Methods of modulating macrophage activity

Abstract

To provide a method for modulating macrophage activity.SOLUTION: CLAIMS What is claimed is: A method of modulating macrophage activity, comprising contacting macrophages with a CD206 - binding agent, thereby modulating the activity of the macrophages, wherein the CD206 - binding agent is a small molecules.SELECTED DRAWING: None

Description

[Background technology]

[0001] Cancer remains one of the leading causes of death worldwide, affecting an estimated 12.7 million men and women worldwide each year, a number that is expected to increase to 21 million by 2030.

[0002] Recent advances in immunotherapy have significantly transformed the treatment of many cancer patients. Immunotherapies, such as checkpoint inhibitor monoclonal antibody (CIMA) therapy or chimeric antigen receptor (CAR) T cell therapy, have become first- or second-line treatments, and some patients have achieved durable and long-lasting responses that are generally not achieved with standard systemic chemotherapy. To date, these positive findings have been limited to a small number of immunologically "hot" cancers. This contrasts with the majority of solid organ cancers, classified as immunologically "cold," in which the vast majority of patients fail to respond to promising T cell-activating immunotherapies. These tumors create an immune environment that induces an exhausted T cell phenotype through the elimination of cytotoxic T cells and abundant immune evasion factors, frequently involving innate immune cells such as tumor-associated macrophages (TAMs) and immature myeloid-derived suppressor cells (MDSCs).

[0003] Macrophages are one of the major innate immune cell populations, and tumor-associated macrophages (TAMs) are a key driver of cancer biology and play pivotal roles in many human diseases, including cancer. Tumor cells attract and reprogram myeloid cells to support tumor growth and metastasis. While the dichotomous M1 / M2 classification fails to capture the purpose of macrophages, tissue-specific factors, and stress responses, generally speaking, TAMs in the early stages of tumorigenesis are predominantly M1-like, secreting proinflammatory type I cytokines and inhibiting immune evasion. In contrast, M2 tumor-associated macrophages often become the dominant phenotype during tumor progression and in more advanced tumor stages. M2-like TAMs can promote tumor growth directly through the excretion of type II tumor-promoting factors, or indirectly through promoting angiogenesis, fostering cancer stem cells resistant to cytotoxic chemotherapy, and creating a tumor microenvironment that evades the immune system. Summary of the Invention

[0004] Aspects of the present disclosure include methods for modulating macrophage activity. In certain embodiments, the method comprises contacting a macrophage with a mannose receptor (CD206)-binding agent in a manner sufficient to modulate macrophage activity. Methods for converting the phenotype of a macrophage from an M2 phenotype to an M1 phenotype are also provided. Methods for inhibiting the proliferation of CD206-expressing cells, as well as methods for treating a subject with a neoplastic condition (e.g., cancer) or a condition associated with chronic inflammation, are described. As used herein, "inhibiting the proliferation of CD206-expressing cells" includes killing the cells or reprogramming the cells. In some embodiments, the method involves killing the CD206-expressing cells. In other embodiments, the method involves reprogramming the CD206-expressing cells. Immunomodulatory peptides suitable for use in the methods of the present disclosure are also disclosed. Aspects of the present disclosure also include active agents for binding to the activity-regulating domain of CD206. Methods for determining whether a compound binds to the activity-regulating domain of CD206 are also provided.

[0005] In certain embodiments, the methods include modulating macrophage activity. The methods according to certain embodiments include contacting a macrophage with a CD206-binding agent to modulate macrophage activity. In these embodiments, the CD206-binding agent binds to CD206. The CD206-binding agent binds to a site selected from the fibronectin II domain of CD206, the C-type lectin carbohydrate recognition domain 3 (CRD3) of CD206, the C-type lectin carbohydrate recognition domain 4 (CRD4) of CD206, and the C-type lectin carbohydrate recognition domain 5 (CRD5) of CD206. In some cases, the CD206-binding agent binds to CD206 with a binding energy of at least -650 kcal / mol. In some cases, the modulated macrophage activity is macrophage polarization. In other cases, the viability of the macrophage is reduced. According to certain embodiments, the macrophage is a macrophage having an M2 phenotype. In other embodiments, the macrophage is a tumor-associated macrophage. In some embodiments, the CD206-binding agent inhibits macrophage activity. In other embodiments, the CD206-binding agent induces apoptosis of the macrophage. In still other embodiments, the CD206-binding agent stimulates phagocytosis. The macrophages can be contacted in vivo or in vitro.

[0006] In other embodiments, the method comprises inhibiting the proliferation of CD206-expressing cells. Methods of the present invention include contacting target CD206-expressing cells with a CD206-binding agent to inhibit cell proliferation. In some cases, the target CD206-expressing cells are cancer cells. For example, the cancer cells may be pancreatic cancer cells, prostate cancer cells, colon cancer cells, skin cancer cells, or breast cancer cells. In certain embodiments, the methods include treating a subject for a neoplastic condition. In certain embodiments, the methods include administering a therapeutically effective amount of a CD206-binding agent to a subject diagnosed with a neoplastic condition to treat the subject's neoplastic condition. In these embodiments, the neoplastic condition may be a solid tumor cancer. For example, the neoplastic condition may be a cancer selected from pancreatic cancer, prostate cancer, colon cancer, breast cancer, and skin cancer. In some cases, the methods further include administering an effective amount of a chemotherapeutic agent, antibody agent, or cell therapy to the subject. For example, the chemotherapeutic agent, antibody agent, or cell therapy may be selected from steroids, anthracyclines, thyroid hormone replacement agents, thymidylate-targeting drugs, antibodies, checkpoint inhibitors, chimeric antigen receptor / T-cell therapies, and other cell therapies. In some embodiments, the chemotherapeutic agent is a non-peptide compound that reduces cancer cell proliferation. For example, the chemotherapeutic agent may be a compound selected from alkylating agents, metal complexes, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, hormone-modulating agents, and steroid hormones. In some cases, the antibody agent is a chemotherapeutic antibody agent. For example, the antibody agent may be an antibody expressed against a tumor-associated antigen selected from the group consisting of CD20, CD30, CD33, CD52, CD47, EpCAM, CEA, gpA33, mucin, TAG-72, CAIX, PSMA, folate-binding protein, gangliosides (e.g., GD2, GD3, GM2, etc.), Ley, VEGF, VEGFR, integrin αVβ3, integrin α5β1, EGFR, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, and tenascin. In certain embodiments, the method comprises administering a checkpoint inhibitor.For example, the checkpoint inhibitor may be an inhibitory compound that targets one or more of PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFRβ.

[0007] In yet other embodiments, the methods include treating a subject for a condition associated with chronic inflammation. In certain embodiments, the methods include administering a therapeutically effective amount of a CD206-binding agent to a subject to treat chronic inflammation. In some embodiments, the condition associated with chronic inflammation includes treating a subject for a condition associated with chronic inflammation. In some embodiments, the condition associated with chronic inflammation is scleroderma or multiple sclerosis, irritable bowel disease, ulcerative colitis, colitis, Crohn's disease, idiopathic pulmonary fibrosis, asthma, keratitis, arthritis, osteoarthritis, rheumatoid arthritis, autoimmune disease, 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, and the like. The therapeutic agent is selected from the group consisting of 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.

[0008] In yet another embodiment, the method comprises changing the phenotype of macrophages from an M2 phenotype to an M1 phenotype. In certain embodiments, the method includes contacting macrophages having an M2 phenotype with a CD206-binding agent in a manner sufficient to convert the phenotype of the macrophages to an M1 phenotype. In some cases, contact with the CD206-binding agent induces macrophage proliferation. Conformation of the CD206 receptor on macrophages is sufficient to convert the phenotype of the macrophage to an M1 phenotype. In some cases, converting the phenotype of the macrophage includes inducing expression of CD86 by the macrophage. In other cases, converting the phenotype of the macrophage includes decreasing expression of CD206 by the macrophage. In other instances, converting the phenotype of the macrophage includes inducing expression of CD163 by the macrophage. In yet other instances, converting the phenotype of the macrophage includes inducing expression of CD163 by the macrophage. Converting the macrophage phenotype includes converting the macrophage to a phenotype that exhibits upregulation of M1 cytokines and markers. For example, the M1 cytokines and markers are selected from the group consisting of IL-1β, IL-12, TNFα, and nitric oxide synthase. In other instances, converting the macrophage phenotype includes converting the macrophage to a phenotype that exhibits downregulated expression of signal-regulatory protein alpha (SIRPα).

[0009] In certain embodiments, active agents that bind to the activity-modulating domain of CD206 are provided. In these embodiments, the active agent is the fibronectin II domain of CD206, the C-type domain of CD206, In some cases, the active agent binds to an activity-modulating domain of CD206 selected from C-type lectin carbohydrate recognition domain 3 (CRD3), C-type lectin carbohydrate recognition domain 4 (CRD4) of CD206, and C-type lectin carbohydrate recognition domain 5 (CRD5) of CD206. In some cases, the active agent binds to the CRD5 domain of CD206. In some cases, the active agent binds to the fibronectin II domain of CD206. In some other cases, the active agent binds to the CRD3 domain of CD206.

[0010] In certain embodiments, the method comprises determining whether an active agent binds to an activity-modulating domain of CD206. In these embodiments, the method includes determining whether a macrophage comprising CD206 is present. contacting the phage with a compound, and the compound binding to the activity-regulating domain of CD206; In some cases, the method includes determining whether an activity-modulating domain of CD206 binds to the compound. In certain cases of these methods, the macrophage is a macrophage comprising one or more variants in the activity-modulating domain of CD206. In certain embodiments, the activity-modulating domain of CD206 is selected from a fibronectin II domain of CD206, a C-type lectin carbohydrate recognition domain 3 (CRD3) of CD206, a C-type lectin carbohydrate recognition domain 4 (CRD4) of CD206, and a C-type lectin carbohydrate recognition domain 5 (CRD5) of CD206. In certain cases, the activity-modulating domain of CD206 is a CRD5 domain. In certain cases, the activity-modulating domain of CD206 is a fibronectin II domain. In certain other cases, the activity-modulating domain of CD206 is a CRD3 domain.

[0011] According to certain embodiments of the present disclosure, the CD206-binding agents are immunomodulatory peptides. In some cases, the immunomodulatory peptide is 5 to 18 amino acid residues in length and is expressed under physiological conditions. The striatal region comprises alternating hydrophilic and hydrophobic modules that adopt an amphipathic conformation under certain conditions. In these cases, the striatal region comprises three or more hydrophobic modules, and two or more hydrophilic modules, each containing at least one cationic residue. In some embodiments, the immunomodulatory peptide is defined by one of the following formulas: The sequence includes the sequence to be [J 1a J 1b ]-[X 1a X 1b ]-[J 2a J 2b ]-[X 2a X 2b ]-[J 3a ]-[X 3a ]; and [X 3a ]-[J 3a ]-[X 2b X 2a ]-[J2b J 2a ]-[X 1b X 1a ]-[J 1b J 1a ]; Here, J 1a , J 1b , J 2a , J 2b and J. 3a are each independently selected from phenylalanine, tryptophan, alanine, valine, and glycine; 1a , X 1b , X 2a , X 2b oh Call X 3a are independently lysine, arginine, histidine, aspartic acid, and glutamic acid. In some instances, the amino acid is selected from the group consisting of thiamin, thiamin, asparagine, and glutamine. 1a , J 1b , J 2a , J 2b and J. 3a are phenylalanine, and X 1a , X 1b , X 2a , X 2b Oh and X 3a are each independently selected from lysine and arginine. In one embodiment, the immunomodulatory peptide comprises the following sequence: a) a sequence selected from: KFRKAFKRFF(RP182); FFRKFAKRFK(RP183); FFKKFFKKFK(RP185); FFKKFFKKFK(RP186); and FFKKFFKKFK(RP233); or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a). In another embodiment, the immunomodulatory peptide comprises the following sequence: a) a sequence selected from: RWKFGGFKWR(RP832C); FKWRGGRWKF(RP837C); FWKRGGRKWF(RP837A); FWKRFV(RP837N); FVRKWR(RP837C1); FAOOFAOOFO(RP850); FWKRFVRKWR(RP837); FWKKFVKKWK(RP841); WWHHWWHHWH(RP847); WWRHWWHRWR(RP848); WWKHWWHKWK(RP849); GDRGIKGHRGF(RP842); LYKKIIKKLL(RP846); FYPDFFKKFF(RP844); FFRKSKEKIG(RP853); FFRHFATHLD(RP845); and EKLSAFRNFF(RP843); or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a).

[0012] CD206-binding agents according to some embodiments of the present disclosure also include immunomodulatory peptides comprising a sequence defined by one of the following formulas: [X 1a ]-[J 2a ]-[X 2a ]-[J 2a ]-[X 3a ]-[J 3a ] [J 3a ]-[X 3a ]-[J 2a ]-[X 2a ]-[J 1a ]-[X 1a ] [J 1a J 1b ]-[X 1a X 1b ]-[J 2aJ 2b ]; [J 1a J 1b ]-[X 1a X 1b ]-[J 2a ]-[X 2a ]; [X 3a ]-[J 3a ]-[X 2b X 2a ]-[J 2b J 2a ]; [J 1a J 1b ]-[X 1a ]-[J 2a J 2b ]-[X 2a ]; and [X 1a ]-[J 1a J 1b ]-[X 2a ]-[J 2a J 2b ]; Here, J 1a , J 1b , J 2a , J 2b and J. 3a are each independently selected from phenylalanine, tryptophan, alanine, valine, leucine, isoleucine, methionine, tyrosine, threonine, serine, cysteine, proline, and glycine; 1a , X 1b , X 2a , X 2b and X 3a are independently lysine, arginine, histidine, and asparagine. In certain embodiments, the immunomodulatory peptide comprises the following sequence: a) Sequence selected from: AFKRFF(182-FN6); FFKKFF(185-FN6); FWKRFV(837-FN6); WVRRVV(WLUB-F1-N6); IFKKIE(CEC-F1-N6)FLRNLV(LL37F-3-N6); FLHSAK(MAG-F1-N6); FFHHIF(PISC-F-N6); FFKKAA(PLEU-F-N6); ALKKVF(PSEU-F-N6); LYKKII(CXCL4-F-N6); LFRRAF(IL24-FN6); FLKRLL(IL7-FN6); FFRRFA(ABCP-FN6); FFRHFA(E1P-FN6); AIRRIP(gP120-FN6); AFHRFF(GP2B-FN6); FFNRFA(MCPH-FN6); AFKRFF(SPRA-FN6); AFKRFF(TPRO-FN6); IVRRAD(COL18-FN6); FWRWFK(HX5 / CPAP); KFWRWF(HX6 / YJPA); WFRFWK(HX7 / CLPB)KWFRFW(HX8 / ATG1); AFHHFF(HEX16F / STPK); FFRNFA(HEXF13 / SIF1); AFHRFF(HEX9F / THIF); FFRQFA(HEXF1 / ATPB); AFNRFF(HEX2F / AATF); WIQRMM(CXCL13-FN6); WVQRVV(CXCL8-FN6); AFRNFF(HEX3F / FBNA); and TLRRFM(HEX18 / HSHK); or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a). In other embodiments, the immunomodulatory peptide comprises the following sequence: a) a sequence selected from: DVRMRL (MCMV-FN6); and RRAELG (TONB-FN6); or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a). In some other embodiments, the immunomodulatory peptide comprises the following sequence: a) a sequence selected from: DVRMRL (MCMV-FN6); and RRAELG (TONB-FN6).a) a sequence selected from: FWRWFA(HX1 / MMPL); AFWRWF(HX2 / ABCT); WFRFWA(HX3 / GTRF); AWFRFW(HX4 / AXES); VAVRIW(HX9 / IDRF / AMIA); FFRFFA(HEXF2 / AMT1); and AFFRFF(HEX13F / TGME); or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a). In other embodiments, the immunomodulatory peptide comprises the following sequence: a) a sequence selected from: FFKKFF; WWKKFF; FWKKWF; FFKKWW; WWKKWW; YYKKYY; IIKKYY; YIKKIY; YYKKII; IIKKII; MMKKMM; LLKKMM; MLKKLM; MMKKLL; LLKKLL; VVKKVV; AAKKVV; VAKKAV; VVKKAA; AAKKAA; GGKKGG; TTKKGG; GTKKTG; GGKKTT; TTKKTT; SSKKSS; CCKKSS; SCKKCS; SSKKCC; and CCKKCC; or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a). In certain other cases, the immunomodulatory peptide comprises the following sequences: a) a sequence selected from: FKFKFK; WKWKWK; YKYKYK: IKIKIK; MKMKMK; LKLKLK; VKVKVK; AKAKAK; GKGKGK; TKTKTK; SKSKSK; CKCKCK; KFKFKF; KWKWKW; KYKYKY; KIKIKI; KMKMKM; KLKLKL; KVKVKV; KAKAKA; KGKGKG; KTKTKT; KSKSKS; and KCKCKC; or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a).

[0013] CD206-binding agents according to some embodiments of the present disclosure also include immunomodulatory peptides comprising a sequence defined by one of the following formulas: [J 1a ]-[X 2a ]-[J 2a ]-[X 3a ]-[J 3a ] [X 1a ]-[J 1a ]-[X 2a ]-[J 2a ]-[X 3a ] [X 1a ]-[J 1a ]-[X 2a ]-[J 2a J 2b ]; [J 1a J 1b ]-[X 1a ]-[J 2a ]-[X 2a ]; [X 1a ]-[J 1a J 1b ]-[X 2a ]-[J 2a ]; [J 1a ]-[X 1a ]-[J 2a J 2b ]-[X 2a ]; and [J 1a J 1b ]-[X 2a ]-[J 2a J 2b ]; Here, J 1a , J 1b , J 2a , J 2b and J. 3a are each independently selected from phenylalanine, tryptophan, alanine, valine, leucine, isoleucine, methionine, tyrosine, threonine, serine, cysteine, proline, and glycine; 1a , X 1b , X 2a , X 2b and X 3a are independently lysine, arginine, histidine, and asparagine. In certain embodiments, the immunomodulatory peptide comprises the following sequence: a) a sequence selected from the following: AFKRF; FFKKF; FWKRF; WVRRV; IFKKI; FLRNL; FLHSA; FFHHI; FFKKA; ALKKV; LYKKI; LFRRA; FLKRL; FFRRF; FFRHF; AIRRI; AFHRF; FFNRF; IVRRA; FWRWF; KFWRW; WFRFW; KWFRF; AFHHF; FFRNF; FFRQF; AFNRF; WIQRM; WVQRV; AFRNF; TLRRF; FKRFF; FKKFF; WKRFV; VRRVV; FKKIE; LRNLV; LHSAK; FHHIF; FKKAA; LKKVF; YKKII; FRRAF; LKRLL; FRRFA; FRHFA; IRRIP; FHRFF; FNRFA; VRRAD; WRWFK; FRFWK; In another embodiment, the immunomodulatory peptide comprises the following sequences: FHHFF; FRNFA; FRQFA; FNRFF; IQRMM; VQRVV; FRNFF; LRRFM; DVRMR; VRMRL; RRAEL; RAELG; and RWKFG; or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a). In another embodiment, the immunomodulatory peptide comprises the following sequences: a) a sequence selected from: AFWRW; AWFRF; VAVRI; FFRFF; AFFRF; WRWFA; FRFWA; AVRIW; and FRFFA; or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a). In another embodiment, the immunomodulatory peptide comprises the following sequences:a) a sequence selected from the following: FFKKF; WWKKF; FWKKW; FFKKW; WWKKW; YYKKY; IIKKY; YIKKI; YYKKI; IIKKI; MMKKM; LLKKM; MLKKL; MMKKL; LLKKL; VVKKV; AAKKV; VAKKA; VVKKA; AAKKA; GGKKG; TTKKG; GTKKT; GGKKT; TTKKT; SSKKS; CCKKS; SCKKC; SSKKC; and CCKKC; FKKFF; WKKFF; WKKWF; FKKWW; WKKWW; YKKYY; IKKYY; IKKIY; YKKII; IKKII; MKKMM; LKKMM; LKKLM; MKKLL; LKKLL; VKKVV; AKKVV; AKKAV; VKKAA; AKKAA; GKKGG; TKKGG; TKKTG; GKKTT; TKKTT; SKKSS; CKKSS; CKKCS; SKKCC; and CKKCC; or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a). In certain other cases, the immunomodulatory peptide comprises the following sequences: a) a sequence selected from: FKFKF; WKWKW; YKYKY: IKIKI; MKMKM; LKLKL; VKVKV; AKAKA; GKGKG; TKTKT; SKSKS; CKCKC; KFKFK; KWKWK; KYKYK; KIKIK; KMKMK; KLKLK; KVKVK; KAKAK; KGKGK; KTKTK; KSKSK; and KCKCK; or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a).

[0014] CD206-binding agents according to some embodiments of the present disclosure also include immunomodulatory peptides comprising a sequence defined by one of the following formulas: [J 1a ]-[X 1a ]-[J 2a ]-[X 2a ] [X 1a ]-[J 1a ]-[X 2a ]-[J 2a ] [X 1a X 2a ]-[J 2a J 2b ]; and [J 1a J 1b ]-[X 1a X 2a ]; Here, J 1a , J 1b , J 2a , and J. 2b are independently phenylalanine, triphosphatase X is selected from threonine, alanine, valine, leucine, isoleucine, methionine, tyrosine, threonine, serine, cysteine, proline, and glycine; 1a , X 1b , X 2a , and X 2b are independently lysine, arginine, histidine, aspartic acid, and glutamic acid. In certain embodiments, the immunomodulatory peptide comprises the sequence: a) a sequence selected from the following: AFKR; FFKK; FWKR; WVRR; IFKK; FLRN; FLHS; FFHH; ALKK; LYKK; LFRR; FLKR; FFRR; FFRH; AIRR; AFHR; FFNR; IVRR; FWRW; KFWR; WFRF; KWFR; AFHH; FFRN; FFRQ; AFNR; WIQR; WVQR; AFRN; TLRR; KRFF; KKFF; KRFV; RRVV; KKIE; RNLV; HSAK; HHIF; KKAA; KKVF; KKII; RRAF; KRLL; RRFA; RHFA; RRIP; HRFF; NRFA; RRAD; RWFK; RFWK; HHFF; RNFA; RQFA; NRFF; QRMM; QRVV; RNFF; RRFM; VRMR; RMRL; RAEL; AELG; and WKFG; or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a). In other embodiments, the immunomodulatory peptide comprises the following sequences: a) a sequence selected from: FWRW; AFWR; WFRF; AWFR; VAVR; FFRF; AFFR; RWFA; WRWF; RFWA; FRFW; VRIW; RFFA; and FRFF; or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a). In other embodiments, the immunomodulatory peptide comprises the following sequences: a) a sequence selected from: FWRW; AFWR; WFRF; AWFR; VAVR; FFRF; AFFR; RWFA; WRWF; RFWA; FRFW; VRIW; RFFA; and FRFF; a) a sequence selected from the following: FFKK; WWKK; FWKK; YYKK; IIKK; YIKK; MMKK; LLKK; MLKK; VVKK; AAKK; VAKK; GGKK; TTKK; GTKK; SSKK; CCKK; SCKK; KKFF; KKWF; KKWW; KKYY; KKIY; KKII; KKMM; KKLM; KKLL; KKVV; KKAV; KKAA; KKGG; KKTG; KKTT; KKSS; KKCS; and KKCC; or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a).In certain other cases, the immunomodulatory peptide comprises a) a sequence selected from: FKFK; WKWK; YKYK; IKIK; MKMK; LKLK; VKVK; AKAK; GKGK; TKTK; SKSK; CKCK; KFKF; KWKW; KYKY; KIKI; KMKM; KLKL; KVKV; KAKA; KGKG; KTKT; KSKS; and KCKC; or b) a sequence having one or two amino acid substitutions relative to a sequence defined in a).

[0015] CD206-binding agents according to certain embodiments of the present disclosure also include small molecule active agents. In instances, the small molecule active agent comprises formula (I):

[0016] [ka]

[0017] where: R 1 -R 4 are each independently selected from hydrogen, alkyl, and substituted alkyl; X 1 is alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted aryl selected from ralkyl, heterocycle, substituted heterocycle, heteroaryl, and substituted heteroaryl; X 2 is alkyl, substituted alkyl, aryl, substituted aryl, amino, substituted amino , heteroaryl, substituted heteroaryl, heterocycle, substituted heteroaryl; X 3 is alkyl, substituted alkyl, aryl, substituted aryl, naphthyl, substituted naphthyl, aryl, heterocyclic, substituted heterocyclic, heteroaryl, substituted heteroaryl, arylheterocyclic aryl heterocycles, and substituted aryl heterocycles; n is an integer from 1 to 10, or a pharmaceutically acceptable salt or solvate thereof.

[0018] The CD206-binding agent according to certain embodiments of the present disclosure also includes a low molecular weight compound represented by the following formula (II): Contains a molecular active agent.

[0019] [ka]

[0020] where: R 7a , R 7b , R 8 , R 9 and R 10 are each independently selected from hydrogen, alkyl, and substituted alkyl; X 4 is an alkyl, aryl, aralkyl, heterocycle, and heteroaryl, Selected from sill, where X 4 is alkyl, substituted alkyl, aryl, substituted aryl, Optionally further substituted with one or more groups selected from amino, substituted amino, carboxamido, substituted carboxamido, heterocycle, substituted heterocycle, and a second compound of formula (II). or a pharmaceutically acceptable salt or solvate thereof.

[0021] CD206-binding agents according to certain embodiments of the present disclosure also include small molecule active agents represented by (III):

[0022] [ka]

[0023] where: R 13 is selected from hydrogen, alkyl and substituted alkyl; X 5 is alkyl, substituted alkyl, aryl, substituted aryl, amino, substituted amino , heteroaryl, substituted heteroaryl, heterocycle, substituted heteroaryl; X 6 is alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted aryl selected from ralkyl, heterocycle, substituted heterocycle, heteroaryl, and substituted heteroaryl; X 7 is alkyl, substituted alkyl, aryl, substituted aryl, naphthyl, substituted naphthyl, selected from alkyl, heterocycle, substituted heterocycle, heteroaryl, substituted heteroaryl, aryl heterocycle, substituted aryl heterocycle; p is an integer from 1 to 10, or a pharmaceutically acceptable salt or solvate thereof.

