCR amplification can be used to verify the
integration of the transgene. The level of mRNA expression can then be analyzed using
techniques such as in situ hybridization, Northern blot analysis, PCR, or immunocytochemistry.
The animals may be further examined for signs of appropriate pathology, such as
cardiovascular disease pathology, for example by histological examination and/or imaging or
ultrasound analysis to determine atherosclerotic plaque burden and vascular function (see
Examples below). Blocking experiments can also be performed in which the transgenic
animals are treated with IL-22, IL-22 Fc fusion protein or a candidate agonist to determine the
extent of effects on atherosclerotic plaque formation, including the size, number, and degree of
plaque formation. In these experiments, blocking antibodies which bind to the polypeptide of
the description are administered to the animal and the biological effect of interest is monitored.
Alternatively, "knock out" animals can be constructed which have a defective or altered
gene encoding IL-22, as a result of homologous recombination between the endogenous gene
encoding the IL-22 polypeptide and altered genomic DNA encoding the same polypeptide
introduced into an embryonic cell of the animal. For example, cDNA encoding IL-22 can be
used to clone genomic DNA encoding IL-22 in accordance with established techniques. A
portion of the genomic DNA encoding IL-22 can be deleted or replaced with another gene, such
as a gene encoding a selectable marker which can be used to monitor integration. Typically,
several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the
vector [see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologous
recombination vectors]. The vector is introduced into an embryonic stem cell line (e.g., by
electroporation) and cells in which the introduced DNA has homologously recombined with the
endogenous DNA are selected [see e.g., Li et al., Cell, 69:915 (1992)]. The selected cells are
then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras
[see e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J.
Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implanted
into a suitable pseudopregnant female foster animal and the embryo brought to term to create a
"knock out" animal. Progeny harboring the homologously recombined DNA in their germ cells
can be identified by standard techniques and used to breed animals in which all cells of the
animal contain the homologously recombined DNA. Knockout animals can be characterized
for instance, for their ability to defend against certain pathological conditions and for their
development of pathological conditions due to absence of the IL-22 polypeptide.
Thus, the biological activity of IL-22 or its potential agonists can be further studied in
murine IL-22 knock-out mice.
The foregoing written description is considered to be sufficient to enable one skilled in
the art to practice the invention. The following Examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present invention in any way. Indeed,
various modifications of the invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing description and fall within the
scope of the appended claims.
EXAMPLES
The following are examples of methods and compositions of the invention. It is
understood that various other embodiments may be practiced, given the general description
provided above, and the examples are not intended to limit the scope of the claims.
Example 1 Cloning, Expression and Purification of the IL-22 Fc Fusion Protein
General molecular cloning and protein purification techniques can be applied in the
following experiments.
i. Cloning
Full-length human IL-22 was cloned from a human colon cDNA library (Genentech).
Constructs expressing human IgG1 or IgG4 IL-22Fc fusion protein were generated for this
experiment using overlapping PCR technique using the following primers: IL-22 Fc fusion
IgG1 forward primer:
TTGAATTCCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACT
GGAGTACATTCAGCGCCCATCAGCTCCCACTGCAGGC (SEQ ID NO:52),
IL-22 Fc fusion IgG1 reverse primer AGGTCGACTCATTTACCCGGAGACAGGGAGAGG
(SEQ ID NO:53), IL-22 Fc fusion IgG4 forward primer:
TTGAATTCCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACT
GGAGTACATTCAGCGCCCATCAGCTCCCACTGCAGGC (SEQ ID NO:54),
IL-22 Fc fusion IgG4 reverse primer: AGGTCGACTTATTTACCCAGAGACAGGGAGAGG
(SEQ ID NO:55). The PCR products were cloned into expression vectors pRK5.sm
(Genentech). The leader sequence (or signal peptide) was cleaved in the cell and the mature IL-
22 Fc fusion did not contain the leader sequence. The clones carrying artificial linkers were
cloned with primers containing the linker sequences. The N297G mutation was further
introduced by mutagenesis PCR using the following primers: IgG1 N297G forward primer:
GCG GGA GGA GCA GTA CGG AAG CAC GTA CCG TGT GG (SEQ ID NO:56), IgG1
N297G reverse primer: CCA CAC GGT ACG TGC TTC CGT ACT GCT CCT CCC GC (SEQ
ID NO:57), IgG4 N297G forward primer: ACA AAG CCG CGG GAG GAG CAG TTC GGA
AGC ACG TAC CGT GTG GTC AGC GTC (SEQ ID NO:58), and IgG4 N297G reverse
primer: GAC GCT GAC CAC ACG GTA CGT GCT TCC GAA CTG CTC CTC CCG CGG
CTT TGT (SEQ ID NO:59). Sequences of all IL-22Fc constructs were confirmed by DNA
sequencing.
ii. Cell Culture
CHO cells were grown in suspension by splitting the culture 2 times per week to 0.3 x
cells/ml in an incubator set at 37°C and 5%CO
iii. Transfection of IL-22 Fc fusion protein into CHO cells and protein expression
CHO cells were seeded at 1.23 x10 cells/ml in 720 mL culture medium. The
transfection complex (1.6 mL PEI + 800 ug DNA in 80 mL serum free media) was incubated
for 10 min before added to the cells. The culture was incubated at 33°C, 5% CO for 24 hours.
After further culturing for 14 days, the supernatant of the culture was harvested via
centrifugation. Transient CHO conditioned media (supernatant from above) was purified using
the MabSelect Sure (GE Healthcare) protein A affinity column. The eluate at low pH was
neutralized to pH5.0 and further purified through a gel filtration column (GE Healthcare). The
eluted peak was pooled, formulated and sterile filtered. The glycosylation status of the Fc
region of the fusion protein was analyzed by Mass Spectrometry as discussed below.
iv. Establishment of stable clones expressing IL-22 Fc fusion protein
The plasmid encoding IL-22 Fc fusion protein was introduced into CHO cells by
transfection using Lipofectamine 2000 CD (Invitrogen). After transfection, the cells were
centrifuged and re-plated into serum-free selective medium. Isolates were selected for secretion
of IL-22 Fc. Clones with the highest titer, as identified by ELISA, were then pooled and scaled
for production.
v. Expression of IL-22 Fc fusion protein in E. coli
E. coli fermentation feedstock was homogenized and conditioned to 0.4% w/w PEI pH
6.7 and centrifuged. Centrate was purified using a MabSelect Sure (GE Healthcare) protein A
affinity column. The eluate at low pH was neutralized to pH 5.0 and further purified through an
ion exchange chromatography. Fractions were pooled, formulated and sterile filtered.
Example 2 IL-22 Fc Fusion Protein Exhibited High Percentage of Afucosylation in the
Fc Region
In this study, the glycosylation status of the Fc portion of the IL-22 Fc fusion proteins
was examined. Samples of purified IL-22 Fc fusion proteins from transiently transfected cells
were digested with trypsin (1:25 trypsin: IL-22 Fc, w/w) for 2 hrs at 37 C. Samples were
acidified with trifluoroacetic acid to a final concentration of 0.1% and injected onto a heated
C18 column (PLRP-S, 1000A 8um, Agilent) equilibrated with 0.05% TFA in water. The
digestion products were separated by a linear acetonitrile gradient (5 to 60%) over 20 min time.
The column was directly connected to the electrospray orifice of an Agilent 6520B TOF Mass
Spectrometer and the masses of the eluted fractions were determined in positive ion mode.
Since the Fc portions of these fusion constructs are stable in trypsin under these digestion
conditions, a direct comparison of the carbohydrate status of various IL-22 fusions could be
made.
As shown in Figure 2, both IL-22 IgG1 and IgG4 Fc fusion proteins showed abnormally
high levels of afucosylation. The expected masses for a glycosylated Fc of a typical
monoclonal IgG1 antibody would be those labeled as 53296, 53458 and 53620 Da of panel A in
Figure 2. Typically the core carbohydrate species on each arm of the Fc would each consist of
the following carbohydrate composition: 4 N-acetyl glucosamine, 3 mannose and 1 fucose
sugar species (as on the peak labeled 53296 in Panel A). The addition of one or two galactose
sugars would produce the peaks labeled 53458 and 53620 Da, respectively (Panel A). A
negligible amount of molecules containing sugar moieties that was missing fucose on one arm
of the Fc was detected (“-1 fucose”).
Surprisingly, human IL-22 IgG1 Fc fusion proteins of different constructs in which the
CH2 domain is glycosylated all exhibited high level of afucosylation, including sugar moieties
missing fucose on one arm (“-1 fucose”) and both arms of Fc (“-2 fucose”). See Figure 2,
Panels B-D. These afucosylated molecules comprised as high as about 30% of the total species
observed. Afucosylation can increase the undesirable effector activities of the IL-22 IgG1 Fc
fusion.
IgG4 is known to have less effector function as compared to IgG1. Unexpectedly,
results of Mass Spectrometry analysis also showed the “-1 fucose” and “-2 fucose” glycosylated
species in the trypsin-digested Fc regions of human IL-22 IgG4 Fc fusion protein. These
afucosylated molecules comprised more than 50% of the total species observed. Figure 2,
Panel E. Afucosylated antibodies have much enhanced ADCC or CDC cytotoxicity activities, a
property not desirable with these IL-22 Fc fusion proteins.
Subsequently, two additional IL-22 Fc molecules, one containing IgG1 Fc and the other
IgG4 Fc were constructed in which the residue in the Fc that would normally be glycosylated
(N297) was mutated to a glycine (N297G) thereby preventing attachment of the normal core
sugar. These were shown to be devoid of any sugar on their Fc portions and both had their
expected Fc molecular weights based on their amino acid sequences (Figure 2, Panels F and G).
In summary, the Fc region of the human IL-22 Fc fusion proteins, either IgG1 or IgG4
Fc fusion, showed high levels of afucosylation, which can result in increased ADCC or CDC
activities, a property not desirable for use as IL-22 therapeutics. Thus, the non-glycosylated
variants were tested in further studies.
Example 3 IL-22 IgG1 and IgG4 Fc Fusion Protein in vitro Activity Assay
IL-22 engages IL-22 receptor complex and activates Jak-Stat signaling pathway. STAT3
activation is a predominant event in IL-22 mediated signaling pathway. In this experiment, the
in vitro activities of IL-22 Fc fusion proteins were measured using a luciferase reporter assay.
