U.S. patent application number 16/806620 was filed with the patent office on 2020-09-03 for n-terminally truncated interleukin-38.
The applicant listed for this patent is FRAUNHOFER-GESELLSCHAFT ZUR FORDERUNG DER ANGEWANDTEN FORSCHUNG E.V.. Invention is credited to Bernhard BRUNE, Christina DILLMANN, Gerd GEISSLINGER, Javier MORA, Michael John PARNHAM, Andreas WEIGERT.
Application Number | 20200277349 16/806620 |
Document ID | / |
Family ID | 1000004838261 |
Filed Date | 2020-09-03 |
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United States Patent
Application |
20200277349 |
Kind Code |
A1 |
WEIGERT; Andreas ; et
al. |
September 3, 2020 |
N-TERMINALLY TRUNCATED INTERLEUKIN-38
Abstract
The present invention pertains to an N-terminally truncated
interleukin (IL)-38 protein, or functional variants thereof, as
well as to nucleic acids and vectors encoding the truncated IL-38
peptide and recombinant cells comprising these nucleic acids or
vectors. The invention shows that IL-38 is N-terminally processed
and that the truncated version of the cytokine acts as an
antagonist of immune activation in macrophages. This indicates a
use of the truncated cytokine in the treatment and prevention of
autoimmune disorders. The invention further provides pharmaceutical
compositions comprising the truncated IL-38 protein, and method for
screening modulators of the function of truncated IL-38.
Inventors: |
WEIGERT; Andreas; (Hofheim
am Taunus, DE) ; MORA; Javier; (Frankfurt am Main,
DE) ; BRUNE; Bernhard; (Schoneck, DE) ;
DILLMANN; Christina; (Frankfurt, DE) ; PARNHAM;
Michael John; (Bad Soden am Taunus, DE) ;
GEISSLINGER; Gerd; (Bad Soden, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
FRAUNHOFER-GESELLSCHAFT ZUR FORDERUNG DER ANGEWANDTEN FORSCHUNG
E.V. |
Munich |
|
DE |
|
|
Family ID: |
1000004838261 |
Appl. No.: |
16/806620 |
Filed: |
March 2, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15328690 |
Jan 24, 2017 |
10618946 |
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PCT/EP2015/066084 |
Jul 14, 2015 |
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16806620 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/5041 20130101;
G01N 33/6869 20130101; G01N 2333/54 20130101; C07K 14/54 20130101;
A61K 38/00 20130101; G01N 2500/10 20130101 |
International
Class: |
C07K 14/54 20060101
C07K014/54; G01N 33/68 20060101 G01N033/68; G01N 33/50 20060101
G01N033/50 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 25, 2014 |
EP |
14178478.5 |
Claims
1. An isolated truncated IL-38 protein, or a functional variant
thereof, wherein said truncated IL-38 protein is N-terminally
truncated compared to the amino acid sequence according to SEQ ID
NO: 1, and wherein said truncation comprises at least 10 adjoining
amino acids between positions 1 to 30 of SEQ ID NO: 1.
2. The isolated truncated IL-38 protein according to claim 1,
wherein said truncated IL-38 protein has 2-50 amino acids truncated
at its N-terminus as compared with wild type IL-38 protein (SEQ ID
NO: 1).
3. The isolated truncated IL38 protein according to claim 2,
wherein said truncated IL-38 protein has 11, 12, 13, 14, 15, 16,
17, 18, 19 or 20 amino acids truncated at its N-terminus as
compared to the protein shown in SEQ ID NO: 1.
4. The truncated IL-38 protein according to claim 1, having an
N-terminus that is not identical to the first 100, 50, 30, 20, or
19 amino acids of SEQ ID NO: 1.
5. A nucleic acid comprising a sequence coding for a truncated
IL-38 protein according to claim 1.
6. The nucleic acid according to claim 5 comprising a sequence that
when expressed produces a polypeptide consisting of the truncated
IL-38 protein and not a full length IL-38 protein according to SEQ
ID NO: 1, wherein said truncated IL-38 protein is N-terminally
truncated compared to the amino acid sequence according to SEQ ID
NO: 1, and wherein said truncation comprises at least 10 adjoining
amino acids between positions 1 to 30 of SEQ ID NO: 1.
7. A vector comprising a nucleic acid according to claim 5.
8. The vector according to claim 7, wherein the expressible
sequence is operable linked to a promoter.
9. A recombinant cell, comprising a nucleic acid according to claim
5, or a vector comprising said nucleic acid.
10. A pharmaceutical composition comprising the truncated IL-38
protein according to claim 1, or a nucleic acid encoding the
truncated IL-38 protein, or a vector comprising the nucleic acid,
or a recombinant cell comprising the truncated IL-38 protein, the
nucleic acid or the vector.
11. An in-vitro method for modulating the immune response of a
cell, the method comprising contacting said cell with a truncated
IL-38 protein according to claim 1, or by expressing in said cell a
nucleic acid comprising a sequence encoding said truncated IL-38
protein.
12. The method according to claim 11, wherein modulating the immune
response is an inhibition of JNK signaling, in particular the
inhibition of IL-6 release and TH17 generation.
13. A method for screening for modulators of the activity of
truncated IL-38, comprising the steps of a. Providing a cell, b.
Contacting said cell with microbe-associated molecular pattern
(MAMP), pathogen-associated molecular patterns (PAMP) or apoptotic
cell supernatants (ACM), c. Further contacting said cell with a
truncated IL-38 protein according to claim 1 and a candidate
modulator, d. Determining JNK activation in said cell, wherein an
increase of JNK activation in said cell compared to a control cell
or reference value indicates that the candidate modulator is an
antagonist of truncated IL-38, and a decrease of JNK activation
compared to a control cell or reference indicates that the
candidate modulator is an agonist of truncated IL-38.
14. The method according to claim 13, wherein said cell expresses
on the cell surface a receptor of truncated IL-38, for example by
ectopically expressing IL-1RAPL1 in said cell.
15. The method according to claim 13, wherein said JNK activation
is determined by means of an AP-1 reporter construct.
16. A method for the treatment or prevention of an immune or
inflammatory disease in a subject in need of such treatment, the
method comprising a step of administering a therapeutically
effective amount of any one or a combination of: a. an isolated
truncated IL-38 protein, or a functional variant thereof, wherein
said truncated IL-38 protein is N-terminally truncated compared to
the amino acid sequence according to SEQ ID NO: 1, and wherein said
truncation comprises at least 10 adjoining amino acids between
positions 1 to 30 of SEQ ID NO: 1; and/or b. a nucleic acid, or a
vector or recombinant cell comprising the nucleic acid, wherein the
nucleic acid comprises a sequence coding for a truncated IL-38
protein or a functional variant thereof, wherein said truncated
IL-38 protein is N-terminally truncated compared to the amino acid
sequence according to SEQ ID NO: 1, and wherein said truncation
comprises at least 10 adjoining amino acids between positions 1 to
30 of SEQ ID NO: 1.
17. The method according to claim 16, wherein the immune or
inflammatory disease is selected from autoimmune diseases, such as
septic shock, hemorrhagic shock, arthritis, for example
spondyloarthritis, rheumatoid arthritis, psoriatic arthritis or
osteoarthritis, inflammatory bowel disease, multiple sclerosis and
meta-bolic diseases such as arteriosclerosis and type I diabetes;
or is Muckle-Wells syndrome, cryopyrin-associated periodic fever
syndromes (CAPS), familial Mediterranean fever, Still's disease,
Behcet's disease or diabetes mellitus.
Description
FIELD OF THE INVENTION
[0001] The present invention pertains to an N-terminally truncated
interleukin (IL)-38 protein, or functional variants thereof, as
well as to nucleic acids and vectors encoding the truncated IL-38
peptide and recombinant cells comprising these nucleic acids or
vectors. The invention shows that IL-38 is N-terminally processed
and that the truncated version of the cytokine acts as an
antagonist of immune activation in macrophages. This indicates a
use of the truncated cytokine in the treatment and prevention of
autoimmune disorders. The invention further provides pharmaceutical
compositions comprising the truncated IL-38 protein, and method for
screening modulators of the function of truncated IL-38.
