Collagen hybridization peptides having a sequence comprising citrullinated lair binding sequence and use thereof

Abstract

Disclosed are a series of novel collagen hybridizing peptides including citrullinated LAIR binding sequences and uses thereof.

Description

[0001] MLB Docket No. 127033-5006-WO

[0002] COLLAGEN HYBRIDIZATION PEPTIDES HAVING A SEQUENCE COMPRISING CITRULLINATED LAIR BINDING SEQUENCE AND USE THEREOF

[0003] BACKGROUND

[0004] The present disclosure relates to a series of novel collagen hybridizing peptides comprising citrullinated LAIR binding sequences and uses thereof.

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

[0006] Meanwhile, 4 out of 10 Americans develop cancer during their lifetime. Some tumors respond to current treatment modalities, but some patient groups exhaust therapeutic options, resulting in fatal outcomes. Research on biomarkers has elucidated the quality of tumors associated with aggressive diseases and poor clinical outcomes. For example, overexpression of tumor-associated collagens is associated with lower overall survival in lung, colorectal, and uterine cancers. Tire protumor effects of collagen are multifaceted. Collagen directly promotes tumor cell invasion, acts as a physical barrier to immune cell infiltration, and attenuates antitumor immune responses through binding of the LAIR1 receptor on immune cells. Immune cell infiltration and activation are important for the functions of immunotherapies in oncology , such as immune checkpoint blockade (i.e., Keytruda or Opdivo) and chimeric antigen receptor T-cell (CAR-T) therapy. Unfortunately, Keytruda is effective in only a minority of patients. One reason for Keytruda’s ineffectiveness is LAIR1 signaling on antitumor T-cells when bound to tumor-associated collagen. Therefore, there is a need for novel adjuvant therapies to support T-cell-mediated immunotherapy in collagen-rich tumors.

[0007] PROBLEM TO BE SOLVED

[0008] Therefore, the present disclosure has been made in view of the above problems, and it is an object of the present disclosure to provide a series of novel collagen hybridizing peptides comprising citrullinated LAIR binding sequences.

[0009] It is another object of the present disclosure to provide a method of reducing or preventing downregulation of immune cells using the collagen hybridizing peptide.

[0010] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO

[0011] It is another object of the present disclosure to provide a pharmaceutical composition for reducing or preventing downregulation of immune cells containing the collagen hybridizing peptide.

[0012] It is another object of the present disclosure to provide a method of preventing or treating cancer using the collagen hybridizing peptide.

[0013] It is another object of the present disclosure to provide a pharmaceutical composition for preventing or treating cancer containing the collagen hybridizing peptide.

[0014] MEANS TO SOLVE PROBLEM

[0015] Hereinafter, exemplary embodiments of the present disclosure will be described in detail. However, the present disclosure is not limited to the embodiments disclosed below and may be implemented in various forms. The embodiments described below are provided to enable those skilled in the art to implement and practice the embodiments of the present disclosure.

[0016] Materials, compositions, and components that may be used for the disclosed methods and compositions, may be used in conjunction therewith, may be used in the preparation of the compositions, or are products thereof, are disclosed. These and other materials are disclosed herein and it is understood that, when combinations, subsets, interactions, groups, and the like of these materials are disclosed, each of the various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, but each is specifically considered and described herein. For example, when peptide conjugates are disclosed and discussed, and a number of modifications that may be made on a number of molecules comprising the peptide conjugates are discussed, each and every combination and permutation and possible modification of the peptide conjugate is specifically considered unless specifically stated other ise. Thus, when not only the classes of molecules A, B, C are disclosed, but also the classes of molecules D, E, F and the combination molecules, for example A-D, are disclosed, each is considered individually and collectively, although not individually mentioned. Thus, in this example, the combinations A-E, A- F, B-D, B-E, B-F, C-D, C-E, and C-F are each specifically considered and should be considered disclosed from the disclosure of the exemplary combinations of A, B and C; D, E, and F; and A-D. Similarly, any subset or combination thereof is also specifically considered and disclosed. Thus, for example, the subsets of A-E, B-F and C-E are specifically considered and should be considered disclosed from the disclosure of the exemplary combinations of A, B and C; D, E, and F; and A-D. This applies to all aspects of the present disclosure, including, but are not limited to, the steps of preparing and using the disclosed compositions. Accordingly, it is understood that, when there are various additional steps that may be performed, each of these additional steps may be performed by any particular embodiment or combination of embodiments of the disclosed method, and it is

[0017] DB1 / 161378021.4 MLB Docket No. 127033-5006-WG understood that each such combination should be considered specifically and disclosed.

[0018] SUMMARY

[0019] In accordance with one aspect of the present disclosure, there is provided a collagen hybridizing peptide (CHP), wherein the CHP has a sequence represented by Formula I:

[0020] Ac-S-(Gly-X-Y)n-bioactive-(Gly-X-Y)m (Formula I) in which Ac is an acetyl capping group; S is zero or more spacer molecules; X is proline or modified proline; Gly is glycine; Y is hydroxyproline or any other amino acid; n is a number from 0 to 20; m is a number from 0 to 20; and the bioactive is an amino acid sequence from 3 to 27 amino acid residues having bioactivity, wherein the bioactive comprises at least one leukocyte-associated immunoglobulin-like receptor (LAIR-1 and / or LAIR-2) binding sequence, and wherein the amino acid sequence of the bioactive includes at least one citrulline residue.

[0021] In one embodiment, the at least one bioactive may comprise a LAIR-1 binding sequence.

[0022] In another embodiment, the at least one bioactive may comprise a LAIR-2 binding sequence.

[0023] In the CHP of any one of the previous embodiments, the at least one bioactive may comprise a LAIR-1 and / or LAIR-2 binding sequence comprising a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-27, 56-64, 93, 94, 98-100 and 107-187.

[0024] In the CHP of any one of the previous embodiments, the CHP may comprise a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-27, 56-64, 93, 94, 98-100 and 107-187.

[0025] In the CHP of any one of the previous embodiments, the CHP may comprise a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1-27, 56-64, 93, 94, 98-100 and 107-187.

[0026] In the CHP of any one of the previous embodiments, the CHP may comprise a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 1-27, 56-64, 93, 94, 98-100 and 107-187.

[0027] In the CHP of any one of the previous embodiments, the CHP may comprise a sequence selected from the group consisting of SEQ ID NOs: 107-157.

[0028] In the CHP of any one of the previous embodiments, S may be greater than 0.

[0029] In the CHP of any one of the previous embodiments, the CHP may be a first CHP, wherein the first CHP is bound to one other bioactive collagen hybridizing peptide to form a dimeric structure.

[0030] In the CHP of any one of the previous embodiments, the CHP may be a first CHP, wherein the first CHP is bound to two other bioactive collagen hybridizing peptides to fonn a trimeric

[0031] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO structure; and wherein the first CHP has a monomeric bioactive portion extending beyond the trimeric structure, the monomeric portion configured to bind to collagen alpha chains.

[0032] In the CHP of any one of the previous embodiments, the CHP may be a first CHP, wherein the first CHP is bound to two other bioactive collagen hybridizing peptides to form a trimeric structure, and wherein a monomeric "‘Tail” CHP is conjugated to an N-terminal or a C-terminal of the first CHP of the trimeric structure using copper-free click chemistry or condensation reactions.

[0033] In accordance with another aspect of the present disclosure, there is provided a composition comprising the CHP of any one of the previous embodiments.

[0034] In an embodiment, each individual CHP may not form a triple helix with other CHPs.

[0035] In another embodiment, the composition further comprises a carrier.

[0036] In the composition of any one of the previous embodiments, the carrier may comprise 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 matrix, extracellular matrix, and artificial extracellular matrix.

[0037] In the composition of any one of the previous embodiments, the composition may be a topical cream, a saline solution, gel, polymer, or a solution for systemic injection within a subject.

[0038] In the composition of any one of the previous embodiments, a CHP may be bound directly to collagen matrix.

[0039] In accordance with another aspect of the present disclosure, there is provided a method of reducing or preventing downregulation of immune cells in a subject, comprising administering the CHP or the composition of any one of the previous embodiments.

[0040] In an embodiment, the administration may be performed by injection.

[0041] In another embodiment, the administration may be performed by micro-dermal injection.

[0042] In the method of any one of the previous embodiments, the administration may be performed by intravenous injection.

[0043] In the method of any one of the previous embodiments, the administration may be performed by intratumoral injection.

[0044] In the method of any one of the previous embodiments, the administration may be performed by subcutaneous injection.

[0045] In the method of any one of the previous embodiments, the administration may be performed by topical application.

[0046] In the method of any one of the previous embodiments, the administration may be oral administration.

[0047] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO

[0048] In accordance with another aspect of the present disclosure, there is provided a method of treating cancer in a subject, comprising administering the CHP of any one of the previous embodiments.

[0049] In an embodiment, the administration may be performed by injection.

[0050] In another embodiment, the administration may be performed by micro-dermal injection.

[0051] In the method of any one of the previous embodiments, the administration may be performed by intravenous injection.

[0052] In the method of any one of the previous embodiments, the administration may be performed by intratumoral injection.

[0053] In the method of any one of the previous embodiments, the administration may be performed by subcutaneous injection.

[0054] In the method of any one of the previous embodiments, the administration may be performed by topical application.

[0055] In the method of any one of the previous embodiments, the administration may be oral administration.

[0056] In the method of any one of the previous embodiments, the CHP may be administered in combination with an immunotherapy.

[0057] In the method of any one of the previous embodiments, the immunotherapy may include immune checkpoint blockade therapy.

[0058] In the method of any one of the previous embodiments, the CHP may be administered in combination with a chimeric antigen therapy.

[0059] In the method of any one of the previous embodiments, the chimeric antigen therapy may include chimeric antigen receptor T-cell (CAR-T) therapy.

[0060] In the method of any one of the previous embodiments, the chimeric antigen therapy may include one or more of chimeric antigen receptor macrophage therapy, chimeric antigen receptor NK cell therapy, and chimeric antigen receptor dendritic cell therapy.

[0061] In the method of any one of the previous embodiments, the cancer may comprise one or more of lung cancer, colorectal cancer, ovarian cancer, hematological cancer, breast cancer, pancreatic ductal cell carcinoma, oral squamous cell carcinoma, and melanoma.

[0062] In the method of any one of the previous embodiments, the cancer may comprise multiple myeloma.

[0063] In accordance with another aspect of the present disclosure, there is provided a pharmaceutical composition for reducing or preventing downregulation of immune cells containing the CHP of any one of the previous embodiments as an active ingredient.

[0064] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO

[0065] In accordance with another aspect of the present disclosure, there is provided a pharmaceutical composition for preventing or treating cancer containing the CHP of any one of the previous embodiments as an active ingredient.

[0066] In another aspect, the disclosure provides a method of increasing a level of citrulines in an area of disease with collagen present comprising administering the CHP described herein or the composition described herein.

[0067] BRIEF DESCRIPTION OF THE DRAWINGS

[0068] The above and other objects, features and other advantages of the present disclosure will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0069] FIGURE 1 is a schematic diagram illustrating the design and operation of a CHP according to one embodiment of the present disclosure. FIGURE 1 illustrates the CHP design for delivering the LAIR bioactive sequence in the correct conformation.

[0070] FIGURE 2 is a schematic diagram illustrating the effects of CHP on immune cell activation according to one embodiment of the present disclosure. FIGURE 2 illustrates that T-cells binding to natural collagen get inhibited, while t-cells binding to citrullinated collagen do not.

[0071] FIGURE 3 illustrates the effects of CHP on pancreatic ductal carcinoma according to one embodiment of the present disclosure. FIGURE 3 shows validation of CHPs home to the tumor site in pancreatic ductal adenocarcinoma (PDAC), and remodeling collagen in the tumor tissue, demonstrating CHPs as a good targeting moiety for delivering the citrullinated LAIR sequence to the remodeling collagen at the tumor site. The figure shows an increase of collagen damage at the tumor site when compared to normal tissue that is adjacent to it.

[0072] FIGURE 4 illustrates melting temperature of Arg and Cit CHP using Circular Dichroism (CD). FIGURE 4A shows melting temperature of Arg CHP. FIGURE 4B shows melting temperature of Cit CHP.