HEK 293 cells were engineered to overexpress human IL-22 receptor complex IL22R1 and
IL10R2. On day 1, 1x10 293 cells were seeded in 24-well plates in 0.4 ml Dulbecco's
modified Eagle Medium (DMEM)+10% Fetal Bovine Serum (FBS). On day 2, cells were
transfected with a STAT3-driven luciferase reporter and a Renilla luciferase control using
Lipofectamine 2000 (Invitrogen) in 0.1 ml reduced serum media (Gibco Poti-MEM with
reduced serum reduced by at least 50%). The STAT3 luciferase reporter construct contains
STAT3-responsive luciferase reporter construct containing tandem repeats of the sis-inducible
element (SIE) and the firefly luciferase reporter gene. On day 3, IL-22 Fc fusion proteins
produced by either transient or stable CHO clones were titrated into different concentrations in
0.5 ml media, and added on top of transfected cells. On day 4, media were removed and cells
were lysed with 100ul passive lysis buffer (provided by the Dual-Luciferase Reporter 1000
Assay System). Twenty microliter of cell lysates were transferred into 96-well plate and
analyzed with Dual-Luciferase Reporter 1000 Assay System on luminometer (Promega). The
EC50 was calculated based on the dose-dependent activity in GraphPad Prism software (La
Jolla, CA). The EC50 values for different IL-22 Fc fusion constructs are shown in Table 2
below.
Table 2
IL-22 Fc
Fc isotype Linker Production EC50 (pM)
Constructs
1 huIgG1 DKTHT (SEQ ID NO:32) CHO 150-200
2 huIgG1 EPKSCDKTHT (SEQ ID NO:33) CHO 350-500
3 huIgG1 VEPKSCDKTHT (SEQ ID NO:34) CHO 100-150
4 huIgG1 KVEPKSCDKTHT (SEQ ID NO:35) CHO 50-75
huIgG1 KKVEPKSCDKTHT (SEQ ID NO:36) CHO 25-50
DKKVEPKSCDKTHT (SEQ ID
6 huIgG1 CHO 25-50
NO:37)
VDKKVEPKSCDKTHT (SEQ ID
7 huIgG1 CHO 25-50
NO:38)
KVDKKVEPKSCDKTHT (SEQ ID
8 huIgG1 CHO 2.5-5
NO:39)
9 huIgG1 GGGDKTHT (SEQ ID NO:41) CHO 50-75
huIgG1 GGGSTHT (SEQ ID NO:63) CHO 50-100
11 huIgG1 EPKSSDKTHT (SEQ ID NO:40) CHO 50-100
12 huIgG1 DKKVEPKSSDKTHT (SEQ ID NO:64) CHO 25
KVDKKVEPKSSDKTHT (SEQ ID
13 huIgG1 CHO 25
NO:65)
14 DKTHT (SEQ ID NO:32)
huIgG1 CHO 150-200
N297A
EPKSSDKTHT (SEQ ID NO:40)
huIgG1 CHO 50-100
N297A
16 huIgG1 DKTHT (SEQ ID NO:32) (N297G) CHO 150-200
EPKSSDKTHT (SEQ ID NO:40)
17 huIgG1 CHO 50-100
(N297G)
KKVEPKSSDKTHT (SEQ ID NO:66)
18 huIgG1 CHO 20
(N297G)
19 huIgG4 SKYGPP (SEQ ID NO:43) CHO 150-200
SKYGPP (SEQ ID NO:43)
huIgG4 CHO 75-100
N297G
21 huIgG4 RVESKYGPP (SEQ ID NO:44) CHO 25-50
RVESKYGPP (SEQ ID NO:44)
22 huIgG4 CHO 50-75
N297G
ELKTPLGDTTHT (SEQ ID NO:42)
23 huIgG1 CHO 50-75
(IgG3 linker)
24 huIgG1 EPKSSDKTHT (SEQ ID NO:40) E. coli 16
huIgG1-
monomeric EPKSSDKTHT (SEQ ID NO:40) E. coli 82
IL-22
A large number of IL-22 Fc fusion proteins were constructed with linkers of different
length and sequences to examine the activities, stability and yield of each design. Linkers with
native IgG sequences are preferred to minimize potential risk of immunogenicity; however,
linkers with exogenous sequences that showed good in vitro activity were considered and
encompassed by the current invention.
The IL-22 IgG1 Fc fusion protein containing the DKTHT linker (SEQ ID NO:32) was
tested in the STAT3 luciferase assay. See Table 2. To improve EC50 of the fusion protein, the
linker length was increased from 5 to 10 amino acids containing the native IgG1 sequence
EPKSCDKTHT (SEQ ID NO:33). The resulting IL-22 Fc fusion protein, however, exhibited
reduced in vitro activity. See Table 2. Surprisingly, an increase in the linker length even by one
amino acid VEPKSCDKTHT (SEQ ID NO:34) improved the activity of the IL-22 fusion
protein. Further increases in the linker length resulted in further improvement in activity. See
Table 2.
In separate experiments, the Cys in EPKSCDKTHT was changed to Ser to remove the
potential of disulfide bond formation. As shown in Table 2, IL-22 Fc fusion with the linker
EPKSSDKTHT (SEQ ID NO:40) showed improved activity as compared to the parent linker
sequence with the Cys residue. Longer linker sequence incorporating the upstream sequences
(into the CH1 domain of IgG1) further improved activity. Constructs with N297G mutation
showed similar EC50 values when compared with the wild type counterparts. IL-22 IgG1
(N297G) Fc fusion protein (SQE ID NO:12) and IL-22 IgG4 (N297G) Fc fusion protein (SEQ
ID NO:8) were chosen for further studies.
The in vitro activities of human IL-22 IgG1 (N297G) Fc fusion protein (SQE ID NO:12)
or IL-22 IgG4 (N297G) Fc fusion protein (SEQ ID NO:8) expressed from stable clones were
tested in the same assay. Data in Figure 4 show representative results. Both IL-22 IgG1 and
IgG4 Fc fusion proteins induced STAT3 activity at a dose-dependent manner. Both IL-22 Fc
fusion proteins showed similar potency. IL-22 Fc fusion proteins expressed from transiently
transfected cells showed similar results (data not shown). As a control, native IL-22 protein
produced in CHO cells was tested in the same assay, and exhibited two to three folds higher
potency than the IL-22 Fc fusion proteins.
In summary, both IgG1 and IgG4 IL-22 Fc fusion proteins exhibited in vitro activity
demonstrated by STAT3 luciferase assay. Further, IL-22 Fc fusion proteins with linkers of
different length and sequences were shown to activate IL-22R mediated luciferase activity.
Example 4 IL-22 Fc Fusion Proteins Reduced Symptoms of DSS-Induced Colitis in
Mice
Dextran Sodium Sulfate (DSS)-induced colitis is a commonly-accepted mouse colitis
model. Oral administration of DSS-containing water rapidly damages colon epithelial cells and
causes substantial body weight loss and colon epithelial structure disruption characterized by
either immunohistochemical (IHC) staining or histology clinical score by pathologist. In this
proof of concept study, the effect of IL-22 Fc fusion protein on DSS-induced colitis was tested.
In C57BL/6 mice, colitis was induced with drinking water containing 3.5% DSS for five
consecutive days starting from day 0. Mouse IL-22 IgG2a Fc (SEQ ID NO:60), a surrogate for
human IL-22 Fc fusion protein was dosed through intraperitoneal route at 5mg/Kg on day -1, 1,
4, and 6. Body weight of the animals was measured daily. On day 8, all animals were sacrificed
and colon histology was studied through both IHC staining and manual histological score.
As shown in Figure 5, DSS induced colitis is associated with dramatic body weight loss
(Figure 5A), colonic epithelial damage and colon inflammation (Figure 5B) and high histology
score (Figure 5C). IL-22Fc treatment significantly prevented weight loss, restored epithelial
integrity, diminished inflammation and reduced histology score. See Figure 5. The efficacy of
IL-22 Fc exceeded the effect of dexamethasone, the steroid standard of care (SOC) that caused
significant body weight loss in this study.
Example 5 IL-22 Fc Fusion Protein Pharmacokinetics Study
The pilot safety and PKPD study in cynomolgus monkeys was approved by the
Institutional Animal Care and Use Committee (IACUC). The study was conducted at Charles
River Laboratories (CRL) Preclinical Services (Reno, NV). A total of 15 male cynomolgus
monkeys (4-5 kg) from CRL stock were randomly assigned to five groups (n = 3/group).
Animals in group 1 were given an intravenous (i.v.) dose of the control vehicle on Days 1 and
8. Animals in groups 2 and 3 were given a single i.v. bolus dose of IL22-Fc IgG1 at 0.15 and
1.5 mg/kg, respectively, on Days 1 and 8. Animals in groups 4 and 5 were given a single i.v.
bolus dose of IL22-Fc IgG4 at 0.15 and 1.5 mg/kg, respectively, on Days 1 and 8. Serum
samples were collected at various time points for PK and PD analysis out to Day 43 and
concentrations of IL22-Fc were assessed by ELISA.
For analysis of human ILFc in cynomolgus monkey serum, mouse anti-human IL-
22 mAb (Genentech) was used as a capture antibody in an ELISA assay. The recombinant IL-
22 Fc fusion protein was used to develop a standard curve. Plate-bound ILFc was detected
during a 1 hour incubation with HRP-conjugated anti-human-Fcγ-pan murine mAb (Genentech)
diluted to 500 ng/mL in assay buffer. After a final wash, tetramethyl benzidine peroxidase
substrate (Moss, Inc., Pasadena, MD) was added, color was developed for 15 minutes, and the
reaction was stopped with 1 M phosphoric acid. The plates were read at 450 nm with a 620 nm
reference using a microplate reader. The concentrations of IL-22 Fc fusion were calculated
from a four-parameter fit of the IL-22 Fc fusion standard curve.
For PK data calculations, Study Day 1 was converted to PK Day 0 to indicate the start
of dose administration. All time points after the in life dosing day are calculated as Study Day
minus 1. The serum concentration data for each animal were analyzed using 2 compartment
analysis with WinNonlin®, Version 5.2.1 (Pharsight; Mountain View, CA).
The plasma concentrations of IL22-Fc showed a bi-exponential decline after i.v. dosing
(0.15 mg/kg and 1.5 mg/kg) with a short distribution phase and a long terminal elimination
phase. See Figure 6. The two-compartment model with linear elimination of IL-22 Fc from the
central compartment described the pharmacokinetic profiles for both the doses well, suggesting
negligible target mediated disposition at these dose ranges.
The maximum serum concentration (C ) and area-under-serum-concentration-time-
curve (AUC) estimated by the two-compartmental analysis were roughly linear and dose-
proportional. See Table 3. The dose-proportional kinetics suggested IL-22R saturation at the
doses tested. As shown in Figure 6, the IL-22 IgG4 Fc fusion unexpectedly showed a 2-fold
slower CL and greater than 2-fold higher exposure compared to the IgG1 Fc fusion. Without
limiting to particular mechanisms, the faster clearance (CL) of IgG1 fusion may be due to less
stability of the IgG1 fusion construct because the greater than 2-fold faster CL of the IL-22
IgG1 Fc fusion appeared to be mainly driven by a larger volume of distribution. The Beta half-
lives of 4 - 5 days were similar between IgG1 and IgG4 fusions.
