Anti-inflammatory bioactive collagen hybridizing peptides and methods of use thereof

Abstract

Novel anti-inflammatory bioactive collagen hybridizing peptides (CHPs), compositions containing CHPs, and methods of using CHPs and compositions containing CHPs are disclosed. The novel anti-inflammatory bioactive CHPs can be used to localize collagen anti-inflammatory sequences to directly down-regulate the inflammatory response.

Description

[Background technology]

[0001] background Collagen homeostasis is a complex process involving both collagen synthesis and degradation. Collagen homeostasis is tightly regulated primarily through a specialized cell population called fibroblasts. Fibroblasts produce both collagen and matrix metalloproteinases (MMPs) in response to chemical signals, interactions between cells and the extracellular matrix (ECM), and environmental conditions such as UV radiation and force. Collagen is a major structural protein found in nearly all human tissues. Collagen contains a binding sequence that must be in a triple-helical form to be recognized by cells, including fibroblasts, and other cell types, including immune cells. Cells bind to collagen through cell surface receptors, including integrins, discoidin domain receptors, MMPs, osteoclast-associated receptors (OSCARs), and leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1). Importantly, immune cell activity can be mediated through interaction with triple-helical collagen via cell surface LAIR-1.

[0002] Degraded collagen is present in damaged tissues, which has been shown to be frequently associated with numerous human diseases and injuries. Damaged collagen lacks the triple helix structure that allows it to be recognized by fibroblasts, other cell types, or biological molecules (e.g., MMPs). Damaged collagen sites often exhibit additional inflammation and autoimmune activity (e.g., excessive production of inflammatory cytokines), which can further damage collagen and the ECM. Collagen damage can impair the microenvironment necessary for cell attachment and mechanics, reducing fibroblast collagen production and further exacerbating the condition of the affected area. This can occur under specific conditions, including diseases such as osteoarthritis; as a result of natural conditions such as aging (fibroblast senescence); reduced mechanical stimulation; exposure to environmental factors such as UV damage; or exposure to chemical signals such as reactive oxygen species. Conditions such as dry eye disease (DED), atopic dermatitis (eczema), psoriasis, diabetic conditions, osteoarthritis, rheumatoid arthritis, and other inflammatory diseases can all involve damaged collagen and an excessive inflammatory response near the site of collagen damage, which can lead to barrier dysfunction.

[0003] By providing localized triple helix binding sites for cells to bind at sites of increased collagen damage, both the poor cellular microenvironment and increased inflammatory response can be resolved. Providing new binding sites in the microenvironment can increase collagen production, thereby increasing mechanical stimulation of cells, forming a positive feedback loop. The presence of more native collagen increases binding sites and mechanical stimulation, promoting tissue regeneration and repair by resident cells. Furthermore, increasing collagen anti-inflammatory sequences at sites of collagen damage can help reduce or prevent the activation of immune cells, including, but not limited to, T cells, natural killer cells, macrophages, B cells, neutrophils, and monocytes. Therefore, it may be beneficial to restore a damaged collagen-based cellular environment using methods and compositions that stimulate fibroblasts to increase collagen production, increase barrier function, and downregulate immune responses, thereby restoring the structural integrity of the ECM and ultimately restoring a tissue pathology to a healthy state. Summary of the Invention

[0004] Disclosure Summary The present disclosure relates to novel anti-inflammatory bioactive (bioACTTVE) collagen hybridizing peptides (CHPs; also referred to as "collagen hybridizing peptides"), compositions comprising CHPs, and methods of using CHPs and compositions comprising CHPs. The novel anti-inflammatory bioactive CHPs can be used to localize collagen anti-inflammatory sequences to directly down-regulate immune and inflammatory responses. The disclosure of PCT / US2022 / G77730 is incorporated herein by reference for all purposes. [Brief explanation of the drawings]

[0005] [Figure 1A] 1 illustrates a CHP composed of a triple-helix forming moiety covalently linked to a bioactive sequence. [Figure 1B]The unique binding mechanism of bioactive CHP is shown. CHP may be inactive in a single-chain conformation, but becomes activated upon forming a triple helix structure with damaged collagen. Thus, bioactive CHP can target and localize to remodeling or damaged collagen. Upon binding to damaged collagen, it forms the appropriate triple helix and presents the bioactive sequence in a form suitable for recognition, thereby promoting therapeutic action through interaction with cells that recognize the bioactive sequence and promoting ECM healing and / or regeneration. [Figure 2] Various immune cells possess LAIR-1 receptors and can interact with naturally occurring collagen molecules in triple-helical form. The proposed bioactive CHP contains the LAIR-1 bioactive sequence, and upon binding to damaged collagen, presents the LAIR-1 binding site in the appropriate form for recognition and binding by immune cells, thereby reducing the inflammatory response. [Figure 3] Illustrated are bioactive CHP in monomeric form (with a monomeric binding site), dimeric bioactive CHP (two linked bioactive and folding domains), and bioactive CHP bound to two other CHPs in a trimeric form (presenting a trimeric binding site) with a monomeric "tail" portion of the bioactive CHP according to embodiments disclosed herein. [Figure 4] 1 shows a proposed mechanism of action for LAIR-1 bioactive CHP according to embodiments disclosed herein. [Figure 5] 1 shows the amino acid sequence of an exemplary bioactive CHP according to embodiments disclosed herein. [Figure 6] 1 shows the experimental design of an LAIR-1 plate binding assay for detecting LAIR-1 binding to bioactive CHP of an LAIR-1 monomer according to an embodiment disclosed herein. [Figure 7] 1 shows the results of a plate binding assay at room temperature for bioactive CHP of LAIR-1 monomers with high melting temperatures according to embodiments disclosed herein. [Figure 8]1 shows the results of a plate binding assay at 37° C. of bioactive CHP of LAIR-1 monomers with high melting temperatures according to embodiments disclosed herein. [Figure 9] 1 shows the results of a plate binding assay at 4° C. of bioactive CHP of LAIR-2 monomers with low melting temperatures according to embodiments disclosed herein. [Figure 10] 1 shows the experimental design of an LAIR-1 plate binding assay for detecting LAIR-1 binding to bioactive CHP of an LAIR-1 dimer according to an embodiment disclosed herein. [Figure 11] 1 shows the results of a binding assay of a LAIR-1 dimer with a low melting temperature according to an embodiment disclosed herein to bioactive CHP at 4° C. [Figure 12] 1 shows an experimental design for demonstrating T cell activation / suppression in vitro using LAIR-1 CHP according to embodiments disclosed herein. [Figure 13] 1 shows the experimental conditions used in an assay to demonstrate in vitro T cell activation / suppression using LAIR-1 CHP according to an embodiment disclosed herein. [Figure 14] 1 shows the results of an in vitro T-cell activation assay performed at 37° C. by detecting IL-2 levels 72 hours after stimulation, according to embodiments disclosed herein. [Figure 15] 1 shows the results of an in vitro T-cell activation assay performed at 37° C. by detecting IL-2 levels 72 hours after stimulation, according to embodiments disclosed herein. DETAILED DESCRIPTION OF THE INVENTION

[0006] Detailed Description Hereinafter, exemplary embodiments of the present disclosure will be described in detail. However, the present disclosure is not limited to the disclosed embodiments and may be embodied in various forms. The following embodiments are described to enable those skilled in the art to embody and practice the embodiments of the present disclosure.

[0007] Disclosed herein are materials, compositions, and components that can be used in, with, used in preparation for, or are products of the disclosed methods and compositions. Where these and other materials are disclosed herein, and where combinations, subsets, interactions, groups, etc. of these materials are disclosed, it should be understood that each is specifically contemplated and described herein, even though specific reference to each of the various individual and collective combinations and permutations of such compounds may not be expressly disclosed. For example, where peptide conjugates are disclosed and discussed, and numerous variations that can be made for numerous molecules comprising the peptide conjugates are discussed, each and every combination and permutation of the peptide conjugates and possible variations is specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C is disclosed, and examples of molecules A-D are disclosed in combination with a class of molecules D, E, and F, each is considered individually and collectively, even if not individually mentioned. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F is specifically contemplated and should be considered disclosed from the disclosure of A, B, and C; D, E, and F; and exemplary combinations A-D. Likewise, subsets or combinations thereof are also specifically contemplated and disclosed. Thus, for example, subgroups of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from the disclosure of A, B, and C; D, E, and F; and exemplary combinations A-D. This concept applies to all aspects of this application, including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, where there are various additional steps that can be performed, each such additional step can be performed in any particular embodiment or combination of embodiments of the disclosed methods. It is also understood that each such combination is specifically contemplated and should be considered disclosed.

[0008] definition Terms such as "first," "second," etc. may be used to describe various components, but these components are not limited by these terms. These terms are used to distinguish one component from another. For example, a first component may be termed a second component, and a second component may be termed a first component, without departing from the scope of example embodiments. The term "and / or" includes any and all combinations of one or more of the associated listed items.

[0009] It should be noted that, as used in this specification and the appended claims, the singular includes the plural unless the context clearly dictates otherwise. Thus, for example, a reference to "a peptide" includes a plurality of such peptides, and a reference to "the peptide" is a reference to one or more peptides and equivalents thereof known to those skilled in the art. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise.

[0010] The phrase "at least one," as used herein and in the claims, while referring to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but does not necessarily include at least one of each and every element specifically listed in the list of elements, nor does it exclude any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, to be optionally present, whether or not related to the specifically identified elements. 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") can mean, in one embodiment, at least one (optionally including more than one) A (and, optionally, including other components other than B) in the absence of B; in another embodiment, it can mean at least one (optionally including more than one) B (and, optionally, including other components other than A) in the absence of A; and in yet another embodiment, it can mean at least one (optionally including more than one) A and at least one (optionally including more than one) B (and, optionally, including other components).

[0011] Terms such as "comprises," "having," and the like are used interchangeably and have the same meaning. Similarly, terms such as "comprises," "having," and the like are used interchangeably and have the same meaning. Specifically, each of these terms is defined consistent with the general U.S. patent law definition of "comprises," and thus is interpreted as open-ended, meaning "at least," and not excluding additional features, limitations, aspects, etc. Thus, for example, "an apparatus having components a, b, and c" means that the apparatus includes at least components a, b, and c. Similarly, the phrase "a method including steps a, b, and c" means that the method includes at least steps a, b, and c. Additionally, although steps and processes may be described in a particular order herein, those skilled in the art will recognize that the order of the steps and processes may be varied unless a specific order is clearly indicated by the context.

[0012] As used herein, the term "about" refers to numerical values, including, for example, integers, fractions, and percentages, whether explicitly stated or not. The term "about" generally refers to a range of numbers (e.g., ±5, 6, 7, 8, 9, or 10% of the stated value) that one of ordinary skill 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 numerical values ​​rounded to the nearest significant figure. In general, the term "about" includes values ​​for a given quantity that are within the relevant tolerance for manufacturing, formulation, and / or measurement, to a minimum.

[0013] As used herein, "collagen" can be derived from all tissue types (e.g., bone, dermis, muscle, ligament, cornea, etc.). Collagen can refer to a molecule in which three alpha chains of polyproline II-like structure are folded together into a triple helix. This can also apply to all proteins containing triple helix regions, including types I-XXVIII collagen and bacterial collagen. As used herein, the term "collagen" can refer to all forms of collagen, including artificial collagen and processed or modified collagen. In some embodiments, the collagen is selected from type I collagen, type II collagen, type III collagen, type IV collagen, type V 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.

[0014] As used herein, the term "proline or modified proline" refers to the amino acid proline and its various isomers, analogs, and variants, including all natural and non-natural isomers. In one example, the modified proline contains an electron-withdrawing group. Examples of modified prolines include, but are not limited to, hydroxyproline, methylated proline, 4-fluoroproline, and 4-chloroproline.

