Collagen hybridization peptides having a sequence comprising lair and / or rage binding sequence and use thereof

Abstract

Disclosed are a series of novel collagen hybridizing peptides (CHPs) including LAIR and / or RAGE binding sequences and uses thereof.

Description

[0001] MLB Docket No. 127099-5007-WG

[0002] COLLAGEN HYBRIDIZATION PEPTIDES HAVING A SEQUENCE COMPRISING LAIR AND / OR RAGE BINDING SEQUENCE AND USE THEREOF

[0003] BACKGROUND

[0004] The present disclosure relates to a series of novel collagen hybridizing peptides including Leukocyte-associated immunoglobulin-like receptor (LAIR) and / or Receptor for Advanced Glycation End products) RAGE binding sequences and uses thereof.

[0005] Collagen is a major structural protein found in almost all human tissues. Degraded collagen is present in damaged tissues and is implicated in many human diseases and injuries. A collagen hybridizing peptide (CHP) is a short repeating Gly-X-Y tripeptide that specifically binds to degraded collagen without affinity for intact collagen molecules. CHP is capable of identifying damaged collagen by recognizing a structural motif (poly-proline ii helix of individual alpha chains) that is not available on intact collagen molecules, then forms collagen triple helixes by hybridization, and forms very stable and persistent bonds.

[0006] PROBLEM TO BE SOLVED

[0007] Therefore, the present disclosure has been made in view of the above problems, and it is an object of the present disclosure to provide a series of novel collagen hybridizing peptides (CHPs) including LAIR and / or RAGE binding sequences.

[0008] It is another object of the present disclosure to provide a method of preventing or treating a fibrotic condition using the CHPs.

[0009] It is another object of the present disclosure to provide a pharmaceutical composition for preventing or treating a fibrotic condition containing the CHPs.

[0010] It is another object of the present disclosure to provide a method of preventing or treating a fibrotic condition using the CHPs.

[0011] It is another object of the present disclosure to provide a composition for activating a Resolvin DI (RvDl) and / or Resolvin D2 (RvD2) pathway containing the CHPs. It is another object of the present disclosure to provide a composition for inducing secretion of Resolvin DI (RvDl) and / or Resolvin D2 (RvD2) lipid mediators containing the CHPs.

[0012] It is another object of the present disclosure to provide a method of preventing, alleviating or treating atherosclerotic plaques using the CHPs.

[0013] It is another object of the present disclosure to provide a pharmaceutical composition for preventing, alleviating or treating atherosclerotic plaques containing the CHPs.

[0014] It is another object of the present disclosure to provide a method of inhibiting or reducing DB1 / 161378034.4 MLB Docket No. 127099-5007-WG foam cells in fibrotic cells and / or denatured collagen using the CHPs.

[0015] It is another object of the present disclosure to provide a pharmaceutical composition for inhibiting or reducing foam cells and / or denatured collagen in fibrotic tissues or cells containing the CHPs.

[0016] It is another object of the present disclosure to provide a method of downregulating immune- related cytokines using the CHPs.

[0017] It is another object of the present disclosure to provide a composition for downregulating immune-related cytokines containing the CHPs.

[0018] It is another object of the present disclosure to provide a method of preventing or treating pathologies associated with advanced glycation end products (AGEs) using the CHPs.

[0019] It is another object of the present disclosure to provide a pharmaceutical composition for preventing or treating pathologies associated with AGEs containing the collagen hybridizing peptides.

[0020] It is another object of the present disclosure to provide a method of preventing or treating inflammatory diseases mediated by RAGE binding and / or LAIR binding using the CHPs.

[0021] It is another object of the present disclosure to provide a pharmaceutical composition for preventing or treating inflammatory diseases mediated by RAGE binding and / or LAIR binding containing the CHPs.

[0022] It is another object of the present disclosure to provide a method of downregulating T-cells, B-cells, natural killer cells, macrophages, macrophage differentiation, monocytes, and neutrophils using the CHPs.

[0023] It is another object of the present disclosure to provide a composition for downregulating T- cells, B-cells, natural killer cells, macrophages, macrophage differentiation, monocytes, and neutrophils containing the CHPs.

[0024] It is another object of the present disclosure to provide a method of preventing or treating cancer using the CHPs.

[0025] It is another object of the present disclosure to provide a pharmaceutical composition for preventing or treating cancer containing the CHPs.

[0026] MEANS TO SOLVE PROBLEM

[0027] Hereinafter, exemplary embodiments of the present disclosure will be described in detail. However, the present disclosure is not limited to the embodiments disclosed below and may be implemented in various forms. The embodiments described below are provided to enable those skilled in the art to implement and practice the embodiments of the present disclosure.

[0028] Materials, compositions, and components that may be used for the disclosed methods and

[0029] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO compositions, may be used in conjunction therewith, may be used in the preparation of the compositions, or are products thereof, are disclosed. These and other materials are disclosed herein and it is understood that, when combinations, subsets, interactions, groups, and the like of these materials are disclosed, each of the various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, but each is specifically considered and described herein. For example, when peptide conjugates are disclosed and discussed, and a number of modifications that may be made on a number of molecules containing the peptide conjugates are discussed, each and every combination and permutation and possible modification of the peptide conjugate is specifically considered unless specifically stated otherwise. Thus, when not only the classes of molecules A, B, and C are disclosed, but also the classes of molecules D, E, and F and the combination molecules, for example A-D, are disclosed, each is considered individually and collectively, although not individually mentioned. Thus, in this example, the combinations A-E, A- F, B-D, B-E, B-F, C-D, C-E, and C-F are each specifically considered and should be considered disclosed from the disclosure of the exemplar} combinations of A, B and C; D, E, and F; and A-D. Similarly, any subset or combination thereof is also specifically considered and disclosed. Tirus, for example, the subsets of A-E, B-F and C-E are specifically considered and should be considered disclosed from the disclosure of the exemplary combinations of A, B and C; D, E, and F; and A-D. This applies to all aspects of the present disclosure, including, but are not limited to, the steps of preparing and using the disclosed compositions. Accordingly, it is understood that, when there are various additional steps that may be performed, each of these additional steps may be performed by any particular embodiment or combination of embodiments of the disclosed method, and it is understood that each such combination should be considered specifically and disclosed.

[0030] SUMMARY

[0031] In accordance with one aspect of the present disclosure, there is provided a collagen hybridizing peptide (CHP) comprising one or more sequences represented by Formulas I-V:

[0032] (Gly-X-Y)n-S-RAGE Seq-linker-LAIR Seq-(Gly-X-Y)m (Formula I)

[0033] RAGE Seq-linker-S-(Gly-X-Y)n-LAIR Seq-S-(Gly-X-Y)m (Formula II)

[0034] (Gly-X-Y)n-LAIR Seq-(Gly-X-Y)m (Formula III)

[0035] (Gly-X-Y)n-EAIR Seq-linker-RAGE Seq-linker-(Gly-X-Y)m (Formula IV) (Gly-X-Y)n-LAIR Seq-(Gly-X-Y)m-linker-RAGE Seq (Formula V) in which S is zero to 10 amino acids; X is proline or modified proline; Gly is glycine; Y is hydroxyprolinc or any other amino acid; n is a number from 1 to 20; m is a number from 1-20; and the RAGE Seq is an amino acid sequence from 3 to 27 amino acid residues capable of

[0036] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO binding to cell membrane-bound Receptor for Advanced Glycation End products (RAGE), the linker includes one or more of: one or more glycines, (Gly-Gly-Gly-Ser)3 (SEQ ID NO: 302), GSAGSAAGSGEF (SEQ ID NO: 303), KESGSVSSEQLAQFRSLD (SEQ ID NO: 304), EGKSSGSGSESKST (SEQ ID NO: 305), 6-aminohexanoic acid, 12-aminododecanoic acid, N-Fmoc- N'’-succinyl-4,7,10-trioxa-l,13-tridecanediamine (TTDS), a polymer, an amino acid sequence having from 3 to 35 amino acid residues, (Ala-Pro)p, and (Z- Pro)p, wherein p is a number from 1 to 10 and Z is any amino acid residue, and the LAIR Seq is an amino acid sequence from 3 to 27 amino acid residues capable of binding to a leukocyte-associated immunoglobulin-like receptor (LAIR-1 and / or LAIR-2).

[0037] In one embodiment, the CHP further comprises an acetyl capping group (Ac) at N-terminal of the CHP.

[0038] In another embodiment, the LAIR Seq comprises a LAIR-1 binding sequence.

[0039] In the CHP of any one of the previous embodiments, the LAIR Seq comprises a LAIR-2 binding sequence.

[0040] In the CHP of any one of the previous embodiments, the LAIR Seq comprises a LAIR-1 and / or LAIR-2 binding sequence comprising a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-27, 56-64, 93, 94, 98-100, 114-141 and 148-222.

[0041] In the CHP of any one of the previous embodiments, S is at least one amino acid.

[0042] In the CHP of any one of the previous embodiments, the CHP is a first CHP, wherein the first CHP is bound to two other bioactive collagen hybridizing peptides to form a trimeric structure; and wherein the first CHP has a monomeric portion extending beyond the trimeric structure, the monomeric portion configured to bind to collagen alpha chains.

[0043] In the CHP of any one of the previous embodiments, the CHP is a first CHP, wherein the first CHP is bound to two other bioactive collagen hybridizing peptides to form a trimeric structure, and wherein a monomeric ‘"Tail” CHP is conjugated to an N-terminal or a C-terminal of the first CHP of the trimeric structure using copper-free click chemistry or condensation reactions.

[0044] In the CHP of any one of the previous embodiments, the CHP is a first CHP, wherein the first CHP is bound to one other bioactive collagen hybridizing peptide to form a dimeric structure; and wherein the first CHP has a monomeric portion extending beyond the dimeric structure, the monomeric portion configured to bind to collagen alpha chains.

[0045] In the CHP of any one of the previous embodiments, the CHP is a first CHP, wherein the first CHP is bound to one other bioactive collagen hybridizing peptides to form a dimeric structure, and wherein a monomeric “Tail” CHP is conjugated to an N-tcrminal or a C-tcrminal of the first CHP of the dimeric structure using copper-free click chemistry or condensation reactions.

[0046] DB1 / 161378034.4 MLB Docket No. 127099-5007-WG

[0047] In the CHP of any one of the previous embodiments, each individual CHP does not form a triple helix with other CHPs.

[0048] In accordance with another aspect of the present disclosure, there is provided a method of treating or preventing a fibrotic condition by administering one or more of the CHPs according to any one of the previous embodiments to a subject in need thereof.

[0049] In an embodiment, the fibrotic condition is one or more of atherosclerosis cutaneous Lupus, Duchenne muscular dystrophy, NASH, subretinal fibrosis, and idiopathic pulmonary fibrosis (IPF).

[0050] In accordance with another aspect of the present disclosure, there is provided a method of activating the GPR18 pathway to induce production of Resolvin DI (RvDl) and / or Resolvin D2 (RvD2) pathway in a subject in need thereof by administering one or more of the CHPs according to any one of the previous embodiments to the subject.

[0051] In an embodiment, the RAGE pathway is up regulated in cells of the subject.

[0052] In another embodiment, the RAGE pathway is not activated in cells of the subject.

[0053] In accordance with another aspect of the present disclosure, there is provided a method for reducing, preventing, and / or treating atherosclerotic plaques in a subject in need thereof by administering one or more of the CHPs according to any one of the previous embodiments to the subject.

[0054] In accordance with another aspect of the present disclosure, there is provided a method for inhibiting or reducing foam cells in fibrotic cells and / or denatured collagen of a subject in need thereof by administering one or more of the CHPs according to any one of the previous embodiments to the subject.

[0055] In accordance with another aspect of the present disclosure, there is provided a method for downregulating immune-related cytokines including, but not limited to: TLR ligands, interleukins. Leukotriene B4 (LTB4), IFN-a, TLR-9, TNF-a, IL- ip, IL-6, IL-10, PGE2, interferon regulatory factor 5 (IRF5), CCL3, IL-16, IL-18, and / or TNF-a in a subject in need thereof by administering one or more of the CHPs according to any one of the previous embodiments to the subject.

[0056] In accordance with another aspect of the present disclosure, there is provided a method for treating pathologies associated with advanced glycation end products (AGEs) comprising administering one or more of the CHPs according to any one of the previous embodiments to a subject in need thereof.

[0057] In accordance with another aspect of the present disclosure, there is provided a method for treating inflammatory diseases mediated by RAGE binding and / or LAIR binding comprising administering the one or more of the CHPs according to any one of the previous embodiments to a subject in need thereof.

[0058] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO

[0059] In accordance with another aspect of the present disclosure, there is provided a method for downregulating T-Cells, B-Cells, Natural Killer Cells, Macrophages, macrophage differentiation, Monocytes, and Neutrophils in a subject in need thereof comprising administering the one or more of the CHPs according to any one of the previous embodiments to the subject.

[0060] In accordance with another aspect of the present disclosure, there is provided a method for treating cancer in a subject in need thereof comprising administering the one or more of the CHPs according to any one of the previous embodiments to the subject.

[0061] In an embodiment, the method may comprise administering a composition comprising the one or more CHPs to the subject.

[0062] In another embodiment, the method may further comprise administering a carrier with the CHPs.

[0063] In the method of any one of the previous embodiments, the carrier comprises at least one selected from the group consisting of micelles, dendrites, lipids, microemulsions, nanoemulsions, solid lipid nanoparticles, polymers, gels, lens, surfactants, cyclodextrins, inserts, nanostructured lipid carriers, liposomes, transfersomes, ethosomes, niosomes, collagen matrix, extracellular matrix, and artificial extracellular matrix.

[0064] In the method of any one of the previous embodiments, the composition is administered topically, intravenously, orally, subcutaneously, via injection, or via ocular injection.

[0065] In the method of any one of the previous embodiments, the composition is a topical cream, a saline solution, gel, polymer, or a solution for systemic injection within a subject.

[0066] In the method of any one of the previous embodiments, a CHP may be bound directly to collagen matrix.

[0067] In the method of any one of the previous embodiments, the administering is performed byinjection.