[0024] CD206-binding agents according to certain embodiments of the present disclosure also include specific binding members. In certain cases, the specific binding pair is an antibody, or a binding fragment thereof. In certain cases, the specific binding member targets a sequence of CD206 selected from NFGDLVSIQSESEKK, NDAQSAYFIGLLISL, SKEKETMDNARAF, and EDENCVTMYSNSGFWN. [Brief explanation of the drawings]

[0025] [Figure 1]A biophysical homology screen using a Molly font is shown, identifying the conservation of a 10-mer structural motif across representative HDPs, external virulence factors, and collagens. A. Molly font heuristics showing amino acid chemistry for biophysical homology comparisons. The size of the circle indicates the proportional 3D amino acid volume in water, and the hydrophobicity / hydrophilicity of each amino acid is indicated by a color scale in which the most hydrophobic amino acids are the most intense cyan and those with less hydrophobicity are proportionally less intense cyan. The most hydrophilic amino acids have the deepest magenta, and a less intense magenta scale indicates less hydrophilic amino acids. Mnemonic letters used to capture biophysical relationships and further characterize the chemical properties of amino acids include thicknesses indicating the dissociation constant of their ionizable protons and charges, with charged amino acids having a "+" or "-" sign incorporated within the letter. The numerical value encodes the energy required to move the amino acid side chain from the interior of the lipid bilayer to the external aqueous environment in kcal / mol. [Figure 2] Biophysical homology screening using a moly font identifies conservation of a decameric structural motif across representative HDPs, external virulence factors, and collagens. Conservation of a decameric structural motif with a secondary α-helical structure with an amphipathic surface topology of hydrophobic and hydrophilic faces across representative HDPs, external virulence factors, and collagens is indicated by the moly font. "DHDP" designates a designed host defense peptide. [Figure 3] The secondary α-helical structure, helical hydrophobic wheel projections, and Mollyfont alignment (bottom) of RP-182 and RP-426 are shown. [Figure 4] The relative binding affinities (BE; in kcal / mol) of individual 10-mer homologous sequence ligand-lectin receptor combinations by ClusPro® are shown, identifying MRC1 / CD206 as a top binding target. [Figure 5] Relative binding energies for the top 10 C-type lectin receptors using ClusPro® are shown. [Figure 6] Docking campaigns for biophysical 10-mer homology motifs and C-type lectin receptors are shown. A. Relative binding energies (in kcal / mol) of the top 10-mer peptide motif ligands-C-type lectin receptors using ClusPro®. Origin of the 10-mer peptide sequence is shown below; predicted affinities for individual lectin receptors are annotated above. B. The mannose receptor CD206 exhibits the highest affinity for the 10-mer biophysical peptide sequence across all tested peptide-ligand-receptor combinations. The cumulative scores of the top three peptide ligand-receptor combinations are shown, along with each receptor examined by the 23 peptide ligand-receptor combinations. The receptor with the highest affinity (lowest - BE; in kcal / mol) was assigned 3 points, the second highest 2 points, and the third highest 1 point. Plots show the sum of points for each receptor across all peptides. CD206FL, full-length CD206; CRD, carbohydrate recognition domain. [Figure 7A] A full-length MRC1 / CD206 model is shown. Known functional domains of human MRC1 / CD206. CRD, carbohydrate recognition domain 1-8. [Figure 7B] A full-length MRC1 / CD206 model is shown. The SAXS profile is shown as a function of the concentration of full-length MRC1 / CD206 protein, where I(q) is the scattering intensity and q (in A-1) is the scattering vector. The structural parameters extracted from the SAXS data are listed below. Rg Guinier: radius of gyration generated from a Guinier plot of the SAXS data extrapolated to 0 concentration; Rest Rg and Dmax (maximum particle dimension) are generated from GNOM (https: / / www.embl-hamburg.de / biosaxs / manuals / gnom.html). MW1 and MW2 are molecular weights estimated from the SAXS data using the Porod volume (Vporod) and correlation volume (Vc). For the Vc calculation, qmax = 0.15 A-1 was used. The results suggest that MRC1 / CD206 forms a dimer in solution. [Figure 7C] The full-length MRC1 / CD206 model is shown. C. The top five human MRC1 / CD206 models generated by I-TASSER were tested for the best fit with the experimental SAXS data. The discrepancies between the model and experimental curves (χ2) are shown in the table below. In the fits, MRC1 / CD206 dimer models were used based on models 1–5 as monomers, respectively. [Figure 7D] The full-length MRC1 / CD206 model is shown. C. The top five human MRC1 / CD206 models generated by I-TASSER were tested for the best fit with the experimental SAXS data. The discrepancies between the model and experimental curves (χ2) are shown in the table below. In the fits, MRC1 / CD206 dimer models were used based on models 1–5 as monomers, respectively. [Figure 8A] A model of the conformational bending in CRD4 and CRD5 of MRC1 / CD206 induced by RP-182 is shown. The hydrophobic face of RP-182 bound to CRD5 (cyan). [Figure 8B] Another model of the conformational bending in CRD4 and CRD5 of MRC1 / CD206 induced by RP-182 is shown. The hydrophobic face of RP-182 bound to CRD5 (cyan). [Figure 9A] Negative stain electron microscopy photomicrographs of full-length CD206 protein incubated with vehicle (blue squares) and RP-182 (red squares) and the corresponding 2D classes (inlets) are shown, with schematic diagrams of the open, "elongated" and "closed" conformations shown on the left. [Figure 9B] Another negative stain electron microscopy photomicrograph of full-length CD206 protein incubated with vehicle (blue squares) and RP-182 (red squares) and the corresponding 2D classes (inlets) is shown, with schematic diagrams of the open, "elongated" and "closed" conformations shown on the left. [Figure 10]Figure 1 shows that RP-182 binds to MRC1 / CD206. A. Dose-response relationship between increasing concentrations of RP-182 and induction of the closed conformation of the CD206 receptor. The percentage of closed conformation of 100 CD206 particles examined in negatively stained EM micrographs of full-length CD206 protein was measured with increasing concentrations of RP-182 (μM). [Figure 11] The ratio of closed and open conformations of MRC1 / CD206 is shown (low, CD206:peptide ratio 1:40, high, 1:500). [Figure 12A] Figure 1 shows that RP-182 binds to MRC1 / CD206. Induction of the closed conformation of the CD206 receptor by the 10-mer homology motif. Representative micrographs of full-length CD206 incubated with the 10-mer biophysical homology peptides RP-184, RP-832C, AVP1, LL37F, and the control peptide RP-426, 2D class, are shown below. [Figure 12B] Additional illustration of RP-182 binding of MRC1 / CD206 is shown. Induction of a closed conformation of the CD206 receptor by the 10-mer homology motif. Representative micrographs of full-length CD206 incubated with the 10-mer biophysical homology peptides RP-185, RP-832C, AVP1, LL37F, and the control peptide RP-426, 2D class, are shown below. [Figure 12C] An extra view of RP-182 binding to MRC1 / CD206 is shown. Induction of the closed conformation of the CD206 receptor by the 10-mer homology motif. Representative micrographs of full-length CD206 incubated with the 10-mer biophysical homology peptides RP-185, RP-832C, AVP1, LL37F, and the control peptide RP-426, 2D class, are shown below. [Figure 13] Microscale thermophoresis (MST) shifts of human (left) and mouse (right) MRC1 / CD206 in response to increasing concentrations of RP-182 are shown. KD model response curves are fitted to MST data from three independent experiments with the control peptide RP-426 shown below. Error bars represent the standard deviation of the mean. [Figure 14A] Human peripheral blood monocyte-derived macrophage polarization protocol is shown. [Figure 14B] 1 shows a cellular thermal shift assay (CETSA) of human macrophages. [Figure 14C] Murine bone marrow-derived macrophage (BMDM) polarization protocol is presented. [Figure 14D] 1 shows a cellular thermal shift assay (CETSA) of mouse macrophages. [Figure 15A] Schematic synthesis scheme of diazirine-containing phenylalanine and biotin-containing RP-182 analogs. Synthesis of diazirinyl D-phenylalanine derivatives and Fmoc-diazirine-containing phenylalanine (enantiomers were separated using a chiral column (Chiralpak IB 4.6 x 250 mm, 100% EOH; 1 ml / min)). Fmoc solid-phase peptide synthesis and coupling with a biotinylated linker. [Figure 15B] Derivatives of RP-182 bearing a diazirine group and biotin via a PEG (NCGC-00510433 and -35) or hydrocarbon (NCGC-00510432 and -34) linker were tested for in vitro binding to recombinant MRC1 / CD206 via microscale thermophoresis (MST) assay. The measured KD constants are shown on the right. NCGC-00510434 was used for all further pull-down experiments. [Figure 16]Mapping of the binding region of RP-182 to MRC1 / CD206 by crosslinking followed by pulldown and proteomic analysis identifies the fragment NFGDLVSIQSESEKK within the carbohydrate recognition domain 5 (CRD5; CTLD5) as the binding region of RP-182 to CD206. A. Peptide fragments (highlighted in rectangle) of full-length rMRC1 / CD206 pulled down with biotinylated diazirine RP-182 analog NCGC-00510434 after trypsin digestion. B. Peptide fragments of MRC1 / CD206 identified by mass spectrometry after crosslinking with the RP-182 analog, digestion, and pulldown, as measured by the reduced MRC1 / CD206 fragments in the supernatant. The carbohydrate recognition domain 5 (CRD5; CTLD5) fragment NFGDLVSIQSESEKK (highlighted in red) was identified by both approaches. C. MS / MS spectrum of the MRC1 / CD206 CRD5 peptide NFGDLVSIQSESEKK precipitated by biotin-coupled RP-182. Panel A: LC / MS / MS chromatography showing retention of the CRD5 peptide. Panel B: MS1 full scan showing the triply charged ion m / z = 560.9519 (monoisotopic) of the peptide. Panel C: MS2 fragment scan showing the spectrum of the monoisotopic m / z: 560.95319 Da (+0.34 mmu / +0.61 ppm), RT: 27.01 min. The spectrum was obtained by nano-HPLC-MS / MS analysis. [Figure 17] Volcano plots of RNASeq analysis of vehicle vs. RP-182 treated M2 macrophages are shown. [Figure 18] Gene expression changes induced by RP-182 in M1- and M2-polarized BMDMs are shown. Volcano plots of RNASeq analysis of vehicle- versus RP-182-treated M1 and M2 macrophages, with treatment times indicated at the top. Differentially expressed genes identified by EdgeR analysis with a false discovery rate (FDR) q < 0.05, -1 < Log2[FC] > 1 are shown (FC, fold change). A summary of identified DEGs is shown on the right. [Figure 19]Cytoscape® functional GO enrichment and network analysis of DEGs in vehicle vs. RP-182 treated M2 BMDMs are shown. [Figure 20] PathwayStudio® graph of GO cellular processes of the most common genes across the enriched gene set of RP-182-treated M2 BMDMs. [Figure 21A] Figure 1 shows the protein network of the CD206 interactome and associated cellular processes induced by RP-182 in M2 macrophages. [Figure 21B] RP182 vs. vehicle treated M2 BMDMs are shown. [Figure 21C] RNA-Seq of RP182-treated and untreated macrophages is shown. [Figure 21D] Pulldown of M2 protein following treatment with RP182 is shown. [Figure 21E] RP182 / MS M2 macrophage protein pulldown is shown. [Figure 22] Electron microscopy images (1,000x magnification, zoom 2,400x) of M1- and M2-polarized BMDMs are shown. [Figure 23] Immunofluorescence images of M2-polarized BMDMs stained with anti-Rab5, Rab7, LAMP-1, and CD206 antibodies, and nuclei with DAPI are shown. Quantification of induced fluorescence is shown on the bottom panel. For all figures, data shown represent three independent experiments and are normalized to the corresponding vehicle treatment unless otherwise indicated. [Figure 24]Immunofluorescence of human macrophages polarized into M1 and M2 populations derived from CD14-positive PBMCs of a healthy donor and stained with anti-Rab5, Rab7, LAMP-1, and CD206 antibodies shows that RP-182 activates phagocytosis and phagolysosome formation in M2-polarized human macrophages, but not in M1-polarized human macrophages derived from peripheral blood mononuclear cells (PBMCs) of healthy volunteers. Representative images are at 40x magnification. Quantification of RP-182-induced Rab5, Rab7, LAMP-1, and CD206 expression levels in M1 (blue bars) and M2 (red bars) BMDMs is shown on the left. Fluorescence (bright objects) was normalized to the number of nuclei (DAPI), and the signal in vehicle-treated BMDMs was set to 1. At least 100 cells in ≥5 independent fields were measured. Macrophages were treated with 20 μM RP-182 for 2 h. [Figure 25A] We demonstrate that RAB5a and RAB7 RP-182 activates phagocytosis and phagolysosome formation in M2-polarized human macrophages, but not in M1-polarized human macrophages derived from peripheral blood mononuclear cells (PBMCs) of healthy volunteers. Immunofluorescence of human macrophages polarized into M1 and M2 populations derived from CD14-positive PBMCs of a healthy donor and stained with anti-Rab5, Rab7, LAMP-1, and CD206 antibodies. Representative images are at 40x magnification. Quantification of RP-182-induced Rab5, Rab7, LAMP-1, and CD206 expression levels in M1 (blue bars) and M2 (red bars) BMDMs is shown on the left. Fluorescence (bright objects) was normalized to the number of nuclei (DAPI), and the vehicle-treated signal was set to 1. At least 100 cells in ≥5 independent fields were measured. Macrophages were treated with 20 μM RP-182 for 2 hours. [Figure 25B]MAP1 and CD206 RP-182 activates phagocytosis and phagolysosome formation in M2-polarized human macrophages, but not in M1-polarized human macrophages derived from peripheral blood mononuclear cells (PBMCs) of healthy volunteers. Immunofluorescence of human macrophages polarized into M1 and M2 populations derived from CD14-positive PBMCs of a healthy donor and stained with anti-Rab5, Rab7, LAMP-1, and CD206 antibodies. Representative images are at 40x magnification. Quantification of RP-182-induced Rab5, Rab7, LAMP-1, and CD206 expression levels in M1 (blue bars) and M2 (red bars) BMDMs is shown on the left. Fluorescence (bright objects) was normalized to the number of nuclei (DAPI), and the vehicle-treated signal was set to 1. At least 100 cells in ≥5 independent fields were measured. Macrophages were treated with 20 μM RP-182 for 2 hours. [Figure 26] RP-182 activates phagocytosis, autophagy, and apoptosis in BMDMs cocultured with conditioned medium from PANC1 cells. Representative images are shown at 40x magnification. Quantification of RP-182-induced Rab7, LC-3, LAMP-1, and cleaved caspase 8 expression levels (red bars) in BMDMs is shown below. Fluorescence (bright objects) was normalized to the number of nuclei (DAPI), and the signal in vehicle-treated BMDMs was set to 1. At least 100 cells in ≥5 independent fields were measured. Macrophages were treated with 20 μM RP-182 for 2 h. [Figure 27A] RP-182, but not the control peptide RP-426, induces phagocytosis in M2-polarized BMDMs. A. Rab7 levels by immunocytochemistry in M2-polarized macrophages treated with vehicle, 20 μM RP-182, or RP-426. Fluorescence (bright objects) was normalized to nuclei count (DAPI), which was set to 1 for vehicle treatment. At least 100 cells in ≥5 independent fields were measured. [Figure 27B]Induction of phagosomes in M2 macrophages treated with RP-182, but not RP-426. Electron microscopy images (representative images at 1,000x magnification shown on the left, 2,400x zoom shown on the right) show phagosomes with internal membrane structures. [Figure 28] Immunofluorescence images of M2 BMDM stained with anti-NF-kB / p65 are shown. [Figure 29] Figure 1 shows that RP-182 reduces the cytoplasmic expression of the negative regulator of macrophage activation and inflammation IKKα subunit of the IkappaB kinase (IKK) complex, activating autophagy and caspase 8 in M2-polarized BMDMs. A. Immunofluorescence staining of IKKα in M2 BMDMs after 2 h treatment with vehicle and RP-182. [Figure 30] Quantification of activation of phagocytosis, autophagy, and apoptosis in M1 and M2 macrophages over time. [Figure 31] Figure 1 shows the activation of autophagy and apoptosis in M2 macrophages by RP-182 as measured by immunofluorescence staining of LC3 and cleaved caspase 8. Representative immunofluorescence images of vehicle- and RP-182-treated M2 macrophages at 24 h. [Figure 32] RP-182 activates autophagy and apoptosis in human M2 macrophages. LC3 and cleaved caspase 8 levels were measured by immunocytochemical staining of human M2 macrophages after 24 hours of treatment with RP-182. Measured fluorescence (bright objects) was normalized to nuclei count (DAPI), and the fold change relative to the vehicle control was plotted. Error bars represent the standard deviation of n ≥ 2 independent experiments, measuring ≥ 50 cells in 5 independent fields. [Figure 33] Quantification of cleaved caspase 3 and 7 levels after 24 hours of treatment is shown. [Figure 34] Figure 1 shows cell viability of human and mouse M1 (blue curve) and M2 (red curve) macrophages after 48 h treatment with RP-182 versus vehicle treatment. [Figure 35] Figure 2C shows that RP-182, but not RP-426, induces cell death in M2-polarized macrophages. A. Representative images of M2 BMDMs treated with vehicle or RP-182 and stained 48 hours later using dual calcein AM (green)-ethidium homodimer (red). Viability for the dose-response curve shown in Figure 2K was calculated as the ratio of live cells (calcein AM positive and negative red staining) to dead cells (non-green, positive red staining). [Figure 36] Dose-response curves for the control peptide RP-426 in human (left) and mouse (right) M2-polarized macrophages are shown. The percentage of viable cells is normalized to vehicle-treated controls, and error bars represent the standard deviation of two independent experiments in triplicate. [Figure 37] RP-182 does not inhibit cell proliferation in multipotent progenitor cells, cancer cells, fibroblasts, endothelial cells, or dendritic cells. Dose-response curves for RP-182 in human pancreatic cancer cells PANC1, mouse KPC, human fibroblasts, endothelial cells, and DC2.4 dendritic cells were shown. Cells were stained with dual calcein AM (green)-ethidium homodimer (red) after 48 hours, and parallel experiments were performed to test RP-182 in M2 BMDMs as a positive control. A representative curve is shown from two independent experiments. [Figure 38] Flow cytometry plots of CD86 and CD206 positive CD11B + F4 / 80 + Gr-1 − macrophage fractions of M2 BMDMs after treatment with vehicle or RP-182 at the indicated time points are shown. [Figure 39A] The gating strategy for determining CD86+ and CD206-positive CD11b+F4 / 80+Gr-1- macrophage fractions is shown. A. Representative flow cytometry plots showing the gating strategy for determining CD86- and CD206-positive macrophages after 24 h (center) and 48 h (bottom) treatment with vehicle RP-182. Live cells are circled in black. [Figure 39B]Representative immunocytometry images of M2 BMDMs stained for CD206 after 24 h of treatment with RP-182 or vehicle (left) and quantification of total anti-CD206 immunofluorescence signal intensity in M2 BMDMs (right) are shown. [Figure 40A] Flow cytometric quantification of the fractions of IL-1β, IL-12p40, and TNF-α positive CD11b+F4 / 80+Gr-1− cells and CD86+CD206−, CD86+CD206+ double positive, and CD86−CD206+ subpopulations of M2 BMDMs is shown. [Figure 40B] Flow cytometry plot fractions of IL-1β, IL-12p40, and TNF-α positive CD11b+F4 / 80+Gr-1− cells, and CD86+CD206−, CD86+CD206+ double positive, and CD86−CD206+ subpopulations of M2 BMDMs are shown. [Figure 40C] Flow cytometry plot fractions of IL-1β, IL-12p40, and TNF-α are shown. [Figure 41] M1 and M2 gene expression profiles in CD11b+F4 / 80+Gr-1-CD206+M2 BMDMs isolated by fluorescence-activated cell sorting (FACS) after 2 hours of treatment with vehicle (black bars) and RP-182 (red bars). Relative transcript levels by qRT-PCR after normalization to internal housekeeping genes; vehicle-treated signals were set to 1. N≥3 / group, triplicates. Error bars indicate SEM. [Figure 42] Immune checkpoint and M1 cytokine-positive macrophage populations in vehicle-treated CD86- CD206+ M2 BMDMs and RP-182-treated CD86+ CD206- and CD86+ CD206+ subpopulations are shown. Univariate histograms with percentage of positive cell fractions are shown, quantification of N=3 independent experiments. [Figure 43]Representative flow cytometry plots of the gating strategy used, including univariate histograms (with percentages of positive cell fractions) for determining SIRPα checkpoint-positive and TNFα-positive macrophage cell fractions in vehicle-treated CD86-CD206+ (top row) and RP-182-treated (bottom row) CD86+CD206+ (double-positive) and CD86+CD206-positive M2 BMDMs, are shown. Quantification of N = 3 independent experiments was performed in triplicate as shown below: 1: vehicle-treated CD86-CD206+ fraction; 2: RP-182-treated CD86+CD206+ double-positive fraction; 3: RP-182-treated CD86+CD206-positive cell fraction. [Figure 44] Flow cytometry analysis and quantification of the CD11b+F4 / 80+Gr-1- cell fraction using latex beads coated with phagocytosed E. coli. Quantification of N=3 independent experiments is shown. [Figure 45] Quantification of annexin V-positive (top) and cleaved caspase 3-positive cell fractions (bottom) of CD86+CD206-, CD206+CD86+, and CD86-CD206+M2 BMDMs treated with RP-182 at the indicated time points is shown. [Figure 46] Quantification of vehicle- and RP-182-treated CD86+ (top) and CD206+ (bottom) CD11b+F4 / 80+Gr-1- BMDM-M2 cells incubated with the NF-kB inhibitors JSH-23 and QNZ (EVP4593) and the autophagy inhibitors bafilomycin (BF) and chloroquine (CQ) is shown. The MEK inhibitor AZD6244 (selumetinib; AZD) is shown as a negative control. [Figure 47] Pearson correlation analysis of gene expression matrices between samples using global RNASeq data (left) and BMDM-derived M1-M2 marker sets (right). Values ​​range from 0 to 1, with higher values ​​between samples indicating a higher degree of correlation between the two sample sets. [Figure 48A]The biological activity of RP-182 is CD206-dependent and initiates activation of Rac1 / CDC42 signaling. A. RP-182 dose-response curves for binding to recombinant CD206 (MST assay: red curve), induction of the closed conformation of CD206 (electron microscopy: green curve), induction of phagocytosis in M2 BMDMs (anti-Rab7; purple), and M2 cell viability (blue). [Figure 48B] M1 and M2 gene expression profiles of BMDMs isolated from B6.129P2-Mrc1tm1Mnz / J (CD206- / -) and wild-type C57BL / 6 mice (CD206+ / +) after polarization into M1 and M2 are shown. Similar M1 and M2 gene expression profiles of BMDMs isolated from CD206- / - and wild-type C57BL / 6 mice (CD206+ / +) after polarization into M1 and M2 are shown. Relative gene expression levels measured by qRT-PCR for M1 markers CD86, iNOS, IL-1β, and IL-27 and M2 markers CD206, Fizz1, and YM1 in the M1 (blue) and M2 populations (red) of CD206- / - and CD206+ / + BMDMs. For quantification of relative expression levels, see Materials and Methods. Mean ± SEM of n≥3 per group is shown. [Figure 49] Immunofluorescence images of BMDMs derived from M2-polarized wild-type and CD206 − / − mice stained with anti-Rab7, LC3, and cleaved caspase 8 are shown, with quantification of three independent experiments shown at the bottom. [Figure 50] Figure 1 shows cell viability of M2 BMDMs derived from CD206wt (red curve) and CD206- / - (brown curve) mice. D. Flow cytometry plots of the CD86+ and CD206+ fractions (top) and quantification of M1 marker-positive cells in CD11b+F4 / 80+Gr-1-M2-BMDMs isolated from CD206- / - mice. The mean % positive cells from N=3 independent experiments in triplicate are shown. [Figure 51]Characterization of binding partners of CD206 induced by RP-182 in M2 macrophages. Proteomic analysis of CD206 pulled down from M2 BMDMs incubated with biotinylated RP-182 bound to beads versus beads alone. PSM, peptide spectrum match. Applied cutoff criteria included a RP-182 to control ratio of ≥5, MS-identified peptides covering ≥10% of the protein sequence, and 119 proteins (separately collated) with an FDR <0.01. [Figure 52A] Immunoblots of lysates immunoprecipitated with biotinylated peptide (left) or anti-GRB2 antibody (right) are shown. Proteins visualized using anti-GRB2 and anti-CD206 antibodies, and preloaded with control superoxide dismutase 1 (SOD1) as indicated below. [Figure 52B] Immunoblot analysis of lysates from M2 BMDMs immunoprecipitated for the active GTP-bound form of Rac1 / CDC422 and visualized with anti-CDC42 or anti-Rac1 antibodies (left). Immunoblot of M2 lysates with anti-phospho-Pak1 and anti-phospho-AKT (right). Quantification of band intensity summarizes N=3 independent experiments. [Figure 53] Figure 1 shows that RP-182 induces IQGAP1 binding to CD206 and recruitment to the plasma membrane of M2-polarized macrophages. A. Lysates from M2 BMDMs treated for 10 minutes with biotinylated RP-182, biotinylated control peptide RP-426, or vehicle control were precipitated with streptavidin magnetic beads, and proteins were visualized with an anti-IQGAP1 antibody. Control superoxide dismutase 1 (SOD1) levels are shown below. B. Membrane recruitment of IQGAP1 by RP-182. Immunofluorescence staining of M2 BMDMs for IQGAP1 at the time points after administration of RP-182 indicated at the bottom. [Figure 54A] Immunofluorescence images after anti-IQGAP1 staining of M2 BMDMs treated with RP-182 and preincubated with the indicated inhibitors are shown. [Figure 54B]Quantification of Rab7 immunofluorescence levels in M2 BMDMs treated with RP-182 and pre-incubated with ZCL278 (left), NSC23766 (center), and negative control selumetinib (right) is shown. Data shown are representative of two independent experiments and are normalized to the corresponding vehicle treatment unless otherwise indicated. [Figure 55A] Expression of LC3 and cleaved caspase 8 in M2 macrophages preincubated with chloroquine (CQ) and bafilomycin (BF). N=2, triplicates. [Figure 55B] Quantification of phagocytosis (Rab7) and cleaved caspase 8 levels in M2 BMDMs in the presence of NF-kB inhibitors JSH23 and QNZ. N=2, triplicates. K. Schematic of RP-182 function in M2 macrophages. [Figure 56] Representative images of LC3 and cleaved caspase 8 immunofluorescence levels in M2 BMDMs preincubated with the autophagy inhibitors chloroquine (CQ) and bafilomycin (BF), treated with 20 μM RP-182 for 2 hours, demonstrating that induction of cleaved caspase 8 expression in M2 macrophages is independent of autophagy induction. [Figure 57] Figure 1 shows that inhibition of RP-182-induced TNF signaling suppresses autocrine activation of apoptosis in M2-polarized macrophages. A. Cartoon depicting interference with RP-182-induced TNF signaling. [Figure 58] Immunohistochemical staining of CD206 in CD206 (high) and CD206 (low) PDAC is shown (10X, right portal at 40X). [Figure 59-1]Immunofluorescence images of M2 BMDMs stained with anti-cleaved caspase 3 and anti-cleaved caspase 8 antibodies are shown. The absence of caspase 8 and the absence or reduction of caspase 3 activation upon addition of neutralizing anti-TNFα antibodies, small molecule-mediated inhibition of TNF receptor (TNFR1), and caspase 8 inhibition of caspase 3 activation in M2 BMDMs treated with RP-182 are shown (left panel). Controls with anti-TNFα antibodies, the TNFR1 inhibitor R-7050, and caspase 8 inhibitor alone are shown on top. Cleaved caspase 8 and 3 staining in M2 BMDMs upon addition of conditioned medium (right panel). Addition of conditioned medium from M2 BMDMs treated with RP-182 or recombinant TNFα to M2 BMDMs potently activated caspases 8 and 3, an effect that was reduced by the addition of neutralizing TNFα to the conditioned medium (bottom). TNFα, recombinant TNFα ligand; α-TNF, anti-TNFα antibody; CM, conditioned medium; TNFR1i, TNF receptor inhibitor R-7050. [Figure 59-2] Immunofluorescence images of M2 BMDMs stained with anti-cleaved caspase 3 and anti-cleaved caspase 8 antibodies are shown. The absence of caspase 8 and the absence or reduction of caspase 3 activation upon addition of neutralizing anti-TNFα antibodies, small molecule-mediated inhibition of TNF receptor (TNFR1), and caspase 8 inhibition of caspase 3 activation in M2 BMDMs treated with RP-182 are shown (left panel). Controls with anti-TNFα antibodies, the TNFR1 inhibitor R-7050, and caspase 8 inhibitor alone are shown on top. Cleaved caspase 8 and 3 staining in M2 BMDMs upon addition of conditioned medium (right panel). Addition of conditioned medium from M2 BMDMs treated with RP-182 or recombinant TNFα to M2 BMDMs potently activated caspases 8 and 3, an effect that was reduced by the addition of neutralizing TNFα to the conditioned medium (bottom). TNFα, recombinant TNFα ligand; α-TNF, anti-TNFα antibody; CM, conditioned medium; TNFR1i, TNF receptor inhibitor R-7050. [Figure 60]CD206 expression in human pancreatic cancer. A. CD206 is overexpressed in two of three clinical pancreatic cancer sample sets compared to matched uninvolved normal glandular tissue. MRC1 / CD206 gene expression levels from gene sets GSE15471, GSE16515, and GSE28735. B. Tissue microarrays (TMAs) of 80 cases of pancreatic adenocarcinoma. Representative images at 10x magnification of the original cores are shown above. Waterfall plots show the percentage of CD206-positive cells of all cells ranked from highest (left) to lowest (right) for each case. Below are representative images at 40x magnification of CD206-high (left) and CD206-low (right) cases. [Figure 61] Kaplan-Meier plot of overall survival for 125 patients with PDAC stratified by CD206 expression. Log-rank test, two-tailed. [Figure 62] Kaplan-Meier analysis of patients with CD206 high TAM stratified by CD8 high and CD8 low is shown. [Figure 63A] Correlation of CD8A, INFG expression, 8- and 6-gene CD8 T cell activation signatures (bottom) and M2 marker expression levels in clinical samples from the TCGA cancer dataset. Samples across all cancers were divided into CD206 high and CD86 low using the median value across all samples. [Figure 63B] Correlation of CD8A, INFG expression, 8- and 6-gene CD8 T cell activation signatures (bottom) and M2 marker expression levels in clinical samples from the TCGA cancer dataset. Samples across all cancers were divided into CD206 high and CD86 low using the median value across all samples. Correlation of macrophage subtypes and CD8+ T cell function in the pancreatic cancer TCGA dataset. [Figure 64] Kaplan-Meier analysis of KPC tumors allografted into CD206- / - B6.129P2-Mrc1tm1Mnz / J mice (red curve) and C57B / L wild-type mice (black curve). Log-rank test, two-tailed. [Figure 65]Immunohistochemical staining of KPC wild-type (KPC) and KPC tumors generated in CD206- / - B6.129P2-Mrc1tm1Mnz / J mice (KPC CD206 KO- / -) is shown, with quantification (N≧4 / group) shown on the right. [Figure 66] Flow cytometry quantification of TAM subpopulations in KPC tumors grown in C57B / L6 wild-type and CD206 − / − mice (N≧5 / group) is shown. [Figure 67A] A. Kaplan-Meier analysis of KP16 mice. Log-rank test, two-tailed. B. Waterfall plot of best objective response. [Figure 67B] Survival and antitumor activity in KPC mice are shown. [Figure 68] Images of immunohistochemical staining of tumors from KP16 mice treated with vehicle or RP-182 are shown. Quantification shows the mean percentage of positive cells by computer-based histological analysis, N=4 / group. Arrows indicate membranous staining of CD206-positive cells or CD8+ T cells. [Figure 69] Immunohistochemical staining for E-cadherin and vimentin, quantification of N≧4 per group shown on the right. [Figure 70] Treatment with RP-182 reduces the induction of EMT markers vimentin and SNAIL expression in KPC cancer cells upon coculture with M2-polarized macrophages. Flow cytometry analysis of ecadherin-, vimentin-, and SNAIL2-positive cancer cell fractions after coculture without cells (blue bars), vehicle-treated M2 BMDMs (black bars), and M2 BMDMs pretreated with 20 μM RP-182 for 2 hours. The percentage of positive cells was gated on live CD11b-CK19-9+ cells. Results are shown from two independent experiments performed in triplicate. [Figure 71] The percentages of CD206-positive M2 macrophages, MDSCs, dendritic cells, CD8+, and CD4+ cell fractions in total cells of KP16 tumors treated for 7 days are shown. The percentages of FoxP3-positive Tregs among CD4+ T cells are shown on the right. Representative flow cytometry plots are shown below. [Figure 72] Figure 6G shows the gating strategy for identifying tumor-infiltrating CD8+ T cells (top) and CD206-high M2-like TAM fractions in pancreatic tumor lysates. Representative flow cytometry plots showing the gating strategy for determining tumor-infiltrating CD8a T cells and CD206-positive TAMs from KP16 animals after 1 week of treatment with vehicle and RP-182. For the final percentages shown in Figure 6G, percentages were normalized to total live cells. [Figure 73] Figure 1 shows that RP-182 in combination with gemcitabine reduces CD206-high monocytic MDSCs in spontaneous KPC tumors. Quantification of MDSC cell fractions by flow cytometry of tumor lysates from KPC mice treated with vehicle and RP-182 in combination with gemcitabine for 7 days, showing the % cells for each individual tumor examined. Monocytic MDSCs, M-MDSCs as determined by CD11b+Gr-1+Ly6ChighLy6G- cells; polymorphonuclear (PMN) MDSCs as determined by CD11b+Gr-1+Ly6ClowLy6G+ cells. [Figure 74] INFγ-positive T cells after addition of TAM isolated from KP16 and KPC tumors added to co-cultured cancer and splenic CD8+ T cells from tumor-bearing mice are shown. [Figure 75] Quantification of M1 and M2 gene expression levels in TAMs isolated from KPC and KP16 tumors by qRT-PCR analysis (N≥3 / group, triplicates). Quantification of the percentage of CD206- and CD86-positive macrophages in CD11b+F4 / 80+ / Gr-1- TAMs, analysis of the cell fraction of TAMs expressing M1 cytokines and immune checkpoints as indicated on the right. [Figure 76] Figure 1 shows that RP-182-induced M1-like CD86+ TAM populations in KPC mice have increased M1 cytokines and decreased PD-1- and SIRPα-positive cell fractions. Flow cytometry analysis of KPC mice treated with vehicle (top) and RP-182 (bottom) for 7 days shows the percentage of PD-1-, SIRPα-, and TNFα-positive cells among CD11b+F4 / 80+Gr-1-TAM (N≧5 / group). [Figure 77] Figure 1 shows that RP-182-induced M1-like CD86+ TAM populations in KPC mice increased the M1 cytokine-positive cell fraction. RP-182-induced increased cell fractions staining positive for the M1 cytokines IL-1β, IL-12, and TNFα, and the M1 marker iNOS in double-positive CD86+CD206+ and CD86+CD206- TAM, but not CD86-CD206+ TAM, populations. Flow cytometry analysis of KPC treated with vehicle and RP-182 for 7 days shows the percentage of positive cell fractions in the CD86+CD206-, CD86+CD206+, and CD86-CD206+ macrophage populations (N≧5 / group). [Figure 78] Flow cytometry plots including the gating strategy used to determine the cleaved caspase 3, Rab7, and LAMP1-positive CD11b+F4 / 80+Gr-1-TAM fraction in KPC tumors from vehicle- and RP-182-treated mice are shown. Quantification of the % positive TAM fraction for N≧7 mice per group is shown below. [Figure 79] Flow cytometric analysis of apoptosis, phagocytosis, and phagolysosome formation in CD11b-CK19+ cancer cells in tumor lysates from KPC mice treated with vehicle (top) and RP-182 (bottom) for 7 days. Flow cytometry plots include the gating strategy used to determine the cleaved caspase 3, Rab7, and LAMP1-positive CD11b-CK19+ cell fraction in vehicle- and RP-182-treated mice. Quantification, shown in the bar graph below, represents the average fraction percentage for N≥7 mice per group. [Figure 80]Tumor single-cell sequencing of CD11b+ and t-SNE plots after filtering for KRT19+, CD11c+, Ly6G+ cells (N=4 / group) from vehicle- (light blue) vs. RP-182-treated (dark blue) tumors; color bars indicate log10 [molecules / cell]. The increased expression levels of RP-182-induced DEGs identified in M2 BMDMs in vitro (the "in vitro gene M2 signature") are projected onto the CD11b+KRT19-CD11c-Ly6G- cluster from RP-182-treated tumors (right); color bars indicate log10 [total in vitro gene expression changes]. [Figure 81] Immunofluorescence images of RP-182 (top) and vehicle (bottom) treated KP16 tumors co-stained with anti-CD206 (red) and anti-LC3 (green), with computer-based quantification of co-stained cells (total cell number, N = 4 / group) shown. [Figure 82] Tumor growth after intratumoral cell transplantation of M2 BMDM into KPC allografts is shown (N=5). [Figure 83] Quantification of EliSpots (IFNγ-secreting CD8+ T cells of total CD8+ T cells added) after co-culture of KP16 (left) and KPC cancer cells (right) and CD8+ T cells isolated from tumor and spleen is shown. [Figure 84] Kaplan-Meier analysis of KP16 mice treated with anti-CD8 neutralizing antibody, anti-IgG2 isotype control, and the indicated combinations. [Figure 85] Quantification of flow cytometry analysis of PD-L1-expressing CD45-CK19+ cancer cells (left) and PD-1 expression on CD45+CD3e+CD8+ T cells (right) in KP16 tumors. [Figure 86] Best objective response (BOR) of KP16 tumors treated with vehicle (gray bars), anti-PD-L1 injection (purple), RP-182 (red), and anti-PD-L1 in combination with RP-182 is shown. N≥7 / group. [Figure 87]Tumor growth of CT-26 allografts is shown, with tumor weights at study endpoints shown on the right. Tumor growth of murine B16 melanoma tumors. [Figure 88A] Pancreatic cancer patient-derived xenograft models with high (#133R, #328R) and low (#295R, #057R) CD206 expression levels treated with RP-182, RP-426, and vehicle control are shown. N≥7 / group. H. RP-182 administration rescues mice with chemically induced pulmonary fibrosis. Quantification of total body weight and Kaplan-Meier survival analysis are shown. N=6 / group. Error bars indicate standard deviation. [Figure 88B] Showing pulmonary inflammatory infiltrates after bleomycin administration. H&E stained images, quantification of the measured weight of excised lungs on the right. N=4 / group. [Figure 88C] Masson's trichrome stained images are shown, quantification of surface area (μm 2 ) of the examined 100 mm 2 lung surface on the right. [Figure 89] Figure 1. Decreased CD206-positive alveolar cell infiltration in RP-182-treated mice. Immunofluorescence lung images of uninjected and bleomycin-injected mice treated with vehicle or RP-182 stained with DAPI and anti-CD206 (green). Quantification of the number of CD206-positive cells is shown on the right, N=4 / group. [Figure 90] Immunofluorescence images of KPC tumors treated with biotinylated RP-182 and co-stained with anti-CD206 (green), streptavidin (red), and DAPI (blue) are shown. Laser intensity profile (A1) of a linear scan of a random tissue section measuring intensity (fluorescence intensity, y-axis) over distance (µm, x-axis). Colocalization of CD206-expressing cells (green) and bio-RP-182 (red channel) (B1) produces a yellow / purple emission. [Figure 91]Figure 1 shows the tissue distribution of RP-182 after imaging of indicated organs containing subcutaneously grown CT-26 tumors in BALB / c mice, excised 10 minutes after intraperitoneal injection of Alexa-488-labeled peptide RP-182 at 20 mg / kg. Fluorescence in manually defined regions of interest was quantified by Living Image, a color scale of emitted photon intensity shown on the right. [Figure 92] Kaplan-Meier analysis of KPC wild-type (left) and KPC CD206 KO- / - allografts (right) treated with vehicle (black curve) or RP-182 (red). Phase contrast image of M2 BMDMs incubated with carboxyfluorescein succinimidyl ester (CFSE)-labeled cancer cells (green). The image (100x) shows a representative macrophage with encapsulated green-labeled cancer cells and induced cytoplasmic vacuoles and blebs. [Figure 93] Toxicity measurements after 14 days of daily administration of increasing concentrations of RP-182 are shown. Body weight (left) and red and white blood cell (RBC, WBC) counts (right) of mice treated with vehicle and RP-182 at 30 mg / kg after 14 days of daily administration of RP-182. C. Individual organ weights (in grams) of mice treated with the indicated daily doses of RP-182 for 14 days, n≧7 / group. [Figure 94A] Phase contrast images of M2 BMDMs incubated with carboxyfluorescein succinimidyl ester (CFSE)-labeled cancer cells (green) are shown. The inlet (100x) shows encapsulated green-labeled cancer cells and representative macrophages with induced cytoplasmic vacuoles and vesicles. [Figure 94B] Quantification of the number of CFSE-positive macrophages out of the total number of M2 BMDM (%, phagocytic index) is shown. [Figure 95] Flow cytometry plots of CFSE-positive CD11b+F4 / 80+Gr-1-CD86+ (top) and CD206+ BMDMs are shown after addition of CFSE-labeled KPC cancer cells and treatment with vehicle or RP-182. [Figure 96]Tumor growth of KPC xenografts is shown (≧7 / group). [Figure 97] RP-182 restricts tumor growth of MDA-MB231 breast cancer and C4-2 prostate cancer xenografts grown in T cell-deficient nu / J mice. A. MDA-MB231 tumors (N≥7 / group) grown in nu / J mice treated with vehicle (red), RP-182 (purple), gemcitabine (green), and the combinations (brown) shown on the left. The effect of RP-182 treatment on lymph node metastasis in MDA-MB231 mice after 42 days of treatment is shown on the right. Analysis shows the percentage of cancer-involved lymph nodes per total number of lymph nodes examined in the draining basin. In the representative H&E stain of metastatic cancer involved in a lymph node shown on the right, green arrows highlight cancer cells with abnormal mitotic counts. B. Tumor growth of C4-2 prostate cancer xenografts grown in T cell-deficient nu / J mice. [Figure 98] Transmission electron microscopy of vehicle- and RP-182-treated KP16 tumors shows TAMs with intracellular vesicles indicated by arrows, cancer cell clasping, and partial or complete cancer cell phagocytic events highlighted in red. DETAILED DESCRIPTION OF THE INVENTION

