IL-37 variants

Abstract

The present invention relates to polypeptides comprising variants of interleukin-37 (IL-37), and related therapeutic agents and compositions. The present invention also relates to the use of polypeptides and compositions in methods of treating inflammatory diseases or disorders. The present invention provides monomeric anti-inflammatory polypeptides comprising the amino acid sequence of an IL-37 monomer having a mutation or modification that prevents the anti-inflammatory peptide from forming a homodimer.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to Australian provisional applications 2015902262 and 2016900703, the entire contents of which are incorporated herein by reference. Invention Field

[0003] This invention relates to polypeptides, including variants of interleukin-37 (IL-37), and related therapeutic agents and compositions. The invention also relates to the use of polypeptides and compositions in methods of treating inflammatory diseases or conditions. Background of the Invention

[0005] Inflammation plays a fundamental role in host defense and the progression of immune-mediated diseases. The inflammatory response is initiated in response to tissue damage (e.g., trauma, local ischemia, and foreign particles) and infection, by a complex cascade of events including chemical mediators (e.g., cytokines and prostaglandins) and inflammatory cells (e.g., leukocytes). The inflammatory response is characterized by increased blood flow, increased capillary permeability, and the influx of phagocytes. These events lead to swelling, redness, warmth (altered heat patterns), and purulent formation at the site of injury.

[0006] The interaction between humoral and cellular immune elements in the inflammatory response enables the elimination of harmful substances and the initiation of repair of damaged tissues. When this interaction is disrupted, the inflammatory response can lead to considerable damage to normal tissues due to uncontrolled inflammation, requiring clinical intervention to prevent tissue damage and organ dysfunction.

[0007] Interleukin-37 (IL-37) is a member of the IL-1 cytokine family and possesses unique and broad-spectrum anti-inflammatory effects in both innate and adaptive immunity. IL-37 specifically reduces inflammation through the mediation of pro-inflammatory cytokines such as IL-1β and TNF via their receptors and Toll-like receptor ligands, and has broad-spectrum protective effects against inflammation triggered by infection or other non-infectious attacks. IL-37 initiates signal transduction at the cell membrane through interactions with IL-18 receptor α and IL-1R8 (Sigirr), and also initiates intracellular signal transduction through interactions with Smad3. However, the understanding of the structural, functional, and regulatory relationships of IL-37 activity is currently lacking.

[0008] There is a need for improved anti-inflammatory compositions and treatments.

[0009] Any reference to prior art in this specification is not an admission or implication that such prior art forms part of common general knowledge in any jurisdiction, or that such prior art can be reasonably expected to be understood, considered relevant, and / or combined with other prior art known to those skilled in the art. Invention Overview

[0011] The present invention provides a monomeric anti-inflammatory polypeptide comprising the amino acid sequence of an IL-37 monomer, wherein the amino acid sequence has a mutation or modification that prevents the anti-inflammatory polypeptide from forming a homodimer.

[0012] The present invention provides an anti-inflammatory polypeptide comprising the amino acid sequence of an IL-37 polypeptide, wherein the amino acid sequence has mutations or modifications that reduce the ability of the anti-inflammatory polypeptide to form dimers.

[0013] Preferably, the mutation or modification reduces or prevents the formation of a dimerization interface that enables the dimerization of IL-37 monomers. Typically, the mutation or modification is located in a region of the peptide that has the same amino acid sequence as the dimerization interface forming the IL-37 monomer. The mutation or modification may be located in the β3 or β4 ring of the dimerization interface forming the IL-37 monomer.

[0014] The present invention provides a polypeptide comprising the amino acid sequence of an IL-37 polypeptide or a fragment thereof, wherein the polypeptide has a reduced ability to form dimers compared to a polypeptide having the sequence of SEQ ID NO: 1.

[0015] The present invention provides a polypeptide comprising an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1, wherein the polypeptide has a reduced ability to form dimers compared to the polypeptide having the sequence of SEQ ID NO: 1.

[0016] The present invention provides an IL-37 polypeptide that has a reduced ability to form dimers compared to polypeptides having the sequence of SEQ ID NO: 1.

[0017] This invention provides a polypeptide comprising the amino acid sequence of an IL-37 polypeptide or a fragment thereof, wherein the polypeptide has a reduced ability to form dimers compared to a polypeptide having the sequence of SEQ ID NO: 1, wherein the polypeptide has a mutation or modification of at least one residue at a position equivalent to the β3-β4 ring region in SEQ ID NO: 1. Preferably, the β3-β4 ring region is composed of residues 83 to 91 of SEQ ID NO: 1 or residues equivalent thereto. Preferably, the mutation or modification occurs at or equivalent to residues at positions K83, N84, Y85, I86, R87 and / or P88. Preferably, the mutation is a substitution of a non-conserved residue; more preferably, the mutation is a substitution of an alanine or an amino acid with an opposite charge.

[0018] This invention provides a polypeptide comprising the amino acid sequence of an IL-37 polypeptide or a fragment thereof, wherein the polypeptide has a reduced ability to form dimers compared to a polypeptide having the sequence of SEQ ID NO: 1, wherein the polypeptide has a mutation or modification at residues at or equivalent to the positions of D73, K83, and Y85. Preferably, the mutation is a substitution of a non-conserved amino acid residue; more preferably, the mutation is a substitution of alanine or an amino acid with an opposite charge. More preferably, the mutation is any one or more of D73A, D73K, K83E, K83A, and Y85A.

[0019] This invention provides a polypeptide comprising the amino acid sequence of an IL-37 polypeptide or a fragment thereof, wherein the polypeptide has a reduced ability to form dimers compared to a polypeptide having the sequence of SEQ ID NO: 1, wherein the polypeptide has mutations or modifications at residues at or equivalent to the positions of V71, V80, and I78. Preferably, the mutation is a substitution of a non-conserved residue; more preferably, the mutation is a substitution of an alanine or an amino acid with an opposite charge.

[0020] This invention provides an IL-37 peptide that has a reduced ability to form dimers compared to a peptide having the sequence of SEQ ID NO: 1, wherein the peptide has modifications of a region equivalent to the β3-β4 ring region in SEQ ID NO: 1. Modifications include residues of deleted rings, shortened rings, extended rings, mutant rings, and / or chemically modified ring residues.

[0021] The present invention provides a monomeric polypeptide comprising an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1.

[0022] This invention provides isolated, recombinant, or synthetic IL-37 peptides existing as monomers.

[0023] This invention provides polypeptides comprising a paralogous or orthologous sequence of the sequence shown in SEQ ID NO: 1, wherein the polypeptide exists as a monomer or has a reduced ability to form dimers compared to the polypeptide having the sequence of SEQ ID NO: 1. Preferably, the polypeptide comprising a paralogous or orthologous sequence of the sequence shown in SEQ ID NO: 1 has a mutation or modification in the residues located at the dimerization interface, the dimerization interface being equivalent to the dimerization interface described herein for SEQ ID NO: 1, such as the residues in Table 1.

[0024] The polypeptides of the present invention may be isolated, purified, substantially purified, enriched, synthetic, or recombinant.

[0025] As used in this article, reduced ability to form dimers can refer to reduced ability to form heterodimers and / or homodimers.

[0026] The present invention also provides a polypeptide comprising, substantially consisting of or composed of, the amino acid sequence of the IL-37 polypeptide or a fragment thereof, wherein said amino acid sequence contains at least one mutation or modification at the position of a residue at the dimer interface of SEQ ID NO: 1, or equivalent thereto. The dimer interface associated with the polypeptide having the amino acid sequence of SEQ ID NO: 1 includes the residues in Table 1. Typically, said polypeptide comprises an amino acid sequence containing a mutation or modification at the positions of residues at or equivalent to positions 73, 83, and 85 of SEQ ID NO: 1. More preferably, the mutation is any one or more of D73A, D73K, K83E, K83A, and Y85A.

[0027] This invention provides isolated, recombinant, or synthetic IL-37 peptides that do not have the ability to form dimers or have a reduced ability to form dimers. In this or any other aspect of the invention described herein, the ability of the peptide to form dimers can be determined by any of the methods described herein, including size exclusion chromatography and multi-angle light scattering, analytical gel electrophoresis under non-denaturing conditions, analytical centrifugation, mass spectrometry, or reversed-phase high-performance liquid chromatography (RP-HPLC).

[0028] The reduction in dimer formation can be compared to that of a reference peptide. The reference peptide is typically a natural, wild-type, unmodified, or unmutated IL-37 peptide. Preferably, the reference peptide has the amino acid sequence of SEQ ID NO: 1. Alternatively, if the peptide of the present invention has a paralogous or orthologous amino acid sequence to SEQ ID NO: 1, the reference peptide is a natural, wild-type, unmodified, or unmutated paralogous or orthologous amino acid sequence.

[0029] The present invention provides an amino acid sequence comprising at least 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1, wherein amino acid residues are at or equivalent to the following positions:

[0030] Position 71 in SEQ ID NO:1 is not Val;

[0031] Position 72 in SEQ ID NO:1 is not Leu;

[0032] Position 73 in SEQ ID NO:1 is not Asp;

[0033] Position 74 in SEQ ID NO:1 is not Ser;

[0034] Position 78 in SEQ ID NO:1 is not Ile;

[0035] Position 80 in SEQ ID NO:1 is not Val;

[0036] Position 83 in SEQ ID NO:1 is not Lys;

[0037] Position 84 in SEQ ID NO:1 is not Asn;

[0038] Position 85 in SEQ ID NO:1 is not Tyr;

[0039] Position 86 in SEQ ID NO:1 is not Ile;

[0040] Position 87 in SEQ ID NO:1 is not Arg;

[0041] Position 88 in SEQ ID NO:1 is not Pro; and / or

[0042] Position 184 in SEQ ID NO:1 is not Asn. Preferably, the amino acid residue is a non-conservative substitution relative to the amino acid appearing at that position in SEQ ID NO:1. In one embodiment, the amino acid at position 85 is alanine, the amino acid at position 83 is glutamic acid, the amino acid at position 73 is alanine, and / or the amino acid at position 73 is lysine. In one embodiment, amino acid residues at positions 71, 72, 73, 74, 78, 80, 83, 84, 85, 86, 87, 88, and / or 184 in SEQ ID NO:1, or at equivalent positions, are deleted.

[0043] With respect to any polypeptide of the present invention described herein, the polypeptide may have an N-terminus truncated from residues 1 to 20 or 1 to 45 or equivalent to those in SEQ ID NO: 1.

[0044] Any peptide of the present invention described herein may exhibit greater anti-inflammatory properties compared to peptides having the amino acid sequence of SEQ ID NO: 1, unmodified or unmutated IL-37 amino acid sequences, or IL-37 peptides that do not exhibit a reduced ability to form dimers. Anti-inflammatory properties can be determined by the assays described herein, particularly those in the examples, including Examples 1, 3, and 4. In one embodiment, when tested in the assays described herein, including the LPS-stimulated IL-1β assays described in Examples 1 and 3, the peptides of the present invention exhibit anti-inflammatory properties at concentrations less than or about 1 μg / ml, 100 ng / ml, 10 ng / ml, 100 pg / ml, 10 pg / ml, or 1 pg / ml. Inflammatory lesions may include ligands of Toll-like receptors (TLRs) 2, 4, 7, 8, and / or 9, such as HKLM, imiquimod, CpG-A, or ssRNA40. When tested in the LPS-stimulated IL-1β assays described in Examples 1 and 3, the peptides of the present invention can reduce IL-1β by at least 30% at 10 pg / ml, at least 40% at 10 ng / ml, or at least about 50% at 1 pg / ml, 10 pg / ml, 100 pg / ml, 10 ng / ml, or 100 ng / ml. Reductions in other pro-inflammatory cytokines such as IL-6 can also be observed. When tested at 10 ng / ml and 100 ng / ml in the LPS-stimulated IL-1β assays described herein, including those in Examples 1 and 3, the peptides of the present invention may not show statistically significant differences in anti-inflammatory properties. The peptides of the present invention can exhibit the same or similar anti-inflammatory properties as the mutated IL-37 described in the examples.

[0045] This invention provides pharmaceutical compositions for treating or preventing inflammatory diseases or conditions, comprising the polypeptide of this invention and a pharmaceutically acceptable diluent, excipient, or carrier. In one embodiment, the sole active ingredient present in the composition is the polypeptide of this invention.

[0046] This invention provides pharmaceutical compositions for treating or preventing inflammatory diseases or conditions, comprising a polypeptide of the invention as an active ingredient and a pharmaceutically acceptable diluent, excipient, or carrier. In one embodiment, the only active ingredient present in the composition is the polypeptide of the invention.

[0047] This invention provides pharmaceutical compositions for treating or preventing inflammatory diseases or conditions, comprising a polypeptide of the invention as a main component and a pharmaceutically acceptable diluent, excipient, or carrier. In one embodiment, the only active ingredient present in the composition is the polypeptide of the invention.

[0048] The present invention also provides polypeptides of the present invention for the treatment of inflammatory diseases or conditions.

[0049] The present invention also provides pharmaceutical compositions comprising the polypeptides of the present invention and pharmaceutically acceptable diluents, excipients or carriers for the treatment of inflammatory diseases or conditions.

[0050] The present invention also provides a method for inhibiting inflammation in a subject with such need, the method comprising administering to the subject a therapeutically effective amount of the polypeptide of the present invention or a pharmaceutical composition of the present invention, thereby inhibiting inflammation.

[0051] The present invention provides a method for treating or preventing inflammatory diseases or conditions, the method comprising the step of administering a composition to a subject requiring treatment or prevention, wherein the composition comprises, is substantially composed of, or is composed of, the polypeptides of the present invention and pharmaceutically acceptable diluents, excipients or carriers.