[0073] FIGURE 5 illustrates binding of Arg / Cit CHP to mouse skin tissue that was damaged with heat. FIGURE 5A shows negative control. FIGURE 5B shows binding assay of Arg CHP. FIGURE 5C shows binding assay of Cit CHP.

[0074] FIGURE 6 illustrates binding of Arg / Cit CHP to gelatin. FIGURE 6 shows that both Arg and Cit CHP bind to gelatin comparable to B-CHP, a standard benchmark peptide. It also shows that it binds to gelatin significantly more than gelatin binds to itself.

[0075] FIGURE 7 illustrates binding of Arg / Cit CHP to LAIR1. FIGURE 7 highlights the ability of

[0076] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO the Arg or Cit CHP / Gelatin complex to bind LAIR1 significantly better than Gelatin alone.

[0077] FIGURE 8 illustrates IL-2 production in T cells treated with Cit / Arg CHP. FIGURE 7, paired with FIGURE 8 highlights the ability of Cit CHP to bind to LAIR1 while not resulting in decreased cytokine output.

[0078] FIGURE 9 illustrates viability of Arg / Cit CHP, highlighting the lack of toxicity in these peptides.

[0079] FIGURE 10 illustrates FLAIR3 and Cit-FLAIR3 mouse serum stability.

[0080] FIGURE 11 illustrates FLAIR3 and Cit-FLAIR binding to LAIR1.

[0081] DETAILED DESCRIPTION

[0082] It will be understood that, although the terms “first,” “second,” etc. may be used herein to describe various elements, these terms should not be construed as limiting these elements. These terms are used only to distinguish one element from another. For example, within the scope of the exemplar}' embodiment, a “first” element may be referred to as a “second” element, and similarly, a “second” element may be referred to as a “first” element. In addition, the term “and / or” includes any combination of one or more of the related mentioned items.

[0083] It must be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly indicates otherwise. Thus, for example, the expression “a peptide” includes a plurality of peptides including such peptide, and the expression “the peptide” includes one or more peptides and equivalents thereof and the like known to those skilled in the art. Similarly, the term “or” is intended to include “and” unless the context clearly indicates otherwise.

[0084] As used herein, the term “at least one” in relation to a list of one or more elements means at least one element selected from any one or more of the elements in the list of elements but does not necessarily include at least one of all elements specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows that other elements may optionally be present in addition to the elements specifically identified in the list of elements to which the term “at least one” relates, whether or not it is related to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently, “at least one of A and / or B”) may refer to, in one embodiment, at least one A, optionally including more than one B, where B is not present (and, optionally, including an element other than A); in another embodiment, at least one B, optionally including more than one B, where A is not present (and, optionally, including an element other than A); in yet another embodiment, at least one A, optionally including more than 1, and at least one B,

[0085] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO optionally including more than 1 (and optionally including other elements).

[0086] The terms “comprising,” “including,” “having,” and the like are used interchangeably and have the same meaning. Similarly, the terms “comprises,” “includes,” “has” and the like are used interchangeably and have the same meaning. Specifically, each term is defined consistently with the tenn “comprising” as generally defined in U.S. patent law and thus is construed as an open-ended term meaning “at least the following” while not excluding additional features, limitations, aspects, etc. Thus, for example, “a device having components a, b, and c” means that the device includes at least components a, b, and c. Similarly, the phrase “a method comprising steps a, b, and c” means that the method includes at least steps a, b, and c. Furthermore, steps and processes may be described schematically herein in a particular order, but those skilled in the art will recognize that the order of steps and processes may vary, unless a particular order is clearly mentioned by context.

[0087] As used herein, the term “about” refers to a numerical value, including, for example, integers, fractions, and percentages, whether or not explicitly mentioned. The term “about” generally refers to a range of numerical values (e.g., + / - 5%, 6%, 7%, 8%, 9%, or 10% of the stated value) that those skilled in the art would consider equivalent to the stated value (e.g., having the same function or result). In some cases, the term “about” may include a numerical value rounded to the nearest significant figure. In general, the term “about” includes a value for a given amount that falls at least within a corresponding margin of error for preparation, formulation, and / or measurement.

[0088] As used herein, the term “collagen” may refer to collagen derived from any type of tissue (e.g., bone, dermis, tendon, ligament, or the like). Collagen may refer to a molecule in which three alpha chains of a polyproline Il-like structure fold together to fonn a triple helix. In addition, this may be applied to any protein that contains a triple helical region, including collagen types I-XXVIII and bacterial collagen. As used herein, the tenn “collagen” may refer to any form of collagen, including artificial collagen and processed, or otherwise modified collagen. In some embodiments, the collagen is selected from type I collagen, type II collagen, type III collagen, type IV collagen, type V 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 XXV111 collagen, and combinations thereof.

[0089] As used herein, the term “proline or modified proline” may refer to the amino acid proline, natural and unnatural isomers thereof, various isomers including both natural and unnatural isomers thereof, and analogues and variants thereof. In an embodiment, the modified proline includes an electron withdrawing group. Examples of the modified proline include, but are not limited to,

[0090] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO hydroxyproline, methylated proline, 4-fluoroproline, and 4-chloroproline.

[0091] As used herein, the term “hydroxyproline” may refer to hydroxy group-substituted proline, which is a type of the modified proline.

[0092] As used herein, the term “citrullinated arginine” may refer to deaminated and citrullinated arginine in which arginine is substituted with citrulline. Thus, “citrullinated arginine” may also be interchangeably used with “citrulline”.

[0093] In some embodiments, the method may exclude collecting a sample from a subject. In some embodiments, the term “subject” used herein may refer to a human, an animal, or a microorganism, or a cell culture from any of the groups mentioned above. Examples of the animal may include, but are not limited to, vertebrates such as primates, rodents, domestic animals, or game animals. The primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques (e.g., Rhesus). The rodents may include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic animals and game animals may include cattle, horses, pigs, deer, bison, buffalo, moose, felines (e.g., domestic cats), and canines (e.g., dogs, foxes, wolves). The fish may include Chondrichthyes (cartilaginous fish) and Osteichthyes (bony fish). Tire subject may be a mammal. The mammal may be a human, a nonhuman primate, a mouse, a rat, a dog, a cat, a horse, or a cow, but is not limited thereto. In addition, the methods described herein may be used to diagnose and / or treat livestock or pets. This tenn does not denote a particular age or gender. Tirus, this term is intended to encompass all genders, including male and female, and all ages, including adults, newborns, and fetuses.

[0094] The term “prevention” may refer to any action that inhibits or delays the occurrence, propagation, and recurrence of a specific disease, and the tenn “treatment” may refer to partial or complete relief, alleviation, palliation, delayed onset, inhibition of progression, reduction in severity, and / or reduction in incidence of one or more symptoms or characteristics of a specific disease, disorder, and / or condition. For example, the prevention or treatment may be performed by administration to a subject who does not have signs of the disease, disorder, and / or condition and / or to a subject who has only early signs of the disease, disorder, and / or condition, to reduce the risk of developing pathology associated with the disease, disorder, and / or condition.

[0095] Bioactive CHP targets LAIR1, which may inhibit immune cell activation at the applied site. LAIR1 is a membrane-bound cell receptor expressed by several immune cells, including T-cells, macrophages, natural killer cells, B cells, and dendritic cells. LA1R1 may bind to intact collagen that initiates downregulation of LAIR1 signaling and immune cell activation. Membrane -bound LAIR1, secreted soluble LAIR1, and soluble homolog LAIR2 thereof may bind to other proteins such as adiponcctin, complement protein Cql, and surfactant D protein containing collagcn-mimicking domains, in addition to binding to collagen. CHP -targeting LAIRs developed by the present applicants may

[0096] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO initiate LAIR1 binding and activation, causing knockdown of T-cell activation. The CHPs described herein also target LAIR, but the functionality thereof may differ in that the bioactive ingredient includes a citrullinated arginine residue. Under certain disease conditions, such as rheumatoid arthritis, arginine residues in collagen molecules may be deaminated by the enzyme peptidylarginine deiminase (PAD2 and PAD4 normally act on collagen). Binding of LAIR- 1 to this sequence may not cause LAIR-1 phosphorylation of ITIMs signaling and / or inhibition of immune cell activation, which is a remarkable distinction. Therefore, the CHP may act as a decoy ligand for LAIR1 that competes with endogenous ligands found in collagen, clq protein, surfactant d protein and adiponectin for LAIR1 binding, which induces deactivation of immune cells upon binding. Thus, immune cells may remain activated to more effectively identify and destroy cancer cells in the tumor microenvironment.

[0097] The present disclosure provides novel bioactive CHPs that may be used to mimic collagen anti-inflammatory sequences while preventing dampening of inflammatory responses. In accordance with the present disclosure, collagen supports the sustained activation of immune cells that naturally suppress or resolve immune system activation. That is, the present disclosure relates to novel designs and applications of CHPs.

[0098] In addition, tire citrullinated LAIR CHPs of the present disclosure may be novel adjuvant therapies required to support T cell mediated immunotherapy in collagen-rich tumors.

[0099] In accordance with one aspect of the present disclosure, there is provided a collagen hybridizing peptide (CHP) having at least one sequence represented by Formula I:

[0100] [Formula I]

[0101] Ac-S-(Gly-X-Y)n-bioactive-(Gly-X-Y)m in which

[0102] Ac is an acetyl capping group;

[0103] S is zero or more spacer molecules:

[0104] X is proline or modified proline;

[0105] Gly is glycine;

[0106] Y is hydroxyproline or any other amino acid; n is a number from 0 to 20; m is a number from 0 to 20; and the bioactive is an amino acid sequence including 3 to 27 bioactive amino acid residues, wherein the bioactive comprises at least one leukocyte-associated immunoglobulin-like receptor (LAIR-1 and / or LAIR-2) binding sequence, and wherein the amino acid sequence of the bioactivc includes at least one citrullinc residue.

[0107] In an exemplary embodiment, both n and m are 0. In an exemplary embodiment, n is 0 and m

[0108] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO is 1. In an exemplary embodiment, n is 0 and m is 2. In an exemplary embodiment, n is 0 and m is 3.

[0109] In an exemplary embodiment, n is 0 and m is 4. In an exemplary embodiment, n is 0 and m is 5. In an exemplary embodiment, n is 0 and m is 6. In an exemplary embodiment, n is 0 and m is 7. In an exemplary embodiment, n is 0 and m is 8. In an exemplary embodiment, n is 0 and m is 9. In an exemplary embodiment, n is 0 and m is 10. In an exemplary embodiment, n is 0 and m is 11. In an exemplary embodiment, n is 0 and m is 12. In an exemplary embodiment, n is 0 and m is 13. In an exemplary embodiment, n is 0 and m is 14. In an exemplary embodiment, n is 0 and m is 15. In an exemplary embodiment, n is 0 and m is 16. In an exemplary embodiment, n is 0 and m is 17. In an exemplary embodiment, n is 0 and m is 18. In an exemplary^ embodiment, n is 0 and m is 19. In an exemplary embodiment, n is 0 and m is 20.

[0110] In an exemplary embodiment, both n and m are 1. In an exemplary embodiment, n is 1 and m is 2. In an exemplary embodiment, n is 1 and m is 3. In an exemplary embodiment, n is 1 and m is 4.

[0111] In an exemplary' embodiment, n is 1 and m is 5. In an exemplary embodiment, n is 1 and m is 6. In an exemplary embodiment, n is 1 and m is 7. I ]n an exemplary' embodiment, n is 1 and m is 8. In an exemplary embodiment, n is 1 and m is 9. In an exemplary embodiment, n is 1 and m is 10. In an exemplary embodiment, n is 1 and m is 11. In an exemplary embodiment, n IS 1 and m IS 12. In an exemplary embodiment, n is 1 and m is 13. In an exemplary embodiment, n is 1 and m is 14. In an exemplary embodiment, n is 1 and m is 15. In an exemplary embodiment, n IS 1 and m IS 16. In an exemplary embodiment, n is 1 and m is 17. In an exemplary embodiment, n IS 1 and m IS 18. In an exemplary7embodiment, n is 1 and m is 19. In an exemplary embodiment, n IS 1 and m IS 20.