[0026] Aspects of the present disclosure include methods for modulating macrophage activity. In certain embodiments, the method comprises contacting a macrophage with a mannose receptor (CD206)-binding agent in a manner sufficient to modulate the activity of the macrophage. Methods for converting the phenotype of a macrophage from an M2 phenotype to an M1 phenotype are also provided. Methods for inhibiting the proliferation of CD206-expressing cells, as well as methods for treating a subject with a neoplastic condition (e.g., cancer) or a condition associated with chronic inflammation, are described. Immunomodulatory peptides suitable for use in the methods of the present disclosure are also disclosed. Aspects of the present disclosure also include active agents for binding to the activity-modulating domain of CD206. Methods for determining whether a compound binds to the activity-modulating domain of CD206 are also provided.

[0027] Before the present invention is described in more detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0028] Where a range of values ​​is provided, unless the context clearly dictates otherwise, each intervening value, to the tenth of the unit of the lower limit, shall be included in the upper and lower limits of that range and in any other value within that stated range. Between any stated or intervening value, each intervening value is understood to be included in the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0029] Certain ranges are presented herein with numerical values ​​preceded by the term "about." The term "about" is used herein to provide literal support for the exact number it precedes, as well as a number that is close to or approximately the number it precedes. When determining whether a number is close to or approximately a specifically stated number, the uncited number that is approximately or approximating the number may be a number that, in the context in which it is presented, provides a substantial equivalent to the specifically recited number.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein also can be used in the practice or Representative exemplary methods and materials that may be used for testing are now described.

[0031] All publications and patents cited herein are incorporated by reference to disclose and describe the methods and / or materials to which the publications are cited, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. The citation of a publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0032] As used in this specification and the appended claims, the singular forms "a," "an," and "an" are used interchangeably. Please note that "the" and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claim may be drafted to exclude any element. Thus, this statement is intended to serve as a predicate basis for using exclusive language such as "solely," "only," etc. in connection with the recitation of claim elements or for using a "negative" limitation.

[0033] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0034] Although apparatus and methods have been or will be described with functional descriptions for grammatical fluidity, it is expressly understood that unless expressly recited under 35 U.S.C. 112, the claims should not necessarily be construed as limited by construction of "means" or "step" limitations, but should be given the full scope of meaning and equivalents of the definitions provided by the claims under the doctrine of legal equivalents, and if the claims are expressly recited under 35 U.S.C. 112, they should be given the full legal equivalents under 35 U.S.C. 112.

[0035] Methods for modulating macrophage activity Methods for modulating macrophage activity and converting macrophage phenotype from M2 phenotype to M1 phenotype, methods for inhibiting proliferation of CD206-expressing cells, tumor conditions or or conditions associated with chronic inflammation, as well as combination therapies, are described in more detail in conjunction with the examples set forth below.

[0036] In some embodiments, the methods include modulating macrophage activity, the methods comprising contacting macrophages with a CD206-binding agent (e.g., as described herein). This includes regulating macrophage activity.

[0037] In certain cases, the CD206 binding agent comprises a moiety selected from a fibronectin II domain of CD206, a C-type lectin carbohydrate recognition domain 3 (CRD3) of CD206, a C-type lectin carbohydrate recognition domain 4 (CRD4) of CD206, and a C-type lectin carbohydrate recognition domain 5 (CRD5) of CD206. In certain cases, the site binds to a site in the fibronectin II domain of CD206. In certain instances, the site is the C-type lectin carbohydrate recognition domain 3 (CRD3) of CD206. In certain other instances, the site is the C-type lectin carbohydrate recognition domain 5 (CRD5) of CD206.

[0038] In certain embodiments, the CD206-binding agent has a saturation of at least -700 kcal / mol, such as at least -700 kcal / mol. It binds to CD206 with a binding energy of -650 kcal / mol, and in certain embodiments, binds with at least -750, -800, -900, -1000, -1100, -1200, -1250, -1300, -1350, -1400, -1425, -1450, -1475, -1500, -1525, -1550, -1575, -1600 kcal / mol or more. The energy of binding can be determined, for example, in silico, in vitro, or in vivo, using methods well known in the art (e.g., using the ClusPro™ algorithm).

[0039] In certain embodiments, of the methods for modulating macrophage activity, the modulated macrophage activity is macrophage polarization. In certain embodiments of the methods, the viability of the macrophages is reduced. In certain embodiments, of the methods for modulating macrophage activity, the macrophages are M2 macrophages or tumor-associated macrophages (TAMs). In certain embodiments of the methods for modulating macrophage activity, a CD206-binding agent (e.g., as described herein) inhibits macrophage activity. In certain embodiments of the methods, the CD206-binding agent induces apoptosis of macrophages. In certain embodiments of the methods, the CD206-binding agent stimulates phagocytosis.

[0040] In certain embodiments of the method of modulating macrophage activity, the macrophage is In certain other embodiments, the macrophages are in vivo. In other embodiments, the method includes converting the phenotype of a macrophage from an M2 phenotype to an M1 phenotype. In certain embodiments, the method includes contacting a macrophage having an M2 phenotype with a CD206-binding agent in a manner sufficient to convert the phenotype of the macrophage to an M1 phenotype. In some cases, contact with a CD206-binding agent results in the expression of a macrophage. A conformational change in the CD206 receptor on macrophages is sufficient to convert the phenotype to the M1 phenotype. A change in function is induced. In some cases, converting the phenotype of the macrophage includes inducing expression of CD86 by the macrophage. In other cases, converting the phenotype of the macrophage includes reducing expression of CD206 by the macrophage. In still other cases, converting the phenotype of the macrophage includes converting the macrophage to a phenotype that exhibits upregulation of M1 cytokines and markers. For example, the M1 cytokines and markers may be selected from the group consisting of IL-1β, IL-12, TNFα, and nitric oxide synthase. In other cases, converting the phenotype of the macrophage includes converting the macrophage to a phenotype that exhibits reduced expression of signal-regulatory protein alpha (SIRPα).

[0041] In certain embodiments of the method for converting the phenotype of a macrophage from an M2 phenotype to an M1 phenotype, the CD206-binding agent binds to a site selected from the fibronectin II domain of CD206, the C-type lectin carbohydrate recognition domain 3 (CRD3) of CD206, the C-type lectin carbohydrate recognition domain 4 (CRD4) of CD206, and the C-type lectin carbohydrate recognition domain 5 (CRD5) of CD206. In certain cases, the site is the fibronectin II domain of CD206. In some cases, the site is the C-type lectin carbohydrate recognition domain 3 (CRD3) of CD206. In certain other cases, the site is the C-type lectin carbohydrate recognition domain 5 (CRD5) of CD206. be.

[0042] In certain embodiments of the method for converting the phenotype of a macrophage from an M2 phenotype to an M1 phenotype, the macrophage is contacted with a CD206-binding agent in vitro. In this embodiment, macrophages are contacted with a CD206-binding agent in vivo.

[0043] In some embodiments, the method comprises inhibiting proliferation of CD206-expressing cells, includes contacting target CD206-expressing cells with a CD206-binding agent (e.g., as described herein) to inhibit proliferation of the cells. In certain cases, the cells are cancer cells. Cancer cells include, but are not limited to, pancreatic cancer cells, prostate cancer cells, colon cancer cells, skin cancer cells, or breast cancer cells. In certain cases, the cancer is a solid tumor cancer. Solid tumor cancers include, but are not limited to, pancreatic, prostate, colon, breast, or skin tumors. In certain embodiments, of the methods of inhibiting proliferation of CD206-expressing cells, contacting the target CD206-expressing cells includes administering to a subject in need thereof a therapeutically effective amount of a CD206-binding agent (e.g., as described herein) to treat the cancer in the subject. In some embodiments, the method includes treating the subject for a condition associated with chronic inflammation. According to certain embodiments, the method includes administering to the subject a therapeutically effective amount of a CD206-binding agent to treat the subject for a condition associated with chronic inflammation. In some embodiments, the condition associated with chronic inflammation is selected from scleroderma or multiple sclerosis, irritable bowel disease, ulcerative colitis, colitis, Crohn's disease, idiopathic pulmonary fibrosis, asthma, keratitis, arthritis, osteoarthritis, rheumatoid arthritis, autoimmune diseases, 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, ocular inflammatory conditions (e.g., keratitis), skin diseases (e.g., atopic dermatitis or psoriasis), inflammatory diseases such as sinusitis and otitis media, parasitic infections (e.g., malaria), and chronic viral or microbial infections.

[0044] In certain embodiments of the methods of treating chronic inflammation, a CD206-binding agent (e.g., a compound described herein) is administered. (as described in) is administered in conjunction with another drug known to be effective in treating the condition. In some cases, the condition is cancer. In some cases, the cancer includes, but is not limited to, pancreatic, prostate, colon, breast, or skin cancer. In certain cases, the method further includes administering to the subject an effective amount of a chemotherapeutic agent, antibody agent, or cell therapy. In certain cases, the chemotherapeutic agent, antibody agent, or cell therapy is selected from steroids, anthracyclines, thyroid hormone replacement agents, thymidylate targeting drugs, antibodies, checkpoint inhibitor drugs, chimeric antigen receptor / T cell therapy, and other cell therapies.

[0045] In some embodiments of the method of treating chronic inflammation, the condition associated with chronic inflammation is fibrosis. In some cases, the condition associated with chronic inflammation is scleroderma.

[0046] In some embodiments, the method includes treating a subject for a neoplastic condition, such as cancer (e.g., a solid tumor cancer). The methods of the present disclosure may be used to target and treat a variety of cancers, including, for example, primary cancers, secondary cancers, regrowing cancers, recurrent cancers, refractory cancers, etc. For example, in some cases, the methods of the present disclosure may be used as an initial treatment for a primary cancer identified in a subject. In some cases, the methods of the present disclosure may be used to target and treat a variety of cancers, including, for example, primary cancers, secondary cancers, regrowing cancers, recurrent cancers, refractory cancers, etc. In some cases, the methods of the present disclosure may be used as an initial treatment for a primary cancer identified in a subject. In some cases, the methods of the present disclosure may be used to target and treat a variety of cancers, for example, in subjects with cancer that is refractory to previous treatments, in subjects with cancer that is regrowing after previous treatments, in subjects with a mixed response to previous treatments (e.g., a positive response to at least one tumor in the subject and a positive response to at least one tumor in the subject), In subjects with a negative or neutral response to at least one second tumor, For example, secondary or later).

[0047] In some cases, the methods of the present disclosure can be used to target, treat, or clear subjects with minimal residual disease (MRD) remaining after a previous cancer treatment. Targeting, treating, and / or clearing MRD can be pursued using instant methods regardless of whether MRD is refractory to a previous treatment or has been determined. In some cases, the methods of the present disclosure can be used to target, treat, and / or clear subjects with MRD after determining that MRD is refractory to a previous treatment or one or more available treatment options.

[0048] Cancers of interest associated with commonly mutated genes include, for example, ABI1, ABL1, ABL2, AC KR3, ACSL3, ACSL6, AFF1, AFF3, AFF4, AKAP9, AKT1, AKT2, ALDH2, ALK, AMER1 APC, ARHGAP26, ARHGEF12, ARID1A, ARID2, ARNT, ASPSCR1, ASXL1, ATF1, AT IC, ATM, ATP1A1, ATP2B3, ATRX, AXIN1, BAP1, BCL10, BCL11A, BCL11B, BCL2 BCL3, BCL6, BCL7A, BCL9, BCOR, BCR, BIRC3, BLM, BMPR1A, BRAF, BRCA1, BRCA2 BRD3, BRD4, BRIP1, BTG1, BUB1B, C15orf65, C2orf44, CACNA1D, CALR, CAMT A1, CANT1, CARD11, CARS, CASC5, CASP8, CBFA2T3, CBFB, CBL, CBLB, CBLC, CCD C6, CCNB1IP1, CCND1, CCND2, CCND3, CCNE1, CD274, CD74, CD79A, CD79B, CDC 73 CDH1, CDH11, CDK12, CDK4, CDK6, CDKN2A, CDKN2C, CDX2, CEBPA, CEP89, CH CHD7, CHEK2, CHIC2, CHN1, CIC, CIITA, CLIP1, CLP1, CLTC, CLTCL1, CNBP, CN OT3, CNTRL, COL1A1, COL2A1, COX6C, CREB1, CREB3L1, CREB3L2, CREBBP, CRLF 2, CRTC1, CRTC3, CSF3R, CTNNB1, CUX1, CYLD, DAXX, DCTN1, DDB2, DDIT3, DDX 10, DDX5, DDX6, DEK, DICER1, DNM2, DNMT3A, EBF1, ECT2L, EGFR, EIF3E, EIF4A 2. ELF4, ELK4, ELL, ELN, EML4, EP300, EPS15, ERBB2, ERC1, ERCC2, ERCC3, ER CC4, ERCC5, ERG, ETV1, ETV4, ETV5, ETV6, EWSR1, EXT1, EXT2, EZH2, EZR, FAM4 6C FANCA、FANCC、FANCD2、FANCE、FANCF、FANCG、FAS、FBXO11、FBXW7、FCGR2 B FCRL4 FEV FGFR1 FGFR1OP FGFR2 FGFR3 FH FHIT FIP1L1 FLCN FLI1FLT3、FNBP1、FOXA1、FOXL2、FOXO1、FOXO3、FOXO4、FOXP1、FSTL3、FUBP1、FUS 、GAS7、CAT1、CAT2、CAT3、GMPS、GNA11、GNAQ、GNAS、GOLGA5、GOPC、GPC3、G PHN、H3F3A、H3F3B、HERPUD1、HEY1、HIP1、HIST1H4I、HLA-A、HLF、HMGA1、HMG A2、HNF1A、HNRNP2B1、HOOK3、HOXA11、HOXA13、HOXA9、HOXC11、HOXC13、HOXD 11、HOXD13、HRAS、HSP90AA1、HSP90AB1、IDH1、IDH2、IKZF1、IL2、IL21R、IL6 ST、IL7R、IRF4、ITK、JAK1、JAK2、JAK3、JAZF1、JUN、KAT6A、KAT6B、KCNJ5、KDM 5A、KDM5C、KDM6A、KDR、KDSR、KIAA1549、KIAA1598、KIF5B、KIT、KLF4、KLF6、 KLK2、KMT2A、KMT2C、KMT2D、KRAS、KTN1、LASP1、LCK、LCP1、LHFP、LIFR、LMNA、 LMO1、LMO2、LPP、LRIG3、LSM14A、LYL1、MAF、MAFB、MALT1、MAML2、MAP2K1、MA P2K2、MAP2K4、MAX、MDM2、MDM4、MECOM、MED12、MEN1、MET、MITF、MKL1、MLF1、M LH1、MLLT1、MLLT10、MLLT11、MLLT3、MLLT4、MLLT6、MN1、MNX1、MPL、MSH2、MS H6、MSI2、MSN、MTCP1、MUC1、MUTYH、MYB、MYC、MYCL、MYCN、MYD88、MYH11、MYH9 、MYO5A、NAB2、NACA、NBN、NCKIPSD、NCOA1、NCOA2、NCOA4、NDRG1、NF1、NF2、N FATC2、NFE2L2、NFIB、NFKB2、NIN、NKX2-1、NONO、NOTCH1、NOTCH2、NPM1、NR4A 3、NRAS、NRG1、NSD1、NT5C2、NTRK1、NTRK3、NUMA1、NUP214、NUP98、NUTM1、NUT M2A、NUTM2B、OLIG2、OMD、P2RY8、PAFAH1B2、PALB2、PATZ1、PAX3、PAX5、PAX7、PAX8、PBRM1、PBX1、PCM1、PCSK7、PDCD1LG2、PDE4DIP、PDGFB、PDGFRA、PDGFRB、PER1、PHF6、PHOX2B、PICALM、PIK3CA、PIK3R1、PIM1、PLAG1、PLCG1、PML、PMS1、PMS2、POT1、POU2AF1、POU5F1、PPARG、PPFIBP1、PPP2R1A、PRCC、PRDM1、PRDM16、PRF1、PRKAR1A、PRRX1、PSIP1、PTCH1、PTEN、PTPN11、PTPRB、PTPRC、PTPRK、PWWP2A、RABEP1、RAC1、RAD21、RAD51B、RAF1、RALGDS、RANBP17、RAP1GDS1、RARA、RB1、RBM15、RECQL4、REL、RET、RHOH、RMI2、RNF213、RNF43、ROS1、RPL10、RPL22、RPL5、RPN1、RSPO2、RSPO3、RUNX1、RUNX1T1、SBDS、SDC4、SDHAF2、SDHB、SDHC、SDHD、SEPT5、SEPT6、SEPT9、SET、SETBP1、SETD2、SF3B1、SFPQ、SH2B3、SH3GL1、SLC34A2、SLC45A3、SMAD4、SMARCA4、SMARCB1、SMARCE1、SMO、SOCS1、SOX2、SPECC1、SRGAP3、SRSF2、SRSF3、SS18、SS18L1、SSX1、SSX2、SSX2B、SSX4、SSX4B、STAG2、STAT3、STAT5B、STAT6、STIL、STK11、SUFU、SUZ12、SYK、TAF15、TAL1、TAL2、TBL1XR1、TCEA1、TCF12、TCF3、TCF7L2、TCL1A、TERT、TET1、TET2、TFE3、TFEB、TFG、TFPT、TFRC、THRAP3、TLX1、TLX3、TMPRSS2、TNFAIP3、TNFRSF14、TNFRSF17、TOP1、TP53、TPM3、TPM4、TPR、TRAF7、TRIM24、TRIM27、TRIM33、TRIP11、TRRAP、TSC1、TSC2、TSHR、TTL、U2AF1、UBR5、USP6、VHL、VTI1A、WAS、WHSC1、WHSC1L1、WIF1、WRN、WT1、WWTR1、XPA、XPC、XPO1、YWHAE、These include ZBTB16, ZCCHC8, ZMYM2, ZNF331, ZNF384, ZNF521 and ZRSR2.