[0052] In any of the methods or uses of the invention described herein, the polypeptides of the invention can be applied systemically or directly to the disease site. The polypeptides of the invention can be formulated for oral administration.

[0053] The present invention provides a method for treating or preventing inflammatory diseases or conditions in a subject in need, the method comprising administering to the subject a therapeutically effective amount of the polypeptide or pharmaceutical composition of the present invention, thereby treating or preventing inflammatory diseases or conditions in the subject.

[0054] The present invention also provides a method for relieving or improving symptoms of an inflammatory disease or condition in a subject in need, the method comprising administering a therapeutically effective amount of the polypeptide or pharmaceutical composition of the present invention to the subject in need, thereby relieving or improving symptoms of an inflammatory disease or condition in the subject.

[0055] The present invention also provides the use of therapeutically effective amounts of the polypeptide or pharmaceutical composition of the present invention in the preparation of a medicament for treating or preventing inflammatory diseases or conditions in subjects in need.

[0056] This invention provides a method for treating inflammatory diseases or conditions in subjects, comprising the following steps:

[0057] To identify subjects suffering from inflammatory diseases or conditions; and

[0058] Administer a therapeutically effective amount of the polypeptide or pharmaceutical composition of the present invention to a subject who requires this.

[0059] This is used to treat inflammatory diseases or conditions in the subjects.

[0060] This invention provides a method for treating inflammatory diseases or conditions, comprising the following steps:

[0061] To identify subjects suffering from inflammatory diseases or conditions; and

[0062] Administer a therapeutically effective amount of the polypeptide or pharmaceutical composition of the present invention to subjects who require it.

[0063] This is used to treat inflammatory diseases or conditions in the subjects.

[0064] The present invention also provides nucleic acid molecules encoding polypeptides as described herein.

[0065] The present invention also provides a vector comprising the nucleic acid molecules described herein.

[0066] The present invention also provides cells comprising the vectors or nucleic acid molecules described herein.

[0067] The present invention also provides animals or tissues derived therefrom that contain the cells described herein.

[0068] As used herein, unless the context otherwise requires, the term "comprise" and its variations such as "comprising," "comprises," and "comprised" are not intended to exclude other additives, components, integers, or steps. The term "comprising" may also be used interchangeably with "comprise" and is not intended to exclude other additives, components, integers, or steps.

[0069] Other aspects of the invention, as well as further embodiments of the aspects described in the preceding paragraphs, will become apparent from the following description given by way of example and with reference to the accompanying drawings.

[0070] Brief description of the attached figures

[0071] Figure 1IL-37 forms a novel head-to-head symmetrical homodimer. (A) SDS-PAGE of purified wild-type IL-37 (1-218) and N-terminal truncated variants IL-37 (21-218) and IL-37 (46-218). A small subset of SDS-stabilized IL-37 dimers is shown. (B) SEC-MALS analysis of the IL-37 homodimer relative to monomer IL-18. The SEC trace corresponds to the left y-axis, and the MALS determination of the average molecular weight (right y-axis) is shown as a dashed line below the refractive index trace. (C) Overview of the IL-37 homodimer viewed from two directions, rotated 90° clockwise relative to the horizontal axis. The structures are shown in cartoon format, with IL-37A on the left and IL-37B on the right (A and B are used to distinguish IL-37 molecules in the homodimer).

[0072] Figure 2 Structural and mutational analysis of the IL-37 dimer interface. (A) IL-37 homodimer in animated format, with the C2 symmetry axis marked as a circle. (B) Homodimerized interface with side chains, where key interactions are shown as rods and hydrogen bonds as dashed lines. (C) IL-37 interface rotated 180° horizontally relative to (B), with key side chains showing rods. (D) SEC-MALS analysis of mutant IL-37 dimer interface, with the SEC trace corresponding to the left y-axis and the MALS determination of the average molecular weight (right y-axis) shown as a dashed line below the refractive index trace.

[0073] Figure 3 SEC-MALS analysis of the IL-37 dimer interface. SEC-MALS analysis of wild-type IL-37 and IL-37 interface mutants. SEC was monitored by absorbance at 280 nm (left axis), and the right axis indicates the average molecular weight derived from MALS. *It should be noted that the peak located between the dimer and wild-type peaks is characteristic of dimers that dissociate on column (see Woodbury et al., Protein Science 2002), meaning that the protein is not in equilibrium between monomers and dimers during SEC runs, but dissociates continuously as the concentration decreases with on-column dilution.

[0074] Figure 4 IL-37 isoform 1 (SEQ ID NO: 1). Accession number NP_055254. This is the full-length sequence of the IL-37 isoform used herein and is associated with the numbering mentioned herein. The sequence used for crystallization experiments was derived from residues 46-218 of this sequence.

[0075] Figure 5Different inhibitory effects of recombinant IL-1β variants on LPS-stimulated IL-1β in human PBMCs. Freshly isolated PBMCs from healthy volunteers were incubated with specified concentrations of four variants of recombinant IL-37b (including the native protein (wild-type) and a monomeric mutant (D73K), with two N-terminal truncated variants at amino acid 21 (21-218) or 46 (46-218). After 30 minutes, the cultures were stimulated with LPS (50 pg / ml) or a mediator (without IL-1β induction, not shown). Supernatants were collected 20 hours after LPS addition, and IL-1β was detected by ELISA. The figure shows the percentage change in IL-1β provided by the IL-37b variants ± SEM compared to LPS + mediator (set as 0); n = 15 donors; *P < 0.05, mediator + LPS relative to all other groups; #P < 0.05 and ##P < 0.01, same concentration of monomeric IL-37 relative to all other variants.

[0076] Figure 6 IL-1β was reduced by transfecting THP-1 macrophages with the IL-37b variant. THP-1 macrophages were transfected with the specified IL-37b variant (full-length, 1-218), then differentiated with PMA, followed by stimulation with LPS (250 ng / ml) or a mediator. The supernatant was collected after 24 hours, and IL-1β was measured by ELISA. Supernatant IL-1β ± SEM; n = 11-26; *P < 0.05, control relative to IL-37b transfection; #P < 0.05, native IL-37b relative to monomeric IL-37. P < 0.001 for transfection with the IL-37b variant compared to control transfection.

[0077] Figure 7 Recombinant IL-37 variant (40 μg / kg) or the mediator was injected intraperitoneally into C57Bl / 6 wild-type (WT) mice 60 minutes before administration of 10 mg / kg LPS or the mediator (time 0 h). IL-37tg mice were also injected with either LPS or the mediator for direct comparison. A total of n = 94 mice were used, with 4–24 mice per group for WT and 5 mice per group for IL-37tg. Body temperature was measured at specified time points and ±SEM was plotted; all mediator mice are shown together. *, P<0.05 and ***, P<0.001, LPS relative to all other conditions; #, P<0.05; ##, P<0.01 and ###, P<0.001, D73K or Y85A variants relative to natural recIL-37; ns-tg, not significant relative to IL-37tg; ns-v, not significant relative to the medium; *1-4, 7-hour statistics: *1, * and ns-v; *2, * and ns-v and ns-tg; and *3, *** and ns-tg; *4, * and ns-tg. RecIL-37 21-218Displayed in gray for direct comparison, the statistics for these groups are calculated separately.

[0078] Figure 8 :according to Figure 7 Plasma IL-1β ± SEM in WT mice at 19 hours. Veh, mediator; Nat, natural recIL-37; Mono, D73K; numbers indicate N-terminal truncation of variants.

[0079] Figure 9 Different inhibitory effects of recombinant IL-37 variants in human PBMCs on HKLM-stimulated IL-1β or IL-6. Freshly isolated PBMCs were stimulated as shown. HKLM (heat-inactivated Listeria monocytogenes) was used at 10 μL / mL. 6 Cells were added 30 minutes after adding the recIL-37 variant. Black, HKLM alone; white, HKLM + native recIL-37; light gray, HKLM + D73K; dark gray, HKLM + Y85A (all variants 46-218). Supernatants were collected 20 hours after HKLM addition and IL-1β (ac, g) and IL-6 (df, h) were measured by ELISA. (af) Concentration (conc) 1, 10 ng / ml; Concentration 2, 100 pg / ml; Concentration 3, 10 pg / ml. The figure shows the absolute cytokine concentrations ± SEM in the supernatants of individual donor FC (a, d), JM (b, e), and LM (c, f) cultures. (ef) Data for HKLM + D73K concentration 1 were not available. (g, h) Percentage changes in IL-1β (g) and IL-6 (h) were calculated from the raw data shown in Figure af and plotted as ± SEM. The concentration of recIL-37 is expressed in pg / ml. *, P<0.05; **, P<0.01; ***P<0.001, only HKLM relative to HKLM+recIL-37; ##P<0.01, the same concentration of Y85A relative to natural recIL-37.

[0080] Figure 10Different inhibitory effects of recombinant IL-37 variants on imiquimod stimulation of IL-1β or IL-6 in human PBMCs. Freshly isolated PBMCs were stimulated as shown. Imiquimod was used at 10 μg / ml and added 30 min after the recIL-37b variant. Black, imiquimod alone; white, imiquimod + native recIL-37; light gray, imiquimod + D73K; dark gray, imiquimod + Y85A (all variants 46-218). Supernatants were collected 20 h after imiquimod addition and IL-1β (ac, g) and IL-6 (df, h) were measured by ELISA. (af) Concentration (conc) 1, 10 ng / ml; Concentration 2, 100 pg / ml; Concentration 3, 10 pg / ml. The figure shows the absolute cytokine concentrations ± SEM in the supernatants of individual donor FC (a, d), JM (b, e), and LM (c, f) cultures. (e) Data for imiquimod + natural concentration 2 are unavailable. (g, h) Percentage changes in IL-1β (g) and IL-6 (h) were calculated from the raw data shown in Figure af and plotted as ± SEM. Concentrations of recIL-37 are expressed in pg / ml. *, P < 0.05; **, P < 0.01, imiquimod alone relative to imiquimod + recIL-37; #, P < 0.05, Y85A at the same concentration relative to natural recIL-37.

[0081] Figure 11 As shown, freshly isolated PBMCs were stimulated. CpG-A was used at 3 μM and added 30 min after the recIL-37b variant. Black, CpG-A alone; white, CpG-A + native recIL-37; light gray, CpG-A + D73K; dark gray, CpG-A + Y85A (all variants 46-218). Supernatants were collected 20 h after CpG-A addition and IL-6 (ac) was measured by ELISA. (ac) Concentration (con) 1, 10 ng / ml; Concentration 2, 100 pg / ml; Concentration 3, 10 pg / ml. The figure shows the absolute cytokine concentrations ± SEM in the supernatant of cultures from individual donors FC (a), JM (b), and LM (c). Data for CpG-A + D73K concentration 3 were not available. (d) Percentage change in IL-6 calculated from the raw data shown in Figure ac and plotted as ± SEM. The concentration of recIL 37 is expressed in pg / ml. *, P<0.05; **, P<0.01, CpG-A alone versus CpG-A+recIL 37.

[0082] Figure 12As shown, freshly isolated PBMCs were stimulated. ssRNA40 was used at 0.5 μg / ml and added 30 min after the recIL-37 variant. Black, ssRNA40 alone; white, ssRNA40 + native recIL-37; light gray, ssRNA40 + D73K; dark gray, ssRNA40 + Y85A (all variants 46-218). Supernatants were collected 20 h after ssRNA40 addition and IL-1β was determined by ELISA. (a, b) Concentration (conc) 1, 10 ng / ml; Concentration 2, 100 pg / ml; Concentration 3, 10 pg / ml. The figure shows the absolute cytokine concentrations ± SEM in the supernatant of cultures from individual donors JF (a) and MS (b). Data for ssRNA40 + D73K concentrations 3 (JF) and 2 (MS) are not available. (c) The percentage change in IL-1β was calculated from the raw data shown in Figures a and b, and ±SEM was plotted. The concentration of recIL 37 is expressed in pg / ml. No statistical calculations were performed because only two datasets were available.

[0083] Detailed description of the implementation plan

[0084] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more individual features mentioned or clearly visible from the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0085] Reference will now be made in detail to certain embodiments of the invention. Although the invention will be described in conjunction with embodiments, it should be understood that the invention is not intended to be limited to those embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the invention as defined by the claims.

[0086] Those skilled in the art will recognize that many similar or equivalent methods and materials are available for carrying out the invention. The invention is by no means limited to the methods and materials described herein. It should be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more individual features mentioned or clearly visible from the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0087] All patents and publications cited herein are incorporated herein by reference in their entirety.

[0088] For the purpose of interpreting this specification, terms used in the singular will also include the plural, and vice versa.

[0089] The inventors’ work leading to this invention includes IL-37. High-resolution crystal structures revealed a characteristic β-trefoil fold of the IL-1 cytokine, consisting of 12 β-strands and three α-helices. Unexpectedly, IL-37 formed a unique head-to-head homodimeric arrangement within the IL-1 superfamily. IL-37 is a dimer in solution, and structure-guided point mutations within the dimer interface convert IL-37 into a monomer. Monomeric IL-37 was significantly more effective in inhibiting the release of pro-inflammatory cytokines in a range of cell types, including human blood cells. In summary, these data demonstrate that IL-37 homodimerization constitutes a novel self-regulating mechanism that tightly controls its anti-inflammatory signaling. Significantly, monomeric IL-37 represents a molecule with potential therapeutic benefits for treating a range of inflammatory diseases.

[0090] The peptides of the present invention exhibit significantly increased anti-inflammatory activity. An advantage of the peptides of the present invention is that the loss of efficacy observed in wild-type (i.e., natural, endogenous) IL-37 does not occur at increased concentrations. In other words, the peptides of the present invention consistently exhibit anti-inflammatory activity at high concentrations and in concentrations exceeding at least 4 log₂. This is particularly advantageous because the peptides of the present invention have a considerably wider therapeutic window compared to wild-type recombinant IL-37.