[0068] In the method of any one of the previous embodiments, the administering is performed by micro-dermal injection.

[0069] In the method of any one of the previous embodiments, the administering is performed by topical application.

[0070] In the method of any one of the previous embodiments, the administering is performed intravenously.

[0071] In the method of any one of the previous embodiments, the administering is performed orally.

[0072] In the method of any one of the previous embodiments, the administering is performed via subcutaneous delivery.

[0073] DB1 / 161378034.4 MLB Docket No. 127099-5007-WG

[0074] In the method of any one of the previous embodiments, the administering is performed via ocular injection.

[0075] In accordance with another aspect of the present disclosure, there is provided a pharmaceutical composition for preventing, alleviating or treating atherosclerotic plaques containing the CHP as an active ingredient.

[0076] In accordance with another aspect of the present disclosure, there is provided a composition for inhibiting or reducing foam cells in fibrotic cells and / or denatured collagen containing the CHP as an active ingredient.

[0077] In accordance with another aspect of the present disclosure, there is provided a composition for activating a Resolvin DI (RvDl) and / or Resolvin D2 (RvD2) pathway containing the CHP as an active ingredient.

[0078] In accordance with another aspect of the present disclosure, there is provided a pharmaceutical composition for preventing or treating a fibrotic condition containing the CHP as an active ingredient.

[0079] In accordance with another aspect of the present disclosure, there is provided a composition for downregulating one or more immune-related cytokines selected from the group consisting of TLR ligands, interleukins, Leukotriene B4 (LTB4), IFN-a, TLR-9, TNF-a, IL- 10, IL-6, IL- 10, PGE2, interferon regulatory factor 5 (IRF5), CCL3, IL-16, IL-18, and / or TNF-a containing the CHP as an active ingredient.

[0080] In accordance with another aspect of the present disclosure, there is provided a pharmaceutical composition for preventing or treating pathologies associated with advanced glycation end products (AGEs) containing the CHPs as an active ingredient.

[0081] In accordance with another aspect of the present disclosure, there is provided a pharmaceutical composition for preventing or treating inflammatory diseases mediated by RAGE binding and / or LAIR binding containing the CHP as an active ingredient.

[0082] In accordance with another aspect of the present disclosure, there is provided a composition for downregulating T-cells, B-cells, natural killer cells, macrophages, macrophage differentiation, monocytes, and neutrophils containing the CHP as an active ingredient.

[0083] In accordance with another aspect of the present disclosure, there is provided a pharmaceutical composition for preventing or treating cancer containing the CHP as an active ingredient.

[0084] In another aspect, the disclosure provides a method of polarizing a macrophage to a resolution state, the method comprising costimulating RAGE and LAIR1 of the macrophage. In another aspect, the disclosure provides a method of polarizing a macrophage population to a resolution

[0085] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO state, the method comprising costimulating RAGE and LAIR1 of the macrophage population. In some embodiments, the method further comprising contacting the CHP of any one of claims 1-x with the macrophage prior to the costimulating.

[0086] BRIEF DESCRIPTION OF THE DRAWINGS

[0087] The above and other objects, features and other advantages of the present disclosure will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0088] FIGURE 1 is a schematical diagram illustrating the design of a CHP according to one embodiment of the present disclosure. FIGURE 1 shows the potential CHP designs for the LAIR CHP (top) to deliver LAIR to the sites of inflammation which have high levels of RAGE expressed. Therefore, it may be able to co-activate LAIR and RAGE, thus downregulating the inflammatory response by macrophage polarization from Ml to M2 through the resolving pathway by adding the LAIR site. The RAGE-LAIR CHP (bottom) may also be used when bringing both binding sites to the site of inflammation together, which may have an even larger anti-inflammatory effect.

[0089] FIGURE 2 is a schematical diagram illustrating the mechanism of inhibition of immune cells by binding of the CHP according to one embodiment of the present disclosure to Lair-1. FIGURE 2 illustrates the schematic mechanism of action of the CHP binding to denatured collagen and suppressing the immune cells. Cells only recognize the LAIR site when it is in a triple helix (healthy collagen), then when it gets damaged, they can no longer recognize it and become activated, and since CHPs target damaged collagen and fold into a triple helix, they bring the LAIR site back to the area of damage and help suppress the immune response.

[0090] FIGURE 3 is a schematic diagram illustrating an anti-inflammatory response mechanism by co-activation of RAGE and LAIR- 1. Atherosclerosis is upregulated in diabetic patients, but coactivation of RAGE and LAIR-1 by CHP of the present disclosure mediates the RvD2 pathway to induce an anti-inflammatory response and inhibit formation of foam cells. FIGURE 3 illustrates coactivation of RAGE-LAIR and a therapeutic effect in atherosclerosis via the RvD2 pathway by increasing the amount of LAIR at the target site. This figure describes the importance of the RAGELAIR co-activation and why delivering just LAIR and / or the RAGE-LAIR sites together should produce a reduction of inflammation, especially in atherosclerosis.

[0091] FIGURE 4 illustrates the relationship between the CHP binding and fibrosis. FIGURE 4 illustrates the dense interior of a tumor / established fibrotic scar (blue) while the orange ring represents the remodeling collagen that occurs on the surfacc / around the edges of the fibrotic scar or tumor. The collagen gets remodeled on the edges since it is exposed to a plethora of enzymes and as the fibrotic

[0092] DB1 / 161378034.4 MLB Docket No. 127099-5007-WG scar / tumor grows, it expands from the edges and requires the collagen in the ECM to be remodeled.

[0093] Thus, there are lots of denatured collagen strands on the exterior of these tumors / scars giving CHPs the ability to target, bind, and deliver a therapeutic effect.

[0094] FIGURE 5 illustrates the acceleration of fibrosis due to increased inflammation caused by activated RAGE. FIGURE 5 illustrates the RAGE pathway (without LAIR).

[0095] FIGURE 6 illustrates RvD2 induction by co-activation of RAGE and LAIR-1, and antiinflammatory initiation caused thereby. FIGURE 6 illustrates the RAGE-LAIR pathway which provides resolution in fibrotic conditions. FIGURES 5 and 6 show the use of CHPs to resolve fibrotic scars with the co-activation of the RAGE-LAIR resulting in resolution via the RvD2 pathway.

[0096] FIGURE 7 illustrates RAGE receptor binding assay.

[0097] FIGURE 8 illustrates LAIR-1 receptor binding assay.

[0098] FIGURE 9 illustrates monocyte stimulation assay.

[0099] FIGURE 10 illustrates CellTox Green assay.

[0100] FIGURE 11 illustrates presence of collagen lai.

[0101] FIGURE 12 illustrates TIMP activity assay.

[0102] FIGURE 13 illustrates MCP-1 assay.

[0103] DETAILED DESCRIPTION

[0104] Definition

[0105] It will be understood that, although the terms “first,” “second,” etc. may be used herein to describe various elements, these terms should not be construed as limiting these elements. These terms are used only to distinguish one element from another. For example, within the scope of the exemplary embodiment, a “first” element may be referred to as a “second” element, and similarly, a “second” element may be referred to as a “first” element. In addition, the term “and / or” includes any combination of one or more of the related mentioned items.

[0106] It must be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly indicates otherwise. Thus, for example, the expression “a peptide” includes a plurality of peptides including such peptide, and the expression “the peptide” includes one or more peptides and equivalents thereof and the like known to those skilled in the art. Similarly, the term “or” is intended to include “and” unless the context clearly indicates otherwise.

[0107] As used herein, the term “at least one” in relation to a list of one or more elements means at least one element selected from any one or more of the elements in the list of elements, but docs not necessarily include at least one of all elements specifically listed in the list of elements, and does not

[0108] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO exclude any combination of elements in the list of elements. This definition also allows that other elements may optionally be present in addition to the elements specifically identified in the list of elements to which the term “at least one” relates, whether or not it is related to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently , “at least one of A or B,” or, equivalently, “at least one of A and / or B”) may refer to, in one embodiment, at least one A, optionally including more than one B, where B is not present (and, optionally, including an element other than A); in another embodiment, at least one B, optionally including more than one B, where A is not present (and, optionally, including an element other than A); in yet another embodiment, at least one A, optionally including more than one, and at least one B, optionally including more than one (and optionally including other elements).

[0109] The terms “comprising,” “including,” “having,” and the like may be used interchangeably and have the same meaning. Similarly, the terms “comprises” “includes,” “has” and the like are used interchangeably and have the same meaning. Specifically, each term is defined consistently with the term “comprising” as generally defined in U.S. patent law and thus is construed as an open-ended term meaning “at least the following” while not excluding additional features, limitations, aspects, etc. Thus, for example, “a device having components a, b, and c” means that the device includes at least components a, b, and c. Similarly, the phrase “a method comprising steps a, b, and c” means that the method includes at least steps a, b, and c. Furthermore, steps and processes may be described schematically herein in a particular order, but those skilled in the art will recognize that the order of steps and processes may vary, unless a particular order is clearly mentioned by context.

[0110] As used herein, the term “about” may refer to a numerical value, including for example, integers, fractions, and percentages, whether or not explicitly mentioned. The term “about” generally refers to a range of numerical values (e.g., + / - 5, 6, 7, 8, 9, or 10% of the stated value) that those skilled in the art would consider equivalent to the stated value (e.g., having the same function or result). In some cases, the term “about” may include a numerical value rounded to the nearest significant figure. In general, the term “about” includes a value for a given amount that falls at least within a corresponding margin of error for preparation, formulation, and / or measurement.

[0111] As used herein, the term “collagen” may refer to collagen derived from any type of tissue (e.g., bone, dermis, tendon, ligament, or the like). Collagen may refer to a molecule in which three alpha chains of a polyproline Il-like structure fold together to form a triple helix. In addition, this may be applied to any protein that contains a triple helical region, including collagen types I -XXVIII and bacterial collagen. As used herein, the term “collagen” may refer to any form of collagen, including artificial collagen and processed, or otherwise modified collagen. In some embodiments, the collagen is selected from type I collagen, type II collagen, type III collagen, type IV collagen, type V

[0112] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, type X collagen, type XI collagen, type XII collagen, type XIII collagen, type XIV collagen, type XV collagen, type XVI collagen, type XVII collagen, type XVIII collagen, type XIX collagen, type XX collagen, type XXI collagen, type XXII collagen, type XXIII collagen, type XXIV collagen, type XXV collagen, type XXVI collagen, type XXVII collagen, type XXVIII collagen, and combinations thereof.

[0113] As used herein, the term ‘‘proline or modified proline" may refer to the amino acid proline, natural and unnatural isomers thereof, various isomers including both natural and unnatural isomers thereof, and analogues and variants thereof. In an embodiment, the modified proline includes an electron withdrawing group. Examples of the modified proline include, but are not limited to, hydroxyproline, methylated proline, 4-fluoroproline, and 4-chloroproline.

[0114] As used herein, the tenn “hydroxyproline'’ may refer to hydroxy group-substituted proline, which is a type of the modified proline.

[0115] In some embodiments, the method may exclude collecting a sample from a subject. In some embodiments, the term “subject” used herein may refer to a human, an animal, or a microorganism, or a cell culture from any of the groups mentioned above. Examples of the animal include, but are not limited to, vertebrates such as primates, rodents, domestic animals, or game animals. The primates may include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques (e.g.. Rhesus). The rodents may include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic animals and game animals may include cattle, horses, pigs, deer, bison, buffalo, moose, felines (e.g., domestic cats), and canines (e.g., dogs, foxes, wolves). The fish may include Chondrichthyes (cartilaginous fish) and Osteichthyes (bony fish). The subject may be a mammal. The mammal may be a human, a nonhuman primate, a mouse, a rat, a dog, a cat, a horse, or a cow, but is not limited thereto. In addition, the methods described herein may be used to diagnose and / or treat livestock or pets. This tenn does not denote a particular age or gender. Thus, this term is intended to encompass all genders including male and female, adults, newborns, and fetuses.

[0116] The term “prevention” may refer to any action that inhibits or delays the occurrence, propagation, and recurrence of a specific disease, and the tenn “treatment” may refer to partial or complete relief, alleviation, palliation, delayed onset, inhibition of progression, reduction in severity, and / or reduction in incidence of one or more symptoms or characteristics of a specific disease, disorder, and / or condition. For example, the prevention or treatment may be performed by administration to a subject who does not have signs of the disease, disorder, and / or condition and / or to a subject who has only early signs of the disease, disorder, and / or condition, to reduce the risk of developing pathology associated with the disease, disorder, and / or condition.

[0117] The present disclosure relates to a novel substance and uses thereof, wherein a series of CEIP DB1 / 161378034.4 MLB Docket No. 127099-5007-WG sequences designed based on a combination of LAIR- 1 / 2 binding sequences (capable of forming a triple helix) and / or a RAGE activator are present on the same peptide for use in the treatment of atherosclerotic plaques. The CHPs have been shown to target the advanced remodeling of fibrotic collagen, an important component of the plaque structure. Activation of cell surface receptors to modulate the immune response may be induced using the LAIR- 1 / 2 binding sequences to target the fibrotic remodeling collagen to which endogenous LAIR- 1 / 2 receptors cannot bind. While LAIR- 1 / 2 receptors independently act to reduce immune cell activation, RAGE receptors are pro -inflammatory signals. However, co-activation of LAIR and RAGE facilitates an anti-inflammatory and resolutory pathway via Resolvin D2 (RvD2), resulting in plaque reduction / re sorption. The present disclosure utilizes two components of the platform CHP technology: localization to damaged collagen at the surface of plaques / fibrotic lesions and induction of binding of LAIR receptors to LAIR-targeted bioactive CHP (see PCT / US2024 / 118078). The technology of the present disclosure may be used to treat other fibrotic conditions exhibiting upregulation of RAGE and unidentified inflammatory immune responses.