[0015] The term "treating" means partially or completely alleviating, ameliorating, or alleviating one or more symptoms or characteristics of a particular disease, disorder, and / or condition, delaying the onset, inhibiting progression, reducing the severity, and / or reducing the incidence. For example, "treating" a disease or injury associated with collagen damage can mean reducing or eliminating the amount of damaged or denatured collagen. Treatment can also be administered to subjects who do not exhibit symptoms of the disease, disorder, and / or condition and / or who exhibit only early signs of the disease, disorder, and / or condition, with the goal of reducing the risk of developing pathological symptoms associated with the disease, disorder, and / or condition.

[0016] Introduction Hereinafter, exemplary embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. To facilitate understanding of the present disclosure, the same reference numerals will refer to the same components throughout the description of the drawings, and the description of the same components will not be repeated.

[0017] Collagen is a major structural protein found in nearly all human tissues. Degraded collagen is known to be present in damaged tissue and is frequently associated with many human diseases and injuries, including inflammatory conditions and autoimmune disorders.

[0018] Intact collagen found within the ECM exists as a triple-helical molecule. Cells can bind to specific sequences / sites in intact collagen, and such binding helps maintain the health and structure of the ECM and resident cells. When collagen is damaged, it loses its triple helicity, altering the structure of cell-binding sites and ultimately limiting the cell's ability to bind to that collagen. When immune cells are unable to bind to collagen, which acts as a natural checkpoint inhibitor, the presence of matrix metalloproteinases (MMPs) and inflammatory cytokines can increase at the site of injury. This creates a negative feedback loop in which MMPs and inflammatory cytokines interfere with tissue repair, and subsequent collagen damage can further increase the presence of MMPs and inflammatory cytokines.

[0019] Collagen hybridizing peptides (CHPs) are short, repeating Gly-XY tripeptides that can specifically bind to degraded collagen with little or no affinity for intact collagen molecules. CHPs can identify damaged collagen by recognizing the structural motif of the polyproline II helix in individual alpha chains, which is not present in intact collagen molecules. After identifying damaged collagen, CHPs form highly stable and long-lasting bonds by reforming and restoring the collagen triple helix through hybridization with the damaged collagen. The present disclosure relates to bioactive versions of CHPs that can be used not only to induce cells to produce more collagen, but also to localize collagen-based anti-inflammatory cell-binding sequences to directly down-regulate the inflammatory response in damaged tissues.

[0020] Bioactive CHPs generally contain three major segments: a triple-helix-forming segment, followed by a bioactive sequence (e.g., a cell-binding sequence), followed by a second triple-helix-forming segment (an exemplary diagram of a bioactive CHP is shown in Figure 1A). These CHPs can exist in monomeric, dimeric, or trimeric forms, depending on the needs of the biological receptor to which the bioactive sequence is intended to bind (see Figure 3). Dimeric and trimeric forms of CHP can include linkers between the CHP chains. Such linkers can include 6-aminohexanoic acid and SEQ ID NO: 105 (where z is any integer between 1 and 5). These CHPs target damaged collagen sites by localizing to, hybridizing with, and reforming the appropriate triple-helix structure at sites of collagen damage or remodeling. Upon localization and formation of the triple-helix structure, the CHP can exert its therapeutic effect by presenting the bioactive sequence in a form suitable for binding. The proposed localized mechanism of action for a representative bioactive CHP is shown in Figure 1B.

[0021] Inflammation / immune responses can be downregulated, suppressed, or reduced in a variety of ways by administering the disclosed CHPs and / or compositions. Inflammation can also be reduced, prevented, or treated using the disclosed CHPs and / or compositions. Immune-related cytokines (e.g., TLR ligands, interleukins, IFN-α, TLR-9, TNF-α, IL-1β, IL-6, CCL3, IL-16, IL-18, and / or TNF-α) can be downregulated using embodiments of the disclosed CHPs and / or compositions. Furthermore, embodiments of the disclosed CHPs and / or compositions can prevent differentiation of immune cells (e.g., T cells, B cells, innate killer cells, macrophages, monocytes, neutrophils). Other embodiments of the disclosed CHPs and / or compositions can downregulate the activity of immune cells (e.g., T cells, B cells, innate killer cells, macrophages, monocytes, neutrophils).

[0022] The anti-inflammatory bioactive CHP sequences of the present disclosure utilize specific amino acid sequences derived from anti-inflammatory peptide sequences (e.g., LAIR-1 receptor) that cells recognize and respond to, ultimately reducing inflammatory responses. These sequences act as natural checkpoint inhibitors. By adding these sequences to CHP, the biological functions provided by native, undamaged collagen (e.g., cell binding and ECM-cell signaling) can be reproduced in a novel manner, involving CHP folding into a triple helix structure along with damaged or denatured collagen chains. The bioactive sequences inserted into CHP can be presented in a suitable form to attract cells within the ECM, bind to those cells, down-regulate inflammatory responses, and help treat chronic wounds or disease states involving damaged collagen. Such CHPs and compositions containing them can be used to treat a number of diverse diseases or conditions characterized by damaged collagen and inflammatory responses (e.g., xerophthalmia, atopic dermatitis, osteoarthritis, rheumatoid arthritis, and diabetic pathologies).

[0023] CHP and compositions thereof offer a new method for localizing anti-inflammatory sequences in the proper triple helix conformation while maintaining high aqueous solubility. Due to its high solubility, low immunogenicity, and small size, anti-inflammatory bioactive CHP can be easily formulated into a variety of dosage forms and used to treat a wide variety of conditions or diseases. The anti-inflammatory bioactive CHP and compositions thereof disclosed herein offer an improvement over current anti-inflammatory condition treatments because the disclosed CHP can be localized to sites where inflammation and collagen turnover are most prevalent. Upon binding to damaged collagen chains, the anti-inflammatory CHP amino acid sequence is presented in the proper triple helix conformation for recognition by associated inflammatory cells or ECM-resident cells. Depending on the specific sequence used, the anti-inflammatory bioactive CHP can promote collagen repair, restore tissue barrier function, suppress inflammatory responses, or all of these.

[0024] By treating both the inflammatory and adverse cellular environments, the rate of recovery can be significantly improved. Furthermore, due to the long binding time of CHP (e.g., greater than 5 days), patients do not need to use anti-inflammatory bioactive CHP-containing treatments as frequently, improving patient compliance. This is particularly true for conditions requiring traditional treatments multiple times daily, including, but not limited to, dry eye syndrome (DED) and atopic dermatitis. CHP and compositions containing it can be formulated for use in topical creams, saline solutions (e.g., eye drops), or systemic injections.

[0025] LAIR-1 is known to downregulate T cell-mediated inflammatory pathways. LAIR-1 is an immunoinhibitory receptor broadly expressed across immune cells, including, but not limited to, T cells, B cells, monocytes, neutrophils, innate killer cells, and macrophages. When LAIR-1-expressing cells bind to intact collagen LAIR-1 binding sites, it downregulates immune cell activity and proinflammatory cytokines, including NF-κB and TNF-γ. However, when damaged collagen increases, as observed in diseases such as DED, AD, and OA, the inflammatory response increases due to the lack of collagen binding sites for LAIR-1 present on immune cells. LAIR-1 does not bind to damaged (misfolded) collagen, which can activate T cells. Furthermore, activated immune cells are known to produce MMPs, which further degrade surrounding collagen and cleave any remaining LAIR sites, creating a potential negative feedback loop. FIG. 4 illustrates T cell suppression by intact collagen, T cell activation in the environment of damaged collagen, and a proposed mechanism for T cell suppression using exemplary LAIR-1 bioactive CHPs disclosed herein.

[0026] Without being bound by theory, LAIR bioactive CHP appears to reduce immune activation by generating more potential binding sites for LAIR-1-bearing immune cells (e.g., T cells) on the cell surface. CHP enables LAIR-1 receptors to bind to triple-helical collagen sites, even at sites of damaged or remodeling collagen. Damaged collagen is locally upregulated at disease sites in inflammatory, autoimmune, cancerous, and fibrotic conditions.

[0027] LAIR exists in two forms: membrane-bound LAIR-1 and soluble LAIR-2. The main structural difference between the two variants is that LAIR-2 lacks the transmembrane and cytoplasmic domains present in membrane-bound LAIR-1 and can be exported to the extracellular space. LAIR-1 and LAIR-2 compete for the same site on triple-helical collagen, but in regions with high GPO content, there are only approximately 10 sites per collagen molecule. LAIR-2 appears to preferentially bind to collagen sites with a slightly higher affinity than LAIR-1.

[0028] Increased levels of LAIR-2 correlate with increased survival in some cancers. Furthermore, synovial fluid from rheumatoid arthritis (RA) patients is known to have higher LAIR-2 levels than osteoarthritis patients, and LAIR-2 levels have been reported to be elevated in some tissue-specific autoimmune conditions, such as Graves' disease. This suggests that LAIR-2 is elevated in inflammatory and autoimmune conditions.

[0029] By increasing the number of LAIR-binding sites on damaged collagen, bioactive CHP appears to help overcome the "blocking" properties of LAIR-2 by providing additional sites for LAIR-1-expressing immune cells to bind, despite the preferential binding of LAIR-2. Furthermore, because LAIR-2 is a homolog of LAIR-1 and binds to the same site on intact collagen, bioactive CHP with LAIR-binding sites could bind and sequester excess LAIR-2 around damaged collagen.

[0030] In inflammatory conditions (e.g., rheumatoid arthritis, Graves' disease), collagen damage increases and the number of potential LAIR binding sites decreases, because collagen must be in the proper triple helix conformation to present LAIR binding sites. These inflammatory conditions are also characterized by increased LAIR-2 production, which leads to uncontrolled inflammatory responses. Without being bound by theory, it appears that inflammatory conditions in which LAIR-2 production is upregulated can be treated by administering bioactive CHPs bearing LAIR binding sites. Such administration may increase the total number of LAIR binding sites available at sites of damaged or remodeling collagen. By increasing the total number of LAIR binding sites, such CHPs may act as a "sink" for soluble LAIR-2 molecules, reducing competition for LAIR sites. This may provide an opportunity for cell-bound LAIR-1 to bind to collagen and reduce the inflammatory response.

[0031] Inflammatory conditions A significant number of diseases, disorders, and conditions involve inflammation along with collagen damage. Therefore, to treat these diseases, it is better to interrupt the negative feedback loop in which increased collagen damage leads to increased inflammatory responses, which further exacerbate collagen damage, by enriching damaged collagen with bioactive sites, ultimately reducing inflammation and restoring collagen / ECM. The disclosed CHP and compositions thereof can be used in the clinical treatment of conditions such as DED, atopic dermatitis, osteoarthritis, rheumatoid arthritis, and diabetic disorders.

[0032] xerophthalmia Xerophthalmia (DED) is a common eye disease affecting up to 50% of the population across all age groups. DED is characterized by corneal thinning, nerve damage, cell death, poor tear quality, and a potentially exacerbated inflammatory response. This can lead to blurred vision and even vision loss, and can be a painful chronic condition that is difficult for patients to manage.

[0033] Currently, over-the-counter (OTC) lubricants are available to help relieve pain in patients with mild DED. These OTC solutions have the problem that they are not effective in addressing the underlying cause of DED and only provide temporary relief by increasing moisture content through their lubricating action.

[0034] While most lubricating eye drops are basic saline solutions, others, such as iVIZIA, can be in a more advanced form. iVIZIA uses a hydrating polymer, sodium hyaluronate, and trehalose (a naturally occurring disaccharide that prevents drying). This type of treatment fails to reverse or resolve the underlying cause of DED and appears to only help patients with mild to moderate DED.