[0091] An IL-37 polypeptide is a molecule possessing at least one biochemical or biophysical activity of IL-37, for example, it can bind to the interleukin-18 receptor (IL-18R1 / IL-1Rrp) and can be a ligand of the interleukin-18 receptor. It can also bind to interleukin-18 binding protein (IL-18BP), the inhibitory binding protein of interleukin-18 (IL-18), and subsequently form a complex with the β chain of the IL-18 receptor, through which it can inhibit IL-18 activity. Other biochemical or biophysical activities of IL-37 include binding to IL-1R8 (Sigirr), blocking the production of pro-inflammatory cytokines (rather than anti-inflammatory cytokines) triggered by a broad spectrum of inflammatory attacks (including TLR ligands, IFNγ, TNF and IL-1β) in human or mouse immune cells, inhibiting dendritic cell activation (reducing the surface expression of CD86 and MHC II), triggering specific regulatory patterns of intracellular kinases (including blocking the mTOR, MAPK and NF-κB pathways), and inducing anti-inflammatory kinases such as Mer and PTEN (as described in (5)).

[0092] IL-37 is also known as interleukin-37 (FIL1ζ; IL-1ζ; IL-1F7b (IL-1H4, IL-1H, IL-1RP1); IL-1X protein; IL1F7 (standard product IL-1F7b); interleukin 1 family member 7; interleukin 1,ζ; interleukin-1 homologue 4; interleukin-1 superfamily z; interleukin-1 related protein and interleukin-23). ​​Human IL-37 has five isoforms, a, b, c, d, and e, all of which are included when referring to IL-37 herein unless explicitly stated otherwise. Any isoform or ortholog of human IL-37 polypeptide containing at least one residue equivalent to a dimer interface residue in Table 1 is also included within the scope of this invention. For example, this invention includes polypeptides that are identical to any human IL-37 isoform a, b, c, d, or e.

[0093] The dimerization interface that enables IL-37 monomer dimerization refers to the interface between two IL-37 molecules involved in the binding of one molecule to another. Typically, the interface is not solvent-accessible when the two molecules interact. The interface includes residues that perform key interactions and / or contribute to the embedded surface region of the interface. The interface may be the same or different in terms of surface area size or residue composition on each molecule during the interaction. The interface includes any one or more residues listed in Table 1 below that perform key interactions and / or contribute to the embedded surface region of the interface. The β3-β4 ring (residues 83-91) is a key region controlling the secondary structure of dimerization. Other regions also contribute to the interface. Y85 is packed onto a hydrophobic surface formed by Val71, Val80, and Ile78. Embedding Y85 into this surface region is crucial for dimer formation because the mutation of the side chain to alanine eliminates the dimer.

[0094] Table 1 lists the residues that contribute to the IL-37 dimer interface. Any one or more of these residues can be genetically / chemically modified to interfere with IL-37 dimerization. Data are from PDBePISA (EMBL).

[0095]

[0096]

[0097] Mutations or modifications of any one or more residues in Table 1 that reduce or inhibit their ability to participate in specific interaction types are also considered. For example, any mutation at Asp73 or an equivalent position that reduces or inhibits the ability to form salt bridges is included in this invention. This also applies to hydrogen bonds formed by Ser74, Asn84, Tyr85, Ile86, and Arg87.

[0098] Any modification to the β3-β4 ring (residues 83-91) region can shift the balance from dimer to monomer. This includes, but is not limited to, deletions, shortenings, lengthenings, mutations, and / or chemical modifications of ring residues. Within the ring region, Lys83, Asn84, Tyr85, Ile86, Arg87, and Pro88 appear to be the most critical residues and are therefore considered for mutation or modification.

[0099] The present invention also provides compounds that inhibit the dimerization of IL-37 peptides. Preferably, the compound binds to and / or disrupts the dimerization interface. The compound may be a peptide, peptide mimic, antibody, or small molecule that interacts with at least one amino acid residue in the dimerization interface.

[0100] Furthermore, the present invention provides the use of a compound that inhibits IL-37 peptide dimerization in the preparation of a medicament for suppressing inflammation. Moreover, the present invention provides a method for suppressing inflammation in a subject with this need, comprising administering a compound that inhibits IL-37 peptide dimerization. Preferably, the compound binds to and disrupts the dimerization interface. The compound may be a peptide, peptide mimic, antibody, or small molecule that interacts with at least one amino acid residue in the dimerization interface.

[0101] The present invention also provides a peptide comprising, substantially composed of, or composed of, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 consecutive amino acids of the amino acid sequence at the dimerization interface of SEQ ID NO: 1. Preferably, the consecutive amino acids are V71 to P88 of SEQ ID NO: 1. This peptide can be used to generate antibodies capable of inhibiting the dimerization of IL-37 peptides.

[0102] As used herein, amino acid residues at positions equivalent to those in SEQ ID NO: 1 can be determined by any means known to those skilled in the art. For example, comparison of one or more sequences with the amino acid sequence of SEQ ID NO: 1 will allow those skilled in the art to determine amino acids at positions equivalent to those in SEQ ID NO: 1. Those skilled in the art can compare the three-dimensional structure of a polypeptide with the three-dimensional structure of a polypeptide having the amino acid sequence of SEQ ID NO: 1 and determine the amino acid residues at equivalent positions in SEQ ID NO: 1.

[0103] As used herein, “reduced dimerization ability” refers to a peptide’s lower tendency to form homodimers and / or heterodimers compared to a reference peptide. The dimerization ability of a peptide can be measured by various methods, including those described herein, such as size exclusion chromatography and multi-angle light scattering. Preferably, a peptide with reduced dimerization ability has a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in dimerization compared to a reference peptide. The reference peptide is typically a native, wild-type, unmodified, or unmutated peptide. To assess the proportion of peptides present in a given form (i.e., monomer or dimer), a suitable method is, for example, analytical gel electrophoresis under non-denaturing conditions. In such a method, the peptide solution is run in a polyacrylamide gel along with a set of standard molecular weight markers. If the peptide forms a dimer, protein bands will be observed in the gel corresponding to species whose molecular weight is approximately twice the calculated total amount of amino acids in the peptide. A second band can also be observed, corresponding to substances with an approximate molecular weight of the calculated total amino acid sum of the polypeptide—this represents the sequence in monomeric form. The relative intensities of these bands can be used to quantify the proportion of the polypeptide present in each form. Similar methods can be used to assess molecular weight by alternative means, such as analytical centrifugation, mass spectrometry, or size exclusion chromatography. Alternatively, reversed-phase high-performance liquid chromatography (RP-HPLC) can be used to quantify monomers or dimers, where dimers and higher oligomers are separated from monomers based on their differences in hydrophobicity. Identification of substances can be achieved using mass spectrometry. The same methods can be adapted to assess whether a given polypeptide exhibits a tendency to heterodimerize or homodimerize.

[0104] The anti-inflammatory properties of the peptides of this invention can be determined by any of the methods described herein, particularly those described in the examples.

[0105] When used to describe the various polypeptides disclosed herein, "isolated" means a polypeptide that has been identified and isolated and / or recovered from components of its native environment. Contaminants in its native environment often interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other protein or non-protein solutes. In a preferred embodiment, the polypeptide is purified (1) to the extent that at least 15 N-terminal residues or an internal amino acid sequence can be obtained using a spin cup sequencer, or (2) to homogeneity under non-reducing or reducing conditions by SDS-PAGE using Coomassie blue or preferably silver staining. Isolated proteins include recombinant intracellular in situ polypeptides, as at least one component of the polypeptide's native environment will be absent. However, isolated polypeptides are typically prepared by at least one purification step.

[0106] A “fragment” is a part of the polypeptide of the present invention that retains substantially similar functional activity or substantially the same biological function or activity as the polypeptide, which can be determined using the assays described herein.

[0107] The “percentage of amino acid sequence identity (%)” or “percentage of identity (%)” for a polypeptide sequence (i.e., the polypeptide of the present invention as defined herein) is defined as the percentage of amino acid residues that are identical to those in a candidate sequence after sequence alignment with the amino acid residues in a particular polypeptide of the present invention, if necessary, to introduce gaps to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity.

[0108] Those skilled in the art can determine suitable parameters for measuring alignments, including any algorithms required to achieve maximum alignment on the full-length sequences being compared (non-limiting examples described below). When aligning amino acid sequences, the percentage of amino acid sequence identity of a given amino acid sequence A relative to, and or against, a given amino acid sequence B (which can be alternatively expressed as a given amino acid sequence A having or containing a certain percentage of amino acid sequence identity relative to, and or against, a given amino acid sequence B) can be calculated as follows: Amino acid sequence identity percentage = X / Y × 100, where X is the number of amino acid residues that are identified as identical matches by the alignment score of the sequence alignment program or algorithm for A and B, and Y is the total number of amino acid residues in B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the percentage of amino acid sequence identity of A relative to B will not be equal to the percentage of amino acid sequence identity of B relative to A.

[0109] When calculating the percentage of identity, exact matches are typically counted. The determination of the percentage of identity between two sequences can be accomplished using mathematical algorithms. A non-restrictive example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, which has been modified as in the algorithm of Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such algorithms are incorporated into the BLASTN and BLASTX procedures of Altschul et al. (1990) J. Mol. Biol. 215:403. To obtain gapped alignment for comparison purposes, gapped BLAST (in BLAST 2.0) as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389 can be used. Alternatively, an iterative search can be performed using PSI-Blast, which detects long-distance relationships between molecules. See Altschul et al. (1997), ibid. When using BLAST, GappedBLAST, and PSI-Blast programs, the default parameters of each program (e.g., BLASTX and BLASTN) can be used. Alignment can also be performed manually by inspection. Another non-limiting example of a mathematical algorithm for comparing sequences is the ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences and aligns them to whole amino acid or DNA sequences, thus providing data on sequence conservation for the entire amino acid sequence. The ClustalW algorithm is used in several commercially available DNA / amino acid analysis software packages, such as the ALIGNX module of the Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, CA). After aligning amino acid sequences with ClustalW, the percentage of amino acid identity can be assessed. A non-limiting example of a software program that can be used to analyze ClustalW alignments is GENEDOC. TM Or JalView (http: / / www.jalview.org / ). GENEDOC TMThis allows for the assessment of amino acid (or DNA) similarity and identity between multiple proteins. Another non-limiting example of a mathematical algorithm for sequence comparison is the algorithm by Myers and Miller (1988) CABIOS 4:11-17. Such an algorithm was incorporated into the ALIGN program (version 2.0) as part of the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys, Inc., 9685 Scranton Rd., San Diego, CA, USA). When comparing amino acid sequences using the ALIGN program, a PAM 120 weighted residue table can be used, with a vacancy length penalty of 12 and a vacancy penalty of 4.

[0110] The polypeptide advantageously comprises an amino terminus and a carboxyl terminus. The polypeptide may contain D-amino acids, L-amino acids, or a mixture of D- and L-amino acids. However, the D-amino acid form is particularly preferred because polypeptides comprising D-amino acids are expected to retain greater biological activity in vivo.

[0111] Peptides can be prepared using any of many conventional techniques. Peptides can be isolated or purified from naturally occurring sources or from recombinant sources. Recombinant production is preferred. For example, in the case of recombinant peptides, well-known molecular genetic techniques can be used to subclone a DNA fragment encoding the desired peptide into a suitable vector (see, for example, Maniatis et al., *Molecular Cloning: A Laboratory Manual*, 2nd ed. (Cold Spring Harbor Laboratory, 1982); Sambrook et al., *Molecular Cloning A Laboratory Manual*, 2nd ed. (Cold Spring Harbor Laboratory, 1989)). The fragment can be transcribed and subsequently translated in vitro into a peptide. Commercially available kits can be used (e.g., manufactured by Clontech, Palo Alto, Calif.; Amersham Pharmacia Biotech Inc., Piscataway, NJ; InVitrogen, Carlsbad, Calif., etc.). Polymerase chain reaction can optionally be used to manipulate nucleic acids.

[0112] As used in this article, "conservative substitution" refers to the replacement of a naturally occurring amino acid in a peptide sequence with a naturally occurring or non-naturally occurring amino acid or a peptide mimic with similar spatial properties. When the side chain of the natural amino acid to be substituted is polar or hydrophobic, a conservative substitution should be a naturally occurring amino acid, a non-naturally occurring amino acid, or a peptide mimic that is also polar or hydrophobic (except for having the same spatial properties as the side chain of the amino acid being substituted).

[0113] A table of conserved amino acid substitutions that provides functionally similar amino acids is well known to those skilled in the art. The following six groups are examples of amino acids that can be considered as conserved substitutions for each other:

[0114] 1) Alanine (A), Serine (S), Threonine (T);

[0115] 2) Aspartic acid (D), glutamic acid (E);

[0116] 3) Asparagine (N), glutamine (Q);

[0117] 4) Arginine (R), Lysine (K);

[0118] 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and

[0119] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0120] Since naturally occurring amino acids are typically grouped according to their properties, conservative substitutions of naturally occurring amino acids can be determined, taking into account the fact that the replacement of a charged amino acid with a spatially similar uncharged amino acid is considered a conservative substitution. To generate conservative substitutions from non-naturally occurring amino acids, amino acid analogs (synthetic amino acids) well-known in the art can also be used. Peptide analogs of naturally occurring amino acids are well documented in the literature known to those skilled in the art, and non-natural or unnatural amino acids are further described below. When influencing conservative substitutions, the substituted amino acid should have the same or similar functional groups on its side chain as the original amino acid.

