Fatty acid and cyclized derivatives of the immunomodulatory peptide rp-182 with improved Anti-tumor activity, compositions, and methods of use thereof

Abstract

Aspects of the present disclosure relate to immunomodulatory peptides that modulate CD206 and modulate macrophage activity, compositions containing one or more of the immunomodulatory peptides, and methods of their use.

Description

FATTY ACID AND CYCLIZED DERIVATIVES OF THE IMMUNOMODULATORYPEPTIDE RP-182 WITH IMPROVED ANTI-TUMOR ACTIVITY, COMPOSITIONS,AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional PatentApplication no. 63 / 656535, filed June 5, 2024, which is hereby incorporated herein byreference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH ORDEVELOPMENT

[0002] This invention was made with Government support under contract no. ZIA ВС011267 awarded by the National Cancer Institute, Center for Cancer Research (part of theNational Institutes of Health); and contract no. 1ZIATR000302, awarded by National Centerfor Advancing Translational Sciences (part of the National Institutes of Health). Thegovernment has certain rights in the invention.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (23-1713-US-PRO_ST26_Sequence_Listing.xml; Size: 1,882 bytes; and Date of Creation: June 5, 2024) isherein incorporated by reference in its entirety.FIELD

[0004] The present disclosure is directed to immunomodulatory peptides, and moreparticularly to peptide analog compounds and compositions that modulate CD206 andmodulate macrophage activity, as well as methods of their use and preparation, for thetreatment and / or prevention of solid organ cancer malignancies and to treat and amelioratetissue fibrosis, e.g., lung fibrosis.BACKGROUND

[0005] The innate immune system plays a central role in certain cancers. An abundanceof tumor-educated myeloid immune cells within the tumor microenvironment (TME),including tumor associated macrophages (TAMs), exercise a number of immune suppressive,tumor-promoting functions. Thus, a number of therapeutic strategies that aim to reducetumor-promoting myeloid cell subpopulations or reprogram these cells towards an anti-tumorphenotype have been pursued, thus far without clinical success. One approach is activationof the mannose receptor 1 (Mrc1 or CD206) that targets immune suppressive CD206high M2-like TAMs, which have been identified as a cell population associated with shortenedsurvival, pro-tumor and pro-metastasis function across different solid organ cancers. A tenamino acid therapeutic peptide (RP-182) derived from a conserved motif in naturallyoccurring host defense peptides (HDPs) that binds to the carbohydrate recognition domain 5(CRD5) of the CD206 receptor is known. RP-182 induces a conformational switch thatactivates NF-kB signaling and phagocytosis in CD206high TAMs. RP-182, has dual function:activation of canonical NF-kB signaling triggers TNFa secretion and autocrine activation ofthe tumor necrosis factor receptor 1 (TNFR1), activation of caspase 8, apoptosis and celldeath of CD206high M2-like human (and murine) macrophages, including CD206high TAMs.In cells that do not undergo apoptosis, RP-182, induces phagocytosis, autophagy, andreprogramming towards an M1-like phenotype characterized by secretion of proinflammatorycytokines and induced cancer cell phagocytosis. Despite its activity, RP-182 suffers fromsignificant problems, including enzymatic degradation, renal loss, and high depot effects. Aplasma half-life of less than 10 minutes and concerns for atypical non-IgE-mediatedanaphylaxis make RP-182 an unlikely drug candidate. Thus, a significant need exists fortreatments that target immune suppressive CD206-positive M2-like TAMs.SUMMARY

[0006] Described herein are peptide-based modulators targeting the CD206 receptor,compositions containing the described compounds, methods of using the describedcompounds, and their methods of manufacture.

[0007] In an aspect, a compound of Formula 1 is provided:X-Phe-Arg-Lys-Ala-Phe-Lys-Arg-Phe / H2NFormula I

[0008] or a pharmaceutically acceptable salt thereof, wherein X is H; and Y isrepresented by Formula la:

[0009] wherein PEG is polyethylene glycol according to the following:

[0010] where Q is 0 to 4; R is 0 or 1; S is 0 or 1; V is absent or C6-C20alkyl, C6-C20alkenyl, or C6-C20alkynyl; T is 0 or 1; U is C6-C20alkyl, C6-C20alkenyl, or C6-C20alkynyl;or X and Y are linked to form a cyclic compound, where X and Y are represented by Formula1b or Formula 1c, where Formula 1b is represented as follows:-C-PEG-A-B-PEG-C-Lys-Formula 1b

[0011] where for Formula 1b: A is a methylcarbamate, N-methyl carbamate, N-ethylcarbamate, N-methyl methylcarbamate, or N-ethyl methylcarbamate; B is a 12-Methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene; 12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 5-Methyl-12-methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 5-Methyl-12-ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; and where A comprises a non-carbonyl oxygen(hydroxyl oxygen); and B comprises a cyclopropyl group; and at least one carbon separatesthe non-carbonyl oxygen on the carbamate and the cyclopropyl group; PEGm is polyethyleneglycol represented as follows:

[012] where m is 1 or 2; and PEGn is polyethylene glycol represented as follows:

[0013] where n is 1 or 2; and wherein X corresponds to the carbonyl end of Formula 1band Y corresponds to the -Lys- end of Formula 1b; and the Lys in Formula 1b binds to thecarbonyl carbon (C=O) through a Lys sidechain nitrogen;

[0014] or

[0015] X and Y are linked to form a cyclic compound, where X and Y are represented byFormula 1c:-C-PEGm-D-E-PEG-C-Lys-Formula 1c

[0016] wherein for Formula 1c: D is a 12-Methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 5-Methyl-12-methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 5-Methyl-12-ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; E is a methylcarbamate, N-methyl carbamate,N-ethyl carbamate, N-methyl methylcarbamate, or N-ethyl methylcarbamate; and where Dcomprises a cyclopropyl group; and E comprises a non-carbonyl oxygen (hydroxyl oxygen);and at least one carbon separates the non-carbonyl oxygen on the carbamate and thecyclopropyl group; PEGm is polyethylene glycol represented as follows:

[0017] m is 1 or 2; and PEGn is polyethylene glycol represented as follows:

[0018] where n is 1 or 2; and wherein X corresponds to the carbonyl end of Formula 1cand Y corresponds to the -Lys- end of Formula 1c; and the Lys in Formula 1c binds to thecarbonyl carbon (C=O) through a Lys sidechain nitrogen.

[0019] In an aspect, pharmaceutical compositions comprising a compound or salt ofFormula I with a pharmaceutically acceptable carrier are also provided.

[0020] In another aspect, methods for the treatment of cancer are provided that involveselective targeting of M2 macrophages, e.g., for programmed cell death) and / or thereprogramming of M2 macrophages towards a M1 phenotype in a patient, including the stepof administering to the patient in need thereof a compound of Formula I or a salt thereof, arealso provided.

[0021] In various embodiments targeting CD206 M2 macrophages with compound or saltof Formula I may have a dual effect of reprogramming CD206 M2 macrophages into M1macrophages and directly killing a M2 macrophage (e.g., by programmed cell death).

[0022] In another aspect, methods for the treatment of cancer characterized by thepresence of CD206 positive tumor-associated macrophages (TAM), such as glioma(glioblastoma), sarcoma, astrocytoma, melanoma, non-small cell lung cancer,cholangiocarcinoma, colon cancer, hepatocellular, breast, prostate, gastric, renal cell,endometrial, or pancreatic cancer are also provided, including administering a therapeuticallyeffective amount of a compound or salt of Formula I to a patient in need of such treatment.In various embodiments, the methods further include administering to a patient in needthereof at least one additional therapeutic agent.

[0023] In another aspect, methods for modulating macrophage activity are provided, inwhich a macrophage is contacted with a compound (or salt or composition thereof) ofFormula I to modulate activity of the macrophage. In some embodiments, the activity that ismodulated is, e.g., macrophage polarization, and in embodiments, the macrophage is an M2macrophage or a tumor associated macrophage (TAM).

[0024] In another aspect, methods of treating a subject for a condition associated withchronic inflammation are provided, the methods including administering an effective amountof a therapeutic agent to a patient in need thereof to treat the patient for the conditionassociated with chronic inflammation, the, wherein the therapeutic agent is a compound (orsalt or composition thereof) of Formula I.

[0025] In some embodiments, the condition associated with chronic inflammationincludes one or more of scleroderma or multiple sclerosis, irritable bowel disease, ulcerativecolitis, colitis, Crohn's disease, idiopathic pulmonary fibrosis, asthma, keratitis, arthritis,osteoarthritis, rheumatoid arthritis, auto-immune diseases, a feline or humanimmunodeficiency virus (FIV or HIV) infection, cancer, age-related inflammation and / orstem cell dysfunction, graft-versus-host disease (GVHD), keloids, obesity, diabetes, diabeticwounds, other chronic wounds, atherosclerosis, Parkinson's disease, Alzheimer's disease,macular degeneration, gout, gastric ulcers, gastritis, mucositis, toxoplasmosis, and chronicviral, and microbial infections. In various embodiments, the methods further includeadministering to a patient in need thereof at least one additional therapeutic agent known tobe effective in treating the condition associated with chronic inflammation.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The features, objects and advantages other than those set forth above will becomemore readily apparent when consideration is given to the detailed description below. Suchdetailed description makes reference to the following drawings:

[0027] FIGS. 1A-1D illustrate RP-182 pharmacophore of cyclic derivative 1c retainsbinding and engagement with select CRD5 residues of the CD206 receptor. FIG. 1A showsthe chemical structure of 10mer peptide RP-182. FIG. 1B shows PyMOL three-dimensionalmodel of RP-182, depicting amphipathic surface topology (hydrophobic surfaces shown ingolden-grey, cationic surfaces in blue color). Whole-residue hydrophobicity scale indictingHydrophobicity AG (water to bilayer; in kcal / mol) on right. FIG. 1C shows PyMOL modelof RP-182 and cyclic analogue 1c binding to hydrophobic groove on CRD5 region of CD206.Green rings indicate the position of benzyl groups of phenyl alanine, green tail the guanidinogroup of arginine with deprotonated carboxylic acid in blue within occupied chemical space.FIG. 1D illustrates the enhanced binding of therapeutic peptides after fatty acidderivatization (left) and conjugation to lipid-albumin-binding peptide chimera (right).

[0028] FIGS. 2A-2G illustrate lipid-derivatized and cyclic RP-182 analogues selectivelyinduce apoptosis and cell death in CD206high M2-polarized macrophages. FIGS. 2A and 2Bshow cell viability of M1-polarized (“M1”) and M2-polarized (“M2”) primary murinemacrophages after 48 h dose-response testing [in M] of top hits after initial screening ofsynthesized analogues, along with chemical structures of those analogs. Data shown arerepresentative examples of N=3 independent experiments, in triplicates. FIG. 2C showsimmunofluorescence of M2-polarized macrophages stained with anti-cleaved caspase 8 afterindicated treatments for 24 hours. Nuclei stained with 4',6-diamidino-2-phenylindole (DAPI).Anti-cleaved caspase 8 total fluorescence normalized to DAPI in vehicle-treated cells was setto 1, quantification on right depicts relative change. At least 50 cells of N≥2 independentexperiments were quantified, error bars indicate SEM. FIGS. 2D and 2E show fractions oflate apoptosis and post- apoptotic necrosis after propidium iodide (PI) and annexin Vstaining in CD206high M2 BMDMs after 24h treatment with vehicle, RP-182, 1 and indicatedanalogues determined by flow cytometry. Representative flow cytometry plots shown in FIG.2D and the top portion of FIG. 2E; quantification of late apoptosis (annexin Vhigh / P[high) inCD206-positive CD45+F4 / 80+CD11b+Gr-1-M2-polarized macrophages after indicatedtreatments at bottom of FIG. 2E. N=2 experiments, in triplicates. FIG. 2F shows fold changeTNFa and IFNy secretion in conditioned media after 24 hours treatment of M2 macrophagesby ELISA. N≥2 independent experiments. FIG. 2G shows cell death of RP-182-treated M2-polarized macrophages is dependent on TNFα signaling, NF-κB, and CD206. Dose responsecurves in M2 wild-type and CD206-- knockout macrophages (left), and MyD88- / - and TNFα- / -macrophages (right) after 48 h treatment with indicated RP-182 analogues. CD206expression after polarization was confirmed in BMDMs derived from knockout models byflow cytometry.

[0029] FIGS. 3A-3H show RP-182 analogues effectively activate phagocytosis andinterferon response signaling in M2-polarized macrophages. FIG. 3A shows induction of thepan-phagocytosis marker RAB7 after treatment of CD206high M2-like macrophages. Graphshows relative induction of RAB7 fluorescence normalized to DAPI after 2 h treatment withindicated compounds after anti-RAB7 fluorescence of vehicle-treated cells was set at 1.Representative images shown on left, graph shows representative curves of N=2 independentexperiments. FIG. 3B shows induction of CD206 receptor internalization after treatment withRP-182 analogues. Multicolor confocal microscopy images after staining with depictedmarkers, linear distance analysis (in µm) on right. Cells were treated for 2 hours with 200 nMof indicated analogues. FIG. 3C shows detection of CD206-RAB7 proximity by proximalligation assay (PLA) in M2 BMDM's after 2h treatment with RP-182 analogues. PLA signal(red dots) after indicated treatments using anti-mouse RAB7 and anti-rabbit CD206antibodies. Quantification of in situ PLA signals on the right, signal of number of bright reddots in vehicle-treated cells was set at 1. FIG. 3D shows induction of phosphorylation ofinterferon response factor 7 (pIRF7) after 2 h treatment. FIG. 3E shows RP-182 analogueseffectively induce cancer cell phagocytosis. Phase contrast images of carboxyfluoresceinsuccinimidyl ester (CFSE)-labeled KPC cancer cells co-incubated with M2-polarizedmacrophages which were treated for 2 h with indicated agents. Inset shows a representativemacrophage with engulfed, green-labeled cancer cell and induced cytoplasmatic vacuoles andvesicles. FIG. 3F shows CFSE-positive fractions in CD206+ BMDMs by flow cytometryafter coculture of CFSE-labeled KPC cancer cells and treatment with vehicle, RP-182, 1, and1a. FIG. 3G and FIG. 3H show secreted cytokines IFNa and IL-12 levels in conditionedmedia of M2 macrophages after 24 h of treatment determined by ELISA; in each set of bars,the left-to-right order is 1; 1c; 1f, 1a. FIG. 31 shows Fractions of CD86-positive cells inCD206high M2-like macrophages by flow cytometry. Cells were incubated for 2 hours.

[0030] FIGS. 4A-4E show that fatty acid-derivatized and cyclic RP-182 analogues haveimproved stability. FIG. 4A shows on the top percent intact peptides measured by peak areaof LC after normalization to heat-inactivated murine liver microsomes (MLMs). Table listscalculated half-lives. Bottom, metabolism of RP-182 analogue peptides after 3 hrs ofincubation, y-axis lists % decrease of spiked peptides after normalization to decrease ofspiked peptides in heat-inactivated MLMs. FIG. 4B shows stability parameters ofsynthesized analogues do not correlate with cell-based efficacy measures. Correlation plots ofhalf-life and % metabolized peptide at 3 hrs and induction of cell death (IC50) (left) and EC50values of induction of RAB7 and pIRF7 (right). FIG. 4C shows PyMOL models depictinginterface of docked individual RP-182, 1 analogues and binding region of CRD5 of CD206.FIG. 4D shows determination of equilibrium dissociation constant (Ka) of the interaction ofCD206 and biotinylated RP-182 by Microscale Thermophoresis (MST) after the addition ofincreasing concentrations of indicated RP-182 analogues shows increased competition by1a. FIG. 4E shows competition of cell-based binding of biotinylated RP-182 viaaddition of RP-182 analogues. Left, chemical structure of C-terminally biotinylated RP-182(bio-RP-182), cartoon showing detection of bio-RP-182 bound to CD206 via fluorescentstreptavidin (bottom). Flow cytometry analysis indicating streptavidin (SA)- negativefractions of CD206high M2-polarized macrophages incubated for 10 minutes with biotinylatedRP-182 and indicated RP-182 analogues. % SA-negative fractions are shown, graphsummarizes N=3 independent experiments, performed in triplicates.

[0031] FIG. 5A-5E show RP-182 analogues improve tumor control in autochthonousKPC mice and of B16 melanoma allografts. FIG. 5A shows the best overall response (BOR;% change) in tumor volume (TV) between baseline and on-treatment tumor measurements bytransabdominal USS in KPC mice. Representative USS images shown on right. FIG. 5Bshows Kaplan-Meier plots of overall survival of KPC mice enrolled when TV ≥200mm³ andrandomized to treatment with vehicle, RP-182, 1, and 1a. FIG. 5C shows flow cytometryanalysis of KPC tumor digests after indicated treatments administered for 7 days. Histogramsindicate % CD86-positive CD206high TAMs, bar graphs indicate percent positive cellfractions after flow cytometry analysis. FIG. 5D shows, on top, tumor growth of murineB16 melanomas grown in C57B / L mice, and macroscopic images of tumors harvested fromB16 melanoma-bearing mice after treatment with indicated RP-182 analogues on bottom.FIG. 5E shows flow cytometry analysis of B16 tumors after 2 weeks of treatment.Quantification of fractions of IFNy-, CD40-, IL-12-, and CD86-positive CD206-, CD86-positive macrophages are shown.