[0035] Yet another treatment option for DED is the use of corticosteroids. Kala Pharmaceuticals' EYSUVIS is a corticosteroid treatment for DED designed with a surface coating that helps deliver the steroid to the surface of the eye, rather than trapping it in the protective mucin. This helps locally reduce swelling associated with DED, reducing redness, irritation, and blurred vision. However, this is only effective for short-term relief and can only be used for a short period of time (i.e., only 2 weeks). Furthermore, this treatment only treats symptoms without addressing the underlying mechanism of DED inflammation.

[0036] For more severe DED, several treatments aim to prevent the adhesion of inflammatory cells from producing more cytokines and MMPs, which worsen symptoms. Novartis' XIIDRA (lifitegrast) works by blocking the LF'A-1 receptor on T cells, preventing them from binding to the ICAM-1 receptor on other white blood cells and endothelial cells. Through this mechanism, the treatment reduces the number of activated T cells, prevents their migration to the ocular surface, and provides a way to reduce the inflammatory response. This treatment requires patients to take it twice daily for weeks before seeing results, and sustained administration is necessary to maintain efficacy.

[0037] DED patients prescribed anti-inflammatory therapy generally must use the therapy multiple times over an extended period to see any effect. Effectively neutralizing the inflammatory state commonly present in ocular tissues due to DED requires resolving collagen degradation and localizing anti-inflammatory signaling at the lesion site. Furthermore, anti-inflammatory therapeutic agents do not take into account the deterioration of the cellular microenvironment and impaired barrier function.

[0038] The anti-inflammatory bioactive CHP disclosed herein provides a new method for localizing anti-inflammatory sequences in the proper triple helix conformation of native collagen while maintaining high aqueous solubility, facilitating the formulation of therapeutic compositions such as eye drops. Furthermore, due to their small size and the reduced barrier function associated with DED, CHPs can penetrate the corneal epithelial barrier, which is important for developing therapeutic compositions (e.g., eye drops) to reach inflammatory lesions on the ocular surface. These CHPs directly target unstructured collagen chains found at sites where the cornea thins or corneal ulcers develop due to the presence of inflammatory cytokines and matrix metalloproteinases (MMPs). Through this, the anti-inflammatory bioactive CHPs target the most irritated areas, hybridize with denatured collagen, and remain bound for extended periods (up to several days), allowing cells at the site to interact with the CHP and modulate the inflammatory response and increase collagen production. The long residence time at the injury site means that patients do not have to apply the drops as frequently (e.g., twice daily) but instead only once a week, potentially leading to greater patient compliance. Anti-inflammatory bioactive CHPs may also be combined with other types of bioactive CHPs, such as integrin and DDR, which are designed to increase cell migration to the affected site, decrease the inflammatory response by increasing collagen production, restore barrier function, and repair local injury.

[0039] Atopic dermatitis Atopic dermatitis (or "eczema") is a T-cell-mediated inflammatory skin condition that causes dry, itchy, and inflamed skin. It occurs most frequently in adolescents, especially infants. First-line treatments for atopic dermatitis are moisturizers and strong topical steroid treatments, but these are not recommended for long-term use and are not very effective. Second-line treatment options include non-steroidal creams, which are often strong immunosuppressants designed to reduce immune rejection in transplant patients and may increase the risk of bacterial infection. Second-line treatments are injectable mAbs or oral immunomodulators, which are used only in the most severe cases of atopic dermatitis.

[0040] Anti-inflammatory treatments for atopic dermatitis, such as Pfizer's CIBINQO (abrocitinib), work by blocking adenosine triphosphate binding sites and inhibiting Janus kinase (JAK1). Because Janus kinase activity is thought to contribute to inflammation, blocking its signaling pathway is thought to reduce inflammation and itch and improve skin clarity. However, this mechanism of action has not yet been verified, and the systemic effects of such anti-inflammatory treatments are undesirable. That is, these treatments block the Janus kinase signaling pathway systemically and do not target specific areas of the body where atopic dermatitis occurs.

[0041] There is a clear market gap for a nonsteroidal treatment to treat mild or moderate disease that has fewer side effects than currently available treatments, particularly in adolescents. There is also an urgent need for treatments that are designed to act locally to treat mild relapses and provide patient comfort upon administration. There is also a need to avoid the use of potent immunosuppressants, as they frequently occur in young patients.

[0042] Intact collagen generally contains anti-inflammatory sites such as LAIR-1. LAIR-1 can act as a checkpoint inhibitor by reducing inflammatory cytokines and T cell activation. In inflammatory disorders such as atopic dermatitis / eczema, high T cell and immune activation is often associated with increased MMPs. MMPs reduce barrier function by degrading collagen, thereby reducing natural collagen-based checkpoint inhibitors such as LAIR-1. This increase in MMPs also provides an abundance of damaged collagen for CHP binding. Bioactive CHP can increase the number of anti-inflammatory sites such as LAIR-1 to downregulate the immune response associated with the T cell-mediated condition commonly present in the skin due to atopic dermatitis / eczema and avoid the use of systemic therapeutic agents and potent immunosuppressants. By using the anti-inflammatory bioactive CHPs described herein in conjunction with other types of bioactive CHPs designed to increase cell migration to the affected site, maintain resident cells at the site of injury, or increase collagen production, the CHPs can restore barrier function, down-regulate T cell-mediated inflammatory pathways, and provide rapid relief and improved healing.

[0043] Anti-inflammatory bioactive collagen hybridizing peptide (CHP) The present disclosure relates to anti-inflammatory bioactive collagen hybridizing peptides (CHPs), compositions containing CHPs, and methods of using the CHPs and compositions described herein. Collagen hybridizing peptides (CHPs) are short, repeating Gly-XY tripeptides that have no affinity for intact collagen molecules and can specifically bind to degraded collagen. CHPs can identify damaged collagen by recognizing the structural motif of the polyproline II helix in individual alpha chains that is not present in intact collagen molecules. CHPs can then hybridize with damaged collagen to reform the collagen triple helix, forming highly stable and long-lasting bonds. The present disclosure relates to novel bioactive versions of CHPs that can be used to promote collagen repair by directly down-regulating the inflammatory response at sites of damaged collagen and to localize collagen-based anti-inflammatory sequences to mitigate additional inflammatory damage.

[0044] The present disclosure also relates to methods of administering CHP and compositions comprising CHP to down-regulate inflammatory responses and treat, reduce, prevent, or inhibit disorders, conditions, or diseases associated with excessive inflammation, or to enhance other treatments for such disorders, conditions, or diseases, including xerophthalmia (xeropthalmia), corneal abrasions, diabetic retinopathy, atopic dermatitis (e.g., eczema), psoriasis, vitiligo, bullous pemphigoid, pemphigus vulgaris, Stevens-Johnson syndrome, toxic epidermal necrolysis, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, diabetic wounds, pressure ulcers, wound healing, chemical and other burns, Crohn's disease, ulcerative colitis, ankylosing spondylitis, eosinophilic esophagitis, primary biliary cholangitis, and ascending cholangitis. Any of the conditions and therapies, including, but not limited to, celiac disease, chronic UTI, solid tumor microenvironment signaling, next-generation topical antibiotics, granulomatosis with polyangiitis, eosinophilic granulomatosis, Goodpasture's syndrome, multiple myeloma, scleroderma, CREST syndrome, IPF, dermatitis herpetiformis, keloids, Takayasu's arteritis, giant cell arteritis, and adhesive capsulitis, can be treated, improved, reduced, prevented, or inhibited by the methods described herein. Also provided are methods of increasing immune checkpoints in a subject through administration of the CHPs and compositions disclosed herein. Also provided are methods of downregulating immune-related cytokines, downregulating immune cells, and preventing immune cell differentiation.

[0045] In one aspect, the present disclosure provides a collagen hybridizing peptide (CHP) comprising a sequence represented by Formula I: Ac-S-(Gly-XY) n -Bioactive sequence-(Gly-XY) mwhere 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 another amino acid; n is a number from 1 to 20; and m is a number from 1 to 20. The bioactive sequence may be an amino acid sequence having 3 to 27 amino acid residues. The bioactive sequence may have bioactivity. The bioactive sequence may include at least one sequence selected from the group consisting of a leukocyte-associated immunoglobulin-like receptor sequence (e.g., one or both of the LAIR-1 and LAIR-2 sequences), a G6b-B recognition site sequence, an osteoclast-associated receptor (OSCAR) sequence, a glycoprotein VI (GPVI) sequence, and an FcR-γ domain sequence.

[0046] In one embodiment of the CHPs disclosed herein, X is proline. In other embodiments of the CHPs disclosed herein, X is a modified proline. In some embodiments, the modified proline is the cis form of 2S,4S-4-fluoroproline (flp).

[0047] In one embodiment of the CHPs disclosed herein, Y is hydroxyproline. In other embodiments of the CHPs disclosed herein, Y is an amino acid. In some embodiments, the amino acid is 2S,4R-4-fluoroproline (Flp) in the trans form.

[0048] In one embodiment of the CHP disclosed herein, n is 1. In another embodiment of the CHP disclosed herein, n is 2. In yet another embodiment of the CHP disclosed herein, n is 3. In yet another embodiment of the CHP disclosed herein, n is 4. In yet another embodiment of the CHP disclosed herein, n is 5. In yet another embodiment of the CHP disclosed herein, n is 6. In yet another embodiment of the CHP disclosed herein, n is 7. In yet another embodiment of the CHP disclosed herein, n is 8. In yet another embodiment of the CHP disclosed herein, n is 9. In yet another embodiment of the CHP disclosed herein, n is 10. In yet another embodiment of the CHP disclosed herein, n is 11. In yet another embodiment of the CHP disclosed herein, n is 12. In still other embodiments of the CHP disclosed herein, n is 13. In still other embodiments of the CHP disclosed herein, n is 14. In still other embodiments of the CHP disclosed herein, n is 15. In still other embodiments of the CHP disclosed herein, n is 16. In still other embodiments of the CHP disclosed herein, n is 17. In still other embodiments of the CHP disclosed herein, n is 18. In still other embodiments of the CHP disclosed herein, n is 19. In still other embodiments of the CHP disclosed herein, n is 20.

[0049] In one embodiment of the CHP disclosed herein, m is 1. In another embodiment of the CHP disclosed herein, m is 2. In yet another embodiment of the CHP disclosed herein, m is 3. In yet another embodiment of the CHP disclosed herein, m is 4. In yet another embodiment of the CHP disclosed herein, m is 5. In yet another embodiment of the CHP disclosed herein, m is 6. In yet another embodiment of the CHP disclosed herein, m is 7. In yet another embodiment of the CHP disclosed herein, m is 8. In yet another embodiment of the CHP disclosed herein, m is 9. In yet another embodiment of the CHP disclosed herein, m is 10. In yet another embodiment of the CHP disclosed herein, m is 11. In yet another embodiment of the CHP disclosed herein, m is 12. In still other embodiments of the CHP disclosed herein, m is 13. In still other embodiments of the CHP disclosed herein, m is 14. In still other embodiments of the CHP disclosed herein, m is 15. In still other embodiments of the CHP disclosed herein, m is 16. In still other embodiments of the CHP disclosed herein, m is 17. In still other embodiments of the CHP disclosed herein, m is 18. In still other embodiments of the CHP disclosed herein, m is 19. In still other embodiments of the CHP disclosed herein, m is 20.