[0118] LAIR is a family of membrane-bound cell receptors expressed by several immune cells, including T cells, macrophages, natural killer cells, B cells, and dendritic cells. LAIR binds to intact collagen that initiates downregulation of LAIR1 signaling and immune cell activation. RAGE (Receptor for Advanced Glycation End products) is also a membrane-bound receptor expressed in various types of cells, but is expressed much more highly in inflammatory pathological states. The primary ligand for RAGE is Advanced Glycation End products (AGEs), but is also activated by other ligands such as HMGB1 and S100. Ligand binding and subsequent RAGE activation are implicated in a host of disease states due to upregulation of pro-inflammatory factors. AGEs are naturally produced during the aging process but are markedly upregulated in diabetic patients. AGEs promote vascular damage by directly modifying matrix molecules on the blood vessel wall and by activating RAGE to enhance the release of inflammatory molecules. Normal LAIR activation (which binds to and activates intact collagen) reduces atherosclerotic plaque formation. Normal RAGE activation increases fomiation of atherosclerosis. However, simultaneous activation of LAIR and RAGE reduces formation of atherosclerosis by potent anti-inflammatory actions, including the production of Resolvin DI . Human atherosclerotic plaques contain type I and type III collagen, the sum of which is 60% of the total plaque protein, which supports that human atherosclerotic plaques are a target suitable for the CHP technology of the present disclosure.

[0119] In accordance with one aspect of the present disclosure, there is provided a CHP including at least one sequence represented by Formulas I-V:

[0120] (Formula I)

[0121] DB1 / 161378034.4 MLB Docket No. 127099-5007-WG

[0122] (Gly-X-Y)n-S-RAGE Seq-linker-LAIR Seq-(Gly-X-Y)m

[0123] (Formula II)

[0124] RAGE Seq-linker-S-(Gly-X-Y)„-LAIR Seq-S-(Gly-X-Y)m

[0125] (Formula III)

[0126] (Gly-X-Y)n-LAIR Seq-(Gly-X-Y)m

[0127] (Formula IV)

[0128] (Gly-X-Y)n-LAIR Seq-linker-RAGE Seq-linker-(Gly-X-Y)m

[0129] (Formula V)

[0130] (Gly-X-Y)n-LAIR Seq-(Gly-X-Y)m-linker-RAGE Seq wherein S is 0 to 10 amino acids;

[0131] X is proline or modified proline;

[0132] Gly is glycine:

[0133] Y is hydroxyproline or any other amino acid; n is a number of 1 to 20; m is a number of 1 to 20; the RAGE Seq is an amino acid sequence including 3 to 27 amino acid residues capable of binding to cell membrane -bound Receptor for Advanced Glycation End products (RAGE): the LAIR Seq is an amino acid sequence including 3 to 27 amino acid residues capable of binding to a leukocyte -associated immunoglobulin-like receptor (LAIR-1 and / or LAIR-2); and the linker includes at least one of at least one glycine, (Gly-Gly-Gly-Ser)3(SEQ ID NO: 302), GSAGSAAGSGEF (SEQ ID NO: 303), KESGSVSSEQLAQFRSLD (SEQ ID NO: 304), EGKSSGSGSESKST (SEQ ID NO: 305), 6-aminohexanoic acid, 12-aminododecanoic acid. N-Fmoc- N"-succinyl-4.7. 10-trioxa- l . l 3-tridccancdiaminc (TTDS), a polymer, an amino acid sequence having 3 to 35 amino acid residues. (Ala-Pro)p. or (Z-Pro)p, wherein p is a number of 1 to 10 and Z is any amino acid residue.

[0134] In an exemplary' embodiment, the CHP may include a combination of two or more independent sequences selected from the sequences represented by Formulas I to V. In this case, the two or more sequences may be the same as or different from each other. In addition, the two or more sequences may be a combination of sequences selected from the same or different general fonnula.

[0135] In an exemplary embodiment, both n and m are 0. In an exemplary embodiment, n is 0 and m is 1. In an exemplary embodiment, n is 0 and m is 2. In an exemplary? embodiment, n is 0 and m is 3. In an exemplary' embodiment, n is 0 and m is 4. In an exemplary embodiment, n is 0 and m is 5. In an exemplary’ embodiment, n is 0 and m is 6. In an exemplary’ embodiment, n is 0 and m is 7. In an exemplary embodiment, n is 0 and m is 8. In an exemplary embodiment, n is 0 and m is 9. In an DB1 / 161378034.4 MLB Docket No. 127099-5007-WO exemplary' embodiment, n is 0 and m is 10. In an exemplary embodiment, n is 0 and m is 11. In an exemplary embodiment, n is 0 and m is 12. In an exemplary embodiment, n is 0 and m is 13. In an exemplary embodiment, n is 0 and m is 14. In an exemplary embodiment, n IS 0 and m is 15. In an exemplary embodiment, n is 0 and m is 16. In an exemplary embodiment, n is 0 and m is 17. In an exemplary embodiment, n is 0 and m is 18. In an exemplary embodiment, n is 0 and m is 19. an exemplary embodiment, n is 0 and m is 20.

[0136] In an exemplary embodiment, both n and m are 1. In an exemplary embodiment, n is 1 and m is 2. In an exemplary embodiment, n is 1 and m is 3. In an exemplary’ embodiment, n is 1 and m is 4.

[0137] In an exemplary' embodiment, n is 1 and m is 5 In an exemplary' embodiment, n is 1 and m is 6. In an exemplary embodiment, n is 1 and m is 7. In an exemplary embodiment, n is 1 and m is 8. In an exemplary embodiment, n is 1 and m is 9. In an exemplary embodiment, n is 1 and m is 10. In an exemplary embodiment, n is 1 and m is 11. In an exemplary embodiment, n is 1 and m is 12. an exemplary embodiment, n is 1 and m is 13. In an exemplary embodiment, n is 1 and m is 14. In an exemplary' embodiment, n is 1 and m is 15. In an exemplary embodiment, n is 1 and m is 16. In an exemplary embodiment, n is 1 and m is 17. In an exemplary' embodiment, n is 1 and m is 18. In an exemplary embodiment, n is 1 and m is 19. In an exemplary embodiment, n is 1 and m is 20.

[0138] In an exemplary embodiment, both n and m are 2. In an exemplary embodiment, n is 1 and m is 3. In an exemplary embodiment, n is 2 and m is 4. In an exemplary embodiment, n is 2 and m is 5.

[0139] In an exemplary embodiment, n is 2 and m is 6. In an exemplary embodiment, n is 2 and m is 7. In an exemplary embodiment, n is 2 and m is 8. In an exemplary' embodiment, n is 2 and m is 9. In an exemplary embodiment, n is 2 and m is 10. In an exemplary' embodiment, n is 2 and m is 11. In an exemplary embodiment, n is 2 and m is 12. In an exemplary embodiment, n is 2 and m is 13. an exemplary embodiment, n is 2 and m is 14. In an exemplary embodiment, n is 2 and m is 15. In an exemplary embodiment, n is 2 and m is 16. In an exemplary embodiment, n is 2 and m is 17. In an exemplary embodiment, n is 2 and m is 18. In an exemplary embodiment, n is 2 and m is 19. In an exemplary embodiment, n is 2 and m is 20.

[0140] In an exemplary embodiment, both n and m are 3. In an exemplary' embodiment, n is 3 and m is 4. In an exemplary embodiment, n is 3 and m is 5. In an exemplary embodiment, n is 3 and m is 6. In an exemplary embodiment, n is 3 and m is 7. In an exemplary embodiment, n is 3 and m is 8. In an exemplary embodiment, n is 3 and m is 9. In an exemplary embodiment, n is 3 and m is 10. In an exemplary embodiment, n is 3 and m is 11. In an exemplary embodiment, n is 3 and m is 12. In an exemplary embodiment, n is 3 and m is In an exemplary embodiment, n is 3 and m is 14. an exemplary embodiment, n is 3 and m is 15. In an exemplary’ embodiment, n is 3 and m is 16. In an exemplary embodiment, n is 3 and m is 17. In an exemplary embodiment, n is 3 and m is 18. In an

[0141] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO exemplary embodiment, n is 3 and m is 19. In an exemplary embodiment, n is 3 and m is 20.

[0142] In an exemplary' embodiment, both n and m are 4. In an exemplary embodiment, n is 4 and m is 5. In an exemplary embodiment, n is 4 and m is 6. In an exemplary embodiment, n is 4 and m is 7.

[0143] In an exemplary embodiment, n is 4 and m is 8 In an exemplary embodiment, n is 4 and m is 9. In an exemplary embodiment, n is 4 and m is 10. In an exemplary embodiment, n is 4 and m is 11. In an exemplary embodiment, n is 4 and m is 12. In an exemplary embodiment, n is 4 and m is 13. In an exemplary embodiment, n is 4 and m is 14. In an exemplary embodiment, n is 4 and m is 15. In an exemplary embodiment, n is 4 and m is 16. In an exemplary embodiment, n is 4 and m is 17. In an exemplary embodiment, n is 4 and m is 18. In an exemplary embodiment, n is 4 and m is 19. In an exemplary embodiment, n is 4 and m is 20.

[0144] In an exemplary embodiment, both n and m are 5. In an exemplary embodiment, n is 5 and is 6. In an exemplary embodiment, n is 5 and m is 7. In an exemplary embodiment, n is 5 and m is

[0145] In an exemplary' embodiment, n is 5 and m is 9 In an exemplary embodiment, n is 5 and m is 10. In an exemplary embodiment, n is 5 and m is 11. In an exemplary' embodiment, n is 5 and m is 12. In an exemplary' embodiment, n is 5 and m is 13. In an exemplary embodiment, n is 5 and m is 14. In an exemplary embodiment, n is 5 and m is 15. In an exemplary embodiment, n is 5 and m is 16. In an exemplary embodiment, n is 5 and m is 17. In an exemplary embodiment, n is 5 and m is 18. In an exemplary embodiment, n is 5 and m is 19. In an exemplary embodiment, n is 5 and m is 20.

[0146] In an exemplary embodiment, both n and m are 6. In an exemplary embodiment, n is 6 and m is 7. In an exemplary embodiment, n is 6 and m is 8. In an exemplary embodiment, n is 6 and m is 9.

[0147] In an exemplary embodiment, n is 6 and m is 10. In an exemplary' embodiment, n is 6 and m is 11. In an exemplary embodiment, n is 6 and m is 12. In an exemplary embodiment, n is 6 and m is 13. In an exemplary embodiment, n is 6 and m is 14. In an exemplary embodiment, n is 6 and m is 1 . In an exemplary embodiment, n is 6 and m is 16. In an exemplary embodiment, n is 6 and m is 17. In an exemplary embodiment, n is 6 and m is 18. In an exemplary embodiment, n is 6 and m is 19. In an exemplary embodiment, n is 6 and m is 20.

[0148] In an exemplary embodiment, n and m are both 7. In an exemplary' embodiment, n is 7 and m is 8. In an exemplary embodiment, n is 7 and m is 9. In an exemplary embodiment, n is 7 and m is

[0149] 10. In an exemplary embodiment, n is 7 and m is 11. In an exemplary embodiment, n is 7 and m is

[0150] 12. In an exemplary embodiment, n is 7 and m is 13. In an exemplary embodiment, n is 7 and m is

[0151] 14. In an exemplary embodiment, n is 7 and m is 15. In an exemplary' embodiment, n is 7 and m is

[0152] 16. In an exemplary' embodiment, n is 7 and m is 17. In an exemplary' embodiment, n is 7 and m is

[0153] 18. In an exemplary embodiment, n is 7 and m is 19. In an exemplary’ embodiment, n is 7 and m is

[0154] 20.

[0155] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO

[0156] In an exemplary embodiment, n and m are both 8. In an exemplary embodiment, n is 8 and m is 9. In an exemplary embodiment, n is 8 and m is 10. In an exemplary embodiment, n is 8 and m is

[0157] 11. In an exemplary’ embodiment, n is 8 and m is 12. In an exemplary7embodiment, n is 8 and m is

[0158] 13. In an exemplary embodiment, n is 8 and m is 14. In an exemplary embodiment, n is 8 and m is

[0159] 15. In an exemplary embodiment, n is 8 and m is 16. In an exemplary embodiment, n is 8 and m is

[0160] 17. In an exemplary embodiment, n is 8 and m is 18. In an exemplary embodiment, n is 8 and m is

[0161] 19. In an exemplary embodiment, n is 8 and m is 20.

[0162] In an exemplary? embodiment, both n and m are 9. In an exemplary embodiment, n is 9 and m is 10. In an exemplary7embodiment, n is 9 and m is 11. In an exemplary? embodiment, n is 9 and m is

[0163] 12. In an exemplary embodiment, n is 9 and m is 13. In an exemplary embodiment, n is 9 and m is

[0164] 14. In an exemplary embodiment, n is 9 and m is 15. In an exemplary embodiment, n is 9 and m is

[0165] 16. In an exemplary embodiment, n is 9 and m is 17. In an exemplary embodiment, n is 9 and m is

[0166] 18. In an exemplary embodiment, n is 9 and m is 19. In an exemplary7embodiment, n is 9 and m is

[0167] 20.

[0168] In an exemplary embodiment, both n and m are 10. In an exemplary7embodiment, n is 10 and m is 11. In an exemplary embodiment, n is 10 and m is 12. In an exemplary embodiment, n IS 10 and m is 13. In an exemplary embodiment, n is 10 and m is 14. In an exemplary embodiment, n is 10 and m is 15. In an exemplary embodiment, n is 10 and m is 16. In an exemplary embodiment, n is 10 and m is 17. In an exemplary embodiment, n is 10 and m is 18. In an exemplary? embodiment, n IS 10 and m is 19. In an exemplary embodiment, n is 10 and m is 20.

[0169] In an exemplary embodiment, n and m are both 11. In an exemplary7embodiment, n is 11 and m is 12. In an exemplary embodiment, n is 11 and m is 13. In an exemplary embodiment, n IS 11 and m is 14. In an exemplary embodiment, n is 11 and m is 15. In an exemplary embodiment, n is 11 and m is 16. In an exemplary embodiment, n is 11 and m is 17. In an exemplary embodiment, n is 11 and m is 18. In an exemplary embodiment, n is 11 and m is 19. In an exemplary? embodiment, n IS 11 and m is 20.