[0032] FIG. 6 shows an illustration depicting induction of cancer cell phagocytosis,activation of interferon response signaling, and TNF signaling mediated induction ofapoptosis and cell death after activation of CD206.

[0033] FIGS. 7A-7D show the identification of RP-182 metabolites after incubation for0, 30, and 60 minutes in murine liver microsomes (MLMs). FIG. 7A shows LC-MSchromatograms indicating individual peptide peaks at respective time points. FIG. 7B showsa summary table of RP-182 metabolites after tandem mass spectrometry analysis afterelectrospray ionization (ESI) of indicated LC peaks. Relative peak areas determined fromextracted ion chromatograms of MLM samples at 0, 30, and 60 mins. FIGS. 7C and 7D showthe predicted hydrolysis pathways of RP-182 1, based on detected metabolite profile.Metabolic pathways after initial endopeptidase hydrolysis events shown in FIG. 7C; afterinitial exopeptidase hydrolysis event shown in FIG. 7C.

[0034] FIG. 8A-8C show synthesis schemes for the generation of cyclic RP-182analogues. FIG. 8A and 8B shows macrocyclization of linear peptide RP-182 via clickchemistry to yield 1b and 1c. FIG. 8C shows head-to- side chain macrocyclization of RP-182to yield peptide olefin, 1d.

[0035] FIG. 9A-9F show synthesis schemes of fatty acid derivatized RP-182 analogues.FIG. 9A shows synthesis scheme of RP-182 chimera coupling high-affinity albumin bindinglipidated heptapeptide EYEK(palmitic acid)EYE-NH2, 1e, and lysine side chain coupled topalmitic acid linked via three carbon PEG spacer to the C-terminus of RP-182, 1a. FIG. 9Bshows synthesis of lipidated RP-182 analogues 1f, RP-182-NH-(CH2)10-CONH2 containingan undecanoic acid tail. FIG. 9C shows synthesis schemes of C-terminal polymer-peptideanalogues attaching polyethylenglycol (PEG) linkers of different lengths. FIGS. 9D, 9E and9F show the structure of RP-182, 1, and its analogues. Original RP-182 pharmacophoreoutlined with dashed line.

[0036] FIG. 10A-10C show that M2-polarized bone marrow-derived macrophages(BMDMs), including BMDMs extracted from TNFR1- / - and MyD88- / - knockout mice,express high levels of CD206. Fig. 10A and Fig. 10B show flow cytometry data of BMDMsisolated (from WT, TNFR1- / -, MyD88- / -, and CD206-- knockout mice) and polarized intoindicated differentiation states. Fig. 10C shows quantification of CD206high fractions ofCD45+CD11b+F4 / 80+Gr-1-cells indicates CD206 expression after polarization was notaffected by the lack of MyD88 or TNFR1 expression.

[0037] FIG. 11A-11C show the predicted hydrolysis pathways of compounds 1c (Fig.11B), 1f (Fig. 11C), and 1a (Fig. 11A). Metabolic pathways were estimated based onprevalence of identified metabolite species after 60 min incubation of analogues in MLMsand metabolite identification via LC / MALDI tandem MS / ddMS² analysis.

[0038] FIG. 12A-12E show activation of M2-polarized macrophages by RP-182, 1analogues is dependent on CD206. FIG. 12A shows PLA signals after anti-CD206 and anti-RAB7 staining in CD206- / - macrophages. FIGS. 12B to 12E show representativeimmunofluorescent images stained with anti-CD86 (green) and anti- phospho-IRF7 (red)after treatment with indicated RP-182, 1 analogues in CD206 wild type (FIGS. 12B and12C) macrophages polarized into M2, and in M2-polarized CD206- / --deficient macrophages(FIG. 12D and 12E).

[0039] FIG. 13 shows a gating strategy to detect streptavidin (SA)-negative cell fractionsof CD206- positive macrophages after incubation with biotinylated RP-182 and competingRP-182 analogues.DETAILED DESCRIPTION

[0040] As discussed above, the disclosure herein relates to immune-modulatory peptides,particularly peptides that have immunosuppressive properties, and methods of administeringsuch immune-modulatory polypeptides to a subject, particularly a subject suffering from acancer, at risk for developing a cancer, suffering from a medical condition associated withpersistent inflammation or at risk developing such a medical condition.

[0041] The present disclosure is explained in greater detail below. This description is notintended to be a detailed catalog of all the different ways in which the disclosure may beimplemented, or all the features that may be added to the instant disclosure. For example,features illustrated with respect to one embodiment may be incorporated into otherembodiments, and features illustrated with respect to a particular embodiment may be deletedfrom that embodiment. Numerous variations and additions to the various embodimentssuggested herein will be apparent to those skilled in the art in light of the instant disclosure,which do not depart from the instant disclosure. Hence, the following specification isintended to illustrate some particular embodiments of the disclosure, and not to exhaustivelyspecify all permutations, combinations, and variations thereof.

[0042] Unless otherwise defined, all technical and scientific terms used herein have thesame meaning as commonly understood by one of ordinary skill in the art to which thisdisclosure belongs. The terminology used in the description of the disclosure herein is for thepurpose of describing particular embodiments only and is not intended to be limiting of thedisclosure.

[0043] All publications, patent applications, patents, and other references mentionedherein are incorporated by reference in their entireties for all purposes.

[0044] Definitions

[0045] Compounds are described using standard nomenclature. Unless definedotherwise, all technical and scientific terms used herein have the same meaning as iscommonly understood by one of skill in the art to which this technology belongs.

[0046] The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used herein, the singular forms "a,""an," and "the" are intended to include the plural forms as well, unless the context clearlyindicates otherwise. It will be further understood that the terms "comprises" and / or"comprising," and / or "including" when used in this specification, specify the presence ofstated features, elements, and / or components, but do not preclude the presence or addition ofone or more other features, elements, components, and / or groups thereof. As used herein, theterm "and / or" includes any and all combinations of one or more of the associated listed items.As used herein, phrases such as "between X and Y" and "between about X and Y" should beinterpreted to include X and Y. As used herein, phrases such as "between about X and Y"mean "between about X and about Y." As used herein, phrases such as "from about X to Y"mean "from about X to about Y."

[0047] Moreover, the present disclosure also contemplates that in some embodiments,any feature or combination of features set forth herein can be excluded or omitted.

[0048] Furthermore, the term "about," as used herein when referring to a measurablevalue such as an amount of a compound or agent of this disclosure, dose, time, temperature,and the like, is meant to encompass variations of ±10%, 5%, ±1%, ±0.5%, or even ±0.1% ofthe specified amount.

[0049] In the claims, as well as in the specification above, all transitional phrases such as"comprising," "including," "carrying," "having," "containing," "involving," "holding,""composed of," or the like are to be understood to be open-ended, i.e., to mean including butnot limited to. Only the transitional phrases "consisting of" and "consisting essentially of"shall be closed or semi-closed transitional phrases, respectively, as set forth in the UnitedStates Patent Office Manual of Patent Examining Procedures, Section 2111.03. "Consistingessentially of' is to be interpreted as encompassing the recited materials or steps and thosethat do not materially affect the basic and novel characteristic(s) of the disclosure. The open-end phrases such as "comprising" include and encompass the close-ended phrases.Comprising may be amended to the more limiting phrases "consisting essentially of" of"consisting of" as needed.

[0050] All methods described herein can be performed in any suitable order unlessotherwise indicated herein or otherwise clearly contradicted by context. The use of any andall examples, or exemplary language (e.g., "such as"), is intended for illustration and does notpose a limitation on the scope of the disclosure unless otherwise claimed. No language in thespecification should be construed as indicating any non-claimed element as essential to thepractice of the compounds, compositions and methods of the disclosure.

[0051] Furthermore, the disclosure encompasses all variations, combinations, andpermutations in which one or more limitations, elements, clauses, and descriptive terms fromone or more of the listed claims are introduced into another claim. For example, any claimthat is dependent on another claim can be modified to include one or more limitations foundin any other claim that is dependent on the same base claim. Where elements are presented aslists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and anyelement(s) can be removed from the group.

[0052] All compounds are understood to include all possible isotopes of atoms occurringin the compounds. Isotopes include those atoms having the same atomic number butdifferent mass numbers. By way of general example, and without limitation, isotopes ofhydrogen include tritium and deuterium and isotopes of carbon include 11C, 13C, and 14C.

[0053] "Alkyl" includes both branched and straight chain saturated aliphatichydrocarbon groups, having the specified number of carbon atoms, generally from 6 to about20 carbon atoms. The term C6-C20alkyl as used herein indicates an alkyl group having from6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. Other embodimentsinclude alkyl groups having from 8 to 18 carbon atoms, 10 to 16 carbon atoms or 10 or 14carbon atoms, e.g. C8-C18alkyl, C10-C16alkyl, and C10-C14alkyl. Examples of alkyls include,but are not limited to, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl and eicosyl.

[0054] "Alkenyl" is a branched or straight chain aliphatic hydrocarbon group having oneor more carbon-carbon double bonds that may occur at any stable point along the chain,having the specified number of carbon atoms, e.g., C6-C20alkenyl. Examples of alkenylinclude, but are not limited to, hexeneyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl,dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl,nonadecenyl and eicosenyl.

[0055] "Alkynyl" is a branched or straight chain aliphatic hydrocarbon group having oneor more carbon-carbon triple bonds that may occur at any stable point along the chain, havingthe specified number of carbon atoms, e.g., C6-C20alkynyl.

[0056] "Pharmaceutical compositions" means compositions comprising at least oneactive agent, such as a compound or salt of Formula (I), and at least one other substance, suchas a carrier. Pharmaceutical compositions meet the U.S. FDA's GMP (good manufacturingpractice) standards for human or non-human drugs.

[0057] "Carrier" means a diluent, excipient, or vehicle with which an active compound isadministered. A "pharmaceutically acceptable carrier" means a substance, e.g., excipient,diluent, or vehicle, that is useful in preparing a pharmaceutical composition that is generallysafe, non-toxic and neither biologically nor otherwise undesirable, and includes a carrier thatis acceptable for veterinary use as well as human pharmaceutical use. A "pharmaceuticallyacceptable carrier" includes both one and more than one such carrier.

[0058] The terms "peptide" and "polypeptide" are used synonymously herein to refer topolymers constructed from amino acid residues. The term "amino acid residue,” as usedherein, refers to any naturally occurring amino acid (L or D form), non-naturally occurringamino acid, or amino acid mimetic (such as a peptoid monomer). Amino acid residues maybe referred to by their complete name (e.g., alanine, arginine, phenylalanine), their three lettercode (e.g., Ala, Arg, Phe) or their one letter code (e.g., A, R, F).

[0059] A "patient" or "subject" means a human or non-human animal in need of medicaltreatment. Medical treatment can include treatment of an existing condition, such as adisease or disorder or diagnostic treatment. In some embodiments the patient is a humanpatient.

[0060] "Cancer", as used herein, refers to all neoplastic cell growth and proliferation,whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. Theterms "tumor”, “cancerous”, “cell proliferative disorder", "proliferative disorder" and"tumor" are not mutually exclusive as referred to herein.

[0061] A patient or individual referred to as "suffering from" a cancer, disorder related toelevated inflammation, fibrosis, and the like, as described herein, has been diagnosed withand / or displays one or more symptoms of a cancer or inflammatory condition.

[0062] The term "anti-inflammatory”, as used herein, refers to a property of a peptidereduces or inhibits, or would be expected to reduce or inhibit, a pro-inflammatory signalmediated by a protein target and / or reduces or inhibits inflammation in a subject.

[0063] The term "at risk" for, e.g., a cancer or inflammatory disorder, refers to a subject(e.g., a human) that is predisposed to developing a cancer or inflammatory disorder and / orexpressing one or more symptoms related thereto. This predisposition may be genetic or dueto other factors. It is not intended that the present disclosure be limited to any particular signsor symptoms. Thus, it is intended that the present disclosure encompasses subjects that areexperiencing any range of a cancer or inflammatory disorder, from sub-clinical to advanced,wherein the subject exhibits at least one of the indicia (e.g., signs and symptoms) associatedwith the cancer or inflammatory disorder.

[0064] "Providing" means giving, administering, selling, distributing, transferring (forprofit or not), manufacturing, compounding, or dispensing.

[0065] The terms "treat," "treatment," or "treating", as used herein, refer to any methodused to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of,reduce severity of and / or reduce incidence of one or more symptoms or features of a disease,disorder, and / or condition (e.g., pancreatic cancer). Treatment may be administered to asubject who does not exhibit signs of a disease, disorder, and / or condition. In someembodiments, treatment may be administered to a subject who exhibits only early signs of thedisease, disorder, and / or condition for the purpose of decreasing the risk of developingpathology associated with the disease, disorder, and / or condition.

[0066] A "therapeutically effective amount" or "effective amount" of a peptide orpharmaceutical composition comprising a peptide described herein means an amounteffective, when administered to a patient, to provide a therapeutic benefit such as anamelioration of relevant symptoms. For example, for cancer a decrease in cancerprogression, or cancer regression; for inflammation, a decrease in an abnormal inflammatorystate or response, or a resumption of a normal inflammatory state or response. Atherapeutically effective amount may, in instances, refer to prophylaxis, or prevention of theinitiation of disease (e.g., cancer) or dysregulated state (e.g., inflammation). Atherapeutically effective amount is also one in which any toxic or detrimental effects of thepeptide or composition comprising the peptide are outweighed by the therapeuticallybeneficial effects. An effective amount will also provide a sufficient concentration of acompound of the disclosure when administered to a patient. A sufficient concentration is aconcentration of the compound in the patient's body necessary to provide a therapeuticbenefit. Such an amount may be ascertained experimentally, for example by assaying bloodconcentration of the compound, or theoretically, by calculating bioavailability.

[0067] A significant change is any detectable change that is statistically significant in astandard parametric test of statistical significance such as Student's T-test, where p < 0.05.

[0068] "Pharmaceutically acceptable salts" include derivatives of the disclosedcompounds in which the parent compound is modified by making inorganic and organic,non-toxic, acid or base addition salts thereof. The salts of the present compounds can besynthesized from a parent compound that contains a basic or acidic moiety by conventionalchemical methods. Generally, such salts can be prepared by reacting free acid forms of thesecompounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or Kacetate, trifluoroacetate, hydroxide, carbonate, bicarbonate, or the like), or by reacting freebase forms of these compounds with a stoichiometric amount of the appropriate acid.Peptides typically form salts if they contain a free amino group (found in a free N-terminus oron a side chain that has a free amine, for example, Arg, Lys, and His), and most peptides bindto these counterions and are lyophilized in the form of the corresponding salts. Salts of thepresent compounds further include solvates of the compounds and of the compound salts.

[0069] Acetate salts are a common pharmaceutically acceptable salt for peptides, alongwith HCl and trifluoroacetate salts. Examples of pharmaceutically acceptable salts alsoinclude, but are not limited to, mineral or organic acid salts of basic residues such as amines;alkali or organic salts of acidic residues such as carboxylic acids; and the like. Thepharmaceutically acceptable salts include the conventional non-toxic salts and the quaternaryammonium salts of the parent compound formed, for example, from non-toxic inorganic ororganic acids. For example, conventional non-toxic acid salts include those derived frominorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric andthe like; and the salts prepared from organic acids such as, again, acetic and trifluoroacetic, aswell as propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, maleic,hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic,isethionic, HOOC-(CH2)n-COOH where n is 0-4, and the like. Lists of additional suitablesalts may be found, e.g., in G. Steffen Paulekuhn, et al., Journal of Medicinal Chemistry2007, 50, 6665 and Handbook of Pharmaceutically Acceptable Salts: Properties, Selectionand Use, P. Heinrich Stahl and Camille G. Wermuth Editors, Wiley-VCH, 2002.