DESCRIPTION
[0002] The IL-1 family of cytokines and receptors is a
heterogeneous group of proteins that particularly regulate
immunity. Initially, four IL-1 family cytokines were intensively
characterized (IL-1.alpha., IL-1.beta., IL-1Ra and IL-18),
revealing basal principles of immune regulation, some of which were
already translated into the clinic. The remaining seven IL-1 family
cytokines were identified by in silico analysis of gene databases
(IL-33, IL-36.alpha., IL-36.beta., IL-36.gamma., IL-36Ra, IL-37 and
IL-38). In recent years, important studies were conducted to
investigate their relevance for induction or regulation of the
immune response. Conclusively, IL-1 family cytokines exhibit a
broad spectrum of functions in immunity, including the induction of
Th1 and Th2 inflammation as well as mediating anti-inflammatory or
pro-resolving effects. On a mechanistic level, triggering of
inflammation is mediated by IL-1 family cytokines with receptor
agonist (IL-1.alpha., IL-1.beta., IL-18, IL-33, IL-36) function,
which is counteracted by IL-1 family receptor antagonists (IL-1Ra,
IL-36Ra). Of note, full receptor agonistic or antagonistic function
often requires N-terminal processing of IL-1 family cytokines,
usually creating the mature cytokine from a precursor. The most
prominent of these events is probably IL-1.beta. maturation by the
inflammasome.
[0003] IL-1 family receptors are characterized by the presence of
extracellular immunoglobulin domains and an intracellular TIR
domain that is necessary for signal transduction. The IL-1 receptor
family includes four members with known ligand and function: IL-1R1
(IL-1RI), IL-1R4 (ST2), IL-1R5 (IL-18R), IL-1R6 (IL-1Rrp2); two
co-receptors: IL-1R3 (IL-1RAcP), IL-1R7 (IL-18AcP); one decoy
receptor: IL-1R2 (IL-1RII); and three orphan receptors TIR8
(SIGIRR), TIGIRR-1 (IL-1RAPL2), TIGIRR-2 (IL-1RAPL1). The
nomenclature of the orphan receptors is still ambiguous. IL-1RAPL1,
also known as TIGIRR-2, was originally named IL-1R8. However, it
was recently referred to as IL-1R9 or IL-1R10. To avoid confusion,
the inventors will use the term IL-1RAPL1 in the present
manuscript. IL-1RAPL1 is highly expressed in the brain and is
involved in cerebellar development, mental retardation and
cognitive defects. The main structural difference to other members
of the IL-1 receptor family is a C-terminal 150 amino acid-long
extension in the intracellular domain of IL-1RAPL1, which is also
present in its close homolog IL-1RAPL2 and the regulatory receptor
TIR8. A role of this structure in cellular signaling has not been
described. Functional studies suggest that the mechanism of
activation and downstream signaling of IL-1RAPL1 differs from that
of other members of the IL-1 receptor family. There is evidence
that IL-1RAPL1 selectively activates JNK, which is, among others,
involved in immune activation. Indeed, a functional RNAi screen
revealed that IL-1RAPL1 regulates the macrophage phenotype upon
interaction with apoptotic cells.
[0004] IL-38, also known as IL1F10, is the most recent addition to
the IL-1 family. It shares 41% homology with IL-1Ra and 43% with
IL-36Ra and was therefore proposed as an IL-1 receptor antagonist.
Indeed, it has been recently shown that IL-38 can bind to IL-1R6,
where it reduces cytokine production after C. albicans stimulation
or addition of IL-36 when administered at low concentrations.
Nevertheless, an increase in cytokine produced was noted after LPS
stimulation together with IL-38. In general IL-38 polymorphisms are
associated with increased susceptibility to develop
auto-inflammatory pathologies such as spondyloarthritis,
rheumathoid arthritis or psoriatic arthritis or with CRP levels,
suggesting a role of IL-38 in the regulation of the mechanisms
underlying such conditions.
[0005] Cytokines include a large number of mammalian
immunoregulatory hormones that are secreted by cells of the immune
system. They exert their biological effects through interaction
with specific receptors on cell surfaces. Therefore, the biological
response to a cytokine is regulated both by the presence of the
cytokine and by the expression of its receptor molecule. Many
mammalian diseases, including autoimmune, inflammatory and cancer
diseases, are correlated with increased or otherwise altered levels
of cytokines or cytokine receptors which may contribute to the
misregulation of the immune system and to disease progression.
Compounds which are capable of blocking the immunoregulatory or
inflammatory effects of cytokines should therefore have significant
therapeutic activity with respect to such disease states.
[0006] Conventional strategies for generating immunosuppression
associated with an undesired immune response are based on
broad-acting immunosuppressive drugs. Additionally, in order to
maintain immunosuppression, immunosuppressant drug therapy is
generally a life-long proposition. Unfortunately, the use of
broad-acting immunosuppressants is associated with a risk of severe
side effects, such as tumors, infections, nephrotoxicity and
metabolic disorders. Accordingly, new immunosuppressant therapies
would be beneficial.
[0007] Hence, until this day there is no satisfactory therapeutic
approach for treating or preventing autoimmune disease and there is
a constant need for additional immunosuppressive agents in order to
advance medical care for patients suffering from diseases
associated with a pathological or even chronically activated immune
system.
[0008] The above problem is solved in a first aspect by an isolated
truncated IL-38 protein, or a functional variant thereof, wherein
said truncated IL-38 protein is N-terminally truncated compared to
the amino acid sequence according to SEQ ID NO: 1.
[0009] As used herein, the term "truncated IL-38 protein" refers to
an IL-38 polypeptide in which amino acid residues have been removed
from the amino-terminal (or N-terminal) area of the full length
IL-38 polypeptide. A "truncated IL-38 protein" in the context of
the present invention never comprises the full-length sequence as
shown in SEQ ID NO: 1.
[0010] The term "functional variant" of a protein means herein a
variant protein, wherein the function, in relation to the invention
defined as affinity and stability, is essentially retained. Thus,
one or more amino acids that are not relevant for said function may
have been exchanged. The term `functional variant` should also be
understood to mean homologues from other mammals. Preferably the
functional variants of the present invention retain the immune
suppressive abilities of the herein described truncated IL-38
protein as shown in SEQ ID NO: 2.
[0011] In a preferred embodiment the isolated truncated IL-38
protein, or functional variant thereof, comprises an amino acid
sequence having at least 50% sequence identity to the amino acid
sequence according to SEQ ID NO: 1 (full length IL-38),
characterized in that the truncated IL-38 protein does not comprise
the full length amino acid sequence shown in SEQ ID NO: 1.
[0012] The term "sequence identity" (or "sequence homology")
indicates a quantitative measure of the degree of identity between
two amino acid sequences of equal length or between two nucleotide
sequences of equal length. If the two sequences to be compared are
not of equal length, they must be aligned to the best possible fit
with the insertion of gaps or alternatively truncation at the ends
of the protein sequences.
[0013] A more preferred minimum percentage of sequence identity is
at least 70%, such as at least 80%, at least 85%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, and at
least 99.5%, most preferably 100% compared to the sequence shown in
SEQ ID NO:1, under the proviso that said truncated IL-38 sequence
does not comprise the IL-38 full length sequence as shown in SEQ ID
NO: 1.
[0014] Said truncated IL-38 protein of the invention has in a
preferred embodiment 2-50 amino acids, truncated at its N-terminus,
as compared with wild type IL-38 protein (SEQ ID NO: 1). Preferably
said truncated IL-38 protein has 9, 10 11, 12, 13, 14, 15, 16, 17,
18, 19 or 20 amino acids truncated at its N-terminal as compared to
the protein shown in SEQ ID NO: 1. Most preferred are 19.