[0049] Cancers of interest for treatment with embodiments of the present disclosure include, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancers (e.g., Kaposi's sarcoma, lymphoma, etc.), anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, bile duct cancer (extrahepatic), bladder cancer, bone cancer (e.g., Ewing's sarcoma, osteosarcoma and malignant fibrohistiocytoma, etc.), brain stem glioma, brain tumor (e.g., astrocytoma, central nervous system embryonal tumor, central nervous system germ cell tumor, craniopharyngioma, epithelioma, etc.), breast cancer (e.g., female breast cancer, male breast cancer, pediatric breast cancer, etc.), bronchial tumor, Burkitt's lymphoma, carcinoid tumor (e.g., pediatric, gastrointestinal, etc.), tumor of unknown primary, cardiac (heart) tumor, central nervous system (e.g., atypical teratomas / rhabdoid tumors, embryonal tumors, germ cell tumors, lymphoma, etc.), cervical cancer, childhood cancer, spinal cord tumor, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML) ), chronic myeloproliferative neoplasms, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ducts (e.g., bile duct, extrahepatic, etc.), intraductal carcinoma (DCIS), germinal tumors, endometrial cancer, epithelioma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma, extracranial germ cell tumors, extranasal germ cell tumors, extrahepatic bile duct cancer, eye cancer (e.g., intraocular melanoma, retinoblastoma, etc.), fibrous histiocytoma (e.g., malignant, osteosarcoma, etc.), gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., extracranial, extragonadal, ovarian, testicular, etc.), gestational hyperplasia, glioma, Hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis (e.g., Langerhans cell), Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumors (e.g., pancreatic neuroendocrine tumors), Kaposi's sarcoma, kidney cancer (e.g., renal cell, Wilms' tumor, childhood kidney tumors), Langerhans cell histiocytosis, laryngeal cancer, leukemia (e.g., acute lymphoblastic (ALL), acute myeloid (AML), chronic lymphocytic (CLL), chronic myeloid (CML), hairy cell), lip and oral cavity cancer, liver cancer (primary), primary lobular carcinoma of the liver (LCIS), lung Cancer (e.g., non-small cell, small cell, etc.), lymphoma (e.g., AIDS-related, Burkitt, cutaneous T-cell, Hodgkin, non-Hodgkin, primary central nervous system (CNS), etc.), macroglobulinemia (e.g., primary, etc.), male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma, metastatic squamous cell carcinoma with occult primary, midline tract carcinoma with NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm,Myeloid leukemia (e.g., chronic (CML), etc.), myeloid leukemia (e.g., acute (AML), etc.), myeloproliferative neoplasms (e.g., chronic tumors, etc.), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cavity cancer, oral cavity cancer (e.g., lip), oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma, ovarian cancer (e.g., epithelial, germ cell tumor, low malignant potential tumor, etc.), pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillary tumors, paranasal sinus tumors, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumors, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and urethra Cancer, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (e.g., Ewing, Kaposi, osteosarcoma, rhabdomyosarcoma, soft tissue, uterine, etc.), Sezary syndrome, skin cancer (e.g., childhood, melanoma, Merkel cell carcinoma, non-melanoma, etc.), small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer (e.g., with occult primary, metastatic, etc.), gastric cancer, T-cell lymphoma, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, ureteral and renal pelvic cancer, urethral cancer, uterine cancer (e.g., endometrial cancer, etc.), uterine sarcoma, vaginal cancer, vulvar cancer, primary macroglobulinemia, Wilms' tumor, etc.,

[0050] The treatment methods described herein may, in some cases, be used in patients who have previously undergone one or more conventional treatments. For example, in the case of oncology, the methods described herein may be performed in some cases following conventional cancer therapy, including, but not limited to, conventional chemotherapy, conventional radiation therapy, conventional immunotherapy, surgery, etc. In some cases, the methods described herein may be used when the subject has not responded to or is refractory to conventional therapy.

[0051] With respect to cancer overall, the desired effects of the described treatments may result in a reduction in the number of cancer cells, a reduction in the size of the tumor, a reduction in the overall proliferation of the cancer, a slowing of the overall growth rate of the tumor, etc. For example, effective treatment may, in some cases, require one or more doses to be administered to an individual in need thereof. When administered in amounts that reduce the number of cancer cells in an individual and / or The tumor mass is reduced compared to the number of cancer cells and / or tumor mass in the absence of treatment. A treatment that reduces the level of IL-16 by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, or more than 75%.

[0052] In some embodiments, an effective treatment, when administered alone (e.g., in monotherapy) or in combination (e.g., in combination therapy) with one or more additional therapeutic agents in one or more doses, reduces one or more of tumor growth rate, cancer cell number, and tumor mass in the absence of treatment by more than one or more of the following: at least about 5%, at least about 10%, or less than the tumor mass, cancer cell count, or tumor mass at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40% , at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more.

[0053] In some cases, the treatment may enhance the expression and / or secretion of cytokines by immune cells. Non-limiting examples of cytokines whose expression / secretion may be regulated include interleukins and related cytokines (e.g., IL-1-like, IL-1α, IL-1β, IL-1RA, IL-18, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5, GM-CSF, IL-6-like, IL-6, IL-11, G-CSF, IL-12, LIF, OSM, IL-10-like, IL-10, IL-20, IL-14, IL-16, IL-17, etc.), interferons (e.g., IFN-α, IFN-β, IFN-γ, etc.), TNF family cytokines (e.g., CD154, LT-β, ​​TNF- α, TNF-β, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, etc.), TGF-β family (e.g., TGF-β1, TGF-β2, TGF-β3, etc.), etc. The amount of increase can vary and can be in the range of an increase of 10% or more, including, but not limited to, 10% or more, 25% or more, 50% or more, 75% or more, 100% or more, 150% or more, 200% or more, 250% or more, 300% or more, 350% or more, 400% or more, etc.

[0054] Chemotherapeutic agents and combination therapies As will be readily appreciated, administering a therapeutically effective amount of a CD206-binding agent to a subject described herein In some cases, treatment methods involving the use of steroids may be combined with one or more conventional treatments. For example, in the case of oncology, the methods described herein may, in some cases, be combined with conventional cancer therapies, including, but not limited to, conventional chemotherapy, conventional radiation therapy, conventional immunotherapy, surgery, etc.

[0055] In combination with any of the subject methods, a CD206-binding agent (e.g., as described herein) The CD206-binding agent (or a pharmaceutical composition comprising such a compound) may be administered in combination with another agent designed to reduce or prevent inflammation, treat or prevent chronic inflammation, or treat cancer. In some embodiments, the condition associated with chronic inflammation is fibrosis. In some cases, the condition associated with chronic inflammation is scleroderma. For example, the CD206-binding agent ... For example, the CD206-binding agent may be combined with conventional agents or therapies for treating chronic inflammation or fibrosis, including, but not limited to, pirfenidone, nintedanib, nonsteroidal anti-inflammatory drugs (NSADs), steroids, and standard scleroderma treatments. In each case, the CD206-binding agent may be administered before, simultaneously with, or after the administration of the other agent.

[0056] In some cases, the methods described herein can be used before or after conventional therapy.For example, the methods described herein can be used as adjuvant therapy, for example, after the subject has seen improvement from conventional therapy, or can be used when the subject is not responding to conventional therapy.In some cases, the methods described herein can be used before additional therapy, for example, to prepare the subject for additional therapy (for example, conventional therapy described herein).

[0057] Standard cancer therapies include surgery (eg, surgical removal of cancerous tissue), radiation therapy, bone marrow transplantation, chemotherapy treatment, antibody therapy, biological response modifier therapy, and certain combinations of the foregoing.

[0058] Radiation therapy includes, but is not limited to, x-rays or gamma rays delivered from an externally applied source such as a beam or by implanting small radioactive sources.

[0059] Suitable antibodies for use in cancer therapy include naked antibodies, such as trastuzumab (Herceptin), bevacizumab (Avastin™), cetuximab (Erbitux™), panitumumab (Vectibix™), ipilimumab (Yervoy™), rituximab (Rituxan), alemtuzumab (Lemtrada™), ofatumumab (Arzerra™), oregovomomab (OvaRex™), lambrolizumab (MK-3475), pertuzumab (Perjeta™), ranibizumab (Lucentis™), and the like. and conjugated antibodies, such as, but not limited to, gemtuzumab ozogamicin (Mylortarg™), brentimab vedotin (Adcetris™), 90Y-labeled ibritumabromab tiuctan (Zevalin™), 131I-labeled (Bexxar™), and the like. Antibodies suitable for use in cancer therapy include, but are not limited to, antibodies raised against tumor-associated antigens. Such antigens include, but are not limited to, CD20, CD30, CD33, CD52, EpCAM, CEA, gpA33, mucin, TAG-72, CAIX, PSMA, folate binding protein, gangliosides (e.g., GD2, GD3, GM2, etc.), Ley, VEGF, VEGFR, integrin αVβ3, integrin α5β1, EGFR, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, and the like.

[0060] Conventional cancer treatments include, for example, Ado-trastuzumab, which targets HER2 (ERBB2 / neu). Mutansine (Kadcyla) (approved for use in breast cancer); afatinib (Gilotrif) targeting EGFR (HER1 / ERBB1) and HER2 (ERBB2 / neu) (approved for use in non-small cell lung cancer) Aldesleukin (Proleukin) targets (approved for use in renal cell carcinoma and melanoma); Alectinib (Alecensa) targets ALK (approved for use in non-small cell lung cancer) alemtuzumab (Campath), which targets CD52 (approved for use in B-cell chronic lymphocytic leukemia); atezolizumab (Tecentriq), which targets PD-L1 (approved for use in urothelial carcinoma and non-small cell lung cancer); avelumab, which targets PD-L1 axitinib (Inlyta), which targets KIT, PDGFRβ, and VEGFR1 / 2 / 3, which is approved for renal cell carcinoma; belimumab (Benlysta), which targets BAFF, which is approved for systemic lupus erythematosus; belinumab (Beleodaq), which targets HDAC, which is approved for peripheral T-cell lymphoma; and bevacizumab (Avastin), which targets a VEGF ligand, which is approved for cervical cancer, colorectal cancer, fallopian tube cancer, glioblastoma, non-small cell lung cancer, ovarian cancer, peritoneal cancer, and renal cell carcinoma. Blincyto (blinatumomab), which targets CD19 / CD3 (approved for use in acute lymphoblastic leukemia (precursor B cells)); Velcade (bortezomib), which targets the proteasome (approved for use in multiple myeloma and mantle cell lymphoma); Bosutinib (Bosulif), which targets ABL (approved for use in chronic myeloid leukemia); Adcetris (brentuximab vedotin), which targets CD30 (approved for use in Hodgkin lymphoma and anaplastic large cell lymphoma); and brigatinib (Alunbrig), which targets ALK (approved for use in non-small cell lung cancer (ALK+)). Cabozantinib (Cabometyx, Cometrik), which targets FLT3, KIT, MET, RET, and VEGFR2 (approved for medullary thyroid cancer and renal cell carcinoma); carfilzomib (Kyprolis), which targets the proteasome (approved for multiple myeloma); ceritinib (Zykadia), which targets ALK (approved for non-small cell lung cancer); cetuximab (Erbitux), which targets EGFR (HER1 / ERBB1), (approved for colorectal cancer and squamous cell carcinoma of the head and neck); cobimetinib (Cotellic), which targets MEK (approved for melanoma); crizotinib (Xalkori), which targets ALK, MET, and ROS1 (approved for non-small cell lung cancer); and dabrafenib (Tafinlar), which targets BRAF (approved for melanoma and non-small cell lung cancer).Daratumumab (Darzalex), which targets CD38 (approved for use in multiple myeloma); dasatinib (Sprycel), which targets ABL (approved for use in chronic myeloid leukemia and acute lymphoblastic leukemia); denosumab (Xgeva), which targets RANKL (approved for use in giant cell tumor of bone); dinutuximab (Unituxin), which targets B4GALNT1 (GD2), (approved for use in pediatric neuroblastoma); durvalumab (Imfinzi), which targets PD-L1 (approved for use in urothelial BB); elotuzumab (Empliciti), which targets SLAMF7 (CS1 / CD319 / CRACC), (approved for use in multiple myeloma); enasidenib (Idhifa), which targets IDH2 (approved for use in acute myeloid leukemia); Erlotinib (Tarceva), which targets EGFR (HER1 / ERBB1) (approved for use in non-small cell lung cancer and pancreatic cancer); everolimus (Afinitor), which targets mTOR (approved for use in neuroendocrine tumors of pancreatic, gastrointestinal, or pulmonary origin, renal cell carcinoma, unresectable subependymal giant cell astrocytoma, and breast cancer); gefitinib (Iressa), which targets EGFR (HER1 / ERBB1) (approved for use in non-small cell lung cancer); ibritumomab tiuxetan (Zevalin), which targets CD20 (approved for use in non-Hodgkin's lymphoma); ibrutinib (Imbruvica), which targets BTK (approved for use in mantle cell lymphoma, chronic lymphocytic leukemia, and primary macroglobulinemia); Idelalisib (Zydelig), which targets PI3Kδ (approved for chronic lymphocytic leukemia, follicular B-cell non-Hodgkin's lymphoma, and small lymphocytic lymphoma); imatinib (Gleevec), which targets KIT, PDGFR, and ABL (approved for gastrointestinal stromal tumors (KIT+), dermatofibrosarcoma protuberans, and multiple hematologic malignancies); ipilimumab (Yervoy), which targets CTLA-4 (approved for melanoma); and ixazomib (Ninlaro), which targets the proteasome (approved for multiple myeloma).Lapatinib (Tykerb), which targets HER2 (ERBB2 / neu) and EGFR (HER1 / ERBB1), is approved for breast cancer (HER2+); lenvatinib (Lenvima), which targets VEGFR2, is approved for renal cell carcinoma and thyroid cancer; midostaurin (Rydapt), which targets FLT3, is approved for acute myeloid leukemia (FLT3+); necitumumab (Portrazza), which targets EGFR (HER1 / ERBB1), is approved for squamous non-small cell lung cancer; neratinib (Nerlynx), which targets HER2 (ERBB2 / neu), is approved for breast cancer; and nilotinib (Tasigna), which targets ABL, is approved for chronic myeloid leukemia. Niraparib (Zejula), which targets PARP (approved for use in ovarian cancer, fallopian tube cancer, and peritoneal cancer); nivolumab (Opdivo), which targets PD-1 (approved for use in colorectal cancer, head and neck squamous cell carcinoma, Hodgkin lymphoma, melanoma, non-small cell lung cancer, renal cell carcinoma, and urothelial carcinoma); obinutuzumab (Gazyva), which targets CD20 (approved for use in chronic lymphocytic leukemia and follicular lymphoma); ofatumumab (Arzerra, HuMax-CD20), which targets CD20 (approved for use in chronic lymphocytic leukemia); olaparib (Lynparza), which targets PARP (approved for use in ovarian cancer); olaratumab (Lartruvo), which targets PDGFRα (approved for use in soft tissue sarcoma); osimertinib (Tagrisso), which targets EGFR (approved for use in non-small cell lung cancer); palbociclib (Ibrance), which targets CDK4 and CDK6 (approved for use in breast cancer) panitumumab (Vectibix), which targets EGFR (HER1 / ERBB1) and is approved for use in colorectal cancer; panobinostat (Farydak), which targets HDAC (multiple tumors) approved for use in multiple myeloma; pazopanib (Votrient), which targets VEGFR, PDGFR, and KIT (approved for use in renal cell carcinoma); pembrolizumab (Keytruda), which targets PD-1 (approved for use in classical Hodgkin lymphoma, melanoma, non-small cell lung cancer (PD-L1+), head and neck squamous cell carcinoma, and solid tumors (MSI-H)); pertuzumab (Perjeta), which targets HER2 (ERBB2 / neu), (approved for use in breast cancer (HER2+)); ponatinib (Iclusig), which targets ABL, FGFR1-3, FLT3, and VEGFR2 (approved for use in chronic myeloid leukemia and acute lymphoblastic leukemia); Ramucirumab (Cyramza) targets VEGFR2 (approved for use in colorectal cancer, gastric cancer or gastroesophageal junction (GEJ) adenocarcinoma, and non-small cell lung cancer); regorafenib (Stivarga) targets KIT, PDGFRβ, RAF, RET, and VEGFR1 / 2 / 3 (approved for use in colorectal cancer, gastrointestinal stromal tumors, and hepatocellular carcinoma); ribociclib (Kisqali) targets CDK4 and CDK6 (approved for use in breast cancer (HR+, HER2-)); rituximab (Rituxan, Mabthera) targets CD20 (approved for use in non-Hodgkin's lymphoma, chronic lymphocytic leukemia, rheumatoid arthritis, and granulomatosis with polyangiitis); and rituximab / hyaluronidase human (Rituxan) targets CD20. Hycela) (approved for use in chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and follicular lymphoma); romidepsin (Istodax) which targets HDAC (approved for use in cutaneous T-cell lymphoma and peripheral T-cell lymphoma); rucaparib (Rubraca) which targets PARP (approved for use in ovarian cancer); ruxolitinib (Jakafi) which targets JAK1 / 2 (approved for use in myelofibrosis); siltuximab (Sylvant) which targets IL-6 (approved for use in multicentric Castleman disease); sipuleucel T (Provenge) which targets (approved for use in prostate cancer);Sonidegib (Odomzo), which targets smoothened cytokinin (approved for basal cell carcinoma); sorafenib (Nexavar), which targets VEGFR, PDGFR, KIT, and RAF (approved for hepatocellular carcinoma, renal cell carcinoma, and thyroid cancer); temsirolimus (Torisel), which targets mTOR (approved for renal cell carcinoma); tositumomab (Bexxar), which targets CD20 (approved for non-Hodgkin lymphoma); trametinib (Mekinist), which targets MEK (approved for melanoma and non-small cell lung cancer); trastuzumab (Herceptin), which targets HER2 (ERBB2 / neu), (approved for breast cancer (HER2+) and gastric cancer (HER2+)); Targeted cancer therapies include, but are not limited to, vandetanib (Caprelsa), which targets EGFR (HER1 / ERBB1), RET, and VEGFR2 (approved for medullary thyroid cancer); vemurafenib (Zelboraf), which targets BRAF (approved for melanoma); venetoclax (Venclexta), which targets BCL2 (approved for chronic lymphocytic leukemia); vismodegib (Erivedge), which targets PTCH and smoothened (approved for basal cell carcinoma); vorinostat (Zolinza), which targets HDAC (approved for cutaneous T-cell lymphoma); and Ziv-aflibercept (Zaltrap), which targets PIGF and VEGFA / B (approved for colorectal cancer).

[0061] Suitable biological response modifiers for use in connection with the methods of the present disclosure include: (1) tyrosine (1) inhibitors of rhesus mast cell kinase (RTK) activity, (2) inhibitors of serine / threonine kinase activity, (3) tumor-associated antigen antagonists, e.g., antibodies that specifically bind to tumor antigens, (4) apoptosis receptor agonists, (5) interleukin-2, (6) interferon-α, (7) interferon-γ, (8) colony-stimulating factors, (9) angiogenesis inhibitors, and (10) tumor necrosis factor antagonists.

[0062] Chemotherapeutic agents are non-peptide (i.e., non-proteinaceous) compounds that reduce the proliferation of cancer cells, and include cytotoxic agents and cytostatic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones.

[0063] Drugs that act to reduce cell proliferation are known in the art and are widely used. Such drugs include mechlorethamine, cyclophosphamide (Cytoxan (trade name)), and cyclophosphamide (Cytoxan (trade name)). alkylating agents such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkylsulfonates, and triazenes, including, but not limited to, melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.

[0064] Anti-metabolites include cytarabine (CYTOSAR-U), cytosine arabinoside, and fluorouracil. These include folate analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, 5-fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-diazaphorene 62 (PDDF, CB3717), 5,8-diazatrahydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatin, and gemcitabine.

[0065] Suitable natural products and derivatives thereof (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins) include Araa-C, paclitaxel (Taxol®), docepaclitaxel (Taxotere®), deoxycoformycin, mitomycin-C, L-asparagine, azathioprine; brequinar; alkaloids such as vincristine, vincristine, vincristine, vincristine, vincristine, vincristine, vincristine, vincristine, vincristine, and the like; podophyllotoxins such as etoposide, teniposide, and the like; antibiotics such as anthracyclines, daunomycin hydrochloride (daunomycin, daunomycin, cerbidine), idarubicin, doxorubicin, epirubicin, and morpholino derivatives, and the like; These include, but are not limited to, phenoxyzolidinic cyclopeptides such as dactinomycin; basic glycopeptides such as bleomycin; anthraquinone glycosides such as plicamycin (mithramycin); anthraquinones such as mitoxantrone; azirinopyrroloindole diones such as mitomycin; macrocyclic immunosuppressants such as cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, and the like.

[0066] Other antiproliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.

[0067] Microtubule-affecting agents with antiproliferative activity are also suitable for use, such as epothilone A, epothilone including, but not limited to, benzodiazepine B, discodamolide, allocolchicine (NSC 406042), halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC 361792), trityl cysterin, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones; estramustine, nocodazole, and the like.

[0068] Suitable hormone regulators and steroids (including synthetic analogues) for use include corticosteroids, such as prednisone, dexamethasone, and the like; estrogens and progesterone; progesterones, such as hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; and adrenal cortical suppressants, such as aminoglutethimide; 17α-ethinylestradiol; diethylstyryl These include, but are not limited to, rubestrol, testosterone, fluoxymesterone, dromostanolone propionate, testolactone, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide (Drogenil), troifen (Fareston), and Zoladex.Since estrogen stimulates proliferation and differentiation, compounds that bind to estrogen receptors are used to inhibit this activity.Corticosteroids can inhibit T cell proliferation.

[0069] Other chemotherapeutic agents include metal complexes such as cisplatin (cis-diaminedichloroplatinum), carboplatin, and the like; ureas such as hydroxyurea; and hydrazines such as N-methylhydrazine; epidophyllotoxins; topoisomerase inhibitors; procarbazine; mitoxantrone; leucovorin; tegafur, and the like. Other antiproliferative agents of interest include immunosuppressants such as mycophenolic acid, thalidomide, desoxyspergualin, azasporin, leflunomide, mizoribine, azaspirane (SKF 105685), Iressa® (ZD1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline), and the like.

[0070] "Taxane" includes paclitaxel, as well as any active taxane derivative or prodrug. "Paclitaxel" (as used herein, e.g., docetaxel, TAXOL (trademark), TAXOTERE (formulation of docetaxel), 10-desacetylated paclitaxel analogs, and the 3'N-desbenzoyl-3'Nt-butoxycarbonyl analog of paclitaxel and the like) can be readily prepared utilizing techniques known to those skilled in the art (see also WO 94 / 07882, WO 94 / 07881, WO 94 / 07880, WO 94 / 07876, WO 93 / 23555, WO 93 / 10076; U.S. Patent Nos. 5,294,637, 5,283,253, 5,279,949, 5,274,137, 5,202,448, 5,200,534, 5,229,529, and EP 590,267), or can be purchased from, for example, Sigma Chemical Co. (St. Louis, Missouri) (T7402 from Taxus brevifolia, or Taxus It is obtained from a variety of commercial sources, including T-1912 from T. yannanensis.

[0071] Paclitaxel should be understood to refer not only to the common chemically available forms of paclitaxel, but also to analogs and derivatives (e.g., Taxotere™ docetaxel, discussed above) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

[0072] The term "taxane" also includes a variety of known derivatives, including both hydrophilic and hydrophobic derivatives. Taxane derivatives include those described in International Patent Application No. WO 99 / 18113. Galactose and mannose derivatives as described; piperazines as described in WO 99 / 14209 and other derivatives; as described in WO 99 / 09021, WO 98 / 22451, and U.S. Pat. No. 5,869,680. 6-thio derivatives described in WO 98 / 28288; sulfenamide derivatives described in U.S. Pat. No. 5,821,263; and taxane derivatives described in U.S. Pat. No. 5,415,869. Additionally, the present invention includes, but is not limited to, those described in WO 98 / 58927, WO 98 / 13059, and U.S. Pat. No. 5,824,701. Prodrugs of paclitaxel include, but are not limited to, prodrugs of paclitaxel.

[0073] In some cases, the method for treating a cancer subject may further comprise administering an agent that enhances the activity of the treatment. Such agents that enhance the activity of the treatment vary widely and may include, but are not limited to, agents that inhibit inhibitor molecules. Suitable inhibitor molecules that can be targeted include, but are not limited to, PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta.