Table 3
AUC C CL Beta_HL*
Group
(day • μg/mL) (ug/mL) (mL/day/kg) (day)
0.15 mg/kg IgG1 4.47 ± 0.603 2.70 ± 0.607 34.0 ± 4.26 4.02 ± 0.478
1.5 mg/kg IgG1 51.1 ± 9.70 30.5 ± 4.14 30.1 ± 6.18 5.33 ± 0.580
0.15 mg/kg IgG4 11.3 ± 0.752 3.99 ± 0.432 13.3 ± 0.853 4.61 ± 0.394
1.5 mg/kg IgG4 102 ± 18.9 33.4 ± 4.02 15.0 ± 2.58 5.80 ± 0.770
*Beta half-life
Example 6 Assessment of in vivo Activity of IL-22Fc in Cynomolgus Monkey
Cynomolgus monkeys (Macaca fascicularis) were dosed intravenously with IL-22 Fc
fusion of isotype IgG1 or IgG4 as indicated, at doses of 0.15mg/kg or 1.5mg/kg. IL-22 binding
to IL-22 receptor triggers the expression of several genes including Serum Amyloid A (SAA),
RegIII/Pancreatitis Associated Protein (PAP, also called PancrePAP), and Lipopolysaccharide
Binding Protein (LPS-BP). In this study, IL-22 Fc fusion protein in vivo activities were
analyzed by measuring the expression of SAA, PancrePAP, and LPS-BP. Serum samples were
obtained over a time course pre- and post-dose, as indicated in the graph. Circulating levels of
monkey SAA were quantified in serum using a commercial enzyme-linked immunosorbent
assay (ELISA) kit (catalog # 3400-2) available from Life Diagnostics (West Chester, PA).
Circulating levels of RegIII/PAP were quantified in serum using a commercial ELISA kit
(catalog PancrePAP) produced by Dynabio (Marseille, France).
Levels of Lipoprotein Binding Protein (LBP) in serum samples were determined by
using a qualified ELISA. Biotinylated-Lipoprotein (Enzo Life Sciences, Farmingdale, NY) was
coated on a Streptavidin coated microtiter plate (Thermo; Rockland, IL). Recombinant human
LBP (R&D Systems, Inc., Minneapolis, MN) was used as a standard in the assays. Bound LBP
analyte was detected with an anti-LBP mouse monoclonal antibody (Thermo, Rockland, IL).
Horseradish peroxidase (HRP)-conjugated F(ab’) fragment goat anti−mouse IgG, Fc (Jackson
ImmunoResearch, West Grove, PA) was used for detection. The colorimetric signals were
visualized after addition of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate
(Kirkegaard & Perry Laboratories, Gaithersburg, MD). The reaction was stopped by addition
of 1 M phosphoric acid and absorbance was measured at 450 nm using 650 nm as reference on
a plate reader (Molecular Devices, Sunnyvale, CA). All ELISA samples were run according to
manufacturer's specifications and were prepared either at a single dilution in duplicate or at four
serial dilutions in singlicate and concentrations were interpolated from a standard curve. The
mean value of each sample was reported.
As shown in Figure 7, SAA, LPS-BP, and RegIII/PAP serum protein levels were
induced by IL-22Fc in vivo. Dose-dependent responses were observed in vivo in non-human
primates, indicating IL-22R engagement and suggesting saturation by IL-22Fc. In the majority
of cases, no increase in the serum protein levels was observed 24 hours after the second dose,
suggesting that serum SAA, LPS-BP, and RegIII/PAP proteins had reached the maximal levels.
Serum levels of all three proteins declined slowly over the 35-day recovery period, returning to
baseline in most animals. The exception being the RegIII/PAP levels in the IgG4 high dose
group, which appeared to stay elevated throughout the 42-day course. This may reflect
improved PK and increased exposure by AUC for the IL-22 IgG4 Fc fusion protein as
compared to IL-22 IgG1 Fc fusion protein.
Example 7 - IL-22 Treatment of Atherogenic Prone Mice (Ldlr-/-Apobec1-/-)
Recent studies have revealed IL-22’s role in host defense against pathogenic microbes.
Its beneficial effects on mucosal tissue homeostasis and immunity led us to speculate that IL-22
treatment could alleviate endotoxemia and its pathological consequences including atherogenic
dyslipidemia, systemic inflammation and ultimately slowing the progression of atherosclerotic
disease and related disorders including diabetes.
To test this hypothesis atherogenic prone mice (Ldlr-/-Apobec1-/-) were treated with an
ILFc construct. These mice lack the LDL receptor and synthesis exclusively apoB100.
This model is unique in that it recapitulates much of the pathophysiology associated with
human familial hypercholesterolemia. Specifically, on a chow diet, these mice develop
elevated LDL cholesterol, a lipid profile with a distribution of cholesterol similar to humans,
and progressive plaque formation. Further, Ldlr-/-Apobec-/- mice have measurable risk factors
that contribute to its cardiovascular disease, including insulin resistance, systemic
inflammation, progressive plague burden, and endothelial cell dysfunction. Here we
demonstrate that the 3 months of treatment with the ILFc fusion protein can dramatically
improve the cardiovascular health of these animals and reduce atherosclerotic progression.
Material and Methods
Mouse ILFc Constructs. The ILFc construct and polypeptide used herein was
typically a mouse ILmouse-Fc fusion protein (SEQ ID NO:73) as shown in Figure 32A
(and DNA sequence encoding it as shown in Figure 32B, SEQ ID NO:72). Protein was
produced in CHO cells by transient transfections of plasmid DNA. The fusion protein was
purified by running the cell supernatant over a protein A column followed by ion-exchange
chromatography to eliminate aggregates. Serum half-life was estimated by injecting a single
dose of 10 mg/kg ILFc in a C57B6 mouse followed by obtaining serum from the mice at
specified time intervals. The serum levels of ILFc was determined by a sandwich ELISA
using anti IL-22 mAbs. For the in vivo studies using the Lrlr-/-Apobec1-/- double KO mice a
mouse ILFc construct was utilized. While mouse sequences are presented and have been
used in the examples, it is expected that in various embodiments human sequences can replace
the mouse sequences.
Mouse Studies. Ldlr-/-Apobec1-/- double KO mice were bred in the Genentech
breeding facility and the WT C57BL/6 mice were purchased from Jackson Laboratory. Mice
were maintained in a pathogen-free animal facility at 21°C under standard 12 hr light/12 hr dark
cycle with access to chow: a standard rodent chow (Labdiet 5010, 12.7% calories from fat) or a
high fat, high carbohydrate diet (Harlan Teklad TD.03584, 58.4% calories from fat) and water
ad libitum. db/db mice in C57BLKS/J background were females and other mice used in the
study were all males. The mouse ILFc or Control IgG antibody were administered through
intraperitoneal (ip) route starting at the age 6 months at 50 µg/week for three months (total of
12 weekly doses).
Analysis of Atherosclerotic Burden. High resolution x-ray micro computed tomography
was used to quantify atherosclerotic lesion volume and atherosclerotic plaque composition.
Animals were euthanized with inhalation of carbon dioxide, then perfused via the cardiac left
ventricle with ten milliliters of phosphate buffered saline then ten milliliters of ten percent
neutral buffered formalin. The aortas were dissected and immersed in ten percent neutral
buffered formalin for a minimum of twenty four hours and transferred to a solution of twenty
percent iodine based x-ray contrast agent, Isovue 370 (Bracco Diagnostics Inc., Princeton, NJ)
in ten percent neutral buffered formalin for a minimum of twelve hours. After blotting dry, the
aortas were perfused and immersed in soy bean oil (Sigma-Aldrich, St. Louis, MO), a low x-ray
intensity background imaging media. Micro computed tomography images were obtained using
the µCT40 (Sanco Medical, Basserdorf, Switzerland) with image acquisition energy of 45kV, a
current of 160µA, an integration time of 300 milliseconds with three averages and image
resolution of twelve micrometers. The resulting images were analyzed with Analyze
(AnalyzeDirect Inc., Lenexa, KS) by employing semi-automated morphological filtering and
user defined regions to determine object volumes and object composition.
Assessment of Vascular Function. Vascular function was determined by ultrasound
examination of the femoral artery to flow mediated dilatation and nitroglycerin mediated
dilatation. Animals were anesthetized with two percent isoflurane, and kept at thirty seven
degrees Celsius for twenty minute ultrasound exam. Nair was used to remove the hair from the
ventral surface of the hind limbs and allow for ultrasound imaging using the Vevo770 with a
fifty five megahertz imaging probe (VisualSonics, Toronto, Canada). For flow mediated
dilatation, a baseline image of the femoral artery was collected then a rubber band was used as a
temporary tunicate to occlude femoral artery blood flow for four minutes. The rubber band was
then released for reflow of the femoral artery and an image was acquired every minute for four
minutes and analyzed for femoral artery maximum diameter using manufactures supplied
software tools. For nitroglycerin mediated dilatation, a baseline image of the femoral artery
was collected then an intraperitoneal injection of 20 micrograms of nitroglycerin (Baxter,
Deerfield, IL) was administered and an image was acquired every minute for four minutes and
analyzed for femoral artery maximum diameter using manufactures supplied software tools.
Total Cholesterol, Triglyceride and Lipoprotein Determination. Fresh sera samples
were used to determine the total cholesterol, triglyceride, and lipoprotein distribution per
manufactures instructions using the Cholestech LDX analysis system (Inverness Medical,
Princeton, NJ).
Sera Lipopolysaccharide Measurement. Frozen sera samples were thawed and diluted
one hundred fold in endotoxin free water and incubated at ninety degrees Celsius for ten
minutes in a hot water bath. Samples were then run per manufactures instructions on the
Endosafe-PTS system (Charles River Laboratories, Wilmington MA).
GTT: Glucose Tolerance Test. The Glucose Tolerance Test (GTT) was conducted at
the end of the dosing period with 1g/kg i.p. glucose injection after overnight fast (14 hrs).
Glucose levels were measured using One Touch Ultra glucometer. Food consumption was
calculated during the study by individually housing the mice over 4 days of acclimatizing
period followed by the measurement of one week period.
Measurement of Serum Cytokine Levels. Serum cytokine levels were measured using
Luminex 23 Multiplex panel (BioRad) through automated method. Some of the results were
independently confirmed by Individual ELISA kits (R&D). Total cholesterol and free fatty
acids (FFA) (Roche) were determined by using enzymatic reactions.