[0170] In an exemplary embodiment, both n and m are 12. In an exemplary7embodiment, n is 12 and m is 13. In an exemplary embodiment, n is 12 and m is 14. In an exemplary embodiment, n IS 12 and m is 15. In an exemplary embodiment, n is 12 and m is 16. In an exemplary embodiment, n is 12 and m is 17. In an exemplary embodiment, n is 12 and m is 18. In an exemplary? embodiment, n is 12 and m is 19. In an exemplary embodiment, n is 12 and m is 20.

[0171] In an exemplary7embodiment, both n and m are 13. In an exemplary? embodiment, n is 13 and m is 14. In an exemplary7embodiment, n is 13 and m is 15. In an exemplary7embodiment, n is 13 and m is 16. In an exemplary embodiment, n is 13 and m is 17. In an exemplary embodiment, n is 13 and

[0172] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO m rs 18. In an exemplary embodiment, n is 13 and m is 19. In an exemplary' embodiment, n is 13 and is 20.

[0173] In an exemplary embodiment, both n and m are 14. In an exemplary' embodiment, n is 14 and m is 15. In an exemplary embodiment, n is 14 and m is 16. In an exemplary embodiment, n is 14 and m is 17. In an exemplary embodiment, n is 14 and m is 18. In an exemplary embodiment, n is 14 and m is 19. In an exemplary embodiment, n is 14 and m is 20.

[0174] In an exemplary embodiment, both n and m are 15. In an exemplary embodiment, n is 15 and m is 16. In an exemplary embodiment, n is 15 and m is 17. In an exemplary' embodiment, n is 15 and is 18. In an exemplary' embodiment, n is 15 and m is 19. In an exemplary' embodiment, n is 15 and m is 20.

[0175] In an exemplary embodiment, both n and m are 16. In an exemplary embodiment, n is 16 and m is 17. In an exemplary embodiment, n is 16 and m is 18. In an exemplary embodiment, n is 16 and is 19. In an exemplary embodiment, n is 16 and m is 20.

[0176] In an exemplary embodiment, both n and m are 17. In an exemplary' embodiment, n is 17 and m rs 18. In an exem lary embodiment, n is 17 and m is 19. In an exemplary' embodiment, n is 17 and m is 20.

[0177] In an exemplary embodiment, both n and m are 18. In an exemplary’ embodiment, n is 18 and is 19. In an exemplary embodiment, n is 18 and m is 20. In an exemplary embodiment, n is 19 and is 20.

[0178] In an exemplary embodiment, both n and m are 19. In an exemplary' embodiment, n is 19 and is 20.

[0179] In an exemplary embodiment, both n and m are 20.

[0180] For example, the CHP may further include an acetyl capping group (Ac) at the N-terminus of any sequence selected from Fonnulas I to V. In one embodiment, the acetyl capping group may be directly attached to the N-terminus of the CHP. In another embodiment, the acetyl capping group may be attached to the N-terminus via a spacer, but is not limited thereto.

[0181] For example, in the CHP of some embodiments, the LAIR Seq may include, but is not limited to, a LAIR- 1 binding sequence.

[0182] Alternatively, in the CHP of some embodiments, the LAIR Seq may include, but is not limited to. a LAIR-2 binding sequence.

[0183] Furthermore, in the CHP of some embodiments, the at least one bioactive may include, but is not limited to, both a LAIR-1 binding sequence and a LAIR-2 binding sequence.

[0184] In some embodiments, the LAIR Seq may include a LAIR- 1 binding sequence and / or LAIR-2 binding sequence including a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%.

[0185] DB1 / 161378034.4 MLB Docket No. 127099-5007-WG

[0186] 93%, 94%, 95%, 96%, 97%, 98%, 99% to 100% sequence identity to any one of SEQ ID NOs: 1 to 27, 56 to 64, 98 to 100, and 107 to 109.

[0187] In some embodiments, the RAGE Seq may include a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, 99% to 100% sequence identity to any one of SEQ ID NOs: 93 to 97 to 110 to 113.

[0188] In some embodiments, the sequence may include a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to 100% sequence identity to any one of SEQ ID Nos: 1-106.

[0189] In some embodiments, the sequence may include a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to 100% sequence identity to any one of SEQ ID Nos: 107-222.

[0190] In some embodiments, the sequence may include a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to 100% sequence identity to any one of SEQ ID Nos: 223-298

[0191] In some embodiments, the sequence may include a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99% to 100% sequence identity to any one of SEQ ID Nos: 299-312.

[0192] For example, in some embodiments, the RAGE Seq may include a RAGE binding sequence including a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 110 to 113 and 142 to 147, but is not limited thereto.

[0193] In some embodiments, the collagen hybridization peptide may include, but is not limited to, a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1 to 27, 56 to 64, 93, 94, 98 to 100, 107 to 109, 114 to 141, and 148 to 222.

[0194] The LAIR sequence disclosed in the prior patent of the present applicant and the CHP sequence designed by applying the same are disclosed in Table 1 . Furthermore, specific examples according to some embodiments of the known LAIR Seq, RAGE Seq and CHP sequences applied to the present disclosure are shown in Table 2 below. Furthermore, specific examples according to some embodiments of the CHP sequence having biotin bound to the N-terminus thereof are shown in Table 3 below.

[0195] The LAIR sequence and the CHP sequence designed by applying the same are shown in Table 1. Furthermore, specific examples according to some embodiments of the known bioactive (Cit LAIR) sequences applied to the present disclosure and the citmllinated CHP and non-citrullinated CHP sequences including the same arc shown in Table 2 below. Furthermore, specific examples according to some embodiments of the citrullinated CHP and non-citrullinated CHP sequences having biotin

[0196] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO bound to the N-terminus thereof are shown in Table 3 below.

[0197] Table 1

[0198] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO

[0199] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO

[0200] DB1 / 161378034.4 MLB Docket No. 127099-5007-WG

[0201] The Ac- is an acetyl (capping) group at the N-terminus, the G- is Glycine, the P- is proline, the 0- is hydroxyproline, the f- is 2S.4S-4-fhioroproline. x is any integer from 1-7, y is any integer from 1- 7, and z is any integer from 1-5.

[0202] Table 2

[0203] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO

[0204] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO

[0205] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO

[0206] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO

[0207] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO

[0208] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO

[0209] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO

[0210] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO

[0211] Table 3

[0212] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO

[0213] DBl / 161378034.4 MLB Docket No. 127099-5007-WO

[0214] DBl / 161378034.4 MLB Docket No. 127099-5007-WO

[0215] DBl / 161378034.4 MLB Docket No. 127099-5007-WO

[0216] DBl / 161378034.4 MLB Docket No. 127099-5007-WO

[0217] Table 4. Additional sequences

[0218] DBl / 161378034.4 MLB Docket No. 127099-5007-WO

[0219] In some embodiments, S may include at least one amino acid. S may be any amino acid or modified amino acid, but is not limited thereto. The amino acid may be any of the 20 natural amino acids known in the art. Meanwhile, the modified amino acid may include, but is not limited to, unnatural amino acids corresponding to the natural amino acid, various isomers, analogs, and variants of the natural and unnatural amino acids. For example, the modified amino acid may be an amino acid in which the backbone of the natural amino acid is further substituted with hydroxy, methyl, fluoro, chloro, or the like, but is not limited thereto.

[0220] For example, the CHP is a first CHP and the first CHP may bind to two different bioactive CHPs to form a trimeric structure: the first CHP has a monomeric moiety that extends beyond the trimeric structure, and the monomeric moiety may be configured to bind to a collagen alpha chain, but is not limited thereto.

[0221] For example, the CHP is a first CHP and the first CHP binds to two different bioactive CHPs to form a trimeric structure; the monomeric “tail” CHP may be conjugated to the N-tenninus or C- terminus of the first CHP of the trimeric structure using copper-free click chemistry or a condensation reaction, but is not limited thereto.

[0222] For example, the CHP is a first CHP and the first CHP may bind to one different bioactive CHP to form a dimeric structure; the first CHP has a monomeric moiety that extends beyond the trimeric structure, and the monomeric moiety may be configured to bind to a collagen alpha chain, but is not limited thereto.

[0223] For example, the CHP is a first CHP and the first CHP binds to one different bioactive CHP to form a dimeric structure; the monomeric “tail” CHP may be conjugated to the N-terminus or C-terminus of the first CHP of the dimeric structure using copper-free click chemistry or a condensation reaction, DB1 / 161378034.4 MLB Docket No. 127099-5007-WO but is not limited thereto.

[0224] For example, one CHP does not form a triple helix with another CHP.

[0225] Atherosclerosis is a chronic inflammatory disease characterized by arterial wall lesions that account for approximately 50% of all deaths in Western societies. It is the leading cause of cardiovascular disease, which accounts for 1 in 4 deaths in the United States. An atherosclerotic lesion is asymmetric hypertrophy of the innermost layer of the artery that is composed of cells, ECM proteins, lipids, and debris. These plaques are formed by infdtration and retention of low -density lipoprotein (LDL) in the endothelium which initiate an inflammatory response by immune cells within the arterial wall. Then, this activated endothelium recruits monocytes that differentiate into macrophages that infiltrate the plaques in the arterial wall. These macrophages are then activated by binding of ligands to the receptors thereof, leading to the release of inflammatory cytokines, chemokines, proteases, and radicals. These molecules amplify the inflammatory signaling cascade and enhance plaque formation. An example of such a cascade is the binding of AGEs to the RAGE receptor on macrophages. This binding promotes the polarization of macrophages to the Ml state, which further produces inflammatory7molecules such as IL-1 p, interleukin-6 (IL-6), and TNF-a through the MAPK pathway. Therefore, targeting the RAGE receptor is a promising solution to reprogram these macrophages and block the inflammatory response.

[0226] Meanwhile, another important factor in the onset of atherosclerosis may be the formation of foam cells. This is caused by dysregulation of lipid metabolism, leading to lipid accumulation and foamy appearance of infiltrated macrophages. Silencing LAIR has been shown to modulate the SHP- 1 / CREB / PPAR pathway that promotes the formation of the foam cells. Given that the LAIR protein cannot bind to fibrotic or denatured, non-helical collagen, formation of foam cells is caused by the absence of LAIR signaling in the plaque microenvironment. In addition, LAIR acts as an important checkpoint in the inflammatory immune response that is lost when it cannot bind to triple-helix collagen, which recruits additional immune cells and attenuates the production of inflammatory cytokines.

[0227] Overall, activation of the RAGE pathway and loss of the LAIR regulatory7checkpoint induce polarization of macrophages toward the inflammatory Ml phenotype and foam cells. Uris increases the production of inflammatory cytokines and induces the formation and progression of atherosclerotic plaques. By designing peptides that reprogram the RAGE pathway and restore LAIR function, it is possible to stop and reverse the progression of atherosclerosis.

[0228] This reprogramming is induced by the Resolvin D2 (RvD2) pathway. Resolvin D2 is a pro- rcsolving lipid mediator (SPM) that transmits signals through a specific G protcin-couplcd receptor (GPR18) that promotes inflammation resolution through inhibition of IRF5. IRF5 maintains pro- DB1 / 161378034.4 MLB Docket No. 127099-5007-WG inflammatory macrophages within atherosclerotic lesions and then promotes the Ml phenotype that prefers the growth and development of atherosclerotic lesions. Co-stimulation of RAGE and LAIR has been shown to induce the RvD2 pathway and inhibit IRF5, to reverse atherosclerosis.

[0229] Tire current standard of treatment for atherosclerosis is statin therapy which is used for both secondary and primary prevention therapies. Statin acts to reduce the amount of cholesterol produced in the liver and helps the liver remove cholesterol already present in the blood. However, despite the widespread use thereof, the prevalence of cardiovascular disease remains high and some patients do not respond to statin therapy. Furthermore, although statins can stabilize atherosclerotic plaques, they cannot reverse them. The novel CHP approach of the present disclosure enables patients who do not respond well to statins to be treated through other molecular pathways.

[0230] Although atherosclerotic plaques are used as the primary target indication for the peptides of the present disclosure, the peptides are expected to be useful to treat other fibrotic conditions. Many fibrotic diseases share elements similar to atherosclerosis, such as dysregulated immune responses and upregulation of RAGE.

[0231] An example thereof is nonalcoholic steatohepatitis (NASH), which is a disease characterized by inflammation and damage to liver tissue, causing fibrosis and often cirrhosis. In a high-fat, high- fructose, and high-cholesterol mouse model of NASH, RAGE expression was found to be increased in recruited macrophages. In RAGE knockout mice, activation of these pro-inflammatory macrophages and T-cells was reduced, which indicates that RAGE is a key driver of NASH. On the other hand, another group has shown that LAIR-1 expression on macrophages was reduced in patients with cirrhosis. Thus, this triple-helix LAIR sequence restores the LAIR-1 binding site and promotes canonical immune regulation of LAIR in healthy tissues. Finally, activation of the RvD2 pathway by this combinatory peptide should work especially well in the liver given that treatment with RvD2 has been shown to reduce hepatic fat and collagen accumulation, which are major drivers of NASH progression.

[0232] Other examples include subretinal fibrosis (nAMD) and IPF. The lung is noteworthy since RAGE expression is highly upregulated in lung tissue.

[0233] In accordance with another aspect of the present disclosure, there is provided a pharmaceutical composition for preventing or treating a fibrotic condition containing the collagen hybridizing peptide as an active ingredient.

[0234] In accordance with another aspect of the present disclosure, there is provided a method of preventing or treating a fibrotic condition including administering the pharmaceutical composition to a subject in need thereof.

[0235] For example, the fibrotic condition may include one or more selected from the group

[0236] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO consisting of cutaneous lupus, Duchenne muscular dystrophy, non-alcoholic steatohepatitis (NASH), subretinal fibrosis, and idiopathic pulmonary fibrosis (IPF), but is not limited thereto.

[0237] As a more specific example, the CHP may be used in the treatment of age-related macular degeneration. Tire selective binding of the CHP to fibrotic tissue in the posterior chamber of the eye allows for therapeutic localization and successful reprogramming of the inflammatory response.

[0238] In accordance with another aspect of the present disclosure, there is provided a composition for activating a Resolvin DI (RvDl) and / or Resolvin D2 (RvD2) pathway containing the collagen hybridizing peptide as an active ingredient.