[0121] As used herein, the phrase “non-conservative substitution” or “non-conservative residue” refers to the substitution of an amino acid present in a parent sequence by another naturally occurring or non-naturally occurring amino acid with different electrochemical and / or steric properties. Therefore, the side chain of the substituted amino acid can be significantly larger (or smaller) than the side chain of the substituted natural amino acid, and / or can have functional groups with significantly different electronic properties than the substituted amino acid. Examples of such non-conservative substitutions include phenylalanine or cyclohexylmethylglycine replacing alanine, isoleucine replacing glycine, or -NH-CH[(-CH2)5-COOH]-CO- replacing aspartic acid. Non-conservative substitutions include any mutations that are not considered conserved.

[0122] Non-conservative amino acid substitutions can be caused by changes in: (a) the structure of the amino acid backbone in the substitution region; (b) the charge or hydrophobicity of the amino acid; or (c) the size of the amino acid side chain. Substitutions that typically produce the most significant changes in protein properties are those in which: (a) a hydrophilic residue substitutes for (or is substituted by) a hydrophobic residue; (b) proline substitutes for (or is substituted by) any other residue; (c) a residue with a large side chain, such as phenylalanine, substitutes for (or is substituted by) a residue without a side chain, such as glycine; or (d) a residue with a positively charged side chain (e.g., lysyl, arginyl, or histidine) substitutes for (or is substituted by) an electronegative residue, such as glutamine or aspartate.

[0123] The alteration of the native amino acid sequence to produce mutant peptides can be accomplished by various means known to those skilled in the art, such as by insertion, deletion, and / or substitution. For example, site-specific mutations can be introduced by ligating a synthetic oligonucleotide containing the modification site into an expression vector. Alternatively, oligonucleotide-guided site-specific mutagenesis methods can be used, as disclosed in Walder et al., Gene 42:133 (1986); Bauer et al., Gene 37:73 (1985); Craik, Biotechniques, 12-19 (January 1995); and U.S. Patent Nos. 4,518,584 and 4,737,462. A preferred method for introducing mutations is the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.).

[0124] Recombinant peptides can be produced using any suitable expression vector (e.g., as described in Pouwels et al., Cloning Vectors: A Laboratory Manual (Elsevier, NY: 1985)) and a corresponding suitable host. Expression hosts include, but are not limited to, bacterial species within the genera *Escherichia*, *Bacillus*, *Pseudomonas*, and *Salmonella*, mammalian or insect host cell systems (including baculovirus systems) (e.g., as described in Luckow et al., Bio / Technology 6:47 (1988)), and cell lines such as COS-7, C127, 3T3, CHO, HeLa, and BHK have been established. Those skilled in the art will recognize that the choice of expression host influences the type of peptide produced. For example, the glycosylation of peptides produced in yeast or mammalian cells (e.g., COS-7 cells) will differ from that produced in bacterial cells such as *Escherichia coli*.

[0125] Alternatively, the polypeptides of the present invention can be synthesized using standard peptide synthesis techniques well known to those skilled in the art (e.g., as summarized in Bodanszky, Principles of Peptide Synthesis (Springer-Verlag, Heidelberg: 1984)). In particular, the polypeptides can be synthesized using solid-phase synthesis (see, for example, Merrifield, J. Am. Chem. Soc. 85:2149-54 (1963); Barany et al., Int. J. Peptide Protein Res. 30:705-739 (1987); and U.S. Patent No. 5,424,398). If desired, this can be accomplished using an automated peptide synthesizer. Removal of the tert-butoxycarbonyl (t-BOC) or 9-fluorenylmethoxycarbonyl (Fmoc) amino acid blocking groups and separation of the polypeptide from the resin can be accomplished, for example, by acid treatment at a reduced temperature. The mixture containing the polypeptide can then be extracted with, for example, dimethyl ether to remove non-peptide organic compounds, and the synthesized polypeptide can be extracted from the resin powder (e.g., with about 25% w / v acetic acid). After polypeptide synthesis, optional further purification (e.g., using high-performance liquid chromatography (HPLC)) can be performed to eliminate any incomplete polypeptides or free amino acids. The synthesized polypeptide can be analyzed by amino acid and / or HPLC to verify its identity. For other applications according to the invention, it may be preferred to generate polypeptides as part of a larger fusion protein, for example by the methods described herein or other genetic means, or as part of a larger conjugate, such as by physical or chemical conjugation, as known to those skilled in the art and described herein.

[0126] The peptides of the present invention can also be modified by conjugation or fusion with another portion to facilitate purification, increase the in vivo half-life of the peptide, or for use in immunoassays using methods known in the art. For example, the peptides of the present invention can be modified by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or linkage with cellular ligands or other proteins.

[0127] "Peptide mimics" are synthetic chemical compounds having substantially the same structural and / or functional characteristics as the polypeptides of the present invention, which are further described herein. Typically, peptide mimics have the same or similar structures as the polypeptides of the present invention, such as the same or similar sequences or fragments thereof of SEQ ID NO: 1 with reduced dimerizing ability. Peptide mimics typically contain at least one non-naturally synthesized residue. The non-natural component of a peptide mimic compound can be one or more of the following: a) a residue linking group other than a natural amide bond ("peptide bond"); b) a non-natural residue replacing a naturally occurring amino acid residue; or c) a residue that induces or stabilizes secondary structures, such as β-turns, γ-turns, β-sheets, α-helical conformations, etc.

[0128] Peptide mimics can be synthesized using a variety of procedures and methods described in scientific and patent literature, such as Organic Syntheses Collective Volumes, Gilman et al. (Eds), John Wiley & Sons, Inc., NY, al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby (1997) Curr. Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol. Divers. 3:17-27; Ostresh (1996) Methods Enzymot. 267:220-234.

[0129] Modifications considered herein include, but are not limited to, modifying side chains, incorporating non-natural amino acids and / or their derivatives during peptide synthesis, and other methods using cross-linking agents and imposing conformational constraints on the peptides of the present invention. Any modifications that reduce the ability of the molecule to form dimers are considered herein, including post-translational modifications. Examples include modifications known in the art through click chemistry incorporation. Exemplary modifications include polyethylene glycolation and glycosylation.

[0130] Examples of side-chain modifications considered in this invention include modifications of the amino group, such as reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidation with methyl acetylimine ester; acylation with acetic anhydride; carbamylation of the amino group with cyanate ester; trinitrobenzylation of the amino group with 2,4,6-trinitrobenzenesulfonic acid (TNBS); acylation of the amino group with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxalation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH4.

[0131] The guanidinyl group of arginine residues can be modified by forming heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal, and glyoxal.

[0132] The carboxyl group can be activated by carbodiimide formed via O-acylisourea, and then subsequently modified by derivatization into, for example, the corresponding amide.

[0133] The thiol group can be modified by methods such as carboxylation with iodoacetic acid or iodoacetamide; oxidation of performic acid to sulfoalanine; formation of mixed disulfides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercury derivatives using 4-chloromercuric benzoic acid, 4-chloromercuric benzenesulfonic acid, phenylmercuric chloride, 2-chloromercuric-4-nitrophenol and other mercury preparations; and carbamylation with cyanate esters at alkaline pH.

[0134] Tryptophan residues can be modified, for example, by oxidation with N-bromosuccinimide or by alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfonylphenyl halides. Alternatively, tyrosine residues can be modified by nitration with tetranitromethane to form 3-nitrotyrosine derivatives.

[0135] Modification of the imidazole ring of histidine residues can be accomplished by alkylation with iodoacetic acid derivatives or by N-ethoxycarbonylation with diethyl pyrocarbonate.

[0136] Examples of incorporation of non-natural amino acids and derivatives during protein synthesis include, but are not limited to, the use of leucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, tert-butylglycine, valine, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine, and / or D-isomers of amino acids. A list of non-natural amino acids considered herein is shown in Table 2.

[0137] Table 2

[0138]

[0139]

[0140]

[0141]

[0142] By using bifunctional crosslinking agents, such as bifunctional imide esters having (CH2)n spacer groups of n=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters, and heterobifunctional agents that typically contain an amino reactive moiety such as N-hydroxysuccinimide and another group-specific reactive moiety, crosslinking agents can be used, for example, to stabilize 3D conformations.

[0143] Nucleic acid molecules encoding any polypeptide of the present invention are also within the scope of the present invention. Nucleic acids can be used, for example, to prepare the polypeptides of the present invention and as therapeutic agents. They can be administered to cells in culture or in vivo and can include secretion signals that direct or promote the secretion of the polypeptides of the present invention from cells. Also within the scope of the present invention are expression vectors and host cells that contain or include the nucleic acids of the present invention (described further below). Although the nucleic acids of the present invention may be referred to as “isolated” by definition, the polypeptides of the present invention are not wild-type polypeptides and therefore are not encoded by naturally occurring nucleic acids. Thus, while the polypeptides and nucleic acids of the present invention may be “purified,” “substantially purified,” “isolated,” “recombinant,” or “synthetic,” they are not necessarily required to be distinguished from naturally occurring materials.

[0144] "Isolated" nucleic acid molecules are those identified and isolated from at least one contaminating nucleic acid molecule that is typically associated with a natural source of nucleic acid encoding a polypeptide (typically IL-37). Isolated nucleic acid molecules are not in a form or environment found in nature. Therefore, isolated nucleic acid molecules differ from those present in natural cells. However, isolated nucleic acid molecules include those typically found in cells expressing IL-37, for example, where the nucleic acid molecule is located at a different chromosomal location than in natural cells.

[0145] The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides or similar compounds) of any length. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The polynucleotides of the present invention can be provided in isolated or purified form. A nucleic acid sequence "encoding" a selected polypeptide is a nucleic acid molecule that, when placed under the control of a suitable regulatory sequence, is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxyl) end. For the purposes of this invention, such nucleic acid sequences may include, but are not limited to, cDNA from viral, prokaryotic, or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, or even synthetic DNA sequences. The transcription termination sequence may be located at the 3' end of the coding sequence.

[0146] The polynucleotides of the present invention can be synthesized according to methods well known in the art, such as those described in Sambrook et al. (1989, Molecular Cloning—a laboratory manual; Cold Spring Harbor Press).

[0147] The polynucleotide molecules of the present invention can be provided in the form of expression cassettes, which include control sequences effectively linked to an inserted sequence, thereby allowing the polypeptides of the present invention to be expressed in a target subject. These expression cassettes are then typically provided within a vector (e.g., a plasmid or recombinant viral vector) suitable for use as a reagent for nucleic acid immunization. Such expression cassettes can be administered directly to a host subject. Alternatively, a vector containing the polynucleotides of the present invention can be administered to a host subject. Preferably, a genetic vector is used to prepare and / or administer the polynucleotides. A suitable vector can be any vector capable of carrying a sufficient amount of genetic information and allowing the expression of the polypeptides of the present invention.

[0148] Therefore, the present invention includes expression vectors comprising such polynucleotide sequences. Thus, the present invention provides vectors for the prevention or treatment of inflammatory diseases or conditions, said vectors comprising a polynucleotide sequence encoding a polypeptide of the present invention and optionally one or more additional polynucleotide sequences encoding different polypeptides as defined herein.

[0149] Furthermore, it should be understood that the compositions and products of the present invention may comprise a mixture of peptides and polynucleotides. Therefore, the present invention provides compositions or products as defined herein, wherein a polynucleotide capable of expressing the peptide replaces any single peptide.

[0150] Expression vectors are routinely constructed in the field of molecular biology and can, for example, involve the use of plasmid DNA and suitable initiators, promoters, enhancers, and other elements, such as a possible polyadenylation signal, and are oriented in the correct manner to allow expression of the peptides of the present invention. Other suitable vectors will be apparent to those skilled in the art. In this regard, we refer, for example, to Sambrook et al.

[0151] Therefore, the polypeptides of the present invention can be provided by delivering such a vector into cells and allowing transcription from the vector. Preferably, the polynucleotides of the present invention, or the polynucleotides used in the vectors of the present invention, are effectively linked to a control sequence that enables the host cell to express the coding sequence, i.e., the vector is an expression vector.

[0152] "Effectively linked" refers to the arrangement of elements in which the components described herein are configured to perform their normal functions. Thus, a given regulatory sequence (e.g., a promoter) effectively linked to a nucleic acid sequence can influence the expression of that sequence when a suitable enzyme is present. The promoter does not need to be contiguous with the sequence, as long as it serves to guide its expression. Therefore, for example, an inserted, non-translated but transcribed sequence can exist between the promoter sequence and the nucleic acid sequence, and the promoter sequence can still be considered "effectively linked" to the coding sequence.

[0153] Numerous expression systems have been described in the art, each typically consisting of a vector containing a target gene or nucleotide sequence efficiently linked to expression control sequences. These control sequences include transcription promoter sequences and transcription initiation and termination sequences. The vector of the present invention can be, for example, a plasmid, viral, or phage vector provided with an origin of replication, optionally a promoter for expressing the polynucleotide, and optionally a regulator of the promoter. A “plasmid” is a vector in the form of an extrachromosomal genetic element. The vector may contain one or more selectable marker genes, such as an ampicillin resistance gene in the case of a resistance gene in a bacterial plasmid or fungal vector. The vector can be used in vitro, for example for the production of DNA or RNA, or for transfection or transformation of host cells, such as mammalian host cells. The vector may also be adapted for in vivo use, for example, to allow for the in vivo expression of polypeptides.

[0154] A promoter is a nucleotide sequence that initiates and regulates the transcription of a polynucleotide encoding a polypeptide. Promoters can include inducible promoters (where the expression of the polynucleotide sequence effectively linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressive promoters (where the expression of the polynucleotide sequence effectively linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters. The terms "promoter" or "control element" are intended to include both the full-length promoter region and the functional (e.g., controlling transcription or translation) segment of these regions.