[0070] Compounds

[0071] Peptide compounds that modulate CD206 are provided herein, as well aspharmaceutically acceptable salts thereof and pharmaceutical compositions thereof (e.g., oneor more CD206 modulating compounds or pharmaceutically acceptable salts thereof, togetherwith a pharmaceutically acceptable carrier). In various aspects and embodiments, suchcompounds, salts and / or compositions can be utilized in the methods of the disclosure. In anaspect, a compound of Formula 1 is provided:

[0072] or a pharmaceutically acceptable salt thereof, in which X is H; and Y isrepresented by Formula la:

[0073] where PEG is polyethylene glycol according to the following:

[0074] where Q is 0 to 4; R is 0 or 1; S is 0 or 1; V is absent or C6-C20alkyl, C6-C20alkenyl, or C6-C20alkynyl; T is 0 or 1; U is C6-C20alkyl, C6-C20alkenyl, or C6-C20alkynyl;or X and Y are linked to form a cyclic compound, where X and Y are represented by Formula1b or Formula 1c, where Formula 1b is represented as follows:-C-PEGm-A-B-PEG-C-Lys-Formula 1b

[0075] where for Formula 1b: A is a methylcarbamate, N-methyl carbamate, N-ethylcarbamate, N-methyl methylcarbamate, or N-ethyl methylcarbamate; B is a 12-Methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 5-Methyl-12-methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 5-Methyl-12-ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; and where A comprises a non-carbonyl oxygen(hydroxyl oxygen); and B comprises a cyclopropyl group; and at least one carbon separatesthe non-carbonyl oxygen on the carbamate and the cyclopropyl group; PEGm is polyethyleneglycol represented as follows:

[0076] where m is 1 or 2; and PEGn is polyethylene glycol represented as follows:

[0077] where n is 1 or 2; and wherein X corresponds to the carbonyl end of Formula 1band Y corresponds to the -Lys- end of Formula 1b; and the Lys in Formula 1b binds to thecarbonyl carbon (C=O) through a Lys sidechain nitrogen; or X and Y are linked to form acyclic compound, where X and Y are represented by Formula 1c:-C-PEG-D-E-PEG-C-Lys-Formula 1c

[0078] where for Formula 1c: D is a 12-Methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene;12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 5-Methyl-12-methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 5-Methyl-12-ethyl-10,11,12-triazatricyclo[7.3.0.04, 6]dodeca-1(9), 10-diene; E is a methylcarbamate, N-methylcarbamate, N-ethyl carbamate, N-methyl methylcarbamate, or N-ethyl methylcarbamate; andwhere D comprises a cyclopropyl group; and E comprises a non-carbonyl oxygen (hydroxyloxygen); and at least one carbon separates the non-carbonyl oxygen on the carbamate and thecyclopropyl group; PEGm is polyethylene glycol represented as follows:

[0079] m is 1 or 2; and PEGn is polyethylene glycol represented as follows:

[0080] where n is 1 or 2; and wherein X corresponds to the carbonyl end of Formula 1cand Y corresponds to the -Lys- end of Formula 1c; and the Lys in Formula 1c binds to thecarbonyl carbon (C=O) through a Lys sidechain nitrogen.

[0081] In various embodiments, X is H; and Y is represented by Formula la.

[0082] In various embodiments where X is H; and Y is represented by Formula la, Q is 1to 4, R is 1, and S is 1, V is absent, T is 1, and U is C6-C20alkyl, C6-C20alkenyl, or C6-C20alkynyl.

[0083] In various embodiments where X is H; and Y is represented by Formula la, Q is 2to 4, R is 1, and S is 1, V is absent, T is 1, and U is C8-C18alkyl, C8-C18alkenyl, or C8-C18alkynyl.

[0084] In various embodiments where X is H; and Y is represented by Formula la, Q is 3to 4, R is 1, and S is 1, V is absent, T is 1, and U is C10-C16alkyl, C10-C16alkenyl, or C10-C16alkynyl.

[0085] In various embodiments where X is H; and Y is represented by Formula la, Q is3, R is 1, and S is 1, V is absent, T is 1, and U is C10-C16alkyl, C10-C16alkenyl, or C10-C16alkynyl.

[0086] In various embodiments where X is H; and Y is represented by Formula la, Q is 1to 4, R is 0 or 1, and S is 0, V is C6-C20alkyl, C6-C20alkenyl, or C6-C20alkynyl and T is 0 or 1.

[0087] In various embodiments where X is H; and Y is represented by Formula la, Q is 2to 4, R is 0 or 1, and S is 0, V is C C8-C18alkyl, C8-C18alkenyl, or C8-C18alkynyl, and T is 0 or1.

[0088] In various embodiments where X is H; and Y is represented by Formula la, Q is 3to 4, R is 0 or 1, and S is 0, V is C10-C16alkyl, C10-C16alkenyl, or C10-C16alkynyl, and T is 0 or1.

[0089] In various embodiments where X is H; and Y is represented by Formula la, Q is3, R is 0 or 1, and S is 0, V is C10-C16alkyl, C10-C16alkenyl, or C10-C16alkynyl, and T is 0 or 1.

[0090] In various embodiments where X is H; and Y is represented by Formula la, Q is0, R is 1, and S is 1, V is absent, and T is 1, and U is C6-C20alkyl, C6-C20alkenyl, or C6-C20alkynyl.

[0091] In various embodiments where X is H; and Y is represented by Formula la, Q is0, R is 1, and S is 1, V is absent, and T is 1, and U is C8-C18alkyl, C8-C18alkenyl, or C8-C18alkynyl.

[0092] In various embodiments where X is H; and Y is represented by Formula la, Q is0, R is 1, and S is 1, V is absent, and T is 1, and U is C10-C16alkyl, C10-C16alkenyl, or C10-C16alkynyl.

[0093] In various embodiments where X is H; and Y is represented by Formula la, Q is0, R is 1, S is 0, V is C6-C20alkyl, C6-C20alkenyl, or C6-C20alkynyl, and T is 0 or 1.

[0094] In various embodiments where X is H; and Y is represented by Formula 1a, Q is0, R is 1, S is 0, V is C8-C18alkyl, C8-C18alkenyl, or C8-C18alkynyl, and T is 0 or 1.

[0095] In various embodiments where X is H; and Y is represented by Formula la, Q is0, R is 1, S is 0, V is C10-C16alkyl, C10-C16alkenyl, or C10-C16alkynyl, and T is 0 or 1.

[0096] In various embodiments where X is H; and Y is represented by Formula la, Q is0, R is 0, S is 0, V is C6-C20alkyl, C6-C20alkenyl, or C6-C20alkynyl, and T is 0 or 1.

[0097] In various embodiments where X is H; and Y is represented by Formula la, Q is0, R is 0, S is 0, V is C8-C18alkyl, C8-C18alkenyl, or C8-C18alkynyl, and T is 0 or 1.

[0098] In various embodiments where X is H; and Y is represented by Formula la, Q is0, R is 0, S is 0, V is C10-C16alkyl, C10-C16alkenyl, or C10-C16alkynyl, and T is 0 or 1.

[0099] In an embodiment, the compound is 1a (NCGC00857001), represented by thefollowing structure:Compound 1a (NCGC00857001).

[0100] In an embodiment, the compound is 1f (NCGC00591015), represented by thefollowing structure:Compound 1f (NCGC00591015).

[0101] In an embodiment, the compound is le (NCGC00856998), represented by thefollowing structure:Compound le

[0102] In other embodiments, X and Y are linked to form a cyclic compound and X andY are represented by Formula 1b.

[0103] In various embodiments where X and Y form a cyclic compound and arerepresented by Formula 1b, A is a methylcarbamate, N-methyl carbamate, N-ethyl carbamate,N-methyl methylcarbamate, or N-ethyl methylcarbamate; and B is a 12-Methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 5-Methyl-12-methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 5-Methyl-12-ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; where A comprises a non-carbonyl oxygen(hydroxyl oxygen); and B comprises a cyclopropyl group; and at least one carbon separatesthe non-carbonyl oxygen on the carbamate and the cyclopropyl group; PEGm and PEGn arepolyethylene glycol according to their recited structures herein, where m is 1 or 2; n is 1 or 2;and where X corresponds to the carbonyl end of Formula 1b and Y corresponds to the -Lys-end of Formula 1b.

[0104] In various cyclic embodiments m is 2 and n is 1.

[0105] In various cyclic embodiments, A is an N-methyl methylcarbamate, or N-ethylmethylcarbamate; B is a 12-Methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-dieneor12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; wherein A and B connect atthe 5 position of the 12-Methyl- or 12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene or 12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; m is 2; andn is 1.

[0106] In various cyclic embodiments, A is an N-methyl carbamate, or N-ethylcarbamate;

[0107] B is a 5-Methyl-12-methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene; or 5-Methyl-12-ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; where Aand B connect at the 5 position of the 5-Methyl-12-methyl- or 5-methyl-12-ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; m is 2; and n is 1.

[0108] In an embodiment, the compound is 1c (NCGC01147950), represented by thefollowing structure:Compound 1c (NCGC01147950).

[0109] In other embodiments, X and Y are linked to form a cyclic compound and X andY are represented by Formula 1c.

[0110] In various cyclic embodiments where X and Y are represented by Formula 1c, Dis a 12-Methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene; 5-Methyl-12-methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene; 5-Methyl-12-ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; E is a methylcarbamate, N-methyl carbamate,N-ethyl carbamate, N-methyl methylcarbamate, or N-ethyl methylcarbamate; where Dcomprises a cyclopropyl group; and E comprises a non-carbonyl oxygen (hydroxyl oxygen);and at least one carbon separates the non-carbonyl oxygen on the carbamate and thecyclopropyl group; PEGm and PEGn are polyethylene glycol according to their recitedstructures herein, where m is 1 or 2; n is 1 or 2; and where X corresponds to the carbonyl endof Formula 1c and Y corresponds to the -Lys- end of Formula 1c.

[0111] In various cyclic embodiments where X and Y are represented by Formula 1c, mis 1 and n is 2.

[0112] In various cyclic embodiments where X and Y are represented by Formula 1c, Dis a 12-Methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene or 12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; E is an N-methyl methylcarbamate, or N-ethylmethylcarbamate; where D and E connect at the 5 position of the 12-Methyl- or 12-Ethyl-10,11,12-triazatricyclo[7.3.0.04, 6]dodeca-1(9), 10-diene or 12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene; m is 1; and n is 2.

[0113] In various cyclic embodiments where X and Y are represented by Formula 1c, Dis a 5-Methyl-12-methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; or 5-Methyl-12-ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; E is an N-methylcarbamate, or N-ethyl carbamate; where D and E connect at the 5 position of the 5-Methyl-12-methyl- or 5-methyl-12-ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene; m is1; and n is 2.

[0114] In an embodiment, the compound is 1b (NCGC00886866), represented by thefollowing structure:Compound 1b (NCGC00886866).

[0115] In an embodiment, the compound is 1d (NCGC00887030), represented by thefollowing structure:Compound 1d (NCGC00887030)

[0116] Compositions

[0117] In another aspect, a pharmaceutical composition comprising at least onecompound or salt of Formula I with a pharmaceutically acceptable carrier is also provided.Such pharmaceutical compositions can comprise one or more peptides of Formula I and apharmaceutically acceptable carrier. Pharmaceutical compositions can further include aprotein other than one or more peptides of the disclosure and / or a small molecule agent. Theother protein can be a therapeutic agent, such as a therapeutic antibody. The therapeuticprotein or antibody can have anti-cancer or anti-inflammatory properties or other propertiesthat the peptides of the disclosure augment or are augmented by, as can the small moleculeagent. Alternatively, the other protein can be a carrier protein, such as serum albumin (e.g.,HSA). The serum albumin (e.g., HSA, BSA, etc.) can be purified or recombinantly produced.By mixing the peptide(s) in the pharmaceutical composition with serum album, the peptidesof Formula I can be effectively "loaded" onto the serum albumin, allowing a greater amountof peptide to be successfully delivered to a site of cancer or inflammation. Thechemotherapeutic agent can be, for example, an anti-cancer chemotherapeutic agent. Suchchemotherapeutic agents include, but are not limited to, Gemcitabine, Docetaxel, Bleomycin,Erlotinib, Gefitinib, Lapatinib, Imatinib, Dasatinib, Nilotinib, Bosutinib, Crizotinib, Ceritinib,Trametinib, Bevacizumab, Sunitinib, Sorafenib, Trastuzumab, Ado-trastuzumab emtansine,Rituximab, Ipilimumab, Rapamycin, Temsirolimus, Everolimus, Methotrexate, Doxorubicin,Abraxane, Folfirinox, Cisplatin, Carboplatin, 5-fluorouracil, Teysumo, Paclitaxel,Prednisone, Levothyroxine, and Pemetrexed.

[0118] Compositions can include a single peptide of Formula I, or combinations thereof.In some embodiments, the compositions can include at least 1 mg (e.g., at least 5, 10, 20, 30,40, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000 mg, or more) ofpeptide of Formula I. Thus, for example, the compositions can include an amount of peptideequal to about 1 mg to about 1000 mg (e.g., about 5 mg to about 900 mg, about 5 mg to about800 mg, about 5 mg to about 700 mg, about 5 mg to about 600 mg, about 10 mg to about 500mg, about 10 mg to about 400 mg, about 10 mg to about 300 mg, about 10 mg to about 250mg, about 10 mg to about 200 mg, about 10 mg to about 150 mg, about 10 mg to about 100mg, about 50 mg to about 500 mg, about 50 mg to about 400 mg, about 50 mg to about 300mg, about 50 mg to about 250 mg, about 50 mg to about 200 mg, about 50 mg to about 150mg, about 50 mg to about 100 mg, about 75 mg to about 500 mg, about 75 mg to about 400mg, about 75 mg to about 300 mg, about 75 mg to about 250 mg, about 75 mg to about 200mg, about 75 mg to about 150 mg, about 75 mg to about 100 mg, about 100 mg to about 500mg, about 100 mg to about 400 mg, about 100 mg to about 300 mg, about 100 mg to about250 mg, about 100 mg to about 200 mg, or any other range encompassed therein).

[0119] The compositions provided herein can include a solution that contains at least 1mg / ml (e.g., at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100 mg / ml or more) of peptide of Formula I. Thus, for example, the compositions caninclude a solution having a peptide concentration of about 1 mg / ml to about 1000 mg / ml(e.g., about 5 mg / ml to about 900 mg / ml, about 5 mg / ml to about 800 mg / ml, about 5 mg / mlto about 700 mg / ml, about 5 mg / ml to about 600 mg / ml, about 5 mg / ml to about 500 mg / ml,about 10 mg / ml to about 500 mg / ml, about 10 mg / ml to about 400 mg / ml, about 10 mg / ml toabout 300 mg / ml, about 10 mg / ml to about 250 mg / ml, about 10 mg / ml to about 200 mg / ml,about 10 mg / ml to about 150 mg / ml, about 10 mg / ml to about 100 mg / ml, about 50 mg / ml toabout 500 mg / ml, about 50 mg / ml to about 400 mg / ml, about 50 mg / ml to about 300 mg / ml,about 50 mg / ml to about 250 mg / ml, about 50 mg / ml to about 200 mg / ml, about 50 mg / ml toabout 150 mg / ml, about 50 mg / ml to about 100 mg / ml, about 75 mg / ml to about 500 mg / ml,about 75 mg / ml to about 400 mg / ml, about 75 mg / ml to about 300 mg / ml, about 75 mg / ml toabout 250 mg / ml, about 75 mg / ml to about 200 mg / ml, about 75 mg / ml to about 150 mg / ml,about 75 mg / ml to about 100 mg / ml, about 100 mg / ml to about 500 mg / ml, about 100 mg / mlto about 400 mg / ml, about 100 mg / ml to about 300 mg / ml, about 100 mg / ml to about 250mg / ml, about 100 mg / ml to about 200 mg / ml, about 10 mg / ml to about 150 mg / ml, or anyother range encompassed therein).

[0120] Methods

[0121] The disclosure provides methods of using compounds of Formula I andpharmaceutically acceptable salts thereof, or a pharmaceutical composition comprising theone or more compounds of Formula I or pharmaceutically acceptable salts thereof togetherwith a pharmaceutically acceptable carrier to treat a cancer. In various embodiments, thepatient is suffering from a cell proliferative disorder or disease. The cell proliferativedisorder can be cancer, tumor (cancerous or benign), neoplasm, neovascularization, ormelanoma. Cancers for treatment can include both solid and disseminated cancers, andparticularly solid tumors / cancers characterized by the presence of CD206 positive tumor-associated macrophages (TAMs). Exemplary solid cancers (tumors) that may be treated bythe methods provided herein include e.g. cancers of the lung, prostate, breast, liver, colon,breast, kidney, pancreas, brain, skin including malignant melanoma and Kaposi's sarcoma,testes or ovaries, carcinoma, kidney cancer (renal cell), and sarcoma. Cancers that may betreated with a compound of this disclosure, or pharmaceutically acceptable salts thereof, or apharmaceutical composition comprising the one or more compounds of Formula I orpharmaceutically acceptable salts thereof together with a pharmaceutically acceptable carrieralso include bladder cancer, breast cancer, colon cancer, endometrial cancer, lung cancer,bronchial cancer, melanoma, Non-Hodgkin lymphoma, cancer of the blood, pancreaticcancer, prostate cancer, thyroid cancer, brain or spinal cancer, and leukemia. Particularlyincluded herein are methods of treating cancer by providing a compound of this disclosure, orpharmaceutically acceptable salts thereof, or a pharmaceutical composition comprising theone or more compounds of Formula I, or pharmaceutically acceptable salts thereof, togetherwith a pharmaceutically acceptable carrier to a patient wherein the cancer is a cancercharacterized by the presence of CD206 positive tumor-associated macrophages, such asglioma (glioblastoma), sarcoma, astrocytoma, melanoma, non-small cell lung cancer,cholangiocarcinoma, colon cancer, hepatocellular, breast, prostate, gastric, renal cell,endometrial, or pancreatic cancer.

[0122] The methods of treating a cancer, including preventing a significant progressionof a cancer, causing a significant regression of a cancer or causing a cancer to be eradicatedor otherwise become undetectable, comprise providing to a patient a therapeutically effectiveamount of a compound, salt or composition of Formula I.

[0123] Compounds of Formula I may bind to the carbohydrate recognition domain 5(CRD5) of the CD206 receptor and induce a conformational switch that activates NF-κBsignaling and phagocytosis in CD206high TAMs. Compounds described here can have a dualfunction: activation of NF-kB signaling triggers via TNFa secretion and autocrine activationof the tumor necrosis factor receptor 1 (TNFR1), activation of caspase 8, apoptosis and celldeath of CD206high M2-like human (and, e.g., and murine) macrophages including CD206highTAMs. In parallel, in cells that do not undergo apoptosis, compounds of the disclosure mayinduce phagocytosis, autophagy, and reprogramming towards an M1-like phenotypecharacterized by secretion of proinflammatory cytokines (by the macrophages) and cancercell phagocytosis.