[0074] Inhibition of inhibitory molecules can be achieved by any convenient method, including, but not limited to, administering a direct inhibitor of the inhibitory molecule (e.g., an antibody that binds to the inhibitory molecule, a small molecule antagonist of the inhibitory molecule, etc.), administering an agent that inhibits expression of the inhibitory molecule (e.g., an inhibitory nucleic acid, e.g., dsRNA, e.g., an siRNA or shRNA that targets a nucleic acid encoding the inhibitory molecule), an indirect inhibitor of inhibitory signaling, etc. In some cases, the agent that can be administered may be an antibody or antibody fragment that binds to the inhibitory molecule. For example, the agent may be an antibody or antibody fragment that binds to PD1, PD-L1, PD-L2, or CTLA4 (e.g., ipilimumab (also referred to as MDX-010 and MDX-101, commercially available as Yervoy (Bristol-Myers Sukibb)), tremelimumab (Pfizer, formerly known as ticilimumab, CP-675,206), TIM3, LAG3, etc.).

[0075] In some embodiments, the method comprises administering to the subject an immune checkpoint inhibitor, such as an anti-CTLA4 or anti-PD-1 and anti-PD-1L agent. The immune system relies on multiple checkpoints to avoid excessive activation of the immune system on healthy cells, and tumor cells often exploit these checkpoints to evade detection by the immune system. CTLA-4, which has been shown to be abnormally upregulated in certain cancers and present on the surface of T cells, and PD-1, which has also been found to be upregulated in certain tumors and inhibit T cell function, are checkpoints being investigated as targets for cancer therapy (Pardoll, DM2012 Nat Rev Cancer 12 (4): 252-264; Sharma, et al.2011 Nat Rev Cancer 11 (11): 805-812).

[0076] In some cases, the methods of the present disclosure may be used without any additional conventional therapy, including, for example, when the methods described herein are the only methods used to treat a subject. For example, in the case of oncology, the methods described herein may in some cases be the only methods used to treat a subject's cancer.

[0077] For example, combination therapy involving the administration of one or more agents that ameliorate one or more side effects of the therapies described herein or that enhance the therapies described herein. It is indicated that determining the timing and details of administering such combination therapy are within the skill of the relevant physician. In some cases, the dosing regimen and treatment schedule of the combination therapy may be determined through clinical trials.

[0078] In some cases, the subject may, in a particular context, undergo one or more of the following diagnostic procedures: It may be evaluated through: 3D CT angiography, angiography, angioscopy, autofluorescence bronchoscopy Examination / fluorescence bronchoscopy, barium swallow or enema, biopsy, bone marrow aspiration and biopsy, bone scrub Scan, bronchoscopy, CA-125 test, CAD for mammography, CTC test, chest X-ray, colonoscopy These include endoscopy, complete blood count, computed tomography, CT-guided biopsy, DEXA scan, breast digital tomosynthesis, electrocardiogram, endobronchial ultrasound, endoscopic ultrasound, ERCP, flow cytometry, full-field digital mammography, genetic testing, large-bore CT scanner / RT with simulation, lumbar puncture, magnetic resonance imaging, mammography, Miraluma breast imaging, MRI-guided breast biopsy, multi-detector CT scanner, multi-gated acquisition (MUGA) scan, navigation bronchoscopy, nuclear medicine imaging, Oncotype DX test, Pap test, pelvic exam, PET scan, PET-CT scan, radiofrequency ablation, sentinel lymph node biopsy, spiral CT, tumor marker testing, tumor molecular profiling, ultrasound, video capsule endoscopy, and X-ray.

[0079] Diagnostic procedures include, for example, screening for cancer or precancerous conditions before a person has symptoms of the disease; aiding in the diagnosis of cancer; providing information about the stage of cancer; providing information about the grade of a tumor; providing information about the size and / or extent of a primary tumor; providing information about whether the tumor has spread; planning treatment; monitoring the patient's overall health during treatment; and checking for potential side effects of treatment. The tumor may be examined for a variety of reasons, including, but not limited to, to check the tumor; to determine if the cancer is responding to treatment; to find out if the cancer has recurred; etc.

[0080] activator Active agents for binding to the activity-modulating domain of CD206, also referred to herein as CD206-binding agents, can include any convenient compound. According to certain embodiments disclosed herein, the active agent can be an immunomodulatory peptide, a small molecule active agent, or a specific binding member.

[0081] Immunomodulatory peptides In certain embodiments of the present disclosure, the CD206-binding agent is an immunomodulatory peptide. The terms "immunomodulatory" and "immunomodulatory" are used interchangeably herein. In some cases, an immunomodulatory peptide described herein may be referred to as an anti-inflammatory peptide, and vice versa. In certain cases, an immunomodulatory peptide (e.g., as described herein) is an anti-inflammatory peptide, e.g., the peptide has at least one anti-inflammatory effect. It has disease properties.

[0082] Certain embodiments of immunomodulatory polypeptides of interest that may be applied to or adapted for use with the peptides of the present disclosure are described by Jaynes et al. in WO2016 / 061133, the disclosure of which is incorporated herein by reference in its entirety.

[0083] The terms "peptide" and "polypeptide" are used interchangeably herein to refer to polymers constructed from amino acid residues. The term "amino acid residue," as used herein, refers to any natural amino acid, unnatural amino acid, or amino acid mimetic (such as a peptoid monomer). The amino acid residue may be in the L- or D-form.

[0084] The present disclosure includes immunomodulatory peptides having a striatal region that is at least 25% of the length of the polypeptide and that comprises at least one immunomodulatory property. The term "striatal region" refers to a region or portion of a peptide sequence that consists of a sequence of alternating hydrophobic and hydrophilic modules. A "hydrophobic module" is a peptide sequence that consists of 1 to 5 (e.g., 1 to 3 or 1 to 2) hydrophobic amino acid residues, e.g., 1, 2, 3, 4, or 5 hydrophobic amino acid residues. A "hydrophilic module" is a peptide sequence that consists of 1 to 5 (e.g., 1 to 3 or 1 to 2) hydrophilic amino acid residues, e.g., 1, 2, 3, 4, or 5 hydrophilic amino acid residues.

[0085] Therefore, the striatal region is 1-5 Y 1-5 ) n or (J 1-5 J 1-5 ) nwhere each X represents a hydrophilic amino acid residue, each J represents a hydrophobic amino acid residue, and each n is an integer from 1 to 10, e.g., 2 to 10, 2 to 8, 3 to 8, 4 to 8, or 5 to 10. As described in more detail below, aspects of the present disclosure include immunomodulatory peptides having striatal regions with a particular degree of cationic charge. The immunomodulatory peptides of the present disclosure can include striatal regions with a cationic surface. In certain embodiments, the striatal region has a cationic charge (i.e., a charge >0, e.g., +1, +2, +3, +4, +5, +6, or more). In certain embodiments, the immunomodulatory peptide includes a tail region (e.g., a hydrophobic tail sequence). In certain embodiments, the immunomodulatory peptide includes two or more striatal regions. In such embodiments, the two amphipathic regions of the peptide are in the form of a dimer, and the two amphipathic regions can have the same or different amino acid sequences (i.e., a homodimer or a heterodimer). In certain embodiments, two or more (or more ) The striatal regions are connected via a linker or linking region. The linker can be a contiguous (or in-line) amino acid sequence or a non-amino acid moiety, as desired.

[0086] Hydrophobic amino acid residues are characterized by side chain groups with predominantly non-polar chemical or physical properties in, for example, physiological conditions where the peptide is used. Such hydrophobic amino acid residues can be natural or unnatural. Hydrophobic amino acid residues can be mimics of natural amino acids characterized by side chain groups with predominantly non-polar chemical or physical properties. Conversely, hydrophilic amino acid residues are characterized by side chain groups that are predominantly polar (e.g., charged or neutrally hydrophilic) in, for example, physiological conditions where the peptide is used. Such hydrophilic amino acid residues can be natural or unnatural. Hydrophilic amino acid residues can be mimics of natural amino acids characterized by side chain groups that are predominantly hydrophilic (charged or neutrally polar). Examples of hydrophilic and hydrophobic amino acid residues are shown in Table 1 below. Suitable unnatural amino acid residues and amino acid mimetics are known in the art. See, e.g., Liang et al. (2013), “An Index for Characterization of Natural and Non-Natural Amino Acids for Peptidomimetics,” PLoS ONE 8(7):e67844.

[0087] Although most amino acid residues can be considered either hydrophobic or hydrophilic, some can behave as either hydrophobic or hydrophilic depending on their context. For example, due to their relatively weak non-polar character, glycine, proline, serine and / or cysteine ​​can function as hydrophilic amino acid residues. Conversely, Due to their bulky, slightly hydrophobic side chains, histidine and arginine can function as hydrophobic amino acid residues.

[0088] [Table 1]

[0089] In some cases, the immunomodulatory peptide is 5 to 18 amino acid residues in length; They contain striatal regions of alternating hydrophilic and hydrophobic modules that adopt amphipathic conformations under physiological conditions. In these cases, the striatal region contains three or more hydrophobic modules. In some embodiments, the immunomodulatory peptide may comprise a hydroxyl group, a hydroxyl group, and two or more hydrophilic modules, each comprising at least one cationic residue. In some embodiments, the immunomodulatory peptide may comprise one of the following formulas: This includes an array defined as [J 1a J 1b ]-[X 1a X 1b ]-[J 2a J 2b ]-[X 2a X 2b ]-[J 3a ]-[J 3a ]; and [X 3a ]-[Y 3a ]-[X 2b X 2a ]-[J 2b J 2a ]-[X 1b X 1a ]-[J 1b J 1a ];

[0090] Here, J 1a , J 1b , J 2a , J 2b and J. 3a are each independently selected from phenylalanine, tryptophan, alanine, valine, and glycine; 1a , X 1b , X 2a , X 2b and X 3a are independently lysine, arginine, histidine, aspartic acid, and glutamic acid. In some instances, the amino acid is selected from the group consisting of cis- and trans-amino acids, ... 1a , J 1b , J 2a , J 2b and J. 3a are phenylalanine, and X 1a , X1b , X 2a , X 2b and X 3a are each independently selected from lysine and arginine.

[0091] In certain embodiments, the immunomodulatory peptide comprises a sequence selected from: The sequences defined in a) are: KFRKAFKRFF(RP182); FFRKFAKRFK(RP183); FFKKFFKKFK(RP185); FFKKFFKKFK(RP186); and FFKKFFKKFK(RP233); or b) a sequence having one or two amino acid substitutions relative to the sequences defined in a). In certain embodiments, the immunomodulatory peptide comprises the amino acid sequence KFRKAFKRFF(RP182). In certain instances, the immunomodulatory peptide comprises the amino acid sequence FFRKFAKRFK(RP183). In certain instances, the immunomodulatory peptide comprises the amino acid sequence FFKKFFKKFK(RP185).

[0092] In other embodiments, the immunomodulatory peptide comprises the following sequence: a) selected from: Sequences: RWKFGGFKWR(RP832C); FKWRGGRWKF(RP837C); FWKRGGRKWF(RP837A); FWKRFV(RP837N); FVRKWR(RP837C1); FAOOFAOOFO(RP850); FWKRFVRKWR(RP837); FWKKFVKKWK(RP841); WWHHWWHHWH(RP847); WWRHWWHRWR(RP848); WWKHWWHKWK(RP849); GDRGIKGHRGF(RP842); LYKKIIKKLL(RP846); FYPDFFKKFF(RP844); FFRKSKEKIG(RP853); FFRHFATHLD(RP845); and EKLSAFRNFF(RP843); or b) sequences having one or two amino acid substitutions relative to the sequences defined in a). In certain embodiments, the immunomodulatory peptide comprises an amino acid sequence selected from the following: RWKFGGFKWR (RP832C), FKWRGGRWKF (RP837C), and FWKRGGRKWF (RP837A). In certain cases, the immunomodulatory peptide comprises an amino acid sequence selected from FWKRFV (RP837N) and FVRKWR (RP837C1). In certain cases, the immunomodulatory peptide comprises an amino acid sequence selected from FAOOFAOOFO (RP850), FWKRFVRKWR (RP837), and FWKKFVKKWK (RP841). In certain cases, the immunomodulatory peptide comprises an amino acid sequence selected from WWHHWWHHWH, WWRHWWHRWR, and WWWHWWHKWK (RP847-849).

[0093] In certain embodiments, the immunomodulatory peptide comprises the amino acid sequence LYKKIIKKLL (RP846). In certain instances, the immunomodulatory peptide comprises the amino acid sequence FYPDFFKKFF (RP844). In certain instances, the immunomodulatory peptide comprises the amino acid sequence FFRKSKEKIG(RP853). In certain instances, the immunomodulatory peptide comprises the amino acid sequence FFRHFATHLD(RP845) In certain instances, the immunomodulatory peptide comprises the amino acid sequence FFRKSKEKIG(RP853) Includes.

[0094] In some embodiments, the immunomodulatory peptide has a sequence defined by one of the following formulas: Contains columns. [X 1a ]-[J 2a ]-[X 2a ]-[J 2a ]-[X 3a ]-[J 3a ] [J 3a ]-[X 3a ]-[J 2a ]-[X 2a ]-[J 1a ]-[X 1a ] [J 1a J 1b ]-[X 1a X 1b ]-[J 2a J 2b ]; [J 1a J 1b ]-[X 1a X 1b ]-[J 2a ]-[X 2a ]; [X 3a ]-[J 3a ]-[X 2b X 2a ]-[J 2b J 2a ]; [J 1a J 1b ]-[X 1a ]-[J 2a J 2b ]-[X 2a ]; and [X 1a ]-[J 1a J 1b ]-[X 2a ]-[J 2a J 2b ];

[0095] Here, J 1a , J1b , J 2a , J 2b and J. 3a are independently phenylalanine, tryptophan, alanine, valine, leucine, isoleucine, methionine, tyrosine, and threonine. is selected from cysteine, serine, cysteine, proline, and glycine; 1a , X 1b , X 2a , X 2b and X 3a are independently lysine, arginine, histidine, and asparagine. In certain embodiments, the immunomodulatory peptide comprises the following sequence: a) Sequence selected from: AFKRFF(182-FN6); FFKKFF(185-FN6); FWKRFV(837-FN6); WVRRVV(WLUB-F1-N6); IFKKIE(CEC-F1-N6)FLRNLV(LL37F-3-N6); FLHSAK(MAG-F1-N6); FFHHIF(PISC-F-N6); FFKKAA(PLEU-F-N6); ALKKVF(PSEU-F-N6); LYKKII(CXCL4-F-N6); LFRRAF(IL24-FN6); FLKRLL(IL7-FN6); FFRRFA(ABCP-FN6); FFRHFA(E1P-FN6); AIRRIP(gP120-FN6); AFHRFF(GP2B-FN6); FFNRFA(MCPH-FN6); AFKRFF(SPRA-FN6); AFKRFF(TPRO-FN6); IVRRAD(COL18-FN6); FWRWFK(HX5 / CPAP); KFWRWF(HX6 / YJPA); WFRFWK(HX7 / CLPB)KWFRFW(HX8 / ATG1); AFHHFF(HEX16F / STPK); FFRNFA(HEXF13 / SIF1); AFHRFF(HEX9F / THIF); FFRQFA(HEXF1 / ATPB); AFNRFF(HEX2F / AATF); WIQRMM(CXCL13-FN6); WVQRVV(CXCL8-FN6); AFRNFF(HEX3F / FBNA); and TLRRFM(HEX18 / HSHK); or b ) a) a sequence having one or two amino acid substitutions relative to the sequence defined in a). In other embodiments, the immunomodulatory peptide comprises the following sequence: a) a sequence selected from: DVRMRL (MCMV-FN6); and RRAELG (TONB-FN6); or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a). In some other embodiments, the immunomodulatory peptide comprises the following sequence: a) a sequence selected from: DVRMRL (MCMV-FN6); and RRAELG (TONB-FN6); or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a).a) a sequence selected from: FWRWFA(HX1 / MMPL); AFWRWF(HX2 / ABCT); WFRFWA(HX3 / GTRF); AWFRFW(HX4 / AXES); VAVRIW(HX9 / IDRF / AMIA); FFRFFA(HEXF2 / AMT1); and AFFRFF(HEX13F / TGME); or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a). In other embodiments, the immunomodulatory peptide comprises the following sequence: a) a sequence selected from: FFKKFF; WWKKFF; FWKKWF; FFKKWW; WWKKWW; YYKKYY; IIKKYY; YIKKIY; YYKKII; IIKKII; MMKKMM; LLKKMM; MLKKLM; MMKKLL; LLKKLL; VVKKVV; AAKKVV; VAKKAV; VVKKAA; AAKKAA; GGKKGG; TTKKGG; GTKKTG; GGKKTT; TTKKTT; SSKKSS; CCKKSS; SCKKCS; SSKKCC; and CCKKCC; or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a). In certain other cases, the immunomodulatory peptide comprises the following sequences: a) a sequence selected from: FKFKFK; WKWKWK; YKYKYK: IKIKIK; MKMKMK; LKLKLK; VKVKVK; AKAKAK; GKGKGK; TKTKTK; SKSKSK; CKCKCK; KFKFKF; KWKWKW; KYKYKY; KIKIKI; KMKMKM; KLKLKL; KVKVKV; KAKAKA; KGKGKG; KTKTKT; KSKSKS; and KCKCKC; or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a).

[0096] In certain embodiments, the immunomodulatory peptide comprises a peptide sequence set forth in Tables 2A-2C. include.

[0097] [Table 2-1]

[0098] Table 2-2

[0099] Table 2-3

[0100] Table 2-4

[0101] Table 2-5

[0102] Table 2-6

[0103] Table 2-7

[0104] Table 2-8

[0105] Table 2-9

[0106] Table 2-10

[0107] Table 2-11

[0108] In some embodiments, the immunomodulatory peptide has a sequence defined by one of the following formulas: Contains columns. [J 1a ]-[X 2a ]-[J 2a ]-[X 3a ]-[J 3a ] [X 1a ]-[J 1a ]-[X 2a ]-[J 2a ]-[X 3a ] [X 1a ]-[J 1a ]-[X 2a ]-[J 2a J 2b ]; [J 1a J 1b ]-[X 1a ]-[J 2a ]-[X 2a ]; [X 1a ]-[J 1a J 1b ]-[X 2a ]-[J 2a ]; [J 1a ]-[X 1a ]-[J 2a J 2b ]-[X 2a ]; and [J 1a J 1b ]-[X 2a ]-[J 2a J 2b ];

[0109] Here, J 1a , J 1b , J 2a , J 2b and J. 3a are each independently selected from phenylalanine, tryptophan, alanine, valine, leucine, isoleucine, methionine, tyrosine, threonine, serine, cysteine, proline, and glycine; 1a , X 1b , X 2a , X 2b and X 3a are independently lysine, arginine, histidine, and asparagine. In certain embodiments, the immunomodulatory peptide comprises the following sequences: a) a sequence selected from: AFKRF; FF KKF; FWKRF; WVRRV; IFKKI; FLRNL; FLHSA; FFHHI; FFKKA; ALKKV; LYKKI; LFRRA; FLKRL; WIQRM; WVQRV; AFRNF; TLRRF; FKRFF; FKKFF; WKRFV; VRRVV; FKKIE; LRNLV; LHSAK; FHHIF; FRQFA; FNRFF; IQRMM; VQRVV; FRNFF; LRRFM; DVRMR; VRMRL; RRAEL; RAELG; and RWKFG; or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a). In other embodiments, the immunomodulatory peptide comprises the following sequences: a) a sequence selected from: AFWRW; AWFRF; VAVRI; FFRFF; AFFRF; WRWFA; FRFWA; AVRIW; and FRFFA; or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a). In other embodiments, the immunomodulatory peptide comprises the following sequences: a) a sequence selected from: AFWRW; AWFRF; VAVRI; FFRFF; AFFRF; WRWFA; FRFWA; AVRIW; and FRFFA;a) sequences selected from: FFKKF; WWKKF; FWKKW; FFKKW; WWKKW; YYKKY; IIKKY; YIKKI; YYKKI; IIKKI; MMKKM; LLKKM; MLKKL; MMKKL; LLKKL; VVKKV; AAKKV; VAKKA; VVKKA; AAKKA; GGKKG; TTKKG; GTKKT; GGKKT; TTKKT; SSKKS; CCKKS; SCKKC; SSKKC; and CCKKC; FKKFF; WKKFF; WKKWF; FKKWW; WKKWW; YKKYY; IKKYY; IKKIY; YKKII; IKKII; MKKMM; LKKMM; LKKLM; MKKLL; LKKLL; VKKVV; AKKVV; AKKAV; VKKAA; AKKAA; GKKGG; TKKGG; TKKTG; GKKTT; TKKTT; SKKSS; CKKSS; CKKCS; SKKCC; and CKKCC; or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a). In certain other cases, the immunomodulatory peptide comprises the following sequence: a) a sequence selected from: FKFKF; WKWKW; YKYKY: IKIKI; MKMKM; LKLKL; VKVKV; AKAKA; GKGKG; TKTKT; SKSKS; CKCKC; KFKFK; KWKWK; KYKYK; KIKIK; KMKMK; KLKLK; KVKVK; KAKAK; KGKGK; KTKTK; KSKSK; and KCKCK; or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a).

[0110] In certain embodiments, the immunomodulatory peptide comprises a peptide sequence set forth in Table 2 truncated by one amino acid at the N-terminus. , truncated by one amino acid at the C-terminus, and comprising the peptide sequence set forth in Table 2.

[0111] In some embodiments, the immunomodulatory peptide has a sequence defined by one of the following formulas: Contains columns. [J 1a ]-[X 1a ]-[J 2a ]-[X 2a ] [X 1a ]-[J 1a ]-[X 2a ]-[J 2a ] [X 1a X 2a ]-[J 2a J 2b ]; and [J 1a J 1b ]-[X 1a X 2a ];

[0112] Here, J 1a , J 1b , J 2a , and J. 2b are independently phenylalanine, triphosphatase X is selected from threonine, alanine, valine, leucine, isoleucine, methionine, tyrosine, threonine, serine, cysteine, proline, and glycine; 1a , X 1b , X 2a , and X 2b are independently lysine, arginine, histidine, aspartic acid, and glutamic acid. In certain embodiments, the immunomodulatory peptide comprises the sequence: a) a sequence selected from the following: AFKR; FFKK; FWKR; WVRR; IFKK; FLRN; FLHS; FFHH; ALKK; LYKK; LFRR; FLKR; FFRR; FFRH; AIRR; AFHR; FFNR; IVRR; FWRW; KFWR; WFRF; KWFR; AFHH; FFRN; FFRQ; AFNR; WIQR; WVQR; AFRN; TLRR; KRFF; KKFF; KRFV; RRVV; KKIE; RNLV; HSAK; HHIF; KKAA; KKVF; KKII; RRAF; KRLL; RRFA; RHFA; RRIP; HRFF; NRFA; RRAD; RWFK; RFWK; HHFF; RNFA; RQFA; NRFF; QRMM; QRVV; RNFF; RRFM; VRMR; RMRL; RAEL; AELG; and WKFG; or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a). In other embodiments, the immunomodulatory peptide comprises the following sequences: a) a sequence selected from: FWRW; AFWR; WFRF; AWFR; VAVR; FFRF; AFFR; RWFA; WRWF; RFWA; FRFW; VRIW; RFFA; and FRFF; or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a). In other embodiments, the immunomodulatory peptide comprises the following sequences: a) a sequence selected from: FWRW; AFWR; WFRF; AWFR; VAVR; FFRF; AFFR; RWFA; WRWF; RFWA; FRFW; VRIW; RFFA; and FRFF; a) a sequence selected from: FFKK; WWKK; FWKK; YYKK; IIKK; YIKK; MMKK; LLKK; MLKK; VVKK; AAKK; VAKK; GGKK; TTKK; GTKK; SSKK; CCKK; SCKK; KKFF; KKWF; KKWW; KKYY; KKIY; KKII; KKMM; KKLM; KKLL; KKVV; KKAV; KKAA; KKGG; KKTG; KKTT; KKSS; KKCS; and KKCC; or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a).In certain other cases, the immunomodulatory peptide comprises a) a sequence selected from: FKFK; WKWK; YKYK; IKIK; MKMK; LKLK; VKVK; AKAK; GKGK; TKTK; SKSK; CKCK; KFKF; KWKW; KYKY; KIKI; KMKM; KLKL; KVKV; KAKA; KGKG; KTKT; KSKS; and KCKC; or b) a sequence having one or two amino acid substitutions relative to a sequence defined in a).

[0113] In certain embodiments, the immunomodulatory peptide comprises a peptide sequence set forth in Table 2 truncated at the N-terminus by two amino acids. , truncated at the C-terminus by two amino acids, and the peptide sequence set forth in Table 2. The exemplary immunomodulatory peptide sequences described herein are merely examples and are not the only immunomodulatory polypeptides provided herein. Indeed, fragments and variants of the disclosed peptide sequences are also within the scope of the present disclosure.

[0114] The present disclosure provides immunomodulatory polypeptides (optionally The present disclosure provides immunomodulatory polypeptides, or variants thereof, or fragments thereof, that share minimal homology with any of the exemplary RP peptides disclosed herein. Thus, a peptide or polypeptide of the present disclosure is an immunomodulatory peptide that satisfies one of the formulas set forth herein or shares minimal homology with any of the exemplary RP peptides disclosed herein.

[0115] A "fragment" of the present invention is a fragment of at least 4, 5, 6, 7, 8, 9 or 10 of a peptide disclosed herein. , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive amino acid residues (or up to one less than the number of amino acid residues in the target peptide), The fragments of the present invention retain at least one immunomodulatory property of the peptide. One or two amino acids from the N-terminus and / or C-terminus of the parent immunomodulatory peptide disclosed herein , including peptides lacking three, four, or more amino acids.

[0116] A "variant" of the present invention is a polypeptide that is substantially similar to a polypeptide disclosed herein and retains at least one immunomodulatory property of the subject polypeptide. The compound may comprise one or more amino acids at the N-terminus or C-terminus of a subject polypeptide disclosed herein. Deletion (i.e., truncation) of amino acid residues in one of the subject polypeptides disclosed herein Deletion and / or addition of one or more amino acid residues at the above internal sites, and / or or may include substitution of one or more amino acid residues (e.g., one, two, three, or even more) at one or more positions of the subject polypeptides disclosed herein. For a subject polypeptide of 10 or fewer amino acid residues, variant polypeptides may be located internally, at the N-terminus, and / or at the C-terminus. Regardless, it may contain three or fewer (eg, three, two, one, or none) deleted amino acid residues.

[0117] Thus, the present invention further provides immunomodulatory polypeptides at least 50% identical (i.e., at least 50% sequence identity) to any one of the immunomodulatory polypeptides disclosed herein (e.g., Table 2). For example, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or more) and still retain at least one immunomodulatory property. Sequence identity is based on a comparison of two peptide sequences or fragments thereof of the same or similar length.

[0118] Thus, in certain embodiments, the present disclosure provides a method for the preparation of a polypeptide as disclosed herein. 1 to 10 amino acid differences (e.g., 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 amino acid difference) in any one of and still retain at least one immunomodulatory property. As used herein, "amino acid difference" refers to an amino acid substitution, an amino acid insertion, or the like. amino acid insertions, terminal amino acid additions, amino acid deletions, terminal amino acid truncations, or any combination thereof.

[0119] In some embodiments, any of the peptides disclosed herein may be shorter than one or two amino acids at the N-terminus. Any of the peptides may be shorter than one or two amino acids at the C-terminus. In some cases, any of the peptides disclosed in Table 2 may be shorter than one or two amino acids at the N-terminus. In some other cases, any of the peptides disclosed in Table 2 may be shorter than one or two amino acids at the C-terminus.

[0120] In some embodiments, the peptides disclosed herein can include deletions, additions, and / or substitutions of amino acid residues, as described herein. The amino acid residue may be unrelated to the amino acid residue it replaces (e.g., unrelated in terms of hydrophobicity / hydrophilicity, size, charge, polarity, etc.), or the replacing amino acid residue may constitute a similar, conservative, or highly conservative amino acid substitution. As used herein, "similar," "conservative," and "highly conservative" amino acid substitutions are defined as shown in Table 3 below. The determination of whether an amino acid residue substitution is similar, conservative, or highly conservative is based only on the side chains of the amino acid residues, and not on the peptide backbone, which may be modified to increase the stability of the peptide, as discussed below.