[0015] The N-terminal truncation in accordance to the herein
described invention preferably involves at least 2, preferably 5,
most preferably 10, most preferably 20 adjoining amino acids
between positions 1 to 30 of SEQ ID NO: 1.
[0016] In other embodiments the truncated IL-38 protein of the
invention has an N-terminus that is not identical to the first 100,
50, 30, 20 amino acids of SEQ ID NO: 1.
[0017] In other embodiments the truncated IL-38 protein of the
invention has an N-terminus, wherein the first 50 amino acids of
the N-terminal end are not identical to the first 50 amino acids of
SEQ ID NO: 1.
[0018] In other embodiments the truncated IL-38 protein of the
invention has an N-terminus, wherein the first 20 amino acids of
the N-terminal end are not identical to the first 20 amino acids of
SEQ ID NO: 1.
[0019] Alternatively a truncated IL-38 protein of the invention
does not comprise a sequence that is at least 80% identical to the
sequence between positions 1 to 20 of SEQ ID NO: 1.
[0020] Yet most preferred is a truncated IL-38 protein, consisting
of an amino acid sequence having at least 80%, 85%, 90%, 95% or
100% sequence identity to SEQ ID NO: 2 (20-152 IL-38). If produced
by recombinant expression of a truncated IL-38 protein of the
invention, the sequence of the truncated IL-38 protein is
characterized by the presence of an additional methionine at the
N-terminus. Such proteins may be after expression subjected to a
purification process to obtain isolated truncated IL-38 protein of
the invention.
[0021] "Isolated" and "purified" refer to any molecule or compound
that is separated from its natural environment and is from about
60% to about 99% free, preferably 80% to 99% free from other
components with which it is naturally associated.
[0022] Therefore, in this respect particularly preferred truncated
IL-38 proteins of the invention comprise an N-terminal methionine
at position 1; such proteins are purely artificial and not present
in nature.
[0023] The term "recombinant" as used herein refers to a protein or
nucleic acid construct, generated recombinantly or synthetically,
e.g., in the case of a protein, through the translation of the RNA
transcript of a particular vector- or plasmid-associated series of
specified nucleic acid elements or of an expression cassette in a
host cell. The term "recombinant" as used herein does not encompass
the alteration of the cell or vector by naturally occurring events
(e.g., spontaneous mutation, natural
transformation/transduction/transposition) such as those occurring
without deliberate human intervention.
[0024] By "host cell" is meant a cell, which contains a vector or
expression cassette and supports the replication and/or expression
thereof. Host cells may be prokaryotic cells such as E. coli, or
eukaryotic cells such as yeast, insect, amphibian, plant cells or
mammalian cells. Preferably, host cells are bacterial or
prokaryotic cells.
[0025] As used herein, "vector" includes reference to a nucleic
acid used in transfection of a host cell and into which can be
inserted a polynucleotide. Vectors are often replicons.
[0026] The term "protein" or "proteins" as used herein refers to a
polypeptide or any portion thereof.
[0027] The term "polypeptide" refers to a polymer of amino acids
and does not refer to a specific length of the product; thus,
peptides, oligopeptides, and proteins are included within the
definition of polypeptide. This term also does not refer to or
exclude post-expression modifications of the polypeptide, for
example, glycosylations, acetylations, phosphorylations and the
like. Included within the definition are, for example, polypeptides
containing one or more analogs of an amino acid (including, for
example, unnatural amino acids, etc.), polypeptides with
substituted linkages, as well as the modifications known in the
art, both naturally occurring and non-naturally occurring.
[0028] The term "recombinant protein" as used herein refers to (1)
a polypeptide of semisynthetic or synthetic origin resulting from
the expression of a combination of DNA molecules of different
origin that are joined using recombinant DNA technologies; (2) a
polypeptide of semisynthetic or synthetic origin that, by virtue of
its origin or manipulation, is not associated with all or a portion
of a protein with which it is associated in nature; (3) a
polypeptide of semisynthetic or synthetic origin that is linked to
a polypeptide other than that to which it is linked in nature; or
(4) a polypeptide of semisynthetic or synthetic origin that does
not occur in nature.
[0029] The present invention also contemplates chemically or
otherwise modified truncated IL-38 proteins. Modified versions of
the herein described polypeptides are for example IL-38 proteins
which were post-translational modified. A post-translational
modification may be the glycosylation of the expressed protein.
[0030] The above described problem of the prior art is in one
additional aspect thereof solved by providing a nucleic acid
comprising a sequence coding (or encoding) for a truncated IL-38
protein as described herein. The term "coding" or "encoding" refers
to the ability of a nucleotide sequence to code for one or more
amino acids. The term does not require a start or stop codon. An
amino acid sequence can be encoded in any one of six different
reading frames provided by a polynucleotide sequence and its
complement.
[0031] The nucleic acid of the invention may comprise a sequence
that when expressed produces a polypeptide consisting of the
truncated IL-38 protein of the invention, but which is not the full
length IL-38 protein according to SEQ ID NO: 1.
[0032] Yet another aspect of the invention pertains to a vector
comprising a nucleic acid as described herein before. Most
preferred is that the vector of the invention is an expression
vector.
[0033] An "expression vector" is a nucleic acid construct or
sequence, generated recombinantly or synthetically, with specific
nucleic acid elements that permit transcription and/or expression
of another nucleic acid in a host cell. An expression vector can be
part of a plasmid, virus, or nucleic acid fragment. In one example,
an expression vector is a DNA vector, such as a plasmid, that
comprises at least one promoter sequence and at least one
terminator sequence (e.g., a polyadenylation sequence), and
optionally an origin of replication (ori) sequence, and optionally
a selection or selectable marker sequence. Optionally, the
expression vector may further comprise at least one nucleotide
coding sequence of interest that codes for at least one
polypeptide, wherein the at least one promoter sequence is operably
linked with the at least one coding sequence. The term "expression"
includes any step involved in the production of the polypeptide
including, but not limited to, transcription, post-transcriptional
modification, translation, post-translational modification, and/or
secretion.
[0034] Also provided is a recombinant cell, comprising a nucleic
acid or a vector or expression vector as described herein. A
recombinant cell of the invention is preferably not a human
embryonic stem cell. Preferred recombinant cells of the invention
are for example bacterial cells such as E. coli or other expression
systems, or also animal cells such as insect cells, mammalian cells
and human cells.
[0035] The compounds and compositions as described herein may be
applied in various medical fields, in particular as active
therapeutics in the treatment or prevention of a disease
characterized by the pathological activation of the immune response
in a subject in need of such a treatment. Preferred disease to be
treated by the compounds of the invention will be described herein
below.
[0036] Hence, in an additional aspect the invention also provides a
pharmaceutical composition comprising a truncated IL-38 protein, or
a nucleic acid, a vector or a recombinant cell according to the
afore described embodiments of the invention, for use in medicine,
preferably for use in the treatment or prevention of an immune- or
inflammatory disease.
[0037] In context of the present invention an immune or
inflammatory disease is preferably selected from autoimmune
diseases, such as septic shock, hemorrhagic shock, arthritis, for
example spondyloarthritis, rheumatoid arthritis, psoriatic
arthritis or osteoarthritis, inflammatory bowel disease, multiple
sclerosis and metabolic diseases such as arteriosclerosis and type
I diabetes. Further indications include those responsive to
treatment with inhibitors of IL-1.beta., such as Muckle-Wells
syndrome, cryopyrin-associated periodic fever syndromes (CAPS),
familial Mediterranean fever, Still's disease, Behcet's disease and
diabetes mellitus.