[0124] In various embodiments, Compounds of Formula I that have such a dual functionmay improve tumor control, including solid organ cancers. Compounds of Formula I mayalso leverage several advantages of peptide-based therapeutics over macromolecules, such ashumanized monoclonal antibodies (mAbs). Low molecular weight, flexible peptides are moreeasily able to overcome perfusion barriers like long diffusion distances, a hostile extracellularmatrix, or cellular partitions in desmoplastic, difficult to perfuse malignancies like pancreascancer. Membrane translocation and cell penetration of therapeutic peptides of Formula I maybe facilitated by the introduction of amphipathic features, opposing poles of hydrophilic andhydrophobic charges, as already present in compounds of Formula I, to overcome perfusionbarriers by cell partitions. Therapeutic peptides of the disclosure may bind with highspecificity to their targets and do not induce or induce negligible Fc receptor-mediated T celldepletion, which is known to limit the efficacy of mAb-based ICIs. Compounds of Formula Ialso may reduce or eliminate problems of linear peptides, such as RP-182, including rapidenzymatic degradation, renal loss and / or high depot effects.

[0125] Thus, in an aspect, the methods for the treatment of cancer involve modulatingmacrophage activity by contacting a macrophage with a compound of Formula I. In someembodiments, the macrophage is a M2 macrophage or a tumor associated macrophage(TAM). In some embodiments, the methods include selective targeting of CD206 M2macrophages, which, in various embodiments, includes modulating macrophage polarization.In some embodiments, modulating macrophage polarization includes reprogramming a M2macrophage towards a M1 phenotype (characterized, e.g., by secretion of proinflammatorycytokines and induced cancer cell phagocytosis). In various embodiments, the targeting ofmacrophages includes reducing the viability of the macrophage or inducing macrophage celldeath. In some embodiments, the compound of Formula I stimulates or induces macrophageactivity, e.g., phagocytosis and / or autophagy. In some embodiments, the compound ofFormula I binds to a mannose receptor (e.g., carbohydrate recognition domain 5 (CRD5)) tomodulate activity of signal regulatory protein (SIRP)-alpha to stimulate phagocytosis ordestruction of a cancer cell.

[0126] In various embodiments, the disclosure provides methods for increasing CD86expression, increasing CD40 expression, increasing IFNy expression, increasing IL-12ẞexpression, increasing IL-1ẞ expression, increasing IL-1ẞ secretion, increasing TNFaexpression, increasing TNFa secretion, increasing activation of caspase-8 (i.e., the tumornecrosis factor receptor 1 (TNFR1)-FADD-caspase 8 axis), decreasing inhibitoryphagocytosis checkpoint SIRPa expression, decreasing inhibitory phagocytosis checkpointPD-1 expression, increasing RAB7 expression, increasing expression of phosphorylated(active) interferon response factor 7 (pIRF7), and increasing annexin V expression, each ofwhich in CD206 expressing M2 macrophages (TAMs).

[0127] In some embodiments, one or more compounds of Formula I are present in anamount sufficient to increase CD86, CD40, IFNy, IL-12ẞ, IL-1ẞ, TNFα, RAB7, annexin Vand / or pIRF7 expression by an average of at least about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70,80, 90, more than 90%, 2-fold, 3-fold, 5-fold, or 10-fold as compared to a subject not beingtreated with a method of the disclosure.

[0128] In some embodiments, one or more compounds of Formula I are present in anamount sufficient to increase IL-1ẞ and / or TNFa secretion by an average of at least about 5,10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, more than 90%, 2-fold, 3-fold, 5-fold, or 10-fold ascompared to a subject not being treated with a method of the disclosure.

[0129] In some embodiments, one or more compounds of Formula I are present in anamount sufficient to decrease or inhibit SIRPa and / or PD-1 expression by an average of atleast about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, more than 90%, 2-fold, 3-fold, 5-fold,or 10-fold as compared to a subject not being treated with a method of the disclosure.

[0130] In addition to their effects on CD206 positive tumor-associated macrophages,which can include, e.g., localized (to the M2 macrophages) increases in pro-inflammatorymarker expression and / or activity, the peptides of Formula I may also reduce inflammationand / or treat conditions associated with excessive inflammation (whether acute or chronic).Accordingly, the disclosure provides methods of reducing the expression level and / or activityof at least one (e.g., 2, 3, 4, 5, or more) pro-inflammatory cytokine(s) at a site ofinflammation in a subject. The methods include administering a peptide of Formula I (or,e.g., salt or pharmaceutical composition thereof) to a subject. The pro-inflammatory cytokinecan include, e.g., NF-kB, TNFα, IL-1, IL-6, IL-8, IL-12, IL-17, IL-23, MCP-1, MMP-1, andMMP-9. The reduction can be a reduction of at least 10% (e.g., 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, or more) in the expression or activity of the cytokine.

[0131] The disclosure also provides methods of inhibiting an increase in the expressionlevel and / or activity of at least one (e.g., 2, 3, 4, 5, or more) pro-inflammatory cytokine(s) ata potential site of inflammation in a subject. The methods include administering a peptide ofFormula I (or, e.g., salt or pharmaceutical composition thereof) to the subject. The pro-inflammatory cytokine can include, e.g., NF-kB, TNFα, IL-1, IL-6, IL-8, IL-12, IL-17, IL-23,MCP-1, MMP-1, and MMP-9. The methods can inhibit increased cytokine expression and / oractivity by limiting such increases to no more than 20% (e.g., 15%, 12.5%, 10%, 7.5%, 5%,4%, 3%, 2%, 1%, or less).

[0132] The disclosure also provides a method of treating or preventing a conditionassociated with chronic inflammation. The condition associated with chronic inflammationcan be, for example, irritable bowel disease, ulcerative colitis, colitis, Crohn's disease,idiopathic pulmonary fibrosis, asthma, keratitis, arthritis, osteoarthritis, rheumatoid arthritis,auto-immune diseases, a feline or human immunodeficiency virus (FIV or HIV) infection,cancer, age-related inflammation and / or stem cell dysfunction (e.g., age-related increases inNlrp3 expression, age-related elevation of SOCS3 in muscle stem cells, etc.), graft-versus-host disease (GVHD), keloids, scleroderma, obesity, diabetes, diabetic wounds, other chronicwounds, atherosclerosis, multiple sclerosis, Parkinson's disease, Alzheimer's disease, maculardegeneration, gout, gastric ulcers, gastritis, mucositis, toxoplasmosis, and chronic viral ormicrobial infections (e.g., such as chronic bacterial or protozoan infections). The methodsinclude administering an effective amount of a peptide of Formula I (or, e.g., salt orpharmaceutical composition thereof) to a subject suffering from or likely to develop thecondition associated with chronic inflammation.

[0133] The disclosure also provides a method of treating or preventing fibrosis. Fibrosis,as used herein, refers to the excessive formation of connective tissue in an organ or tissue,usually in response to damage or injury. A hallmark of fibrosis is the production of excessextracellular matrix after local trauma. While a normal physiological response to injuryresults in deposition of connective tissue, this initially beneficial repair process can becomeongoing and pathological, altering tissue architecture and function. At the cellular level,epithelial cells and fibroblasts proliferate and differentiate into myofibroblasts, resulting inmatrix shrinkage, increased stiffness, microvascular compression, and hypoxia. The influx ofinflammatory cells, including macrophages and lymphocytes, results in cytokine release andamplifies deposition of collagen, fibronectin and other molecular markers of fibrosis.

[0134] Exemplary fibroses include lung and hepatic fibroses. Lung fibrosis, which refersto a group of diseases associated with loss of lung functions due to a lesion regarding thereconstruction of an alveolar region, is caused by the phenomenon whereby the alveoloarstructure is destroyed by an inflammatory reaction, and, as a result, growth of fibroblasts andan excessive increase in extracellular matrix mainly composed of collagen take place, so thatthe lung becomes hardened. Hepatic fibrosis is a pathologic condition associated withfibrosis of the liver, which is caused by the phenomenon whereby hepatic cells are necrotizedby various types of hepatopathy such as chronic viral hepatitis or alcoholic hepatitis, andthereafter, extracellular matrix increases to replenish the necrotized portion, resulting in suchfibrosis of the liver. This pathologic condition finally leads to hepatic cirrhosis, in which theentire hepatic fibers shrink and become hardened.

[0135] The disclosure also provides methods of treating or preventing fibrosis. Thefibrosis can be, for example, pulmonary fibrosis, dermal fibrosis, hepatic fibrosis, renalfibrosis, or fibrosis caused by ionizing radiation. The methods include administering apeptide of Formula I (or, e.g., salt or pharmaceutical composition thereof) to a subjectsuffering from or likely to develop fibrosis.

[0136] For any of the methods herein, the patient or subject can be a human or non-human animal, such as a domesticated animal (e.g., a horse, cow, pig, goat, sheep, rabbit,chicken, turkey, duck, etc.), a pet (e.g., a dog, cat, rabbit, hamster, gerbil, bird, fish, etc.), alab animal (e.g., a mouse, rat, monkey, chimpanzee, owl, fish, etc.), a zoo animal (e.g., agorilla, orangutan, chimpanzee, monkey, elephant, camel, zebra, boar, lion, tiger, giraffe,bear, bird, etc.), a wild animal (e.g., a deer, wolf, bird, etc.).

[0137] In conjunction with any of the foregoing methods, the peptide(s) with anti-inflammatory activity can be administered at a dose and frequency that depends on the typeof animal, the size of the animal, and the condition being treated. Typically, the peptide isadministered daily (or every other day, or weekly), in an amount between about 1 mg andabout 1000 mg (e.g., about 5 mg to about 900 mg, about 5 mg to about 800 mg, about 5 mgto about 700 mg, about 5 mg to about 600 mg, about 10 mg to about 500 mg, about 10 mg toabout 400 mg, about 10 mg to about 300 mg, about 10 mg to about 250 mg, about 10 mg toabout 200 mg, about 10 mg to about 150 mg, about 10 mg to about 100 mg, about 50 mg toabout 500 mg, about 50 mg to about 400 mg, about 50 mg to about 300 mg, about 50 mg toabout 250 mg, about 50 mg to about 200 mg, about 50 mg to about 150 mg, about 50 mg toabout 100 mg, about 75 mg to about 500 mg, about 75 mg to about 400 mg, about 75 mg toabout 300 mg, about 75 mg to about 250 mg, about 75 mg to about 200 mg, about 75 mg toabout 150 mg, about 75 mg to about 100 mg, about 100 mg to about 500 mg, about 100 mg toabout 400 mg, about 100 mg to about 300 mg, about 100 mg to about 250 mg, about 100 mgto about 200 mg, or any other range encompassed therein). The daily dose can beadministered once during the day, or broken up into smaller doses that are taken at multipletime points during the day. For a human (and other similarly-sized mammals), a dose of 5mg / kg every other day can be administered. The peptide can be administered for a fixedperiod of time (e.g., for 2-3 weeks), at intervals (e.g., administer polypeptide for 2-3 weeks,wait 2-3 weeks, then repeat the cycle), or until such time as the pro-inflammatory cytokinelevels have been reduced or stabilized, the chronic inflammatory condition or fibrosis hasameliorated, or the cancer has gone into remission.

[0138] The administration of the peptides (or, e.g., salt or pharmaceutical compositionthereof) in conjunction with any of the foregoing methods can be performed intravenously,intraperitoneally, parenteral, orthotopically, subcutaneously, topically, nasally, orally,sublingually, intraocularly, by means of an implantable depot, using nanoparticle-baseddelivery systems, microneedle patch, microspheres, beads, osmotic or mechanical pumps,and / or other mechanical means.

[0139] In conjunction with any of the foregoing methods, the peptides (or, e.g., salt orpharmaceutical composition thereof) can be administered in combination with one or moreother drug designed to reduce or prevent inflammation, treat or prevent chronic inflammationor fibrosis or scleroderma, or treat cancer. In each case, the peptide can be administered priorto, at the same time as, or after the administration of the other drug. For the treatment ofcancer, the peptide(s) can be administered in combination with a chemotherapeutic agent,including steroids, anthracyclines, thyroid hormone replacement drugs, thymidylate-targeteddrugs, checkpoint inhibitor drugs (e.g., anti-PD-1 / PD-L1, anti-CTLA-4 drugs), ChimericAntigen Receptor / T cell therapies, and other cell therapies. Specific chemotherapeutic agentsinclude, for example, Gemcitabine, Docetaxel, Bleomycin, Erlotinib, Gefitinib, Lapatinib,Imatinib, Dasatinib, Nilotinib, Bosutinib, Crizotinib, Ceritinib, Trametinib, Bevacizumab,Sunitinib, Sorafenib, Trastuzumab, Ado-trastuzumab emtansine, Rituximab, Ipilimumab,Rapamycin, Temsirolimus, Everolimus, Methotrexate, Doxorubicin, Abraxane, Folfirinox,Cisplatin, Carboplatin, 5-fluorouracil, Teysumo, Paclitaxel, Prednisone, Levothyroxine, andPemetrexed.

[0140] Alternatively, for the methods of treating cancer, the peptide(s) (or, e.g., salt orpharmaceutical composition thereof) can be administered in combination with radiationtherapy. Again, the peptide(s) can be administered prior to, or after the administration of theradiation therapy.EXAMPLES

[0141] Mannose receptor 1 (Mrc1; CD206)-positive TAMs have been increasinglyrecognized as a bona fide drug target for cancer therapy. Tumors that harbor a high numberof infiltrating CD206-positive TAMs are associated with adverse clinical outcome across avariety of solid organ cancers; whereas, patients with tumors classified as CD206low have amore favorable prognosis. The heterogeneity of TAMs, as evidenced by various pro-inflammatory gene expression programs in CD206-positive TAMs, and CD206-positiveTAMs cross-presenting tumor antigens and mediating anti-tumor immune surveillance,suggest that simple ablation of these cells may not fully harness their therapeutic potential.

[0142] The synthetic, amphipathic 10mer peptide RP-182 (compound 1), was developedfrom an in silico screen which probed large databases of antimicrobial peptides (AMPs) andhost defense peptides (HDPs) for the presence of common, conserved amphipathic motifs.The most common motif was examined in silico for binding to first-line innate defenseregulators, which identified the mannose receptor CD206 as its target. Thus, RP-182, 1,which entailed a series of medicinal chemistry optimizations of the evolutionary conservedamphipathic motif to improve the hydrophobic moment is a biosimilar of natural ligand(s) ofCD206. RP-182, 1, activates the CD206 receptor to effectively induce apoptosis, as wellas reprogram and activate CD206high TAMs to induce tumor control across murine andhuman models of cancer. However, the linear, unprotected RP-182 peptide is not a drugcandidate due to, among other characteristics, its metabolic instability and short in vivo half-life.

[0143] To improve its stability, modifications in the backbone of the a-helix were made.All amide bonds of RP-182 were identified as proteolysis sites in metabolic stabilityexperiments, so a number of analogues inserting D-amino acids into the a-helix weresynthesized. Unexpectedly, and contrary to a large body of research suggesting otherwise, D-amino acid substitutions did not show any biological activity (data not shown). Given thatresult, further internal modifications to the 10 residue pharmacophore, KFRKAFKRFF (SEQID NO:1), were not conducted. The original pharmacophore model of RP-182 suggestsengagement of more than half of the circumference of the a-helix involved is involved in theinterface with the CRD5 domain of CD206. Therefore, introduction of natural amino acidanalogues such as ẞ-phenylalanine or homoarginine as part of backbone modifications tointernally stabilize the secondary structure of RP-182 via backbone-to-tail or side chain-to-side chain cyclization was not pursued. While the use of such protease-resistant analogueshas been successfully employed, for instance, in the development of GLP-1 peptideanalogues like liraglutide and semaglutide, which act as GLP-1 receptor agonists and arecurrently used for the treatment of type II diabetes, a substantial risk that the introduction ofnew amino acids, or amino acid moieties, into the KFRKAFKRFF sequence was predicted,which could lead to loss of the unique binding mode, and activity. The unexpected,significantly different activities of the three synthesized cyclic peptides compounds 1b, 1c,and 1d, which showed, despite increased stability in the liver microsome assay for all three,no improvement in biological activity in case of the less constrained 1d, and to a lesserdegree 1b, indicated a very narrow space for medicinal chemistry modifications of acompound targeting CD206 with a pharmacophore including KFRKAFKRFF. Subtlechanges in linkers, ring size, and flexibility of the a-helix were found herein to deeply impactthe secondary structure of the pharmacophore, and its ability to activate CD206, inunexpected ways, leading to the compounds of Formula I.