Results:
Ldlr-/-Apobec1-/- mice accurately modeled atherogenic dyslipedia and were sensitive
to inflammatory challenges. The Ldlr-/-Apobec1-/- mouse model displays lipoprotein levels
and extensive atherosclerotic lesions characteristic of atherosclerotic disease in humans
(Powell-Braxton et al. (1998). Nat Med 4(8): 934-8). MicroCT analysis of the aortic arch of
Ldlr-/-Apobec1-/- mice revealed signs of atherosclerotic disease as determined using an
automated image processing techniques on prepared samples that included the ascending aorta,
arch of the aorta, descending aorta and part of the brachiocephalic artery. This technique also
demonstrated a high degree of heterogeneity reflecting the regional variation in severity and
progression of atherosclerosis burden that included lipid core, regions of ruptured plaque and
calcification (Figure 8). The heterogeneity of the CT signal reflects the underlying pathology
of the lesions consistent with the complex plaque pathology of the human disease. To
characterize this model and demonstrate its sensitivity to diet induced atherogenesis, the cohort
of mice were treated with either a high fat diet or adding fructose to their drinking water (8%
w/v) for 2 months. The Ldlr-/-Apobec1-/- mice demonstrated sensitivity to these dietary
alterations with only modestly increased serum LDL but with a significant increase in total
atherosclerosis burden as compared to mice on standard chow diet (Figure 9). This
demonstrates that the increase in atherosclerosis burden is likely due to inflammation rather
than LDL increase. Further, an acute low grade inflammation stimulation with LPS challenge
(0.025mg/Kg) resulted in a marked elevation of proinflammatory markers in the Ldlr-/-
Apobec1-/- compared with wt controls (Figure 10). The Ldlr-/-Apobec1-/- mice were also
exposed to chronic LPS dosing (750ng, ip) for 8 weeks and assessed for serum lipid profile and
plaque burden. As shown in Figure 11, chronic endotoxin exposure results in dyslipidemia and
greater plaque instability.
Upon treatment with ILFc, improvements in atherogenic dyslipidemia and
symptoms of metabolic syndrome were seen in the Ldlr-/-Apobec1-/- mice. These mice
develop characteristics of metabolic syndrome, including insulin resistance, on a chow diet.
With ILFc treatment, fasting blood glucose was reduced compared to controls and glucose
clearance was improved in the treatment group compared to control group (Figure 12). Thus,
glucose homeostasis was improved with a normalization of glucose tolerance (GTT) and
improvement in fasting glucose (Figure 12). Both fasted and fed hypercholesterolemia were
reduced (Figure 13A) as were fed TG levels (Figure 13B) and the lipid profiles were improved
(Figure 14). Plasma LPS levels were reduced after ILFc treatment (Figure 15). In addition
to the reduction in dyslipidemia and insulin sensitization, improvement in endothelial function
measured by vascular reactivity was seen (Figure 16). Consistent with an improvement in
dyslipidemia, CT analysis of plaque volume showed a reduction in total atherosclerotic burden
in the aortic arch and in the brachiocephalic artery and aorta valves (Figures 17A-C). The
improvement in lipid profile and insulin resistance was not due to a reduction in caloric intake
since the food intake, measured over a 7 day period, increased despite a modest but statistically
significant reduction in body weight that occurred during the 3 months treatment (Figure 18).
Body weight in the control group did not change during the 3 month treatment protocol and the
ILFc treatment group showed a significant reduction of body weight between the start and
end of study (Figure 18A). The average daily food intake measured over a 7 day period during
the course of the treatment study was elevated in the ILFc treatment group compared to
control group (Figure 18B).
Example 8 - Peripheral Artery Disease Model
Stimulation of IL-22 regulated pathways by ILFc to reduce atherosclerotic
progression is a potentially novel form of therapy for subjects with cardiovascular disease and
related disorder including diabetes and chronic kidney disease. Because cardiovascular disease,
typically, is not limited to one region of a subject's vasculature, a subject who is diagnosed as
having or being at risk of having coronary artery disease is also considered at risk of developing
or having other forms of CVD such as cerebrovascular disease, aortic-iliac disease, and
peripheral artery disease. The same strategy described above can be used to validate IL-22 as a
target using a mouse peripheral artery disease model. The ILFc constructs are prepared and
evaluated as described above. All necessary controls are also used. IL-22 agonists/antagonists
are evaluated and the results will validate IL-22 pathways as a target for drug discovery and
development.
A peripheral artery disease (PAD) model based upon femoral artery ligation to create
ischemic damage is used. The efficacy of the ILFc constructs are evaluated similar to the
procedures described previously (Couffinhal et al., Am. J. Pathol. 152:1667 (1998); Takeshita
et al., Lab. Invst. 75:487 (1996); Isner et al., Human Gene Therapy 7:959(1996)). To test the
ability of an ILFc to modulate such a peripheral arterial disease, the following experimental
protocol is used: a) Using a rodent (as in the above described method), one side of the femoral
artery is ligated to create ischemic damage to a muscle of the hindlimb (the other non-damaged
hindlimb functions as the control); b) an ILFc polypeptide (or fragment thereof) is
delivered to the animal either intravenously and/or intramuscularly (at the damaged limb) at
least 3x per week for 2-3 weeks at a range of dosages; and c) the ischemic muscle tissue is
collected after at 1, 2, and 3 weeks post-ligation for an analysis of biomarkers and histology.
Generally, (as above) parameters for evaluation include determining viability and
vascularization of tissue surrounding the ischemia, while more specific evaluation parameters
may include, e.g., measuring skin blood flow, skin temperature, and factor VIII
immunohistochemistry, and/or endothelial alkaline phosphatase reaction. Polypeptide
expression during the ischemia, is studied using any art known in situ hybridization technique.
Biopsy is also performed on the other side of normal muscle of the contralateral hindlimb for
analysis as a control.
Alternatively, other mouse models are used (Pownall et al. US 2011/0118173 A1).
There are several mouse models of atherosclerosis that will be used to test atheroprotection.
These include the apo A-I KO, apo E KO, cystathionine beta-synthase and apolipoprotein E,
and the apo A-I/SR-BI double KO. These mouse models of atherosclerosis will be treated with
ILFc by injection, oral dosage, or ex vivo treatment. Measurement of blood cholesterol
levels after treatment with ILFc will show an immediate decrease in total plasma
cholesterol and an increased amount of neo HDL and the subsequent appearance of mature
forms of HDL, which contains cholesterol extracted from peripheral tissue over an appropriate
period of hours.
Example 9 - Effect of recombinant IL-22 Fc in diabetic mouse models
In our initial studies to look at the effect of ILFc in metabolic syndromes, we noted
that IL-22R KO mice were more susceptible to diet induced obesity and insulin resistance. In
subsequent experiments we observed a loss of body fat following treatment with recombinant
ILFc. In view of these data we chose to test the role of recombinant ILFc in diabetic
mouse models. Efficacy end points such as fed and fasted glucose, body weight and glucose
and insulin tolerance were evaluated in this study.
Mice (10 animals/group) were treated with either Recombinant ILFc or anti
Ragweed antibody as an isotype IgG control, giving 2 doses/week for 3 weeks (Figure 19):
Group 1: db/db mice (BKS.Cg-Dock7(m)+/+ Lepr(db)/J FAT): anti-Ragweed antibody
(50 µg)
Group 2: db/db mice: Recombinant ILFc (50 µg)
Group 3: Diet Induced Obesity (DIO) mice: anti-Ragweed antibody (50µg)
Group 4: Diet Induced Obesity (DIO) mice: Recombinant ILFc (50µg)
12 week old female db/db were purchased from Jackson Laboratory and used in the experiment.
Prior to the study mice were acclimated (daily handling) for 7-10 days after arrival and housed
single before the start of the experiment. Over days -5 to day -1 blood was collected (3-5µl) via
tail nick for base-line glucose measurement daily. On day 0 proteins were administered by i.p.
injection (150 µl) in PBS, followed by twice weekly doses for 3 weeks. Blood (3-5ul) was
again collected via tail nick for glucose measurement on day 2, 4, 8, 10, 14, 18 and 21. For
measuring pK, 30ul of blood was collected via orbital bleed under anesthesia on Days 2, 7, 13
and 20.
Recombinant ILFc or isotype IgG control antibody was dosed twice a week through
Intraperitoneal route for three weeks. The body weight and fed glucose were measured every 2
days until the end of study at day 23 and glucose measurements were done through tail nick and
measured using glucometer (Figures 20A-B). In order to access the fed and fasting glucose
level, on day 10 the fed glucose measurement was done in the morning and mice from both
groups were fasted for 4 hours (hrs) and glucose measurements were taken using Glucometer
(Figure 20C). ILFc exposure resulted in a significant glucose lowering effect in db/db mice.
Glucose Tolerance Test (GTT) was performed after 2 weeks of treatment with ILFc
or IgG control at 50µg/dose twice a week. The mice were fasted overnight (14 hrs). Fasting
glucose level were measured in the morning and served as a baseline. Body weight was
measured and blood was collected (3-5µl) via tail nick for glucose measurement. Glucose
solution at 1.5mg/Kg body weight was administered intraperitoneally and glucose measurement
was taken every 30 mins. The glucose values were represented in the graph for 30,120,180 and
220 mins. One more GTT was performed on day 21 following overnight fasting on day 20.
Mice were weighed daily. All the groups were euthanized on day 23 and tissues were collected
for histology. ILFc treatment demonstrated significant improvement in glucose tolerance
and insulin sensitivity (Figure 21).
Insulin Tolerance Test (ITT) was performed after on Day 20 of the mice treated with IL-
22-Fc or IgG control at 50µg/dose twice a week. The mice were fasted for 4 hrs and baseline
glucose level was taken. 1mU/Kg body weight was administered intraperitoneally and blood
glucose levels were monitored by tail nicks every 30 mins. In order to calculate % glucose
reduction, baseline glucose level following 4 hrs fasting is normalized to 100%. ILFc
treatment was shown to significantly improve insulin sensitivity measured through Insulin
Tolerance test (Figures 22A-B).
IL-22R is highly expressed in pancreas especially in acinar cells, although its expression
status in β islet cells is still unclear. The insulin signal in pancreas from IL-22 Fc or control
protein treated db/db mice was examined. Histological assessment of the diabetic mice was also
carried out to evaluate insulin expression in the islet cells and the level of hepatic periportal
steatosis in ILFc treated animals. Immunohistochemistry for insulin and glucagon was
performed on formalin fixed paraffin embedded pancreas tissues as previously reported (Wu et
al. 2011, Science translational medicine 3, 113ra126, doi:10.1126/scitranslmed.3002669) using
rabbit anti-glucagon (Cell Signaling Technologies #2760) with Alexa Fluor 555-conjugated
goat anti-rabbit secondary antibody, or guinea pig anti-insulin (DAKO A0564) with Alexa
Fluor 647-conjugated goat anti-guinea pig secondary antibody. The percent insulin area per
islet area was calculated by dividing the insulin positive area by the islet area minus the nuclear
area.