[0112] In an exemplary embodiment, both n and m are 2. In an exemplary' embodiment, n is 1 and m is 3. In an exemplary embodiment, n is 2 and m is 4. In an exemplary embodiment, n is 2 and m is 5. In an exemplary embodiment, n is 2 and m is 6. In an exemplary embodiment, n is 2 and m is 7. In an exemplary embodiment, n is 2 and m is 8. In an exemplary embodiment, n is 2 and m is 9. In an exemplary embodiment, n is 2 and m is 10. In an exemplary embodiment, n IS 2 and m IS 11. In an exemplary embodiment, n is 2 and m is 12. In an exemplary embodiment, n IS 2 and m IS 13. In an exemplary embodiment, n is 2 and m is 14. In an exemplary' embodiment, n IS 2 and m IS 15. In an exemplary embodiment, n is 2 and m is 16. In an exemplary embodiment, n IS 2 and m IS 17. In an exemplary embodiment, n is 2 and m is 18. In an exemplary embodiment, n is 2 and m is 19. In an exemplary embodiment, n is 2 and m is 20.

[0113] In an exemplary embodiment, both n and m are 3. In an exemplary embodiment, n is 3 and m is 4. In an exemplary embodiment, n is 3 and m is 5. In an exemplary' embodiment, n is 3 and m is 6. In an exemplary embodiment, n is 3 and m is 7. In an exemplary’ embodiment, n is 3 and m is 8. In an exemplary embodiment, n is 3 and m is 9. In an exemplary embodiment, n is 3 and m is 10. In an

[0114] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO exemplary embodiment, n is 3 and m is 11. In an exemplary embodiment, n is 3 and m is 12. In an exemplary embodiment, n is 3 and m is 13. In an exemplary embodiment, n is 3 and m is 14. In an exemplary embodiment, n is 3 and m is 15. In an exemplary embodiment, n is 3 and m is 16. In an exemplary embodiment, n is 3 and m is 17. In an exemplary embodiment, n is 3 and m is 18. In an exemplary embodiment, n is 3 and m is 19. In an exemplary embodiment, n is 3 and m is 20.

[0115] In an exemplary embodiment, both n and m are 4. In an exemplary embodiment, n is 4 and m is 5. In an exemplary embodiment, n is 4 and m is 6. In an exemplary embodiment, n is 4 and m is 7.

[0116] In an exemplary7embodiment, n is 4 and m is 8. I n an exemplary embodiment, n is 4 and m is 9. In an exemplary7embodiment, n is 4 and m is 10. In an exemplary7embodiment, n is 4 and m is 11. In an exemplary embodiment, n is 4 and m is 12. In an exemplary embodiment, n is 4 and m is 13. In an exemplary embodiment, n is 4 and m is 14. In an exemplary embodiment, n is 4 and m is 15. In an exemplary embodiment, n is 4 and m is 16. In an exemplary embodiment, n is 4 and m is 17. In an exemplary embodiment, n is 4 and m is 18. In ai r exemplary embodiment, n is 4 and m is 19. In an exemplary7embodiment, n is 4 and m is 20.

[0117] In an exemplary embodiment, both n and m are 5. In an exemplary7embodiment, n is 5 and m is 6. In an exemplary embodiment, n is 5 and m is 7. In an exemplary embodiment, n is 5 and m is 8.

[0118] In an exemplary embodiment, n is 5 and m is 9 In an exemplary embodiment, n is 5 and m is 10. In an exemplary embodiment, n is 5 and m is 11. In an exemplary embodiment, n is 5 and m is 12. In an exemplary embodiment, n is 5 and m is 13. In an exemplary embodiment, n is 5 and m is 14. In an exemplary embodiment, n is 5 and m is 15. In an exemplary embodiment, n is 5 and m is 16. In an exemplary7embodiment, n is 5 and m is 17. In an exemplary embodiment, n is 5 and m is 18. In an exemplary embodiment, n is 5 and m is 19. In an exemplary embodiment, n is 5 and m is 20.

[0119] In an exemplary embodiment, both n and m are 6. In an exemplary embodiment, n is 6 and m is 7. In an exemplary embodiment, n is 6 and m is 8. In an exemplary embodiment, n is 6 and m is 9.

[0120] In an exemplary7embodiment, n is 6 and m is 10. In an exemplary7embodiment, n is 6 and m is 11. In an exemplary embodiment, n is 6 and m is 12. In an exemplary7embodiment, n is 6 and m is 13. In an exemplary embodiment, n is 6 and m is 14. In an exemplary embodiment, n is 6 and m is 15. In an exemplary embodiment, n is 6 and m is 16. In an exemplary embodiment, n is 6 and m is 17. In an exemplary embodiment, n is 6 and m is 18. In an exemplary embodiment, n is 6 and m is 19. In an exemplary embodiment, n is 6 and m is 20.

[0121] In an exemplary embodiment, n and m an 3 both 7. In an exemplary embodiment, n is 7 and m is 8. In an exemplary embodiment, n is 7 and m is 9. In an exemplary7embodiment, n is 7 and m is

[0122] 10. In an exemplary embodiment, n is 7 and m is 11. In an exemplary embodiment, n is 7 and m is

[0123] 12. In an exemplary embodiment, n is 7 and m is 13. In an exemplary embodiment, n is 7 and m is

[0124] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO

[0125] 14. In an exemplary embodiment, n is 7 and m is 15. In an exemplary' embodiment, n is 7 and m is

[0126] 16. In an exemplary' embodiment, n is 7 and m is 17. In an exemplary' embodiment, n is 7 and m is

[0127] 18. In an exemplary embodiment, n is 7 and m is 19. In an exemplary' embodiment, n is 7 and m is 20.

[0128] In an exemplary embodiment, n and m are both 8. In an exemplary embodiment, n is 8 and m is 9. In an exemplary embodiment, n is 8 and m is 10. In an exemplary embodiment, n is 8 and m is

[0129] 11. In an exemplary embodiment, n is 8 and m is 12. In an exemplary embodiment, n is 8 and m is

[0130] 13. In an exemplary embodiment, n is 8 and m is 14. In an exemplary' embodiment, n is 8 and m is

[0131] 15. In an exemplary' embodiment, n is 8 and m is 16. In an exemplary' embodiment, n is 8 and m is

[0132] 17. In an exemplary embodiment, n is 8 and m IS 18. In an exemplary' embodiment, n is 8 and m is

[0133] 19. In an exemplary embodiment, n is 8 and m is 20.

[0134] In an exemplary embodiment, both n and m are 9. In an exemplary embodiment, n is 9 and m is 10. In an exemplary embodiment, n is 9 and m is 11. ] an exemplary embodiment, n is 9 and m is

[0135] 12. In an exemplary embodiment, n is 9 and m is 13. In an exemplary' embodiment, n is 9 and m is In an exemplary' embodiment, n is 9 and m is 15. In an exemplary' embodiment, n is 9 and m is

[0136] 16. In an exemplary embodiment, n is 9 and m is In an exemplary embodiment, n is 9 and m is

[0137] 18. In an exemplary embodiment, n is 9 and m is 19. In an exemplary embodiment, n is 9 and m is

[0138] 20.

[0139] In an exemplary embodiment, both n and m are 10. In an exemplary embodiment, n is 10 and m is 11. In an exemplary embodiment, n is 10 and m is 12. In an exemplary embodiment, n is 10 and is 13. In an exemplary embodiment, n is 10 and m IS 14. In an exemplary' embodiment, n IS 10 and m is 15. In an exemplary' embodiment, n is 10 and m is 16. In an exemplary' embodiment, n is 10 and is 17. In an exemplary embodiment, n is 10 and m is 18. In an exemplary embodiment, n is 10 and m is 19. In an exemplary embodiment, n is 10 and m is 20.

[0140] In an exemplary embodiment, n and m are both 11. In an exemplary' embodiment, n is 11 and is 12. In an exemplary embodiment, n IS 11 and m is 13. In an exemplary' embodiment, n is 11 and m IS 14. In an exemplary embodiment, n is 11 and m is 15. In an exemplary' embodiment, n is 11 and m is 16. In an exemplary' embodiment, n is 11 and m IS 17. In an exemplary' embodiment, n is 11 and m is 18. In an exemplary embodiment, n is 11 and m is 19. In an exemplary embodiment, n is 11 and m is 20.

[0141] In an exemplary embodiment, both n and m are 12. In an exemplary' embodiment, n is 12 and m is 13. In an exemplary embodiment, n IS 12 and m IS 14. In an exemplary' embodiment, n is 12 and m is 15. In an exemplary embodiment, n is 12 and m is 16. In an exemplary' embodiment, n is 12 and is 17. In an exemplary’ embodiment, n is 12 and m IS 18. In an exemplary’ embodiment, n is 12 and m is 19. In an exemplary embodiment, n is 12 and m is 20.

[0142] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO

[0143] In an exemplary embodiment, both n and m are 13. In an exemplary' embodiment, n is 13 and is 14. In an exemplary embodiment, n is 13 and m IS 15. In an exemplary' embodiment, n is 13 and m is 16. In an exemplary' embodiment, n is 13 and m is 17. In an exemplary' embodiment, n is 13 and is 18. In an exemplary embodiment, n is 13 and m is 19. In an exemplary embodiment, n is 13 and is 20.

[0144] In an exemplary embodiment, both n and m are 14. In an exemplary embodiment, n is 14 and is 15. In an exemplary embodiment, n is 14 and m is 16. In an exemplary' embodiment, n is 14 and m is 17. In an exemplary embodiment, n is 14 and m is 18. In an exemplary' embodiment, n is 14 and m is 19. In an exemplary' embodiment, n is 14 and m is 20.

[0145] In an exemplary embodiment, both n and m are 15. In an exemplary embodiment, n is 15 and is 16. In an exemplary embodiment, n is 15 and m is 17. In an exemplary embodiment, n is 15 and m is 18. In an exemplary embodiment, n is 15 and m is 19. In an exemplary embodiment, n is 15 and is 20.

[0146] In an exemplary' embodiment, both n and m are 16. In an exemplary' embodiment, n is 16 and is 17. In an exemplary' embodiment, n is 16 and m is 18. In an exemplary' embodiment, n is 16 and m is 19. In an exemplary embodiment, n is 16 and m is 20.

[0147] In an exemplary embodiment, both n and m are 17. In an exemplary embodiment, n is 17 and m is 18. In an exemplary embodiment, n is 17 and m is 19. In an exemplary embodiment, n is 17 and is 20.

[0148] In an exemplary embodiment, both n and m are 18. In an exemplary' embodiment, n is 18 and m is 19. In an exemplary embodiment, n is 18 and m is 20. In an exemplary' embodiment, n is 19 and is 20.

[0149] In an exemplary embodiment, both n and m are 19. In an exemplary embodiment, n is 19 and is 20.

[0150] In an exemplary embodiment, both n and m are 20.

[0151] For example, the CHP may include, but is not limited to, a CHP containing citrullinated arginine. Overall, this produces a CHP that localizes and anchors the LAIR1 / 2 site to the extracellular matrix (ECM), particularly in areas where there are excessive denatured collagen strands available for CHP binding. Denatured collagen is commonly found in areas of fibrosis and around solid tumors. The CHP may target and bind to each alpha strand of damaged collagen molecules within the ECM, thereby localizing the decoy site to the damaged collagen and preventing immune cell silencing by LAIR1.

[0152] For example, in the CHP of some embodiments, the at least one bioactivc may include, but is not limited to, a LAIR-1 binding sequence.

[0153] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO

[0154] Alternatively, in the CHP of some embodiments, the at least one bioactive may include, but is not limited to, a LAIR-2 binding sequence.

[0155] Furthermore, in the CHP of some embodiments, the at least one bioactive may include, but is not limited to, both a LAIR-1 binding sequence and a LAIR-2 binding sequence.

[0156] In some embodiments, the at least one bioactive may include a LAIR-1 binding sequence, a LAIR-2 binding sequence, or both thereof, including a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 107 to 123.

[0157] For example, the collagen hybridizing peptide may include, but is not limited to, a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NOs. 1 to 27, 56 to 64, 93. 94. 98 to 100, and 107 to 187.

[0158] For example, the collagen hybridizing peptide may include, but is not limited to, a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NOs. 188 to 219. For example, the collagen hybridizing peptide may include, but is not limited to, a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NOs. 220 to 226.

[0159] Specifically, the collagen hybridizing peptide may include, but is not limited to, a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1 to 27, 56 to 64, 93, 94, 98 to 100, and 107 to 187.