[0155] The polynucleotides, expression cassettes, or vectors according to the invention may additionally include a signal peptide sequence. Typically, the signal peptide sequence is inserted at a site effectively linked to a promoter, such that the signal peptide is expressed and promotes the secretion of a polypeptide also encoded by a coding sequence effectively linked to the promoter.

[0156] Typically, signal peptide sequences encode peptides of 10 to 30 amino acids, such as 15 to 20 amino acids. These amino acids are usually predominantly hydrophobic. Typically, the signal peptide targets the endoplasmic reticulum of the expressing cell with the growth polypeptide chain carrying the signal peptide. The signal peptide is cleaved in the endoplasmic reticulum, allowing the polypeptide to be secreted via the Golgi apparatus. Therefore, the peptides of the present invention can be provided to an individual through cellular expression within the individual and secretion from those cells.

[0157] The phrase “therapeutic effective amount” generally refers to the amount of one or more polypeptides or polynucleotides of the present invention that (i) treat a particular disease, symptom or disorder, (ii) reduce, improve or eliminate one or more symptoms of a particular disease, symptom or disorder, or (iii) delay the onset of one or more symptoms of a particular disease, symptom or disorder described herein.

[0158] This invention can be used to treat various inflammatory diseases or conditions. As used herein, "inflammatory diseases or conditions" include acute or chronic inflammation and inflammatory disorders, such as inflammation associated with autoimmune diseases, cardiovascular inflammation (e.g., atherosclerosis, stroke), gastrointestinal inflammation, liver inflammatory disorders, lung inflammation (e.g., asthma, ventilator-induced lung injury), kidney inflammation, eye inflammation (e.g., uveitis), pancreatic inflammation, genitourinary inflammation, neuroinflammatory disorders (e.g., multiple sclerosis, Alzheimer's disease), allergic reactions (e.g., allergic rhinitis / sinusitis), skin allergies and disorders (e.g., urticaria). (icaria / hives), angioedema, atopic dermatitis, contact dermatitis, psoriasis), food allergies, drug allergies, insect allergies, mastocytosis), bone inflammation (e.g., arthritis, osteoarthritis, rheumatoid arthritis, spondyloarthritis), infections (e.g., bacterial or viral infections); oral inflammatory disorders (i.e., periodontitis, gingivitis, or somatitis); and transplants (e.g., allogeneic or xenograft rejection, maternal-fetal tolerance, graft-versus-host disease).

[0159] This invention can be used to treat various inflammatory diseases or conditions for which treatment with recombinant wild-type IL-37 is recommended, such as those mentioned by Dinarello et al., Eur. J. Immunol. (2016) 46: 1067-1081, particularly those mentioned in Table 3.

[0160] Inflammatory diseases or conditions considered for treatment in the methods or applications of the present invention, or by means of the peptides or pharmaceutical compositions of the present invention, include those primarily mediated by activation of Toll-like receptors (TLRs) 2, 4, 7, 8, and / or 9. An overview of those diseases and conditions associated with TLR activation can be found in Connolly et al., Current Opinion in Pharmacology 2012, 12:510-518. Figure 1 And in Table 1. Exemplary indications contemplated for treatment by the methods or uses of the present invention, or by the polypeptides or pharmaceutical compositions of the present invention, and by TLRs that primarily mediate inflammation of the indication, are:

[0161] • Ischemia / reperfusion injury, myocardial ischemia, delayed graft function (TLR 2);

[0162] • Rheumatoid arthritis (TLR 2);

[0163] • Systemic lupus erythematosus (TLR7, 8 and / or 9);

[0164] • Sepsis (TLR 4 and other TLRs); and

[0165] • Acute and chronic inflammation (TLR 4).

[0166] The methods or uses of the present invention, or the polypeptides or pharmaceutical compositions of the present invention, may be used to reduce, inhibit or prevent inflammation induced by other mediators such as other cytokines (IL-1, IFNγ, etc.), other mediators (e.g. complement, leukotrienes, etc.), and chemical and physical damage.

[0167] Autoimmune diseases include, for example, acquired immunodeficiency syndrome (AIDS, a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, celiacsprue-herpetic dermatitis; chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating diseases. Multiple neuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, malignant atrophic papulosis, juvenile dermatomyositis, discoid lupus, primary cryoglobulinemia, fibromyalgia, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Stiel's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple myeloma, myasthenia gravis, pernicious anemia (pemacious anemia). (anemia), polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Leter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive systemic sclerosis (PSS), also known as systemic sclerosis (SS)), Sjögren's syndrome, stiff-person syndrome, systemic lupus erythematosus, aortitis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vitiligo, and Wegener's granulomatosis.

[0168] Preferably, the inflammatory disease or condition is multiple sclerosis, rheumatoid arthritis, skin allergies such as atopic dermatitis, contact dermatitis, psoriasis, inflammatory bowel disease, uveitis, dry eye disease, systemic sclerosis (scleroderma), periodontal disease, vitiligo, SLE / discular lupus / Graves' disease, atherosclerosis, asthma, or delayed-type hypersensitivity reaction.

[0169] Multiple sclerosis (MS) is an inflammatory disease involving demyelination of the myelin sheaths surrounding the axons of the brain and spinal cord. MS symptoms include, but are not limited to, white matter scarring in the brain and / or spinal cord and a wide variety of neurological symptoms, including, but not limited to, altered sensations such as loss of sensitivity or tingling, tingling, or numbness (decreased sensation and paralysis), muscle weakness, clonus, muscle spasms, or difficulty moving; difficulties with coordination and balance (ataxia); problems with speech (dysarthria) or swallowing (dysphagia); visual problems (nystagmus, optic neuritis, etc.); fatigue; acute / chronic pain; and bladder and bowel problems. Different degrees of cognitive impairment and depression are also common. Symptoms of MS typically appear during sporadic acute exacerbations of progressively worsening neurological function, or both.

[0170] Rheumatoid arthritis is a chronic, systemic inflammatory disease that can affect many tissues and organs, but primarily attacks the synovial joints. The process involves synovial cell proliferation, excessive synovial fluid, and the development of fibrous synovial tissue, leading to an inflammatory response in the synovial bursa surrounding the joint. The pathology of the disease often results in the destruction of articular cartilage and joint ankylosis. Rheumatoid arthritis can also cause diffuse inflammation in the lungs, pericardium, pleura, sclera, and nodular lesions, most commonly in the subcutaneous tissues.

[0171] Other inflammatory diseases or conditions include acute inflammation associated with stroke, myocardial infarction, ischemia-reperfusion injury, and transplantation. In addition, chronic inflammation is associated with autoinflammatory diseases or autoimmune disorders such as multiple sclerosis and diabetes (type 1 or type 2 diabetes) mentioned above.

[0172] Further inflammatory diseases or conditions include sepsis, septic shock, or endotoxin shock. Sepsis or endotoxin shock can be caused by bacteria, fungi, viruses, or parasites. Bacterial pathogens can be lipopolysaccharides (LPS), also known as lipotoxins and endotoxins, which are large molecules composed of lipids and polysaccharides with O-antigens, their outer and inner nuclei linked by covalent bonds; they are present in the outer membrane of Gram-negative bacteria and elicit a strong immune response in animals.

[0173] For any inflammatory disease or condition described herein, when the polypeptide of the present invention is administered topically to a human, the therapeutically effective amount of the compound preferably corresponds to about 0.01 to about 10% (w / w), or about 0.1 to 10% (w / w), or about 1.0 to about 10% (w / w), about 0.1 to about 5% (w / w), or about 1.0 to about 5% (w / w). In any inflammatory disease or condition described herein, when the polypeptide of the present invention is administered orally to a subject, the therapeutically effective amount of the compound preferably corresponds to about 1 to about 50 mg / kg, or about 1 to about 25 mg / kg, or about 1 to about 10 mg / kg, about 5 to about 25 mg / kg, or about 10 to about 20 mg / kg.

[0174] In the compositions of the present invention, the proportion of the polypeptides present as monomers can be at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, about 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total polypeptides present in the composition, typically stored in solution for a suitable period of time under suitable conditions. Suitable time periods and conditions include the range and conditions of time and conditions that a person skilled in the art would reasonably expect to keep the polypeptides in solution prior to use. For example, time periods of about 24 hours, about 48 hours, or about 72 hours are typical, although some solutions can keep them for longer periods, such as at least one week, one month, six months, one year, two years, three years, or longer. Storage conditions are typically room temperature and relatively humid, or typically 25°C and 60% relative humidity, but can include any standard storage conditions encountered by a person skilled in the art, such as about 4°C, -20°C, or -80°C.

[0175] The frequency of application can be once daily, or two or three times daily. The treatment period can be the duration of detectable disease.

[0176] Typically, therapeutically effective doses are formulated to contain concentrations of at least about 0.1% up to about 50% or higher (by weight), and all combinations and sub-combinations within such ranges. The composition can be formulated to contain one or more polypeptides of the invention at concentrations from about 0.1% to less than about 50%, such as about 49, 48, 47, 46, 45, 44, 43, 42, 41, or 40%, and at concentrations from greater than about 0.1%, such as about 0.2, 0.3, 0.4, or 0.5%, to less than about 40%, such as about 39, 38, 37, 36, 35, 34, 33, 32, 31, or 30%. Exemplary compositions may contain from about 0.5% to less than about 30%, such as about 29, 28, 27, 26, 25, 25, 24, 23, 22, 21, or 20%, wherein the concentration is from more than about 0.5%, such as about 0.6, 0.7, 0.8, 0.9, or 1%, to less than about 20%, such as about 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10%. Compositions may contain from more than about 1%, such as about 2%, to less than about 10%, such as about 9% or 8%, including more than about 2%, such as about 3% or 4%, to less than about 8%, such as about 7% or 6%. The active agent may be present, for example, at a concentration of about 5%. In all cases, these amounts may be adjusted to compensate for variations in the amount of active ingredient actually delivered to the treated cells or tissues.

[0177] Although this invention is intended for human use, it can also be used for therapeutic veterinary purposes. This invention is useful for livestock or farm animals such as cattle, sheep, horses, and poultry; for companion animals such as cats and dogs; and for zoo animals.

[0178] Pharmaceutical compositions can be formulated for any suitable route of administration, including, for example, external (e.g., transdermal or ocular), oral, buccal, nasal, vaginal, rectal, or parenteral administration. The term "parenteral" as used herein includes subcutaneous, intradermal, intravascular (e.g., intravenous), intramuscular, spinal, intracranial, intrathecal, intraocular, periorbital, intra-orbital, intrasynovial, and intraperitoneal injections, as well as any similar injection or infusion techniques. In some embodiments, compositions in forms suitable for oral or parenteral use are preferred. Suitable oral forms include, for example, tablets, troche, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups, or elixirs. In other embodiments, the compositions provided herein can be formulated as lyophilized products.

[0179] Various dosage units are preferably provided as discrete-dose tablets, capsules, lozenges, sugar-coated pills, gels, or other types of solid dosage forms. Capsules may encapsulate powders, liquids, or gels. The solid dosage form may be swallowed or may be suckable or chewable (fragile or gel-like). The invention contemplates dosage unit holding devices other than blister packaging; for example, packaging such as bottles, tubes, cans, or packets. Dosage units may also include conventional excipients well-known in pharmaceutical formulation practice, such as binders, gelling agents, fillers, tableting lubricants, disintegrants, surfactants, and colorants; and suckable or chewable formulations.

[0180] Compositions intended for oral use may further comprise one or more components, such as sweeteners, flavoring agents, coloring agents, and / or preservatives, to provide an appealing and palatable formulation. Tablets contain the active ingredient mixed with physiologically acceptable excipients suitable for the manufacture of tablets. Such excipients include, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrants such as corn starch or alginate; binders such as starch, gelatin, or gum arabic; and lubricants such as magnesium stearate, stearic acid, or talc. Tablets may be uncoated, or they may be coated using known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a prolonged duration of action. For example, delaying materials such as glyceryl monostearate or glyceryl distearate may be used.

[0181] Preparations intended for oral use may also be presented as hard gelatin capsules, wherein the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules, wherein the active ingredient is mixed with an aqueous or oily medium such as peanut oil, liquid paraffin or olive oil.

[0182] Aqueous suspensions contain a mixture of active ingredients and excipients suitable for making aqueous suspensions. These excipients include suspending agents such as sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum arabic, and gum arabic; and dispersants or wetting agents such as naturally occurring phospholipids (e.g., lecithin), condensation products of alkylene oxides and fatty acids such as polyoxyethylene stearate, condensation products of ethylene oxide and long-chain fatty alcohols such as heptadecaethyleneoxycetyl alcohol, condensation products of ethylene oxide and esters derived from fatty acids and hexitols such as polyoxyethylene sorbitan monooleate, or condensation products of ethylene oxide and esters derived from fatty acids and hexitol anhydrides such as polyvinyl dehydrated sorbitan monooleate. Aqueous suspensions may also contain one or more preservatives, such as ethyl or n-propyl parabens, one or more colorants, one or more flavoring agents, and one or more sweeteners, such as sucrose or saccharin.

[0183] Oily suspensions can be formulated by suspending the active ingredient in vegetable oils such as peanut oil, olive oil, sesame oil, or coconut oil, or in mineral oils such as liquid paraffin. Oily suspensions may contain thickeners such as beeswax, hard paraffin, or cetyl alcohol. Sweeteners and / or flavorings, as described above, can be added to provide a palatable oral formulation. Such suspensions can be preserved by adding antioxidants such as ascorbic acid.