[0144] Fatty acid derivatizations of the KFRKAFKRFF pharmacophore were performed.Lipidation of such therapeutic peptides may alter pharmacokinetics and biodistribution,which can be advantageous where the KFRKAFKRFF pharmacophore is present. Couplingof fatty monoacids with longer hydrocarbon tails, like the 16-carbon palmitic acid tail ofanalogue RP-182-PEG3-K(palm) 1a, facilitated self-association towards the formation ofmicelle-like aggregates. In the case of RP-182, 1, micelle formation is further enhanced by itshydrophobic surfaces which are part of RP-182's amphipathic structure. While the largermolecular size of such aggregates will slow crossing of capillary and cell membranes in theTME, these aggregates are more likely to be taken up by lymphatic vessels and betransported to tumor-draining lymph nodes (TDLNs), which are frequently populated byCD206-positive macrophages. Upon increased RP-182, or compound of Formula I, deliveryand activation of CD206high macrophages in TDLNs, increased cytokine secretion in the formof IFNy, IL-12, or IL-1ẞ, and elevated antigen presentation as part of CD206highmacrophage reprogramming function may retool, and increase, anti-tumor function ofresiding T cells. With regard to connecting the lysine-palmitic acid moiety to RP-182, athree carbon PEG spacer was selected. While short hydrophilic spacer elements including3,8-dioxa-aminooctanoic acid (Ado) or aminoethyl ethanolamine (AEEA) have dramaticallyincreased half-lives of, for example, the long-acting insulin analogue icodec or the GLP-1 / gastric inhibitory peptide (GIP) dual agonist tirzepatide, a three carbon PEG chain as spacerwould maintain the hydrophilic character and short length. Spacer selection comprises abalance between (1) a too short spacer, which is binding the peptide close to albuminprotecting it efficiently against proteolysis but is impairing interaction with the receptor,versus (2) a too long spacer, which provides freedom to engage the target receptor but doesnot shield the peptide sufficiently from degradation. The lack of activity of the RP-182-EYEK(palmitic acid)EYE chimera 1e compared to the RP-182-fatty acid hybrid 1a notcontaining the heptapeptide, which show similar stability in the MLM assay, is likely due toimpaired binding or steric hindrance of CD206 folding by the added heptamer sequencecompared to 1a, which only carries the palmitic acid tail.

[0145] In contrast, a series of synthesized PEG polymer conjugates with hydrophilicPEG tails showed minimal activity compared to parent RP-182. The more hydrophilicnature of PEG-conjugated analogs may prevent association, or binding, to the hydrophobicgroove on CD206, whereas hydrophobic fatty acid tails of la and 1f improve affinity andbinding. However, compound 1c possesses very similar chemical characteristics on the C-terminal end of the KFRKAFKRFF pharmacophore and exhibits strong activity. Compound1c is characterized by a lysine and PEG monomers on its C-terminus, similar in character to,e.g., compounds 1h, 1i, and 1j, which lacked activity, illustrating the surprising andunexpected results described herein.

[0146] The RP-182-PEG3-K(palm)-NH2 analogue (1a) exhibited not only improvedinternalization of CD206, but improved induction of phagocytosis and cell killing ofCD206high M2-polarized macrophages, and enhanced CD206 binding as compared to RP-182(1). Remarkably, la has several fold improved activity skewed towards macrophageactivation and reprogramming, including phagocytosis, compared to M2 cell killing, which isa particularly attractive pharmacological profile not demonstrated elsewhere. Reinvigorationand recruitment of these abundant cells towards anti-tumor phenotypes and function is thepreferred strategy compared to ablating CD206high TAMs for at least the following reasons.First, CD206high TAM populations harbor pro-inflammatory, anti-tumor cues in the form ofeffective tumor antigen presentation which contributes to tumor restriction. Our previouswork (data not shown) has demonstrated that transferring CD206high M2-polarizedmacrophages into murine tumors does not accelerate tumor growth; whereas, transfer of M1-polarized macrophages significantly reduced tumor progression. Thirdly, single cell RNA-Seq analyses of human cancers have identified CD206 expression on TAM populations withshared pro-inflammatory transcriptional programs including antigen presentation, Th1and Th2 activation pathways, phagocytosis maturation, and interferon signaling overallattesting to the plasticity of these cells and the inability to capture function via single surfacemarker expression. In light of above, immuno-oncology drug development efforts targetingthe mannose receptor CD206 that preserve, and enhance, these signals are likely to moresuccessful than indiscriminately abrogating this heterogenous cell population.

[0147] Thus, a selection of compounds that comprise certain judicious medicinalchemistry modifications described and / or claimed herein demonstrate improved stability and,in embodiments, improved the activity over the CD206 modulator RP-182 targetingCD206high macrophages, despite the unexpected failure of certain compounds withchemically related modifications that one of ordinary skill in the art would assumepreemptive success. Fatty acid RP-182-PEG3-K(palm) derivative 1a shows improvedeffector functions of CD206 receptor activation which translates into improved tumorcontrol, and it appears to be a promising candidate towards further development and clinicaltranslation.EXAMPLE 1Synthesis of fatty acid derivatized, cyclic, and polymer-conjugated RP-182 analogues.

[0148] RP-182, 1, is a linear, a-helical peptide that binds to the CD206 receptor. Thepeptide (H-KFRKAFKRFF-OH; H-Lys-Phe-Arg-Lys-Ala-Phe-Lys-Arg-Phe-Phe-OH)consists of alternating hydrophobic and hydrophilic amino acids, which form opposingcationic and hydrophobic surfaces within the a-helix (FIG. 1A). The amphipathic feature ofRP-182, 1, is essential for the activation of the mannose receptor CD206 and is dependent ona strong hydrophobic moment (FIG. 1B). In order to design RP-182 analogues withimproved metabolic and functional properties, the in vitro metabolism of RP-182 wasexamined. RP-182 underwent a series of rapid hydrolysis reactions which were initiatedeither by exopeptidase-mediated hydrolysis of N- and C-terminal residues or byendopeptidase-mediated cleavage that generated di- or tripeptides (FIGS. 7A to 7D). Τοimprove its stability, whether modifications in the backbone of the alpha helix can yieldsufficient improvements against proteolytic degradation was explored. Considering that allamide bonds have been identified as proteolysis sites in the metabolic stability studies, anumber of RP-182 analogues inserting D-amino acids into the a-helix were synthesized.Unfortunately, RP-182 with D-amino acid substitutions did not show any biological activity(data not shown).To increase resistance of RP-182 to hydrolysis, the generation ofcyclic derivates was pursued (FIG. 8A). Due to increased rigidity and decreased flexibility toaccommodate secondary structures of the RP-182 pharmacophore, cyclic RP-182 derivativesmight be both more resistant to peptidase cleavage if the rigid ring structure preventsendopeptidases from accessing internal cleavage sites as well as have increased affinity to theCD206 receptor and activity. RP-182, 1, binds to a hydrophobic groove in the proximalcarbohydrate recognition domain 5 (CRD5) region of CD206 (FIG. 1C). Repeat docking ofthe cyclic peptide 1c showed that head-to-tail cyclization did not interfere with the fit of theRP-182 pharmacophore into the hydrophobic groove (FIG. 1C). To exploit these findings,head-to-tail closing was employed to synthesize two additional cyclic RP-182 derivatives, 1band 1d with alternate linkers and slightly lower restraint (Table 1; FIG. 8A to 8C).

[0149] Table 1. Analytical data of peptide analogues. Pharmacophore of original parentpeptide RP-182 underlined.Peptide NamePeptideclassSequenceM(HLPC)tr (min) / %B(CH3CN)1(RP-182)LinearKFRKAFKRFF(SEQ ID NO:1)13742.473 / 80la(NCGC00857001)Fatty acidderivativeRP-182-PEG3-K(palmiticacid)-NH219299.016 / 581b(NCGC00886866)Cycliccyclic click-[PEG1-RP-182-K(PEG2)]19789.773 / 401c(NCGC01147950)Cycliccyclic click-[PEG2-RP-182-K(PEG1)]1978 10.908 / 311d(NCGC00887030)Cycliccyclic olefin-[NH-RP-182-Lys-NH2]16388.495 / 33le(NCGC00856998)Fatty acidderivativeRP-182-PEG3-EYEK(palm)-EYE-NH22772 11.907 / 551f(NCGC00591015)Fatty acidderivativeRP-182-NH(CH2)10-CONH2 1556 14.433 / 441g(NCGC00591012)PEGylatedanalogRP-182-PEG2-CONH21532 9.701 / 301h(NCGC00591018)PEGylatedanalogRP-182-PEG3-CONH21562 3.077 / 46li(NCGC00591014)PEGylatedanalogRP-182-PEG5-CONH21665 10.538 / 311j(NCGC00591016)PEGylatedanalogRP-182-PEG8-CONH217838.738 / 321k(NCGC00591017)PEGylatedanalogRP-182-PEG11-CONH21915 11.961 / 33

[0150] tr: elution after HPLC with linear gradient of 20-100%, buffer B in buffer A over30 minutes with flow rate of 1 mL / min on what type of C18-silane packed with octadecylsilylsilica gel, 100A, 5µM. Buffer solution A: 0.1% TFA in acetonitrile; buffer B: 0.1% TFA inwater.

[0151] As a second strategy fatty acid tails were added to RP-182, 1, to stabilize and aiddelivery via albumin binding. Albumin, the main fatty acid binding protein in human plasma,contains a total of seven high and low affinity fatty acid binding sites. Fatty acidderivatization, or lipidation, reduces proteolytic degradation of albumin-bound peptides andminimizes rapid filtration in the glomeruli of the kidneys. Similar to long-acting, depoformulations of insulin which added myristic or palmitic acids to delay absorption andincrease plasma half-life, synthesized peptide derivative la contained a palmitic acid lysinesubstitution connected via a three-carbon atom spacer to RP-182's C-terminus (FIG. 9A).Derivative le conjugated the fatty acid-heptapeptide EYEK(palmitic acid)EYE chimera toRP-182's C-terminus (FIG. 9A). Compared to simple lipidation, the addition of this 7merpeptide sequence is known to increase fatty acid binding to albumin several-fold andconsiderably prolong circulation time (FIG. 1D). RP-182 analogue 1f (RP-182-NH-(CH2)10CONH2) carried a shorter undecanoic acid tail connected to RP-182's C-terminus(FIG. 9B). Thirdly, a series of C-terminal polymer-conjugated peptide analogues weresynthesized to leverage reduction of renal clearance, improved solubility, and resistance tohydrolysis for improved stability and activity (FIG. 9C). Synthesis schemes of the differentclasses of analogues are disclosed in FIGS. 8A-8C and FIGS. 9A-9F. Table 1 summarizesanalytical data of synthesized analogues including HPLC data of investigated RP-182derivatives.EXAMPLE 2RP-182 analogues elicit increased cell killing of CD206high M2-like macrophages.

[0152] To investigate the impact of the medicinal chemistry modifications of RP-182, 1,on biological activity, the synthesized RP-182 analogues were subjected to 10-concentrationdose response testing in alternatively activated, CD206high M2-like macrophages. Activationof CD206 by RP-182, 1, induces a confirmational change in CD206 which activates an NF-KB-mediated inflammatory gene response and induces secretion of pro-inflammatorycytokines including TNFa and IL-1ẞ among others. TNFa activates the tumor necrosis factorreceptor 1 (TNFR1)-FADD-caspase 8 axis which selectively induces cell death in M2-like,CD206-positive, but not CD206-negative, M1-like macrophages. Using a dual calcein-AMethidium homodimer-1 live / dead stain, the series of synthesized analogues was screened forcell killing of CD206high M2-like macrophages, counter screening the same compounds inparallel for induction of cell death in pro-inflammatory, M1-like macrophages as control.Half-maximal inhibitory concentration (IC50; µM) values of the RP-182-PEG3-K(palmiticacid) derivative 1a were 3.2, RP-182-NH-(CH2)10CONH2 derivative 1f, 4.01, and the cyclicpeptide 1c, 11.1 which were up to 5-fold more potent than RP-182's IC50 and were selectedas top hits for further study (FIGS. 2A and 2B; Table 2). Remarkably, contrary to C-terminal substitutions with hydrophobic fatty acid tails, none of the hydrophilic PEG polymeradditions to the C-terminus 1g-1k was permissive possibly interfering with the hydrophobicchemical space of RP-182's binding site on CRD5. Treatment of CD206high M2-likemacrophages with 1c, 1f, and 1a resulted in activation of caspase 8, as well as increasedlevels of the apoptosis marker annexin V, with 1a showing the strongest induction (FIGS. 2Cto 2E). Notably, induced TNFa and IFNy secretion was significantly enhanced in CD206highM2-like macrophages treated with 1a compared to RP- 182, 1 (FIG. 2F). To show that theactivity of selected analogues was dependent on CD206 and TNF signaling, response to RP-182, 1 was examined, and selected analogues in M2-polarized macrophages isolated fromCD206- / -, MyD88- / -, and TNFR1- / - knockout mice in comparison to wild type bone marrow-derived macrophages (BMDMs). CD206 expression after polarization was not affected by thelack of MyD88 or TNFR1 expression (FIGS. 10A to 10C). Treatment with RP-182, 1 orselected analogues failed to induce cell death in macrophages lacking CD206, MyD88, orTNFR1 suggesting that the activity of designed analogues remains dependent on RP- 182'soriginal target CD206 and TNF signaling (FIGS. 2G). In summary, select modifications inform of fatty acid derivatization or cyclization increase cytotoxicity of RP-182, 1, inCD206high M2-polarized macrophages.

[0153] Table 2. Cytotoxicity of synthesized RP-182, 1 analogs in CD206high M2-polarized macrophages after 10-concentration dose response testing. IC50, values in µM, M2selectivity indicates activity absence of activity in M1-polarized macrophages.Peptide IDPeptide ClassIC50 (µM) M2 selectiveLinear1(RP-182)KFRKAFKRFF(SEQ ID NO:1)17.6Yes1b(NCGC00886866)cyclic click-[PEG1-RP-182-K(PEG2)]11.96Yes1c(NCGC01147950)cyclic click-[PEG2-RP-182-K(PEG1)]11.1Yes1d(NCGC00887030)cyclic olefin-[NH-RP-182-Lys-NH2]>100YesPEG-RP-182 conjugates1g(NCGC00591012)RP-182-PEG2-CONH2>100Yes1h(NCGC00591018)RP-182-PEG3-CONH2>100Yesli(NCGC00591014)RP-182-PEG5-CONH2>100Yes1j(NCGC00591016)RP-182-PEG8-CONH2>100Yes1k(NCGC00591017)RP-182-PEG11-CONH2>100YesFatty acid RP-182 analogsla(NCGC00857001)RP-182-PEG3-K(palmiticacid)-NH23.2Yesle(NCGC00856998)RP-182-PEG3-EYEK(palm)-EYE-NH2>100Yes1f(NCGC00591015)RP-182-NH(CH2)10-CONH24.03YesEXAMPLE 3Medicinal chemistry modifications of RP182 enhance CD206 internalization and cancercell phagocytosis of CD206high M2-like macrophages.

[0154] In addition to induction of programmed cell death, CD206 agonism by RP-182, 1,also activates phagocytosis including cancer cell phagocytosis in M2-polarized macrophagesnot undergoing apoptosis. The percentage of M2 killing versus cells responding withactivation of phagocytosis was concentration dependent, with a high level of killing observedat higher concentrations of RP-182, 1. To investigate whether the synthesized analoguesretain, and possibly improve, RP182- mediated phagocytosis, induction of the pan-phagocytosis markers RAB7 was measured in CD206high M2-polarized macrophages treatedwith vehicle, RP-182, 1, and RP-182 analogues 1c, 1f, and 1a next. The RP-182-PEG3-K(palmitic acid) derivative 1a showed significantly increased potency in the induction ofphagocytosis in 10-concentration dose-response testing compared to RP-182, 1 and otheranalogues (FIG. 3A). To show that increased levels of phagocytosis are associated withCD206 function, co-localization of RAB7, a marker located in the membrane of early andlate endosomes, and internalized CD206 in the cytoplasm of CD206high M2 macrophages wasexamined via confocal microscopy. Linear distance analysis of cytosolic endosomalstructures showed co-localization of RAB7 and internalized CD206 in induced phagosomesof M2-polarized macrophages after treatment with RP-182 and 1a (FIG. 3B). To quantifyCD206 internalization as a function of CD206 activation, proximity ligation assays (PLAs) ofRAB7 and CD206 was performed. The assay gives a positive signal via the intercalation of ared fluorophore into an amplified circular DNA product generated when two proteinsdetected by species-specific antibodies are in < 40 nm proximity. Treatment with 1a inducedthe strongest colocalization signal of CD206 and RAB7, (FIG. 3C). There was no inductionof phagocytosis and no PLA signal in M2-polarized macrophages derived from CD206- / -mice (FIG. 12A). Interferon response factors (IRFs), including IRF7, are activated as part ofa cytosolic pattern recognition response which involves pattern recognition receptor (PRR)signaling after internalization from the endosomal surface. IRF activation is thus linked toPRR ligation and the initial endophagosomal response induced by the PRR activation.Similar to the induction of RAB7, the induction of the phosphorylated, active form of IRF7,pIRF7, was strongest in CD206high M2-like macrophages treated with 1a and tied to thepresence of CD206 (FIG. 3D; FIGS. 12B and 12C). Notably, for la the EC50 for RAB7(FIG. 3A) and pIRF7 induction (FIG. 3D) was in the range of >10 times lower than 1a'sIC50 for M2 killing (FIG. 2A), whereas EC50 values for RAB7 and pIRF7 induction and IC50values for induced cell death were in the same range for RP-182, 1c, and 1f, possiblyindicating that 1a is an improved molecule with skewed activity towards the induction ofphagocytosis at concentrations that do not induce M2 apoptosis. Next, whether increasedinduction of phagocytosis translated into elevated rates of cancer cell phagocytosis wasinvestigated, an important mechanism of RP-182's anti-tumor phenotype. After co-incubationof CSFE-labeled cancer cells and CD206high M2-like macrophages treated with RP-182'sanalogues, la induced the highest rates of cancer cell phagocytosis (FIGS. 3E and F). In linewith pIRF7 activation, RP-182 analogues increased secretion of inflammatory cytokinesIFNa and IL-12 regulated by interferon regulatory factor signaling and increasedexpression of the M1 macrophage activation marker CD86 (FIGS. 3G to 3I; FIGS. 12B to12E). In summary, medicinal chemistry modifications of RP-182, 1 yielded analogues withenhanced effector functions in CD206high M2-polarized macrophages, including the inductionof both phagocytosis and interferon response signaling.EXAMPLE 4Fatty acid derivatized and cyclic RP-182 analogues show increased activity.