ILFc appears to increase insulin expression in islets in db/db mice (Figure 23A) and
quantitative analysis revealed a significant increase of both insulin-signal intensity (Figure 23 B
and Figure 24) and insulin positive area in ILFc treated animals (Figures 25), while IL-22
Fc did not increase glucagon-signal intensity (Figure 23 C). The insulin positive area showed a
2.16 fold increase with ILFc treatment compared to treatment with Herceptin control (95%
confidence interval 1.25 to 3.72). The number and area of islet were not affected by IL-22 Fc
treatment. But the β cell area per islet and the intensity of insulin staining from IL-22 Fc treated
pancreas was significantly elevated (Figures 23 and 52).
The pancreas beta cells of obese mice showed signs of degranulation and degeneration
(data not shown). Statistically significant higher insulin staining was observed in beta cells of
obese mice treated with IL22, as compared to untreated obese mice (Figure 23A, B). The
increase was probably due to increased insulin storage in the IL22 treatment group. Despite the
higher level of pancreas insulin seen in IL22 treated obese mice, serum insulin levels in these
mice were actually reduced as compared to obese mice without IL22 treatment, either in fed or
fasted condition (Figure 23D, E). But the IL22 treated obese mice responded to glucose by
releasing insulin in a pattern more resembling wild type mice on chow diet, as compared to
untreated obese mice (Figure 23F). Thus, IL22 improved glucose homeostasis in obese mice
potentially by increasing granulation and improving the control mechanism of insulin release in
the obese mice.
Next, the effect of IL-22 Fc on insulin homeostasis was examined. HFD-fed mice were
treated with IL-22 Fc twice per week for 8 weeks. The results show that (Figure 23D and E).
The data presented in Figure 23F show insulin levels in mice 0 or 30 min after glucose
injection. HFD-fed mice treated with IL-22 Fc, but not control HFD mice, responded to glucose
injection by increasing serum insulin levels, similar to wild type mice on Chow diet (normal
diet). See Figure 23 F. Thus, IL-22 improved glucose homeostasis in obese mice and improved
insulin secretion in response to glucose.
As a comparison, we looked at IL-22 receptor KO mice and their susceptibility to diet
induced obesity (DIO) and insulin resistance. The IL-22 R KO mouse is described in Figure 43
and below. IL-22 receptor KO mice and littermate control mice were put on 60% High Fat Diet
from week 7 of age for 10 weeks. To assess the high fat diet (HFD) induced glucose tolerance,
mice were fasted overnight and glucose tolerance test was performed next day morning. For
this experiment, seven week old IL-22 R KO mice and littermate age matched control animals
(WT: served as wildtype) were put on 60% HFD for 10 weeks. Mice were intraperitoneally
injected with 1.5mg/kg body weight of glucose and blood glucose levels were monitored every
mins for a period of 2 hrs. Total area under curve for individual mice were calculated and
graphically represented. The data demonstrate that glucose levels are significantly higher in the
IL-22R KO mice based on the total area under the curve (Figure 27A-B), suggesting that the
IL-22 receptor plays a role in HFD induced glucose tolerance. The IL-22 receptor KO mice did
in fact put on more body weight following HFD feeding compared to Littermate WT control
mice (Figure 28).
Example 10 - IL-22 Treatment of Atherogenic Prone Mice (Ldlr-/-Apobec1-/-), Resulting
in Reduction in Serum LPS and Serum LDL/HDL
Nine month old Ldlr -/-, Apobec1 -/- (dko) mice were injected intraperitoneally with
50ug of fusion protein IL-22Fc or 50 µg anti-ragweed control antibody (n=6 per group). Forty
eight hours later, the animals were euthanized and serum was harvested. Lipid profiles were
analyzed using Cholestech LDX assay, and Endotoxin was analyzed using the Limulus
amebocyte lysate assay. Serum LPS was reduced by 50% (p=0.0052) and serum LDL/HDL was
reduced by 30% (p=0.049) with ILFc as compared to anti-ragweed Fc control antibody
(Figure 29).
In summary, mice treated with IL-22 Fc fusion protein had rapid positive changes in
lipid profile and reduction in circulating endotoxin.
Example 11 IL-22Fc Accelerated Wound Closure in Murine Diabetic Wound Healing
Model, by either Systemic or Topical Administration
Protocol
The ILFc constructs were typically a mouse ILmouse-IgG2a fusion protein (SEQ ID
NOs:72 and 73) as shown in Figures 32A-B.
Mice used in the study: IL-22R KO mice and littermate control wild-type (WT) mice were bred
in the Genentech animal facility. The IL-22R KO mice is described in Figure 43 and below.
The 9 weeks old Diabetic female mice BKS.Cg-Dock7(m)+/+ Lepr(db)/J FAT (db/db) and
BKS.Cg-Dock7(m)+/- Lepr(db)/J lean (control BKS) were used. Mice were randomized in the
study based on body weight and fed glucose level.
The wound healing protocol was strictly followed according to IACUC Rodent Survival
Surgery Guidelines. Sterile technique was used through-out the procedure (including sterile
gloves, mask, gown, and drape). Following induction of a surgical plane of anesthesia, the
dorsal portion of the animals back (from the scapular area to the lumbar area) was shaved,
stubble removed with hair remover lotion (Nair or equivalent), following rinse off with sterile
water and prepped with betadine scrub followed by alcohol rinse. The animal was placed in
ventral recumbency then using a 6mm punch to mark the area of skin to be removed (with
sterile marker on the tip of the punch, then touch to skin). One 6 mm diameter full thickness
skin wounds was made 1cm left and right of midline. The underlying perichondrium was
removed with periosteal elevator and a fine scissors.
Following this a 0.5mm thick silicone frame, 10-12mm inside diameter, was placed around the
circular wound with superglue). Then a 2cm square of Tegaderm™ (3M, St. Paul, MN) or
Opsite® (Smith & Nephew, Inc., St. Petersburg, FL) adhesive was placed over the wound and
frame and the animal is allowed to recover from anesthesia.
Opsite® dressings were removed every other day, wounds were inspected, treatments applied
topically (20uL of test material or saline), and fresh dressing applied. Wound gap was
calculated by measuring wound diameter from day 0 through end of the study.
In some studies fed glucose level was recorded following tail nick and using commercial
Onetouch® glucometer (lifeScan, Inc., Milpitas, CA).
Results
IL-22R-/- mice exhibited defects in dermal wound healing response
The role of IL-22 signaling in dermal wound healing response was studied in IL-22R KO
(lacking signaling of IL-22 and its family members IL-20 and IL-24). Figure 33 shows the
wound gap curve of both IL-22RKO mice (n=10) and IL-22RWT control mice (n=10) over 14
days. A 6mm diameter wound was generated on day 0 and the gap was measured every 2 days
staring from Day4. Wound gap of IL-22R KO mice showed significant delay in the closure
compared to WT littermate control at day 8 through day 14. At the end of the study (day 14)
100% of the WT mice wounds were closed, compared to only 30% of mice in the IL-22RKO
mice (p=0.005). The differences in the wound gap between IL-22RKO and WT control mice
are deemed statistically significant at P ≤0.05.
Wound healing defect in obese Diabetic mice
The dermal wound healing response in diabetic condition was modeled in the preclinical study
using leptin receptor KO diabetic mice (BKS.Cg-Dock7(m)+/+ Lepr(db)/J FAT) (db/db) and
WT control lean mice. Circular wounds (6mm) were generated at the back of a mouse and the
wound gap closure was recorded every 2 days starting from day 4. Figure 34 shows the wound
gap closure (in mm) measured from day 0 through Day 27. Throughout the study period,
diabetic, obese db/db mice wounds displayed significant delay statistically (P ≤ 0.0001) in the
wound closure compared to Lean mice. By day 14 100% of WT mice wounds were closed
while none of the db/db mice wounds are closed even at day 27 (Figure 35A). IL-22 expression
was induced as measured by RNA levels in wild type mice days after wound excision, but not
in db/db mice. See Figure 35B,
IL-22Fc accelerated wound closure in the Diabetic wound healing model
As IL-22R-/- mice display defects in the wound closure, it was hypothesized that IL-22 may
influence in the wound closure. Figure 36 showed the schematic diagram of the study design.
9-week-old female obese db/db mice were used to model diabetic wound healing. In addition
to IL-22Fc (murine), anti-ragweed antibody as Fc control protein and anti-FGFR1 antibody
were used as positive control. Since anti-FGFR1 antibody has been demonstrated to normalize
blood glucose level in this preclinical model, it was used as a control antibody. Figure 36
shows schematic diagram of the study design. Treatment groups were:
• Anti-Ragweed antibody (intra peritoneal (i.p.) 50µg/dose, 8 dose)
• IL-22Fc (intra peritoneal (i.p.) 50µg/dose, 8 dose)
• Anti-FGFR1 antibody (intra peritoneal (ip) 0.5mg/kg on day 0 and day 14).
Both IL-22Fc and anti FGFR1 showed statistically significant (P ≤ 0.001) effect in lowering
glucose level in the diabetic mice compared with anti-ragweed treatment (Figure 37). The data
(Figure 38) shows that systemic administration of IL-22 Fc had striking effect in wound closure
rate compared to control anti Ragweed antibody treatment. The differences in the wound gap
was significant from starting from day 16 (P ≤ 0.05) and the wounds in IL-22Fc treated mice
was completely covered by day 27. The Fc control antibody as well as anti FGFR1 treated
mice failed to close wounds completely even at day 27. Figure 39 shows the wound gap
measurements of individual mice at day 19, 21 and 27 where the differences in the wound gap
between IL-22 Fc treated groups compared to other 2 groups are very significant statistically (P
≤ 0.001).
Comparison of IL-22 Fc topical vs. Systemic treatment
Figure 40 shows the schematic diagram of study design. In this study we compared 2 modes of
treatment -- topical vs. systemic treatment. The groups were:
• Anti-Ragweed antibody (topical 50µg/dose, 8 doses)
• IL-22Fc (topical 50ug/dose, 8 doses)
• IL-22Fc (intra peritoneal (i.p.) 50µg/dose, 8 doses).
The Graph in Figure 41 shows both ILFc topical as well as ILFc systemic
administration accelerated the wound closure compared to control antibody treatment. The
wound gap measurements were statistically significantly (P ≤ 0.001) different from day 16
through day 22. No significant difference was observed with wound closure rate between Il-22
Fc topical and systemic treatment groups. See also Figure 42.
Example 12 Obese Mice Exhibited Reduced IL-22 Induction
In the following experiments, the regulation of IL-22 during immune responses was
examined in obese mice. The major leukocyte sources of IL-22 are innate lymphoid cells
(ILCs) and T helper subsets, especially Th17 and Th22 cells. The IL-22 production from CD4+
T cells upon antigen challenge in leptin receptor deficient db/db mice was examined.