[0038] Arthritis, including osteoarthritis, rheumatoid arthritis,
arthritic joints as a result of injury or crystal deposits, and the
like, are common inflammatory conditions which would benefit from
the therapeutic use of the anti-inflammatory proteins, such as
truncated IL-38 proteins of the present invention. For example,
rheumatoid arthritis (RA) is a systemic disease that affects the
entire body and is one of the most common forms of arthritis. It is
characterized by the inflammation of the membrane lining the joint,
which causes pain, stiffness, warmth, redness and swelling.
Inflammatory cells release enzymes that may digest bone and
cartilage. As a result of rheumatoid arthritis, the inflamed joint
lining, the synovium, can invade and damage bone and cartilage
leading to joint deterioration and severe pain amongst other
physiologic effects. The involved joint can lose its shape and
alignment, resulting in pain and loss of movement.
[0039] Rheumatoid arthritis (RA) is an immune-mediated disease
particularly characterized by inflammation and subsequent tissue
damage leading to severe disability and increased mortality. A
variety of cytokines are produced locally in the rheumatoid joints.
Numerous studies have demonstrated that IL-1 and TNF-alpha, two
prototypic pro-inflammatory cytokines, play an important role in
the mechanisms involved in synovial inflammation and in progressive
joint destruction. Indeed, the administration of TNF-alpha and IL-1
inhibitors in patients with RA has led to a dramatic improvement of
clinical and biological signs of inflammation and a reduction of
radiological signs of bone erosion and cartilage destruction.
However, despite these encouraging results, a significant
percentage of patients do not respond to these agents, suggesting
that other mediators are also involved in the pathophysiology of
arthritis (Gabay, Expert. Opin. Biol. Ther. 2(2):135-149. 2002;
Astry, J. Interferon Cytokine Res. 31(12):927-40. 2011).
[0040] For pharmaceutical use, the truncated IL-38 proteins of the
invention are formulated for parenteral, particularly intravenous
or subcutaneous, delivery according to conventional methods.
Intravenous administration will be by bolus injection, controlled
release, e.g, using mini-pumps or other appropriate technology, or
by infusion over a typical period of one to several hours. In
general, pharmaceutical formulations will include a hematopoietic
protein in combination with a pharmaceutically acceptable vehicle,
such as saline, buffered saline, 5% dextrose in water or the like.
Formulations may further include one or more excipients,
preservatives, solubilizers, buffering agents, albumin to provent
protein loss on vial surfaces, etc. When utilizing such a
combination therapy, the cytokines may be combined in a single
formulation or may be administered in separate formulations.
Methods of formulation are well known in the art and are disclosed,
for example, in Remington's Pharmaceutical Sciences. Gennaro, ed.,
Mack Publishing Co., Easton Pa., 1990, which is incorporated herein
by reference.
[0041] Therapeutic doses will generally be in the range of 0.1 to
100 mg/kg of patient weight per day, preferably 0.5-20 mg/kg per
day, with the exact dose determined by the clinician according to
accepted standards, taking into account the nature and severity of
the condition to be treated, patient traits, etc. Determination of
dose is within the level of ordinary skill in the art. The proteins
will commonly be administered over a period of up to 28 days. More
commonly, the proteins will be administered over one week or less,
often over a period of one to three days. In general, a
therapeutically effective amount of truncated IL-38 protein of the
present invention is an amount sufficient to produce a clinically
significant decrease of the pathological inflammatory response.
[0042] Generally, the dosage of administered truncated IL-38
protein will vary depending upon such factors as the patient's age,
weight, height, sex, general medical condition and previous medical
history. Typically, it is desirable to provide the recipient with a
dosage of truncated IL-38 protein which is in the range of from
about 1 pg/kg to 10 mg/kg (amount of agent/body weight of patient),
although a lower or higher dosage also may be administered as
circumstances dictate. Specific embodiments of the pharmaceutical
compositions of the invention are provided herein below.
[0043] In an additional aspect the present invention provides a
method of treating or preventing a pathological inflammatory
disorder in a subject in need of such a treatment. The method of
the invention may comprise the step of administering to said
subject a therapeutically active amount of any one of the herein
described compounds or compositions of the invention.
[0044] Another aspect of the invention pertains to a method for
modulating the immune response of a cell, the method comprising
contacting said cell with a truncated IL-38 protein of the
invention, or by expressing in said cell a nucleic acid according
to the invention.
[0045] In a preferred embodiment the method is an ex-vivo or
in-vitro method.
[0046] A "modulating the immune response" may be an inhibition of
JNK signalling, in particular the inhibition of IL-6 release and
TH17 generation. Inhibition of JNK signalling may be observed by
the use of a JNK reporter construct in said cell. One widely used
reporter is an AP-1 promoter driven reporter.
[0047] A cell to be used in the described method of the invention
is preferably a mammalian, most preferably a human cell. It is also
preferred that the cell is an immune cell, most preferably a
leucocyte, even more preferably a macrophage.
[0048] Yet another aspect of the invention relates to a method for
screening for modulators of the activity of truncated IL-38,
comprising the steps of [0049] a. Providing a cell, [0050] b.
Contacting said cell with microbe-associated molecular pattern
(MAMP), pathogen-associated molecular patterns (PAMP) or apoptotic
cell supernatants (ACM), [0051] c. Further contacting said cell
with a truncated IL-38 protein of the invention and a candidate
modulator, [0052] d. Determining JNK activation in said cell,
wherein an increase of JNK activation in said cell compared to a
control cell or reference value indicates that the candidate
modulator is an antagonist of truncated IL-38, and a decrease of
JNK activation compared to a control cell or reference indicates
that the candidate modulator is an agonist of truncated IL-38.
[0053] PAMPs or MAMPs in accordance with the invention may be
selected from bacterial lipopolysaccharide (LPS), bacterial
flagellin, lipoteichoic acid from Gram positive bacteria,
peptidoglycan, and nucleic acid variants normally associated with
viruses, such as double-stranded RNA (dsRNA), or unmethylated CpG
motifs.
Said cell to be used in the screening method of the invention
preferably expresses on the cell surface a receptor of truncated
IL-38, for example by ectopically expressing IL-1RAPL1 in said
cell.
[0054] A candidate modulator is preferably a small molecule, a
small nucleic acid, such as a small RNA, or a protein, such as an
antibody.
[0055] Said JNK activation is preferably determined by means of an
AP-1 reporter construct. Such reporter constructs may be luciferase
based, enzyme based or fluorescent protein based. Also the direct
JNK target gene expression may be determined, for example by
quantitative PCR (qPCR).
[0056] Furthermore the invention provides a modulator of the
activity of truncated IL-38 as identified by the herein described
method.
Diseases and Conditions
[0057] The present invention provides a truncated IL-38 protein
which can be used as a therapeutic in the treatment or prevention
of various diseases. In accordance with the present invention the
compounds and compositions are particularly useful for the
treatment and or prevention of a condition characterized by a
pathological activated immune or inflammatory response. In
particular the invention seeks to provide a treatment for
conditions characterized by a pathological activity of cytokines
such as interleukin-6 and interleukin-17, which were shown to be
involved in a wide series of chronic inflammatory and autoimmune
disorders.
[0058] In context of the present invention an autoimmune disease is
as a disorder that results from an autoimmune response. An
autoimmune disease is the result of an inappropriate and excessive
response to a self-antigen, or a pathological activation of immune
response signalling for example via cytokines. Examples of
autoimmune diseases include but are not limited to, Addision's
disease, alopecia greata, ankylosing spondylitis, autoimmune
hepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type
I), dystrophic epidermolysis bullosa, epididymitis,
glomerulonephritis, Graves' disease, Guillain-Barr syndrome,
Hashimoto's disease, hemolytic anemia, systemic lupus
erythematosus, multiple sclerosis, myasthenia gravis, pemphigus
vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis,
sarcoidosis, scleroderma, Sjogren's syndrome,
spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,
pernicious anemia, ulcerative colitis, among others.