[0155] Next, whether the improved activity of RP-182 analogues 1c, 1f, and la wasassociated with improved metabolic stability was examined. Intrinsic half-life was measuredin liver microsomes via a sensitive LC-MS / MS assay. Stability of 1c, 1a, and 1f wassignificantly increased compared to RP-182 (FIG. 4A). Projected half-lives for 1c, 1a, and 1fwere calculated as 122.2, 140.7 and 41.63 min, respectively, compared to 23.02 min for RP-182, 1. Of note, stability of the less active cyclic peptide 1d or the non-active RP-182-EYEK(palmitic acid)EYE peptide chimera 1e was improved compared to RP-182, 1 andsimilar to the undecanoic acid derivatized analogue 1f suggesting that these modifications arenot permitted and impair proper interaction of the pharmacophore with the CD206 receptor.Relaxation of the RP-182 pharmacophore with a longer linker in case of 1d, or interferenceof the added albumin-binding heptapeptide EYEK(palmitic acid)EYE with CD206 receptorbinding or induction of the conformational switch, appear to have offset advantages ofincreased stability.

[0156] Next, to confirm that measured increased stability of the studied analogues wasdue to changes in metabolism compared to the linear RP-182, 1, the three analogues 1c, 1f,and la were subjected to detailed metabolite identification using the same incubation times asfor RP-182, 1. Cyclization of RP-182 and lipidation of the C-terminus of RP-182protected analogues from degradation by exopeptidase activity with cyclic peptide 1c alsoshowing increased resistance to endopeptidase cleavage (FiIGS. 11A to 11C). In contrast,the metabolite profile of lipidated RP-182 analogue 1f carrying a capric acid tail, RP-182-HYD (C10)COOH2, was only marginally different from RP-182, 1. Overall, there was nocorrelation between cell-based activities IC50 values of M2 cell killing or EC50 values ofRAB7 and pIRF7 induction and stability parameters of the RP-182 analogues suggesting thatfactors other than improvement of stability are likely involved in the observed improvedactivity profiles (FIG. 4B). Thus, to examine whether medicinal chemistry modificationsaltered affinity and binding of synthesized analogues to the CD206 receptor, RP-182analogues were first docked to the CRD5 region of the CD206 receptor and determined insilico affinities. Estimated Ka values were higher (3.4 µM) for 1a compared to parent RP-182, 1 and other analogues (FIG. 4D). Next, binding of RP-182 analogues to recombinantCD206 was measured using a sensitive microscale thermophoresis (MST) assay, and lashowed the greatest affinity to CD206 (FIG. 4D). 1k, a PEGylated RP-182 analogue whichhas no impact on viability of CD206high M2 macrophages, was used as a negative control andfound not to bind to CD206 (Table 2, FIG. 4D). To confirm these findings, the interaction ofRP-182 analogues with CD206 was investigated in a cell-based context through evaluation ofthe ability of RP-182 analogues to outcompete RP-182 bound to CD206 expressed on M2-polarized macrophages. Fractions of CD206high macrophages with bound biotinylated RP-182were detected via fluorophore-labeled streptavidin (FIG. 4E; FIG. 13). In comparison to 1cand 1f, the addition of 1a increased the level of non-fluorescently labelled cells mostefficiently suggesting increased competition due to higher affinity of the fatty acid derivative1a to the CD206 receptor compared to RP-182, 1 (FIG. 4E). In summary, whilemedicinal chemistry modifications of RP-182 improved stability, some modifications alsoimproved CD206 receptor binding and activation.EXAMPLE 5RP-182 analogues suppress tumor growth of murine B16 melanoma and autochthonousKPC tumors.

[0157] Subsequently, whether the improved stability and cell-based activities of the RP-182 analogues would translate into improved tumor control was examined using thesyngeneic B16 melanoma and the immunologically cold, Ras-driven autochthonous KPCpancreatic cancer model. Spontaneously occurring pancreatic tumors in geneticallyengineered KPC mice are immunologically cold tumors and harbor a large infiltrate ofCD206high TAMs. After tumors in KPC mice were confirmed by transabdominal ultrasoundscan (USS) to be ≥200 mm³ in size, mice were randomized to treatment with vehicle, RP-182, 1, and 1a. Tumor volume was recorded by USS once weekly. Mice randomized totreatment with 1a showed superior suppression of tumor growth and extension of survivalcompared to RP-182, 1 (FIGS. 5A and 5B). In line with the activation and reprogrammingeffects of RP-182 analogs in CD206high M2-like macrophages in cell-based assay, 1aincreased the fraction of CD206-positive TAMs expressing the M1 activation markers CD86and CD40, inflammatory cytokines IFNy, IL-12ẞ, IL-1ẞ, and TNFa, and decreased theinhibitory phagocytosis checkpoints SIRPa and PD-1 but increased rates of cancer cellphagocytosis (FIGS. 5C). Overall, the M2-to-M1 switch was more pronounced in KPCtumors treated with la than in the RP-182, 1, group (FIG. 5C). Next, RP-182, 1, andanalogues 1c, 1f, and 1a were evaluated in B16 melanomas generated after intradermalinjection of B16 cells. 1a showed the most effective tumor control compared to RP-182, 1,and other analogues which were less effective (FIG. 5D). Flow cytometry analysis oftumors harvested after 14 days of treatment showed an increase in TAMs expressing IFNy,IL-12ẞ, and CD40, all of which were higher in the la compared to RP-182, 1, arm, with theexception of IL- 12B (FIG. 5E).

[0158] In summary, select modifications of the innate immune checkpoint modulator RP-182, 1, a linear, amphipathic peptide activating the mannose receptor CD206, in the form oflipidation increased stability and biological activity. The resulting activation of CD206highmacrophages was defined by a propensity towards cancer cell phagocytosis rather than TNFsignaling- mediated cell death, which translated into improved tumor control (Fig. 6). Thederivative 1a carrying a palmitic acid lysine substitution coupled to the C-terminus appears tobe a promising preclinical candidate for further development.MethodsPeptide synthesis

[0159] De RP-182, 1, and its analogues were synthesized using solid-phase peptidesynthesis on a Liberty Blue synthesizer. Peptides were coupled to the Rink amide resin withS-tritylmercaptopropionic acid as a linker using N,N,N',N'-tetramethyl-O-(6-chloro-1H-benzotriazol-1-yl)uronium hexafluorophosphate (HCTU) for amino acid activation. LinearRP-182 peptide analogues were first synthesized using Fmoc chemistry using the Rinkamide resin. Peptides were cleaved using a mixture of 95% trifluoroacetic acid (TFA) / 2.5%tri-isopropylsilane / 2.5% H2O. TFA was removed and the residue partitioned between 50%acetonitrile in water containing 0.1% TFA and cold diethyl ether. All grafted peptides weresynthesized on a 0.6 mmol scale. After cleavage, the aqueous fractions were lyophilized, andthe resulting crude peptides were purified using reverse-phase HPLC (RP-HPLC) andanalyzed by LCMS.Cyclic RP-182 peptide analogues

[0160] Head-to-side-chain closing using orthogonal click chemistry for RP-182analogues 1b and 1c, was carried out as shown in FIG. 8A. The peptide 1b was synthesizedmanually on Rink Amide resin, using a standard Fmoc synthesis protocol, adapted forassembling the monomers used for cyclization through the click chemistry reaction.

[0161] Specifically, Dde-Lys(Fmoc)-OH was the first amino acid to load onto the resin.After the removal of the protecting group Fmoc with a DMF solution of 20% piperidine,Azido-PEG1-acid was coupled to the ɛ-NH2 of Lys. Afterwards, the protecting group Ddewas removed with a DMF solution containing 2% hydrazine. Fmoc-Phe-OH was thencoupled to the a-NH2 of Lys. The rest of amino acids were assembled sequentially.

[0162] While keeping the Fmoc group of last amino acid intact, the click chemistryreaction was conducted between endo-BCN-PEG2-acid in a DMF solution and the on-resinazido-PEG1-acid for 4 hours. The last Fmoc group was then de-protected, and thecyclization reaction between the freshly exposed a-NH2 of the last amino acid and thecarboxyl group of endo-BCN-PEG2- acid was initiated and allowed to continue overnight.

[0163] The crude peptide was cleaved off the resin with a TFA cocktail (95%TFA,1%H2O, 2%EDT, 2%TIS), and precipitated by adding ice-chilled diethyl ether. Thecrude peptide was freeze-dried, loaded onto a prep-HPLC column and purified with agradient of 20%-45% Acetonitrile within 40 minutes at a flow rate of 10 ml / min. Theproduct was analyzed, confirmed to have >95% HPLC purity, and freeze-dried.

[0164] In a similar manner, peptide 1c was prepared as shown in FIG. 8A where afterassembling the peptide on the Rink Amide resin and attaching the PEG azide moiety to theN-terminal Lys, the Dde group was removed as previously shown and coupled to the Fmoc-PEG2-acid. Removal of the Fmoc followed by simultaneously click / coupling tobicyclo[6.1.0]non-4-yn-9-ylmethyl (2,5-dioxopyrrolidin-1-yl) carbonate gave 1c which wasdeprotected and cleaved from the resin to give 1c.

[0165] In a similar manner, the olefin peptide 1d was prepared as shown in FIGS. 8Band 8C. After the removal of the Dde group from the ɛ-NH2 of Lys, the intramolecularcyclization was carried out with the acid group coming from the N-terminal residue usingHATU. The protecting groups were removed as shown above and cleaved from the resin.The peptide was purified by HPLC.Fatty acid-derivatized RP-182 analogues

[0166] RP-182-PEG3-EYEK(palmitic acid)EYE-NH2, 1e, RP-182-PEG3-K(palmiticacid)-NH2 1a, and RP-182-HYD (CH2)10-COOH, 1f, were synthesized on a Rink Amideresin as shown in FIGS. 9A and 9B using the appropriate Fmoc-NH-amino acid, Fmoc-NH-HYD(C10)-COOH, or Fmoc-Lys(palm)-COOH followed by the growth of the peptide, finaldeprotection, removal from the resin and purification by reversed-phase HPLC. All purifiedpeptides were confirmed by LCMS.PEG-substituted RP-182 analogues

[0167] C-terminal conjugated PEG analogues, 1e, 1f, 1g, 1h, li, 1j, 1k, of RP-182, 1,were synthesized as shown in FIG. 9C, with the exception that the corresponding Fmoc-NH-PEGn-OH was attached to the Rink amide resin before the deprotection and coupling to thecorresponding Fmoc AAs in the RP-182, 1, sequence. The final deprotection and removalfrom the resin were carried out as described above. The peptides were purified by reversed-phase HPLC. The final products were confirmed by LCMS.Microsomal stability and peptide metabolism

[0168] Stability of RP-182, 1, and analogues of 1 was evaluated in murine livermicrosomal (MLM) fractions (Male CD1 Mouse Liver Microsomes [Catalog# M1000;Xenotech LLC)]. One mL of MLM suspension in water was stirred at 65 °C for 1 h. Thesolution was allowed to cool to room temperature. All peptides were first dissolved separatelyin sterile H2O to make a 7.0 mM solution (solution 1). Α 10 µL aliquot of solution 1 wasdiluted with 490 µL of water (140 μΜ; solution 2). A 10 µL aliquot of solution 2 was dilutedseparately with (a) 440 µL of denatured MLM suspension (solution 2a) and (b) 440 µL ofMLM suspension in water (solution 2b) resulting in a 3.11 µM solution of solutions 2a and2b. Solutions 2a and 2b were further diluted by taking a 152 µL aliquot separately anddiluting it with (c) 68 µL of water or (d) 68 µL of NADPH solution to make 2.15 μΜsolutions of (1) peptide with denatured MLMs and (2) peptide with activated MLMs,respectively. Peptide samples were subjected to HPLC-TOF MS analysis with a proteincolumn for better separation purposes. Liquid chromatography (LC) was performed on anAgilent 1290 Infinity II LC system (Agilent Technologies, Wilmington, DE). The mobilephases used for the separation were MS-grade water with 0.1% formic acid (solvent A) andMS-grade acetonitrile with 0.1% formic acid (solvent B). Gradient elution of peptides fromthe analytical column (HALO BioClass Protein Diphenyl, 1000 Å, 2.7 µm, 2.1 x 100 mm)was performed using a gradient starting at 5% B at a flow rate of 0.4 mL / min. The mobilephase was then 5-15% B for 1 min, 15-45% B for 3.5 min, 45 to 90% B for 0.5 min, andmaintained at 90% B for 1.5 min, followed by 5% B for 0.5 min and re-equilibration of thecolumn with 5% B for 1.4 min. Separations were performed at a column temperature of 60°C with a total run time of 8 min. Mass spectrometry (MS) experiments were conducted onan Agilent 6230 TOF system (Agilent Technologies), equipped with a DUAL AJS ESIsource operating in positive ion mode. HPLC-TOF data analysis was performed by AgilentMassHunter Qualitative Analysis (B.07.00).Metabolite identification

[0169] Relative peak areas were determined from extracted ion chromatograms of livermicrosome samples (males, strain ICR / CD-1; cat.# 452701, Corning, NY). 10 µM MLM (1mg / mL) as biological matrix and cofactor NADPH (2 mM) were incubated for 0, 30, 60 minat 37 °C. Incubations were quenched with 1 / 4 volumes of 15% TFA followed bycentrifugation for 15 min at 16,000 xg. Supernatants were analyzed by LC-MS / MS using anACQUITY UPLC Peptide BEH C18 column in a Dionex UltiMate 3000 UHPLC system(ThermoFisher Scientific) with solvents: A, ACN (0.1% formic acid); B, water (0.1% formicacid), flow rate: 500 µL / min for 0-1.5 min, 5% A, 1.5-7.0 min, 5%-40% A, 7.0-8.0 min,40%-100% A, 8.0-9.0 min, 100% A, 9.0-10.0 min, 5% A followed by MS analysis using a QExactive™ Plus Hybrid Quadrupole- Orbitrapтм Mass Spectrometer operated under aninternal analysis algorithm (Pharmaron, Louisville, KY).In silico docking

[0170] Protein-protein interaction of RP-182 and RP-182 analogues with the CRD5region of CD206 was evaluated by in silico docking utilizing the MOE program(www.chemcomp.com). The default parameters in the protein-protein dock module in MOEwith hydrophobic patch potential were applied. The 3D structure of CRD5 was extractedfrom the model of full length CD206, which was generated using AlphaFold 2(alphafold.ebi.ac.uk). A total of 100 poses of binding complexes were generated,respectively. Binding interactions with the predicted models were refined with energyminimization and analyzed based on experimental data.Microscale thermophoresis (MST)

[0171] The binding of RP-182, 1, fatty acid-derivatized analogues 1f and 1a, and cyclicanalogue 1c to purified recombinant CD206 protein was evaluated by microscalethermophoresis (MST). Recombinant polyhistidine-tagged human CD206 was purchasedfrom Acro Biosystems and labeled with RED-tris-NTA 2nd generation dye (NanotemperTechnologies, Munich, Germany) in MST buffer (PBS, pH 7.4, 0.05% Tween-20) followingthe manufacturer's protocol. Two-fold serial dilutions of the peptides were prepared in MSTbuffer and incubated with the same volume of labeled CD206 (final concentration: 50 nM)for 30 min at room temperature (RT). Measurements were carried out in premium capillariesusing a Monolith NT.automated instrument (Nanotemper Technologies, Munich, Germany)with medium MST power and 5% excitation power. Ka values were calculated usingMOAffinity analysis software (Nanotemper Technologies, Munich, Germany).Bone marrow-derived macrophage retrieval, culture, and polarization