Protocol
In vivo treatment with OVA and flagellin. To activate CD4 T cell in vivo, 100 μg
OVA emulsified in complete Freund’s adjuvant (CFA) was injected subcutaneously at lower
back of the animals, and the inguinal lymph nodes were harvested on day 7. To activate TLR5,
3 μg ultra-pure flagellin (InvivoGen) was injected intravenously, and serum samples were
harvested at 2 h.
m db
Mice. Leptin receptor deficient mice (db/db; BKS.Cg-Dock7 +/+ Lepr /J or
db ob
B6.BKS(D)-Lepr /J), Leptin deficient mice (ob/ob; B6.Cg-Lep /J), and their respective lean
control mice, as well as high-fat diet mice (C57BL/6J 60%DIO) and the chow-diet control mice
were purchased from Jackson Laboratory. IL-22 deficient mice (Zheng et al, 2007, Nature 445,
648-651) and IL-22Rα1 deficient mice (described in Figure 43 and below) were generated by
Lexicon Pharmaceuticals and backcrossed with C57BL/6 stain more than 10 times. Where
indicated, mice were fed with adjusted calories diet (HFD, containing 60% fat, Harlan) starting
at the age of 4–6 weeks old. For metabolism studies 12–18 weeks old mice were used, whereas
–6 weeks old mice were used for C. rodentium infection studies. All animal experiments were
approved by the Genentech Institutional Animal Care and Use Committee.
Naïve CD4 T cell purification and differentiation. Naïve CD4 T cells were sorted
and stimulated as previously described (Rutz, et al. 2011, Nature Immunol. 12:1238-45), and
cultured under specific condition for each subset similarly to the way as described previously.
Id. For IL-22 induction, anti-IL-4 (10 μg/ml), anti-IFN-γ (10 μg/ml), and recombinant IL-6 (20
ng/ml) were used; where indicated, recombinant mouse leptin (1 μg/ml, R&D systems) was
added.
Intracellular staining and IL-22 ELISA. Lymphocytes purified from draining lymph
nodes were stained for IL-22 and IL-17A as previously described (Zheng et al., supra) using
phycoerythrin (PE)-anti-IL-22 (1H8PWSR, eBioscience) and fluorescein isothiocyanate
(FITC)-anti-IL-17A (17B7, eBiosceince). IL-22 ELISA was performed as previously described
(Zheng et al., supra) using monoclonal anti-IL-22 antibodies (20E5 and 14B7, Genentech).
RNA isolation and real-time PCR. Colon were harvested and processed, and mRNA
was isolated with RNeasy mini plus kit (Qiagen). Il22, Il22ra1, and Reg3b mRNA level were
evaluated using real-time PCR analysis as previously reported (Ota et al. 2011, Nature
immunol. 12, 941-948). Results were normalized to those of the control housekeeping gene
Rpl19 (encoding ribosomal protein L19) and are reported as 2 . The primer and probe
sequence for Il22 and Reg3b were reported previously. Id. For Il22ra1, 5’–AGG TCC ATT
CAG ATG CTG GT–3’(SEQ ID NO:74), 5’–TAG GTG TGG TTG ACG TGG AG–3’ (SEQ
ID NO:75) and 5’–FAM–CCA CCC CAC ACT CAC ACC GG–TAMRA–3’ (SEQ ID NO:76)
were used.
Statistical analysis All statistical analysis was done with two-tailed unpaired Student’s
t-test. P value less than 0.05 was considered as statistically significant.
Results
After immunizing the mice with ovalbumin (OVA) in Complete Freund’s Adjuvant
(CFA), the IL-22 expressing CD4 T cells were detected ex vivo with intracellular cytokine
staining. IL-22 T cells were significantly reduced in db/db mice (Figures 44 A-B). Consistent
with previous reports, IL-17 CD4 T cells were also significant reduced in db/db mice (Figure
45A). Similar results were observed in leptin deficient ob/ob mice as well (Figure 45B). Leptin
can regulate Th cells, such as Th1 cells and Treg cells. However, a direct effect of Leptin on IL-
22 production from in vitro differentiated Th22 cells was not observed (Figure 45C). Moreover,
similar reduction of IL-22 producing T cells was also observed in immunized DIO (diet-
induced obesity, or HFD-fed) C57BL/6 (Figures 44C and D), suggesting obesity but not lack of
Leptin signaling might be accountable for the reduced IL-22 production in CD4 T cells.
Activation TLR5 pathway by flagellin could stimulate IL-22 production from ILCs.
In db/db mice (Figure 44E), ob/ob mice (Figure 45E), and DIO mice (Figure 44F),
serum IL-22 level was significantly lower than that of WT mice upon in vivo challenge with
flagellin. Consistent with the results from T cells, leptin itself did not enhance IL-22 production
from ILCs in vitro (Figure 45D). Taken together, these data suggested that there is a general
defect in IL-22 induction from both ILCs and T cells in obese mice.
Example 13 The Mucosal Defense was Compromised in Leptin Deficient Mice and
Restored by IL-22 Fc Fusion Protein
IL-22 produced by ILCs and T cells is essential for host defense against Citrobecter
rodentium infection in colon. The IL-22 induction in the colon from db/db and ob/ob mice
infected with C. rodentium was analyzed. C. rodentium was cultured overnight and mice were
orally inoculated with 2x10 CFU of bacteria as described (Zheng et al. 2008, Nature medicine
14, 282-289, doi:10.1038/nm1720). Bacterial burden was analyzed as follows: the spleen and
liver of infected mice were harvested, weighted, and homogenized in 0.1% NP40/PBS in C-
tube with gentleMACS (Miltenyi Biotec). Serially diluted homogenates were plated on
MacConkey agar (Remel), and C. rodentium colonies were identified as pink colonies after
overnight incubation at 37C. Where indicated, the mice were injected intramuscularly with IL-
22–Fc (150 μg/dose) or equivalent amount of mouse isotype control 3 times per week.
Histology analysis of colon from mice infected with C. rodentium was performed as reported
previously (Ota et al. 2011, Nature immunology 12, 941-948, doi:10.1038/ni.2089), and scored
for epithelial changes (proliferation, blebbing, enterocyte shedding), inflammation, and mucosal
thickening. Clinical scores were determined for four anatomic regions – proximal, middle and
distal colon and rectum – on a scale from 0-5 with 0 = normal colon and 5 = severe disease.
Regional scores were summed to get a final colon disease severity score for each animal.
Corroborating with above results, the peak induction of IL-22 on day 4 in the colon in
db/db and ob/ob mice was also significantly reduced, but not completely abolished (Figure
46A). In db/db mice after oral inoculated with C. rodentium there was no significant weight
loss (Figure 46B). Surprisingly, the infected db/db mice started to die 10 days after bacterial
inoculation, and about 60% to 100% db/db mice succumbed during the second week of the
infection in repeated experiments (Figure 46C). Histological analysis of the colon sections from
db/db mice revealed increased inflammatory cell infiltration and severe epithelial damages,
including epithelial shedding at the mucosal surface (Figures 46 D-F). In addition, these mice
showed patchy submucosal edema and multifocal bacterial colonies, which were often
associated with localized necrosis. Significantly elevated bacterial burdens were also detected
in both the liver and spleen of db/db mice (Figures 46 G-H). Similar defects in mucosal defense
were observed in ob/ob mice as well (Figure 54). It was unexpected that db/db mice had such a
significant defect in controlling C. rodentium infection; especially given the induction IL-22 by
C. rodentium infection was only partially defective in these mice (see Figure 46A).
It has been reported that Leptin deficient mice also have defects in B cell functions, and
antibody against C. rodentium is required for eventually eliminating the bacteria from the host
during the later phase of the infection. The production of anti-C. rodentium antibody in these
mice was thus examined. The serum samples were harvested by bleeding from submandibular
vein on day 10 after the infection. ELISA plate was coated with heat-killed C. rodentium or
with a goat anti-mouse Ig capturing antibody. Coated plate was washed with washing buffer
(0.05% Tween 20 in PBS), blocked for 2 h with blocking buffer (0.5% BSA, 15ppm Proclin in
PBS), and washed prior to the addition of serially diluted standard mouse monoclonal IgG
(SouthernBiotech), or serum samples. After 2 h incubation at room temperature, plate was
washed and the Ig were detected with goat anti-mouse IgG conjugated with horseradish
peroxidase (HRP) (SouthernBiotech), diluted 1/4,000 in assay diluent (0.5% BSA, 0.05%
Tween 20, 15ppm Proclin in PBS), and incubated for 2 h at room temperature. After washing,
TMB peroxidase substrate (Sigma-Aldrich) was added to each well. Absorbance was read at
650 nm in plate reader (Molecular Devices).
The titer of anti-C. rodentium IgG antibody was significantly reduced in the survived
db/db mice on day 14 after the infection (Figure 46I). However, the reduced anti-C. rodentium
IgG production alone should also not result in the observed early mortality, since Rag2
deficient mice, which completely lack B cells and antibody production, can survive much
longer after infection (Zheng et al. 2008, Nature medicine 14, 282-289). Therefore, the failed
host defense against C. rodentium in db/db mice were likely caused by defects in both the
adaptive antibody response and the induction of IL-22 from ILCs. Next, experiment was carried
out to examine whether IL-22 was able to restore the mucosal immunity in db/db mice during
C. rodentium infection with the administration of exogenous IL-22–Fc. As shown in Figure 46
J, while the majority of the control IgG-treated db/db mice perished, almost all IL-22 Fc treated
db/db mice survived the infection (Figure 46J), supporting that IL-22 Fc was able to
therapeutically restore the mucosal immune defects in db/db mice.
Example 14 IL-22 Fc Reduced Glucose Levels in Obese Mice and High Fat Diet-Fed
Normal Mice
As described in Example 9 above, IL-22 Fc reduced glucose levels in db/db mice that
already developed hyperglycemia (Figure 20A). The therapeutic benefit was persistent during
the course of IL-22–Fc administration. After 3 weeks of treatment, the glucose level in these
mice dropped below 200 mg/dl, close to the normal glucose level in WT mice, while the control
protein treated db/db mice sustained their high glucose level. The reduction of glucose in IL-22
Fc treated mice was more obvious when the mice were fasted (Figure 20C). IL-22 Fc treatment
also resulted in a trend of weight loss or delayed weight gain compared to control treatment.
However, at the end of this study, the weight difference between the two groups did not reach
statistical significance in these mice (Figure 20B). Corroborating with these data, IL-22 Fc
treatment led significantly improved glucose tolerance and insulin sensitivity in glucose
tolerance test and insulin tolerance test (Figures 21 and Figure 22, respectively).
To confirm general beneficial functions of exogenous IL-22 in modulation of metabolic
disorders, IL-22 Fc was administered for 4 weeks to C57BL/6 mice that had been fed with HFD
for at least 8 weeks to induce glucose intolerance. For the glucose tolerance test (GTT), mice
were fasted overnight, and injected i.p. with glucose solution at 1.5 mg/kg. For the insulin
tolerance test (ITT), mice were injected i.p. with insulin solution at 1.0 unit/kg. Blood glucose
was measured before and after the injection. Blood glucose was measured by Contour (Bayer).