[0184] Dispersible powders and granules suitable for preparing aqueous suspensions by adding water provide an active ingredient that can be mixed with a dispersant or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersants or wetting agents and suspending agents include those already mentioned above. Additional excipients, such as sweeteners, flavoring agents, and coloring agents, may also be present.

[0185] Pharmaceutical compositions may also be in the form of oil-in-water emulsions. The oil phase may be vegetable oils such as olive oil or peanut oil, mineral oils such as liquid paraffin, or mixtures thereof. Suitable emulsifiers include naturally occurring gums such as gum arabic or tragacanth, naturally occurring phospholipids such as soybean lecithin, and esters or metaesters derived from fatty acids and hexitols, acid anhydrides such as sorbitan monooleate, and condensation products of fatty acid metaesters and hexitols with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion may also contain one or more sweeteners and / or flavoring agents.

[0186] Syrups and elixirs can be formulated with sweeteners such as glycerin, propylene glycol, sorbitol, or sucrose. Such formulations may also contain one or more modifiers, preservatives, flavoring agents, and / or coloring agents.

[0187] The polypeptides of the present invention can be formulated for local or topical administration, such as external application to the skin. Formulations for external application typically contain a topical carrier in combination with one or more active agents, with or without additional optional components.

[0188] Suitable topical media and adjuncts are well known in the art, and it is apparent that the choice of media will depend on the specific physical form and delivery method. Topical media include organic solvents such as alcohols (e.g., ethanol, isopropanol, or glycerol), glycols such as butanediol, isopentyl glycol, or propylene glycol, aliphatic alcohols such as lanolin, mixtures of water and organic solvents, and mixtures of organic solvents such as alcohols and glycerol, lipid-based substances such as fatty acids, acylglycerols including oils such as mineral oils, fats of natural or synthetic origin, glyceryl phosphates, sphingolipids, and waxes, protein-based substances such as collagen and gelatin, silicone-based substances (both non-volatile and volatile), and hydrocarbon-based materials such as microsponges and polymer matrices.

[0189] The composition may further comprise one or more components suitable for improving the stability or effectiveness of the applied formulation, such as stabilizers, suspending agents, emulsifiers, viscosity modifiers, gelling agents, preservatives, antioxidants, skin penetration enhancers, humectants, and sustained-release materials. Examples of these components are described in Martindale – The Extra Pharmacopoeia (Pharmaceutical Press, London 1993) and Martin (ed.), Remington's Pharmaceutical Sciences. The formulation may comprise microcapsules, such as hydroxymethyl cellulose or gelatin microcapsules, liposomes, albumin microspheres, microemulsions, nanoparticles, or nanocapsules.

[0190] Topical formulations can be prepared in various physical forms, including, for example, solids, pastes, creams, foams, lotions, gels, powders, aqueous liquids, emulsions, sprays, and patches. The physical appearance and viscosity of such forms can be controlled by the presence and amount of one or more emulsifiers and one or more viscosity modifiers in the formulation. Solids are typically firm and non-pourable, and are usually formulated in strips or rods, or in granule form. Solids can be opaque or transparent, and optionally may contain solvents, emulsifiers, moisturizers, lubricants, fragrances, dyes / colorants, preservatives, and other active ingredients that enhance or improve the efficacy of the final product. Creams and lotions are generally similar to each other, differing primarily in viscosity. Both lotions and creams can be opaque, translucent, or transparent, and typically contain emulsifiers, solvents, and viscosity modifiers, as well as moisturizers, lubricants, fragrances, dyes / colorants, preservatives, and other active ingredients that enhance or improve the final effect. Gels can be prepared in a range of viscosities, from thick or high viscosity to thin or low viscosity. These formulations, such as lotions and creams, may also contain solvents, emulsifiers, moisturizers, lubricants, fragrances, dyes / colorants, preservatives, and other active ingredients that enhance or increase the efficacy of the final product. Liquids are thinner than creams, lotions, or gels and typically do not contain emulsifiers. Liquid topical products generally contain solvents, emulsifiers, moisturizers, lubricants, fragrances, dyes / colorants, preservatives, and other active ingredients that enhance or increase the efficacy of the final product.

[0191] Emulsifiers for topical formulations include, but are not limited to, ionic emulsifiers, cetearyl alcohol, and nonionic emulsifiers such as polyoxyethylene oleyl ether, PEG-40 stearic acid, ceteareth-12, ceteareth-20, ceteareth-30, ceteareth alcohol, PEG-100 stearic acid, and glyceryl stearate. Suitable viscosity modifiers include, but are not limited to, protective colloids or nonionic colloids such as hydroxyethyl cellulose, xanthan gum, magnesium aluminum silicate, silica, microcrystalline wax, beeswax, paraffin, and hexadecyl palmitate. Gel compositions can be formed by adding gelling agents such as chitosan, methylcellulose, ethylcellulose, polyvinyl alcohol, polyquaternium salts, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, carbomer, or aminated glycyrrhizate. Suitable surfactants include, but are not limited to, nonionic, amphoteric, ionic, and anionic surfactants. For example, one or more of the following can be used in topical preparations: polydimethylsiloxane copolyol, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, lauramide DEA, cocamide DEA and cocamide MEA, oil-based betaine, cocamidopropyl phosphatidyl PG-dimethylammonium chloride and lauryl ether ammonium sulfate.

[0192] Preservatives include, but are not limited to, antimicrobial agents such as methylparaben, propylparaben, sorbic acid, benzoic acid, and formaldehyde, as well as physical stabilizers and antioxidants such as vitamin E, sodium ascorbate / ascorbic acid, and propyl gallate. Suitable humectants include, but are not limited to, lactic acid and other hydroxy acids and their salts, glycerin, propylene glycol, and butylene glycol. Suitable lubricants include lanolin alcohol, lanolin, lanolin derivatives, cholesterol, petrolatum, isostearate neopentyl ester, and mineral oil. Suitable fragrances and colors include, but are not limited to, FD&C Red No. 40 and FD&C Yellow No. 5. Other suitable adjuncts that may be included in topical formulations include, but are not limited to, abrasives, absorbents, anti-caking agents, defoamers, antistatic agents, astringents (such as witch hazel), alcohols and herbal extracts such as chamomile extract, binders / excipients, buffers, chelating agents, film-forming agents, thickeners, sprays, sunscreens, pH adjusters, and protectants.

[0193] Typical delivery methods for topical compositions include application using fingers, application using physical applicators such as cloths, tissues, swabs, sticks, or brushes, spraying including mists, aerosols, or foam sprays, dropper application, spraying, soaking, and rinsing. Controlled-release media can also be used, and compositions can be formulated for transdermal application (e.g., as transdermal patches).

[0194] Pharmaceutical compositions can be formulated as inhaled preparations, including sprays, nebulizers, or aerosols. This may be particularly preferred for treating certain inflammatory diseases or conditions. For inhaled preparations, the compositions or combinations provided herein can be delivered via any inhalation method known to those skilled in the art. Such inhalation methods and devices include, but are not limited to, metered-dose inhalers with propellants such as CFCs or HFAs, or physiologically and environmentally acceptable propellants. Other suitable devices are breathing-operated inhalers, multi-dose dry powder inhalers, and aerosol nebulizers. Aerosol formulations used in this method typically comprise a propellant, a surfactant, and a co-solvent, and can be filled into a conventional aerosol container that closes via a suitable metering valve.

[0195] Inhalation compositions may comprise liquid or powder compositions containing an active ingredient suitable for nebulization and intrabronchial use, or aerosol compositions administered by dispensing metered doses of aerosol units. Suitable liquid compositions contain the active ingredient in a pharmaceutically acceptable aqueous inhalation solvent such as isotonic saline or antibacterial water. The solution is administered via a pump or squeeze-driven nebulizer or by any other conventional means to result in or bring the desired dose of the liquid composition into the patient's lungs. Suitable formulations for administration, wherein the carrier is a liquid, such as, for example, nasal sprays or nasal drops, comprising an aqueous or oily solution of the active ingredient.

[0196] Pharmaceutical compositions can also be prepared in suppository form, for example, for rectal administration. Such compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, and thus melts in the rectum to release the drug. Suitable excipients include, for example, cocoa butter and polyethylene glycol.

[0197] Pharmaceutical compositions can be formulated into sustained-release formulations, such as capsules that produce a slow release of a modifier upon administration. Such formulations are typically prepared using well-known techniques and administered, for example, via oral, rectal, or subcutaneous implantation, or by implantation at the desired target site. The carrier used in such formulations is biocompatible and may also be biodegradable. Preferably, the formulation provides a relatively constant level of modifier release. The amount of modifier contained in the sustained-release formulation depends, for example, on the implantation site, the rate of release, the expected duration of release, and the nature of the condition to be treated or prevented.

[0198] In another embodiment, a kit or product comprising one or more polypeptide or polynucleotide and / or pharmaceutical compositions of the present invention as described above is provided.

[0199] In other embodiments, a kit is provided for the above-described treatment or preventative application, the kit comprising:

[0200] - A container for containing the polypeptide, polynucleotide, or pharmaceutical composition of the present invention;

[0201] - A label or packaging insert with instructions for use.

[0202] In some implementations, the kit may contain one or more other effective substances or ingredients for treating inflammatory diseases or conditions.

[0203] The kit or "article" may include a container and a label or packaging insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister packs, etc. Containers can be formed from various materials, such as glass or plastic. The container contains a therapeutic composition that effectively treats a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). The label or packaging insert indicates that the therapeutic composition is intended to treat the selected condition. In one embodiment, the label or packaging insert includes instructions for use and indicates that the therapeutic or preventative composition may be used to treat the inflammatory disease or condition described herein.

[0204] The kit may comprise (a) a therapeutic or preventative composition; and (b) a second container containing a second active substance or ingredient. The kit in this embodiment of the invention may further comprise a packaging insert indicating that the composition and other active ingredients can be used to treat a condition or prevent complications arising from the inflammatory disease or condition described herein. Alternatively or additionally, the kit may further comprise a second (or third) container containing a pharmaceutically acceptable buffer solution, such as bactericidal water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. It may further include other materials required from a commercial and user perspective, including other buffer solutions, diluents, filters, needles, and syringes.

[0205] In some embodiments, the therapeutic composition may be provided in the form of a disposable or reusable device, including a container for containing a therapeutic agent, preventative agent, or pharmaceutical composition. In one embodiment, the device is a syringe. The device can contain 1-2 mL of the therapeutic composition. The therapeutic or preventative composition may be provided in the device in a ready-to-use state or in a state where mixing or adding other components is required.

[0206] The peptides or compositions of the present invention can be used as anti-inflammatory coatings for implantable materials and devices, such as stents. The peptides or compositions of the present invention can be coated onto or integrated with implantable materials or devices. The peptides or compositions of the present invention can be part of a polymer coating.

[0207] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more individual features mentioned or clearly visible from the text or drawings. All these different combinations constitute various alternative aspects of the invention. Example

[0208] Example 1

[0209] Cloning and protein purification

[0210] Codon-optimized IL-37 (46-218) was cloned into a modified tobacco-etched viral protease-cleavable form of pGEX-4T-1 (GE Healthcare) (7). Recombinant protein expression was induced in BL21-CodonPlus (DE3)-RIL cells (Stratagene) at 18 °C via IPTG. Cells expressing the GST-IL-37 variant were lysed by high-pressure cavitation (10–15 Kpsi) in 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, and 3 mM β-mercaptoethanol in tablets containing two completely EDTA-free protease inhibitors (Roche). Cells were clarified by centrifugation, filtered through a 0.45 μm membrane, and bound to glutathione Sepharose 4B resin (GE Healthcare) at 4 °C for 1 h. The resin was washed with 500 ml of 20 mM Tris-HCl (pH 8.0), 200 mM NaCl, and 3 mM β-mercaptoethanol. The protein was released from the GST-tag by incubation with His-TEV protease overnight at 4°C. The protein was further purified on a HiLoad Superdex 75 16 / 60 preparative-grade column (GE Healthcare) in 20 mM Hepes (pH 7.2), 100 mM NaCl, 2 mM DTT, and 1 mM EDTA.

[0211] Crystallization and structural determination

[0212] IL-37 crystals were grown at 20 °C via pendant vapor diffusion in a 1:1 drop ratio of 2.1 M ammonium sulfate and 0.1 M sodium acetate (pH 4.5). The crystals were rapidly cooled in liquid nitrogen in a mother liquor containing 20% ​​(v / v) glycerol. The crystals were then grown at the MX2 beamline (microfocus) of the Australian Synchrotron. X-ray data were collected at a wavelength of 1° oscillation. The data were processed and normalized (scaled) using programs within the XDS and CCP4 suites (8). The structure was resolved by molecular substitution using MRage and Phaser (10,11) with mouse IL-F5 (PDB-coded 1MD6 (9)) as the search model in PHENIX. The structure was automatically built in PHENIX AutoBuild (11). The R-factor was 18.22% (R) by iterative loops of local reconstruction in COOT (13) using Buster (12) with corrections. 自由 (R free The model has a good geometric structure (Table 3). The structure has no Ramachandran outliers, 98.64% of the residues are in favorable regions, and the final MolProbity score is 0.95 (percentile) (14).

[0213] Size exclusion chromatography and multi-angle light scattering (SEC-MALS)

[0214] SEC-MALS measurements were performed on a Superdex 7510 / 300 column (GE Healthcare) equilibrated with 10 mM HEPES (pH 7.3), 100 mM NaCl, 1 mM EDTA, and 2 mM DTT. All experiments were performed at 25°C at a flow rate of 0.4 mL / min in the above buffers. Each run used an injection volume of 110 μL of 6 mg / mL protein. A test injection of 2 mg / mL bovine serum albumin (Thermo Scientific Pierce) was used for calibration purposes. The SEC-MALS system consisted of a Shimadzu DGU-20A5 degasser, an LC-20AD liquid chromatograph, and a SIL-20A... HT An autosampler, a CBM-20A communication bus module, an SPD-20AUV / VIS detector, and a CTO-20AC column oven were coupled to a DAWN HELEO-II multi-angle light scattering detector (Wyatt Technology) equipped with an Optilab T-rEX refractive index detector. Molar mass was calculated by measuring the intensity of scattered light at 18 different scattering angles. Molecular weight was calculated using Astra 6.1 software (Wyatt Technology).