[0160] More specifically, the collagen hybridizing peptide may comprise a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 1 to 27, 56 to 64, 93, 94, 98 to 100, and 107 to 187, but is not limited thereto.

[0161] Even more specifically, the collagen hybridizing peptide may comprise a sequence selected from the group consisting of SEQ ID NOs: 1 to 27, 56 to 64, 93, 94, 98 to 100, and 107 to 157, but is not limited thereto.

[0162] The LAIR sequence and the CHP sequence designed by applying the same are shown in Table 1. In addition, specific examples according to some embodiments of the known bioactive (Cit LAIR) sequences applied to the present disclosure and the citrullinated CHP and non-citrullinated CHP sequences including the same are shown in Table 2 below. Furthermore, specific examples according to some embodiments of the citrullinated CHP and non-citrullinated CHP sequences having biotin bound to the N-terminus thereof are shown in Table 3 below.

[0163] Tabic 1

[0164] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO

[0165] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO

[0166] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO

[0167] DB1 / 161378021.4 MLB Docket No. 127033-5006-WG

[0168] The Ac- is an acetyl (capping) group at the N -terminus. the G- is Glycine, the P- is proline, the 0- is hydroxyproline, the f- is 2S,4S-4-fluoroproline, x is any integer from 1-7, y is any integer from 1- 7, and z is any integer from 1-5.

[0169] Table 2

[0170] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO

[0171] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO

[0172] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO

[0173] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO

[0174] Table 3

[0175] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO

[0176] In some embodiments, S may include one or more spacer molecules. The spacer molecules may be any amino acid or modified amino acid, but are not limited thereto. The amino acid may be any of the 20 natural amino acids known in the art. Meanwhile, the modified amino acid may include, but is not limited to, unnatural amino acid corresponding to the natural amino acid, various isomers, analogs, and variants of the natural and unnatural amino acids. For example, the modified amino acid DB1 / 161378021.4 MLB Docket No. 127033-5006-WO may be an amino acid in which the backbone of the natural amino acid is further substituted with hydroxy, methyl, fluoro, chloro, or the like, but is not limited thereto.

[0177] For example, the CHP is a first CHP and the first CHP may bind to two different bioactive collagen hybridizing peptides to form a dimeric structure, but is not limited thereto.

[0178] For example, the CHP is a first CHP and the first CHP may bind to one different bioactive collagen hybridizing peptide to form a dimeric structure; the first CHP has a bioactive monomer moiety that extends beyond the trimer structure, and the monomer moiety may be configured to bind to a collagen alpha chain, but is not limited thereto.

[0179] For example, the CHP is a first CHP and the first CHP binds to two different bioactive collagen hybridizing peptides to form a trimer structure; the monomeric “tail” CHP may be conjugated to the N -terminus or C-tenninus of the first CHP of the trimer structure using copper-free click chemistry or a condensation reaction, but is not limited thereto.

[0180] Solid tumors are surrounded by a dense ECM and the matrix is actively degraded and remodeled by the coordinated efforts of cancer cells, tumor associated macrophages, and fibroblasts. Fibrosis is characterized by an abundance of remodeled collagen. CHP has been shown to bind to damaged collagen within the tumor microenvironment. During ECM turnover, the collagen strands that constitute the major components of the ECM are enzymatically cleaved by the matrix metalloproteinases (MMP) family. Hie cleavage destabilizes the triple helical structure of collagen, thereby inducing additional denaturation at body temperature, and providing ample hybridization sites for CHP. The ECM is biologically active as it interacts and communicates with cells via a number of receptors, including integrins, OSCAR, GPIV, DDR, LAIR, and UPARAP / ENDO180. Like LAIR1 binding, a number of these receptor binding sites require a triple helical conformation so that they are recognized by and may interact with cells. Thus, the CHP provides a perfect delivery vehicle for a decoy LAIR binding site, and binds to damaged collagen, thereby providing the decoy LAIR site in the form of a triple helical conformation.

[0181] In addition, by embedding a citrullinated LAIR1 binding sequence into the CHP that targets and binds to damaged collagen, the local concentration of decoy LAIR binding sites can be increased at areas of high collagen turnover or disease states. Upon recognition by LAIR 1 -expressing immune cells, these decoy sites occupy LAIR1 and prevent downstream signaling associated with LAIR1 phosphorylation, activation, and attenuation of immune cell activation. Thus, the overall effect of the CHP is to prevent and / or cease local immune weakening at the site of application.

[0182] In another aspect, the present disclosure provides a pharmaceutical composition for reducing or preventing downregulation of immune cells containing the collagen hybridizing peptide (CHP) as an active ingredient.

[0183] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO

[0184] In another aspect, the present disclosure provides a method of reducing or preventing downregulation of immune cells including administering the pharmaceutical composition to a subject in need thereof.

[0185] In another aspect, the present disclosure provides a pharmaceutical composition for preventing or treating cancer containing the collagen hybridizing peptide (CHP) as an active ingredient.

[0186] In another aspect, the present disclosure provides a method of preventing or treating cancer, including administering the pharmaceutical composition to a subject in need thereof.

[0187] For example, in an embodiment, the composition is characterized in that each CHP contained therein does not form a triple helix with another CHP.

[0188] For example, in an embodiment, the composition may include one or more CHPs, but is not limited thereto.

[0189] In addition, the composition may further contain a pharmacally acceptable carrier. In this case, the carrier may include, but is not limited to, at least one selected from the group consisting of micelles, dendrites, lipids, microemulsions, nanoemulsions, solid lipid nanoparticles, polymers, gels, lenses, surfactants, cyclodextrins, inserts, nanostructured lipid carriers, liposomes, transfersomes, ethosomes, niosomes, collagen matrices, extracellular matrices, and artificial extracellular matrices.

[0190] As used herein, the tenn "administration" may mean introducing a predetennined substance into a patient in an appropriate manner and the composition may be administered through any general route as long as it can reach the target tissue. Specifically, the composition may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, topically, intranasally, intrapulmonarily, or rectally, but is not limited thereto.

[0191] For this purpose, the composition may be provided in the form of a topical cream, saline solution, gel, polymer, or solution for systemic injection into a subject, but is not limited thereto.

[0192] The composition may be prepared into the formulation described above and may be administered by injection, micro-dermal injection, intravenous injection, intratumoral injection, subcutaneous injection, topical application, or oral administration, but is not limited thereto.

[0193] Meanwhile, the composition may be administered in a pharmaceutically effective amount, and the tenn “pharmacally effective amount” refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to all medical treatments, without causing side effects. The effective dose may be easily determined by those skilled in the art. based on factors including the gender, age, weight, health condition, and type and severity of the disease of the patient, activity of the dmg, sensitivity of the patient to the drug, administration method, administration time, administration route, excretion rate, treatment period, drugs used in combination or simultaneously, and other factors well known in the pharmaceutical field. In general, the active ingredient may be administered at a dose

[0194] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO of about 0.001 mg / kg / day to 1,000 mg / kg / day. For example, the active ingredient may be administered at a dose of 0.01 mg / kg / day to 100 mg / kg / day, or 0.01 mg / kg / day to 35 mg / kg / day, but is not limited thereto. For oral administration, the active ingredient is preferably in the range of 50 to 500 mg / kg and may be administered once or more per day. Specifically, the active ingredient is generally administered to an adult patient weighing 70 kg at a dose of 0.07 to 7,000 mg / day, preferably 0.7 to 2,500 mg / day and may be administered once or several times per day in divided doses at regular intervals depending on the judgment of physicians or pharmacists.

[0195] In the composition, the CHP directly binds to the collagen matrix.

[0196] Meanwhile, the composition for preventing or treating cancer may be administered in combination with immunotherapy. Specifically, the immunotherapy may include, but is not limited to, immune checkpoint blockade therapy.

[0197] In addition, the composition for preventing or treating cancer may be administered in combination with chimeric antigen therapy. Specifically, the chimeric antigen therapy may include chimeric antigen receptor T-cell (CAR-T) therapy. In addition, the chimeric antigen therapy may include at least one selected from the group consisting of chimeric antigen receptor macrophage therapy, chimeric antigen receptor NK cell therapy, and chimeric antigen receptor dendritic cell therapy, but is not limited thereto.

[0198] The cancer disease that may be prevented or treated using the composition may include at least one solid cancer selected from the group consisting of lung cancer, colorectal cancer, ovarian cancer, hematological cancer, breast cancer, pancreatic ductal cell carcinoma, oral squamous cell carcinoma, and melanoma, but is not limited thereto. In addition, the cancer disease may include any fibrosis based cancer without limitation. Furthermore, the cancer disease may include hematological cancer, such as multiple myeloma, that results in severe collagen damage. The treatment using the CHP may be used as a supplemental treatment or in combination with other immunotherapies including pembrolizumab (Keytruda), nivolumab (Opdivo) and chimeric antigen therapy (i.e., ABECMA and CARVYKTI™) in T cells, macrophages, natural killer cells and dendritic cells.

[0199] In some embodiments, cancer may include bladder cancer, bone cancer, brain tumors, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, and kidney cancer are all types of cancer. In some embodiments, other forms of cancer may include leukemia, liver cancer, and various lymphomas, such as non-Hodgkin lymphoma. In some embodiments, cancers may be a skin like melanoma and non-melanoma skin cancer, as well as cancers affecting the reproductive and urinary systems like ovarian, prostate, testicular, and uterine cancer. In some embodiments, cancers may develop in the oral cavity, stomach, pancreas, thyroid, and as sarcomas and myelomas.

[0200] Specifically, the composition for preventing or treating cancer containing the CHP may be

[0201] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO useful for cancer diseases having high LAIR expression in tumor-associated immune cells, such as oral squamous cell carcinoma. High LAIR1 expression in tumor-associated immune cells may weaken anti-tumor immune responses. Therefore, blocking LAIR1 signaling and activation by the CHP may enable anti-tumor immune responses from endogenous tumor-associated immune cells, such as T cells, macrophages, and natural killer cells.

[0202] Ultimately, the composition containing the CHP may be generally applied to all LAIR- expressing cells, i.e., all immune cells.

[0203] In an embodiment, the composition may be provided in the form of a food composition for preventing or ameliorating the aforementioned diseases.

[0204] For example, the composition may be used before or after the onset of the disease, simultaneously with or separately from a drug for treating the disease, to prevent or ameliorate the aforementioned diseases.

[0205] As used herein, the term “amelioration” means any action that at least reduces a parameter related to the treatment condition, such as the degree of symptoms.

[0206] As used herein, the term “food” includes meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, health functional foods, and health foods, and includes all foods in the conventional sense.

[0207] For example, since the food composition is consumed on a daily basis, it is expected to be highly effective in amelioration of diseases and is very useful to improve health. In addition, the food composition may further contain a bromatologically acceptable food additive.

[0208] As used herein, the tenn “functional food” has the same meaning as the term “food for special health use (FoSHU)” and refers to a food having strong medical and pharmaceutical effects that has been processed to efficiently provide bioregulatory functions as well as nutrition supply functions. As used herein, the temr “functional” means obtaining beneficial effects for health purposes, such as controlling nutrients or exhibiting physiological effects with regard to the structures and functions of the human body. The food may be prepared by a method commonly used in the art and the preparation may be performed using raw materials and ingredients commonly added in the art. Hie food composition may be prepared into various formulations and, unlike general drugs, it is advantageously free of side effects that may occur upon long-term intake of drugs since it uses food as a raw material, and is highly portable, and thus can be consumed as a supplement to enhance the effect of ameliorating diseases.

[0209] The tcmi “health food” may refer to a food having an effect of actively maintaining or improving health beyond that of a general food, and the tenn “health supplement food” refers to a food

[0210] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO ingested for the purpose of health supplement. In some cases, the terms “health functional food,” “health food,” and “health supplement food” are used interchangeably.

[0211] Specifically, the term “health food” means a food product that is prepared by adding the compound to food materials such as beverages, teas, spices, gums, and confectionery , or by preparing the compound in the fonn of a capsule, powder, suspension, or the like, and has a specific health effect, when consumed, and is advantageously free of side effects that may occur when taking drugs for a long period of time because it is prepared from food as a raw material, unlike general drugs.