Consistent with the results from db/db mice, IL-22 Fc treatment significantly reduced
serum glucose level, especially after fasting (Figure 47A). There was also a reduced body
weight (or delayed weight gain) in the IL-22 Fc treated group at the end of the study (Figure
47B). In addition, IL-22 Fc reduced glucose intolerance and insulin resistance in HFD-fed
C57BL/6 mice (Figures 47C and D). Similar results were obtained when mice were
concurrently administrated with IL-22 Fc at the beginning of feeding with HFD (Figure 48).
Taken together, the data demonstrated that IL-22 Fc was a potential therapy to normalize serum
glucose concentration, and alleviate glucose intolerance and insulin resistance in obese mice.
Example 15 IL-22 Fc Reduced Food Consumption and Increased Expression of PYY in
Obese and HFD-Fed Mice
The reduction of food consumption could reverse hyperglycemia and insulin resistance
in diabetic mice. Indeed, db/db mice treated with IL-22 Fc showed significant reduction of food
intake in comparison with the control group (Figure 49A). Pair-feeding experiments were
performed to ensure the same food intakes in the IL-22 Fc and control treated mice (Figure 50).
Food consumption was measured for ad lib-fed group daily during the study. The supplied food
for pair-fed group was restricted to match the previous day food consumption of ad lib-fed
group. Correspondingly, the treatment and measurement of pair-fed group was one day after ad
lib-fed group.
Even under this condition, IL-22 Fc significantly reduced serum glucose although at a
later time point (Figure 49B), and reversed glucose tolerance in db/db mice (Figure 49C),
suggesting that modulating food consumption by IL-22 was not the only mechanism for its
therapeutic effect in metabolic disease. Similar results were observed in HFD-fed mice (data
not shown). To further understand how IL-22 regulated food consumption and metabolism, the
expression of intestine hormones, PYY, which is known to inhibit food intake was examined.
Mice were injected i.p. with 50 μg IL-22–Fc on day 0 and 2. On day 4 mice were fasted
overnight and re-fed for 1 h on day 5. Blood samples were collected on day 2 before treatment
and on day 5 after feeding. All serum samples were mixed with Protease inhibitor (Sigma),
DPPIV inhibitor (Millipore) and Pefabloc (Roche) immediately after collection. PYY was
measured with PYY ELISA kit (Abnova) following manufacture’s instruction. The results
show that IL-22 Fc treatment significantly increased PYY concentration in the serum of db/db
and HFD-fed mice (Figures 49 D and E). To demonstrate that IL22’s effect on food intake was
mediated through promoting PYY production, food intake in mice treated with PYY inhibitor
BIIE0246 was examined. C57BL/6 mice on normal diet were either untreated or treated with
IL-22 Fc on day 2 and day 4. After overnight fasting, food intake during a 4-hour feeding was
measured. The results show that the reduction of food intake in IL-22 Fc treated mice was
reversed by BIIE0246 (data not shown), indicating that the effect of IL-22 Fc on reduced food
intake was mediated through the induction of PYY.
Example 16 IL-22 Fc Reduced Serum LPS and Liver ALT and AST and Increased Lipid
Metabolism in obese Mice
Since IL-22 receptor is expressed in many organs including liver and pancreas that
regulate metabolism, the therapeutic benefits of IL-22 in metabolic diseases are likely mediated
by various mechanisms. Metabolic endotoxemia contributes to inflammatory status and insulin
resistance and modulation of gut microbiota enhance glucose tolerance. Serum endotoxin was
measured by Limulus Amebocyte Lysate assay kit, QCL-1000 (Lonza), following
manufacture’s instruction. ALT and AST were measured by Cholestech LDX (Alere). The
results shown in Figure 49F demonstrate that IL-22 Fc treatment resulted in significant
reduction of the LPS amount in the serum from db/db mice.
IL-22 can repress genes involved in lipogenesis and ameliorate liver steatosis. Serum
ALT and AST levels were next examined. Blood glucose was measured by Contour (Bayer).
ALT and AST were measured by Cholestech LDX (Alere).As shown in Figures 51A and B, IL-
22 Fc treatment lowered ALT and AST levels in the serum in db/db (Figure 51A) and HFD-fed
(Figure 51B) mice. The abdominal fat was also significantly dropped with IL-22 Fc treatment
in HFD-fed mice (Figure 51C). In addition, genes responsible for lipid metabolism were
induced by IL-22 in primary adiopocytes (Figure 51D). Next, the effect of IL-22 on triglyceride
and cholesterol in liver and adipose tissue were examined. The results show that IL-22 Fc
reduced triglyceride, cholesterol, and free fatty acid (FFA) (Figure 51E), as well as hepatic
triglyceride (Figure 51F), hepatic cholesterol (Figure 51G) and triglyceride in white adipose
tissue (Figure 51H) in HFD-fed mice. Similarly, IL-22 reduced triglyceride in the liver and
white adipose tissue in db/db mice (Figure 51I and Figure 51J). Further experiments show that
IL-22 Fc treatment reduced inflammatory cytokines such as TNFα and IL-1β as compared to no
treatment in obese mice (data not shown). H&E staining of liver sections revealed a decrease in
hepatic periportal steatosis with IL-22 Fc fusion protein treatment (Figure 26).
IL-22 signals through IL-22R1 and IL-10R2 chains. IL-22R1 can also be paired with IL-
20R2 chain and be utilized by IL-20 and IL-24. It has been shown that all these ligands induced
very similar downstream biological effects from skin epidermis (Sa et al., 2007, J Immunol
178, 2229-2240). Thus, both the IL-22 and IL-22R1 deficient mice were examined to avoid
potential redundancy of other cytokines in HFD induced diabetes. The generation of IL-22R
knock out mice is illustrated in Figure 43 A. The deletion of IL-22R1 in the KO mice was
confirmed by the absence of IL-22R1 mRNA in the IL-22R KO mice, and the lack of RegIIIb
mRNA expression in response to IL-22 Fc in the IL-22R KO mice. See Figures 43 B and C. In
addition, administration of ILFc to IL-22R KO mice did not induce pStat3 (data not
shown).
No difference was observed in glucose tolerance and body weight in IL-22 deficient
mice from those of WT littermate controls (Figure 53). When IL-22R1 deficient mice were
treated with high fat diets for three months, however, these mice developed significantly more
severe glucose tolerance and gained more weight (Figures 49G, H and I), supporting a critical
role of IL-22R pathway in controlling metabolism. The possibility of IL-20 and IL-24
redundancy in reducing metabolic syndrome was examined. In this experiment, db/db mice
were treated with IL-20 Fc, IL-22 Fc or IL-24 Fc. The result indicates that only IL-22 Fc
reduced serum glucose level (Figure 55B) and improved glucose tolerance in a GTT assay on
day 20 (Figure 55C) in db/db mice, while treatment of db/db mice with IL-20 Fc or IL-24 Fc
did not. The reduction of body weight was not statistically significant. Further experiments
show that although IL-20 Fc and IL-24 induced pStat3 in primary adipocytes, these cytokines
failed to induce pStat3 in liver tissue from db/db mice that had become insensitive to insulin
(data not shown). Treatment of IL-22 Fc in the IL-22R KO mice had no effect in a glucose
tolerance test, confirming that the effect of IL-22 Fc was exerted through the IL-22 R signaling
(data not shown).
The studies presented here indicate critical functions of IL-22 in regulating metabolic
processes. IL-22R1 deficient mice were predisposed to development of metabolic syndromes.
Exogenous IL-22 was not only able to restore the mucosal immune defects in preclinical
diabetic models, but also helped to normalize glucose and lipid metabolisms. IL-22, thus, can
provide a novel therapeutic approach to treat human metabolic disorders.
Example 17 Comparison of VGEF and IL-22 in Promoting Wound Healing in db/db
Mice
In this experiment, the effect of IL-22 on promoting or improving wound healing was
m db
analyzed and compared with that of VEGF. Female BKS.Cg-Dock7 +/+ Lepr /J db/db mice
of 11 weeks of age were purchased from Jackson Laboratory, Bar Harbor, ME. All
experimental animal studies were conducted under the approval of the Institutional Animal
Care and Use Committees of Genentech Lab Animal Research. Under isoflurane anesthesia the
dorsal skin was shaved then depilatory cream was applied to remove the remaining stubble.
After the skin is cleaned and prepped with povidone-iodine followed by alcohol swabs, a
circular, full-thickness wound was created on the dorsal skin of each mouse using a disposable
6 mm biopsy punch (Miltex, Inc.). The wound was covered with a Tegaderm film before and
after treatment.
The results in Figure 56 show that VEGF appeared to achieve faster surface closure as
compared with IL-22; however, when the dermis side of the skin was examined, wounds treated
with VEGF remained open even on day 21 (Figure 56B). The ability of VEGF and IL-22 Fc in
promoting angiogenesis at the wound site was also analyzed. In this experiment, two 6 mm
wounds were excised in db/db mice on day 0. On day 2, 4, 6, 8, 10 and 12, either control anti-
ragweed antibody or IL-22 Fc (50 μg) or VEGF (20 μg) in saline was administered topically
onto the wounds. On day 6 and 12, three mice from each group were taken down for histology
and immunohistochemistry analysis and BrdU staining. On day 16, one mouse was taken down
for BrdU staining. On day 18, 20 and 22, one mouse from each group was taken down for each
time point for immunohistochemistry analysis and CD31 whole tissue staining. The results
indicate that both VEGF and IL22-Fc, but not the control anti-ragweed antibody, promoted
blood vessel formation at the wound site as analyzed by CD31 tissue immunostaining (data not
shown).
Next, we analyzed ILinduced and other IL-10 family member-induced cytokine and
chemokine expression in reconstituted epidermis. The reconstituted epidermis was EpiDerm
RHE tissue models maintained in EPINMM medium purchased from MatTek. See Sa et
al. 2007, J. Immunol. 178:2229-2240. The results show that IL-22 prominently induced
expression of IL-8, CXCL-1, MIP 3a, DMC, and MCP-1 in reconstituted human epidermis,
though inductions by IL-19, IL-20 or IL-24 were also observed (Figure 57). In view of the
effect of IL-22 on wound healing described herein, IL-19, IL-20, and IL-24 may also play a role
in accelerating wound healing.
Example 18 IL-22 Provides Superior Efficacy in the Treatment of Infected Wound than
VEGF and PDGF in a Splinted Wound Model in db/db Mice
In the mouse wound healing model, contraction accounts for a large part of wound
closure in mice because mice skin is mobile. To more closely resemble the wound healing
process in human, a mouse splinted wound model was established in which a silicon ring was
glued to the skin and anchored with sutures around the wound to prevent local skin contraction
(see representative images in Figure 59B). See e.g., Zhao et al., 2012, Wound Rep. Reg.