[0050] In one embodiment of the CHP disclosed herein, the bioactive sequence has bioactivity. In another embodiment of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 3 amino acid residues. In yet another embodiment of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 4 amino acid residues. In yet another embodiment of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 5 amino acid residues. In yet another embodiment of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 6 amino acid residues. In yet another embodiment of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 7 amino acid residues. In yet another embodiment of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 8 amino acid residues. In yet another embodiment of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 9 amino acid residues. In yet another embodiment of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 10 amino acid residues. In yet another embodiment of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 11 amino acid residues. In yet another embodiment of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 12 amino acid residues. In yet another embodiment of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 13 amino acid residues. In yet another embodiment of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 14 amino acid residues. In yet another embodiment of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 15 amino acid residues. In yet another embodiment of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 16 amino acid residues. In yet another embodiment of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 17 amino acid residues. In still other embodiments of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 18 amino acid residues.In yet another embodiment of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 19 amino acid residues. In yet another embodiment of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 20 amino acid residues. In yet another embodiment of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 21 amino acid residues. In yet another embodiment of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 22 amino acid residues. In yet another embodiment of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 23 amino acid residues. In yet another embodiment of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 24 amino acid residues. In yet another embodiment of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 25 amino acid residues. In yet another embodiment of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 26 amino acid residues. In still other embodiments of the CHP disclosed herein, the bioactive sequence is an amino acid sequence having 27 amino acid residues.

[0051] In one embodiment of the CHPs disclosed herein, the bioactive sequence can include a sequence derived from a leukocyte-associated immunoglobulin-like receptor sequence (e.g., one or both of LAIR-1 and LAIR-2 sequences), a G6b-B recognition site sequence, an osteoclast-associated receptor (OSCAR) domain sequence, a glycoprotein VI (GPVI) sequence, and an FcR-γ domain sequence. In other embodiments of the CHPs disclosed herein, the bioactive sequence includes an LAIR-1 sequence. In yet other embodiments of the CHPs disclosed herein, the bioactive sequence includes an LAIR-2 sequence. In yet other embodiments of the CHPs disclosed herein, the bioactive sequence includes a G6b-B recognition site sequence. In yet other embodiments of the CHPs disclosed herein, the bioactive sequence includes an osteoclast-associated receptor (OSCAR) domain sequence. In yet other embodiments of the CHPs disclosed herein, the bioactive sequence includes a glycoprotein VI (GPVI) sequence. In still other embodiments of the CHP disclosed herein, the bioactive sequence comprises an FcR-γ domain sequence.

[0052] In embodiments of the CHP disclosed herein, the CHP comprises any one of SEQ ID NOs: 1 to 64, as shown in Table 1 below. [Table 1] JPEG2026507835000003.jpg210149 JPEG2026507835000004.jpg137149

[0053] Ac- is an N-terminal acetyl (capping) group, G- is glycine, P- is proline, O- is hydroxyproline, f- is 2S,4S-4-fluoroproline, x is any integer from 1 to 7, y is any integer from 1 to 7, and z is any integer from 1 to 5.

[0054] In some embodiments, the acetyl group (Ac) attached to the N-terminus of the CHPs disclosed herein is replaced with one or more therapeutic drugs, fluorescent dyes, and other imaging molecules or modalities. The therapeutic drug replacing the N-terminal acetyl group is not part of the bioactive amino acid sequence within the CHP. In such embodiments, the N-terminal modifications do not inhibit or interfere with the bioactive amino acid sequence of the CHP and do not prevent or inhibit the CHP from binding to damaged collagen.

[0055] In other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 1. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 2. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 3. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 4. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 5. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 6. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 7. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 8. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 9. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 10. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 11. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 12. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 13. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 14. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 15. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 16. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 17. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 18. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 19. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 20. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 21. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 22. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 23. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 24.In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 25. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 26. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 27. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 28. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 29. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 30. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 31. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 32. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 33. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 34. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 35. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 36. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 37. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 38. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 39. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 40. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 41. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 42. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 43. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 44. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 45. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 46. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 47. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 48.In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 49. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 50. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 51. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 52. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 53. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 54. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 55. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 56. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 57. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 58. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 59. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 60. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 61. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 62. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 63. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 64. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 65. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 66. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 67. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 68. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 69. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 70. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 71. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 72.In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 73. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 74. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 75. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 76. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 77. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 78. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 79. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 80. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 81. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 82. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 83. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 84. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 85. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 86. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 87. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 88. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 89. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 90. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 91. In still other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 92. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 93. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 94. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 95. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 96.In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 97. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 98. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 99. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 100. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 101. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 102. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 103. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 104.

[0056] In one embodiment of the CHP disclosed herein, the bioactive sequence comprises an LAIR-1 and / or LAIR-2 receptor sequence comprising a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-27, 56-64, 93, 94, and 98-100.

[0057] In one embodiment of the CHP disclosed herein, the biologically active sequence comprises a G6b-B recognition site sequence having at least 85% sequence identity to any one of SEQ ID NOs: 28-46, 95, and 96.

[0058] In other embodiments of the CHPs disclosed herein, the bioactive sequence comprises an FcR-γ domain sequence comprising a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 47-55 and 97.

[0059] In yet another embodiment of the CHP disclosed herein, the bioactive sequence comprises a GPVI domain sequence comprising a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 65-83 and 101-103.

[0060] In still other embodiments of the CHP disclosed herein, the bioactive sequence comprises an OSCAR domain sequence comprising a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 84-92 and 104.

[0061] In one embodiment of the CHP disclosed herein, the bioactive sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 93-104.

[0062] In other embodiments of the CHP disclosed herein, the bioactive sequence comprises SEQ ID NO: 93. In yet other embodiments of the CHP disclosed herein, the bioactive sequence comprises SEQ ID NO: 94. In yet other embodiments of the CHP disclosed herein, the bioactive sequence comprises SEQ ID NO: 95. In yet other embodiments of the CHP disclosed herein, the bioactive sequence comprises SEQ ID NO: 96. In yet other embodiments of the CHP disclosed herein, the bioactive sequence comprises SEQ ID NO: 97. In yet other embodiments of the CHP disclosed herein, the bioactive sequence comprises SEQ ID NO: 98. In yet other embodiments of the CHP disclosed herein, the bioactive sequence comprises SEQ ID NO: 99. In yet other embodiments of the CHP disclosed herein, the bioactive sequence comprises SEQ ID NO: 100. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 101. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 102. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 103. In yet other embodiments of the CHP disclosed herein, the CHP comprises SEQ ID NO: 104.

[0063] In one embodiment of the CHPs disclosed herein, S is at least one spacer molecule. In other embodiments, S includes greater than zero spacer molecules. In some embodiments, the spacer molecule is a linear PEG molecule, which can have an unlimited number of PEG repeats. In some embodiments, the spacer molecule is aminohexanoic acid. In other embodiments, the spacer molecule is an unlimited number of aminohexanoic acids linked sequentially. In other embodiments, the spacer molecule is one or more amino acids.

[0064] In the specific embodiments of CHPs disclosed herein, a first bioactive CHP is bound to two other bioactive CHPs to form a trimeric structure presenting a trimeric cell-binding site, with the first bioactive CHP being a monomeric "tail" extending from the trimeric cell-binding site. The monomeric "tail" of the first CHP is localized to damaged collagen through structural recognition of individual collagen alpha chains, while the trimeric cell-binding site comprising the first CHP bound to two other CHPs presents the bioactive site in a triple-helical configuration for easy binding to cellular receptors. In some cases, the monomeric bioactive CHP may not be accessible or "visible" to immune cells due to its 3D orientation within tissues or within damaged collagen chains that form triple helices with the monomeric bioactive CHP. This may weaken the binding affinity of the bioactive sequence with the cellular receptor, thereby reducing the efficacy of the monomeric bioactive CHP. Therefore, a first bioactive CHP that binds with two other bioactive CHPs to form a trimeric structure that displays cell receptor binding sites in a triple-helical configuration (see Figure 3) can help solve this problem. The bioactive sequence binding site becomes available regardless of the 3D orientation or the CHP binding location on damaged collagen. Thus, a high-affinity binding site becomes available for cell interaction. The monomeric "tail" portion of the first bioactive CHP recognizes exposed collagen α-chains and maintains its ability to fold with denatured collagen chains, allowing CHP to localize and bind to damaged collagen in wounds / lesions.

[0065] Additionally, in the CHP embodiments disclosed herein, designing a triple-helical cell-binding site with a "tail" of monomeric CHP can involve generating a triple-helical cell-binding site using three bioactive CHPs, then folding the three bioactive CHPs together before attaching the monomeric "tail" to the N- or C-terminus of one of the three bioactive CHP sequences in the triple-helical form via conjugation chemistry. The three bioactive CHPs in triple-helical form have the same amino acid sequence.

[0066] In one embodiment, two bioactive CHPs with identical amino acid sequences are synthesized using standard Fmoc-mediated SPPS to attach a monomeric "tail" to the C-terminus of the triple-helical cell-binding site. A single bioactive sequence (with the same amino acid sequence and cell-binding site as the two bioactive CHPs synthesized using Fmoc-mediated SPPS) is synthesized using Fmoc-mediated SPPS but with Wang resin instead of Rink-amide resin. This allows the single bioactive CHP sequence generated using Wang resin to generate a free carboxylic acid (-COOH) at the C-terminus when cleaved from the resin. The bioactive CHPs are then mixed in a 2:1 ratio (bioactive sequence without -COOH: bioactive sequence with -COOH) and allowed to fold at 4°C for 72 hours to form a triple helix. Next, a monomeric CHP "tail" without the bioactive sequence cell-binding site is separately synthesized using Rink-amide resin using standard Fmoc-mediated SPPS procedures. The N-terminus of the monomeric CHP "tail" is left uncapped and has a primary amine (NH2-) group available for conjugation. The monomeric "tail" CHP is then attached through solution-phase coupling using an activation reagent that allows for the formation of an amide bond between the -COOH of a single bioactive CHP sequence generated using Wang resin in the triple-helical cell-binding site and the free amine group (NH2-) at the N-terminus of the monomeric "tail" CHP. Once the monomeric CHP "tail" is attached, the bioactive CHP is purified through dialysis using a MWCO of 3000-5000 Da to remove any unconjugated CHP.

[0067] In yet another embodiment, to attach the "tail" of a monomeric CHP to its N-terminus, the monomeric "tail" CHP is synthesized on Wang resin, generating a reactive -COOH group at the C-terminus. To form a triple-helical cell-binding site, three bioactive CHP sequences are synthesized on Rink-amide resin, with one of the three bioactive CHPs remaining uncapped (no acetyl group) and leaving a free amine (NH2-) at the N-terminus. The bioactive CHP sequences are then mixed in a 2:1 ratio (acetyl-capped bioactive CHP:uncapped bioactive CHP) and allowed to fold at 4°C for 72 hours to form a triple helix. Finally, the monomeric "tail" CHP is mixed with the already folded triple-helical cell-binding site in solution using appropriate reagents, allowing the NH2 and COOH groups to form an amide bond through a condensation reaction. In both cases, the CHP with a carboxylic acid group must be added in excess of the amine group during the peptide bond formation process. Once the monomeric CHP "tail" is attached, the bioactive CHP is purified through dialysis using a MWCO of 3000-5000 Da to remove any unbound CHP.

[0068] In additional embodiments, a monomeric "tail" CHP is attached using copper-free click chemistry. First, three bioactive CHPs are synthesized, all of which have the same amino acid sequence. Two of the bioactive CHP sequences are identical and N-terminally capped with an acetyl group, while the other bioactive CHP has a dibenzocyclooctyne (DBCO) group attached instead of an acetyl group at the N-terminus. This is achieved by reacting commercially available DBCO (e.g., DBCO-PEG4-acid, or many other commercially available forms of the DBCO group with various linker sizes or conjugation chemistries) with the free amine (NH2-) at the N-terminus of the uncapped bioactive CHP. The bioactive CHP sequences are then mixed in a 2:1 ratio (biologically active CHP with acetyl:biologically active CHP with DBCO) and allowed to fold into a triple helix at 4°C for 72 hours. Monomeric "tail" CHP is synthesized on Wang resin without a bioactive cell-binding sequence, so that its C-terminus has a reactive carboxylic acid group (-COOH) that can react with an azide group (e.g., azidoacetic acid NHS-ester). (There are many commercially available forms of azide groups with various linker sizes or conjugation chemistries that can be used.) Once attached, the DBCO group at the N-terminus of the triple-helical cell-binding site specifically binds to the azide group at the C-terminus of the monomeric "tail" CHP. Once the monomeric CHP "tail" is attached, the bioactive CHP is purified through dialysis using a MWCO of 3000-5000 Da to remove any unbound CHP.