Compositions and Kits for Treating or Preventing Autoimmune
Diseases
[0059] Another aspect of the present application relates to
compositions and kits for treating or preventing autoimmune or
inflammatory diseases. In one embodiment, the composition comprises
a compound such as a protein, nucleic acid, or recombinant cell as
described herein, optionally together with a pharmaceutically
acceptable carrier.
[0060] As used herein the language "pharmaceutically acceptable
carrier" is intended to include any and all solvents, solubilizers,
fillers, stabilizers, binders, absorbents, bases, buffering agents,
lubricants, controlled release vehicles, diluents, emulsifying
agents, humectants, lubricants, dispersion media, coatings,
antibacterial or antifungal agents, isotonic and absorption
delaying agents, and the like, compatible with pharmaceutical
administration. The use of such media and agents for
pharmaceutically active substances is well-known in the art. Except
insofar as any conventional media or agent is incompatible with the
active compound, use thereof in the compositions is contemplated.
Supplementary agents can also be incorporated into the
compositions. In certain embodiments, the pharmaceutically
acceptable carrier comprises serum albumin.
[0061] The pharmaceutical composition of the invention is
formulated to be compatible with its intended route of
administration. Examples of routes of administration include
parenteral, e.g., intrathecal, intra-arterial, intravenous,
intradermal, subcutaneous, oral, transdermal (topical) and
transmucosal administration.
[0062] Solutions or suspensions used for parenteral, intradermal,
or subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine; propylene glycol or other
synthetic solvents; antibacteial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfate; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
[0063] Pharmaceutical compositions suitable for injection use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the injectable
composition should be sterile and should be fluid to the extent
that easy syringability exists. It must be stable under the
conditions of manufacture and storage and must be preserved against
the contaminating action of microorganisms such as bacteria and
fungi. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyetheylene glycol, and
the like), and suitable mixtures thereof. The proper fluidity can
be maintained, for example, by the use of a coating such as
lecithin, by the maintenance of the requited particle size in the
case of dispersion and by the use of surfactants. Prevention of the
action of microorganisms can be achieved by various antibacterial
and antifungal agents, for example, parabens, chlorobutanol,
phenol, ascorbic acid, thimerosal, and the like. In many cases, it
will be preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, and sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0064] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a neuregulin) in the
required amount in an appropriate solvent with one or a combination
of ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the active compound into a sterile vehicle which contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and freeze-drying which yields a
powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0065] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Stertes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0066] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0067] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the
pharmaceutical compositions are formulated into ointments, salves,
gels, or creams as generally known in the art.
[0068] In certain embodiments, the pharmaceutical composition is
formulated for sustained or controlled release of the active
ingredient. Biodegradable, biocompatible polymers can be used, such
as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
e.g. Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions (including liposomes targeted to infected cells with
monoclonal antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art.
[0069] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein includes physically discrete units suited as unitary dosages
for the subject to be treated; each unit containing a predetermined
quantity of active compound calculated to produce the desired
therapeutic effect in association with the required pharmaceutical
carrier. The specification for the dosage unit forms of the
invention are dictated by and directly dependent on the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0070] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50. Compounds which exhibit
large therapeutic indices are preferred. While compounds that
exhibit toxic side effects may be used, care should be taken to
design a delivery system that targets such compounds to the site of
affected tissue in order to minimize potential damage to uninfected
cells and, thereby, reduce side effects.
[0071] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration utilized.
For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose may be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC50 (i.e., the concentration of the test compound which achieves a
half-maximal inhibition of symptoms) as determined in cell culture.
Such information can be used to more accurately determine useful
doses in humans. The pharmaceutical compositions can be included in
a container, pack, or dispenser together with instructions for
administration.
[0072] The present invention will now be further described in the
following examples with reference to the accompanying figures and
sequences, nevertheless, without being limited thereto. For the
purposes of the present invention, all references as cited herein
are incorporated by reference in their entireties. In the
Figures:
[0073] FIG. 1: IL-38 is secreted from apoptotic cells. (A) A549
human lung cancer cells, MDA-231 breast cancer cells, human primary
PBMCs and human primary neutrophils remained viable, were treated
with TNF-alpha (20 ng/ml)/CHX (10 .mu.M) to incudce apoptosis or
were incubated at 60.degree. C. for 30 min to induce necrosis.
Respective supernatants of viable (VCM), apoptotic (ACM) or
necrotic (NCM) cells were harvested and IL-38 levels were analyzed
by ELISA. Data are means.+-.SEM, n=5. (B) Secretion of IL-38 from
apoptotic A549 cells was analyzed by ELISA at the times indicated.
Data are means.+-.SEM, n=5. *p<0.05, ANOVA with Bonferroni's
correction.
[0074] FIG. 2: Apoptotic tumor cell-released IL-38 regulates
cytokine production in macrophages. Human macrophages were
stimulated for 24 h with (A) LPS (1 ng/ml) alone or in combination
with recombinant human IL-38 (rhIL-38) long/ml or with (B)
supernatants of apoptotic A549 cells (ACM) alone or in combination
with recombinant human IL-38 (rhIL-38) 10 ng/ml. Cytokine
production was measured using cytometric bead array, normalized
results are shown. Data are means.+-.SEM, n=5 (C) Human macrophages
were stimulated with supernatants of viable (VCM) or apoptotic A549
cells (ACM), which were previously transfected with non-targeting
siRNA (siCtrl), siRNA directed against IL-38 (siIL38) or an IL-38
overexpression vector (oeIL-38). Cytokine production was measured
using cytometric bead array, normalized results are shown. Data are
means.+-.SEM, n=5 (D) Human macrophages were transfected with an
AP1 reporter construct and luciferase activity was measured after
24 h stimulation with ACM from siCtrl and siIL-38 A549 cells.
Background measurements obtained from mock-transfected cells were
subtracted from each experimental value. Normalized results are
shown. Data are means.+-.SEM, n=5.*p<0.05, ANOVA with
Bonferroni's correction.
[0075] FIG. 3: IL-38 binds to IL-1R6 and IL-1RAPL1. (A,B) Human
macrophages were stimulated with LPS or ACM for 6 and 24 h. mRNA
expression (A) and cell surface protein expression (B) of IL-1R6
and IL-1RAPL1 were measured by RT-qPCR and FACS respectively. Data
are means.+-.SEM, n=5. (C,D) Macrophages were controls (Ctrl) or
incubated with 50 ng/ml IL-38 for 15 min on ice before staining
with anti-IL-1R6 or anti-IL-1RAPL1 and their respective
PE-conjugated secondary antibodies. Representative flow cytometry
histograms (C) and statistical quantification of median PE
intensity (D) are displayed. Data are means.+-.SEM, n=5. (E)
Binding kinetics of IL-38 to immobilized IL-1R6 and IL-1RAPL1. 96
well plates were coated with 0.5 .mu.g of human IL-1R6 and
IL-1RAPL1 extracellular domain-Fc chimeras and incubated with
increasing amounts of human recombinant IL-38 as indicated. IL-38
binding to the extra-cellular domain of the receptors was detected
using biotinylated monoclonal IL-38 antibody. Data are
means.+-.SEM, n=5.*p<0.05, ANOVA with Bonferroni's
correction.
[0076] FIG. 4: Role of IL-1R6 and IL-1RAPL1 in cytokine production.
Human macrophages were transfected with non-targeting siRNA
(siCtrl) or siRNA directed against (A) IL-1RAPL1 (siIL1RAPL1) or
(B) IL-1R6 (siIL1R6) and stimulated with VCM and ACM for 24 h.
Cytokine production was measured using cytometric bead array,
normalized results are shown. Data are means.+-.SEM, n=5.
*p<0.05, ANOVA with Bonferroni's correction.