:342-352 and Brubaker et al., 2013, J. Immunol., 190:1746-57. In this model, wounds healed
through granulation and re-epithelialization processes, similar to the wound healing processes
in humans. To splint the wound, Krazy glue (Elmer’s Products, Inc.) was applied to one side of
the sterile silicone splint (Grace Bio-Labs, Inc.) and the splint was carefully placed around the
wound with the glue side down so that the wound was centered within the splint. The glue
bonded to the skin on contact and served as a splint for the entire course of the study. The
splint was further anchored to the skin with four interrupted 6.0 monofilament nylon suture
(Ethicon, Inc.). Digital image of the wound was taken before the wound was covered with a
Tegaderm transparent film. Further, microbial infection on the open wound can delay wound
healing, and chronic wounds, such as chronic wounds observed in diabetic patients, are often
infected wounds.
Using the splinted wound model, the effect of IL-22 Fc on infected wound was
examined in db/db mice. Wounds excised as described above in wild type or db/db mice were
6 6 6
inoculated topically with 0.5 x 10 CFU, 1 x 10 CFU (plaque forming unit) or 2 x 10 CFU of
Staphylococcus aureus two days after wound excision. As shown in Figure 58, db/db mice
exhibited delayed wound healing as compared to wild type mice, and wound healing was
further delayed when the wound was infected by bacteria in these mice as compared to control.
In separate experiments, IL-22’s wound healing effect was compared with other agents
in the splinted infected wound model. Two days after wound excision, the methicillin-resistant
S. aureus strain USA300 NRS 384 (NARSA) at 1 x 10 CFU in 30ul saline was inoculated onto
the wound surface and covered again with a Tegaderm film. Topical treatment began 48 hours
after S. aureus infection with 30ug of either IL22-Fc or VEGF (Lot #110308, Genentech) or
PDGF (Lot #0507CY420, PeproTech, Inc.) in 30ul of saline 3 times a week thereafter. Digital
images of the wound were recorded before treatment and twice a week after treatment until
closure of the wounds. Percentage of wound closure was calculated from the wound images
using ImageJ, a java-based image processing program developed at the NIH.
As shown in Figure 59, IL-22 Fc promoted faster wound healing than VEGF when same
amount of the compounds was applied to infected wounds in the splinted wound model, which
more closely resembled wound healing in human. Next, different doses of VEGF and IL-22 Fc
were tested on infected wounds. In this experiment, one 6 mm diameter splinted excisional
wound was created in db/db mice with blood glucose > 300 mg/dl. At each wound 1 x 10 CFU
of S. aureus USA 300 was inoculated. Varying doses of VEGF or IL-22 Fc in saline were
administered topically three times per week until wound closure. Saline was used as a control.
At wound closure, mice were sacrificed and samples were subjected to histology, immune-
histochemistry, and PCR analysis and CFU count. The results in Figure 60 show that IL-22 Fc
at the amount of 30 μg demonstrated better infected wound healing efficacy than 60 μg VEGF.
Thus, the faster surface closure by VEGF observed in a non-splinted wound model was likely
due to mouse skin contraction and the effects of IL-22 Fc on promoting keratinocyte
proliferation and re-epithelializatin were likely masked by mouse skin contraction.
Similar results are shown in Figure 61, in which IL-22 Fc was demonstrated of having
superior efficacy than VEGF and PDGF when the same amount (30 μg) of each compound was
applied to the wound. Complete wound closure in IL-22 Fc treated infected splinted wound was
seen on day 15. In VEGF- or PDGF-treated mice, however, complete closure of infected
splinted wound was not seen until day 25, same as untreated uninfected wound. Wound closure
in the control group, i.e., untreated infected wound, was not seen until day 29. Without being
limited to specific mechanism(s), the superiority of IL-22 Fc in promoting wound healing than
VEGF or PDGF can be due to its effects on re-epithelialization, promoting keratinocyte
proliferation, induction of neovascularization, induction of proteases to facilitate tissue
remodeling and repair and the antimicrobial activities.
Next, we tested whether IL-22 Fc can be administered in a gel formulation for wound
healing. The exemplary gel formulation used in this experiment contained 10 mM sodium
phosphate at pH 7.1 with 0.5 mg/g Methionine and 3% Hydroxypropyl methylcellulose (HPMC
E4M premium from Dow Chemicals), with or without 1mg/g IL-22 Fc. The gel solution and
IL-22 Fc solution were mixed prior to being applied topically to the splinted wound. The
formulation containing IL-22 Fc also contained a small amount of sucrose (< 20 mM) and P20
(< 0.002%) carried from the original protein formulation. The results shown in Figure 62
demonstrate that IL-22 Fc in both solution and gel formulation promoted wound healing in a
non-infected splinted wound.
The specification is considered to be sufficient to enable one skilled in the art to practice the
invention. Although the foregoing invention has been described in some detail by way of illustration
and example for purposes of clarity of understanding, the descriptions and examples should not be
construed as limiting the scope of the invention. Indeed, various modifications of the invention in
addition to those shown and described herein will become apparent to those skilled in the art from
the foregoing description and fall within the scope of the appended claims.
Certain statements that appear herein are broader than what appears in the statements of
the invention. These statements are provided in the interests of providing the reader with a
better understanding of the invention and its practice. The reader is directed to the
accompanying claim set which defines the scope of the invention.
In this specification where reference has been made to patent specifications, other
external documents, or other sources of information, this is generally for the purpose of
providing a context for discussing the features of the invention. Unless specifically stated
otherwise, reference to such external documents is not to be construed as an admission that such
documents, or such sources of information, in any jurisdiction, are prior art, or form part of the
common general knowledge in the art.
Claims (25)
1. An interleukin (IL)-22 Fc fusion protein comprising an IL-22 polypeptide linked to an IgG1 Fc region by a linker, wherein the Fc region is not glycosylated.
2. The IL-22 Fc fusion protein of claim 1, wherein the IL-22 Fc fusion protein comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:12.
3. The IL-22 Fc fusion protein of claim 2, wherein the amino acid sequence has at least 96% sequence identity to the amino acid sequence of SEQ ID NO:12.
4. The IL-22 Fc fusion protein of claim 2 or claim 3, wherein the amino acid sequence has at least 97% sequence identity to the amino acid sequence of SEQ ID NO:12.
5. The IL-22 Fc fusion protein of any one of claims 2-4, wherein the amino acid sequence has at least 98% sequence identity to the amino acid sequence of SEQ ID NO:12.
6. The IL-22 Fc fusion protein of any one of claims 2-5, wherein the amino acid sequence has at least 99% sequence identity to the amino acid sequence of SEQ ID NO:12.
7. The IL-22 Fc fusion protein of any one of claims 1-6, wherein the Fc region comprises a hinge region comprising the amino acid sequence of CPPCP (SEQ ID NO:31).
8. The IL-22 Fc fusion protein of any one of claims 1-7, wherein the Fc region comprises an altered glycosylation consensus site.
9. The IL-22 Fc fusion protein of any one of claims 1-8, wherein the Fc region comprises an insertion, a deletion, or a substitution mutation that results in an aglycosylated Fc region.
10. The IL-22 Fc fusion protein of any one of claims 1-9, wherein the amino acid residue at position 297 as in the EU index of the Fc region is changed and/or the amino acid residue at position 299 as in the EU index of the Fc region is changed.
11. The IL-22 Fc fusion protein of any one of claims 1-10, wherein the amino acid residue at position 297 as in the EU index of the Fc region is Gly or Ala.
12. The IL-22 Fc fusion protein of any one of claims 1-11, wherein the amino acid residue at position 297 as in the EU index of the Fc region is Gly.
13. The IL-22 Fc fusion protein of any one of claims 1-12, wherein the amino acid residue at position 299 as in the EU index of the Fc region is Ala, Gly or Val.
14. The IL-22 Fc fusion protein of any one of claims 1-13, wherein the linker is 8-20 amino acids long.
15. The IL-22 Fc fusion protein of claim 14, wherein the linker is 8-16 amino acids long.
16. The IL-22 Fc fusion protein of claim 14, wherein the linker is 10-16 amino acids long.
17. The IL-22 Fc fusion protein of any one of claims 1-13, wherein the linker comprises the amino acid sequence DKTHT (SEQ ID NO:32).
18. The IL-22 Fc fusion protein of claim 17, wherein the linker is at least 11 amino acids long and comprises the amino acid sequence EPKSCDKTHT (SEQ ID NO:33).
19. The IL-22 Fc fusion protein of claim 18, wherein the linker comprises the amino acid sequence VEPKSCDKTHT (SEQ ID NO:34), KVEPKSCDKTHT (SEQ ID NO:35), KKVEPKSCDKTHT (SEQ ID NO:36), DKKVEPKSCDKTHT (SEQ ID NO:37), VDKKVEPKSCDKTHT (SEQ ID NO:38), or KVDKKVEPKSCDKTHT (SEQ ID NO:39).
20. The IL-22 Fc fusion protein of any one of claims 1-13, wherein the linker comprises the amino acid sequence EPKSSDKTHT (SEQ ID NO:40).
21. The IL-22 Fc fusion protein of claim 20, wherein the linker comprises the amino acid sequence VEPKSSDKTHT (SEQ ID NO:67), KVEPKSSDKTHT (SEQ ID NO:68), KKVEPKSSDKTHT (SEQ ID NO:66), DKKVEPKSSDKTHT (SEQ ID NO:64), VDKKVEPKSSDKTHT (SEQ ID NO:69), or KVDKKVEPKSSDKTHT (SEQ ID NO:65).
22. The IL-22 Fc fusion protein of any one of claims 1-13, wherein the linker does not comprise the amino acid sequence GGS (SEQ ID NO:45).
23. The IL-22 Fc fusion protein of claim 1, wherein the IL-22 Fc fusion protein comprises the amino acid sequence of SEQ ID NO:12.
24. The IL-22 Fc fusion protein of claim 1, wherein the IL-22 Fc fusion protein comprises the amino acid sequence of SEQ ID NO:14.
25. The IL-22 Fc fusion protein of claim 1, wherein the IL-22 Fc fusion protein comprises the amino acid sequence of SEQ ID NO:20.
Applications Claiming Priority (11)
Application Number | Priority Date | Filing Date | Title |
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US201361800795P | 2013-03-15 | 2013-03-15 | |
US201361801144P | 2013-03-15 | 2013-03-15 | |
US201361800148P | 2013-03-15 | 2013-03-15 | |
US61/801,144 | 2013-03-15 | ||
US61/800,795 | 2013-03-15 | ||
US61/800,148 | 2013-03-15 | ||
US201361821062P | 2013-05-08 | 2013-05-08 | |
US61/821,062 | 2013-05-08 | ||
US201361860176P | 2013-07-30 | 2013-07-30 | |
US61/860,176 | 2013-07-30 | ||
NZ711095A NZ711095B2 (en) | 2013-03-15 | 2014-03-14 | Il-22 polypeptides and il-22 fc fusion proteins and methods of use |
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