[0069] In yet another embodiment, to attach a monomeric "tail" CHP to the C-terminus of the triple-helical cell-binding site, the DCBO / azide protocol described above is followed, but the positions of the DBCO and azide groups are reversed (i.e., the DBCO group is attached to the N-terminus of the monomeric "tail" CHP sequence, while the azide group is attached to the C-terminus of one of the three bioactive CHP sequences in the triple-helical configuration). Once the monomeric CHP "tail" is attached, the bioactive CHP is purified via dialysis using a MWCO of 3000-5000 Da to remove any unbound CHP.

[0070] In one aspect, a composition comprising the CHP disclosed herein is provided. In one embodiment of the composition of the present disclosure, the composition further comprises a carrier. In another embodiment of the composition of the present disclosure, the CHP does not form a triple helix with another CHP.

[0071] In one embodiment of a composition comprising a CHP disclosed herein, the composition is a cosmetic and / or skin treatment composition. In another embodiment of the composition, the composition comprises a carrier. In yet another embodiment of the composition, the carrier comprises 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.

[0072] In one embodiment of the compositions comprising the CHPs disclosed herein, the composition is CHP directly bonded to a collagen matrix mesh or sponge.

[0073] In one embodiment of a composition comprising a CHP disclosed herein, the composition is a topical cream, saline solution, gel, polymer, or solution for systemic injection in a subject. In another embodiment, the composition is a topical cream. In yet another embodiment, the composition is a saline solution. In yet another embodiment, the composition is a gel. In yet another embodiment, the composition is a solution for systemic injection in a subject.

[0074] In one aspect, a method of reducing, preventing, or treating inflammation in a subject is provided, the method comprising administering to the subject a CHP disclosed herein or a composition disclosed herein. In one embodiment, the method reduces inflammation in the subject. In one embodiment, the method prevents inflammation in the subject. In another embodiment, the method treats inflammation in the subject.

[0075] In certain embodiments of the methods for reducing, preventing, or treating inflammation in a subject disclosed herein, the administering step is performed by injection. In certain embodiments of the methods for reducing, preventing, or treating inflammation in a subject disclosed herein, the administering step is performed by microdermal injection. In certain embodiments of the methods for reducing, preventing, or treating inflammation in a subject disclosed herein, the administering step is performed by topical application.

[0076] In one aspect, a method of suppressing or reducing an inflammatory response in a subject is provided, the method comprising administering to the subject a CHP disclosed herein or a composition disclosed herein. In one embodiment, the method suppresses an inflammatory response in the subject. In one embodiment, the method reduces an inflammatory response in the subject. In one embodiment of the methods disclosed herein, the administering step is performed by injection. In one embodiment of the methods disclosed herein, the administering step is performed by microdermal injection. In one embodiment of the methods disclosed herein, the administering step is performed by topical application.

[0077] In another aspect, a method for down-regulating immune-related cytokines comprises administering to a subject a CHP disclosed herein or a composition disclosed herein. In one embodiment, immune-related cytokines include, but are not limited to, Toll-like receptor (TLR) ligands, interleukins, IFN-α, TLR-9, TNF-α, IL-1β, IL-6, CCL3, IL-16, and / or IL-18.

[0078] In other embodiments, the method down-regulates one or more TLR ligands, hi yet other embodiments, the method down-regulates TLR-9.

[0079] In other embodiments, the method down-regulates IFN-α. In additional embodiments, the method down-regulates CCL3.

[0080] In other embodiments, the method downregulates interleukins. In other embodiments, the method downregulates IL-1β. In other embodiments, the method downregulates IL-6. In other embodiments, the method downregulates IL-16. In other embodiments, the method downregulates IL-18.

[0081] In yet another aspect, a method for preventing immune cell differentiation includes administering to a subject a CHP disclosed herein or a composition disclosed herein. In one embodiment, the administering step is performed by injection. In one embodiment, the administering step is performed by microdermal injection. In one embodiment, the administering step is performed by topical application.

[0082] In yet another aspect, a method for down-regulating immune cells comprises administering to a subject a CHP disclosed herein or a composition disclosed herein. The immune cells can be one or more of T cells, B cells, natural killer cells, macrophages, monocytes, and neutrophils. In one embodiment, the administering step is performed by injection. In one embodiment, the administering step is performed by microdermal injection. In one embodiment, the administering step is performed by topical application.

[0083] In yet another aspect, a method for preventing immune cell differentiation includes administering a CHP disclosed herein or a composition disclosed herein to a subject. The immune cells can be one or more of T cells, B cells, natural killer cells, macrophages, monocytes, and neutrophils. In one embodiment, the administering step is performed by injection. In one embodiment, the administering step is performed by microdermal injection. In one embodiment, the administering step is performed by topical application.

[0084] In one aspect, a method for preventing or treating dry eye syndrome in a subject includes administering a CHP disclosed herein or a composition disclosed herein to the subject. In one embodiment, administration is by local injection, intravenous injection, topical application, or physical application. In another embodiment, administration is by local injection. In yet another embodiment, administration is by intravenous injection. In yet another embodiment, administration is by topical application. In yet another embodiment, administration is by physical application.

[0085] In other embodiments of the method of preventing or treating dry eye in a subject, the CHP or composition is administered as a putty, mesh, patch, adhesive, cream, suture, gel, or eye drops. In yet other embodiments, the CHP or composition is administered as a putty. In yet other embodiments, the CHP or composition is administered as a mesh. In yet other embodiments, the CHP or composition is administered as a patch. In yet other embodiments, the CHP or composition is administered as an adhesive. In yet other embodiments, the CHP or composition is administered as a cream. In yet other embodiments, the CHP or composition is administered as one or more sutures. In yet other embodiments, the CHP or composition is administered as a gel. In yet other embodiments, the CHP or composition is administered as eye drops.

[0086] In one aspect, a method for preventing or treating atopic dermatitis in a subject includes administering a CHP disclosed herein or a composition disclosed herein to the subject. In one embodiment, administration is by local injection, intravenous injection, topical application, or physical application. In another embodiment, administration is by local injection. In yet another embodiment, administration is by intravenous injection. In yet another embodiment, administration is by topical application. In yet another embodiment, administration is by physical application.

[0087] In other embodiments of the method of preventing or treating atopic dermatitis in a subject, the CHP or composition is administered as a putty, mesh, patch, adhesive, cream, suture, gel, or eye drops. In yet other embodiments, the CHP or composition is administered as a putty. In yet other embodiments, the CHP or composition is administered as a mesh. In yet other embodiments, the CHP or composition is administered as a patch. In yet other embodiments, the CHP or composition is administered as an adhesive. In yet other embodiments, the CHP or composition is administered as a cream. In yet other embodiments, the CHP or composition is administered as one or more sutures. In yet other embodiments, the CHP or composition is administered as a gel.

[0088] In one aspect, a method for preventing or treating osteoarthritis in a subject includes administering a CHP disclosed herein or a composition disclosed herein to the subject. In one embodiment, administration is by local injection, intravenous injection, topical application, or physical application. In another embodiment, administration is by local injection. In yet another embodiment, administration is by intravenous injection. In yet another embodiment, administration is by topical application. In yet another embodiment, administration is by physical application.

[0089] In other embodiments of the method of preventing or treating osteoarthritis in a subject, the CHP or composition is administered as a putty, mesh, patch, adhesive, cream, suture, gel, or eye drops. In yet other embodiments, the CHP or composition is administered as a putty. In yet other embodiments, the CHP or composition is administered as a mesh. In yet other embodiments, the CHP or composition is administered as a patch. In yet other embodiments, the CHP or composition is administered as an adhesive. In yet other embodiments, the CHP or composition is administered as a cream. In yet other embodiments, the CHP or composition is administered as one or more sutures. In yet other embodiments, the CHP or composition is administered as a gel.

[0090] In one aspect, a method for preventing or treating rheumatoid arthritis in a subject includes administering a CHP disclosed herein or a composition disclosed herein to the subject. In one embodiment, administration is by local injection, intravenous injection, topical application, or physical application. In another embodiment, administration is by local injection. In yet another embodiment, administration is by intravenous injection. In yet another embodiment, administration is by topical application. In yet another embodiment, administration is by physical application.

[0091] In other embodiments of the method of preventing or treating rheumatoid arthritis in a subject, the CHP or composition is administered as a putty, mesh, patch, adhesive, cream, suture, gel, or eye drops. In yet other embodiments, the CHP or composition is administered as a putty. In yet other embodiments, the CHP or composition is administered as a mesh. In yet other embodiments, the CHP or composition is administered as a patch. In yet other embodiments, the CHP or composition is administered as an adhesive. In yet other embodiments, the CHP or composition is administered as a cream. In yet other embodiments, the CHP or composition is administered as one or more sutures. In yet other embodiments, the CHP or composition is administered as a gel.

[0092] In one aspect, a method for preventing or treating a diabetic condition in a subject includes administering a CHP disclosed herein or a composition disclosed herein to the subject. In one embodiment, administration is by local injection, intravenous injection, topical application, or physical application. In another embodiment, administration is by local injection. In yet another embodiment, administration is by intravenous injection. In yet another embodiment, administration is by topical application. In yet another embodiment, administration is by physical application.

[0093] In other embodiments of the method of preventing or treating a diabetic pathology in a subject, the CHP or composition is administered as a putty, mesh, patch, adhesive, cream, suture, gel, or eye drops. In yet other embodiments, the CHP or composition is administered as a putty. In yet other embodiments, the CHP or composition is administered as a mesh. In yet other embodiments, the CHP or composition is administered as a patch. In yet other embodiments, the CHP or composition is administered as an adhesive. In yet other embodiments, the CHP or composition is administered as a cream. In yet other embodiments, the CHP or composition is administered as one or more sutures. In yet other embodiments, the CHP or composition is administered as a gel.

[0094] In one aspect, a method for preventing or treating psoriasis in a subject includes administering a CHP disclosed herein or a composition disclosed herein to the subject. In one embodiment, administration is by local injection, intravenous injection, topical application, or physical application. In another embodiment, administration is by local injection. In yet another embodiment, administration is by intravenous injection. In yet another embodiment, administration is by topical application. In yet another embodiment, administration is by physical application.

[0095] In other embodiments of the method of preventing or treating psoriasis in a subject, the CHP or composition is administered as a putty, mesh, patch, adhesive, cream, suture, gel, or eye drops. In yet other embodiments, the CHP or composition is administered as a putty. In yet other embodiments, the CHP or composition is administered as a mesh. In yet other embodiments, the CHP or composition is administered as a patch. In yet other embodiments, the CHP or composition is administered as an adhesive. In yet other embodiments, the CHP or composition is administered as a cream. In yet other embodiments, the CHP or composition is administered as one or more sutures. In yet other embodiments, the CHP or composition is administered as a gel.

[0096] In one aspect, a method for increasing an immune checkpoint in a subject comprises administering a CHP disclosed herein or a composition disclosed herein to the subject. In one embodiment, administration is by local injection, intravenous injection, topical application, or physical application. In another embodiment, administration is by local injection. In yet another embodiment, administration is by intravenous injection. In yet another embodiment, administration is by topical application. In yet another embodiment, administration is by physical application.