[0212] The food composition may further contain a physiologically acceptable carrier. There is no particular limitation as to the type of carrier and any carrier may be used as long as it is commonly used in the relevant art.

[0213] In addition, the food composition may contain additional ingredients commonly used in food compositions to improve fragrance, flavor, aesthetics and the like. For example, the food composition may contain vitamins A, C, D, E, Bl, B2, B6, B12, niacin, biotin, folate, pantothenic acid, or the like. Further, the food composition may contain: minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chromium (Cr), and amino acids such as lysine, tryptophan, cysteine, and valine.

[0214] In addition, the food composition may contain food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, and the like), bactericides (bleaching powder and highly bleaching powder, sodium hypochlorite, and the like), antioxidants (butylated hydroxyanisole, BHA), butylated hydroxytoluene (BHT and the like), colorants (coal tar dyes, and the like), color developers (sodium nitrite, sodium nitrite, and the like), bleaching agents (sodium sulfite), seasonings (MSG. monosodium glutamate, and the like), sweeteners (dulcin, cyclamate, saccharin, sodium, and the like), flavorings (vanillin, lactones, and the like), leavening agents (alum, D-potassium hydrogen tartrate, and the like), reinforcing agents, emulsifiers, thickeners (glutinating agents), coating agents, gum bases, de-foaming agents, solvents, and enhancers. The additives may be selected depending on the type of food and used in an appropriate amount.

[0215] When the food composition is used as a food additive, the composition may be added as it is or used in combination with other foods or food ingredients, and may be used appropriately according to a conventional method. Hie amount of the active ingredient may be appropriately determined depending on the purpose of use. such as prevention, health, or therapeutic treatment. In general, the composition is added in an amount of 15 wt% or less, preferably 35 wt% or less, based on the weight of raw material in the process of preparing a food or beverage. However, in case of long-term intake for health and hygiene or health control, the amount of active ingredient may be below the range defined above.

[0216] In an embodiment, the food composition may be used as a health beverage composition. In DB1 / 161378021.4 MLB Docket No. 127033-5006-WO this case, the food composition may contain various flavoring agents or natural carbohydrates as additional ingredients, like a conventional beverage. The natural carbohydrates include: monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, or erythritol. Sweeteners may include: natural sweeteners such as thaumatin and stevia extracts, and synthetic sweeteners such as saccharin or aspartame, and the like. The content of the natural carbohydrate may be generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g, with respect to 100 mL of the health beverage composition.

[0217] Furthermore, the health beverage composition may contain various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners. pH regulators, stabilizers, preservatives, glycerin, alcohol, or carbonating agents. In addition, the health beverage composition may contain fruit pulp for the preparation of natural fruit juice, fruit juice beverage, or vegetable beverage. These ingredients may be used alone or in combination. The content of these additives is not particularly important, but is typically selected within the range of 0.01 to 0.1 parts by weight with respect to 100 parts by weight of the health beverage composition.

[0218] In another aspect, the disclosure provides a method of increasing a level of citrulines in an area of disease with collagen present comprising administering the CHP described herein or the composition described herein.

[0219] Hereinafter, the present disclosure will be described in more detail with reference to the following examples. However, it will be obvious to those skilled in the art that the following examples are provided only for illustration of the present disclosure and should not be construed as limiting the scope of the present disclosure.

[0220] EXAMPLES

[0221] Example 1: Design of Sequence

[0222] Another bioACTIVE CHP design that includes a LAIR active site in the middle of the sequence is disclosed in PCT / US2024 / 18078. The bioACTIVE CHP disclosed in the aforementioned PCT application carries an active LAIR1 site that is required for a triple-helix conformation that may be recognized by cells in a microenvironment. While both designs have similar binding sites for the LAIR1 receptor, the bioactivity of the novel CHP design of the present disclosure is impaired due to the inclusion of citrulline. Therefore, the target of the CHP is the same, but the biological impact of binding to each CHP is significantly different. Thus, the sequence of the present disclosure is distinguished from conventional sequences.

[0223] DB1 / 161378021.4 MLB Docket No. 127033-5006-WG

[0224] Another technology having a similar strategy is NC410 from Nextcure (PCT / US2017 / 045310). NC410 is a LAIR2 mimetic chimeric antibody that binds to the LAIR1 / 2 binding site and has anticancer activity. The CHP of the present disclosure differs from NC410 in that it is smaller and does not include an Fc IgG antibody domain. The NextCure's technology is based on blocking LAIR1 binding sites such as collagen, whereas the approach according to the present disclosure is based on blocking downstream LAIR1 signaling after the corresponding binding.

[0225] Finally, a collagen mimetic peptide from Stewart Therapeutics known as ST- 100 has a similarity to the CHP design disclosed herein (U.S. Patent No. 10,632,168). ST-100 is useful for treatment of ocular diseases, disorders or wounds including cataracts, comeal ulcerations, scleritis, keratoconus and post-operative eye wounds and is limited thereto. Since not all LAIR binding sites are defined, LAIR1 / 2 may also bind to the collagen mimetic peptide ST-100. Importantly, this binding has the opposite effect of the CHP according to the present disclosure and thus activates LAIR1. rather than keeping LAIR1 inactive. Therefore, the citrullinated CHP of the present disclosure is a unique technology with novel applications.

[0226] Example 2: Example of Specific Sequence Design

[0227] The prior LAIR bioACTIVE CHP developed by the present applicant includes the following sequence:

[0228] Ac-S-(Gly-X-Y)n-bioactive-(Gly-X-Y)m, wherein Ac is an acetyl capping group; S is 0 to 10 spacer molecules; X is proline or modified proline; Gly is glycine; Y is hydroxyproline or any other amino acid; n is a natural number of 1 to 20; m is a natural number of 1 to 20; and bioactive is an amino acid sequence having 3 to 27 amino acid residues having bioactivity, wherein the bioactive includes at least one sequence selected from the group consisting of a leukocyte-associated immunoglobulin-like receptor sequence (LAIR-1 and / or LAIR-2) sequence, a G6b-B recognition site sequence, an OSCAR domain sequence, a GPVI sequence, and an FcR-gamma domain sequence.

[0229] The novel LAIR decoy CHPs of the present disclosure may have the same configuration except that the bioactive site is a citrullinated LAIR binding site including a deaminated arginine residue.

[0230] Currently, a known LAIR sequence including arginine, disclosed in the original LAIR patent of the present applicant, is only one. Such a sequence should be extended to all CHPs that bind to LAIR having citrullinated amino acids. Not all LAIR sequences are known.

[0231] Example 3: Conditions of Sequence Design

[0232] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO

[0233] The collagen hybridizing peptide (CHP) according to the present disclosure satisfies the following requirements:

[0234] 1. There are several aspects of changeable CHP designs that still achieve the ultimate goal of binding to the damaged site and bringing the biologically active LAIR binding site to the application site. Hie triple helix CHP region that binds to damaged collagen may be changed as long as the sequence includes a Gly-X-Y motif. For hybridization with damaged collagen, it can be seen that the sequence has a very high affinity for binding when X is proline and Y is hydroxyproline. However, the amino acid residues at the positions X and Y may be regulated to control the overall Tm of the CHP depending on a specific purpose. The number (n) of Gly-X-Y repeats may be selected from 1 to 20 depending on the desired properties of the CHP.

[0235] 2. The CHP must have a self-association temperature of 37°C or less, which may be 0°C to 37°C.

[0236] 3. CHP must be able to bind to damaged collagen at a temperature which may be 37°C to 100°C or 37°C.

[0237] 4. CHP may be a dimer.

[0238] 5. CHP may be a trimer with a tail.

[0239] 6. CHP must have a bioACTIVE CHP sequence within the entire sequence, but doesn't have to be in the middle. CHP may be located at any one of both ends as long as it binds to damaged collagen.

[0240] 7. Unnatural amino acids may be introduced to increase the binding affinity of the sequence. Some residues of interest are 2S,4R-4 fluoroprolines as the trans form. When the amino acid is disposed at the Y position, it actually supports proper pre-alignment of the phi-psi angle of poly-proline, such as a type-II helix, which allows for better trimerization. This is due to the fluorine atom having a stronger electron-withdrawing functionality than the hydroxyproline residues typically found at the position Y. Another unnatural amino acid that could be tested is the aza-glycine residue, which increases the stability of the triple helix formed by increasing the formation of cross-strand hydrogen bonds. In addition to inclusion of these residues in the CHP sequence, the number of residues included, the position thereof within the CHP sequence, and the mix of these residues in a single CHP sequence may be varied.

[0241] 8. There are several possible LAIR binding sites that act along with the CHPs of the present disclosure and still have the same effect. These LAIR binding sites include several shortened renditions such as GAOGLRGGAGPOGPEGGKGAAGPOGPOXXX (SEQ ID NO: 223), GAOGLRGGAGPOGPEGGKGAA (SEQ ID NO: 224), GLRGGAGPOGPEGGKGAA (SEQ ID NO: 225), GLRGGAGPOGPE, and ARGLTGRPGDA (SEQ ID NO: 226), but a number thereof remain undefined. Any change in the binding site sequence for any citrullinated form of the sequence should

[0242] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO allow LAIR1 / 2 binding while preventing biological activity of the LAIR1 receptor.

[0243] 9. All sequences disclosed in the first LAIR patent of the present application may be incorporated herein, along with the citrullinated amino acids that move throughout the sequence.

[0244] Experimental Example 1: Human LAIR1 Binding Plate Assay

[0245] First, black opaque Nunc amino flat-bottom plates (Thermo, 436008) are coated with 100 pL of human placenta collagen or porcine gelatin (pH 8) at a concentration of 100 pg / mL for 1-4 hours in a shaker at room temperature. The protein solution is removed and washed four times with phosphate- buffered saline with IxTween 20 (PBST). The attached protein is further fixed with 150 pL of ice-cold methanol at -20°C for 30 minutes. The methanol is removed and the plates are washed four times with phosphate-buffered saline (PBS). The amine-active groups on the plates are then blocked with 1 M Tris HC1 at room temperature for 30 minutes. The plates are washed four times with PBS and nonspecific protein binding is blocked with 1% BSA in PBS for 30 minutes at room temperature. Meanwhile, CHP solutions of citrullinated LAIR-CHP and standard LAIR-CHP (same sequence in which citrulline is substituted with arginine) are preheated to 80°C for 10 minutes to release the CHP from the trimer and facilitate gelatin binding. After blocking with BSA, the wells are washed four more times with PBS and 0 pL of 0 to 200 pM CHP in PBS is added to each well. CHP is allowed to bind to the plate at 37°C overnight. Controls used herein include human IgG Alexa fluor 488 antibody (Thermo, A10631) added to wells that do not contain recombinant human LAIR1 Fc chimeric protein and thus do not have any signal. Further, scrambled citrullinated and scrambled non-citrullinated LAIR-CHP are used as negative binding controls.

[0246] The following day, the CHP is removed from the wells, washed four times with PBS, and 100 pL of 100 pg / mL recombinant human LAIR1 Fc chimeric protein (R&D Systems, 9964-LR-050) in 1% BSA solution in PBST is added to each well and incubated for 1 hour at room temperature. Unbound LAIR1 is removed and washed four times with PBS. Bound human LAIR1 is detected by adding 100 pL of 5 pg / mL anti-human IgG Alexa fluor 488 antibody (Thermo, A10631) in 1% BSA solution in PBST and incubated at room temperature for 1 hour. Then, unbound antibodies are washed four times with PBS and the signal at 488 nm is read using a plate reader.

[0247] Experimental Example 2: Mouse LAIR1 Plate Binding Assay

[0248] First, black opaque Nunc amino flat-bottom plates (Thermo, 436008) are coated with 100 pL of human placenta collagen or porcine gelatin (pH 8) at a concentration of 100 pg / mL for 2 hours in a shaker at room temperature. The protein solution is removed and washed four times with phosphate- buffered saline with IxTween 20 (PBST). Tire attached protein is further fixed with 150 pL of ice-cold DB1 / 161378021.4 MLB Docket No. 127033-5006-WO methanol at -20°C for 30 minutes. The methanol is removed and the plates are washed four times with phosphate-buffered saline (PBS). Tire amine-active groups on the plates are then blocked with 1 M Tris HC1 at room temperature for 30 minutes. The plates are washed four times with PBS and nonspecific protein binding is blocked with 1% BSA in PBS for 30 minutes at room temperature.