[0077] FIG. 5: IL-38 regulates the Th17 response. Human T-cells
were activated with anti-CD.sup.3/anti-CD28 beads, stained with
eFluor 670 and stimulated with the supernatant of macrophages
previously stimulated with ACM from control A549 cells
(ACMshCtrl/M.PHI.) or IL-38 knock-down A549 cells
(ACMshIL-38/M.PHI.). After 7 days (A) cytokine production and (B,C)
cell proliferation was measured. (A) Cytokines were quantified
using cytometric bead array, normalized results are shown. Data are
means.+-.SEM, n=10. T-cell proliferation was determined by
following eFluor 670 dilution. (B) Statistical quantification of
all proliferating T cells and (C) representative flow cytometry
histograms are displayed. Data are means.+-.SEM, n=10. *p<0.05,
ANOVA with Bonferroni's correction.
[0078] FIG. 6: IL-38 is truncated at the N-Terminus. (A) The
characteristic consensus motif of the IL-36 family in IL-38, which
defines the putative cleavage site of this cytokine at the
N-terminus, is displayed. (B) C-terminally myc-tagged IL-38 was
over-expressed in A549 cells, which were then used for ACM
production. After immunoprecipitating the over-expressed IL-38
using anti-myc coated beads, 2D gel electrophoresis was performed
(isoelectric focusing at pH 4-7, followed by polyacrylamide gel
separation), and a monoclocal anti-myc antibody was used to detect
the immunoprecipitated IL-38 upon protein transfer onto
nitrocellulose. (C) Coomasie-stained 2D gels were used for picking
putative IL-38 spots, which were analyzed by mass spectrometry.
Identified IL-38 N-terminal peptides for the different IL-38 spots
are displayed.
[0079] FIG. 7: Full length and truncated IL-38 have opposite roles
in cytokine production and bind to IL-1RAPL1. (A,C) Human
macrophages were (A) untreated or (C) previously transfected with
non-targeting siRNA (siCtrl) or siRNA directed against IL-1RAPL1
(siIL1RAPL1) and stimulated for 6 h with recombinant human
IL-1.beta. 50 ng/ml alone or in combination with different
concentrations of recombinant human full length (IL-38aa1-152) or
cleaved (IL-38aa20-152) IL-38. After 24 h IL-6 concentration in the
supernatants was measured using cytometric bead array, normalized
results are shown. Data are means.+-.SEM, n=7. (B) Binding kinetics
of full length and cleaved IL-38 to immobilized IL-1RAPL1. 96 well
plates were coated with 0.5 .mu.g of human IL-1RAPL1 extra-cellular
domain-Fc chimeras and incubated with increasing amounts of human
recombinant IL-38aa1-152 or IL-38aa20-152 as indicated. IL-38
binding to the extracellular domain of the receptors was detected
using biotinylated monoclonal IL-38 antibody. Data are
means.+-.SEM, n=5.*p<0.05, ANOVA with Bonferroni's
correction.
[0080] FIG. 8: IL-1RAPL1-induced signalling pathways regulated by
IL-38 in HEK cells. HEK cells were co-transfected with an IL-1RAPL1
over-expression plasmid in combination with (A,D) AP1, (B,E)
NF.kappa.B or (C) IL-6 reporter constructs. HEK cells transfected
with the reporter constructs together with an empty plasmid instead
the IL-1RAPL1 overexpression plasmid were used as controls (Mock).
(A,B) HEK cells were stimulated with IL-1.beta. (50 ng/ml) for 24 h
and the AP1 or NF.kappa.B activity was measured. Normalized results
are shown. Data are means.+-.SEM, n=15. (C) IL-6 reporter
constructs with point mutations in the indicated transcription
binding sites were used. After transfection, cells were incubated
for additional 24 h and IL-6 promoter-dependent luciferase activity
was measured. Results are expressed as fold induction relative to
Mock transfected cells. Data are means.+-.SEM, n=10. (D,E) After
transfection, fresh medium (Ctrl) or different concentrations of
IL-38aa1-152 or IL-38aa20-152 were added and cells were incubated
for additional 24 h. Results are expressed as fold induction
relative to Mock transfected cells. Data are means.+-.SEM, n=15.
(F) IL1RAPL1 was overexpressed in HEK cells and after transfection
fresh medium (Ctrl) or IL-38aa1-152 or IL-38aa20-152 (25 ng/ml)
were added to the cells followed by incubation for additional 24 h.
Intracellular staining of phosphorylated JNK and p38 was perfomed
and measured by FACS. Results are expressed as fold induction
relative to Mock transfected cells. Data are means.+-.SEM, n=5.
*p<0.05, ANOVA with Bonferroni's correction.
[0081] FIG. 9: IL-38 regulates AP1 activity in macrophages. Human
macrophages were transfected with an empty vector, AP1, NF.kappa.B
or IL-6 reporter constructs. After transfection, macrophages were
stimulated for 24 h with IL-1.beta. (50 ng/ml) alone or in
combination with IL-38aa1-152 or IL-38aa20-152 (20 ng/ml).
Luciferase activity was measured. Background measurements obtained
from mock-transfected cells were subtracted from each experimental
value. Normalized results are shown. Data are means.+-.SEM, n=7.
*p<0.05, ANOVA with Bonferroni's correction.
TABLE-US-00001 SEQ ID NO: 1
MCSLPMARYYIIKYADQKALYTRDGQLLVGDPVADNCCAEKICILPNRGL
ARTKVPIFLGIQGGSRCLACVETEEGPSLQLEDVNIEELYKGGEEATRFT
FFQSSSGSAFRLEAAAWPGWFLCGPAEPQQPVQLTKESEPSARTKFYFEQ SW SEQ ID NO: 2
LYTRDGQLLVGDPVADNCCAEKICILPNRGLARTKVPIFLGIQGGSRCLA
CVETEEGPSLQLEDVNIEELYKGGEEATRFTFFQSSSGSAFRLEAAAWPG
WFLCGPAEPQQPVQLTKESEPSARTKFYFEQSW
EXAMPLES
Example 1
IL-38 is Released From Apoptotic Cells
[0082] When performing an in-house ELISA to determine IL-38 levels
produced by tumor cell lines, the inventors noticed that induction
of apoptotic cell death markedly increased IL-38 secretion into the
supernatant. Compared to the supernatant of viable A549 lung cancer
or MDA.231 breast cancer cells (VCM), apoptotic cell supernatants
(ACM), but not necrotic cell supernatants (NCM) contained
approximately 10 fold higher levels of IL-38 (FIG. 1A). This was
also the case for primary human neutrophils or PBMCs, although the
increase of IL-38 release during apoptosis was not as strong (FIG.
1A). In order to analyze the kinetics of IL-38 secretion, the
concentration of IL-38 in A549 supernatants was measured at
different time points upon apoptosis induction. Enhanced IL-38
secretion was observed after 12 h following apoptosis induction
(FIG. 1B), coinciding with the occurrence of apoptosis markers in
A549 cells (data not shown).
Example 2
IL-38 Regulates Cytokine Production After ACM Stimulation
[0083] Apoptotic cell-derived mediators have the potential to
modulate phagocyte responses, including cytokine production (26).
The inventors analyzed the role of IL-38 in the production of a
panel of cytokines that are produced upon macrophage activation by
LPS or upon interaction with apoptotic cells (27). Of these, IL-6
and IL-8 production were regulated by IL-38. Addition of
recombinant IL-38 to LPS-stimulated macrophages increased IL-6 and
IL-8 production compared with LPS alone (FIG. 2A). Interestingly,
when human macrophages were stimulated with ACM of A549 cells alone
or in combination with recombinant human IL-38, the opposite effect
was observed (FIG. 2B). IL-38 suppressed ACM-induced IL-6 and IL-8
secretion from macrophages. Since ACM already contained IL-38, the
inventors wondered whether endogenous IL-38 affected cytokine
macrophage cytokine production. To answer this question, IL-38 was
over-expressed or knocked down in A549 cells before generating ACM.
Indeed, stimulation of human macrophages with ACM produced from
IL-38-overexpressing A549 cells resulted in reduced secretion of
IL-6 and IL-8, whereas stimulation with ACM of IL-38 knock-down
A549 cells yielded higher IL-6 and IL-8 concentrations (FIG. 2C).