[0097] In other embodiments of the method of increasing immune checkpoints in a subject, the CHP or composition is administered as a putty, mesh, patch, adhesive, cream, suture, gel, or eye drops. In yet other embodiments, the CHP or composition is administered as a putty. In yet other embodiments, the CHP or composition is administered as a mesh. In yet other embodiments, the CHP or composition is administered as a patch. In yet other embodiments, the CHP or composition is administered as an adhesive. In yet other embodiments, the CHP or composition is administered as a cream. In yet other embodiments, the CHP or composition is administered as one or more sutures. In yet other embodiments, the CHP or composition is administered as a gel. In yet other embodiments, the CHP or composition is administered as eye drops.

[0098] Each description and embodiment disclosed in this disclosure may also be applied to other descriptions and embodiments. In other words, all combinations of the various elements disclosed in this disclosure fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific description below. Furthermore, those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents to the specific aspects of this application described herein. Furthermore, such equivalents should be construed as falling within the scope of this application. [Example]

[0099] Hereinafter, exemplary embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. To facilitate understanding of the present disclosure, the same reference numerals throughout the description of the drawings refer to the same components, and the description of the same components will not be repeated.

[0100] Synthesis and in vitro characterization of anti-inflammatory bioactive collagen hybridizing peptides (CHPs) All peptide sequences, including CHP, were synthesized on TentaGel RRAM resin (Peptides International, RTS-9995-PI) using standard Fmoc chemistry on a CS-136X automated synthesizer (CSBio) with Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Hyp(tBu)-OH (EMD Millipore), and Fmoc-cis-4-fluoro-Pro-OH (Chem-Impex, 14494) as the major amino acid residues. Other residues included Fmoc-Lys(boc)-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asp-OH, and Fmoc-Glu(OtBu)-OH. Other commercially available fmoc-, hoc-, or Z-protected amino acids can also be used in the bioactive sequence, and unnatural amino acids can be added to improve the overall peptide's favorable properties, such as increased binding (aza-glycine), hydrophilicity, immune evasion, and stability. The coupling reagents used were HATU (Aapptec) and 1-hydroxy-7-azabenzotriazole (HOAt, Aapptee). To remove the Fmoc protecting group, piperidine (20% by volume) dissolved in N-methyl-2-pyrrolidone was used. Each coupling reaction involved 5 molar equivalents of amino acid residue (0.119 M) and coupling reagent (0.114 M) dissolved in dimethylformamide. The resin was treated with trifluoroacetic acid (TFA) / triisopropylsilane (TIS) / water (95:2.5:2.5) for 2 hours, and the crude peptide was precipitated by adding an excess of cold ether to the TFA solution. Peptides are purified by reversed-phase high-performance liquid chromatography (RP-HPLC) on an Agilent Zorbax SB C-18 column using a linear gradient mixture of water (0.1% TFA) and acetonitrile (0.1% TFA) (5–35% acetonitrile, 30 min) as the mobile phase. The molecular weight of the purified peptides is confirmed using ESI-TOF (Electrospray Ionization Time of Flight) MS.The purity of the peptides is determined by HPLC.

[0101] To confirm whether all of the synthesized CHPs maintain their ability to bind to denatured collagen, we will evaluate the triple helix structure of CHPs using CD spectroscopy and determine the melting temperature (Tm) for dissociation. We will also confirm whether CHPs specifically bind to denatured collagen through gelatin binding assays and tissue histology.

[0102] In vitro evaluation of the effects of LAIR-1 bioactive CHP on T cell activation and suppression To assess the effects of LAIR-1 anti-inflammatory bioactive CHPs, exemplary CHPs are tested (see Figure 5 for a list of CHPs used in in vitro studies).

[0103] Plate assay for LAIR-1 binding First, we performed an LAIR-1 plate-binding assay to detect LAIR-1 binding to bioactive CHP (both monomeric and dimeric forms) (see Figure 6 for the experimental setup). This is a plate assay developed to allow bioactive CHP to bind to gelatin to detect LAIR-1 binding by detecting the bound LAIR-1 protein using a secondary antibody.

[0104] A 96-well black, opaque Nunc Ammo flat-bottom plate was coated with a neutral pH (pH 7) protein solution (porcine skin gelatin or human placenta intact collagen) dissolved in PBS at 100 μg / mL per well. Negative control (blank) wells contained no gelatin or collagen protein solution, leaving an open surface. Both the negative control (gelatin) and bioactive CHP test wells contained gelatin. Positive control wells contained intact human collagen. The plate was then covered with packing tape to reduce evaporation and spillover and incubated on a shaker at room temperature for 2 hours.

[0105] After incubation, each well of the plate was washed consecutively with 200 μL PBS wash solution for approximately 5 minutes, a total of four times, and the final wash solution was discarded.

[0106] The wells were then fixed with 150 μL of 100% ice-cold methanol (stored at -20°C) and incubated at -20°C for 30 minutes. After incubation, each well of the plate was washed consecutively with 200 μL PBS washes for approximately 5 minutes, for a total of four times. The final wash was discarded.

[0107] The plate was then incubated with 1 M TRIS-HCl (pH 7.4) at room temperature for 30 minutes on a shaker (200 μL per well). This was done to block any remaining reactive groups on the surface of the amino capture plate. Again, each well of the plate was washed consecutively with 200 μL PBS washes for approximately 5 minutes, for a total of four times. The final wash was discarded.

[0108] Each well of the plate was blocked with 20 μL of 1% BSA dissolved in 1X PBS pH 7.4 for 30 minutes at room temperature. After blocking, each well of the plate was washed with 200 μL of PBS wash solution for approximately 5 minutes four times in succession. The final wash solution was discarded.

[0109] Bioactive CHP peptide solution was dissolved in 1x PBS at a concentration of 500 μM and heated at 80°C for 10 minutes to release the OTP in the monomeric form required for binding to denatured collagen. The peptide solution was then rapidly cooled in ice water (room temperature) until cold to the touch. Then, 50 μL of CHP dissolved in 1x PBS was added to each test well of the plate (no CHP was added to the negative control (blank) wells, negative control (gelatin) wells, and positive control (collagen) wells).

[0110] The plate was covered with tape and incubated overnight to allow CHP binding at the specified temperature. The final CHP solution was removed, and each well was washed three times consecutively with 200 μL of cold PBS for 10 min. The final wash was discarded.

[0111] Next, recombinant human LAIR-1 Fc chimeric protein (final 5 μg / mL) in 1% BSA dissolved in PBS was added to each well (100 μL per well). The plate was incubated at room temperature (FIG. 7) or 37°C (FIG. 8) for 1 hour, or at 4°C for 2 hours (FIGS. 9 and 11) to allow binding of the LAIR-1 protein.

[0112] After removing the LAIR solution from the wells, the wells were washed successively with 200 μL of cold PBS wash solution for a total of four 10-minute washes, with the final wash solution discarded.

[0113] Anti-human IgG Alexa Fluor 488 antibody (5 μg / mL final) in 1% BSA in PBS was added to each well. Plates were incubated at room temperature or 37°C for 1 hour, or incubated at 4°C for 2 hours covered with foil. After removing the antibody solution from the wells, the wells were washed consecutively with 200 μL of cold PBS wash for a total of four times for 10 minutes. The final wash was discarded.

[0114] All liquid was removed from each well and the wells were allowed to dry. After drying, each well was imaged for fluorescent signal using a Spectra Max iD3 plate reader at an excitation wavelength of 480 nm and a detection wavelength of 520 nm. A total of 32 evenly spaced reads per well were collected and averaged.

[0115] Figure 7 shows the results of plate binding assay of a high-melting-temperature LAIR-1 monomer designated LAIR-1 HTm CHP2 (SEQ ID NO: 2, where x = 4 and y = 4) against bioactive CHP at room temperature. As shown in the graph, the LAIR-1 HTm CHP2 monomer increased LAIR-1 binding by 3.5% (expressed as relative fluorescence units (RFU)) compared to the negative (gelatin) control group upon incubation at room temperature (P value = 0.0446 from a one-tailed type 2 T-test). Three replicates per group were used.

[0116] Figure 8 shows the results of a plate binding assay of a high-melting-temperature LAIR-1 monomer designated LAIR-1 HTm CHP2 (SEQ ID NO: 2, where x = 4 and y = 4) against bioactive CHP at 37°C incubation (approximately human body temperature). As shown in the graph, the LAIR-1 HTm CHP2 monomer increased LAIR-1 binding by 8.5% (expressed as RFU) compared to the negative (gelatin) control group upon incubation at 37°C (P value = 0.0342 from a one-tailed type 2 t-test). Three replicates per group were used. Without being bound by theory, the difference in binding demonstrated by LAIR-1 HTm CHP2 between the room temperature and 37°C incubation assays is likely due to an increased amount of available favorable attachment sites in the gelatin test wells and reduced CHP self-trimerization at 37°C (approximately human body temperature).

[0117] Figure 9 shows the results of a plate binding assay of a LAIR-1 monomer designated LAIR-1 LTm CHP2 (SEQ ID NO: 2, where x = 2 and y = 2), which has a low melting temperature, against bioactive CHP at 4°C incubation. As shown in the graph, the LAIR-1 LTm CHP2 monomer increased LAIR-1 binding by 6.5% (expressed as RFU) compared to the negative (gelatin) control group when incubated at 4°C (P value = 0.0303 from a one-tailed type 2 t-test). Three replicates were used per group. The incubation temperature was chosen due to the low melting temperature of LAIR-1 LTm CHP2.

[0118] FIG. 10 shows the experimental design of the LAIR-1 plate binding assay to detect LAIR-1 binding to bioactive CHP of LAIR-1 dimers.

[0119] FIG. 11 shows the results of a plate binding assay of an LAIR-1 dimer designated as the LAIR-1 LTm CHP2 dimer with a low melting temperature according to an embodiment disclosed herein (two copies of SEQ ID NO: 2 (where x=2 and y=2), each copy attached to a linker, SEQ ID NO: 105 (where z=1)), incubated at 4°C to bioactive CHP. As shown in the graph, the LAIR-1 LTm CHP2 dimer increased LAIR-1 binding by 17.6% (expressed as RFU) compared to the negative (gelatin) control group when incubated at 4°C (P value of 0.0147 from a one-tailed type 2 t-test). Three replicates were used per group. The incubation temperature was chosen due to the low melting temperature of the LAIR-1 LTm CHP2 dimer.

[0120] In vitro immune cell activation analysis To evaluate the effects of anti-inflammatory bioactive CHPs (e.g., CHPs containing the LAIR-1 sequence), several in vitro parameters are evaluated. First, immune cells (e.g., Jurkat T cells, CD4+ cells, CD8+ cells, naïve T cell lines, macrophages, etc.) are cultured in suspension using appropriate cell culture medium and additives. Once the cells are grown, they are placed in tissue culture well plates with or without gelatin (denatured collagen). The thickness of the cross-linked gelatin varies depending on the amount of gelatin solution added, ranging from 50 to 250 μL per well. Because gelatin provides binding sites for CHP binding, well plates with gelatin serve as experimental groups, while blank wells (containing PBS + medium or medium alone) serve as controls.

[0121] Anti-inflammatory bioactive CHP was then added to experimental and control wells and replenished daily with each cell culture medium change. ELISAs were performed on supernatants collected from each well (experimental and control) to assess IL-1, IL-6, IL-7, IL-8, TNF-α, IFN-γ, IL-17, IL-22, and IL-27. Wells treated with gelatin / collagen and CHP showed reduced levels of pro-inflammatory cytokines compared to control wells.