[0239] In accordance with another aspect of the present disclosure, there is provided a method of activating a Resolvin DI (RvDl) and / or Resolvin D2 (RvD2) pathway including administering the composition to a subject in need thereof.

[0240] For example, the composition upregulates the RAGE pathway that is not activated in the cells of the subject when administered to the subject.

[0241] In accordance with another aspect of the present disclosure, there is provided a pharmaceutical composition for preventing, alleviating or treating atherosclerotic plaques containing the collagen hybridizing peptide as an active ingredient.

[0242] In accordance with another aspect of the present disclosure, there is provided a method of preventing, alleviating or treating atherosclerotic plaques including administering the pharmaceutical composition to a subject in need thereof.

[0243] The CHP can prevent, reduce or treat atherosclerotic plaque and thus is useful for the treatment of all arterial diseases where atherosclerosis is present. Examples of the arterial diseases include coronary arterial diseases, peripheral arterial diseases, carotid arterial diseases, renal arterial diseases, vertebral arterial diseases, and mesenteric artery ischemia. This will help prevent further atherosclerotic-induced conditions such as heart attack, stroke, vascular dementia, erectile dysfunction, and limb loss.

[0244] In accordance with another aspect of the present disclosure, there is provided a composition for inhibiting or reducing foam cells in fibrotic cells and / or denatured collagen containing the collagen hybridizing peptide as an active ingredient.

[0245] In accordance with another aspect of the present disclosure, there is provided a method of inhibiting or reducing foam cells in fibrotic cells and / or denatured collagen including administering the composition to a subject in need thereof.

[0246] In accordance with another aspect of the present disclosure, there is provided a composition for downrcgulating one or more immune-related cytokines selected from the group consisting of TLR ligands, interleukins, Leukotriene B4 (LTB4), IFN-a, TLR-9, TNF-a, IL-10, IL-6, IL-10, PGE2,

[0247] DB1 / 161378034.4 MLB Docket No. 127099-5007-WG interferon regulatory factor 5 (IRF5), CCL3, IL-16, IL-18, and / or TNF-a containing the collagen hybridizing peptide as an active ingredient.

[0248] In accordance with another aspect of the present disclosure, there is provided a method of downregulating one or more immune-related cytokines selected from the group consisting of TLR ligands, interleukins, Leukotriene B4 (LTB4), IFN-a, TLR-9, IL-ip, IL-6, IL-10, PGE2, interferon regulatory factor 5 (IRF5), CCL3, IL- 16, IL-18, and / or TNF-a including administering the composition to a subject in need thereof.

[0249] In accordance with another aspect of the present disclosure, there is provided a pharmaceutical composition for preventing or treating pathologies associated with Advanced Glycation End products (AGEs) containing the collagen hybridizing peptide as an active ingredient.

[0250] In accordance with another aspect of the present disclosure, there is provided a method of preventing or treating pathologies associated with AGEs including administering the composition to a subject in need thereof.

[0251] For example, the pathologies associated with AGEs may include, but are not limited to, diabetes, Alzheimer’s disease, and secondary7stages of traumatic brain injury7.

[0252] In accordance with another aspect of the present disclosure, there is provided a pharmacal composition for preventing or treating inflammatory diseases mediated by RAGE binding and / or LAIR binding containing the collagen hybridizing peptide as an active ingredient.

[0253] In accordance with another aspect of the present disclosure, there is provided a method of preventing or treating inflammatory diseases mediated by RAGE binding and / or LAIR binding including administering the composition to a subject in need thereof.

[0254] The CHP induces upregulation of RAGE and LAIR in tissues and thus is useful for the treatment of a wide range of lung diseases. For example, the inflammatory diseases mediated by the RAGE binding and / or LAIR binding include, but are not limited to, IPF, allergic airway inflammation, asthma, pulmonary fibrosis, lung cancer, chronic obstructive pulmonary artery disease, acute lung injury, pneumonia, cystic fibrosis, and bronchopulmonary7dysplasia.

[0255] In accordance with another aspect of the present disclosure, there is provided a composition for downregulating T-cells, B-cells, natural killer cells, macrophages, macrophage differentiation, monocytes, and neutrophils containing the collagen hybridizing peptide as an active ingredient.

[0256] In accordance with another aspect of the present disclosure, there is provided a method of downregulating T-cells, B-cells, natural killer cells, macrophages, macrophage differentiation, monocytes, and neutrophils including administering the composition to a subject in need thereof.

[0257] In accordance with another aspect of the present disclosure, there is provided a pharmacal composition for preventing or treating cancer containing the collagen hybridizing

[0258] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO peptide as an active ingredient.

[0259] In accordance with another aspect of the present disclosure, there is provided a method of preventing or treating cancer including administering the composition to a subject in need thereof.

[0260] For example, in an embodiment, the composition may include one or more CHPs, but is not limited thereto.

[0261] In addition, the composition may further contain a pharmacally acceptable carrier. In this case, the carrier may include, but is not limited to, at least one selected from the group consisting of micelles, dendrites, lipids, microemulsions, nanoemulsions, solid lipid nanoparticles, polymers, gels, lenses, surfactants, cyclodextrins, inserts, nanostructured lipid carriers, liposomes, transfersomes, ethosomes, niosomes, collagen matrices, extracellular matrices, and artificial extracellular matrices.

[0262] As used herein, the tenn “administration” means introducing a predetennined substance into a patient in an appropriate manner and the composition may be administered through any general route as long as it can reach the target tissue. Specifically, the composition may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, topically, intranasally, intrapulmonarily, or rectally, but is not limited thereto.

[0263] For example, the composition may be administered topically, intravenously , orally, subcutaneously, via injection, or via ocular injection, or may be administered via micro-dermal injection, such as using a microneedle array, but is not limited thereto.

[0264] For this purpose, the composition may be provided in the form of atopical cream, saline solution, gel, polymer, or solution for systemic injection into a subject, but is not limited thereto.

[0265] Meanwhile, the composition may be administered in a pharmaceutically effective amount, and the tenn “pharmacally effective amount” refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to all medical treatments, without causing side effects. The effective dose may be easily determined by those skilled in the art based on factors including the gender, age, weight, health condition, and type and severity of the disease of the patient, activity of the drug, sensitivity of the patient to the drug, administration method, administration time, administration route, and excretion rate, treatment period, drugs used in combination or simultaneously with, and other factors well known in the pharmaceutical field. In general, the active ingredient may be administered at a dose of about 0.001 mg / kg / day to 1,000 mg / kg / day. For example, the active ingredient may be administered at a dose of 0.01 mg / kg / day to 100 mg / kg / day, or 0.01 mg / kg / day to 35 mg / kg / day, but is not limited thereto. For oral administration, the active ingredient is preferably the range of 50 to 500 mg / kg and may be administered once or more per day. Specifically, the active ingredient is generally administered to an adult patient weighing 70 kg at a dose of 0.07 to 7,000 mg / day, preferably 0.7 to 2,500 mg / day and may be administered once or several times per day in divided doses at regular

[0266] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO intervals depending on the judgment of physicians or pharmacists.

[0267] In the composition, the CHP directly binds to the collagen matrix.

[0268] In an embodiment, the composition may be provided in the form of a food composition for preventing or ameliorating the aforementioned diseases.

[0269] For example, the composition may be used before or after the onset of the disease, simultaneously with or separately from a drug for treating the disease, to prevent or ameliorate the aforementioned diseases.

[0270] As used herein, the term “amelioration” means any action that at least reduces a parameter related to the treatment condition, such as the degree of symptoms.

[0271] As used herein, the term “food” includes meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, teas, drinks, alcoholic beverages, vitamin complexes, health functional foods, and health foods, and includes all foods in the conventional sense.

[0272] For example, since the food composition is consumed on a daily basis, it is expected to be highly effective in amelioration of diseases and is very useful to improve health. In addition, the food composition may further contain a bromatologically acceptable food additive.

[0273] As used herein, the tenn “functional food” has the same meaning as the term “food for special health use (FoSHU)” and refers to a food having strong medical and pharmaceutical effects that has been processed to efficiently provide bioregulatory functions as well as nutrition supply functions. As used herein, the term “functional” means obtaining beneficial effects for health purposes, such as controlling nutrients or exhibiting physiological effects with regard to the structures and functions of the human body. The food may be prepared by a method commonly used in the art and the preparation may be performed using raw materials and ingredients commonly added in the art. Hie food composition may be prepared in various formulations and, unlike general drugs, it is advantageously free of side effects that may occur upon long-term use of drugs since it uses food as a raw material, and is highly portable, and thus can be consumed as a supplement to enhance the effect of ameliorating diseases.

[0274] The tenn “health food” refers to a food having an effect of actively maintaining or improving health beyond that of a general food, and the term “health supplement food” refers to a food ingested for the purpose of health supplement. In some cases, the terms “health functional food”, “health food”, and “health supplement food” are used interchangeably.

[0275] Specifically, health food means a food product that is prepared by adding the compound to food materials such as beverages, teas, spices, gums, and confectionery , or by preparing the compound in the form of a capsule, powder, suspension, or the like, and has a specific health effect, when

[0276] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO consumed, and is advantageously free of side effects that may occur when taking drugs for a long period of time because it is prepared from food as a raw material unlike general drugs.

[0277] The food composition may further contain a physiologically acceptable carrier. There is no particular limitation as to the type of carrier and any carrier may be used as long as it is commonly used in the relevant art.

[0278] In addition, the food composition may contain additional ingredients commonly used in food compositions to improve fragrance, flavor, aesthetics and the like. For example, the food composition may contain vitamins A, C, D, E, Bl, B2, B6, B12, niacin, biotin, folate, pantothenic acid, or the like. Further, the food composition may contain: minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), and copper (Cu); and amino acids such as lysine, tryptophan, cysteine, and valine.

[0279] In addition, the food composition may contain food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, and the like), bactericides (bleaching powder and highly bleaching powder, sodium hypochlorite, and the like), antioxidants (butylated hydroxyanisole, BHA), butylated hydroxytoluene (BHT and the like), colorants (coal tar dyes, and the like), color developers (sodium nitrite, sodium nitrite, and the like), bleaching agents (sodium sulfite), seasonings (monosodium glutamate (MSG), and the like), sweeteners (dulcin, cyclamate, saccharin, sodium, and the like), flavorings (vanillin, lactones, and the like), leavening agents (alum, D-potassium hydrogen tartrate, and the like), reinforcing agents, emulsifiers, thickeners (glutinating agents), coating agents, gum bases, de-foaming agents, solvents, and enhancers. The additives may be selected depending on the type of food and may be used in an appropriate amount.

[0280] When the food composition is used as a food additive, the composition may be added as it is or used in combination with other foods or food ingredients, and may be used appropriately according to a conventional method. Tire amount of the active ingredient may be appropriately determined depending on the purpose of use, such as prevention, health, or therapeutic treatment. In general, the composition is added in an amount of 15 wt% or less, preferably 35 wt% or less, based on the weight of raw material in the process of preparing a food or beverage. However, in case of long-term intake for health and hygiene or health control, the amount of active ingredients may be below the range defined above.

[0281] In an embodiment, the food composition may be used as a health beverage composition. In this case, the food composition may contain various flavoring agents or natural carbohydrates as additional ingredients, like a conventional beverage. The natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodcxtrin; and sugar alcohols such as xylitol, sorbitol, or ery thritol. Sweeteners may include natural sweeteners such as thaumatin and stevia extracts; synthetic

[0282] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO sweeteners such as saccharin or aspartame and the like. The content of the natural carbohydrate may be generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g, with respect to 100 mL of the health beverage composition.

[0283] Furthermore, the health beverage composition may contain various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners. pH regulators, stabilizers, preservatives, glycerin, alcohol, or carbonating agents. In addition, the health beverage composition may contain fruit pulp for the preparation of a natural fruit juice, a fruit juice beverage, or vegetable beverage. These ingredients may be used alone or in combination. The content of these additives is not particularly important, but is typically selected within the range of 0.01 to 0.1 parts by weight with respect to 100 parts by weight of the health beverage composition.

[0284] An important feature of the novel peptides of the present disclosure is the ability thereof to localize the composition in damaged collagen and LAIR-1 binding sites. Systemic activation of RAGE is likely? to result in adverse pathological outcomes. However, the peptide of the present disclosure ensures that the therapeutic agent is localized to damaged collagen sites in disease models while providing a binding site for LAIR- 1 at the precise target site, thus providing a LAIR- 1 sequence capable of forming a triple helix, thereby acting as a stable and effective binding site for the receptor to promote anti-inflammatory responses.

[0285] In some embodiments, Fibrosis may be a formation of excess fibrous connective tissue in an organ or tissue, which is a reparative or reactive process. It may be often a response to injury, and if it becomes excessive, it may impair the function of the affected organ. In some embodiments, a fibrotic disease may include Idiopathic Pulmonary Fibrosis (IPF), which is a chronic, progressive lung disease with an unknown cause that leads to irreversible scarring of the lungs; Cystic Fibrosis (CF), which is a genetic disorder that causes thick, sticky mucus to build up. leading to repeated lung infections and progressive lung damage, including fibrosis; asbestosis, a lung fibrosis caused by' inhaling asbestos fibers; silicosis, a lung disease caused by inhaling crystalline silica dust; chronic hypersensitivity pneumonitis, a form of lung fibrosis resulting from an allergic reaction to inhaled organic dusts; and pulmonary fibrosis from autoimmune diseases which conditions like rheumatoid arthritis and scleroderma may cause inflammation and scarring in the lungs. In some embodiments, a fibrotic disease may include cirrhosis which is the final stage of liver fibrosis caused by chronic liver damage, common causes include chronic hepatitis B and C, chronic alcohol abuse, and non-alcoholic fatty liver disease (NAFLD); Chronic Kidney Disease (CKD), a progressive kidney fibrosis may lead to cnd-stagc renal disease (ESRD) that may be caused by various conditions, including chronic high blood pressure and diabetes; myocardial fibrosis, a scarring of the heart muscle that can occur after a

[0286] DB1 / 161378034.4 MLB Docket No. 127099-5007-WG heart atack or from chronic conditions like hypertension or heart failure. In some embodiments, a fibrotic disease may include scleroderma (systemic sclerosis), an autoimmune disease characterized by the hardening and tightening of the skin. It can also affect internal organs; keloids, raised scars caused by an overgrowth of fibrous tissue at the site of a skin injury; hypertrophic scars, raised scars that stay within the boundary of the original wound: nyelofibrosis, a bone marrow disorder where the marrow is replaced by fibrous scar tissue, impairing the production of blood cells; Peyronie's Disease, a condition where fibrous scar tissue forms in the penis, causing a curved and painful erection; Crohn's Disease, a type of inflammatory bowel disease that can lead to intestinal fibrosis and strictures (narrowing) of the bowel; and cystic fibrosis-related pancreatitis, fibrosis of the pancreas can occur due to the thick mucus blocking the ducts, leading to inflammation and damage.