[0249] Meanwhile, CHP solutions of citrullinated LAIR-CHP and standard LAIR-CHP are preheated to 80°C for 10 minutes to release the CHP from the trimer and facilitate gelatin binding. After blocking with BSA, the wells are washed four more times with PBS and 50 pL of 0 to 200 pM CHP in PBS is added to each well. After blocking with BSA, the wells are further washed four times with PBS and 50 pL of 0 to 200 pM CHP in PBS is added to each well. CHP is allowed to bind to the plate at 37°C overnight. Controls used herein include goat anti-mouse IgG2a Alexa fluor 488 antibody (Thermo, A-21131) added to wells that do not contain recombinant human LAIR1 Fc chimeric protein and thus do not have any signal. Further, scrambled citrullinated and scrambled non -citrullinated LAIR-CHP are used as negative binding controls.

[0250] The following day, CHP is removed from the wells, washed four times with PBS, and 100 pL of 100 pg / mL recombinant mouse LAIR1 Fc chimeric protein (R&D Systems, 10092-LR-050) in 1% BSA solution in PBST is added to each well and incubated for 1 hour at room temperature. Unbound LAIR1 is removed and washed four times with PBS. Bound human LAIR1 is detected by adding 100 pL of goat anti-mouse IgG2a Alexa fluor 488 antibody (Thermo. A-21131) in 1% BSA solution in PBST and incubated at room temperature for 1 hour. Then, unbound antibodies are washed four times with PBS and the signal at 488 nm is read using a plate reader.

[0251] Experimental Example 3: Thl T-Cell Activation Assay

[0252] Jurkat E6-1 cells (ATCC TIB- 152) are cultured in a RPMI 1640 medium containing GlutaMAX (Thermo, 61870036) supplemented with 10% heat inactivated fetal bovine serum (Thermo, 16140071) and 1% penicillin and streptomycin (Thermo, 15070063) at 37°C, 5% CO2. CHP and gelatin solutions are prepared in a medium containing 5 to 25 pM citrullinated LAIR-CHP, or normal LAIR CHP (previously heated to 80°C for 10 minutes) at 0.52 mg / mL of porcine gelatin (Millipore Sigma, 48724). CHP and gelatin solutions are incubated at 37°C for 3 days prior to combination with cells.

[0253] On the day of the experiment. Nunc 6-well tissue culture plates (Thermo. 140675) are coated with 3 pg / mL of anti-human CD3 monoclonal antibody (Thermo, clone OKT3, 16-003781) in sterile PBS for 2 hours at 37°C. Inactivated control wells are left blank and not coated with an antibody. T cells arc resuspended in a gelatin CHP solution containing 3 pg / mL of an anti-human CD28 antibody (Thermo 16-0281-82) or 10 ng / mL ofphorbol 12-myristate 13-acetate (PMA, Sigma, P1585).

[0254] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO

[0255] Controls are suspended in gelatin without CHP in the presence or absence of an activator. T cells are incubated with CHP and gelatin solution, and media samples are collected for analysis by Alamar Blue (Thermo, DAL 1025) to determine the level of activation and for IL-2 expression by ELISA (abeam, ab270883).

[0256] Experimental Example 4: LAIR-1 Phosphorylation Assay

[0257] Tyrosine phosphorylation of LAIR-1 is determined using human phosphoimmunoreceptor array (Proteome Profiler Array; R&D Systems) according to the manufacturer’s protocol. Human t- cells (5 * 106cells) are activated as described above. Next, t-cells are incubated along with LAIR-CHP, citrullinated LAIR-CHP, or scrambled control peptide at 37°C for 15 minutes. Phosphory lated proteins are detected using a horseradish peroxidase-conjugated pan anti-phosphotyrosine antibody. Array signals are visualized using a Sapphire Biomolecular Imager (Azure Biosystems).

[0258] Experimental Example 5: In Vivo and Ex Vivo Validation

[0259] The Onco-Hu mouse models (Jackson Laboratory ) are used to evaluate the therapeutic effects of citrullinated CHP (Cit-CHP) on tumor resolution. These models are humanized NOD-scid IL2Rynull (NSG) mice having patient-derived xenografts (PDX) and enable studies of the efficacy of checkpoint inhibitors such as LAIR-1. In the presence of isoflurane (3-5%), high (25-100 pM) or low (1-20 pM) doses of LAIR-CHP in combination with pembrolizumab, cit-LAIR-CHP in combination with pembrolizumab, pembrolizumab alone, or PBS blank are injected locally into mice on day 0 (n = 3 for each group). Each mouse is dosed in situ every' 7 days and up to 200 pL of blood is collected to evaluate the level of T-cell activation between the respective groups. Tumor size is monitored every 3 days over 21 days (total of 7 measurements). At the end of the experiment, mice are sacrificed according to the approved IACUC protocol, and tumors are resected, fixed, and sectioned for histological analysis. To measure collagen content, sections are stained with Masson’s Trichrome or basic CHP stain to determine the amount of total collagen or denatured collagen due to tumor remodeling. CD3 antibody is used to stain T cells in the samples and anti-IL2R mAb is used to stain activated T cells.

[0260] Experimental Example 6: Sequence Used in the Experiments

[0261] Sequences used in the experiments are shown in Table 4.

[0262] Table 4

[0263] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO

[0264] Melting temperature of Arg CHP and Cit CHP are shown in Figure 4. Arg CHP has a melting temperature of 29.5°C (Figure 4A), and Cit CHP has a melting temperature of 30.6°C (Figure 4B).

[0265] Experimental Example 7: Tissue Binding

[0266] This example describes that arginated and citrullinated CHPs can bind to damaged collagen in heated tissue sections.

[0267] PDAi enzymes, including PDAi2 and PDAi4, convert arginine residues in collagen to citrullinc 1. This experiment showed that Arg CHP and Cit CHP can bind to damaged collagen in rat skin tissue. To remove paraffin, slides were rinsed with 100% xylenes, 100% ethanol, 95% ethanol, 50% ethanol, and deionized water for two 5 minutes cycles with shaking of each solvent in consecutive order. The de-paraffinized tissue can be used directly for staining without any antigen-retrieval process. Each washing step required ~100 mL (fill to line in jar) of fresh reagent between washes. A tissue steamer was used to perform heat-induced epitope retrieval (HIER) for damaging collagen or for co-staining with an antibody. IX Citrate buffer (pH 6) was prepared by mixing 5 mL of 10X Citrate buffer (back of fridge) with 45 mL of DI or MilliQ water, then transferred the slides into the tissue steamer vials containing the IX Citrate buffer. When the knob was turned to 20 minutes, the steamer started and reached temperatures between 98°C-105°C. HIER treatment was performed, the slides were taken out and washed 3X 5 minutes in distilled water with shaking. CHP solution was prepared (5-20 pM of B- CHP, R-CHP, or F-CHP, 50-250 pL per section; 100 pL of Arg CHP; 100 pL of Cit CHP) in lx PBS or lx PBS containing 1% BSA (when co-staining with an antibody). Next, it was heated for 10 minutes in an 80°C water bath to dissociate the trimeric peptides, followed by immediate incubation in an ice / water bath (for 15-90 seconds depending on the solution volume) to quench the hot solution to room temperature. When needed, an antibody can be diluted into this quenched CHP solution for co-staining. The solution containing the quenched (cool) CHP monomers was quickly pipetted to each slide (usually 50-250 pL per section) within 1 minute.

[0268] To prepare slides for staining, a single slide was taken from the slide-staining jar containing water (the rest was left in water so they did not dry out) and the back and sides were dried with a kim wipe. Then the slide was shaken / flicked to remove as much water from the tissue section as possible without damaging them. After shaking, a kim wipe was carefully used to dry off areas between the tissue sections to remove excess water. A hydrophobic pen was used to outline the tissue sections (do not press down on the pen or the fluid will run over the slide and ruin the staining). 100 pL of CHP solution was added to each slide and tissue sections were incubated in a humidity chamber at 4°C overnight. After staining, the CHP solution was knocked off onto a paper towel by tapping it. Then, the DB1 / 161378021.4 MLB Docket No. 127033-5006-WO slides were washed 2x for 5 minutes in lx PBST (lx PBS +tween 20) at room temperature and shaken to remove unbound material and for the final washing step that used distilled water for 5 minutes.

[0269] Thetissue sections were subsequently incubated with AlexaFluor-labeled streptavidin (5-10 pg / ml) in a lx PBS solution containing 1% BSA. To detect the co-stained primary antibody, a labeled secondary antibody was diluted into the AlexaFluor647-streptavidin solution.The tissues were incubated with the streptavidin and / or secondary antibody solution in a humidity chamber for 1 h at room temperature. After staining with the dye or secondary antibody, the streptavidin dye solution was knocked off onto a paper towel by tapping it. Then, the slides were washed 2x for 5 minutes in lx PBST (lx PBS +tween 20) at room temperature with shaking to remove unbound material and for the final washing step, using distilled water for 5 minutes. The slides were left in water while the sections were coverslipped one at a time using fluoroshield mounting media with DAPI. This stains the cell nuclei blue and allows reversible coverslip mounting. One slide was taken out from the water and the back and sides were dried with a kim wipe. The slide was shaken / flicked to remove as much water from the tissue section as possible without damaging it. After shaking, a kim-wipe was used to carefully dry off areas between the tissue sections to remove excess water. Once the slide was dried, a single small drop of fluoroshield and DAPI was applied to each section. Then the solution was added to the coverslip. The slide containing the tissue section was then lowered onto the coverslip containing the fluoroshield and DAPI. Once coverslipped, the slide was allowed to mount for 20-30 minutes covered from light at room temperature before imaging. Slides were imaged with a fluorescent microscope.

[0270] For succinct methods, slides were washed with xylenes and ethanol and heated in citrate buffer using a tissue steamer. Then CHP solution was applied and incubated overnight. Once the slides were washed, streptavidin was applied to conjugate to biotin.

[0271] As shown in Figure 5, both that Arg and Cit CHPs have the capability of binding to damaged collagen.

[0272] Experimental Example 8: Gelatin Binding

[0273] This example describes that binding of arginated and citrullinated CHPs to gelatin.

[0274] 96-well black opaque Nunc Amino flat-bottom plate was coated with lOOpg / mL NEUTRAL pH gelatin solution in PBS with magnesium chloride and calcium chloride. pH was approximately 8 with pH strips before plating. IOOUL per well was added according to the plate map. The plate was covered with packing tape to reduce evaporation and spillover. It was then incubated on a shaker at room temperature for 1 hour. After the plates were coated, the appropriate wells were crosslinked in a 250: 100: 1 ratio of EDC:NHS:COOH. 20uL of crosslinkcr per well at room temperature for 1 hour (229 mg EDC, 55 mg NHS in 8 mL of 75% EtOH / 25% MES in pH 5.5). Plates were washed with TBST2 3x DB1 / 161378021.4 MLB Docket No. 127033-5006-WO

[0275] 200 JJ.L and further blocked with 1% BSA in TBST2 (200pL per well added for 30 minutes at RT). Plate wells were then washed with TBST2 3x (200uL per well). CHPs were added and were bound overnight at 37°C. Controls included IX PBS and 200 uM biotin, along with 200 uM BCHP and 200 uM of the CHP LAIR2 L scramble. Plates were washed 3x with lx TBST2 at room temperature, then incubated with AlexaFluor-labeled (488) streptavidin (1 pg / ml) in a lx TBST2 solution containing 1% BSA, lOOuL per well fori hour in the dark. Diluted streptavidin (320 uL lOug / mL streptavidin into 2.88 mL lx TBST) was used. Slides were washed with TBST2 for 3 times and imaged the plate with plate reader for a 488-labeled secondary antibody. Statistics were calculated using a One-way ANOVA and a Dunnett’s multiple comparisons test. Tire gelatin group was used as a control, ns: not significant, **** p < .0001.

[0276] As shown in Figure 6, Arg CHP and Cit CHP bound to gelatin better than it binds to itself and bound well compared to thebenchmark B-CHP. Specifically, Arg CHP (R) bound 5.5x better than gelatin and Cit CHP (Cit) bound 4.8x better than gelatin. The result demonstrated that Arg CHP and Cit CHP bind to gelatin comparable to B-CHP, and better than gelatin binding to itself.