[0122] To evaluate the effect of the anti-inflammatory bioactive LAIR-1 CHP on T cells, a specific in vitro activation assay was performed. A schematic diagram of this T cell activation / suppression assay is shown in Figure 12. The experimental conditions for the T cell assay using Jukat T cells are shown in Figure 13. As shown, Jukat T cells were incubated with activating antibodies against CD3 and CD28 receptors to initiate activation. Negative control T cell groups did not receive anti-CD3 or anti-CD28 antibodies. T cell activation was then assessed by ELISA, and IL-2 secretion was assessed in the culture supernatant. Activated T cells express high levels of IL-2.

[0123] Secreted IL-2 was measured for the following conditions: a negative control group of unactivated T cells; a positive control group of T cells to which anti-CD3 and anti-CD28 antibodies were added; a positive control group of T cells to which stimulating antibodies were added in the presence of soluble gelatin; and a CHP test condition to which stimulating antibodies were added in the presence of LAIR1-CHP.

[0124] For the T cell activation / suppression experimental protocol, three monomeric CHPs (LAIR-1 HTm CHP [SEQ ID NO: 87, where x=4 and y=4], LAIR-1 HTm CHP2 [SEQ ID NO: 2, where x=4 and y=4], and LAIR-1 HTm CHP3 [SEQ ID NO: 106]) were tested for T cell activation / suppression. First, CHPs were incubated with gelatin under test conditions at 31°C for at least 24 hours. The final concentration of gelatin in the incubation medium was 0.5 mg / mL.

[0125] Next, activated wells (positive control and CHP test conditions) of Nunc cell culture-treated 6-well plates were coated with anti-CD3 antibody (anti-human CD3 (OKT3)) at a concentration of 3 μg / mL per 2 mL. Negative control wells were left uncoated.

[0126] The plates were then incubated for 2 hours at 37° C. and 5% CO 2. The anti-CD3 mixture was then aspirated from each coated well (without rinsing).

[0127] 900,000 Jurkat T cells (ATCC E6-1) were added to each well of each plate in a total volume of 3 mL of medium containing the respective therapeutic agent and a CD28 antibody (anti-human CD28 monoclonal antibody (CD28.2)) at 3 μg / mL. The complete medium contains RPMI 1640 medium, fetal bovine serum, L-glutamine, and penicillin-streptomycin.

[0128] The resulting 6-well plate contains: 3 μg / mL anti-CD28 (anti-human CD28 monoclonal antibody) for all wells except the negative control group 5 μM CyA (cyclosporine) as a known therapeutic control when both Anti-CD28 and Anti-CD3 are present in one well For wells containing T-cells where gelatin is present, 0.5 mg / mL gelatin · 0.5 mg / mL gelatin with CHP at a final concentration of 500 μM in LAIR-1 HTm CHP1 and LAIR-1 HTm CHP2 test wells, and 100 μM in LAIR-1 HTm CHP3 test wells.

[0129] The 6-well plates containing T cells were then incubated at 37°C for 4 days to activate the T cells. On day 3, 500 μL of cell culture medium was removed from each well and tested using an IL-2 ELISA kit. The 6-well plates were further incubated at 37°C for 1 day to further activate the T cells, for a total of 4 days of incubation. 500 μL of cell culture medium was then removed from each well and tested using an IL-2 ELISA kit according to the kit manufacturer's instructions.

[0130] Figure 14 shows the results of an in vitro T cell activation assay performed at 37°C using ELISA to detect IL-2 levels 72 hours after stimulation. Because CHP requires damaged collagen for localization and binding, gelatin (damaged collagen) served as a second positive control. Cyclosporine A (CyA) inhibited T cell activation by suppressing the production of interleukins, including IL-2. Compared to the gelatin positive control, LAIR-1 HTm CHP1 reduced IL-2 production by 68.2% (P = .0035), and LAIR1 HTm CHP2 reduced IL-2 production by 53.8% (P = .031).

[0131] Figure 15 shows the results of an in vitro T cell activation assay performed at 37°C, using ELISA to detect IL-2 levels 96 hours after stimulation. T cell activation was confirmed by high IL-2 levels 96 hours after stimulation. Because CHP requires damaged collagen for localization and binding, gelatin (damaged collagen) served as a second positive control. Cyclosporine A (CyA) inhibited T cell activation by suppressing the production of interleukins, including IL-2. Compared to the gelatin positive control, LAIR1 HTm CHP1 reduced IL-2 production by 97.3% (P = .0022), and LAIR-1 HTm CHP2 reduced IL-2 production by 58.0% (P = .004). LAIR-1 HTm CHP3 had no effect on IL-2 production.

[0132] In vitro characterization in atopic dermatitis, psoriasis, and xerophthalmia models In addition, in vitro characterization will be performed to confirm the results of the in vitro characterization of anti-inflammatory bioactive CHP in various immune cell lines (e.g., Jurkat T cells, macrophages) cultured in suspension. Psoriasis / atopic dermatitis model

[0133] GenoSkin's psoriasis model, "Inflammaskin," is used as an in vitro inflammatory model of atopic dermatitis. Because the mechanisms of action of atopic dermatitis / eczema and psoriasis are very similar, and both conditions exhibit nearly identical symptoms of red, itchy, scaly, and inflamed skin lesions, such a model provides a suitable method for testing anti-inflammatory bioactive CHPs and their modulation of inflammatory pathways. This model is used to evaluate the overall therapeutic response and preventative effects of CHPs.

[0134] For treatment evaluation, the Inflammaskin model is left untreated for 3-4 days, followed by topical application of CHP for an additional 3-4 days. For the preventative model, CHP is administered topically on the skin model, as this model receives an activation cocktail. The HypoSkin model, lacking T cell activation, serves as a healthy control group.

[0135] The evaluation of the skin model was based on histopathological analysis at the end of the study (day 7), and a quantitative analysis of the skin structural integrity was performed by assigning a score according to defects detected in H&E stained sections of the model as follows: 0: Uncultured skin with no change 1: Early signs of keratinocyte separation (spongiosis) 2: Progression of keratinocyte separation and early signs of keratinocyte volume increase (hypertrophy) / dermal disorganization 3: Progression of pyknosis / dermal breakdown 4: Cytoplasmic vacuolization 5: Separation of basal layer / spinous layer from dermis

[0136] Additionally, the supernatants will be collected and the levels of inflammatory cytokines will be assessed by ELISA, similar to the protocol for in vitro cell culture models. Dry eye syndrome model

[0137] For dry eye syndrome (DED), the ex vivo eye irritation test (EVEIT) is used. This model measures the ability of substances to induce corneal damage and assesses reversibility. Rabbit corneas are extracted from rabbit eyes and the cells are maintained for 72 hours. Damage to the epithelial barrier function is detected using sodium fluorescein staining.

[0138] The therapeutic effects of anti-inflammatory CHP will be examined by comparing corneas treated with bioactive CHP with those treated with PBS (PBS sham model). Bioactive CHP or PBS sham will be applied to the corneas four to six times daily. Corneal barrier integrity will then be assessed multiple times throughout the experimental period using optical coherence tomography (OCT) to noninvasively evaluate epithelial and stromal damage and recovery. After the experiment is completed, the corneas will be fixed and sectioned, and histopathological examination of corneal recovery will be performed using H&E and fluorescently labeled CHP to detect collagen during remodeling. These sections will then be assessed by a pathologist for accuracy and consistency. Cells will also be harvested from one corneal section per group and subjected to rtPCR for evaluation of inflammatory markers. Diseased models will be compared with the PBS sham model.

[0139] In vivo characterization in atopic dermatitis and xerophthalmia models In vivo xerophthalmia model Because there is no perfect model that effectively mimics all xerophthalmia (DED) conditions, various in vivo models can be utilized to thoroughly understand the anti-inflammatory effects of bioactive CHP as a DED treatment. We used the airflow and scopolamine model. This model mimics the dry environmental stress imposed on the ocular surface of the eye, which is often considered one of the initiating factors of xerophthalmia (DED). In this model, C57BL / 6 female mice are exposed to an evaporative environment with constant low humidity flow on the face. The longer the exposure time, the longer the period of decreased tear secretion. In addition to environmentally induced ocular surface dryness, this model also uses subcutaneous administration of scopolamine to inhibit muscarinic receptors, which reduces tear secretion. This model provides a good imitation of anhydrous xerophthalmia (DED), which is characterized by decreased tear production, tear film stability, ocular surface epithelial cell death, and increased cytokine levels.

[0140] Two studies were conducted to test the therapeutic and preventive capabilities of anti-inflammatory bioactive CHP using this mouse model. Three groups of mice were used: a group treated with each bioactive CHP, a sham (PBS) control group, and a healthy control group. For the therapeutic evaluation study, experimental mice were housed in a controlled-environment chamber (CEC) for 14 days, where the relative humidity was maintained at less than 1.5%, the airflow was 10 L / min, and the temperature was kept constant between 21 and 23°C. DED symptoms were induced by subcutaneous injection of 0.1 mL of 5 mg / mL scopolamine hydrobromide into the dorsum of the mice three times daily (9:00 AM, 1:00 PM, and 5:00 PM). Once DED symptoms were established, a 5 nmol dose of bioactive CHP (0.1 mL) was administered daily via intraorbital injection. However, other methods that could be tested include eye drop administration and systemic administration via tail vein injection.

[0141] The mice were then returned to their normal habitat and their response to CHP was assessed by how quickly they returned to normal after discontinuing the scopolamine injections (measured using corneal fluorescein staining (CFS)), which is used to assess corneal epithelial damage caused by DED. A 1 μL dose of 2.5% fluorescein was applied to the lateral conjunctival sac of the mice, and after 3 minutes, the corneas were examined under cobalt blue light using a slit lamp biomicroscope (Topcon SL-D7; Topcon Corp., Tokyo, Japan). Punctate staining was scored using the National Eye Institute's grading system in a masked manner, with a score of 0 to 3 assigned to each of five corneal regions (central, superior, inferior, nasal, and temporal).

[0142] In addition, tears are collected from each mouse to assess the inflammatory response by measuring the levels of IL-1, IL-6, IL-7, IL-8, TNF-α, IFN-γ, IL-17, IL-22, and IL-27 via ELISA.

[0143] In the preventative study, mice will undergo the same procedures as above with daily administration of CHP. The same evaluation variables and inflammatory assessments will also be performed. The same endpoints and inflammatory assessments will also be performed. In vivo atopic dermatitis model

[0144] For an in vivo atopic dermatitis (eczema) model, an oxazolone-induced model was used in SKH1 hairless mice. This model allows for disease response monitoring through ear measurements (antemortem), histopathological analysis, and cytokine analysis using ELISA. The anti-inflammatory bioactive CHP was tested in comparison with a control peptide using two CHP administration routes. Four groups of mice were used: a group administered bioactive CHP using each of the two administration routes, a PBS control group, and a healthy control group without atopic dermatitis.

[0145] One method of administering anti-inflammatory bioactive CHP is by systemic tail vein injection of a single bolus of 1–5 nmol every 3 days. Another method evaluated is topical application of CHP to the site of eczema lesions. These various administration methods are used to evaluate the therapeutic effect of CHP on the inflammatory response and to determine which route of administration is more effective. Evaluation is performed by measuring the size of eczema lesions in the ears of nude mice. During treatment with CHP, the size of the lesions decreases.

[0146] At the end of the experiment, mice are sacrificed and skin histology is performed. Unaffected areas are compared with lesioned areas from the same mice, as well as between experimental groups. Skin is stained with H&E, Masson's Trichrome, and biotin-labeled CHP. A veterinary pathologist evaluates sections to assess the degree of inflammation and the beneficial effects of the anti-inflammatory bioactive CHP. Blood samples are also collected and tested for inflammatory biomarkers via ELISA: IL-1, IL-6, IL-7, IL-8, TNF-α, IFN-γ, IL-17, IL-22, and IL-27.