[0287] A method of polarization of macrophage population to a resolution state comprising costimulating RAGE and LAIR1 .

[0288] In some embodiment, the peptide may be aerosolized. In some embodiments, a peptide product may be administered pulmonarily, such as by oral inhalation or nasal inhalation. A pulmonarily administered drug can treat a lung disorder or / and a systemic disorder. Oral or nasal inhalation may be achieved by means of, e g., a metered-dose inhaler (MDI), a nebulizer or a dr powder inhaler (DPI). For example, a peptide product may be formulated for aerosol administration to the respiratory tract by oral or nasal inhalation. The peptide in the aerosol formulation may be dissolved, or more often suspended, in the propellant for delivery to the lungs. The aerosol may contain excipients such as a surfactant (which enhances penetration into the lungs by reducing the high surface tension forces at the air-water interface within the alveoli, may also emulsify, solubilize or / and stabilize the drug, and can be, e.g., a phospholipid such as lecithin) or / and a stabilizer.

[0289] In another aspect, the disclosure provides a method of polarizing a macrophage to a resolution state, the method comprising costimulating RAGE and LAIR1 of the macrophage. In another aspect, the disclosure provides a method of polarizing a macrophage population to a resolution state, the method comprising costimulating RAGE and LAIR1 of the macrophage population. In some embodiments, the method further comprising contacting the CHP of any one of claims 1-x with the macrophage prior to the co stimulating.

[0290] Hereinafter, the present disclosure will be described in more detail with reference to the following examples. However, it will be obvious to those skilled in the art that the following examples are provided only for illustration of the present disclosure and should not be construed as limiting the scope of the present disclosure.

[0291] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO

[0292] EXAMPLES

[0293] Example 1: Design of Sequence

[0294] Another bioactive CHP design that includes a LAIR active site in the middle of the sequence is disclosed in PCT / US2024 / 18078. The bioactive CHP disclosed in the aforementioned PCT application carries an active LAIR1 site that is required for a triple helix conformation that may be recognized by cells in a microenvironment. While both designs have similar binding sites for the LAIR1 receptor, the bioactivity of the novel CHP design of the present disclosure requires activation of the RAGE element. Therefore, the target of the CHP is the same, but the biological repercussions of binding to each CHP are significantly different. Thus, the sequence of the present disclosure is distinguished from conventional sequences.

[0295] Other researchers have a similar technique that acts by co-targeting RAGE and LAIR (Liu T et al. HMGBl-Clq complexes regulate macrophage function by switching between leukotriene and specialized proresolving mediator biosynthesis. Proc Natl Acad Sci, 2019, Nov. 12; 1 16(46):23254- 23263). However, these peptides do not have a targeting sequence to localize the therapeutic agent to sites of damaged collagen. These peptides also do not have a triple helix binding sequence for LAIR and are thus much inferior. With current designs thereof, they do not target sites of fibrosis / damaged collagen that could potentially cause the broad actions of RAGE, and would likely exacerbate the inflammatory response they are intended to treat. Products using the platform CHP technique of the present disclosure would be much safer to use, as they specifically bind to plaques and fibrotic tissue, and prevent broad activation of RAGE.

[0296] Finally, a collagen mimetic peptide from Stewart Therapeutics known as ST- 100 has similarity to the CHP design disclosed herein (U.S. Patent No. 10,632,168). ST-100 is useful for treatment of ocular diseases, disorders or wounds including cataracts, corneal ulcerations, scleritis, keratoconus and post-operative eye wounds and is limited thereto. The peptide does not contain a RAGE binding sequence and thus cannot activate the RvD2 pathway and induce an anti-inflammatory response that is possible with the combinatorial peptides of the present disclosure.

[0297] Example 2: Example of Specific Sequence Design

[0298] LAIR bioACTIVE CHP is comprised of the following sequence: Ac-S-(Gly-X-Y)n-bioactive- (Gly-X-Y)m in which Ac is an acetyl capping group; S is zero or more spacer molecules; X is proline or modified proline; Gly is glycine; Y is hydroxyproline or any other amino acid: n is a number from 1 to 20: m is a number from 1 to 20; and tire bioactive is an amino acid sequence from 3 to 27 amino acid residues having bioactivity, and comprises at least one sequence selected from the group consisting of a leukocyte associate immunoglobulin-like receptor sequence (LAIR-1 and / or LAIR-2).

[0299] Additionally, when LAIR plus RAGE binding domains are combined in a sequence, a CHP

[0300] DB1 / 161378034.4 MLB Docket No. 127099-5007-WG contains the same components, plus a linker (triple helical or not), and the N-terminal side of the peptide will contain the RAGE binding sequence. The RAGE binding sequence may be analogous to a High Mobility Group family protein (HMGB1), members of the SlOO / calgranulin family, and advanced glycation end products. These sequences are KLKEKYEKDIAAYRAKGKPDAAKKGVVKAEKSKK (HMGBl-like; SEQ ID NO: 306) and SPQGGGPWDSVARVL (SlOOB-like; SEQ ID NO: 307). A standard GPO6 sequence containing an AGE product can be used since collagen is naturally glycated.

[0301] Example 3: Conditions of Sequence Design

[0302] The collagen hybridizing peptide (CHP) according to the present disclosure satisfies the following requirements:

[0303] 1. There are several aspects of changeable CHP designs that still achieve the ultimate goal of binding to the damaged site and bring the biologically active LAIR and RAGE binding sites to the application site. The triple helix CHP region that binds to damaged collagen may be changed as long as the sequence includes a Gly-X-Y motif. For hybridization with damaged collagen, it can be seen that the sequence has a very high affinity for binding when X is proline and Y is hydroxyproline. However, the amino acid residues at the positions X and Y may be regulated to control the overall Tm of the CHP depending on a specific purpose. The number (n) of Gly-X-Y repeats may be selected from 1 to 15 depending on the desired properties of the CHP.

[0304] 2. The CHP must have a self-association temperature of 37°C or less, which may be 0°C to 37°C.

[0305] 3. CHP must be able to bind to damaged collagen at a temperature which may be 37°C to 100°C, or 37°C.

[0306] 4. CHP may be a dimer.

[0307] 5. CHP may be a trimer with a tail.

[0308] 6. CHP must have a bioactive CHP sequence within the entire sequence, but doesn’t have to be in the middle. CHP may be located at any one of both ends as long as it binds to damaged collagen.

[0309] 7. Unnatural amino acids may be introduced to increase the binding affinity of the sequence. Some residues of interest are 2S,4R-4 fluoroproline as a trans form. When the amino acid is disposed at the Y position, it actually supports proper pre-alignment at the phi-psi angle of poly-proline, such as a poly-proline type II helix, which allows for better trimerization. This is due to the fluorine atom having a stronger electron-withdrawing functionality than the hydroxyproline residues typically found at the position Y. Another unnatural amino acid that could be tested is the aza-glycine residue, which increases the stability of the triple helix formed by increasing the formation of cross-strand hydrogen bonds. In addition to inclusion of these residues in the CHP sequence, the number of residues

[0310] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO included, the position thereof within the CHP sequence, and the mix of these residues in a single CHP sequence may be varied.

[0311] 8. There are several possible LAIR binding sites that act along with the CHPs of the present disclosure and still have the same effect. These LAIR binding sites include several shortened renditions such as GAOGLRGGAGPOGPEGGKGAAGPOGPOXXX (SEQ ID NO: 308) and GAOGLRGGAGPOGPEGGKGAA (SEQ ID NO: 309), GLRGGAGPOGPEGGKGAA (SEQ ID NO: 310), GLRGGAGPOGPE (SEQ ID NO: 311), and ARGLTGRPGDA (SEQ ID NO: 312), but a number thereof remain undefined.

[0312] 9. There are several possible RAGE binding sites that act along with the CHP of the present disclosure and still have the same effect. These include, but are not limited to, KLKEKYEKDIAAYRAKGKPDAAKKGVVKAEKSKK (HMGBl-like; SEQ ID NO: 306) and SPQGGGPWDSVARVL (SlOOB-like; SEQ ID NO: 307) (there are other sequences that act just as well).

[0313] 10. Any sequence disclosed in the first LAIR patent of the present applicant may be incorporated herein with a mobile RAGE binding site included at the end of the sequence.

[0314] Experimental Example 1 : LAIR- 1 Phosphorylation Assay

[0315] Tyrosine phosphorylation of LAIR- 1 is determined using human phospho immunoreceptor array (proteome profiler array; R&D Systems) according to the manufacturer’s protocol. Human monocytes (5 x 106cells) are activated in the presence ofHMGBl protein and then incubated along with LAIR-CHP, RAGE-LAIR-CHP, or control peptide at 37°C for 15 minutes. Also, monocytes are incubated along with LAIR-CHP, RAGE-LAIR CHP, or control peptide without activation by HMGB 1 protein. Phosphorylated proteins are detected using a horseradish peroxidase -conjugated pan anti- phosphotyrosine antibody. Array signals are visualized using a Sapphire Biomolecular Imager (Azure Biosystems).

[0316] Experimental Example 2: Thl T-cell Activation Assay

[0317] Jurkat E6-1 cells (ATCC TIB- 152) are cultured in RPMI 1640 medium containing GlutaMAX (Thenno, 61870036) supplemented with 10% heat inactivated fetal bovine serum (Thermo, 16140071) and 1% penicillin and streptomycin (Thenno, 15070063) at 37°C, 5% CO2. CHP and gelatin solutions are prepared in medium containing 5 to 25 pM LAIR-CHPs, RAGE-LAIR CHPs, or control peptides (previously heated to 80°C for 10 minutes) at 0.52 mg / mL of porcine gelatin (Millipore Sigma, 48724). CHP and gelatin solutions are incubated at 37°C for three days prior to combination with cells.

[0318] On the day of the experiment, T-cells are resuspended in CHP solution containing 3 pg / mL of a high-mobility group box-1 protein (HMGB1) in Nunc 6-wcll tissue culture plates (Thermo, 140675). The concentration ofHMGBl protein is matched to the RAGE-LAIR CHP concentration. Controls are

[0319] DB1 / 161378034.4 MLB Docket No. 127099-5007-WG suspended in gelatin without CHP in the presence or absence of activators. T-cells are incubated along with CHP and gelatin solution and media samples are collected for analysis by Alamar Blue (Thermo, DAL1025) and for expression of other T-cell markers including those from T-reg cells, especially Foxp3 by ELISA (abeam, ab270883) to determine the level of activation from each experimental group with respect to IL-2 and controls.

[0320] Experimental Example 3: Polarization of Macrophages

[0321] As positive and negative controls, overnight cultured monocytes are polarized into Ml macrophages using LPS and IFNy, and into M2 macrophages by treatment with different combinations of IL-4, IL-10, IL-13, and TGF-P or without treatment with the same during the culture period for 24 hours. The dose of the cytokines is 20 ng / mL, and LPS is used at a concentration of 50 ng / mL. In addition, monocytes are cultured in six-well culture plates at a cell concentration of 2x l06cells / mL in RPMI / 10% FCS supplemented with 50 ng / mL of M-CSF or GM-CSF for 6 days to induce predifferentiation into macrophages. Macrophage colony-stimulating factor (M-CSF) and granulocytemacrophage colony-stimulating factor (GM-CSF) are important hematopoietic growth factors as well as potent cytokines required for cell survival, proliferation, differentiation, and activation. As an alternative positive control for activation and polarization into Ml macrophages, monocytes are incubated along with 3 pg / mL HMGB 1. The levels of secreted cytokines are recorded to determine polarization, and nitric oxide is assayed from the supernatant by Griess assay and IL- 10. IL- 12, IRF5, and RvD2 are analyzed by ELISA.

[0322] To test the anti-inflammatory response of CHP, macrophages are cultured on well plates coated with denatured collagen (gelatin) and the content of nitric oxide is measured by Griess assay from the content of the supernatant of IL-10, IL- 12. IRF5, and RvD2 measured by ELISA to determine Ml macrophage activation levels. Cells cultured in wells containing collagen and LAIR-CHP or RAGE -LAIR CHP exhibited lower Ml polarization and higher M2 polarization compared to macrophages cultured on denatured collagen in the absence of CHP or in wells containing control DHP. This supports the anti-inflammatory effect of LAIR-CHP and RAGE-LAIR CHP and activation of the Resolvin pathway .

[0323] Experimental Example 4: Atherosclerosis Mouse Model

[0324] To confirm the therapeutic effect in vivo, apolipoprotein E knockout (ApoE KO) mice fed a high-fat diet (HFD) for 16 weeks are used as an atherosclerosis model. Tire body weight of each mouse is tracked to confirm that the HFD acts and to ensure that plaque formation occurs. The CHP is administered to mice at various concentrations ranging from 1 pM to 250 pM by IV injection in the presence of isofluranc (3-5%). LAIR-CHP, RAGE-LAIR CHP, control peptide, and control group injected with PBS blank are retested. Then, at predetermined time points within 6 hours to 10 days, DB1 / 161378034.4 MLB Docket No. 127099-5007-WG

[0325] 200 pL of blood is collected from the mice, total cholesterol is analyzed using the Mouse Total Cholesterol ELISA kit (Abeam), and scRNA-sequencing is performed. At the end of the experiment, mice are sacrificed by CO2 asphyxiation (according to the approved IACUC protocol), and the aortas are excised, frozen, and sectioned for histology. Plaque identification is performed using Oil-Red-O. which stains lipid-rich plaques red and non-plaque areas pale. In addition, the aortas are sliced open (en-face preparation) and stained with Oil-Red-O to quantitatively assess the distribution of atherosclerotic lesions. Serial sections are also stained with Masson’s Trichome and CHP to assess the level of fibrosis and collagen turnover in the lesions.