[0277] Experimental Example 9: LAIR1 Binding

[0278] This example describes that binding of arginated and citrullinated CHPs to LAIR1.

[0279] Stock solutions were prepared with 2mg / mL BSA in PBS, 2mg / mL gelatin in PBS. Img / mL or 0.5mg / mL human collagen in 0.5M Acetic acid, IX PBS, lx PBS-Tween20 (PBST), 1% (w / v) BSA in PBST, and 5% (w / v) BSA in PBS.

[0280] 96-well black opaque Nunc Amino flat-bottom plate was coated with 1 OOpg / mL NEUTRAL pH protein solution in PBS. pH was approximately 8 with pH strips before plating. lOOuL per well was added according to the plate map and the plate was covered with packing tape to reduce evaporation and spillover, then incubated on a shaker at room temperature for 1-2 hours. Protein solutions were prepared according to Tables 5-7.

[0281] Table 5. Collagen I from human placenta (neutralize with -50% volume of collagen stock with IN NaOH)

[0282] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO

[0283] Table 7. BSA

[0284] Removed protein solution by flicking plate into the sink and washed with PBST 4x 200pL, then patted plate on a paper towel between washes to remove all liquid. Removed all PBST and fixed wells with 150uL of 100% ice-cold methanol (stored in -20°C). Incubated at -20°C for 30 minutes. Removed methanol and washed 4x 200uL PBS. patting plate on a paper towel between washes to remove all liquid. Removed all PBS and added block amine reactive plate surface with IM TRIS-HCL for 30 minutes at RT on a shaker (200pL per well). Removed TRIS-HC1 and washed with 4x 200uL PBS. Flicked and smacked wells dry on paper towel between washes. Turned on water bath and set to 80°C for later. Removed PBS and further blocked with 200pL 1% BSA in PBS for 30 minutes at RT. Removed blocking solution and washed wells with PBS 4x 200uL per well. Flicked and smackede wells dry on paper towel between washes. Heated the peptide solutions at 80°C for 10 min. Quenched in ice water until cool to touch (room temperature) and added 50 pL of 200 pM of CHPs in lx PBS or PBS alone for control samples. Covered with tape and incubated at 4°C overnight to allow CHPs to bind.

[0285] Removed CHP solution and washed each well 3x with 200uL lx PBS for 10 minutes each on a shaker. Patted plate on a paper towel in between washes to remove all liquid from each wash. Removed PBS and replaced with 1: 100 dilution of recombinant human LAIR1 Fc Chimera protein in 1% BSA in PBST (5pg / mL final from 500pg / mL stock in -20°C. IOOUL per well). Made sure liquid covered all of the well surface. Tapped the sides of the plate as needed to cover well bottom completely. Incubated at room temperature for 1 hour to allow LAIR protein to bind. Made a fresh LAIR1 - Reconstituted 50 ug in 100 uL lx PBS to make 500 ug / mL solution. Then, mixed 35 uL of the 500 ug / mL stock solution with 3.465 mL BSA in PBST to make a 5 ug / mL solution at 3.5 mL. Removed LAIR solution from wells then washed with 200uL PBS 4x. Flicked and smacked wells dry on paper towel between washes. Removed PBS and replaced with 1:200 dilution of anti-human IgG Alexa Fluor 488 antibody (stored in 4°C) in 1% BSA in PBST (5pg / mL from Img / mL stock) (lOOpL per well). Incubated for 1 hour at RT DB1 / 161378021.4 MLB Docket No. 127033-5006-WO covered in foil. Protected plate from light from this point forward. Removed antibody solution and washed wells with PBS 4x 200uL. Flicked and smacked wells dry on paper towel between washes. Removed all liquid from wells and patted dry on a paper towel. Once dry, it was imaged on Spectra Max iD3 plate.

[0286] As shown in Figure 7, Arg CHP and Cit CHP bind better than gelatin to LAIR1. Statistics were calculated using a One-way ANOVA and a Dunnett’s multiple comparisons test, p < .01, *** p < .001.

[0287] Experimental Example 10: IL-2Cit / Arg CHP and Citrullinated Collagen T Cells

[0288] This example describes IL-2 Production in T Cells with arginated and citrullinated CHP.

[0289] A 2mg / mL solution of porcine gelatin was prepared in media and the solution was incubated at 37°C in the water bath. Vortexed often until gelatin dissolved completely. Filter was sterilized with a 0.2um filter. Stored at 4°C until ready for use. CHP stocks were prepared by heating the stock CHPs at 80°C for 10 minutes to force into monomeric form and sterilized with a 0.2um filter by syringe. Then, CHPs, gelatin, and media were combined and CHP and gelatin solutions were incubated at 37°C in the cell culture incubator for 72 hours to allow CHPs to hybridize with gelatin. PMA stock was diluted to 1:500 in media to bring a 5mg / mL solution to lOug / mL. Activated T cell plates were coated with the anti-CD3 antibody at 3 pg / mL in 2 mL and incubated at 37°C in 5% CO2 for 2 hours. Stock cell solution was prepared by counting and resuspending Jurkat E6-1 at 9xl0bper mL. Cells, media, gelatin solution, PMA antibody and other drugs were combined and plated 3mL per well in the coated 6 well plate. Final concentrations of cells were 300,000 / mL, PMA was 10 ng / mL, and gelatin was 0.5 mg / mL. Plates were inoculated for 3 days to allow for activation. Then, media was collected for IL -2 ELISA (without any dilution). 250uL of cell suspension were collected, centrifuged at 2000xg for 10 minutes at RT, transferred to a fresh tube and frozen at -80°C until ready for ELISA. As shown in Figure 8, treatment of activated Jurkat cells with Arg CHP reduces IL-2, and treatment with Cit CHP partially rescues these IL-2 levels. Viability was measured using Alamar Blue. Jurkat cells were treated with Arg, and Cit CHP do not have growth reduction compared to the positive control (Figure 9).

[0290] Experimental Example 11: Serum Stability of FLAIR3 and Cit-FLAIR3

[0291] This example describes serum stability of FLAIR3 and Cit-FLAIR3.

[0292] Sequences used in the Experimental Examples 11 and 12 were shown on Table 8.

[0293] Table 8.

[0294] DBl / 161378021.4 MLB Docket No. 127033-5006-WO

[0295] Peptide stock solutions were prepared in 50 pL deionized water at a concentration of 1 mM. Then, mouse serum was diluted to 25% by mixing 750 pL of 1 x PBS and 250 pL of serum. Tire reaction mixture was prepared to final concentration of 50 pM of peptide by adding 50 pL of peptide stock solution to 950 pL of diluted serum solution. Peptide was heated to 80 °C for 10 min followed by quenching on ice for ~1 min to break up any triple helix formation prior to mixing with the serum. The Mixture was placed in an incubator at 37 °C. A 100 pL aliquot of the peptide-serum mixture were removed at specified time points (0, 0.5, 1, 2, 4, 6, 8, and 24 h), and 200 pL of ice-chilled ethanol were added. Samples were vortexed and centrifugated at 13,800g for 2 min at 4 °C to precipitate the serum proteins. Then, 150 pL of the supernatant were transferred and placed it in a HPLC vial so that tire proteins could not interact with the peptides in solution. Sample were ran on an RP-HPLC with UV / Vis capabilities. UV detection was at 214nm. To determine the level of degradation, use the integration of the area under the HPLC peak corresponding to the intact peptide. The peak area for ’’zcro' -minutc time point is designated as 100% peptide and each peak area at successive time points was normalized to the zero-minute peak in order to calculate the percent peptide remaining.

[0296] As shown on Figure 10, the % of original peptide remaining at the indicated time points. % remaining was calculated as Area Under Curve (AUC) compared to the zero time point.

[0297] Experimental Example 12: LAIR1 Binding Assay of FLAIR3 and Cit-FLAIR3

[0298] This example describes LAIR1 binding assay of FLAIR3 and Cit-FLAIR3.

[0299] A 96-well black opaque Nunc Amino flat-bottom plate was coated with lOOpg / mL neutral pH protein solution in PBS. pH was approximately 8 with pH strips before plating. Samples were added lOOuL per well according to plate map. Plate was covered with packing tape to reduce and the protein solution was removed by flicking plate into the sink. Samples were washed with PBST 4x 200pL, patting plate on a paper towel between washes to remove all liquid. Samples were further washed with 4x 200uL PBS. Flick and smack wells dry on paper towel between washes. PBS wea removed and further blocked with 200pL 1% BSA in PBS for 30 minutes at RT. The blocking solution was removed and washed wells with PBS 4x 200uL per well. Flick and smack wells dry on paper towel between washes. The peptide solutions were heated at 80 °C for 10 min, quenched in ice water until cool to touch (room temperature) and 50 pL of 200 pM of CHPs in lx PBS or PBS alone for control samples were added. The plate was covered with tape and incubated at 4C for 3 days to allow CHPs to bind. Then. CHP solution was removed and washed each well 3x with 200uL lx PBS for 10 minutes each on a shaker. Pat plate on a paper towel in between washes to remove all liquid each wash. PBS was removed and replaced with 1: 100 dilution of recombinant human LAIR1 Fc Chimera protein in 1% BSA in PBST (5pg / mL final from 500pg / mL stock in -20C, lOOuL per well). Tap the sides of the plate as needed to

[0300] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO cover well botom completely. The plate was incubate at room temp for 1 hour to allow LAIR protein to bind. LAIR solution was removed from wells then wash with 200uL PBS 4x. Flick and smack wells dry on paper towel between washes. PBS solution was removed and replaced with 1:200 dilution of antihuman IgG Alexa Fluor 488 antibody (stored in 4C) in 1% BSA in PBST (5pg / mL from Img / mL stock)(100pL per well). Plate was incubate for 1 hour at RT covered in foil, protected from light from this point forward. An antibody solution was removed and washed wells with PBS 4x 200uL. Flick and smack wells dry on paper towel between washes. All liquid were removed from wells and pat dry on a paper towel. Once dry. imaged on Spectra Max iD3 plate

[0301] As shown on Figure 11, data shows that Cit-FLAIR3 is more stable in serum compared to FLAIR3, and that Cit-FLAIR3 binds significantly beter to LAIR1 than the gelatin control. The background signal of gelatin or CHP binding to the Alexa Fluor Antibody was subtracted from the experimental groups to obtain the Relative Fluorescence Units (RFU) (Background Subtracted) Statistics were calculated using a One-Way ANOVA with an Uncorrected Fisher’s LSD. * p < .05, ** p < .01.

[0302] Unless otherwise specified, it should be understood that all numbers expressing quantities of ingredients, properties, such as molecular weights and reaction conditions, used in this disclosure and claims are modified in all cases by the tenn “about.” Accordingly, unless otherwise specified, the numerical parameters set forth in this disclosure and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an atempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed by applying ordinary' rounding techniques in consideration of the number of significant digits reported.

[0303] Notwithstanding that the numerical ranges and parameters seting forth the broad scope of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0304] At the ve ' least, and not as an atempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0305] The grouping of alternative elements or embodiments of the present disclosure should not be construed as limiting. Each group member may be referred to and claimed individually or in combination yvith the other members of the group or other elements herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the disclosure is deemed to satisfy

[0306] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO all Markush group descriptions used in the appended claims, including the modified group.

[0307] Specific embodiments of the present disclosure including the best mode known to the present inventors for carrying out the present disclosure are described herein. Of course, variations of the described embodiments will be apparent to those skilled in the art who read the foregoing descriptions. The present inventors expect that those skilled in the art employ such variations as appropriate and intend that the present disclosure is implemented otherwise than as specifically described herein. Accordingly, the present disclosure encompasses all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by the corresponding law. Furthermore, any combination of all possible variations of the elements described above is encompassed by the present disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

[0308] The specific embodiments disclosed herein may be further limited in the claims by the use of the phrases “consisting of’ or “consisting essentially of.” When used in the claims, whether as filed or added by amendment, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of’ limits the scope of the claim to those materials or steps specified and those that do not substantially affect the basic and novel characteristic(s). The embodiments of the present disclosure so claimed are essentially or explicitly described and enabled herein.