[0121] [Table 3]

[0122] The "length" of a subject peptide or polypeptide is the number of amino acid residues connected end-to-end that comprise the peptide or polypeptide, excluding any non-peptide linkers and / or modifications that the peptide or polypeptide may contain. , peptides may be 5 to 30 amino acid residues (e.g., 5 to 25, 10 to 20, or 5 to 18, 5 to 12, or 5 to 10, or 6 to 30, 6 to 25, 6 to 20, 6 to 18 In some embodiments, the peptide is 4 to 12 amino acid residues (e.g., 4, 5, 6, 7, 8, 9, or 10 amino acid residues) in length and comprises a striatal region of alternating hydrophilic and hydrophobic modules that adopt an amphipathic conformation under physiological conditions (e.g., as described herein). In some embodiments, the peptide is 4 to 12 amino acid residues (e.g., 4, 5, 6, 7, 8, 9, or 10 amino acid residues). In certain instances, the striatal region of the peptide is 5 to 18 amino acid residues in length (e.g., 6 to 18, 6 to 10 amino acid residues) and comprises a striatal region of alternating hydrophilic and hydrophobic modules that adopts an amphipathic conformation under physiological conditions. 1 to 14, 6 to 12, 7 to 12, or 5, 6, 7, 8, 9, 10, 11, or 12 amino acids in length ), where the peptide is optionally further modified (e.g., as described herein). The striatal region can include two or more (e.g., three or more or four or more) of the following: and one or more (e.g., two or more, three or more, or four or more) hydrophilic modules (e.g., each containing at least one cationic residue). In examples, the striatal region of the peptide has a length of 4 to 10 amino acid residues, such as 4 to 6. In some cases, the striatal region of the peptide has a length of 2 to 3 amino acid residues.

[0123] The hydrophobic module can comprise any convenient residue. In certain instances, the hydrophobic module comprises phenylalanine, tryptophan, alanine, valine, and glycolipid. The striatal region can comprise a total of one, two, or more cationic amino acid residues, for example, three or more, four or more, five or more, six or more, or even more. The immunomodulatory peptide can comprise two, three, or more hydrophilic modules of any convenient residues. In some cases, the hydrophilic module comprises an amino acid residue selected from lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, and glutamine.

[0124] In the formulas described herein, J(N) is used to refer to a particular hydrophobic module, where N indicates its position within the linear equation. Similarly, X(N) is used to refer to a particular hydrophilic module, where N indicates its position within the linear equation.

[0125] In the formulas described herein, J (nx) is used to refer to specific hydrophobic amino acid residues where n indicates which module the residue is located in and x indicates the position within that module. (nx) is used to refer to a particular hydrophilic amino acid residue, n indicates which module the residue is located in, and x indicates the position within that module. .

[0126] low molecule In certain embodiments of the present disclosure, the CD206-binding agent is a small molecule. Molecules include, but are not limited to, small organic or inorganic compounds having a molecular weight (MW) of more than 50 and less than about 2,500 Daltons (Da), e.g., more than 50 and less than about 1,000 Da, or more than 50 and less than about 500 Da. "Small molecules" encompass numerous biological and chemical classes, including synthetic, semi-synthetic, or naturally occurring inorganic or organic molecules, including synthetic, recombinant, or naturally occurring nucleic acids. Small molecules of interest can contain functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and can include at least amine, carbonyl, hydroxyl, or carboxyl groups, and can contain at least two of the functional chemical groups. Small molecules can include cyclic carbon or heterocyclic structures substituted with one or more of the above functional groups, and / or aromatic or polyaromatic structures. Small molecules are also found among biomolecules, including sugars, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.

[0127] Some molecules of interest contain a backbone containing one or more carboxamide functional groups. In some cases, small molecules of interest may have a backbone containing one or more urea functional groups. In some cases, the small molecule of interest may contain one or more carboxamide functional groups. and one or more urea functional groups. In certain instances, small molecules of interest include one or more optionally substituted aryl groups. In certain instances, small molecules of interest include one or more In certain instances, small molecules of interest include the above optionally substituted naphthyl groups. In certain cases, small molecules of interest contain one or more optionally substituted carbazole groups.

[0128] In some embodiments, the small molecule active agent is described by formula (I):

[0129] [ka]

[0130] where: R 1 -R 4 are each independently selected from hydrogen, alkyl, and substituted alkyl; X 1 is alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted aryl selected from ralkyl, heterocycle, substituted heterocycle, heteroaryl, and substituted heteroaryl; X 2 is alkyl, substituted alkyl, aryl, substituted aryl, amino, substituted amino , heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle; X 3 is alkyl, substituted alkyl, aryl, substituted aryl, naphthyl, substituted naphthyl, selected from alkyl, heterocycle, substituted heterocycle, heteroaryl, substituted heteroaryl, aryl heterocycle, substituted aryl heterocycle; n is an integer from 1 to 10, or a pharmaceutically acceptable salt or solvate thereof.

[0131] In certain embodiments of compounds of Formula (I), X 1 is alkyl or substituted alkyl. In certain cases, X 1 is aryl or substituted aryl. In certain cases, X 1is selected from heterocycle, substituted heterocycle, heteroaryl, and substituted heteroaryl. 1 is an optionally substituted carbazole. In certain cases, X 1 is optionally substituted naphthyl. In certain cases, X 1 is a phenol. In some cases, X 1 is phenyl. In certain cases, X 1 is aralkyl or substituted aralkyl. In certain cases, X 1 contains one or more aryl groups In certain cases, aralkyl is an aralkyl that is optionally substituted with one or more C1-C containing phenyl groups 10 Contains alkyl chains. In certain cases, X 1 is at least 2 C1-C containing two optionally substituted phenyl groups 10 In certain cases, the alkyl chain terminates in at least two optionally substituted phenyl groups. In certain cases, the phenyl group is unsubstituted. In certain cases, the phenyl group is substituted with one or more groups selected from hydroxyl, amino, carboxamide, guanidine, acyl, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, substituted heterocycle, heteroaryl, substituted heteroaryl, nitrile, and nitro. In certain cases, X 1 is a C1-C6 alkyl chain substituted with two optionally substituted phenyl groups. In certain cases, the alkyl chain terminates in two optionally substituted phenyl groups.

[0132] In certain embodiments of compounds of Formula (I), X 2 is alkyl or substituted alkyl. In certain cases, X 2 is C1-C6 alkyl. In certain cases, X 2 is methyl In a particular case, X 2is a C1-C6 alkyl group substituted with one or more groups In certain cases, the alkyl group is substituted with one or more groups selected from hydroxyl, amino, carboxamide, guanidine, acyl, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, substituted heterocycle, heteroaryl, substituted heteroaryl, nitrile, and nitro. In certain cases, the alkyl group is substituted with a guanidine group. In certain cases, X 2 is aryl or substituted aryl. In certain cases, X 2 is selected from heterocycle, substituted heterocycle, heteroaryl, and substituted heteroaryl. 2 is an optionally substituted carbazole. In certain cases, X 2 is optionally substituted naphthyl. In certain cases, X 2 is a phenol. In some cases, X 2 is phenyl. In certain cases, X 2 is amino or substituted amino. In certain cases, X 2 is an amino group substituted with one or more aryl groups. In certain cases, the amino group is substituted with one or more optionally substituted phenyl groups. In certain cases, X 2 is an amino group substituted on one or more phenolic groups.

[0133] In certain embodiments of compounds of Formula (I), X 3 is alkyl or substituted alkyl. In certain cases, X 3 is aryl or substituted aryl. In certain cases, X 3 is selected from heterocycle, substituted heterocycle, heteroaryl, and substituted heteroaryl. 3 is an aryl heterocycle or a substituted aryl heterocycle. In certain cases, X 3 is an optionally substituted carbazole. In certain cases, X 3is optionally substituted naphthyl. In certain cases, X 3 is a phenol. In some cases, X 3 is phenyl. In certain cases, X 3 The group is substituted with one or more groups selected from hydroxyl, amino, carboxamide, guanidine, acyl, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, substituted heterocycle, heteroaryl, substituted heteroaryl, nitrile, and nitro. 3 is a carbazole substituted with one or two hydroxyl groups. In certain cases, X 3 is an unsubstituted carbazole. In certain cases, X 3 is naphthyl substituted with one or two hydroxyl groups. In certain cases, X 3 is unsubstituted naphthyl.

[0134] In certain embodiments of compounds of Formula (I), n is less than 10, e.g., 9 or less, 8 or less, 7 or less, n is an integer from 1 to 6, e.g., 1 to 3 or 1 to 2. In certain cases, n is 1.

[0135] In some embodiments, the compound of formula (I) is described by formula (Ia):

[0136] [ka]

[0137] where: R 1 -R 4 are each independently selected from hydrogen and alkyl; R 5 -R 6 are each independently selected from aryl and substituted aryl; X 2is alkyl, substituted alkyl, and NR 2a R 2b Selected from R 2a and R 2b teeth, independently selected from hydrogen, aryl, and substituted aryl; X 3 is aryl, substituted aryl, naphthyl, substituted naphthyl, carbazole, and and substituted carbazoles; n is an integer from 1 to 6, m is an integer from 1 to 6.

[0138] In certain embodiments of Formula (I) or (Ia), R 1 -R 4 are hydrogen. In certain cases, R 1 -R 4 At least one of R is alkyl. 1 is alkyl, and R 1 -R 3 are hydrogen. In certain cases, R 2 Is Alki R 1 , R 3 and R 4 are hydrogen. In certain cases, R 3 is alkyl, and R 1 , R 2 and R 4 are hydrogen. In certain cases, R 4 is alkyl, and R 1 -R 3 are hydrogen. In certain cases, R 1 -R 2 is alkyl, and R 3 -R 4 is hydrogen. In certain cases, R 1 and R 3 is alkyl, and R 2 and R 4 is hydrogen. In certain cases, R 2 -R 3 is alkyl, and R1 and R 4 is hydrogen. In the case of R 3 -R 4 is alkyl, and R 1 -R 2 is hydrogen. In certain cases, R 1 teeth hydrogen, R 1 -R 3 are each alkyl. In certain cases, R 2 is hydrogen and R 1 , R 3 and R 4 are each alkyl. In certain cases, R 3 is hydrogen and R 1 , R 2 and R 4 are each alkyl. In certain cases, R 4 is hydrogen and R 1 -R 3 are each alkyl. In certain cases, R 1 -R 4 are each alkyl. In certain cases, R 1 -R 4 is alkyl, the alkyl is C-C alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, or hexyl). 1 -R 4 If at least one of is alkyl, then alkyl is In certain cases, R 4 is alkyl, the compound is enantiomerically pure, and R 4 The carbon to which is attached is in the R configuration. In certain cases, R 4 When R is alkyl, the compound is enantiomerically pure and 4 The carbon to which is bonded is in the S configuration. In certain cases, R 4 When is alkyl, the compound is a racemic mixture.

[0139] In certain embodiments of Formula (Ia), R 5 and R 6 are each aryl. In certain cases, R 5 and R 6 are each phenyl. In certain cases, R 5 or R e At least one of R is substituted aryl. 5 or R 6 At least one of the following is selected from the group consisting of hydroxyl, amino, carboxamide, guanidine, acyl, halogen, and alkoxy. substituted with one or more groups selected from alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, substituted heterocycle, heteroaryl, substituted heteroaryl, nitrile, and nitro. In certain cases, R 5 and R 6 are both substituted aryl. In certain cases, R 5 and R 6 are both substituted phenyl. In some cases, R 5 and R 6 Both are phenols.

[0140] In certain embodiments of compounds of Formula (Ia), m is less than 6, e.g., 5 or less, 4 or less, 3 or less. In some cases, n is an integer from 1 to 4, e.g., from 1 to 3 or from 1 to 2. In certain cases, n is 1.

[0141] In certain embodiments of Formula (Ia), X 2 is alkyl or substituted alkyl. In certain cases, X 2 is C1-C6 alkyl. In certain cases, X 2 is methyl. In certain cases, X 2is a C1-C6 alkyl group substituted with one or more groups. In certain cases, the alkyl group is one selected from hydroxyl, amino, carboxamide, guanidine, acyl, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, substituted heterocycle, heteroaryl, substituted heteroaryl, nitrile, and nitro. In certain cases, the alkyl group is substituted with a guanidine group. In certain cases, X 2 is NR 2a R 2b and R 2a and R 2b are independently hydrogen, In certain cases, R is selected from aryl, aryl, and substituted aryl. 2a and R 2b both Both are hydrogen. In certain cases, R 2a and R 2b are both aryl or substituted aryl. In certain cases, R 2a is an optionally substituted aryl group, and R 2b is H. In certain cases, the aryl group is substituted with one or more groups selected from hydroxyl, amino, carboxamide, guanidine, acyl, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, substituted heterocycle, heteroaryl, substituted heteroaryl, nitrile, and nitro. 2a is phenyl and R 2b is H. In certain cases, R 2a is phenol and R 2b is H.

[0142] In certain embodiments of compounds of Formula (Ia), X 3 is aryl or substituted aryl. In certain cases, X 3 is an optionally substituted carbazole. In certain cases, X 3 is optionally substituted naphthyl. In certain cases, X 3 is a phenol. In some cases, X3 is phenyl. In certain cases, X 3 The group is substituted with one or more groups selected from hydroxyl, amino, carboxamide, guanidine, acyl, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, substituted heterocycle, heteroaryl, substituted heteroaryl, nitrile, and nitro. In this case, X 3 is a carbazole substituted with one or two hydroxyl groups. In certain cases, X 3 is an unsubstituted carbazole. In certain cases, X 3 is one or naphthyl substituted with two hydroxyl groups. In certain cases, X 3 is non It is a substituted naphthyl.

[0143] In certain embodiments of compounds of Formula (Ia), n is less than 6, e.g., 5 or less, 4 or less, 3 or less. In some cases, n is an integer from 1 to 4, e.g., from 1 to 3 or from 1 to 2. In certain cases, n is 1.

[0144] In certain embodiments, the small molecule active agent is a compound selected from the group consisting of:

[0145] [ka]

[0146] [ka]

[0147] In certain other embodiments, the small molecule active agent is described by (II):

[0148] [ka]

[0149] where: R 7a , R 7b , R 8 , R 9 and R 10 are each independently selected from hydrogen, alkyl, and substituted alkyl; X 4 is an alkyl, aryl, aralkyl, heterocycle, and heteroaryl, Selected from sill, where X 4 is optionally further substituted with one or more groups selected from alkyl, substituted alkyl, aryl, substituted aryl, amino, substituted amino, carboxamido, substituted carboxamido, heterocycle, substituted heterocycle, and a second compound of formula (II). or a pharmaceutically acceptable salt or solvate thereof.

[0150] In certain embodiments of compounds of Formula (II), X 4 is optionally substituted alkyl. In certain cases, X 4 is optionally substituted aryl. In certain cases, X 4 is selected from heterocycle, and heteroaryl, either of which is optionally substituted. In certain cases, X 4 is an optionally substituted aralkyl. In certain cases, X 4 is optionally substituted acyl. In certain cases, X 4 is aralkyl or alkyl containing one or more aryl groups. In certain cases, X 4 is a C1-C aryl group containing one or more optionally substituted phenyl groups 20 In certain cases, X may be an aralkyl group containing an alkyl chain. 4 is a C1-C aryl group containing at least two optionally substituted phenyl groups 20 Alkyl chain In a particular case, X 4 is attached to the N atom at a central point and has a small number at each end of the alkyl chain. It is an alkyl chain terminating in at least two optionally substituted phenyl groups. In certain cases, the phenyl group is unsubstituted. In certain cases, the phenyl group is substituted with one or more groups selected from hydroxyl, amino, carboxamide, guanidine, acyl, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, substituted heterocycle, heteroaryl, substituted heteroaryl, nitrile, and nitro. In certain cases, X 4 is a C1-C substituted with two optionally substituted carboxamide groups 20 In certain cases, the alkyl chain is attached to the N atom at a central point and terminates at each end of the alkyl chain in two optionally substituted carboxamide groups. In certain cases, the carboxamide groups are substituted with aryl groups. In certain cases, X 4 is a C1-C substituted with two optionally substituted acyl groups 20 In certain cases, the alkyl chain is attached at a central point to the N atom and terminates at each end of the alkyl chain in two optionally substituted acyl groups. In certain cases, the acyl group is substituted with an aryl group. In certain cases, X 4 is a C1-C substituted with at least one additional compound of formula (II) 20 Alkyl chain In some cases, C1- 20 The alkyl chain may be further substituted with an optionally substituted aryl group. In some cases, X 4 is an optionally substituted acyl group. In some cases, the acyl group is substituted with at least one additional substituent comprising a compound of formula (II). In some cases, the acyl group is substituted with a substituent that includes a heterocyclic group.

[0151] In certain embodiments of compounds of formula (II), X 4 In certain cases, the chelating group is a group that connects to a metal (e.g., In certain cases, the chelating group is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), triethylenediamine, thiazolinone ... ethylenetetramine (TETA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) In certain cases, X may be selected from any of 1,4,7-triazacyclononane-1,4-diacetic acid (NODA), (tert-butyl)2NODA, NETA, C-NETA, L-NETA, S-NETA, NODA-MPAA, and NODA-MPAEM. 4 includes chelators derived from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (also known as DOTA or tetraxetane), which can be attached to compounds of formula (II) via the attachment of one of the pending acetate groups. In certain cases, X 4 comprises a chelator derived from DOTA, which is linked to one, two, three or four compounds of formula (II) via the accommodation of a pending acetate group.

[0152] In certain embodiments, the compound of formula (II) is described by formula (IIa):

[0153] [ka]

[0154] where: R 7a , R 7b , R 8 , R 9 , R 10 and R 10a are each independently selected from hydrogen and alkyl; R 11 and R 12 each independently represents an aryl, a substituted aryl, a heteroaryl, selected from substituted heteroaryl, naphthyl, substituted naphthyl, carbazole, and substituted carbazole; n1 and m1 each independently represent an integer from 1 to 10; n2 and m2 are each independently 0 or 1; n3 and m3 each independently represent 0 or 1.

[0155] In certain embodiments of compounds of Formula (II) or (IIa), R 7a , R 7b , R 8 , R 9 , R 10 and R 10a are hydrogen. In certain cases, R 7a , R 7b , R 8 , R 9 , R 10 and R 10a of At least one of R is alkyl. 10 is alkyl, and R 7 a、R7b , R 8 , R 9 , and R10 a are hydrogen. In certain cases, R 9 is alkyl, and R 7a , R 7b , R 8 , R 10 and R 10a are hydrogens. In certain cases, , R 7a is alkyl, and R 9 , R 7b , R 8 , R 10 and R 10a are hydrogen. In certain cases, R 8 at least one of R is alkyl; 7a , R 7b , R 9 , R 10 and R 10a are hydrogen. In certain cases, R 10a at least one of R is alkyl; 7a , R 7b , R 9, R 10 and R 8 are hydrogen. In certain cases, R 7a , R 7b , R 8 , R 9 , R 10 and R 10a is alkyl, the alkyl is methyl. 10a is alkyl, the compound is enantiomerically pure, and R 10a The carbon to which is attached is in the R configuration. In certain cases, R 10a is an alkyl group, The compound is enantiomerically pure and R 10a The carbon to which is bonded is in the S configuration. In this case, R 10a When is alkyl, the compound is a racemic mixture.

[0156] In certain embodiments of compounds of Formula (IIa), n1 and m1 are each independently an integer from 1 to 8, e.g., from 1 to 7, from 1 to 6, or from 1 to 5. In some cases, n1 and m1 are each 10 or less, e.g., 9, 8, 7, 6, 5, 4, or less. In some cases, n1 and m1 are each 4 to 8, e.g., from 5 to 7, e.g., from 5 to 6. In some cases, n1 and m1 are both 5. In certain cases of Formula (IIa), n2 and m2 are each 0. In some cases, at least one of n2 and m2 is 1. In some cases, n2 and m2 are each 1. In some cases of Formula (IIa), n3 and m3 are each 0. In some cases, at least one of n3 and m3 is 1. In some cases, n3 and m3 are each 1. In some cases, each of n1 and m1 is an integer from 1 to 10, each of n2 and m2 is 0, and each of n3 and m3 is 0. In some cases, each of n1 and m1 is an integer from 1 to 10, each of n2 and m2 is 1, and each of n3 and m3 is 0. In some cases, each of n1 and m1 is an integer from 1 to 10, each of n2 and m2 is 0, and each of n3 and m3 is 1. In some cases, each of n1 and m1 is an integer from 1 to 10, each of n2 and m2 is 1, and each of n3 and m3 is 1.

[0157] In certain embodiments of compound formula (IIa), R 11 and R 12 are each independently selected from aryl, or substituted aryl, and in certain instances, R 11 and R 12 are each independently selected from heteroaryl, and substituted heteroaryl. 11 and R 12 At least one of R is an optionally substituted carbazole. 11 and R 12At least one of R is optionally substituted naphthyl. 11 and R 12 At least one of R is a phenol. 11 and R 12 At least one of R is phenyl. 11 and R 12 are each phenyl. In certain cases, R 11 and R 12 are each independently substituted with one or more groups selected from hydroxyl, amino, carboxamide, guanidine, acyl, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, substituted heterocycle, heteroaryl, substituted heteroaryl, nitrile, and nitro. 11 and R 12 At least one of R is a carbazole substituted with one or two hydroxyl groups. 11 and R 12 At least one of R is an unsubstituted carbazole. 11 and R 12 At least one of R is naphthyl substituted with one or two hydroxyl groups. 11 and R 12 At least one of is unsubstituted naphthyl.

[0158] In certain embodiments, the small molecule active agent is a compound selected from the group consisting of: be.

[0159] [ka]

[0160] In certain embodiments, the small molecule active agent is a compound selected from the group consisting of: be.

[0161] [ka]

[0162] In certain other embodiments, the small molecule active agent is described by (III):

[0163] [ka]

[0164] In certain embodiments of compounds of Formula (III), R 13 is hydrogen. In other examples, R 13 is alkyl or substituted alkyl, e.g., C1-C6 alkyl. In some cases, R 13 is methyl.

[0165] In certain embodiments of compounds of formula (III), X 5 is alkyl or substituted alkyl. In certain cases, X 5 is aryl or substituted aryl. In certain cases, X 5 is selected from heterocycle, substituted heterocycle, heteroaryl, and substituted heteroaryl. 5 is an optionally substituted carbazole. In certain cases, X 5 is optionally substituted naphthyl. In certain cases, X 5 is a phenol. In some cases, X 5 is phenyl. In certain cases, X 5 is amino or substituted amino. In certain cases, X 5 is an amino group substituted with one or more aryl groups. In certain cases, the amino group is substituted with one or more optionally substituted phenyl groups. In certain cases, X 5 is an amino group substituted on one or more phenolic groups. In certain cases, X 5The group is substituted with one or more groups selected from hydroxyl, amino, carboxamide, guanidine, acyl, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, substituted heterocycle, heteroaryl, substituted heteroaryl, nitrile, and nitro.

[0166] In certain embodiments of compounds of formula (III), X 6 is alkyl or substituted alkyl In certain cases, X 6 is C1-C6 alkyl. In certain cases, X 6 is methyl. In certain cases, X 6 is a C1-C6 alkyl group substituted with one or more groups. In certain cases, the alkyl group is substituted with one or more groups selected from hydroxyl, amino, carboxamide, guanidine, acyl, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, substituted heterocycle, heteroaryl, substituted heteroaryl, nitrile, and nitro. In certain cases, the alkyl group is substituted with a guanidine group. In certain cases, X 6 is aryl or substituted aryl. In certain cases, X 6 is selected from heterocycle, substituted heterocycle, heteroaryl, and substituted heteroaryl. 6 is an optionally substituted carbazole. In certain cases, X 6 is optionally substituted naphthyl. In certain cases, X 6 is a phenol. In some cases, X 6 is phenyl. In certain cases, X 6 is aralkyl or substituted aralkyl. In certain cases, X 6 is an aralkyl containing one or more aryl groups. In certain cases, an aralkyl is a C-C group containing one or more optionally substituted phenyl groups. 10 Contains alkyl chains. In certain cases, X 6 is a C1-C aryl group containing at least one optionally substituted phenyl group 10In certain cases, the alkyl chain is an alkyl chain. In certain cases, the alkyl chain is terminated with at least one optionally substituted phenyl group. In certain cases, the phenyl group is unsubstituted. In certain cases, the phenyl group is substituted with one or more groups selected from hydroxyl, amino, carboxamide, guanidine, acyl, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heterocyclic, substituted heterocyclic, heteroaryl, substituted heteroaryl, nitrile and nitro.

[0167] In certain embodiments of compounds of Formula (I), X 7 is alkyl or substituted alkyl. In certain cases, X 7 is aryl or substituted aryl. In certain cases, X 7 is selected from heterocycle, substituted heterocycle, heteroaryl, and substituted heteroaryl. 7 is an aryl heterocycle or a substituted aryl heterocycle. In certain cases, X 7 is an optionally substituted carbazole. In certain cases, X 7 is optionally substituted naphthyl. In certain cases, X 7 is a phenol. In some cases, X 7 is phenyl. In certain cases, X 7 The group is substituted with one or more groups selected from hydroxyl, amino, carboxamide, guanidine, acyl, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heterocycle, substituted heterocycle, heteroaryl, substituted heteroaryl, nitrile, and nitro. 7 is a carbazole substituted with one or two hydroxyl groups. In certain cases, X 7 is an unsubstituted carbazole. In certain cases, X 7 is naphthyl substituted with one or two hydroxyl groups. In certain cases, X 7 is unsubstituted naphthyl.

[0168] In certain embodiments of compounds of Formula (III), p is less than 10, e.g., 9 or less, 8 or less, 7 or more. In some cases, p is an integer from 1 to 6, e.g., from 1 to 3 or from 1 to 2. In certain cases, p is 1 or less. be.

[0169] In certain embodiments, the small molecule active agent is the following compound:

[0170] [ka]

[0171] It is understood that any compound disclosed herein can exist in salt form.In some cases, the salt form of the compound is a pharmaceutically acceptable salt.It is understood that any compound disclosed herein can exist in prodrug form.

[0172] Embodiments of the present disclosure include small molecule active agents (e.g., as described herein), their salts (e.g., pharmaceutically acceptable salts), and / or their solvates, hydrates, and / or prodrug forms. Furthermore, in any compounds described herein that possess one or more chiral centers, unless the absolute stereochemistry is explicitly indicated, it is understood that each center may independently be in the R or S configuration or a mixture thereof. It is understood that all permutations of salts, solvates, hydrates, prodrugs, and stereoisomers are intended to be encompassed by the present disclosure.

[0173] In some embodiments, the small molecule active agents of the present invention, or their prodrug forms, are provided in the form of pharmaceutically acceptable salts. Compounds containing amine or nitrogen-containing heteroaryl groups may be basic in nature and therefore may react with any number of inorganic and organic acids to form pharmaceutically acceptable acid addition salts. Acids include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and phosphoric acid, as well as paratoluenesulfonic acid, methanesulfonic acid, oxalic acid, parabromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, and benzoic acid. It is commonly used to form salts with organic acids such as acetic acid, and related inorganic and organic acids. Accordingly, such pharmaceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, Included are fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, hippurate, gluconate, lactobionate, and similar salts. In certain embodiments, pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as fumaric acid and maleic acid.