[0215] Cell culture and transfection.

[0216] PBMC assays were performed with the approval of Monash Health Human Research Ethics Committee B and with the explicit written consent of all volunteers. PBMCs were isolated from peripheral venous blood of healthy volunteers by density gradient centrifugation as described in (15). PBMCs were seeded into RPMI medium containing 1% v / v human serum and 1:500 MycoZap PR, and then pretreated with the medium or recIL-37 as indicated for 30 min, followed by treatment with 50 pg / ml LPS or HKLM, imiquimod, CpG-A, or ssRNA40 at concentrations described herein for 20 h. The supernatant was then analyzed for cytokines. THP-1 cells were derived from ATCC. They were always cultured in the presence of MycoZap Plus-CL (Lonza) containing antibiotics, antifungal agents, and antimycoplasma agents.

[0217] As described in (16, 1, and 5), THP1 cells were transfected with constructs encoding either the native IL-37b protein or a monomeric D73K variant. Briefly, each IL-37b variant was inserted into a pIRES vector containing a GFP expression sequence and a constitutively active CMV promoter, and the C-terminus of IL-37b was ligated to a FLAG. Cells were transfected using the Amaxa Nucleofector KitV (THP 1) and program V001, followed by overnight recovery. Twenty hours post-transfection, cells were counted and plated. Transfected THP1 cells were differentiated into macrophages by incubation with 50 ng / ml PMA for 24 hours. The medium was then replaced with RPMI containing penicillin / streptomycin and 1% human serum, and the stimulant was added. After the incubation period indicated in the legend, the supernatant was collected and stored at -80°C until analysis. Sample collection and analysis were performed in a blinded manner.

[0218] ELISA and multiplex ELISA.

[0219] Cytokines were measured using a standard ELISA (BD, elisakit.com). Both ELISA methods were performed according to the manufacturer's recommendations.

[0220] Statistical analysis.

[0221] First, the normality and homoscedasticity of the test dataset (original data) were determined using SigmaPlot 12.5 (Systat Software Inc.) (P-value to rejection = 0.05). Subsequently, appropriate statistical tests were applied, including unpaired t-tests (two-tailed test, α = 0.05), Mann-Whitney rank-sum tests, one-way ANOVA, or one-way ANOVA on ranks.

[0222] Animal experiments.

[0223] All procedures involving mice were approved by the Monash Health Animal Ethics Committee. C57Bl / 6 wild-type mice received an intraperitoneal injection of 40 μg / kg of the recombinant IL-37 variant or the vector, followed by an intraperitoneal injection of LPS (10 mg / kg) 60 minutes later. Homozygous mice transgenic with IL-37b (1,5) were also treated with LPS (10 mg / kg) or the vector for direct comparison. Room temperature and humidity were continuously monitored. Body temperature was measured as described in (1). Twenty-four hours after LPS injection, mice were anesthetized and plasma was obtained via orbital hemorrhage and placed in heparinized tubes.

[0224] reagents

[0225] Human IL-1β (beta) (ELISA): Catalog No. 557953, Human IL-6 ELISA: Catalog No. 555220, Mouse IL-1β (beta) ELISA: Catalog No. 559603 (BD ​​Biosciences, New Jersey, USA), Escherichia coli LPS 055:85, #L4005-100mg (Sigma, St Louis MO, USA), CpG-A ODN 2216Innaxon#INAX-200-005 (Adipogen, Switzerland), Imiquimod VacciGrade#vac-img, HKLM#Tlr1-hklm (Invivogen, San Diego, CA, USA).

[0226] Example 2

[0227] The precursor and mature IL-37 form a homodimer in solution.

[0228] Size exclusion chromatography (SEC) analysis of the bacterial purified precursor IL-37 (isotype b, referred to as IL-37 in this paper) showed that the protein formed homodimers in solution. Figure 1A and 1B, left axis). To confirm that the protein is a homodimer, multi-angle light scattering (MALS) coupled to SEC was performed (…). Figure 1 (B, right axis). The measured molecular weight of the precursor IL-37 (residues 1-218) was 43.9 kDa, consistent with the theoretical molecular weight of 48 kDa for the IL-37 dimer. These data are relevant to previous studies detecting dimeric IL-37 (~45 kDa) in human PBMCs and reconstituted IL-37 (1,2) by ultracentrifugation. Figure 1 In B, IL-37(1-218) was eluted with 9.8 ml, IL-37(21-218) with 10 ml, IL-37(46-218) with 11 ml, and IL-18(37-193) with 12.6 ml.

[0229] To investigate whether N-terminal truncation from the precursor to the mature form prevented dimerization, IL-37 (residues 21-218) and IL-37 (residues 46-218) were purified from bacterial cells and subjected to MALS analysis. Figure 1 (A and 1B). Mature IL-37 (21-218) and IL-37 (46-218) each formed dimers in solution with molecular weights of 40.7 kDa and 37.3 kDa, respectively. Compared to IL-37, mature IL-18 (residues 37-193) has a measured molecular weight of 17.3 kDa, consistent with the monomer structure. Figure 1 B)(3).

[0230] Crystal structure of IL-37 homodimer

[0231] To elucidate the molecular basis of IL-37 dimerization, mature IL-37 (residues 46-218) was crystallized via hanging drop vapor diffusion. The structure was resolved and corrected through molecular substitution. Resolution, R 工作 / R 自由 (R work / R free The concentrations were 18.22% and 21.74%, respectively, and they possessed excellent geometry (Table 3). The asymmetric unit contained two copies of IL-37, designated IL-37A and IL-37B, which formed a head-to-head symmetric homodimer. Figure 1 C). To our knowledge, this arrangement of the IL-37 subunit into a symmetrical homodimer is unique in the structures of IL-1 family cytokines resolved to date. These two subunits are located on 142 Cα atoms. The RMS deviations show high structural homology. IL-37A is the most complete subunit, containing residues 48-207, with only residue 126 missing in the final plot. Overall, IL-37B has a higher B-factor and is less complete, containing residues 49-206, 125-128, 161, and 194-196 missing from the loop region. The secondary structure of the β-trefoil fold begins at Lys58, and the N-terminal residues 48-57 are mostly unstructured, with no electron density at residues 46-48. Two potential cleavage sites for mature IL-37 have been identified at residues 21 and 46, and each is compatible with maintaining the β-trefoil fold of cleaved IL-37 based on this structure.

[0232] Each IL-37 subunit consists of 12 β strands and three α helices, forming the characteristic β-trilobal folds of the IL-1 superfamily. Figure 1 C). Three pseudo-repeats of four β strands are bundled together, with two strands of each repeat forming a six-strand β-bucket and the remaining two strands forming a six-strand capping region. The interaction between the N-terminus and C-terminus strands β1 and β12 in the antiparallel β-bucket closes the β-trefoil fold. Three α-helices decorate the outer side of the β-trefoil fold, with helices α1 and α2 located between strands β7 and β8, and a short 3-helix following strand β11. 10 spiral.

[0233] Table 3. Statistics on Data Collection and Correction

[0234]

[0235]

[0236] The values ​​in parentheses represent the highest resolution shell, and the dataset is from a single crystal.

[0237] IL-37 homodimers have head-to-head symmetry and highly organized interfaces.

[0238] The symmetrical head-to-head IL-37 dimer interface has a total of The surface area is formed by β3-β4 rings and three strands of β-sheets (β2-β3-β11) in each subunit. Figure 2 A). The dimer interface appears highly organized, with numerous mirror interactions centered between the β3-β4 rings of each IL-37 subunit, forming across the C2 symmetry axis. Within the core of the interface, two main-chain hydrogen bonds are formed between Tyr85 and Arg87 from each IL-37 monomer. Figure 2B). The partially hydrophobic Tyr85 side chain of each subunit is embedded in a hydrophobic pocket formed on the surface of the β2-β3-β11β sheet. The hydrophobic core of this interface is shielded by ionic interactions between Lys83 and Asp73 at the solvent-exposed edges. Hydrogen bonds formed by the carbonyl group of the Ile86 main chain and the Arg87 side chain of each molecule further strengthen the β3-β4 ring interface. Figure 2 C).

[0239] To verify the molecular details of the interface and generate the monomer IL-37, several structure-guided mutations targeting and interfering with IL-37 dimerization were designed. Asp73 forms an ionic interaction with Lys83 within the interface, and the charge-switching mutation (D73K) on Asp73 effectively eliminates the interface, resulting in a molecular weight of 18.8 kDa corresponding to the IL-37 monomer. Figure 2 D). Tyr85 is located at the hydrophobic core and contributes a large buried surface area to the interface. The mutation to alanine (Y85A) also disrupts the dimer interface. It has a molecular weight of 18.2 kDa as measured by SEC-MALS. Figure 2 D; IL-37WT was eluted in 11 ml, IL-37D73A in 11.6 ml, IL-37D73K in 12.7 ml, and IL-37Y85A in 12.6 ml. Due to the symmetry of the IL-37 dimer interface, each point mutation effectively targets two interacting sites within the interface and may further contribute to the disruption of the dimer interface.

[0240] To assess changes at the monomer-dimer interface, five IL-37 mutants have been tested using SEC-MALS. Figure 3 IL-37D73A+K83A was eluted with 11.1 ml, and IL-37K83E was eluted with 11.6 ml. Elution times for other mutants and wild-type were the same as above. Figure 2The D-related mutation, located at the core of the interface, transforms into alanine (Y85A), converting IL-37 into a monomer. Asp73, situated at the interface boundary, forms ionic interactions with Lys83. The Asp73 mutation into alanine (D73A) partially disrupts the dimer interface, shifting the equilibrium towards monomer. It is speculated that a charge-transformation mutation in Asp73 (D73K) eliminates the dimer interface through charge repulsion with Lys83. A charge-transformation mutation in Lys83 (K83E) shifts the equilibrium towards monomer, but is less effective than D73K in eliminating the interface. Interestingly, the complete removal of the gating interface by ionic interactions (D73A / K83A) appears to have only a small effect on the equilibrium. These mutations suggest that ionic gating interactions are not necessary for interface formation. Instead, the buried Tyr85 side chains and hydrogen bonds in the β3-β4 ring region may be more critical.

[0241] Example 3

[0242] Two methods were used to assess the functional impact of IL-37 monomerization. First, fresh human PBMCs were treated with different variants of recombinant IL-37, including the native protein (wild-type) that spontaneously dimers even at very low concentrations, and a variant in which dimerization was prevented by mutations in amino acids 73 through D to K. Both variants were tested with N-terminal truncation at amino acids 21 and 46.

[0243] As expected by previous studies, natIL-37 (wild-type recIL-37) conferred a modest reduction in LPS-induced IL-1β protein (natIL-37(21-218) up to 40%, natIL-37(46-218) up to 52%). Figure 5 (Left group). Interestingly, increasing the concentration of natIL-37 to above 10 ng / ml, such as 100 ng / ml, significantly reduced the effectiveness of natIL-37. Figure 5 In fact, in all three donors, 100 ng / ml natIL-37 (46-218) induced a pro-inflammatory response (IL-1β increased by up to 36%).

[0244] Monomerization of IL-37 significantly increased these anti-inflammatory activities. At all tested concentrations, MonoIL-37 (D73K) was more effective than natIL-37 in blocking IL-1β release in LPS-treated PBMCs. Figure 5At 10 pg / ml, monoIL-37b (46-218) reduced IL-1β by 59%, nearly twice that of natIL-37 (46-218), and at 100 ng / ml, monoIL-37 (46-218) was more than five times more effective than natIL-37b (46-218). Furthermore, monoIL-37b maintained its anti-inflammatory activity at higher concentrations; therefore, dimerization may contribute to reducing IL-37 activity at such concentrations, or actually convert it into pro-inflammatory activity, as seen in the case of the 21-218 variant at 100 ng / ml (monoIL-37b reduced IL-1β by 21%, compared to a 26% increase in IL-1β by natIL-37). For monoIL-37 (46-218), the concentration range was further extended, with anti-inflammatory effects observed at 1 pg / ml (52% reduction in IL-1β) and 1 μg / ml. Furthermore, the loss of anti-inflammatory function occurred at concentrations 10 times higher than natIL-37 (1 μg / ml). Notably, at the lower end of the 1 pg / ml concentration range, monomeric 46-218recIL-37b still exhibited 1.6 times the activity of the natural dimer recIL-37 at 10 pg / ml. Figure 5 N-terminal truncation also affects IL-37 function: by comparing the two, the natural one, and the D73K mutant with their counterparts ( Figure 5 Group 1 is relative to Group 3 and Group 2 is relative to Group 4 (as designated from left to right as Group 1 to Group 4). Variant 46-218 is much more effective than variant 21-218 in blocking IL-1β production.

[0245] To investigate the intracellular mechanism of action of IL-37, the effect of IL-37 monomerization on its anti-inflammatory efficacy was assessed by transfecting THP-1 macrophages. Therefore, for the first time, the anti-inflammatory activities of the dimer native IL-37b (natIL-37) and the mutant monomeric IL-37b (D73K, monoIL-37) in THP-1 macrophages were compared. Figure 6 Compared to control transfection, transfection with full-length natIL-37 reduced LPS-induced IL-1β protein abundance by 71% (271 to 80 pg / ml). MonoIL-37 exhibited 2.7-fold stronger anti-inflammatory activity, reducing IL-1β by 89% (271 to 30 pg / ml). Unlike natIL-37, monoIL-37 also reduced IL-1β in media-treated cultures. These data suggest that the anti-inflammatory activity of monoIL-37 is greater than that of the dimer natIL-37, and that IL-37 bioactivity does not require dimerization.