Among the prominent transcription factors that regulate cytokine
production and are regulated by the IL-1 family are NF.kappa.B and
AP1. As NF.kappa.B is blocked after interaction of macrophages with
apoptotic cells (28), the inventors asked whether endogenous IL-38
regulated AP1 activation in response to ACM. When applying ACM of
IL-38 knock-down A549 cells in comparison to control ACM to
macrophage transfected with an AP1 luciferase reporter construct,
the inventors noticed that ACM containing lower levels of IL-38
induced a more pronounced AP1 activation (FIG. 2D). In conclusion,
endogenous IL-38 restricted inflammatory macrophage activation in
response to apoptotic cell supernatants.
Example 3
IL-38 Antagonizes IL1RAPL1-Dependent Cytokine Production in
Response in ACM
[0084] The inventors hypothesized that IL-38 inhibits ACM-induced
cytokine production by acting as a receptor antagonist. Therefore,
the inventors analyzed candidates of the IL-1 receptor family for
their association with IL-38. It was shown that IL-38 binds to the
IL-1R6 (19) and the inventors observed that the orphan receptor
IL-1RAPL1 regulates cytokine production in macrophages after ACM
stimulation (16). The inventors first determined the expression of
IL1R6 and IL-1RAPL1 in macrophages was determined at mRNA level
using qPCR (FIG. 3A) and at the level of cell surface availability
by FACS (FIG. 3B) after ACM or LPS stimulation. IL1R6 expression
was generally low (FIG. 3C) and was further down-regulated at the
mRNA level after ACM or LPS stimulation, which was nevertheless not
apparent at the cell surface expression level. Contrarily,
IL-1RAPL1 expression was abundant (FIG. 3C) and was further induced
both at the mRNA level as well as on the cell surface at 6 h
following LPS and at 6 h and 24 h following ACM treatment (FIG. 3A,
B). Moreover, IL-1RAPL1 expression at the cell surface was reduced
after 24 h stimulation with LPS. These experiments suggested
IL-1RAPL1 at least as an additional candidate for the action of
IL-38. Next, the inventors analyzed whether IL-38 would bind to
IL-1RAPL1 by performing both competition assays and receptor
binding assays. For competition assays, human macrophages were
incubated with recombinant human IL-38 before analyzing surface
expression of IL-1R6 or IL-1RAPL1. Based on the low level of IL-1R6
surface expression, it was difficult to see to observe differences
in cell surface expression due to IL-38 pre-incubation (FIG. 3C,
D). However, for IL-1RAPL1 the inventors observed that IL-38
competed with the antibody used for the FACS staining (FIG. 3C, D),
indicating that IL-38 may bind to IL-1RAPL1. To validate these
results, direct receptor binding assays were performed. Plates were
coated with IL-1R6-Fc and IL-1RAPL1-Fc chimeras, different IL-38
concentrations were added to the wells and the bound IL-38 was
visualized. As shown recently (19) IL-38 indeed bound to IL-1R6
(FIG. 3E). Moreover, IL-38 also bound to IL-1RAPL1 (FIG. 3E). As
these results suggested that IL-38 might regulate cytokine
production by binding to IL-1RAPL1, the inventors asked for the
role of IL-1RAPL1 in ACM-induced cytokine production. Transient
knock-down of IL-1R6 or IL-1RAPL1 was performed in human
macrophages and IL-6 and IL-8 levels in macrophage culture
supernatants were measured after ACM stimulation. IL-6 and IL-8
production after ACM stimulation were IL-1RAPL1 dependent (FIG.
4A), whereas IL-1R6 was not involved in cytokine production in the
inventor's model (FIG. 4B).
Example 4
IL-38 Regulates Th17 Responses
[0085] Next the inventors asked for downstream consequences of
IL-38-dependent suppression of cytokine production from macrophages
by analyzing the effect of macrophages supernatants on T cell
activation. The inventors isolated primary human T-cells,
stimulated them with antiCD.sup.3/antiCD28 beads and incubated them
repeatedly with supernatants of macrophages previously stimulated
with ACM and with ACM of IL-38 knock-down A549 cells. IL-10, IL-17
and IFN-.gamma. levels were measured in the supernatants of the
T-cells after seven days of culture. When macrophages were
stimulated with ACM, their supernatants reduced IFN-.gamma. and
IL-10 production by T cells and slighty elevated IL-17 levels (FIG.
5A). Nevertheless, when macrophages were stimulated with ACM
lacking IL-38, their supernatants strongly elevated IL-17
production by T cells and further decreased IL-10 concentrations
(FIG. 5A). These effects were independent of differences in T cell
proliferation. Treatment with ACM did not affect the number of
proliferating T cells (FIG. 5B), although it affected the number of
divisions pre dividing T cells, which might explain the reduced
IFN-.gamma. and IL-10 levels (FIG. 5C). However, there was no
difference in T cell proliferation whether ACM contained IL-38 or
not (FIG. 5B, C). These data show that IL-38 from apoptotic cells
restricts the macrophage-dependent generation of Th17 cells and
maintains IL-10 expression.
Example 5
IL-38 is N-Terminally Processed in Apoptotic Cells
[0086] Except for IL-1Ra all members of the IL-1 family are
produced as precursors, which need to be cleaved at the N-terminus
in order to reach full activity. Recently, according to the size of
the N-terminal pro-domain, IL-38 was classified into the IL-36
subfamily (4, 19). IL-38, as the other members of this subfamily,
possesses a consensus motif, which putatively determines the
N-terminal cleavage site (FIG. 6A). In order to determine whether
or not apoptosis induced IL-38 processing, C-terminally myc-tagged
IL-38 was overexpressed in tumor cells and ACM was produced from
these cells. After immunoprecipitating IL-38 2D gel electrophoresis
was performed to visualize IL-38 isoforms. Two IL-38 isoforms were
successfully identified in the gel, indicating that IL-38 is indeed
processed during apoptosis (FIG. 6B). The two spots representing
putative IL-38 isoforms were picked and analyzed by mass
spectrometry (MS). In the spot with higher molecular weight,
predicted as full length IL-38, two N-terminal peptides were found
in the MS analysis, one from amino acid 9 to 18, and the second one
from amino acid 24 to 41, whereas in the sample with lower
molecular weight only the peptide from amino acid 24 to 41 was
found (FIG. 6C). Thus, IL-38 is N-terminally processed in apoptotic
cells.
Example 6
Full Length and Truncated IL-38 Exert Opposite Roles on the
Regulation of Cytokine Production Through IL-1RAPL1
[0087] In order to determine whether full-length and truncated
IL-38 have a different biological activity, IL-6 concentration in
the supernatants of human macrophages stimulated with IL-1(3, alone
or in combination with different concentrations of the full-length
(IL-38aa1-152) or truncated (IL-38aa20-152) IL-38, was determined.
After IL-.beta. stimulation, higher concentrations of IL-38aa1-152
(20 ng/ml, 10 ng/ml) significantly increased IL-6 production,
whereas IL-38aa20-152 decreased IL-6 production even when applied
at low concentration (FIG. 7A). Since IL-38 in ACM regulated IL-6
production by interacting with IL-1RAPL1, the inventors asked
whether both IL-38 isoforms, which have opposite roles in cytokine
production, bind to IL-1RAPL1. The inventors performed a receptor
binding assay as explained above. Both IL-38 isoforms bound to
IL-1RAPL1 in this assay (FIG. 7B). However, binding kinetics seemed
to differ slightly. When considering that even though IL-38aa1-152
and IL-38aa20-152 exert opposite roles on cytokine production, they
are both able to bind to IL-1RAPL1, another key point to analyze
was whether or not the effects on IL-6 production were both
IL-1RAPL1 dependent. To achieve this, a transient IL-1RAPL1
knock-down was performed in macrophages and IL-6 concentration in
the supernatants was measured after stimulation with IL-1.beta.
alone or in combination with IL-38aa1-152 or IL-38aa20-152.