[0326] In control versus treated groups, scRNA-seq of whole aortas is performed at the University of Utah facility according to standard protocols. This provides information on the cells activated and genes expressed in each cell population. In the binding to scRNA-seq, flow cytometry is performed at the University of Utah facility to identify the formation of foam cells, which is a characteristic of early- atherosclerotic lesions and a major factor in the development of atherosclerotic plaques. Identification of this cell population provides another marker for inflammation in these lesions and plaques, and downregulation of these cells indicates the anti-inflammatory effects of LAIR and RAGE-LAIR CHP.

[0327] RAGE Receptor Binding Assay: Ac-RAGE-FLAIR2 (Ac-

[0328] KLKEKYEKDIAAYRAKGKPDAAKKGVVKAEKSKK-C6-C6-(GPO)4-GQDGLAGPK-(GPO)3- GFF, SEQ ID NO: 299) was used for the experiments. A Nunc Amino 96-well plate was coated with various proteins, including bovine semm albumin, recombinant human HMBG1, and Ac-RAGE- FLAIR2 peptide. Wells were washed with phosphate buffered saline with Tween 20 (PBST) to remove excess protein. Wells were then blocked with a 1% BSA solution to reduce non-specific protein binding to the plate. Next, 5pg / mL of recombinant human RAGE Fc Chimera protein was added. Again, wells were washed with PBST to remove excess protein. Wells were then incubated with an anti-human IgG Alexa Fluor 488 secondary antibody to detect bound RAGE protein. Lastly, the wells were washed with PBST and read on a fluorescent plate reader at 485nm. As shown on Figure 7, both Ac-RAGE-FLAIR2 and HMGB 1 (RAGE receptor ligand) demonstrated high affinity for binding of RAGE receptor chimeric protein. This indicated that the Ac-RAGE-FLAIR2 peptide was a ligand for the RAGE receptor. Higher fluorescent signal was detected for the RAGE-FLAIR2 peptide over HMGB1, likely due to the lower molecular weight of the RAGE-FLAIR2 peptide leading to higher ligand density per well.

[0329] LAIR-1 Receptor Binding Assay: A 96-well black opaque Nunc Amino flat bottom plate was coated with either a 100 ug / mL gelatin or Bovine Seram Albumin (BSA) protein in a neutral pH buffer and incubated at room temperature for 4 hours. The protein solution was then eluted from the plate. The plate was washed with Phosphate Buffered Saline (PBS) 4 times, and then blocked with 1% BSA in

[0330] DB1 / 161378034.4 MLB Docket No. 127099-5007-WG

[0331] TBST for 30 minutes. The liquid was eluted and the plate was washed 4x with PBS-Tween (PBST). Then, 200uM AC-RAGE-FLAIR2 was deposited on the plate for three days at 37 C. The liquid was then eluted from the plate, the plate was washed 4x in PBST, and 5 ug / mL Recombinant human Leukocyte- Associated Immunoglobulin-like Receptor 1 (LAIR1) Fc Chimera protein in a 1% BSA in TBST solution was then added to the plate for one hour. Background control wells for each group were not incubated with Fc rLAIR-1 to determine background fluorescence and non-specific binding from the antibody. After removal of the LAIRl-Fc protein, wells were washed with 4x PBS. Anti-human IgG Alexa Fluor 488-Fc antibody in a 1% BSA in TBST solution was added to all wells. Tire antibody solution was eluted from the plate, and fluorescence was read by a plate reader at 485nm. Background controls were subtracted from each group. As shown on Figure 8, Ac-RAGE-FLAIR2 bound to gelatin exhibited a higher binding affinity for recombinant LAIR-1 than Gelatin alone. This indicated that the peptide promoted LAIR-1 binding.

[0332] Monocyte Stimulation Assay: RAGE-FLAIR2 peptide (0.05uM) was incubated with gelatin for three days prior to cell experiment. THP-1 monocytes were plated and then dosed with either the peptide-gelatin mixture or gelatin alone. At three and sixteen hours, media was collected, centrifuged, and the supernatant was frozen. An Enzyme-Linked Immunosorbent Assay (ELISA) for human IL- 10 was subsequently performed on these samples. As shown on Figure 9, incubation with RAGE-FLAIR2 increased IL- 10 expression after 16 hours in monocytes. Crosslinking of the RAGE and LAIR-1 receptors lead to an increase in IL- 10 expression in the '‘M2-like state .” Expression of IL- 10 could also suggest reduction of inflammatory cytokines and promotion of immune homeostasis, resulting in decreased inflammation and increased wound healing. This data indicated that RAGE-FLAIR2 was able to bind and crosslink the receptors in vitro and drive the intended cellular response.

[0333] Human Ex-vivo Liver Fibrosis Assay: The study used tissue from a single human donor (PHS 1.1) who was histologically confirmed to have F2 fibrosis and SO steatosis. Precision-cut liver slices (PCLS) with a thickness of 3 mm and a diameter of 250 pm were prepared for a 96-well culture platform. A total of 96 PCLS from the single donor were divided into 12 study groups (n=8 PCLS per group). Tire groups were treated with either a vehicle (0.1% DMSO) or various interventions, including IKK2 inhibitor VI (IKKi), ALK5 inhibitor SB-525334 (ALK5i), Resmetirom (MGL-3196), and Ac- RAGE-FLAIR2, at different concentrations. PCLS were assessed for function and viability using two assays: the CellTox™ Green Cytotoxicity Assay to measure cell membrane integrity as a marker for viable cells. The assay was read using a Tecan Infinite M Nano plate reader. In addition, fibrotic and inflammatory markers, specifically Collagen lai, TIMP-1, and MCP-1, were quantified in the culture media using R&D Duoset ELISA kits. These assays were performed using an automated liquid handling robot, and samples with a coefficient of variation (CV) of less than 15% were accepted for analysis.

[0334] DB1 / 161378034.4 MLB Docket No. 127099-5007-WG

[0335] Histological analysis with Haematoxylin & Eosin (H&E) and PicroSirius Red (PSR) staining was also performed on FFPE tissue to confirm the donor’s fibrosis and steatosis grade. As shown on Figure 10, CellTox Green assay was used to determine if Ac-RAGE-FLAIR2 possessed cytotoxic properties. CellTox Green levels for Ac-RAGE-FLAIR2 were comparable to vehicle control and treatment positive controls, indicating that the peptide did not significantly affect cell viability after 96 hours.

[0336] Figure 11 shows increased presence of collagenlal, or pro-alpha 1 collagen. It indicates active fibrotic activity through excess collagen deposition. The dysregulation of COL1A1 is a hallmark of both lung and liver fibrosis. All concentrations of RAGE-FLAIR2 tested were able to significantly reduce the amount of COL1A1 compared to the vehicle control. At 48 hours, Ac -RAGE-FLAIR outperformed inhibitors of IKK and ALK5. This reduction was noted as early as 48 hours and was sustained through 72 and 96 hours of culture.

[0337] TIMP-1. tissue inhibitor of metalloproteinases- 1, is an inhibitor of MMP activity. In fibrosis, high levels of TIMP-1 activity prevent the breakdown of ECM components leading to fibrotic deposition accumulation. Ac-RAGE-FLAIR2 significantly reduced TIMP-1 levels at all concentrations and timepoints except for lOuM at 96 hours. Additionally, Ac-RAGE-FLAIR2 performed as well as a IKKi, ALKi, and Resmetirom at reducing TIMP-1 at 48 and 72 hours. In combination with reduction of COL1A1, these results demonstrate the anti-fibrotic capabilities of Ac-RAGE-FLAIR2 (Figure 12). MCP-1 is a chemokine that promotes macrophage differentiation and increased tissue remodeling. Ac- RAGE-FLAIR2 significantly reduced MCP-1 secretion at all concentrations and timepoints except for lOuM at 96 hours. This demonstrated that RAGE-FLAIR2 had significant anti-inflammatory capabilities (Figure 13). In all, this data suggests that RAGE-FLAIR2 promotes a resolution state through both anti- fibrotic and anti-inflammatory capabilities.

[0338] Unless otherwise specified, it should be understood that all numbers expressing quantities of ingredients, properties, such as molecular weights and reaction conditions, used in this disclosure and claims are modified in all cases by the term “about”. Accordingly, unless otherwise specified, the numerical parameters set forth in this disclosure and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed by applying ordinary rounding techniques in consideration of the number of significant digits reported.

[0339] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in the respective testing measurements.

[0340] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO

[0341] At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0342] Tire grouping of alternative elements or embodiments of the present disclosure should not be construed as limiting. Each group member may be referred to and claimed individually or in combination with the other members of the group or other elements herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the disclosure is deemed to satisfy all Markush group descriptions used in the appended claims, including the modified group.

[0343] Specific embodiments of the present disclosure, including the best mode known to the present inventors for carry ing out the present disclosure are described herein. Of course, variations of the described embodiments will be apparent to those skilled in the art who read the foregoing descriptions. The present inventors expect that those skilled in the art employ such variations appropriately and intend that the present disclosure is implemented otherw ise than as specifically described herein.

[0344] Accordingly, the present disclosure encompasses all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by the corresponding law. Furthennore, any combination of all possible variations of the elements described above is encompassed by the present disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

[0345] The specific embodiments disclosed herein may be further limited in the claims by the use of the phrases “consisting of’ or “consisting essentially of’. When used in the claims, whether as filed or added by amendment, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of’ limits the scope of the claim to those materials or steps specified and those that do not substantially affect the basic and novel characteristic(s). The embodiments of the present disclosure so claimed are essentially or explicitly described and enabled herein.

[0346] It should be understood that the embodiments of the present disclosure disclosed herein are illustrative of the principles of the present disclosure. Other modifications that may be employed fall within the scope of the present disclosure. Tirus, by way of example, and not limitation, alternative configurations of the present disclosure may be employed in accordance with the teachings herein. Accordingly, the present disclosure is not limited to what has been precisely set forth and described.

[0347] While the present disclosure has been described and illustrated herein with reference to various specific materials, procedures, and examples, it should be understood that the present disclosure is not limited to the specific combinations of materials and procedures selected for the purposes for which they are intended. Many variations of these details are contemplated, as will be appreciated by those skilled

[0348] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO in the art. It is intended that the disclosure and examples be considered illustrative only, with the true scope and spirit of the present disclosure being indicated by the claims that follow. All references, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

[0349] From the foregoing description, it will be apparent to those skilled in the art that the present disclosure may be implemented in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present disclosure should be construed as encompassing within the scope of the present disclosure all changes or modifications derived from the meaning and scope of the following claims and equivalents thereof, rather than the foregoing detailed description.

[0350] EMBODIMENTS

[0351] Embodiment 1. A collagen hybridizing peptide (CHP) comprising one or more sequences represented by Formulas I-V:

[0352] (Gly-X-Y)n-S-RAGE Seq-linker-LAIR Seq-(Gly-X-Y)m (Formula I)

[0353] RAGE Seq-linker-S-(Gly-X-Y)n-LAIR Seq-S-(Gly-X-Y)m (Formula II)

[0354] (Gly-X-Y)n-LAIR Seq-(Gly-X-Y)m (Formula III)

[0355] (Gly-X-Y)n-LAIR Seq-linker-RAGE Seq-linker-(Gly-X-Y)m (Formula IV)

[0356] (Gly-X-Y)n-LAIR Seq-(Gly-X-Y)m-linker-RAGE Seq (Formula V) in which S is zero to 10 amino acids; X is proline or modified proline; Gly is glycine; Y is hydroxyproline or any other amino acid; n is a number from 1 to 20; m is a number from 1 to 20; and the RAGE Seq is an amino acid sequence from 3 to 27 amino acid residues capable of binding to cell membrane-bound Receptors for Advanced Glycation End products (RAGE), the linker includes one or more of: one or more glycines, (Gly-Gly-Gly-Ser)3, GSAGSAAGSGEF, KESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, 6-aminohexanoic acid, 12-aminododecanoic acid, N-Fmoc-N”-succuinyl-4,7,10-trioxa-l,13-tridecanediamine (TTDS), a polymer, an amino acid sequence having from 3 to 35 amino acid residues, (Ala- Pro)p, and (Z-Pro)p, wherein p is a number from 1 to 10 and Z is any amino acid residue, and the LAIR Seq comprises an amino acid sequence from 3 to 27 amino acid residues capable of binding to a leukocyte-associated immunoglobulin-like receptor (LAIR-1 and / or LAIR-2).

[0357] Embodiment 2. The CHP according to any one of the preceding embodiments, further

[0358] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO comprising an acetyl capping group (Ac) at N-terminal of the CHP.

[0359] Embodiment 3. The CHP according to any one of the preceding embodiments, wherein the LAIR Seq comprises a LAIR-1 binding sequence.

[0360] Embodiment 4. The CHP according to any one of the preceding embodiments, wherein the LAIR Seq comprises a LAIR-2 binding sequence.

[0361] Embodiment 5. The CHP according to any one of the preceding embodiments, wherein the LAIR Seq comprises a LAIR-1 and / or LAIR-2 binding sequence comprising a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-27, 56-64, 98-100, and 107-109.

[0362] Embodiment 6. The CHP according to any one of the preceding embodiments, wherein the LAIR Seq comprises a LAIR-1 and / or LAIR-2 binding sequence comprising a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-27, 56-64, 98-100, and 107-109.

[0363] Embodiment 7. The CHP according to any one of the preceding embodiments, wherein the RAGE Seq comprises a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 93-97 and 110-113.

[0364] Embodiment 8. The CHP according to any one of the preceding embodiments, wherein S is at least one amino acid.

[0365] Embodiment 9. The CHP according to any one of the preceding embodiments, wherein the CHP is a first CHP, wherein the first CHP is bound to two other bioactive CHPs to form a trimeric structure; and wherein the first CHP has a monomeric portion extending beyond the trimeric structure, the monomeric portion configured to bind to collagen alpha chains.