[0174] In some embodiments, the subject compound is provided in a prodrug form. "Prodrug" refers to a derivative of an active agent that requires conversion in the body to release the active agent. In certain embodiments, the conversion is enzymatic. Prodrugs are often, but not necessarily, pharmacologically inactive until converted to the active agent. "Promoiety" refers to a form of protecting group that, when used to mask a functional group in an active agent, converts the active agent into a prodrug. In some cases, the promoiety is attached to the drug via bond(s) that are cleaved in vivo by enzymatic or non-enzymatic means. Any convenient prodrug form of the subject compound can be prepared according to the strategies and methods described, for example, by Rautio et al. ("Prodrugs: Design and Clinical Applications," Nature Reviews Drug Discovery 7, 255-270 (February 2008)). In some cases, the promoiety is attached to a hydroxy group of the reference compound. In certain cases, the promoiety is an acyl or substituted acyl group. In certain cases, the promoiety is, for example, an alkyl or substituted alkyl group that forms an ester functionality when attached to a hydroxyl functionality of the subject compound.

[0175] In some embodiments, the small molecule active agents, prodrugs, stereoisomers or salts thereof of the present invention are provided in the form of a solvate (e.g., a hydrate). As used herein, the term "solvate" refers to a complex or aggregate (e.g., a hydrate) formed by one or more molecules of a solute. For example, a prodrug or a pharmaceutically acceptable salt thereof, and one or more molecules of a solvent. Such solvates are typically crystalline solids having a substantially fixed molar ratio of solute and solvent. Representative solvates include water, methanol, ethanol, isopropanol, acetic acid, etc. When the solvent is water, the solvate formed is a hydrate.

[0176] In some embodiments, the small molecule active agent is administered orally and absorbed into the bloodstream. In some embodiments, the oral bioavailability of the subject compound is 30% or greater. The subject compounds or their formulations can be modified using any convenient method to enhance absorption or their bioavailability throughout the intestinal lumen.

[0177] In some embodiments, the subject compounds are metabolically stable (e.g., remain substantially intact in vivo for the half-life of the compound). , 5 minutes or more, e.g., 10 minutes or more, 12 minutes or more, 15 minutes or more, 20 minutes or more, 30 minutes or more, 60 minutes or more, 2 hours or more, 6 hours or more, 12 hours or more, 24 hours or more, or even longer half-lives (e.g., For example, in vivo half-life.

[0178] Specific connecting members In certain embodiments of the present disclosure, the CD206-binding agent is a specific binding member. The term "binding member" refers to one member of a pair of molecules that have binding specificity for one another. One member of the pair of molecules may have a region on its surface or a cavity that specifically binds to a region on the surface or a cavity within the other member of the pair of molecules. Thus, the members of the pair have the property of specifically binding to each other to produce a binding complex. In some embodiments, the affinity between the specific binding members in the binding complex is greater than 10. -6 M or less, e.g. 10 -7 M or less, 10 -8 M or less, e.g. 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 Below, 10 -13 M or less, 10 -14 Below, 10 -15 K of M dIn some embodiments, a particular binding member specifically binds with high avidity. High avidity means that the binding member specifically binds with a dissociation constant of 1x10 -9 M or less, 3x10 -10 M or less, 1x10 -10 M or less, 3x10 -11 M or less, 1x10 -11 M or less, 3x10 -12 M or smaller, or 1x10 -12 10x10 such as M or smaller -9 M or less apparent K d This means that the antibody specifically binds with an apparent affinity characterized by:

[0179] In some embodiments, the specific binding member is proteinaceous (e.g., composed of amino acid residues). In certain cases, the proteinaceous specific binding member is an antibody. In certain embodiments, the proteinaceous specific binding member is an antibody fragment, e.g., an antibody binding fragment that specifically binds to the activity-modulating domain of CD206. As used herein, the terms "antibody" and "antibody molecule" are used interchangeably and refer to a protein consisting of one or more polypeptides substantially encoded by all or part of recognized immunoglobulin genes. For example, recognized immunoglobulin genes in humans include the kappa (k), lambda (l), and heavy chain gene lcoi, which together comprise numerous variable region genes, and the constant region genes mu (u), delta (d), gamma (g), sigma (e), and alpha (a), which encode the IgM, IgD, IgG, IgE, and IgA isotypes, respectively. An immunoglobulin light or heavy chain variable region consists of a "framework" region (FR) interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs." The extent of the framework region and CDRs has been precisely defined (see "Sequences of Proteins of Immunological Interest," E. Kabat et al., US Department of Health and Human Services, (1991)). The numbering of all antibody amino acid sequences discussed herein conforms to the Kabat system. The sequences of framework regions of different light or heavy chains are relatively conserved within species. The framework region of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. CDRs are primarily responsible for binding to an antigen epitope.

[0180] The term "antibody" is meant to include full-length antibodies and may refer to natural antibodies from any organism, engineered antibodies, or antibodies recombinantly produced for experimental, therapeutic, or other purposes, as further defined herein. Antibody fragments of interest include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, or other antigen-binding fragments of antibodies, either produced by modification of a whole antibody or synthesized de novo using recombinant DNA technology. Antibodies may be monoclonal or polyclonal and may have other specific activities on cells (e.g., antagonist, agonist, neutralizing, inhibitory, or stimulatory). It is understood that antibodies may have additional conservative amino acid substitutions that do not substantially affect antigen binding or other antibody functions.

[0181] In certain embodiments, the specific binding member is an antibody. In certain embodiments, the specific binding member is a Fab fragment, F(ab')2, fragment, scFv, diabody, or triabody. In some cases, the specific binding member is a murine antibody or binding fragment thereof. In certain cases, the specific binding member is a recombinant antibody or binding fragment thereof.

[0182] In certain embodiments, the specific binding member is an antibody or antibody thereof that targets the sequence of CD206. In certain cases, the specific binding member targets a sequence of CD206 selected from the group consisting of NFGDLVSIQSESEKK, NDAQSAYFIGLLISL, SKEKETMDNARAF, and EDENCVTMYSNSGFWN. In some cases, the antibody or fragment thereof targets the NFGDLVSIQSESEKK sequence of CD206. In some cases, the antibody or binding fragment thereof targets the NDAQSAYFIGLLISL sequence of CD206. In some cases, the antibody or binding fragment thereof targets the SKEKETMDNARAF sequence of CD206. In some cases, the antibody or binding fragment thereof targets the EDENCVTMYSNSGFWN sequence of CD206.

[0183] In the context of the present disclosure, antibodies capable of binding to the activity-regulating domain of CD206 include monoclonal antibodies (monoclonal antibodies) capable of binding to the activity-regulating domain of CD206. Monoclonal antibodies, polyclonal antibodies, bispecific antibodies, Fab fragments, F(ab)2 antibody fragments, Fv antibodies Body fragments (e.g., V H or V L ), single chain Fv antibody fragments, and dsFv antibody fragments. The antibody molecule can be, but is not limited to, a fully human antibody, a humanized antibody, or a chimeric antibody. Furthermore, the antibody molecule can be a fully human antibody, a humanized antibody, or a chimeric antibody. Antibodies that can be used in connection with the present disclosure can include any antibody variable region, mature or native, linked to any immunoglobulin constant region. Minor variations in the amino acid sequence of an antibody or immunoglobulin molecule are encompassed by the present disclosure, provided that the variations in the amino acid sequence maintain 75% or more of the sequence, for example, 80% or more, 90% or more, 95% or more, or 99% or more.

[0184] An "antibody fragment" comprises a portion of an intact antibody, e.g., the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; Di; linear antibody (Zapata et al., Protein Eng. 8(10): 1057-1062(1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Pepsin treatment produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0185] "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association. The three CDRs of each variable domain interact to form a single CDR on the surface of the VH-VL dimer. It is this configuration that defines the antigen-binding site of an antibody. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site.

[0186] The "Fab" fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments contain the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. F(ab')2 antibody fragments differ from Fab' fragments by the addition of a few residues at the carboxyl terminus of the fragment. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0187] The "light chains" of antibodies (immunoglobulins) from any vertebrate species are of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Immunoglobulins can be assigned to one of the constant domains of their heavy chains. Depending on the amino acid sequence of the antibody, it can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these They can be further divided into subclasses (isotypes), eg, IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

[0188] "Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of an antibody, The domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0189] Thus, antibodies that may be used in connection with the present disclosure include monoclonal antibodies, polyclonal antibodies, bispecific antibodies, Fab antibody fragments, F(ab)2 antibody fragments, Fv antibody fragments (e.g., VH or or VL), single chain Fv antibody fragments, and dsFv antibody fragments. Furthermore, the antibody molecule may be fully human, humanized, or chimeric. In some embodiments, the antibody molecule is a monoclonal fully human antibody.

[0190] Antibodies that can be used in connection with the present disclosure can include any antibody variable region, mature or unprocessed, linked to any immunoglobulin constant region. When a light chain variable region is linked to a constant region, it can be a kappa chain constant region. When a heavy chain variable region is linked to a constant region, it can be a human gamma 1, gamma 2, gamma 3, or gamma 4 constant region, more preferably gamma 1, gamma 2, or gamma 4, even more preferably gamma 1 or gamma 4. It is possible.

[0191] Minor variations in the amino acid sequence of an antibody or immunoglobulin molecule are contemplated as long as the variations in the amino acid sequence maintain at least 75%, e.g., at least 80%, 90%, 95%, or 99% of the sequence. Conservative amino acid substitutions are encompassed by the present invention, provided that: In particular, conservative amino acid substitutions are contemplated (e.g., as described herein). Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative. Fragments (or analogs) of antibody or immunoglobulin molecules can be readily prepared by those skilled in the art. The amino and carboxy termini of preferred fragments or analogs occur near the boundaries of functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs that occur in other proteins of known structure and / or function or predicted protein sequences. Identifying Protein Conformational Domains. Methods are known for identifying protein sequences that fold into known three-dimensional structures. Sequence motifs and structural conformations can be used to define structural and functional domains according to the present invention.

[0192] Non-limiting examples of antibodies that may be used in the present disclosure include: adecatumumab, asclinbacumab, cixutumumab, conatumumab, daratumumab, dorozitumumab, durigotumumab, durvalumab, dusigitumumab, enfortumab, enotitumumab, figitumumab, ganitumumab, glenbatumumab, intetumumab, ipilimumab, iratumumab, icrucurcumab, lexatumumab, lucatumumab, mapatumumab, narunatumumab, necitumumab, nesbacumab, ofatumumab, olatumumab, panitumumab, patritumumab, pritumumab, radletumumab, ramucirumab, rilotumumab, lobatumumab, Seribantumab, talexumab, teprotumumab, tovtumumab, vanticumab, besencumab, votumumab, zalutumumab, framvotumumab, altumomab, anatumomab, arcitumomab, bectumomab, blinatumomab, detumomab, ibritumomab, ibritumomab, minletumomab, mitumomab, mo Xetumomab, naptumomab, nofetumomab, pemtumomab, pintumomab, racotumomab, satumomab, solitomab, taplitumomab, tenatumomab, tositumomab, tremelimumab, abagovomab, igovomab, oregovomab, capromab, edrecolomab, nacolomab, amatuximab, bavituximab, brentuximab, cetuximab Mab, dellotuximab, dinutuximab, encituximab, futuximab, girentuximab, indatuximab, isatuximab, margetuximab, rituximab, siltuximab, ublituximab, ecloneximab, abituzumab, alemtuzumab, bevacizumab, bivatuzumab, brontuzumab, cantuzumab, cant Tuzumab, sitatuzumab, clivatuzumab, dacetuzumab, demcizumab, dalotuzumab, denintuzumab, elotuzumab, emactuzumab, emibetuzumab, enoblitzumab, etaracizumab, farletuzumab, ficlatuzumab, gemtuzumab, imgatuzumab, inotuzumab, labetuzumab, rifastuzumab, lintuzumab,Lorvotuzumab, lumletuzumab, matuzumab, milatuzumab, nimotuzumab, obinutuzumab, ocaratuzumab, otratuzumab, onartuzumab, oportuzumab, parsatuzumab, pertuzumab, pinatuzumab, polatuzumab, siburozumab, simtuzumab, tacatazumab, tigatuzumab, trastuzumab, tucotuzumab, vandaltuzumab, vanucizumab, veltuzumab, borsetuzumab, sofituzumab, catumacat, ertumaxomab, depatuxizumab, ontuxizumab, brontuzumab, tamtubetumab, or antigen-binding variants thereof. As used herein, the term "variant" refers to an antibody that binds to a specific cognate antigen but has fewer or more amino acids than the parent antibody, an antibody that has one or more amino acid substitutions compared to the parent antibody, a single-chain variant of the parent antibody (e.g., an scFv variant), or any combination thereof.

[0193] In certain embodiments, the specific binding member may be an aptamer, a nucleic acid (e.g., DNA, RNA, or nucleic acid analogs).

[0194] In certain embodiments, the specific binding member is an aptamer or a polypeptide that targets the sequence of CD206. or a nucleic acid (e.g., DNA, RNA, or a nucleic acid analog). In certain cases, a particular binding member targets a sequence of CD206 selected from the group consisting of NFGDLVSIQSESEKK, NDAQSAYFIGLLISL, SKEKETMDNARAF, and EDENCVTMYSNSGFWN. In some cases, an aptamer or nucleic acid (e.g., DNA, RNA, or a nucleic acid analog) targets the NFGDLVSIQSESEKK sequence of CD206. In some cases, an aptamer or nucleic acid (e.g., DNA, RNA, or a nucleic acid analog) targets the NDAQSAYFIGLLISL sequence of CD206. In some cases, an aptamer or nucleic acid (e.g., DNA, RNA, or a nucleic acid analog) targets the SKEKETMDNARAF sequence of CD206. In some cases, the aptamer or nucleic acid (e.g., DNA, RNA, or nucleic acid analog) targets the EDENCVTMYSNSGFWN sequence of CD206.

[0195] Conjugated Compounds In certain embodiments of the present disclosure, the CD206-binding agent is conjugated to one or more other active agent compounds (such as, for example, one or more of the active agents described above for combination therapy). In some cases, the CD206-binding agent may be conjugated to two or more other active agent compounds, for example, compounds containing three or more and five or more. The ... It may be conjugated to one or more active agents, such as by interaction. In one embodiment, the CD206-binding agent is conjugated to one or more active agents with one or more covalent bonds. The CD206 binding agent may be directly conjugated to the active agent or may be conjugated to the active agent via one or more linkers, and in certain cases, the CD206 binding agent and the active agent may be conjugated via maleimide / thiol, succinimide ester (NHS ester) / amine, azide chemistry, carboxy / EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) / amine, amine / Sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate) / thiol and amine / BMPH (N-[s-maleimidopropionic acid] hydra The conjugation may be via coupling chemistries including, but not limited to, thiol / dihydrobenzophenone (TFA).

[0196] Screening Method Embodiments of the present disclosure also include assays configured to identify agents that find use in the methods of the present invention, for example, as outlined above. In some instances, the method includes a method for identifying a candidate agent for its ability to bind to a node domain. In some instances, the method includes contacting a CD206-containing macrophage with a compound, and In some cases, the method involves determining whether a substance binds to the activity-modulating domain of CD206. In this case, the method further comprises determining the activity-modulating domain of CD206 that binds to the compound. Evaluating or determining that a given test compound is at least predictive of having the desired binding may allow for further assays, such as animal models and / or clinical assays. This means that further testing of the compound in the presence of HCl may be desirable.

[0197] In certain cases, the macrophage is a macrophage comprising one or more mutations in the activity-regulating domain of CD206. In certain cases, the activity-regulating domain of CD206 binds to a site selected from the fibronectin II domain of CD206, the C-type lectin carbohydrate recognition domain 3 (CRD3) of CD206, the C-type lectin carbohydrate recognition domain 4 (CRD4) of CD206, and the C-type lectin carbohydrate recognition domain 5 (CRD5) of CD206. In certain cases, the compound binds to the CRD5 activity-regulating domain of CD206. In some cases, the compound binds to the fibronectin II activity-regulating domain of CD206. In some cases, the compound binds to the CRD3 activity-regulating domain of CD206.

[0198] The candidate compound can be an immunomodulatory peptide, a small molecule, or a specific binding member (e.g., an antibody) described herein. In some cases, the determining step includes detecting a cellular parameter, wherein a change in the parameter in the cell compared to a cell not contacted with the candidate compound indicates that the candidate compound specifically binds to the activity-modulating domain of CD206.

[0199] Compound screening may be performed using in vitro models, genetically modified cells, microorganisms, or purified CD206 protein. The screening can identify ligands that mimic the CD206 motif. To identify compounds that bind to the domain, a labeled in vitro binding assay is used. A wide variety of assays may be used, including electrophoretic mobility shift assays, immunoassays for protein binding, etc. Knowledge of the three-dimensional structure of CD206, and the experimental data provided herein, allows for the rational design of compounds that specifically bind to the activity-modulating domain of CD206. It can also lead to a calculation.

[0200] As used herein, the term "compound" refers to a compound that binds to the activity-modulating domain of CD206. Any molecule having this ability, such as immunomodulatory peptides, small molecules, specific binding members (e.g., antibodies or their fragments), is described. Generally, multiple assay mixtures are run in parallel with different drug concentrations to obtain differential responses to various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the detection level.

[0201] Candidate compounds encompass numerous chemical classes, including oligonucleotides, antibodies, peptides, polypeptides, and organic molecules, e.g., small organic compounds having a molecular weight greater than 50 and less than about 2,500 daltons. Candidate agents contain functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically contain at least an amine, carbonyl, hydroxyl, or carboxyl group, preferably at least two of the functional chemical groups. Candidate small molecule compounds often contain cyclic carbon or heterocyclic structures, and / or aromatic or polyaromatic structures, substituted with one or more of the above functional groups. Candidate compounds are also found among biomolecules, including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.

[0202] Candidate compounds can be obtained from a variety of sources, including libraries of synthetic or natural compounds.For example, numerous means are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including the expression of randomized oligonucleotides and oligopeptides.Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or can be easily produced.In addition, natural or synthetically produced libraries and compounds can be easily modified through conventional chemical, physical and biochemical means and used to produce combinatorial libraries.Known pharmacological agents can undergo directed or random chemical modification, such as acylation, alkylation, esterification, amidation, etc., to generate structural analogs.In certain embodiments, compounds that cross the blood-brain barrier are of interest.

[0203] If the screening assay is a binding assay, one or more of the molecules may be a signal. A component of the antibody-producing system may be bound to, for example, a label, which can directly or indirectly provide a detectable signal. Various labels include, but are not limited to, radioisotopes, fluorophores, chemiluminescent materials, enzymes, specific binding molecules, particles (e.g., magnetic particles), and the like. Specific binding molecules include pairs such as biotin and streptavidin, digoxin and antidigoxin, and the like. For a particular binding component, the complementary component is typically labeled with a molecule that provides detection, according to known procedures.

[0204] A variety of other reagents may be included in the screening assay. These include reagents such as salts, neutral proteins, e.g., albumin, detergents, etc., used to promote optimal protein-protein binding and / or to reduce nonspecific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc., may be used. The mixture of components is added in any order that provides the required binding. Incubation is carried out at any suitable temperature, typically between 4 and 40°C. The incubation period is selected for optimal activity but may be optimized to facilitate rapid high-throughput screening; in some cases, 1 to 48 hours will be sufficient.

[0205] In some embodiments, the screening step comprises screening from about 1 to about 1000 micromolar samples of the compound. The assay is performed at a single concentration, for example, about 10 to about 500 micromolar or about 10 to about 100 micromolar. In some cases, a dose-response curve is evaluated for each compound. In certain cases, compounds are evaluated for binding at a single concentration.

[0206] composition In embodiments, compositions for use in treating a subject according to the present disclosure can be formulated according to any of the conventional methods known in the art and widely described in the literature. Thus, an active ingredient (e.g., a CD206-binding agent described herein) can be optionally combined with other and one or more conventional pharmaceutically acceptable carriers suitable for the particular use of the composition, together with the active agent. and / or a vehicle, diluent and / or excipient, suitable for administration or suitable for administration. Suitable conventional preparations may be made. These may be formulated as liquids, semi-solids or solids, liquid solutions, dispersions, suspensions, etc., depending on the intended mode of administration and therapeutic application. In some embodiments, the compositions of the invention are prepared in the form of injectable or infusible solutions.

[0207] In certain embodiments, the CD206-binding agent composition comprises a serum albumin (e.g., HSA, BSA). The CD206-binding agent in the pharmaceutical composition may include a carrier protein such as a carrier protein (e.g., a carboxyl group ... By combining the CD206-binding agent with serum albumin, the CD206-binding agent can be effectively "loaded" onto serum albumin. Therefore, larger amounts of the CD206-binding agent may be successful.

[0208] In certain embodiments of the therapeutic methods of the present invention, administration is via any one of a variety of routes, including intravenous (IV), intramuscular (IM), intraarterial, intramedullary, intrathecal, subcutaneous (SQ), intracerebroventricular, transdermal, intradermal, intradermal, intratumoral, intratracheal instillation, bronchial instillation, and / or by inhalation, nasal spray, and / or aerosol, and / or via a portal vein catheter. In certain embodiments, intravenous injection or infusion may be used. Any suitable administration site may be used. For example, the compositions of the present invention may be administered locally and directly to the site where the effect is required, or may be conjugated, for example, to an entity that facilitates targeting to the appropriate location in the body.

[0209] In certain embodiments, any physiologically compatible carrier, excipient, diluent, buffer, or stabilizer can be used in the compositions of the present invention. Examples of suitable carriers, excipients, diluents, buffers, and stabilizers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In some cases, isotonic agents, such as sugars, polyalcohols (e.g., mannitol, sorbitol), or sodium chloride, may be included. In certain embodiments, the compositions of the present invention can be used to prevent the active ingredients (peptide A, peptide B, or variants thereof, and / or additional peptides) from becoming viscous after administration to a subject by using procedures well known in the art. The composition may be formulated to provide rapid, sustained, or delayed release of the additional drug(s). As noted above, in certain embodiments, the composition is in a form suitable for injection, and the suitable carrier may be present in any suitable concentration, although exemplary concentrations are 1% to 20% or 5% to 10%.

[0210] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. Suitable methods for achieving such sterility and stability are well known and described in the art.

[0211] Pharmaceutical compositions are typically formulated in unit dosage form for ease of administration and uniformity of dosage. However, the total daily (or other) usage of the compositions of the present invention may be It will be understood that the specific therapeutically effective dose level for any particular subject will depend on a variety of factors, including the activity of the composition used, the half-life of the composition after administration, the subject's age, weight, general health, sex, and diet, the time of administration, the route of administration, and the excretion rate of peptide A, as well as additional therapeutic agents used (if any), the duration of treatment, drugs used in combination with or concomitantly with the particular compound used, and similar factors well known in the medical arts. Furthermore, effective doses may be extrapolated from dose-response curves derived from in vitro and / or in vivo animal models.

[0212] Therefore, the appropriate dose of the CD206-binding agent and other active ingredients, if any, will depend on the patient's In some embodiments, the dose will comprise a therapeutically effective amount or a prophylactically effective amount, depending on the nature of the treatment involved. The ability to induce steroid hormone-releasing hormone (SHRH) will also be a factor. Exemplary daily doses are 0.1 to 250 mg / kg, Or 0.1 to 200 or 100 mg / kg, or 0.5 to 100 mg / kg, or 1 to 50 or 1 to 10 mg / kg of active ingredient. This can be administered as a single unit dose or as multiple unit doses administered one or more times a day, for example, subcutaneously, intraperitoneally, or intravenously. However, it should be noted that the appropriate dose may vary depending on the patient, and for any particular subject, the specific dosage regimen should be adjusted over time according to the individual needs of the patient. Therefore, the dosage ranges set forth herein should be considered exemplary and are not intended to limit the scope or practice of the claimed compositions or methods.

[0213] kit In one aspect, the present disclosure further provides kits of CD206-binding agents or compositions formulated with CD206-binding agents. The kits can include one or more other components, including, but not limited to, instructions for use, other therapeutic agents (for combination therapy), other reagents, such as diluents, devices or other materials for preparing the composition for administration, pharmaceutically acceptable carriers, and devices or other materials for administration to a subject. The instructions can include instructions for therapeutic application, including recommended dosages and / or modes of administration, for example, in a human subject as described herein. In some embodiments, the kits are for use in the methods and uses described herein, e.g., therapeutic, diagnostic, or imaging methods, or for use in in vitro assays or methods. The peptide(s) or variants in such kits can, in some embodiments, be conjugated, e.g., conjugated to a detectable moiety.

[0214] example Example 1: Biophysical homology screening Natural host defense peptides (HDPs) are short α-helical or β-helical peptides of 10 to 40 amino acids. They frequently exist as sheets, with clusters of polar-charged amino acids arranged along opposing planes of hydrophobic residues, resulting in a bisected amphipathic charge distribution. A database of 431 α-helical antimicrobial peptides (AMPs) and HDPs (http: / / aps.unmc.edu / AP / main.php) was screened using a molybdenum font under the hypothesis that phylogenetically conserved structural domains within naturally occurring HDPs have important innate immune functions and that such selected structure / function paradigm domains could be isolated and optimized for the design of novel therapeutics.

[0215] Instead of homology comparison using primary amino acid alignments, the Molly Font (Molly Hydrophobicity Wheel) evaluates three key biophysical features: hydrophobicity, amino acid electrostatic charge, and 3D amino acid volume, detecting structural homology through their unique conserved biophysical properties (Figure 1A). Of the 431 peptides identified and screened, 129 peptides, or 30%, were found to have a conserved 10-amino acid domain consistent with the structural determinants of a secondary α-helical structure with an amphipathic surface topology (Table 4). Biophysically similar sequences were also identified in human collagen and various microbial virulence factors, indicating peptide structures likely involved in conserved, shared innate immune functions (Figure 2 and Table 4).

[0216] [Table 4]

[0217] [Table 5]

[0218] The synthetically designed RP-182 is hydrophobic (hydrophobic moment vector <μ>) and molybdenum The original conserved 10-mer sequence was optimized for maximum amphipathicity by increasing the positive charge density as visualized in (Figure 3). RP-426 was designed to minimize the effect of hydrophobicity on activity. It was designed as a control to test.

[0219] Next, to identify potential leads for putative binding partners of RP-182 and to investigate whether RP-182 and the original 10-mer homologous sequence share common innate defense regulator functions, we performed in silico docking studies of human C-type lectin receptors, which are target receptors of HDPs and key regulators of innate immune processes in higher organisms. The Animal Lectin Database (http: / / www.imperial.ac.uk / research / animallectins / ctld / mammals / humanvmousedata.html) contains 86 membrane-associated human C-type lectin-like domain (CTLD)-containing proteins, 24 of which had available crystal structures. Using ClusPro®, we examined the crystal structures for binding to RP-182 and biophysically similar 10-mer peptide fragments from 23 representative HDPs, virulence factors, and internal collagen sequences (Figure 4). Figure 5 shows the 10 CTLD-containing proteins with the highest predicted binding affinity for RP-182, and Figure 6 shows the binding affinities of the top receptor / ligand combinations for other 10-mer homology motifs, identifying mannose receptor 1 (MRC1 / CD206) as the target with the highest in silico affinity. MRC1 / CD206 is a member of group 6, the C-type lectin receptor family, and undergoes a conformational change from an open, "elongated," state to a closed state upon ligand binding or as the pH in the surrounding environment decreases (Figure 7A).

[0220] Next, an in silico model was created of full-length human CD206 derived from I-TASSER aligned with small-angle X-ray scattering (SAXS) data (Figure 7B-D). The molecular weight estimated by SAXS data was This revealed that CD206 forms a dimer in solution (Fig. 7B). The CD206 dimer based on model 1 achieved the best fit (lowest χ2) to the SAXS experimental data. Therefore, we chose model 1, which shows that full-length CD206 is the top binding partner of RP-182 and that all but two of the 10-mer peptide sequences share biophysical homology (Figure 7C-D). The model was used to repeat docking studies that confirmed this (Figure 4). Based on Model 1, PR-182 was predicted by ClusPro® to nest within the cavity of CRD5 and engage through three equally spaced prolines (P722, P733, and P744) within CRD4. P760 serves as a fulcrum that bends the "handle" of CRD4 and allows receptor rolling, inducing a closed "globular" state of the receptor (Figure 8A-B).