[0172] The generation of M2-polarized macrophages has been previously described. Inbrief, murine myeloid precursor cells were obtained by flushing out the bone marrow fromfractured femur bones of 6-8-week-old C57B / L6 wild-type mice (Charles RiverLaboratories, Rockville, MD) or mannose receptor 1 (Mrc1; CD206)-deficient B6.129P2-Mrc1tm1Mnz / J (strain# 007620), MyD88 null B6.129P2(SJL)-Myd88tm1.1Defr / J (strain# 009088)and TNFR1 null B6.129- Tnfrsf1atm1Mak / J (strain# 002818) (all from The Jackson Laboratory,Bar Harbor, ME) using IMDM or RPMI media with 10% FBS loaded in a 5 mL syringeconnected with a 26-gauge needle. Both ends of the bones were flushed, and bone marrowaspirates were diluted to 40 mL regular growth media and incubated at 37°C with 5% CO2for 4 hours to remove adherent mature macrophages from the hematopoietic progenitors.Culture supernatant was centrifuged for 5 min at 1,200 rpm, cell pellets were resuspended in5 mL of media, nucleated cells were counted, and 0.5 - 1 x 106 cells seeded into T75 flasks inIMDM or RPMI with 10% FBS, 150 µM monothioglycerol (MTG), and 10 ng / mL of M-CSF(cat.# PMC2044, Thermofisher Scientific). On day 4, the media was replaced with freshMCSF-containing media. On day 7, IL-4 (mouse IL-4 (Cat# 404-ML-050) at 20 ng / mL or 10ng / mL of IFNy was added for polarization into M2 or M1 phenotypes, respectively. After 48hours of IL-4 treatment for the M2 phenotype, the media was replaced with regular growthmedia for 24 hours prior to the treatment of cells. For the M1 phenotype, after treating withIFNy for 24 hours, the media was replaced with regular media for 48 hours. The polarizationwas confirmed by expression of M1 and M2 markers using flow cytometry.Immunofluorescence assays

[0173] Immunocytochemistry analysis was carried out using a Zeiss LSM 880 confocalmicroscope. 50,000 myeloid progenitors were seeded onto 8-well chamber slides (cat.#80807, Ibidi USA Inc, WI), polarized with IL-4 for 48 hours, and incubated for 24 hours aftermedia was changed to non-IL-4 containing growth media. Then, cells were treated with 0.1µM of RP-182, 1, or RP- 182 analogues for 2 hours at 37°C followed by fixation with 4%paraformaldehyde in PBS for 15 min, permeabilization with 0.3% Triton X-100 in PBS for 5min and blocked with 3% BSA in PBS for 1 hour. After blocking, cells were incubated withrespective primary antibodies (1:250 dilution) for 1 hour at RT. Staining with secondaryantibodies was carried out for 1 hour at RT at 1:1000 dilution, followed by washing with PBSand the addition of DAPI with mounting media (cat.# P36931, Invitrogen). Images weretaken at 63x magnification, and three separate images for each treatment group containingabout 200 cells were analyzed using ImageJ software (NIH). The number of automaticallycounted bright objects (fluorescence of secondary antibodies for specific proteins) wasnormalized to the number of DAPI-stained nuclei. For relative comparisons, the fluorescenceratio for vehicle-treated cells was set to 1.Cell viability assay

[0174] Cell viability of seeded macrophages treated with RP-182, 1, or RP-182 analogueswas determined using the Live / Dead Viability / Cytotoxicity Kit (CAT# L3224, ThermoFisherScientific, Grand Island, NY). Macrophages were seeded onto glass bottom 96 well plates(Corning, NY), polarized into M1 and M2 phenotypes, and treated with differentconcentrations of RP-182, 1, and respective analogues ranging from 0.01 µM to 100 µM for48 h. After drug treatment, cells were washed with DPBS, and incubated with 100 µLmixture of 2 µM calcein- AM and 4 µM ethidium homodimer dissolved in PBS for 1 hour.Fluorescence measurements were obtained using a plate reader (Biotek Synergy, Winooski,VT) with an excitation wavelength of 494 nm and an emission wavelength of 517 nm forcalcein. IC50 values were calculated using GraphPad Prism software (La Jolla, CA) aftersetting the number of vehicle- treated macrophages to 100%.Cytokine measurement

[0175] To measure concentrations of secreted chemokines, conditioned media frommacrophages were collected after 2 hours and 24 hours incubation with RP-182, 1, and RP-182 analogues and added into 96-well ELISA plates with detection antibodies against TNFα,IFNa and y, and IL-12ß, standard reagents, and diluents in accordance with themanufacturer's protocol (cat.# BMS607-3, BMS6027, BMS228, BMS616, ThermofisherScientific). Measurements were derived from a standard curve generated by plotting meanabsorbance against protein concentration at serial dilutions and curve fitting.Proximity ligation assay (PLA)

[0176] Proximity ligation assays (PLA) were conducted on macrophages seeded into 8-well chamber slides (cat.# 80807, Ibidi USA Inc, WI) and fixed and permeabilized under thesame protocols as for immunofluorescence studies using the Duolink In Situ Red Starter KitMouse / Rabbit (Millipore Sigma, Burlington, MA) following the manufacturer's instructions.The anti-RAB7 antibody raised in mouse (cat.# sc-376362, Santacruz Biotech) and the anti-CD206 antibody raised in rabbit (cat.# ab64693, Abcam) were used at 1: 250 dilution.Phagocytosis assay

[0177] Primary KPC cancer cells (murine pancreatic cancer) were labeled with CFSE (5-(and-6)- carboxyfluorescein diacetate, succinimidyl ester) dye (cat.# C1157, ThermoFisherScientific, Grand Island, NY) for 1 h at 37°C according to manufacturer's instruction. CFSE-labeled cancer cells were added onto RP-182, 1, or RP-182 analogue-treated M2 polarizedBMDMs (0.1 μΜ; for 2 hours) and co-cultured for 6 hours before excess cancer cellswere washed away and prepared for live microscopy. Images of green-labelled macrophageswere collected on a Zeiss LSM 880 confocal microscope at 63x using green and phasecontrast channels.Animal models

[0178] All experiments were conducted according to protocols and policies approved bythe Institutional Animal Care and Use Committee (ACUC) of the National Institutes ofHealth. 8-12-week old C57B / L6 mice were intracutaneously injected with 1 x 106 B16 cellssuspended in 100 µL HBSS in the right flank. The length and width of tumors were measuredby a caliper twice a week. Tumor size was calculated by the formula: (L x W²) / 2. Tumor-bearing mice were randomized to respective treatment groups including vehicle (PBS), 20mg / kg RP-182 (PolyPeptide Group, San Diego, CA), or respective analogues. Peptides wereinjected intraperitoneally (IP) with a final volume of 200 µL 0.9% N / S at the same dose everyother day. Tumor growth was followed until tumors reached 2,000 mm³ or mice reached thestudy endpoint (determined by 20% weight loss, recognizable signs of morbidity, abnormalposture, loss of ability to ambulate, labored respiration, inability to drink or feed) whereanimals were euthanized in accordance with ACUC animal care guidelines. Geneticallyengineered KPC mice were followed with serial abdominal palpations and subject to weeklytransabdominal ultrasound (USS) examination when an abdominal mass was palpable. Micewere randomized when tumors measured ≥ 200 mm³ to treatment with vehicle, RP-182, 1, or1a. Best objective responses (BORs) recorded a % change in tumor volume at baseline andmaximum tumor control during weekly USS while mice received treatment.Dissociation of tumors and flow cytometry

[0179] After CO2 euthanasia, tumors cut into approximately 1 mm³ cubes were subject todigestion using a combination of enzymatic (Mouse tumor dissociation kit # 130-096-730,MACS Miltenyi Biotec, San Diego, CA) and mechanical dissociation (Gentle Macs Agitator,Miltenyi Biotec) as per company's protocol. Tumor lysates were passed through a 70µmfilter, washed in PBS, and stained with respective fluorophore-coupled antibodies prior toflow cytometry. BMDMs subject to flow cytometry were treated with RP-182, 1 or RP-182analogues for 2 hours or 24 hours at 37°C prior to staining. Cells were stained with theLive / Dead Fixable Blue Dead Cell Stain Kit (ThermoFisher Scientific, Grand Island, NY)and washed with FACS buffer prior to sample acquisition on a BD LSRFortessa SORP I flowcytometer (BD Bioscience). Flow cytometry data was analyzed using FlowJo software(TreeStar, Ashland, OR). For metastasis quantification, lungs at necropsy were individuallyperfused with 4% paraformaldehyde (4% PFA) and allowed to fix for 24 to 48 hours at RT.Specimens were rinsed in distilled H2O, transferred to 70% ethanol (mixed in distilled H2O),and 4 µm sections from paraffin-embedded blocks were stained with hematoxylin and eosin(H&E).Statistical analysis

[0180] Data was statistically analyzed using SPSS software version 16 (IBM, Armonk,NY). Continuous data, including absolute immune fluorescence of total bright objects, tumorvolumes, or immune cell population percentages were compared using Student's t-test inGraphPad Prism. Flow data analysis was performed by one-way ANOVA with the post hocTukey's test. Error bars indicate standard error of the means (SEM) unless otherwiseindicated. Calculated p values were given by number and asterisk(s) with * indicatingp<0.05, ** p<0.01, and *** p<0.001.Materials

[0181] Common chemicals for peptide synthesis of RP-182 backbone included: Fmoc-protected amino acids, Rink amide resin, 1-hydroxy benzotriazole (HOBt), diiodomethane,ethanedithiol (EDT), diethyl ether, and trifluoracetic acid (TFA), N,N'-diisopropylcarbodiimide (DIC) and 2-cyano- 2-(hydroxyimino) acetic acid ethyl ester (oxyma),hexafluoro-16-phosphane, 2-(3H- [1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium salt (HATU), bicyclo[6.1.0]non-4-yn-9-ylmethyl (2,5-dioxopyrrolidin-1-yl) carbonate, Dimethylformamide (DMF), dichloromethane (DCM),acetonitrile (ACN), tetrahydrofuran (THF), tris(2- carboxyethyl)phosphine hydrochloride(TCEP), N,N-diisopropylethylamine (DIPEA), piperidine, thioanisole, N-a-1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl-N-e-Fmoc-L-lysine, 6-Carboxytetramethylrhodamine, Triethylamine (Et3N), triisopropylsilane (TIS), Fmoc-Lys(Palmitoyl)-OH, bicyclo[6.1.0]non-4-yn-9-ylmethyl (2,5-dioxopyrrolidin-1-yl) carbonate,2,5-dioxopyrrolidin-1-yl-1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azatridecan-13-oate werepurchased from Sigma Aldrich (Millipore Sigma, St. Louis, MO). 1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azatridecan-13-oic acid and 3-(2-azidoethoxy)propanoic acid were obtainedfrom piperidine, thioanisole, N-a-1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl-N-ε-Fmoc-L-lysine, 6-Carboxytetramethylrhodamine, Triethylamine (Et3N), triisopropylsilane(TIS), Fmoc- Lys(Palmitoyl)-OH, bicyclo[6.1.0]non-4-yn-9-ylmethyl (2,5-dioxopyrrolidin-1-yl) carbonate, 2,5-dioxopyrrolidin-1-yl-1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azatridecan-13-oate were purchased from Sigma Aldrich (Millipore Sigma, St. Louis, MO).1-(9H-fluoren-9-yl)-3-oxo- 2,7,10-trioxa-4-azatridecan-13-oic acid and 3-(2-azidoethoxy)propanoic acid were obtained from AA Blocks Inc., Endo-BCN-PEG2-acid(CAS# 1993134-72-7) from AxisPharm, and Azido- PEG1-acid (CAS# 1393330-34-1) fromBroadPharm, and 4-Octenedioic acid, (4E) (CAS# 48059-97-8) from Aaron Chemicals LLC(all San Diego, CA). All reagents and solvents were of analytical grade and used withoutfurther purification.

[0182] One skilled in the art will readily appreciate that the present disclosure is welladapted to carry out the objects and obtain the ends and advantages mentioned, as well asthose inherent therein. The present disclosure described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended as limitations on the scope of thepresent disclosure. Changes therein and other uses will occur to those skilled in the art whichare encompassed within the spirit of the present disclosure as defined by the scope of theclaims. In addition, the section headings used herein are for organizational purposes only andare not to be construed as limiting the subject matter described.

[0183] No admission is made that any reference, including any non-patent or patentdocument cited in this specification, constitutes prior art. In particular, it will be understoodthat, unless otherwise stated, reference to any document herein does not constitute anadmission that any of these documents forms part of the common general knowledge in theart in the United States or in any other country. Any discussion of the references states whattheir authors assert, and the applicant reserves the right to challenge the accuracy andpertinence of any of the documents cited herein. All references cited herein are fullyincorporated by reference, unless explicitly indicated otherwise.

[0184] The present disclosure shall control in the event there are any disparities betweenany definitions and / or description found in the cited references.