[0309] It should be understood that the embodiments of the present disclosure disclosed herein are illustrative of the principles of the present disclosure. Other modifications that may be employed fall within the scope of the present disclosure. Thus, by way of example, and not limitation, alternative configurations of the present disclosure may be employed in accordance with the teachings herein. Accordingly, the present disclosure is not limited to what has been precisely set forth and described.

[0310] While the present disclosure has been described and illustrated herein with reference to various specific materials, procedures, and examples, it should be understood that the present disclosure is not limited to the specific combinations of materials and procedures selected for the purposes for which they are intended. Many variations of these details are contemplated, as will be appreciated by those skilled in the art. It is intended that the disclosure and examples be considered illustrative only, with the true scope and spirit of the present disclosure being indicated by the claims that follow. All references, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

[0311] From the foregoing description, it will be apparent to those skilled in the art that the present disclosure may be implemented in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above arc illustrative in all respects and not restrictive. The scope of the present disclosure should be construed as encompassing within the scope of the present disclosure all changes or modifications

[0312] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO derived from the meaning and scope of the following claims and equivalents thereof, rather than the foregoing detailed description.

[0313] EMBODIMENTS

[0314] Embodiment 1. A collagen hybridizing peptide (CHP) comprising a sequence represented by Formula I: a. Ac-S-(Gly-X-Y)n-bioactive-(Gly-X-Y)m(Formula I) b. in which Ac is an acetyl capping group: S is zero or more spacer molecules; X is proline or modified proline; Gly is glycine; Y is hydroxyproline or any other amino acid; n is an integer from 0 to 20; m is an integer from 0 to 20; and c. the bioactive is an amino acid sequence from 3 to 27 amino acid residues having bioactivity, wherein the bioactive comprises at least one leukocyte-associated immunoglobulin-like receptor (LAIR-1 and / or LAIR-2) binding sequence, and wherein the amino acid sequence of the bioactive includes at least one citrulline residue.

[0315] Embodiment 2. The CHP according to embodiment 1, wherein the at least one bioactive comprises a LAIR- 1 binding sequence.

[0316] Embodiment 3. Tire CHP according to embodiment 1, wherein the at least one bioactive comprises a LAIR-2 binding sequence.

[0317] Embodiment 4. The CHP according to any one of the preceding embodiments, wherein the at least one bioactive comprises a LAIR-1 and / or LAIR-2 binding sequence comprising a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-27, 56-64, 93, 94, 98-100 and 107-187.

[0318] Embodiment 5. Tire CHP according to any one of the preceding embodiments, wherein the

[0319] CHP comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1- 27, 56-64. 93. 94. 98-100 and 107-187.

[0320] Embodiment 6. The CHP according to any one of the preceding embodiments, wherein the CHP comprises a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1- 27, 56-64, 93, 94, 98-100 and 107-187.

[0321] Embodiment 7. Tire CHP according to any one of the preceding embodiments, wherein the CHP comprises a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 1- 27, 56-64. 93. 94. 98-100 and 107-187.

[0322] Embodiment 8. The CHP according to any one of the preceding embodiments, wherein the CHP comprises a sequence selected from the group consisting of SEQ ID NOs: 107-157.

[0323] Embodiment 9. The CHP according to any one of the preceding embodiments, wherein S is greater than 0.

[0324] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO

[0325] Embodiment 10. The CHP according to any one of the preceding embodiments, wherein the CHP is a first CHP, wherein the first CHP is bound to one other bioactive collagen hybridizing peptide to form a dimeric structure.

[0326] Embodiment 11. The CHP according to any one of the preceding embodiments, wherein the CHP is a first CHP, wherein the first CHP is bound to two other bioactive collagen hybridizing peptides to form a trimeric structure; and wherein the first CHP has a monomeric bioactive portion extending beyond the trimeric structure, the monomeric portion configured to bind to collagen alpha chains.

[0327] Embodiment 12. The CHP according to any one of the preceding embodiments, wherein the CHP is a first CHP, wherein the first CHP is bound to two other bioactive collagen hybridizing peptides to form a trimeric structure, and wherein a monomeric ‘Tail” CHP is conjugated to an N -terminal or a C-terminal of the first CHP of the trimeric structure using copper-free click chemistry or condensation reactions.

[0328] Embodiment 13. A composition comprising the CHP of any one of the preceding embodiments.

[0329] Embodiment 14. Tire composition according to embodiment 13, wherein each individual CHP does not form a triple helix with other CHPs.

[0330] Embodiment 15. The composition according to embodiment 13 or embodiment 14. further comprising a carrier.

[0331] Embodiment 16. The composition according to any one of embodiments 13-15, wherein the carrier comprises at least one selected from the group consisting of micelles, dendrites, lipids, microemulsions, nanoemulsions, solid lipid nanoparticles, polymers, gels, lens, surfactants, cyclodextrins, inserts, nanostructured lipid carriers, liposomes, transfersomes, ethosomes, niosomes. collagen matrix, extracellular matrix, and artificial extracellular matrix.

[0332] Embodiment 17. The composition according to any one of embodiments 13-16, wherein the composition comprises at least one selected from the group consisting of a topical cream, a saline solution, gel, polymer, or a solution for systemic injections within a subject.

[0333] Embodiment 18. Tire composition according to any one of embodiments 13-17. in which a CHP is bound directly to collagen matrix.

[0334] Embodiment 19. A method of reducing or preventing downregulation of immune cells in a subject, comprising administering the CHP of any one of embodiments 1-12 or the composition of any one of embodiments 13-18.

[0335] Embodiment 20. The method according to embodiment 19, wherein the administration is performed by injection.

[0336] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO

[0337] Embodiment 21. The method according to embodiment 20, wherein the administration is performed by micro-dermal injection.

[0338] Embodiment 22. The method according to embodiment 20, wherein the administration is performed by intravenous injection.

[0339] Embodiment 23. Hie method according to embodiment 20, wherein the administration is performed by intratumoral injection.

[0340] Embodiment 24. The method according to embodiment 20, wherein the administration is performed by subcutaneous injection.

[0341] Embodiment 25. The method according to embodiment 19, wherein the administration is performed by topical application.

[0342] Embodiment 26. The method according to embodiment 19, wherein the administration is oral administration.

[0343] Embodiment 27. A method of treating cancer in a subject, comprising administering the CHP of any one of embodiments 1-12 or the composition of any one of embodiments 13-18.

[0344] Embodiment 28. The method according to embodiment 27, wherein the administration is performed by injection.

[0345] Embodiment 29. The method according to embodiment 28, wherein the administration is performed by micro-dermal injection.

[0346] Embodiment 30. The method according to embodiment 28, wherein the administration is performed by intravenous injection.

[0347] Embodiment 31. The method according to embodiment 28, wherein the administration is performed by intratumoral injection.

[0348] Embodiment 32. The method according to embodiment 28, wherein the administration is performed by subcutaneous injection.

[0349] Embodiment 33. The method according to embodiment 27, wherein the administration is performed by topical application.

[0350] Embodiment 34. The method according to embodiment 27, wherein the administration is oral administration.

[0351] Embodiment 35. The method according to any one of embodiments 27-34, wherein the CHP is administered in combination with an immunotherapy.

[0352] Embodiment 36. The method according to any one of embodiments 27-35, wherein the immunotherapy includes immune checkpoint blockade therapy.

[0353] Embodiment 37. The method according to any one of embodiments 27-36, wherein the CHP is administered in combination with a chimeric antigen therapy.

[0354] DB1 / 161378021.4 MLB Docket No. 127033-5006-WO

[0355] Embodiment 38. The method according to any one of embodiments 27-37, wherein the chimeric antigen therapy includes chimeric antigen receptor T-cell (CAR-T) therapy.

[0356] Embodiment 39. The method according to any one of embodiments 27-38, wherein the chimeric antigen therapy includes one or more of chimeric antigen receptor macrophage therapy, chimeric antigen receptor NK cell therapy, and chimeric antigen receptor dendritic cell therapy.

[0357] Embodiment 40. The method according to any one of embodiments 27-39, wherein the cancer comprises one or more of lung cancer, colorectal cancer, ovarian cancer, hematological cancer, breast cancer, pancreatic ductal cell carcinoma, oral squamous cell carcinoma, and melanoma.

[0358] Embodiment 41. The method according to any one of embodiments 27-39, wherein the cancer comprises multiple myeloma.

[0359] Embodiment 42. A pharmaceutical composition for reducing or preventing downregulation of immune cells comprising the CHP according to any one of embodiments 1-12 as an active ingredient.

[0360] Embodiment 43. A pharmaceutical composition for preventing or treating cancer comprising the CHP according to any one of embodiments 1-12 as an active ingredient.

[0361] Embodiment 44. The CHP according to any one of embodiments 1 to 12, wherein the CHP comprises a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 188- 219.

[0362] Embodiment 45. The CHP according to any one of embodiments 1 to 12, wherein the CHP comprises a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 220- 226.

[0363] Embodiment 46. The method according to any one of embodiments 27-29, wherein the cancer comprises at least one selected from the group consisting of bladder cancer, bone cancer, brain tumors, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, kidney cancer, leukemia, liver cancer, various lymphomas, such as nonHodgkin lymphoma, melanoma, non-melanoma skin cancer, ovarian cancer, prostate cancer, testicular cancer, uterine cancer, sarcomas and myelomas.

[0364] Embodiment 47. A method of increasing a level of citrulines in an area of disease with collagen present comprising administering the CHP of any one of embodiments 1 to 12 or the composition of embodiments 13 to 18.

[0365] DB1 / 161378021.4

Claims

MLB Docket No. 127033-5006-WOCLAIMS1 . A collagen hybridizing peptide (CHP) comprising a sequence represented by Formula I: Ac-S-(Gly-X-Y)n-bioactive-(Gly-X-Y)m (Formula I) in which Ac is an acetyl capping group; S is 0 or more spacer molecules; X is proline or modified proline; Gly is glycine; Y is hydroxyproline or any other amino acid; n is an integer from 0 to 20; m is an integer from 0 to 20; and the bioactive is an amino acid sequence from 3 to 27 amino acid residues having bioactivity, wherein the bioactive comprises at least one leukocyte-associated immunoglobulin-like receptor (LAIR-1 and / or LAIR-2) binding sequence, and wherein the amino acid sequence of the bioactive includes at least one citrulline residue.

2. The CHP according to claim 1, wherein the at least one bioactive comprises a LAIR-1 and / or LAIR-2 binding sequence comprising a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-27, 56-64, 93, 94, 98-100 and 107-187.

3. The CHP according to claim 1 or 2, wherein the CHP comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-27, 56-64, 93, 94, 98-100 and 107-187.

4. The CHP according to any one of the preceding claims, wherein the CHP comprises a sequence selected from the group consisting of SEQ ID NO: 107-157.

5. The CHP according to claim 1 or 2, wherein the CHP comprises a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 188-219.

6. The CHP according to claim 1 or 2, wherein the CHP comprises a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 220-226.

5. The CHP according to any one of the preceding claims, wherein the CHP is a first CHP, wherein the first CHP is bound to one or more other bioactive collagen hybridizing peptides to form a multimeric structure.

6. A composition comprising the CHP of any one of the preceding claims.DB1 / 161378021.4MLB Docket No. 127033-5006-WO7. The composition according to claim 6, further comprising a carrier.

8. A method of reducing or preventing downregulation of immune cells in a subject, comprising administering the CHP of any one of claims 1 to 5 or the composition of claim 6 or 7.

9. A method of treating cancer in a subject, comprising administering the CHP of any one of claims 1 to 5 or the composition of claim 6 or 7.

10. A pharmaceutical composition for reducing or preventing downregulation of immune cells comprising the CHP of any one of claims 1 to 5 as an active ingredient.11 . A pharmaceutical composition for preventing or treating cancer comprising CHP of any one of claims 1 to 5 as an active ingredient.

12. The method according to claim 9, wherein the cancer comprises at least one selected from the group consisting of bladder cancer, bone cancer, brain tumors, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, kidney cancer, leukemia, liver cancer, various lymphomas, such as non-Hodgkin lymphoma, melanoma, non-melanoma skin cancer, ovarian cancer, prostate cancer, testicular cancer, uterine cancer, sarcomas and myelomas.

13. A method of increasing a level of citrulines in an area of disease with collagen present comprising administering the CHP of any one of claims 1 to 5 or the composition of claim 6 or 7.DB1 / 161378021.4

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