IL-1RAPL1 knock-down in macrophages abrogated both, IL-6 induction
by full-length 11-38 and IL-6 suppression by truncated IL-38 (FIG.
7C).
Example 7
IL-38 Regulates the IL-1RAPL1-Activated Pathway JNK/AP1
[0088] The inventors obtained evidence that IL-38 regulates AP-1 in
macrophages upon interaction with apoptotic cells (FIG. 2D). To
analyze the signaling pathways that are affected by IL-38 in
relation to its interaction with IL-1RAPL1, the inventors first
utilized a receptor-over-expression model with HEK 293T cells. The
cells were co-transfected with an over-expression construct for
IL-1RAPL1 and AP1 or NF.kappa.B reporter constructs. HEK cells
transfected with the reporter constructs but without
over-expression of IL-1RAPL1 were used as control. First, to
characterize the model IL-1RAPL1 over-expressing cells and control
cells were stimulated with IL-1.beta., and AP1 (FIG. 8A) or
NF.kappa.B (FIG. 8B) activity was measured. IL-1.beta. was used as
a low-affinity ligand for the orphan receptor IL-1RAPL1 (14). After
IL-1.beta. stimulation, a significant induction of NF.kappa.B but
not AP1 activity was observed in control cells. Nevertheless, when
IL-1RAPL1 was over-expressed an activation of AP1 as well as
enhanced NF.kappa.B activity was observed. Thus, IL-1.beta. alone
induces NF.kappa.B activation in an IL-1RAPL1-independent manner,
but not AP1 activation, which required IL-1RAPL1. Interestingly,
even without any stimulus, the presence of IL-1RAPL1 was sufficient
to increase of AP1 and NF.kappa.B activities compared to control
cells (FIG. 8A,B). IL-1RAPL1 therefore activates AP1, but not for
NF.kappa.B, after IL-1.beta. stimulation in HEK cells, but induces
AP1 and NF.kappa.B activation upon overexpression without addition
of an exogenous ligand. To confirm this, IL-6 promoter constructs
with or without point mutations in different transcription factor
binding sites (AP1, NF.kappa.B, CREB and CEBP.beta.) were used. HEK
cells were co-transfected with an IL1RAPL1 over-expression plasmid
and IL-6 reporter constructs (29). Also in this set-up,
over-expression of IL-1RAPL1 activated the IL-6 promoter compared
with HEK control cells. This IL-6 promoter induction was abrogated
when the AP1 and NF.kappa.B binding sites were mutated (FIG. 8C).
This suggests the presence of an endogenous ligand for IL-1RAPL1
that produced by HEK cells. Next the inventors asked whether
IL-38aa1-152 and IL-38aa20-152 were able to affect AP1 and
NF.kappa.B activity downstream of IL-1RAPL1. NF.kappa.B promoter
activity in this set-up was not regulated by IL-38 (FIG. 8D), but
AP1 induction was negatively regulated by both IL-38 isoforms (FIG.
3E). Importantly, IL-38aa20-152 was able to regulate the AP1
induction at lower concentrations compared to the full-length
protein. To analyze the signaling pathways leading to
IL-38-dependent suppression of IL-1RAPL-induced AP1 activity,
intracellular staining of phosphorylated JNK and p38 was performed
in IL-1RAPL1 over-expressing cells compared with control HEK cells.
After IL-1RAPL1 over-expression an induction in phosphorylated JNK
but not p38 was observed. This induction was significantly reduced
by IL-38aa20-152 but not by IL-38aa1-152, confirming the stronger
regulatory role of truncated IL-38.
Example 8
IL-38 Regulates AP1 Activity in Macrophages
[0089] Next, the inventors transferred the inventor's data from the
HEK model into the macrophage setting. Human macrophages were
transfected with AP1 or NF.kappa.B reporter constructs and
stimulated with IL-1.beta. alone or in combination with
IL-38aa20-152 or IL-38aa1-152. As in HEK cells, IL-38aa20-152
decreased AP1, but not NF.kappa.B activity in macrophages, whereas
IL-38aa1-152 was ineffective (FIG. 9). Thus, only truncated IL-38
suppressed AP-1 activity in macrophages, which is in concordance
with the inventor's finding that macrophages stimulated with
apoptotic cell supernatants lacking IL-38 showed higher levels of
AP1 activity (FIG. 2D). Finally the inventors approached the
question, why IL-38aa1-152 increased IL-6 production after
IL-1.beta. stimulation of macrophages (FIG. 7A), whereas in the HEK
cell model, IL-38aa1-152 did neither increase AN nor NF.kappa.B
activation. To investigate this discrepancy macrophages were
transfected with an IL-6 reporter construct and stimulated with
IL-1.beta. alone or in combination with both IL-38 isoforms. As
expected, IL-38aa20-152 reduced IL-6 promoter activity, but
IL-38aa1-152 did not affect IL-6 promoter induction at all (FIG.
9), suggesting that the IL-38aa1-152 mediated increase of IL-6
production was not transcriptionally regulated.
[0090] In conclusion, the present invention shows an N-terminally
processed IL-38 which can be used in the clinic for limiting
auto-inflammation in general or resulting, e.g., from defective
interaction of macrophages with apoptotic cells.
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Sequence CWU 1
1
21152PRTHomo sapiens 1Met Cys Ser Leu Pro Met Ala Arg Tyr Tyr Ile
Ile Lys Tyr Ala Asp1 5 10 15Gln Lys Ala Leu Tyr Thr Arg Asp Gly Gln
Leu Leu Val Gly Asp Pro 20 25 30Val Ala Asp Asn Cys Cys Ala Glu Lys
Ile Cys Ile Leu Pro Asn Arg 35 40 45Gly Leu Ala Arg Thr Lys Val Pro
Ile Phe Leu Gly Ile Gln Gly Gly 50 55 60Ser Arg Cys Leu Ala Cys Val
Glu Thr Glu Glu Gly Pro Ser Leu Gln65 70 75 80Leu Glu Asp Val Asn
Ile Glu Glu Leu Tyr Lys Gly Gly Glu Glu Ala 85 90 95Thr Arg Phe Thr
Phe Phe Gln Ser Ser Ser Gly Ser Ala Phe Arg Leu 100 105 110Glu Ala
Ala Ala Trp Pro Gly Trp Phe Leu Cys Gly Pro Ala Glu Pro 115 120
125Gln Gln Pro Val Gln Leu Thr Lys Glu Ser Glu Pro Ser Ala Arg Thr
130 135 140Lys Phe Tyr Phe Glu Gln Ser Trp145 1502133PRTHomo
sapiens 2Leu Tyr Thr Arg Asp Gly Gln Leu Leu Val Gly Asp Pro Val
Ala Asp1 5 10 15Asn Cys Cys Ala Glu Lys Ile Cys Ile Leu Pro Asn Arg
Gly Leu Ala 20 25 30Arg Thr Lys Val Pro Ile Phe Leu Gly Ile Gln Gly
Gly Ser Arg Cys 35 40 45Leu Ala Cys Val Glu Thr Glu Glu Gly Pro Ser
Leu Gln Leu Glu Asp 50 55 60Val Asn Ile Glu Glu Leu Tyr Lys Gly Gly
Glu Glu Ala Thr Arg Phe65 70 75 80Thr Phe Phe Gln Ser Ser Ser Gly
Ser Ala Phe Arg Leu Glu Ala Ala 85 90 95Ala Trp Pro Gly Trp Phe Leu
Cys Gly Pro Ala Glu Pro Gln Gln Pro 100 105 110Val Gln Leu Thr Lys
Glu Ser Glu Pro Ser Ala Arg Thr Lys Phe Tyr 115 120 125Phe Glu Gln
Ser Trp 130
* * * * *