[0185] Various aspects and embodiments of the disclosure are provided by the followingenumerated embodiments, which may be combined in any number and any combination notlogically or technically inconsistent.Embodiment 1. A compound of Formula I:X-Phe-Arg-Lys-Ala-Phe-Lys-Arg-Phe / H2NFormula Ior a pharmaceutically acceptable salt thereof, whereinX is H; andY is represented by Formula la:whereinPEG is polyethylene glycol:Q is 0 to 4;R is 0 or 1;S is 0 or 1;V is absent or C6-C20alkyl, C6-C20alkenyl, or C6-C20alkynyl;Tis 0 or 1;U is C6-C20alkyl, C6-C20alkenyl, or C6-C20alkynyl;orX and Y are linked to form a cyclic compound, where X and Y are represented byFormula 1b:-C-PEGm-A-B-PEG-C-Lys-Formula 1bwherein for Formula 1b:A is a methylcarbamate, N-methyl carbamate, N-ethyl carbamate, N-methylmethylcarbamate, or N-ethyl methylcarbamate;B is a 12-Methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene; 5-Methyl-12-methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene; 5-Methyl-12-ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene;whereinA comprises a non-carbonyl oxygen (hydroxyl oxygen); andB comprises a cyclopropyl group; andat least one carbon separates the non-carbonyl oxygen on the carbamate and thecyclopropyl group;PEGm is polyethylene glycol:PEGn is polyethylene glycol:whereinm is 1 or 2;n is 1 or 2;wherein X corresponds to the carbonyl end of Formula 1b and Y corresponds to the -Lys- end of Formula 1b; andthe Lys in Formula 1b binds to the carbonyl carbon (C=O) through a Lys sidechainnitrogen;orX and Y are linked to form a cyclic compound, where X and Y are represented byFormula 1c:-C-PEG-D-E-PEG-C-Lys-Formula 1cwherein for Formula 1c:D is a 12-Methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 5-Methyl-12-methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 5-Methyl-12-ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene;E is a methylcarbamate, N-methyl carbamate, N-ethyl carbamate, N-methylmethylcarbamate, or N-ethyl methylcarbamate;whereinD comprises a cyclopropyl group; andE comprises a a non-carbonyl oxygen (hydroxyl oxygen); andat least one carbon separates the non-carbonyl oxygen on the carbamate and thecyclopropyl group;PEGm is polyethylene glycol:PEGn is polyethylene glycol:whereinm is 1 or 2;n is 1 or 2;wherein X corresponds to the carbonyl end of Formula 1c and Y corresponds to the -Lys- end of Formula 1c; andthe Lys in Formula 1c binds to the carbonyl carbon (C=O) through a Lys sidechainnitrogen.Embodiment 2. The compound or salt of embodiment 1, whereinX is H; andY is represented by Formula la.Embodiment 3. The compound or salt of embodiment 2, whereinQ is 1 to 4, R is 1, and S is 1, V is absent, T is 1, and U is C6-C20alkyl, C6-C20alkenyl,or C6-C20alkynyl.Embodiment 4. The compound or salt of embodiment 2, whereinQ is 2 to 4, R is 1, and S is 1, V is absent, T is 1, and U is C8-C18alkyl, C8-C18alkenyl,or C8-C18alkynyl.Embodiment 5. The compound or salt of embodiment 2, whereinQ is 3 to 4, R is 1, and S is 1, V is absent, T is 1, and U is C10-C16alkyl, C10-C16alkenyl, or C10-C16alkynyl.Embodiment 6. The compound or salt of embodiment 2, whereinQ is 3, R is 1, and S is 1, V is absent, T is 1, and U is C10-C16alkyl, C10-C16alkenyl, orC10-C16alkynyl.Embodiment 7. The compound or salt of embodiment 2, whereinQ is 1 to 4, R is 0 or 1, and S is 0, V is C6-C20alkyl, C6-C20alkenyl, or C6-C20alkynyland T is 0 or 1.Embodiment 8. The compound or salt of embodiment 2, whereinQ is 2 to 4, R is 0 or 1, and S is 0, V is C C8-C18alkyl, C8-C18alkenyl, or C8-C18alkynyl, and T is 0 or 1.Embodiment 9. The compound or salt of embodiment 2, whereinQ is 3 to 4, R is 0 or 1, and S is 0, V is C10-C16alkyl, C10-C16alkenyl, or C10-C16alkynyl, and T is 0 or 1.Embodiment 10. The compound or salt of embodiment 2, whereinQ is 3, R is 0 or 1, and S is 0, V is C10-C16alkyl, C10-C16alkenyl, or C10-C16alkynyl,and T is 0 or 1.Embodiment 11. The compound or salt of embodiment 2, whereinQ is 0, R is 1, and S is 1, V is absent, and T is 1, and U is C6-C20alkyl, C6-C20alkenyl,or C6-C20alkynyl.Embodiment 12. The compound or salt of embodiment 2, whereinQ is 0, R is 1, and S is 1, V is absent, and T is 1, and U is C8-C18alkyl, C8-C18alkenyl,or C8-C18alkynyl.Embodiment 13. The compound or salt of embodiment 2, whereinQ is 0, R is 1, and S is 1, V is absent, and T is 1, and U is C10-C16alkyl, C10-C16alkenyl, or C10-C16alkynyl.Embodiment 14. The compound or salt of embodiment 2, whereinQ is 0, R is 1, S is 0, V is C6-C20alkyl, C6-C20alkenyl, or C6-C20alkynyl, and T is 0 or1.Embodiment 15. The compound or salt of embodiment 2, whereinQis 0, R is 1, S is 0, V is C8-C18alkyl, C8-C18alkenyl, or C8-C18alkynyl, and T is 0 or1.Embodiment 16. The compound or salt of embodiment 2, whereinQ is 0, R is 1, S is 0, V is C10-C16alkyl, C10-C16alkenyl, or C10-C16alkynyl, and T is 0or 1.Embodiment 17. The compound or salt of embodiment 2, whereinQ is 0, R is 0, S is 0, V is C6-C20alkyl, C6-C20alkenyl, or C6-C20alkynyl, and T is 0 or1.Embodiment 18. The compound or salt of embodiment 2, whereinQ is 0, R is 0, S is 0, V is C8-C18alkyl, C8-C18alkenyl, or C8-C18alkynyl, and T is 0 or1.Embodiment 19. The compound or salt of embodiment 2, whereinQ is 0, R is 0, S is 0, V is C10-C16alkyl, C10-C16alkenyl, or C10-C16alkynyl, and T is Oor 1.Embodiment 20. The compound of embodiment 1, wherein the compound is la(NCGC00857001):Embodiment 21. The compound or salt of embodiment 1, wherein the compound is 1f(NCGC00591015):Embodiment 22. The compound or salt of embodiment 1, whereinX and Y are linked to form a cyclic compound andX and Y are represented by Formula 1b.Embodiment 23. The compound or salt of embodiment 22, whereinA is a methylcarbamate, N-methyl carbamate, N-ethyl carbamate, N-methylmethylcarbamate, or N-ethyl methylcarbamate;B is a 12-Methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 5-Methyl-12-methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 5-Methyl-12-ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene;whereinA comprises a non-carbonyl oxygen (hydroxyl oxygen); andB comprises a cyclopropyl group; andat least one carbon separates the non-carbonyl oxygen on the carbamate andthe cyclopropyl group;PEGm is polyethylene glycol:PEGn is polyethylene glycol:whereinm is 1 or 2;n is 1 or 2; andwherein X corresponds to the carbonyl end of Formula 1b and Y corresponds to the -Lys- end of Formula 1b.Embodiment 24. The compound or salt of embodiment 22 or embodiment 23, wherein m is 2and n is 1.Embodiment 25. The compound or salt of embodiment 22, whereinA is an N-methyl methylcarbamate, or N-ethyl methylcarbamate;B is a 12-Methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene or 12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene,wherein A and B connect at the 5 position of the 12-Methyl- or 12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene or 12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene;m is 2; andn is 1.Embodiment 26. The compound or salt of embodiment 22, whereinA is an N-methyl carbamate, or N-ethyl carbamate;B is a 5-Methyl-12-methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; or 5-Methyl-12-ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene;whereinA and B connect at the 5 position of the 5-Methyl-12-methyl- or 5-methyl-12-ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene;m is 2; andn is 1.Embodiment 27. The compound or salt of embodiment 22, wherein the compound is 1c(NCGC01147950):1c (NCGC01147950).Embodiment 28. The compound or salt of embodiment 1, whereinX and Y are linked to form a cyclic compound andX and Y are represented by Formula 1c.Embodiment 29. The compound or salt of embodiment 28, whereinD is a 12-Methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene; 5-Methyl-12-methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene; 5-Methyl-12-ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene;E is a methylcarbamate, N-methyl carbamate, N-ethyl carbamate, N-methylmethylcarbamate, or N-ethyl methylcarbamate;whereinD comprises a cyclopropyl group; andE comprises a non-carbonyl oxygen (hydroxyl oxygen); andat least one carbon separates the non-carbonyl oxygen on the carbamate and thecyclopropyl group;PEGm is polyethylene glycol:PEGn is polyethylene glycol:whereinm is 1 or 2;n is 1 or 2; andwherein X corresponds to the carbonyl end of Formula 1c and Y corresponds to the -Lys- end of Formula 1c.Embodiment 30. The compound or salt of embodiment 28 or embodiment 29, wherein m is 1and n is 2.Embodiment 31. The compound or salt of embodiment 28, whereinD is a 12-Methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene or 12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene,E is an N-methyl methylcarbamate, or N-ethyl methylcarbamate;wherein D and E connect at the 5 position of the 12-Methyl- or 12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene or 12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene;m is 1; andn is 2.Embodiment 32. The compound or salt of embodiment 28, whereinD is a 5-Methyl-12-methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene; or 5-Methyl-12-ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene;E is an N-methyl carbamate, or N-ethyl carbamate;whereinD and E connect at the 5 position of the 5-Methyl-12-methyl- or 5-methyl-12-ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene;m is 1; andn is 2.Embodiment 33. The compound of embodiment 28, wherein the compound is 1b(NCGC00886866):1b (NCGC00886866).Embodiment 34. A pharmaceutical composition comprising a compound or salt of any oneof embodiments 1 to 33, together with a pharmaceutically acceptable carrier.Embodiment 35. A method of treating a cancer characterized by selective targeting M2macrophages and the reprogramming of M2 macrophages towards a Ml phenotype in apatient, comprising the step of providing to a patient in need thereof a therapeutic agent,wherein the therapeutic agent is a compound or salt thereof of any one of embodiments 1 to33.Embodiment 36. The method of embodiment 35, wherein CD206, a large C-type lectinreceptor, targets and modulates the M2 macrophages and induces cell death.Embodiment 37. The method of embodiment 35, wherein the cancer is selected from glioma(glioblastoma), acute myelogenous leukemia, acute myeloid leukemia,myelodysplastic / myeloproliferative neoplasms, sarcoma, chronic myelomonocytic leukemia,non-Hodgkin lymphoma, astrocytoma, melanoma, non-small cell lung cancer,cholangiocarcinomas, chondrosarcoma, colon cancer or pancreatic cancer.Embodiment 38. The method of embodiment 35, wherein the cancer is pancreatic cancer.Embodiment 39. The method of any one of embodiments 35 to 38, further comprisingadministering to the patient in need thereof at least one additional therapeutic agent.Embodiment 40. A method of modulating macrophage activity, the method comprising:contacting a macrophage with a compound or salt thereof of any one of embodiments 1 to 33to modulate activity of the macrophage.Embodiment 41. The method according to embodiment 40, wherein the CD206-binding agentbinds to a mannose receptor to modulate activity of signal regulatory protein (SIRP)-alpha tostimulate phagocytosis or destruction of a cancer cell.Embodiment 42. The method according to embodiment 40, wherein the macrophage activitythat is modulated is macrophage polarization.Embodiment 43. The method according to embodiment 40, wherein viability of themacrophage is reduced.Embodiment 44. The method according to embodiment 40, wherein the macrophage is a M2macrophage or a tumor associated macrophage (TAM).Embodiment 45. The method according to embodiment 40, wherein the CD206-binding agentstimulates macrophage activity.Embodiment 46. The method according to embodiment 45, wherein the CD206-binding agentstimulates macrophage autophagy or phagocytosis.Embodiment 47. A method of treating a subject for a condition associated with chronicinflammation, the method comprising:administering an effective amount of a therapuetic agent to the subject to treat thesubject for the condition associated with chronic inflammation, wherein thetherapeutic agent is a compound or salt thereof of any one of embodiments 1 to 33.Embodiment 48. The method according to embodiment 47, wherein the condition associatedwith chronic inflammation is selected from the group consisting of scleroderma or multiplesclerosis, irritable bowel disease, ulcerative colitis, colitis, Crohn's disease, idiopathicpulmonary fibrosis, asthma, keratitis, arthritis, osteoarthritis, rheumatoid arthritis, auto-immune diseases, a feline or human immunodeficiency virus (FIV or HIV) infection, cancer,age-related inflammation and / or stem cell dysfunction, graft-versus-host disease (GVHD),keloids, obesity, diabetes, diabetic wounds, other chronic wounds, atherosclerosis,Parkinson's disease, Alzheimer's disease, macular degeneration, gout, gastric ulcers, gastritis,mucositis, toxoplasmosis, and chronic viral or microbial infections.Embodiment 49. The method according to embodiment 47, wherein the CD206-binding agentis administered in conjunction with another drug known to be effective in treating thecondition.Embodiment 50. The method according to embodiment 47, wherein the condition is cancer.Embodiment 51. The method according to embodiment 50, further comprising administeringan effective amount of a chemotherapeutic agent or cell therapy to the subject.Embodiment 52. The method according to embodiment 51, wherein the chemotherapeuticagent or cell therapy is selected from steroids, anthracyclines, thyroid hormone replacementdrugs, thymidylate-targeted drugs, checkpoint inhibitor drugs (for example anti-PD-1 / PD-L1,anti-CTLA-4 drugs), Chimeric Antigen Receptor / T cell therapies, and other cell therapies.Embodiment 53. The method according to embodiment 47, wherein the condition associatedwith chronic inflammation is a fibrosis or scleroderma.Embodiment 54. A method of treating a cancer in a patient, comprising the step of providingto a patient in need thereof a therapeutic agent, wherein the therapeutic agent is a compoundor salt thereof of any one of embodiments 1 to 33.Embodiment 55. The method of embodiment 54, wherein the cancer is selected from glioma(glioblastoma), acute myelogenous leukemia, acute myeloid leukemia,myelodysplastic / myeloproliferative neoplasms, sarcoma, chronic myelomonocytic leukemia,non-Hodgkin lymphoma, astrocytoma, melanoma, non-small cell lung cancer,cholangiocarcinomas, chondrosarcoma, colon cancer or pancreatic cancer.Embodiment 56. The method of embodiment 54, wherein the cancer is pancreatic cancer.

Claims

What is claimed is:

1. A compound of Formula I:X-Phe-Arg-Lys-Ala-Phe-Lys-Arg-Phe-H2NFormula Ior a pharmaceutically acceptable salt thereof, whereinX is H; andY is represented by Formula la:-PEGQ-(CH2-C)RwhereinPEG is polyethylene glycol:Q is 0 to 4;R is 0 or 1;S is 0 or 1;V is absent or C6-C20alkyl, C6-C20alkenyl, or C6-C20alkynyl;Tis 0 or 1;U is C6-C20alkyl, C6-C20alkenyl, or C6-C20alkynyl;orX and Y are linked to form a cyclic compound, where X and Y are represented byFormula 1b:-C-PEGm-A-B-PEG-C-Lys-Formula 1bwherein for Formula 1b:A is a methylcarbamate, N-methyl carbamate, N-ethyl carbamate, N-methylmethylcarbamate, or N-ethyl methylcarbamate;B is a 12-Methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene; 5-Methyl-12-methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene; 5-Methyl-12-ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene;whereinA comprises a non-carbonyl oxygen (hydroxyl oxygen); andB comprises a cyclopropyl group; andat least one carbon separates the non-carbonyl oxygen on the carbamate and thecyclopropyl group;PEGm is polyethylene glycol:PEGn is polyethylene glycol:whereinm is 1 or 2;n is 1 or 2;wherein X corresponds to the carbonyl end of Formula 1b and Y corresponds to the -Lys- end of Formula 1b; andthe Lys in Formula 1b binds to the carbonyl carbon (C=O) through a Lys sidechainnitrogen;orX and Y are linked to form a cyclic compound, where X and Y are represented byFormula 1c:-C-PEGm-D-E-PEG-C-Lys-Formula 1cwherein for Formula 1c:D is a 12-Methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene; 5-Methyl-12-methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene;5-Methyl-12-ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene;E is a methylcarbamate, N-methyl carbamate, N-ethyl carbamate, N-methylmethylcarbamate, or N-ethyl methylcarbamate;whereinD comprises a cyclopropyl group; andE comprises a a non-carbonyl oxygen (hydroxyl oxygen); andat least one carbon separates the non-carbonyl oxygen on the carbamate and thecyclopropyl group;PEGm is polyethylene glycol:PEGn is polyethylene glycol:whereinm is 1 or 2;n is 1 or 2;wherein X corresponds to the carbonyl end of Formula 1c and Y corresponds to the -Lys- end of Formula 1c; andthe Lys in Formula 1c binds to the carbonyl carbon (C=O) through a Lys sidechainnitrogen.

2. The compound or salt of claim 1, whereinX is H; andY is represented by Formula la.

3. The compound or salt of claim 2, whereinQis 1 to 4, R is 1, and S is 1, V is absent, T is 1, and U is C6-C20alkyl, C6-C20alkenyl,or C6-C20alkynyl.

4. The compound or salt of claim 2, whereinQ is 1 to 4, R is 0 or 1, and S is 0, V is C6-C20alkyl, C6-C20alkenyl, or C6-C20alkynyland T is 0 or 1.

5. The compound of claim 1, wherein the compound is 1a (NCGC00857001):la (NCGC00857001).

6. The compound or salt of claim 1, wherein the compound is 1f (NCGC00591015):1f (NCGC00591015).

7. The compound or salt of claim 1, whereinX and Y are linked to form a cyclic compound andX and Y are represented by Formula 1b.

8. The compound or salt of claim 7, whereinA is a methylcarbamate, N-methyl carbamate, N-ethyl carbamate, N-methylmethylcarbamate, or N-ethyl methylcarbamate;B is a 12-Methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene; 5-Methyl-12-methyl-10,11,12-triazatricyclo[7.3.0.04,6] dodeca-1(9),10-diene; 5-Methyl-12-ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene;whereinA comprises a non-carbonyl oxygen (hydroxyl oxygen); andB comprises a cyclopropyl group; andat least one carbon separates the non-carbonyl oxygen on the carbamate andthe cyclopropyl group;PEGm is polyethylene glycol:PEGn is polyethylene glycol:whereinm is 1 or 2;n is 1 or 2; andwherein X corresponds to the carbonyl end of Formula 1b and Y corresponds to the -Lys- end of Formula 1b.

9. The compound or salt of claim 7, wherein the compound is 1c (NCGC01147950):1c (NCGC01147950).

10. The compound or salt of claim 1, whereinX and Y are linked to form a cyclic compound andX and Y are represented by Formula 1c.

11. The compound or salt of claim 10, whereinD is a 12-Methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 12-Ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9),10-diene; 5-Methyl-12-methyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene; 5-Methyl-12-ethyl-10,11,12-triazatricyclo[7.3.0.04,6]dodeca-1(9), 10-diene;E is a methylcarbamate, N-methyl carbamate, N-ethyl carbamate, N-methylmethylcarbamate, or N-ethyl methylcarbamate;whereinD comprises a cyclopropyl group; andE comprises a non-carbonyl oxygen (hydroxyl oxygen); andat least one carbon separates the non-carbonyl oxygen on the carbamate and thecyclopropyl group;PEGm is polyethylene glycol:PEGn is polyethylene glycol:whereinm is 1 or 2;n is 1 or 2; andwherein X corresponds to the carbonyl end of Formula 1c and Y corresponds to the -Lys- end of Formula 1c.

12. The compound of claim 10, wherein the compound is 1b (NCGC00886866):1b (NCGC00886866).

13. A pharmaceutical composition comprising a compound or salt of any one of claims 1 to12, together with a pharmaceutically acceptable carrier.

14. A method of treating a cancer a patient, comprising the step of providing to a patient inneed thereof a therapeutic agent, wherein the therapeutic agent is a compound or salt thereofof any one of claims 1 to 12.

15. The method of claim 14, wherein the cancer is characterized by selective targeting M2macrophages and the reprogramming of M2 macrophages towards a M1 phenotype.

16. The method of claim 13, wherein the cancer is pancreatic cancer.

17. The method of claim 13, wherein the cancer is selected from glioma (glioblastoma),acute myelogenous leukemia, acute myeloid leukemia, myelodysplastic / myeloproliferativeneoplasms, sarcoma, chronic myelomonocytic leukemia, non-Hodgkin lymphoma,astrocytoma, melanoma, non-small cell lung cancer, cholangiocarcinomas, chondrosarcoma,or colon cancer.

18. A method of modulating macrophage activity, the method comprising:contacting a macrophage with a compound or salt thereof of any one of claims 1 to 12 tomodulate activity of the macrophage.

19. A method of treating a subject for a condition associated with chronic inflammation, themethod comprising:administering an effective amount of a therapuetic agent to the subject to treat thesubject for the condition associated with chronic inflammation, wherein thetherapeutic agent is a compound or salt thereof of any one of claims 1 to 12.

20. The method according to claim 47, wherein the condition associated with chronicinflammation is selected from the group consisting of scleroderma or multiple sclerosis,irritable bowel disease, ulcerative colitis, colitis, Crohn's disease, idiopathic pulmonaryfibrosis, asthma, keratitis, arthritis, osteoarthritis, rheumatoid arthritis, auto-immune diseases,a feline or human immunodeficiency virus (FIV or HIV) infection, cancer, age-relatedinflammation and / or stem cell dysfunction, graft-versus-host disease (GVHD), keloids,obesity, diabetes, diabetic wounds, other chronic wounds, atherosclerosis, Parkinson'sdisease, Alzheimer's disease, macular degeneration, gout, gastric ulcers, gastritis, mucositis,toxoplasmosis, and chronic viral or microbial infections.