[0246] The protein abundance of TNF was also assessed in the supernatant of PBMC and THP-1 cultures, revealing that IL-37 also blocked this pro-inflammatory cytokine, although its potency was slightly lower than that of IL-1β (data not shown).

[0247] Example 4

[0248] In vivo model of endotoxin shock

[0249] To augment the in vivo dimension of these findings, a mouse model of endotoxin shock was used. C57Bl / 6 wild-type mice received an intraperitoneal injection of either a recombinant IL-37 variant (40 μg / kg) or a vector, followed by an intraperitoneal injection of LPS. IL-37 transgenic mice were also treated with either LPS or the vector for direct comparison. Although each IL-37 variant improved endotoxin shock (reducing hypothermia and plasma IL-1β), Figure 7 and 8 The anti-inflammatory activities of both monomeric IL-37 proteins were higher than those of their dimeric natIL-37 counterparts. Notably, the protection provided by monoIL-37(46-218) was almost as strong as that conferred by the IL-37 transgene. In fact, the difference in body temperature and plasma IL-1β between the monoIL-37(46-218) group and mice not treated with LPS was minimal.

[0250] Example 5

[0251] Mutations at position 85 from tyrosine to alanine (besides the D73K mutation) are another pathway that disrupts the IL-37 dimer interface. Partially hydrophobic tyrosine residues at position 85 of each subunit are buried in hydrophobic pockets formed by the dimer interface. Mutating this residue to alanine significantly reduces these interactions and disrupts the dimer interface. Therefore, recIL-37Y85A cannot form a homodimer. Consistent with the concept that monomeric IL-37 has higher biological activity than the dimer IL-37b, recIL-37D73K, and especially native recIL-37b, shows increased activity in PBMCs stimulated in vitro with heat-inactivated Listeria monocytogenes (HKLM) by a TLR2 agonist. Figure 9 In mice injected in vivo with the TLR4 agonist LPS, the anti-inflammatory effect of recIL 37Y85A was increased. Figure 7 However, in PBMCs stimulated with imiquimod and CpG-A, the ligands of TLR 7 and TLR 9 respectively, there was little difference in bioactivity between recIL-37D73K and Y85A.

[0252] Overall, the data from the Y85A variant support the concept that shifting the IL-37 monomer-dimer balance toward the monomer when used for extracellular treatment increases the anti-inflammatory properties of IL-37.

[0253] Example 6

[0254] Effects of recIL-37 and monomeric variants on inflammation induced by TLR agonists other than LPS

[0255] IL-37 is a potent buffer against inflammation triggered by various inflammatory attacks. In vitro, the inventors have shown that, in addition to LPS, these attacks also include the TLR1 agonist Pam3CSK4 (Nold et al., Nat Immunol 2010). Figure 1 ), IL-1β (Nold et al., Nat Immunol 2010) Figure 3 and 4 LPS+IL-12, TNF and IL-12+IL-18 (Nold et al., Nat Immunol 2010) Figure 7 Along with Nold-Petry et al., in Nat Immunol 2015 Figure 4 In addition, IL-37 plays a protective role in a large number of animal disease models, including DSS colitis, ichthyosis pilaris, contact hypersensitivity reactions, and other diseases, in addition to endotoxin shock models.

[0256] This activity against a wide range of inflammatory triggers has a high translational relevance, so it can be expected that IL-37 will effectively improve inflammation in the same wide range of diseases, from the common cold to psoriasis, myocardial infarction, stroke and many other diseases.

[0257] However, data described in Nold et al., Nat Immunol 2010 and Nold-Petry et al., Nat Immunol 2015, were generated by using cells transfected to express IL-37 or mouse strains transfected with the IL-37 gene. Since IL-37 has a dual mechanism of action, including intracellular and extracellular signaling pathways, it is important to establish that treatment with recIL-37, which may act almost exclusively through the extracellular IL-37 receptor-mediated pathway, not only effectively blocks LPS-induced inflammation but also inflammation induced by other inflammatory agents. The data described in this paper suggest that this is indeed the case.

[0258] In human PBMCs stimulated with the TLR2 agonist HKLM, recIL-37 conferred up to a 51% reduction in IL-1β and up to a 36% reduction in IL-6 protein abundance. Figure 9 In cultures treated with the following method, the abundance of pro-inflammatory cytokines was also lower: TLR7 ligand imiquimod + recIL-37b (reducing IL-1β by up to 36%). Figure 10 TLR9 agonist CpG-A+recIL-37b (reduces IL-6 by up to 30%) Figure 11 ) and TLR8 ligand ssRNA40 (reduces IL-1β by up to 49%) Figure 12 ).

[0259] Furthermore, the data in this paper strongly suggest that the anti-inflammatory effect of the monomeric recIL-37 is greater than that of the recIL-37 that can form homodimers. Figure 5 The results showed that in PBMCs stimulated with the TLR4 ligand LPS, the monomeric D73K variant was significantly more active than its native dimer counterpart at most concentrations. Similar observations were observed in PBMCs stimulated with TLR2 and 7, 8, and 9 ligands: the Y85A variant was significantly more active than native recIL-37 in blocking HKLM (TLR2 ligand) and imiquimod (TLR7 ligand)-induced IL-1β or IL-6 (respectively, respectively). Figure 9 and 10 Furthermore, in cultures stimulated with imiquimod and CpG-A (TLR9 ​​ligand), the inhibition of IL-1β and IL-6 conferred by D73K and Y85A was significant, while that conferred by natIL-37b was not (see [references]). Figure 10 and 11 Although statistical analysis of the TLR8 ligand ssRNA40 was impossible because only two donors were studied, D73K was significantly more effective than natIL-37 in blocking IL-1β at every concentration tested. Figure 12 ).

[0260] Monomeric variants of IL-37 are clinically effective pharmacologically active ingredients that block inflammation in a variety of diseases caused by extensive inflammatory damage.

[0261] References

[0262] 1.Nold, MF, Nold-Petry, CA, Zepp, JA, Palmer, BE, Bufler, P., and Dinarello, CA (2010) IL-37 is a fundamental inhibitor of innateimmunity. Nat. Immunol. 11, 1014–1022

[0263] 2.Kumar,S.,Hanning,CR,Brigham-Burke,MR,Rieman,DJ,Lehr,R,Khandekar,S,Kirkpatrick,RB,Scott,GF,Lee,JC,Lynch,FJ,Gao,W,Gambotto,A,and Lotze,MT(2002)Interleukin-1F7B(IL-1H4 / IL-1F7)is processed bycaspase-1and mature IL-1F7B binds to the IL-18receptor but does not induceIFN-gamma production.Cytokine.18,61–71

[0264] 3.Kato, Z., Jee, J., Shikano, H., Mishima, M., Ohki, I., Ohnishi, H., Li, A., Hashimoto, K., Matsukuma, E., Omoya, K., Yamamoto, Y., Yoneda, T., Hara, T., Kondo, N., andShirakawa, M. (2003)The structure and binding mode of interleukin-18.Nat.Struct.Biol.10,966–971

[0265] 4.Tsutsumi,N.,Kimura,T.,Arita,K.,Ariyoshi,M.,Ohnishi,H.,Yamamoto,T.,Zuo,X.,Maenaka,K.,Park,EY,Kondo,N.,Shirakawa,M.,Tochio,H.,and Kato,Z interleukin-18.NatCommun.5.5340

[0266] 5.Nold-Petry,C.A.,Lo,C.Y.,Rudloff,I.,Elgass,K.D.,Li,S.,Gantier,M.P.,Lotz-Havla,A.S.,Gersting,S.W.,Cho,S.X.,Lao,J.C.,Ellisdon,A.M.,Rotter,B.,Azam,T.,Mangan,N.E.,Rossello,F.J.,Whisstock,J.C.,Bufler,P.,Garlanda,C.,Mantovani,A.,Dinarello,C.A.,and Nold,M.F.(2015)IL-37requires the receptors IL-18RαandIL-1R8(SIGIRR)to carry out its multifaceted anti-inflammatory program uponinnate signal transduction.Nat.Immunol.16,354–365(2015).

[0267] 6.Krissinel,E.,and Henrick,K.(2007)Inference of macromolecularassemblies from crystalline state.J.Mol.Biol.372,774–797

[0268] 7.Matsuura,Y.,and Stewart,M.(2004)Structural basis for the assemblyof a nuclear export complex.Nature.432,872–877

[0269] 8.Collaborative Computational Project,Number 4(1994)The CCP4 suite:programs for protein crystallography.Acta Crystallogr.D Biol.Crystallogr.50,760–763

[0270] 9.Dunn,E.F.,Gay,N.J.,Bristow,A.F.,Gearing,D.P.,O'Neill,L.A.J.,andPei,X.Y.(2003)High-resolution structure of murine interleukin 1 homologue IL-1F5 reveals unique loop conformations for receptor bindingspecificity.Biochemistry.42,10938–10944

[0271] 10.Bunkóczi,G.,Echols,N.,McCoy,A.J.,Oeffner,R.D.,Adams,P.D.,and Read,R.J.(2013)Phaser.MRage:automated molecular replacement.Acta Crystallogr.DBiol.Crystallogr.69,2276–2286

[0272] 11.Adams,P.D.,Afonine,P.V.,Bunkóczi,G.,Chen,V.B.,Davis,I.W.,Echols,N.,Headd,J.J.,Hung,L.-W.,Kapral,G.J.,Grosse-Kunstleve,R.W.,McCoy,A.J.,Moriarty,N.W.,Oeffner,R.,Read,R.J.,Richardson,D.C.,Richardson,J.S.,Terwilliger,T.C.,and Zwart,P.H.(2010)PHENIX:a comprehensive Python-basedsystem for macromolecular structure solution.Acta Crystallogr.DBiol.Crystallogr.66,213–221

[0273] 12.BUSTER version 2.10.0

[0274] 13. Emsley, P., and Cowtan, K. (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132

[0275] 14. Chen, V.B., Arendall, W.B., Headd, J.J., Keedy, D.A., Immormino, R.M., Kapral, G.J., Murray, L.W., Richardson, J.S., and Richardson, D.C. (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21

[0276] 15. Nold M., et al. IL-18BPa:Fc cooperates with immunosuppressive drugs in human whole blood. Biochem Pharmacol 66, 505-510 (2003).

[0277] 16. Nold M.F., et al. Endogenous IL-32 Controls Cytokine and HIV-1 Production. J Immunol 181, 557-565 (2008). Sequence Listing <110> Monash University Prince Henry's Institute of Medical Research <120> IL-37 variants <130> M50134783 <150> 2015902262 <151> 2015-06-15 <150> 2016900703 <151> 2016-02-26 <160> 1 <170> SIPOSequence Listing 1.0 <210> 1 <211> 218 <212> PRT <213> Homo sapiens <400> 1 Met Ser Phe Val Gly Glu Asn Ser Gly Val Lys Met Gly Ser Glu Asp 1 5 10 15 Trp Glu Lys Asp Glu Pro Gln Cys Cys Leu Glu Asp Pro Ala Gly Ser 20 25 30 Pro Leu Glu Pro Gly Pro Ser Leu Pro Thr Met Asn Phe Val His Thr 35 40 45 Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser Ile His Asp 50 55 60 Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu Ile Ala Val 65 70 75 80 Pro Asp Lys Asn Tyr Ile Arg Pro Glu Ile Phe Phe Ala Leu Ala Ser 85 90 95 Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile Leu Leu Gly 100 105 110 Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp Lys Gly Gln 115 120 125 Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met Lys Leu Ala 130 135 140 Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr Arg Ala Gln 145 150 155 160 Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro Gly Trp Phe 165 170 175 Ile Cys Thr Ser Cys Asn Cys Asn Glu Pro Val Gly Val Thr Asp Lys 180 185 190 Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro Val Cys Lys 195 200 205 Ala Glu Met Ser Pro Ser Glu Val Ser Asp 210 215

Claims

1. An anti-inflammatory polypeptide, which is an IL-37 polypeptide variant based on SEQ ID NO: 1 with a D73K or Y85A mutation.

2. The anti-inflammatory polypeptide according to claim 1, wherein the anti-inflammatory polypeptide is truncated at the N-terminus of residues 1 to 20 or 1 to 45.

3. A pharmaceutical composition comprising an anti-inflammatory polypeptide according to claim 1 or 2 and a pharmaceutically acceptable excipient or carrier.

4. The pharmaceutical composition of claim 3, wherein the pharmaceutically acceptable excipient is a diluent.

5. Use of a therapeutically effective amount of the anti-inflammatory peptide according to claim 1 or 2 in the preparation of a medicament for the treatment or prevention of an inflammatory disease or condition in a subject in need.

6. The use according to claim 5, wherein the inflammatory disease is primarily mediated by the activation of TLR2 or TLR4.

7. The use according to claim 5, wherein the inflammatory disease is primarily mediated by activation of TLR7, 8 or 9.

8. The use according to claim 5, wherein the inflammatory disease is an autoimmune disease.

9. The use according to claim 5, wherein the inflammatory disease is endotoxin shock.

10. The use according to claim 5, wherein the inflammatory disease is endotoxin shock and wherein the endotoxin shock is caused by bacteria.

11. A nucleic acid molecule encoding an anti-inflammatory polypeptide according to claim 1 or 2.

12. A vector comprising the nucleic acid molecule according to claim 11.

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