[0147] Unless expressly indicated otherwise, all numbers used in the specification and claims expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and the like, should be understood to be modified in all instances by the term "about." Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained through the present disclosure. To the minimum, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0148] 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, however, all numerical values ​​inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0149] Grouping of alternative elements or embodiments of the present disclosure disclosed herein should not be construed as limiting. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements described 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 such inclusion or deletion occurs, the specification includes the modified group and is deemed to satisfy the written description of all Markush groups used in the appended claims.

[0150] Specific embodiments of the present disclosure have been described herein, including the best mode known to the inventors for carrying out the disclosure. Of course, variations on each of these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors expect that those skilled in the art will apply appropriate variations, and intend the present disclosure to be practiced in ways other than those specifically described herein. Accordingly, the present disclosure includes all modifications and equivalents of the subject matter recited in the appended claims to the extent permitted by applicable law. Furthermore, all possible combinations of the elements described above are included in the present disclosure unless otherwise expressly stated herein or clearly contradicted by context.

[0151] Particular embodiments disclosed herein may be further limited in the claims using "consisting of" or "consisting essentially of." When used in a claim as filed or added by amendment, the transitional term "consisting of" excludes all elements, steps, or ingredients not explicitly stated in the claim. The transitional term "consisting essentially of" limits the scope of the claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics. Embodiments of the present disclosure so claimed may be embodied as implicitly or explicitly described herein.

[0152] It should be understood that each embodiment of the present disclosure disclosed herein exemplifies the principles of the present disclosure. Other variations that may be used are within the scope of the present disclosure. Thus, by way of example and not limitation, each alternative configuration of the present disclosure may be utilized in accordance with the teachings described herein. Thus, the present disclosure is not limited to that precisely as shown and described.

[0153] While the present disclosure has been described and illustrated with reference to various specific materials, procedures, and examples, it should be understood that the disclosure is not limited to the particular combination of materials and procedures selected for that purpose. Various variations of such details may be implied as will be recognized by those skilled in the art. The specification and examples should be considered exemplary only, with the true scope and spirit of the present disclosure being indicated by the following claims. All references, patents, and patent applications mentioned in this application are incorporated herein by reference in their entirety.

Claims

1. A collagen hybridizing peptide (CHP) having the sequence represented by Formula I below: Ac-S-(Gly-X-Y) ｎ -Bioactive sequence- (Gly-X-Y) ｍ (Formula I) In the formula, 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 other amino acid, n is a number from 1 to 20, and m is a number from 1 to 20; The bioactive sequence is an amino acid sequence of 3 to 27 amino acid residues having bioactivity, and includes at least one sequence selected from the group consisting of a leukocyte-associated immunoglobulin-like receptor sequence (LAIR-1 and / or LAIR-2), a G6b-B recognition site sequence, an OSCAR domain sequence, a GPVI sequence, and an FcR-γ domain sequence.

2. 2. The CHP of claim 1, wherein the at least one bioactive sequence comprises a LAIR-1 receptor sequence.

3. 2. The CHP of claim 1, wherein the at least one bioactive sequence comprises a LAIR-2 receptor sequence.

4. The CHP according to any one of claims 1 to 3, wherein the at least one biologically active sequence comprises a G6b-B recognition site sequence.

5. The CHP according to any one of claims 1 to 4, wherein the at least one biologically active sequence comprises an FcR-γ domain sequence.

6. The CHP according to any one of claims 1 to 5, wherein the at least one biologically active sequence comprises an OSCAR domain sequence.

7. The CHP of any one of claims 1 to 6, wherein the at least one bioactive sequence comprises a GPVI sequence.

8. 8. The CHP of any one of claims 1 to 7, wherein the at least one bioactive sequence comprises a LAIR-1 and / or LAIR-2 receptor sequence comprising a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-27, 56-64, 93, 94, and 98-100.

9. The CHP according to any one of claims 1 to 8, wherein the at least one biologically active sequence comprises a G6b-B recognition site sequence comprising a sequence having at least 85% sequence identity with any one of SEQ ID NOs: 28 to 46, 95 and 96.

10. The CHP of any one of claims 1 to 9, wherein the at least one biologically active sequence comprises an FcR-γ domain sequence comprising a sequence having at least 85% sequence identity with any one of SEQ ID NOs: 47 to 55 and 97.

11. The CHP of any one of claims 1 to 10, wherein the at least one biologically active sequence comprises a GPVI domain sequence comprising a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 65 to 83 and 101 to 103.

12. The CHP of any one of claims 1 to 11, wherein the at least one biologically active sequence comprises an OSCAR domain sequence comprising a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 84-92 and 104.

13. The CHP of any one of claims 1 to 12, wherein the CHP comprises a sequence selected from the group consisting of SEQ ID NOs: 1 to 104.

14. The CHP according to any one of claims 1 to 13, wherein S is more than 0.

15. The CHP described in any one of claims 1 to 14, wherein the CHP is a first CHP that is bound to two other bioactive collagen hybridizing peptides to form a trimeric structure, the first CHP having a monomer portion extending from the trimeric structure, and the monomer portion configured to bind to a collagen alpha chain.

16. The CHP of any one of claims 1 to 14, wherein the CHP is a first CHP that is conjugated to two other bioactive collagen-hybridizing peptides to form a trimeric structure, and a monomeric "tail" CHP is attached to the N-terminus or C-terminus of the first CHP of the trimeric structure using copper-free click chemistry or a condensation reaction.

17. A composition comprising the CHP of any one of claims 1 to 16.

18. 18. The composition of claim 17, wherein each individual CHP does not form a triple helix with other CHPs.

19. 19. The composition of claim 17 or 18, further comprising a carrier.

20. 20. The composition of any one of claims 17 to 19, wherein the composition is a cosmetic composition and the carrier comprises 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.

21. The composition of any one of claims 17 to 20, wherein the composition is a topical cream, saline solution, gel, polymer, or solution for systemic injection in a subject.

22. The composition of any one of claims 17 to 21, wherein the CHP is directly bound to the collagen matrix.

23. 23. A method of reducing, preventing or treating inflammation in a subject, comprising administering a CHP according to any one of claims 1 to 16, or a composition according to any one of claims 17 to 22.

24. 24. The method of claim 23, wherein the administration is by injection.

25. 24. The method of claim 23, wherein the administration is by microdermal injection.

26. 24. The method of claim 23, wherein the administration is by topical application.

27. 23. A method of suppressing or reducing an inflammatory response in a subject, comprising administering a CHP according to any one of claims 1 to 16, or a composition according to any one of claims 17 to 22.

28. 28. The method of claim 27, wherein the administration is by injection.

29. 28. The method of claim 27, wherein the administration is by microdermal injection.

30. 28. The method of claim 27, wherein the administration is by topical application.

31. 23. A method of down-regulating immune related cytokines, including but not limited to TLR ligands, interleukins, IFN-α, TLR-9, TNF-α, IL-1β, IL-6, CCL3, IL-16, IL-18 and / or TNF-α activity in a subject, comprising administering a CHP of any one of claims 1-16 or a composition of any one of claims 17-22.

32. A method for preventing immune cell differentiation, comprising administering a CHP according to any one of claims 1 to 16, or a composition according to any one of claims 17 to 22.

33. 23. A method for down-regulating T cells, B cells, natural killer cells, macrophages, monocytes and neutrophils, comprising administering a CHP according to any one of claims 1 to 16 or a composition according to any one of claims 17 to 22.

34. 23. A method for preventing differentiation of T cells, B cells, natural killer cells, macrophages, monocytes and neutrophils, comprising administering a CHP according to any one of claims 1 to 16 or a composition according to any one of claims 17 to 22.

35. The method of any one of claims 31 to 34, wherein the administration is by injection.

36. The method of any one of claims 31 to 34, wherein the administration is by microdermal injection.

37. The method of any one of claims 31 to 34, wherein the administration is by topical application.

38. 23. A method of preventing or treating xerophthalmia in a subject, comprising administering a CHP according to any one of claims 1 to 16, or a composition according to any one of claims 17 to 22.

39. 39. The method of claim 38, wherein the administration is by local injection, intravenous injection, topical application, or physical application.

40. 39. The method of claim 38, wherein the CHP or composition is administered as a putty, mesh, patch, adhesive, cream, suture, gel, or eye drops.

41. 23. A method for preventing or treating atopic dermatitis in a subject, comprising administering a CHP according to any one of claims 1 to 16, or a composition according to any one of claims 17 to 22.

42. 42. The method of claim 41, wherein the administration is by local injection, intravenous injection, topical application, or physical application.

43. 42. The method of claim 41, wherein the CHP or composition is administered as a putty, mesh, patch, adhesive, cream, suture, gel, or eye drops.

44. 23. A method of preventing or treating osteoarthritis in a subject, comprising administering a CHP according to any one of claims 1 to 16, or a composition according to any one of claims 17 to 22.

45. 45. The method of claim 44, wherein the administration is by local injection, intravenous injection, topical application, or physical application.

46. 45. The method of claim 44, wherein the CHP or composition is administered as a putty, mesh, patch, adhesive, cream, suture, gel, or eye drops.

47. 23. A method of preventing or treating rheumatoid arthritis in a subject, comprising administering a CHP according to any one of claims 1 to 16, or a composition according to any one of claims 17 to 22.

48. 48. The method of claim 47, wherein the administration is by local injection, intravenous injection, topical application, or physical application.

49. 48. The method of claim 47, wherein the CHP or composition is administered as a putty, mesh, patch, adhesive, cream, suture, gel, or eye drops.

50. A method for preventing or treating a diabetic pathology in a subject, comprising administering a CHP according to any one of claims 1 to 16, or a composition according to any one of claims 17 to 22.

51. 51. The method of claim 50, wherein the administration is by local injection, intravenous injection, topical application, or physical application.

52. 51. The method of claim 50, wherein the CHP or composition is administered as a putty, mesh, patch, adhesive, cream, suture, gel, or eye drops.

53. 23. A method of preventing or treating psoriasis in a subject, comprising administering a CHP according to any one of claims 1 to 16, or a composition according to any one of claims 17 to 22.

54. 54. The method of claim 53, wherein the administration is by local injection, intravenous injection, topical application, or physical application.

55. 54. The method of claim 53, wherein the CHP or composition is administered as a putty, mesh, patch, adhesive, cream, suture, gel, or eye drops.

56. 23. A method of inhibiting a checkpoint in a subject, comprising administering a CHP according to any one of claims 1 to 16, or a composition according to any one of claims 17 to 22.

57. 57. The method of claim 56, wherein the administration is by local injection, intravenous injection, topical application, or physical application.

58. 57. The method of claim 56, wherein the CHP or composition is administered as a putty, mesh, patch, adhesive, cream, suture, gel, or eye drops.

59. 23. A method of healing wounds, including diabetic lesions, in a subject, comprising administering a CHP according to any one of claims 1 to 16, or a composition according to any one of claims 17 to 22.

60. 60. The method of claim 59, wherein the administration is by local injection, intravenous injection, topical application, or physical application.

61. 60. The method of claim 59, wherein the CHP or composition is administered as a putty, mesh, patch, adhesive, cream, suture, gel, or eye drops.

62. 23. A method of preventing or treating inflammatory conditions of the gastrointestinal tract, such as Crohn's disease and ulcerative colitis, in a subject, comprising administering a CHP of any one of claims 1-16, or a composition of any one of claims 17-22.

63. 63. The method of claim 62, wherein the administration is by local injection, intravenous injection, topical application, or physical application.

64. 63. The method of claim 62, wherein the CHP or composition is administered as a putty, mesh, patch, adhesive, cream, suture, gel, or eye drops.

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