[0366] Embodiment 10. The CHP according to any one of the preceding embodiments, wherein the CHP is a first CHP, wherein the first CHP is bound to two other bioactive CHPs to form a trimeric structure, and wherein a monomeric “Tail’’ CHP is conjugated to an N-terminal or a C-terminal of the first CHP of the trimeric structure using copper-free click chemistry or condensation reactions.

[0367] Embodiment 11. The CHP according to any one of embodiments 1-8, wherein the CHP is a first CHP, wherein the first CHP is bound to one other bioactive CHP to fonn a dimeric structure; and wherein the first CHP has a monomeric portion extending beyond the dimeric structure, the monomeric portion configured to bind to collagen alpha chains.

[0368] Embodiment 12. The CHP according to any one of embodiments 1-8 and 10, wherein the CHP is a first CHP, wherein the first CHP is bound to one other bioactivc CHP to form a dimeric structure, and wherein a monomeric “Tail’’ CHP is conjugated to an N-terminal or a C- DB1 / 161378034.4 MLB Docket No. 127099-5007-WG terminal of the first CHP of the dimeric structure using copper-free click chemistry or condensation reactions.

[0369] Embodiment 13. The CHP according to any one of the preceding embodiments, wherein each individual CHP does not form a triple helix with other CHPs.

[0370] Embodiment 14. A method of treating or preventing a fibrotic condition by administering one or more of the CHPs according to any one of the preceding embodiments to a subject in need thereof.

[0371] Embodiment 15. The method of embodiment 14, wherein the fibrotic condition comprises at least one selected from the group consisting of atherosclerosiscutaneous Lupus, Duchenne muscular dystrophy, NASH, subretinal fibrosis, and idiopathic pulmonary fibrosis (IPF).

[0372] Embodiment 16. A method of activating the Resolvin DI (RvDl) and / or Resol vin D2 (RvD2) pathway in a subject in need thereof by administering one or more of the CHPs according to any one of embodiments 1-13 to the subject.

[0373] Embodiment 17. The method of embodiment 16, wherein the RAGE pathway is upregulated in cells of the subject.

[0374] Embodiment 18. The method of embodiment 16, wherein the RAGE pathway is not activated in cells of the subject.

[0375] Embodiment 19. A method for reducing, preventing, and / or treating atherosclerotic plaques in a subject in need thereof by administering one or more of the CHPs according to any one of embodiments 1-13 to the subject.

[0376] Embodiment 20. A method for inhibiting or reducing foam cells in fibrotic cells and / or denatured collagen of a subject in need thereof by administering one or more of the CHPs according to any one of embodiments 1-13 to the subject.

[0377] Embodiment 21 . A method for downregulating immune-related cytokines including but not limited to; TLR ligands, interleukins, Leukotriene B4 (LTB4), IFN-a, TLR-9, TNF-a, IL- 1 P, IL-6, IL-10, PGE2, interferon regulatory factor 5 (IRF5), CCL3, IL-16, IL-18, and / or TNF-a activity in a subject in need thereof by administering one or more of the CHPs according to any one of embodiments 1-13 to the subject.

[0378] Embodiment 22. A method for treating pathologies associated with advanced glycation end products (AGEs) comprising administering one or more of the CHPs according to any one of embodiments 1-13 to a subject in need thereof.

[0379] Embodiment 23. A method for treating inflammatory7diseases mediated by RAGE binding and / or LAIR binding comprising administering the one or more of the CHPs according to any one of embodiments 1-13 to a subject in need thereof.

[0380] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO

[0381] Embodiment 24. A method for downregulating T-Cells, B-Cells, Natural Killer Cells,

[0382] Macrophages, macrophage differentiation, Monocytes, and Neutrophils in a subject in need thereof comprising administering the one or more of the CHPs according to any one of embodiments 1-13 to the subject.

[0383] Embodiment 25. A method for treating cancer in a subject in need thereof comprising administering the one or more of the CHPs according to any one of embodiments 1-13 to the subject.

[0384] Embodiment 26. The method according to any one of embodiments 14-25, comprising administering a composition comprising the one or more CHPs to the subject.

[0385] Embodiment 27. The method according to any one of embodiments 14-26, further comprising administering a carrier with the CHPs.

[0386] Embodiment 28. The method according to embodiment 27, wherein the carrier comprises at least one selected from the group consisting of micelles, dendrites, lipids, microemulsions, nanoemulsions, solid lipid nanoparticles, polymers, gels, lens, surfactants, cyclodextrins, inserts, nanostructured lipid carriers, liposomes, transfersomes, ethosomes, niosomes, collagen matrix, extracellular matrix, and artificial extracellular matrix.

[0387] Embodiment 29. The method according to any one of embodiments 14-28, wherein the composition is administered topically, intravenously, orally, subcutaneously, via injection, or via ocular injection.

[0388] Embodiment 30. The method according to any one of embodiments 14-29, wherein the composition comprises at least one selected from the group consisting of a topical cream, a saline solution, gel, polymer, or a solution for systemic injection within a subject.

[0389] Embodiment 31. The method according to any one of embodiments 14-30, in which a CHP is bound directly to collagen matrix.

[0390] Embodiment 32. The method according to any one of embodiments 14-31, wherein the administering is performed by injection.

[0391] Embodiment 33. The method according to embodiment 32, wherein the administering is performed by micro-dermal injection.

[0392] Embodiment 34. The method according to any one of embodiments 14-31, wherein the administering is performed by topical application.

[0393] Embodiment 35. The method according to any one of embodiments 14-31, wherein the administering is performed intravenously.

[0394] Embodiment 36. The method according to any one of embodiments 14-31, wherein the administering is performed orally.

[0395] DB1 / 161378034.4 MLB Docket No. 127099-5007-WO

[0396] Embodiment 37. The method according to any one of embodiments 14-31, wherein the administering is performed via subcutaneous delivery.

[0397] Embodiment 38. The method according to any one of embodiments 14-31, wherein the administering is performed via ocular injection.

[0398] Embodiment 39. A pharmaceutical composition for preventing, alleviating or treating atherosclerotic plaques comprising the CHP according to any one of embodiments 1-13 as an active ingredient.

[0399] Embodiment 40. A composition for inhibiting or reducing foam cells in fibrotic cells and / or denatured collagen and the CHP according to any one of embodiments 1-13 as an active ingredient.

[0400] Embodiment 41. A composition for activating a Resolvin D 1 (RvD 1) and / or Resolvin D2 (RvD2) pathway comprising the CHP according to any one of embodiments 1-13 as an active ingredient.

[0401] Embodiment 42. A pharmaceutical composition for preventing or treating a fibrotic condition comprising the CHP according to any one of embodiments 1-13 as an active ingredient.

[0402] Embodiment 43. A composition for downregulating one or more immune-related cytokines selected from the group consisting of TLR ligands, interleukins. Leukotriene B4 (LTB4), IFN- a, TLR-9, IL-10, IL-6, IL-10, PGE2, interferon regulatory factor 5 (IRF5), CCL3, IL-16, IL- 18, and / or TNF-a comprising the CHP according to any one of embodiments 1-13 as an active ingredient.

[0403] Embodiment 44. A pharmaceutical composition for preventing or treating pathologies associated with advanced glycation end products (AGEs) comprising the CHP according to any one of embodiments 1-13 as an active ingredient.

[0404] Embodiment 45. A pharmaceutical composition for preventing or treating inflammatory diseases mediated by RAGE binding and / or LAIR binding comprising the CHP according to any one of embodiments 1-13 as an active ingredient.

[0405] Embodiment 46. A composition for downregulating T-cells, B-cells, natural killer cells, macrophages, macrophage differentiation, monocytes, and neutrophils comprising the CHP according to any one of embodiments 1-13 as an active ingredient.

[0406] Embodiment 47. A pharmaceutical composition for preventing or treating cancer comprising the CHP according to any one of embodiments 1-13 as an active ingredient.

[0407] Embodiment 48. The CHP according to any one of embodiments 1 -13, wherein the sequence comprises a sequence having at least 85% sequence identity to any one of SEQ ID DB1 / 161378034.4 MLB Docket No. 127099-5007-WG

[0408] Nos: 299-312.

[0409] Embodiment 49. A method of treating or preventing a fibrotic disease in a subject in need thereof, the method comprising administering one or more of the CHPs according to any one of embodiments 1-13 to the subject

[0410] Embodiment 50. A method of polarizing a macrophage to a resolution state, the method comprising costimulating RAGE and LAIR1 of the macrophage.

[0411] Embodiment 51. A method of polarizing a macrophage population to a resolution state, the method comprising costimulating RAGE and LAIR1 of the macrophage population.

[0412] Embodiment 52. The method of embodiment 18 or 19, the method further comprising contacting the CHP of any one of claims 1-13 with the macrophage prior to the costimulating.

[0413] DB1 / 161378034.4

Claims

MLB Docket No. 127099-5007-WOCLAIMS1 . A collagen hybridizing peptide (CHP) comprising one or more sequences represented by Formulas I-V:(Gly-X-Y)n-S-RAGE Seq-linker-LAIR Seq-(Gly-X-Y)m (Formula I)RAGE Seq-linker-S-(Gly-X-Y)n-LAIR Seq-S-(Gly-X-Y)m (Formula II)(Gly-X-Y)n-LAIR Seq-(Gly-X-Y)m (Formula III)(Gly-X-Y)n-LAIR Seq-linker-RAGE Seq-linker-(Gly-X-Y)m (Formula IV) (Gly-X-Y)n-LAIR Seq-(Gly-X-Y)m-linker-RAGE Seq (Formula V) in which S is 0 to 10 amino acids; X is proline or modified proline; Gly is glycine; Y is hydroxyproline or any other amino acid; n is a number from 1 to 20; m is a number from 1 to 20; and the RAGE Seq comprises an amino acid sequence from 3 to 27 amino acid residues capable of binding to cell membrane-bound Receptor for Advanced Glycation End products (RAGE), the linker includes one or more of one or more glycines, (Gly-Gly-Gly-Ser)3, GSAGSAAGSGEF, KESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, 6-aminohexanoic acid, 12-aminododecanoic acid, N-Fmoc-N”-succuinyl-4,7,10-trioxa-I,I3-tridecanediamine (TTDS), a polymer, an amino acid sequence having from 3 to 35 amino acid residues, (Ala-Pro)p, and (Z- Pro)p, wherein p is a number from 1 to 10 and Z is any amino acid residue, and the LAIR Seq comprises an amino acid sequence from 3 to 27 amino acid residues capable of binding to a leukocyte-associated immunoglobulin-like receptor (LAIR-1 and / or LAIR-2).

2. The CHP according to claim 1, further comprising an acetyl capping group (Ac) at N- terminal of the CHP.

3. The CHP according to claim 1 or 2, wherein the LAIR Seq comprises a LAIR-1 and / or LAIR-2 binding sequence comprising a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-27, 56-64, 98-100, and 107-109.

4. The CHP according to any one of the preceding claims, wherein the RAGE Seq comprises a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 93-97, 110-1 13.

5. The CHP according to any one of the preceding claims, wherein the sequence comprises a sequence having at least 85% sequence identity to any one of SEQ ID Nos: 299-312.DB1 / 161378034.4MLB Docket No. 127099-5007-WG6. A method of treating or preventing a fibrotic condition by administering one or more of the CHPs according to any one of the preceding claims to a subject in need thereof, wherein the fibrotic condition comprises at least one selected from the group consisting of atherosclerosiscutaneous Lupus, Duchenne muscular dystrophy, NASH, subretinal fibrosis, and idiopathic pulmonary fibrosis (IPF) .

7. A method of activating the Resolvin DI (RvDl) and / or Resolvin D2 (RvD2) pathway in a subject in need thereof by administering one or more of the CHPs according to any one of claims 1-5 to the subject.

8. A method for reducing, preventing, and / or treating atherosclerotic plaques in a subject in need thereof by administering one or more of the CHPs according to any one of claims 1-5 to the subject.

9. A method for inhibiting or reducing foam cells in fibrotic cells and / or denatured collagen of a subject in need thereof by administering one or more of the CHPs according to any one of claims 1-5 to the subject.

10. A method for downregulating immune-related cytokines including, but not limited to, TLR ligands, interleukins, Leukotriene B4 (LTB4), IFN-a, TLR-9, TNF-a, IL-ip, IL-6, IL-10, PGE2, interferon regulator7factor 5 (IRF5), CCL3, IL-16, IL-18, and / or TNF-a in a subject in need thereof by administering one or more of the CHPs according to any one of claims 1-5 to the subject.I L A method for treating pathologies associated with advanced glycation end products (AGEs) comprising administering one or more of the CHPs according to any one of claims 1-5 to a subject in need thereof.

12. A method for treating inflammatory diseases mediated by RAGE binding and / or LAIR binding comprising administering the one or more of the CHPs according to any one of claims 1-5 to a subject in need thereof.

13. A method for downregulating T-Cells, B-Cells, Natural Killer Cells, Macrophages, macrophage differentiation, Monocy tes, and Neutrophils in a subject in need thereof comprising administering the one or more of the CHPs according to any one of claims 1-5 to the subject.DB1 / 161378034.4MLB Docket No. 127099-5007-WG14. A method for treating cancer in a subject in need thereof comprising administering the one or more of the CHPs according to any one of claims 1-5 to the subject.

15. The method according to any one of claims 6-13, comprising administering a composition comprising the one or more CHPs to the subject.

16. The method according to any one of claims 6-14, further comprising administering a carrier with the CHPs.

17. A method of treating or preventing a fibrotic disease in a subject in need thereof, the method comprising administering one or more of the CHPs according to any one of the claims 1 to 5 to the subject.

18. A method of polarizing a macrophage to a resolution state, the method comprising costimulating RAGE and LAIR1 of the macrophage.

19. A method of polarizing a macrophage population to a resolution state, the method comprising costimulating RAGE and LAIR1 of the macrophage population.

20. The method of claim 18 or 19, the method further comprising contacting the CHP of any one of claims 1-x with the macrophage prior to the costimulating.DB1 / 161378034.4

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