[0221] To confirm the above binding studies, the first ratio of open to closed CD206 particles was measured by visualization by electron microscopy after incubation with RP-182 and controls. Incubation with RP-182 induces an open, "elongated" conformation of CD206. The conformation of CD206 switched to a closed "globular" conformation (Fig. 9A-B). The half-maximal effective concentration (EC50) of RP-182 to induce the closed conformation was measured at approximately 11 μM (Figure 10). Peptides LL37F1 or the 10-mer homologous motif derived from AVP1, which were predicted to bind poorly to CD206, had a lower ratio of closed to open conformations (-1,146 and -877 kcal / mol, respectively) compared to peptides RP-832C and RP-182, which were predicted to bind with higher affinity (Figures 11 and 12A-C).

[0222] Next, we measured the binding of RP-182 to human CD206 using microscale thermophoresis (MST) and determined a KD of approximately 8 μM. The binding affinity of RP-426 to CD206 was approximately 10-fold lower (KD = 85 μM) (Figure 13). For RP-182 binding to mouse CD206, we measured a KD of approximately 19 μM. The binding of RP-182, but not the control peptide RP-426, to endogenous CD206 in human and mouse macrophages was confirmed in bone marrow-derived macrophages polarized to the M2 phenotype expressing CD206 by cell-based thermal shift assay (CETSA) (Figures 14A-D). CETSA assesses target engagement of a ligand via a shift in the thermal stability of the target protein(s) in a cell-based context. Human and mouse M2-polarized macrophages incubated with RP-182, but not RP-426, showed a shift in CD206 thermal stability (>4°C) compared to vehicle controls, indicating the interaction of RP-182 with the CD206 receptor in its natural environment (Figure 14A-D).

[0223] To further map the binding region of RP-182, we cross-linked it to the RP-182 derivative NCGC-00510434. Mass spectrometry studies of trypsin-digested CD206 were performed. NCGC-00510434, which showed similar binding to recombinant CD206 as wild-type RP-182, contained a diazirine-substituted phenylalanine and a C-terminally attached biotin (Figure 15A-B). Trypsin-digested CD206 pulled down with NCGC-00510434. Fragment analysis of the identified the CRD5 sequence NFGDLVSIQSESEKK, which aligned with peptide analysis of CD206 covalently cross-linked to NCGC-00510434, followed by digestion, pull-down and sequencing of the peptide fragments. The CRD5 motif previously predicted in in silico studies using the CD206 SAXS structure as well as the CD206 SAXS structure was confirmed to be the binding region for RP-182 (Figure 16).

[0224] In summary, RP-182 is a synthetic HDP derived from conserved homologous sequences found across a diverse range of peptide and protein regulators involved in innate immune processes. It selectively induces a conformational switch from the open to the closed state of the mannose receptor MRC1 / CD206, which is distinct from the conformational change of CRDD3 associated with a decrease in pH or binding to the fibronectin II domain of collagen.

[0225] Example 2: Effect of RP-182 on cellular function PR-182 was found to induce programs of phagocytosis, autophagy, and apoptosis in human and mouse M2 macrophages.

[0226] The effects of RP-182 binding and the induced conformational changes of CD206 on cellular functions To investigate the effects of RP-182 on M2 BMDMs, we first examined its effects by global RNA-Seq analysis. Volcano blot analysis of gene expression changes between vehicle and RP-182-treated M2 BMDMs revealed significant differences in the phenotype. Seven of the top eight DEGs were significantly higher after 2 hours of treatment. Classical pro-inflammatory M1 expression levels increased 10- to 100-fold or more after treatment The changes in transcription factors after treatment with RP-182 in myeloid precursors of mouse bone marrow-derived macrophages (BMDMs) polarized to M1 and M2 phenotypes were predominantly observed in M2-polarized macrophages, and were significantly elevated after 2 hours of treatment with RP-182. There were no differentially expressed genes in M1 macrophages, 6 DEGs after 6 hours, and 8 DEGs after 24 hours (Figure 18). Cytoscape® functional GO enrichment and network Network analysis revealed upregulated inflammation and macrophage expression in M2 BMDMs treated with RP-182, including C-type lectin receptor, NF-kB, TNF, or Toll-like receptor (TLR) signaling. The identified pathways of activation were identified (Figure 19).

[0227] Identified by Leading Edge Analysis after Gene Set Enrichment Analysis (GSEA) The most commonly expressed genes were complemented by Pathway Studio®, which identified the processes of endocytosis, phagocytosis, autophagy, and apoptosis as the top biological pathways affected by RP-182 in M2 macrophages (Figure 1A) (Figure 20). Proteomic analysis of binding partners in the CD206 complex pulled down after 10 min of treatment with RP-182 compared to the bead-only control showed enrichment of proteins involved in similar cellular processes (Figure 21A, Figure 4, and Figure 21B-E).

[0228] To confirm the results of the above analysis, BMDMs were evaluated and polarized into M1 and M2 by electron microscopy. RP-182 induced phagocytosis in M2-polarized BMDMs, but not in M1-polarized BMDMs (Figure 22). The M2-selective induction of phagocytosis was confirmed by upregulation of early and late endosomal markers Rab5a and Rab7, as well as lysosome-associated membrane protein 1 (LAMP-1). (Fig. 23). Treatment with RP-182 resulted in increased detection of CD206 in the cytoplasm and co-staining with CD206. The selective induction of phagosomes in the M2 phenotype upon treatment with RP-182 was consistent with the known internalization of the mannose receptor (Figure 24). This was equally observed in M2-polarized CD14+ peripheral monocytes isolated from healthy volunteers (Figures 25A-B) and in BMDMs polarized to an "in vitro TAM-like" phenotype after co-culture with conditioned medium from cancer cells (Figure 26). The control peptide RP-426 did not induce phagocytosis (Figures 27A-B). RP-182 activated NF-kB signaling (Figures 28 and 29).

[0229] Next, we extended the treatment time to 24 hours and measured the induction of autophagy and apoptosis over several time points. RP-182 sequentially induced phagocytosis, autophagy, and apoptosis in M2 macrophages, while leaving M1 cells unaffected (Figure 30 and Figures 31 and 32). RP-182 also cleaved caspase 8, a known downstream substrate of activated caspase 8. RP-182 also induced phospholipases 3 and 7 (Figure 33). Using a dual-stain cell viability assay, 48-hour exposure to increasing concentrations of RP-182 resulted in a reduction of M2 (but not M1) polarized macrophages with calculated potencies (IC50s) of 1.1 μM and 3.4 μM for human M2 and mouse M2, respectively (Figures 34 and 35). The control peptide RP-426 did not show any activity (Figure 36), and RP-182 had no effect on the proliferation of mesenchymal stem cells, mouse and human cancer cells, fibroblasts, or endothelial cells and D2.4 dendritic cells (Figure 37).

[0230] Example 3: Effect of RP-182 on M2 macrophages We found that RP-182 reprogrammed M2 macrophages toward an M1-like phenotype. The observation that the viable cell fraction after 48 hours of treatment with the highest concentration of RP-182 was greater than the initial fraction of CD206-negative cells (31% viable cells vs. 6.8% CD206-negative cells after the maximum response in human M3 macrophages; 17.2% viable cells vs. 12.7% CD206-negative cells in M2 BMDMs) led us to explore a possible second mechanism of action of RP-182. We speculated that M2 macrophages reprogrammed by RP-182 toward an M1-like phenotype might lose CD206 expression and be immune to the cell-killing function of RP-182.

[0231] Flow cytometry experiments of CD11b+F4 / 80+Gr-1- macrophages gated on live cells using the M1 marker CD86 and the M2 marker CD206 showed a rapid induction of CD86 expression and an increase in the fraction of CD86+CD206+ double-positive macrophages (87.8% vs. 10.3% in vehicle-treated controls) within 30 min after treatment with RP-182 (Figure 38 and Figure 39A-B). CD86 expression was followed by a loss of CD206 expression, resulting in a 10.6% CD86+ M1-like fraction that did not express the M2 marker CD206 after 24 h of treatment (Figure 38). Increased CD86 expression in RP-182-treated M2 BMDMs was accompanied by upregulation of M1 cytokines and markers, including IL-1β, IL-12, TNFα, and inducible nitric oxide synthase (iNOS) expressed by M1 macrophages (Figure 40A-C).

[0232] The induction of M1 and loss of M2 markers was also observed in RP-182-treated M2 BMDMs isolated by fluorescence-activated cell sorting (FACS) (Figure 41). Increased M1 cytokine expression , was selective for CD86+ macrophages and not observed in CD206+CD86- cells (Figures 40A-C). The induced M1-like CD86+CD206- and double-positive CD206+CD86+ macrophage cell populations showed reduced numbers of cells staining positive for immune checkpoints PD-1 (8.52% and 18.6% vs. 66.2%) and inhibitory regulatory membrane glycoprotein signal-regulatory protein alpha (SIRPα) (2.81% and 7.84% vs. 16.9%) compared to vehicle-treated CD86-CD206+ M2 cells (Figures 42 and 43). The phenotypic switch induced to M1 by RP-182 was accompanied by an increase in M1 function as the fraction of macrophages involved in bacterial phagocytosis, a function more commonly associated with the M1 phenotype, increased compared with vehicle controls (19.4% and 16.0% vs. 4.97%, and 17.1% and 12.7% vs. 5.74%, respectively). The RP-macrophage fraction stained positive for the M1 cytokines TNFα and IL-12 from 20.4 to 81.3 percent after treatment.

[0233] The RP-182-induced phenotypic switch to M1 was accompanied by increased bacterial phagocytosis, a function more commonly associated with the M1 phenotype (Figure 44). Notably, the rate of apoptosis in reprogrammed M1-like CD86+CD206- macrophages after treatment with RP-182 was significantly lower than the rate of apoptosis in CD206+CD86- and CD206+CD86+ double-positive cells, potentially indicating that (1) CD206-negative cells escape direct cell killing by RP-182 and (2) RP-182 reprograms M2 macrophages toward an M1-like phenotype (Figure 45). Pharmacological inhibition of both RP-182-induced NF-kB signaling and autophagy, previously shown to be induced by RP-182, suppressed the reprogramming effect of RP-182 toward the M1 phenotype (Figure 46).

[0234] RP-182-induced gene expression changes reprogram M2 BMDMs to an M1-like phenotype To investigate whether this supports ramification, gene expression matrices were analyzed from RNASeq data. Pearson correlation analysis of gene expression matrices derived from global RNASeq data of RNA isolated from M1, M2, and M2 BMDMs treated with RP-182 showed significant correlation across the three data sets. Using the M1-M2 marker set previously described for characterizing macrophage phenotype in BMDMs, M2 macrophages were identified after treatment with RP-182. Compared to untreated M1 cells, untreated M2 cells showed greater similarity (Figure 47). In summary, induction of phagocytosis, autophagy, and apoptosis in M2 macrophages was observed. In addition, the synthetic HDP RP-182 induces a shift towards an M1-like phenotype.

[0235] Example 4: Mechanism of action of RP-182 The mechanism of action of RP-182 was investigated and it was found that its action was dependent on CD206 and involved activation of Rac1 / Cdc42 and IQGAP1. It indicated that mobilization would begin.

[0236] When the pharmacodynamic readouts of treatment with RP-182 were overlaid, the EC50 and IC50 activities of RP-182 suggested a shared mechanism of action mediated by a common CD20 target (Figure 48A). To demonstrate that the M2-selective action of RP-182 is indeed MRC1 / CD206-dependent, BMDMs were isolated from B6.129P2-Mrc1tm1Mnz / J mice, which are deficient in CD206 (24). We first confirmed that, with the exception of CD206, there were no differences in the expression profiles of M1 and M2 markers between CD206wt and CD206- / - BMDMs upon polarization into M1 and M2 populations (Figure 48B). In contrast to M2-polarized macrophages from wild-type mice, M2-polarized macrophages isolated from B6.129P2-Mrc1tm1Mnz / J mice failed to demonstrate induction of phagocytosis, autophagy, or apoptosis (Figure 49), were unresponsive to RP-182 (Figure 50), and showed no induction of M1 cytokines upon treatment with RP-182 (Figure 50).

[0237] To better understand the downstream MRC1 / CD206 signaling mechanisms induced by RP-182, proteomic analysis of the MRC1 / CD206 complex was revisited from M2-polarized BMDMs treated with vehicle or biotinylated RP-182 (Figure 50, Figure 51). Growth factor receptor-binding protein 2 Previous studies have identified GRB2 (GRB2) as an intracellular signaling adaptor molecule for mycobacterial activation of the CD206 receptor, and have shown that it induces phagocytosis via GRB2 recruitment and is highly enriched in CD206 pulldowns from BMDMs treated with RP-182. GRB2 was found to induce activation of the Rac1 / CDC42 / Pak1 signaling cascade, a regulatory factor Co-immunoprecipitation and immunoblotting studies in M2 macrophages showed that binding of RP-182 to CD206 recruits GRB2 and activates Rac1 / CDC42 / Pak1 signaling (Figure 52A-B). As a control, we examined phospho-AKT levels in M2 macrophages treated with RP-182 and did not identify activation of this pathway. Compared to controls, IQ motif-containing GTPase-activating proteins 1 and 2 (IQGAP1 and 2) were enriched 9-fold and 76-fold in CD206 complexes pulled down from RP-182-treated cells. These small GTPases stabilize the active state of GTP-bound Rac1 / CDC42, and are effectors of Rac1 / CDC42 in cytoskeletal dynamics, previously shown to be involved in endocytosis / phagocytosis. In M2 BMDMs, RP-182 increased IQGAP1 binding to the CD206 complex and induced its membrane recruitment within 10 min (Figure 53). Inhibition of Rac1 / CDC42 signaling suppressed RP-182-induced IQGAP1 membrane translocation and phagocytosis (Figure 54A-B). Treatment with an autophagy inhibitor prevented RP-182-induced LC3 expression but did not affect caspase-8 induction, which did not occur in the presence of an NF-kB inhibitor. This suggests that caspase-8 activation is not part of the autophagolysosomal cascade but is driven by NF-kB activation mediated by Rac1 / CDC42-Pak1 signaling activation (Figures 55A-B and 56). Indeed, apoptosis induction was mediated by RP-182-induced autocrine TNFα signaling triggered by NF-kB activation. Inhibition of TNFα signaling suppressed the induction of caspase 8 and 3 activation, whereas conditioned medium from M2 BMDMs treated with RP-182 activated apoptosis that was not observed in the presence of anti-TNFα antibodies (Figure 57).

[0238] These data suggest that RP-182 binding to CD206 recruits GRB2 and the Rac1 / CDC42 effector IQGAP1, activates Rac1 / CDC42 / Pak1 signaling, promotes phagocytosis and autophagy, and costimulates NF-kB signaling, which is associated with the induction of apoptosis via autocrine TNFα signaling (Figure 55A-B).

[0239] Example 5: Association between CD206 expression status and intratumoral immunity When we evaluated the relationship between CD206 expression status and intratumoral immunity, we found that CD206 (high) expression status , was found to be associated with reduced intratumoral immunity in human and mouse pancreatic cancer.

[0240] CD206 expression status as a surrogate for M2 macrophage populations was assessed in clinical pancreatic cancer resection specimens. CD206 was significantly altered across the tumors (Figures 58 and 59). Clinically significant differences were observed in the 100% CI, 0.01 to 0.25, 0.15, 0.25, 0.35, 0.45, 0.50, 0.60, 0.75, 0.80, 0.90, 0.95 ... It was found to be overexpressed in two of three available independent gene expression sets in bed pancreatic cancer specimens (gene sets GSE15471, GSE16515, and GSE28735; Figure 60).

[0241] Overall survival (OS) of patients with pancreatic cancer was more favorable in CD206-rich clinical cases There was no association with urinary tract infection (HR 1.87, 95% confidence interval (CI) 1.165 to 2.813; log-rank test; p=0.003). (Figure 61). Infiltrating CD8+ T cells, as measured by CD8 transcript levels, resulted in a high M2-like Outcomes of clinical cases with clusters were further separated (HR 6.09, 95% CI, 1.338 to 10.16; Long rank test; p=0.0006) (Figure 62).

[0242] Adverse disease outcomes in CD206-high patients are explained by immune subpopulation correlations in human pancreatic cancer. To explore whether this correlation is supported by tumor size and across solid organ cancers, we investigated the correlation between intratumoral macrophage subsets and surrogates of intratumoral CD8+ T cell function in the TCGA pan-cancer and pancreatic adenocarcinoma datasets. After selecting tumors with low abundance of CD206, there was also a negative correlation with CD8 transcripts, as well as measures of CD8+ T cell function, including low expression of two previously described T cell activation response signatures (Figure 6A-B). To further explore the relevance of CD206 and clinical outcome, we generated murine pancreatic cancer in CD206-deficient B6.129P2-Mrc1tm1Mnz / J mice.

[0243] There was a discernible difference in survival between KPC CD206- / - vs. CD206 wild-type allografts bearing CD206-deficient tumors, demonstrating prolonged overall survival (median OS of KPC CD206- / - vs. CD206 wild-type: 32 days vs. 25 days, p=0.0278; Figure 64). KPC tumors in CD206- / - mice lacked CD206 expression and significantly increased intratumoral CD8+ T cell numbers compared with KPC tumors generated in CD206-proficient C57B / L6 wild-type mice, in line with the negative correlation between CD206-high and CD8+ T cell function in human cancer specimens (Figure 65). In summary, CD206-positive M2-like TAMs negatively correlate with intratumoral T cell function. Pancreatic cancer allografted into CD206-deficient B6.129P2-Mrc1tm1Mnz / J mice exhibited attenuated tumor progression compared with tumors generated in CD206 wild-type mice and shared immunogenic traits, including increased intratumoral CD8+ T cells, observed in human CD206-low tumors. Note that although KPC tumors in CD206- / - mice attracted similar numbers of TAMs compared with KPC tumors grown in wild-type mice, there was a significant shift toward an M1-like phenotype in the TAM population of CD206- / - mice (Figure 66).

[0244] Example 6: Effects of RP-182 on tumors and tumor microenvironment We investigated the effects of RP-182 on tumors and found that it mediates antitumor activity and reprograms the tumor microenvironment. RP-182 was tested in genetically engineered, Ras-driven KP16 and KPC models of spontaneous pancreatic cancer. Kaplan-Meier analysis and tumor growth measurements demonstrated prolonged survival and antitumor activity of RP-182 monotherapy, resulting in increased survival and tumor suppression similar to gemcitabine (20.5 days in vehicle vs. 10 days in RP-182-treated KPC models). Median overall survival (OS) was 32 days in KP16 animals; p=0.0125, and 27 days vs. 31.5 days in KP16 animals; p=0.0241) (Figures 67A-B). Animals treated with the combination of RP-182 and gemcitabine had the greatest survival benefit in both models, with improved outcomes in the combination cohort compared with single-agent treatment (34 days vs. 44 days for gemcitabine in KP16 mice, p=0.0006, and 24.5 days vs. 42.6 days for KP16 mice, p=0.0002, respectively) (Figures 67A-B). Tumor tissue harvested at the study endpoint showed a decrease in stromal CD206-positive macrophages and decreased nuclear Ki67 expression (Figure 68). RP-182 induced E-cadherin expression and reduced expression of the epithelial-mesenchymal transition (EMT) marker vimentin (Figure 69).

[0245] In vitro, the expression of EMT markers vimentin and SNAIL in mouse pancreatic cancer cells induced by co-culture with M2 BMDMs was significantly enhanced when macrophages were pretreated with RP-182. The IL-182 expression was reduced compared to the control group (Figure 70). Flow cytometry studies of tumor digests from RP-182 in combination with gemcitabine demonstrated a reduction in the M2-like TAM fraction in RP-182 and RP-182 in combination with gemcitabine-treated mice. We confirmed a reduction in tumor CD8+ T cells (10.3% vs. 4.61%, p=0.001 and 10.3% vs. 3.91%, p=0.0003, respectively) (Figure 71). RP-182 also reduced immunosuppressive CD4+ T regulatory cells (Tregs) in combination with gemcitabine myeloid-derived suppressor cells (MDSCs) (8.75 vs. 4.99%, p=0.015). Either alone or in combination with gemcitabine, RP-182 increased intratumoral CD8+ T cells (1.74 vs. 3.40%, p=0.032 and 1.74 vs. 4.99%, p=0.020, respectively) (Figures 71 and 72). The reduction in the MDSC population occurred almost exclusively in the CD206-high monocytic MDSC subset, whereas CD206-low polymorphonuclear MDSCs showed no change (Figure 73).

[0246] We then isolated equal numbers of TAMs from treated murine KPC and KP16 pancreatic tumors and analyzed their effects on T cell function. The effects of interferon gamma on TAMs isolated from vehicle-treated animals were assessed. In combination with RP-182 or gemcitabine, IFN-γ did not induce an increase in INF-γ release. TAMs isolated from RP-182-treated animals showed activated T cell function (Figure 74), suggesting a switch of the TAM population to an antitumor, proinflammatory M1-like phenotype. Indeed, gene expression analysis of TAMs isolated from tumors of RP-182-treated animals and flow cytometry analysis of the TAM population confirmed a switch from a decreased M2 fraction to an increased M1 fraction in RP-182-treated animals (Figures 75 and 76). An increase in the fraction of macrophages staining positive for the M1 cytokines IL-1β, IL-12β, and TNFα and the M1 marker iNOS was observed in RP-182-induced double-positive CD86+CD206+ and CD86+CD206- M1-like cells, but not in CD86-CD206+ M2-like TAMs (Figure 77), a finding consistent with the reprogramming effect of RP-182 observed in M2 BMDMs in vitro.

[0247] Consistent with the mechanism of action of RP-182 in vitro, cleaved caspase 3, Rab7, and LAMP-1 positivity The soluble TAM fraction was significantly higher in RP-182 compared to vehicle-treated tumors (10.9 vs. 72.1%, 2.7 vs. 19.8%, and 3.9 vs. 9.2%, respectively) (Figure 78). CD11b+F4 / 80+Gr-1- macrophages showed little or no change compared with CD11b-CK19-9+ cancer cells. Therefore, the induction of apoptosis and phagocytosis was selective (Fig. 79). Whole-transcriptome analysis of single cells derived from RP-182 revealed differences between RP-182-treated M2 BMDMs and untreated BMDMs. Applying a previously obtained set of differentially expressed genes (DEGs) from treated M2 BMDMs, we found that RP-182-induced TAM cell clusters in vitro were significantly increased in the TAM cell clusters formed by the treated cohort. Genes altered by RP-182 were significantly enriched (Figure 80). Dual staining of RP-182-treated tumors with the markers LC3 and CD206 showed that RP-182 induced autophagosome formation in CD206-positive TAMs and LC3 expression in human and mouse M2-like macrophages in vitro (Figure 81). RP-182-induced changes in M2 macrophages were associated with tumor growth, limiting the impact on intratumoral migration of RP-182-pretreated M2 BMDMs in vivo (Figure 82).

[0248] Example 7: RP-182 Immune Checkpoint Inhibition and Its Effect on Anti-Tumor Immunogenicity and Disease Outcome Collaboration with Influence The effect of RP-182 on intratumoral T cell function was examined by measuring antigen recognition and T cell activation by interferon gamma (INFγ) release upon co-culture with KPC and KP16 cancer cells (EliSpot assay). Intratumoral T cells from animals treated with RP-182, and RP-182 in combination with gemcitabine, were significantly increased compared to T cells isolated from animals treated with vehicle control. Compared to T cells isolated from tumors, RP-182 showed significantly stronger activation when co-cultured with cancer cells, suggesting improved tumor antigen recognition after treatment with RP-182 (Figure 83). Improved T cell function was observed in T cells isolated from tumors. It was selective for T cells isolated from the spleen and was not observed in T cells isolated from the spleen.

[0249] To link the increased tumor cell recognition described above to the observed antitumor activity in vivo, efficacy studies were performed using RP-182 and gemcitabine treatment in CD8+ T cell-depleted mice. The study was repeated in mice that had CD8+ T cells neutralized and were treated with RP-182 and gemcitabine. showed reduced survival compared to mice treated with an isotype control, indicating the involvement of CD8+ T cells in the mechanism of action of RP-182 (Figure 84). Murine pancreatic cancer showed increased PD-L1 expression on CK19-positive cancer cells upon treatment with RP-182 (Figure 85). To test whether these elevated levels of checkpoint expression could be utilized in combination therapy and whether RP-182-mediated anti-TAM therapy could cooperate with PD-L1 immune checkpoint blockade in pancreatic cancers not known to respond to single-agent anti-PD-1 / PD-L1 therapy, RP-182 was combined with anti-PD-L1 therapy. The antitumor activity of the combination was enhanced compared to single-agent therapy (p=0.0215) (Figure 86).

[0250] We next investigated whether the above antitumor activity extended to additional cancer models, including patient-derived xenograft models. RP-182 reduced the growth of CT-26 colon tumors and murine B16 melanoma, demonstrating efficacy comparable to standard anti-CTLA4 checkpoint therapy (Figure 87). Using previously genotyped human pancreatic cancer tissue from the NCI's Patient-Derived Model Repository (PDMR; https: / / pdmr.cancer.gov / ), we generated patient-derived xenografts (PDXs) with CD206-high and CD206-low expression levels and treated them with vehicle, the control peptide RP-426, or RP-182. RP-182 reduced tumor growth in the CD206-high PDX models compared to vehicle and RP-426 controls, but had no effect in the CD206-low models.

[0251] Given that CD206-positive, or activated, macrophages are involved in other disease processes, we next tested RP-182 in a bleomycin-activated lung fibrosis model. Treatment with RP-182 resulted in increased animal weight, improved overall survival, and reduced lung fibrosis (Figures 88A-C). Correlative lung histology studies demonstrated a reduction in M2-like macrophages, as measured by CD206 expression levels (Figure 89). These findings suggest that RP-182 modulates macrophage activity across several mouse and human cancer models, potentially including non-cancer disease models driven entirely by CD206-positive macrophages, suggesting broad applicability. CD206 expression status may aid in the future selection of tumors most likely to respond.

[0252] Example 8: Effect of RP-182 on cancer cell phagocytosis by M1-like macrophages RP-182 efficiently engages CD206-positive target cells in pancreatic tumors after systemic administration. To confirm this, 20 mg / kg of biotin-loaded RP-182 was administered to KPC mice. Tumors were harvested, implanted, and probed with anti-CD206 antibody and AlexaFluor streptavidin. Intratumoral RP-182 was detected by co-staining with avidin (NCGC-00510434; Figure 15B). Multicolor confocal microscopy, measuring staining intensity over a distance (μm), demonstrated significant colocalization of RP-182 with CD206-positive cells in the pancreatic KPC tumor microenvironment, confirming that RP-182 is targeting CD206-positive cells. The photon-mediated heterologous determination of AlexaFluor 480-RP-182 in organs was Quantification showed significant enrichment in tumors and kidneys compared to other organs (Figure 91). A 14-day toxicity study of RP-182 administered continuously at doses up to 30 mg / kg daily showed no hematological changes in whole blood or changes in total body or selected organ weights in treated animals.

[0253] In line with its target CD206 and selectivity for CD206 expressing M2 macrophages, treatment with RP-182 mediated improved survival in C57BL / 6 wild-type mice allografted with KPC tumors, but not in CD206-deficient B6.129P2-Mrc1tm1Mnz / J mice bearing KPC tumors lacking the RP-182 target receptor (Figure 92). The smaller effect of RP-182 on overall survival compared to spontaneous KPC tumors shown in Figure 67B may be due to differences between spontaneous KPC tumors and KPC tumors generated from allograft cells. RP-182 did not produce discernible hematological changes in whole blood or changes in total body weight or selective organ weights in treated animals during preliminary toxicity studies (Figure 93).

[0254] The loss of SIRPα receptors, involved in innate immune cell "don't eat me" signaling, on macrophages isolated from RP-182-treated mouse pancreatic tumors (Figure 75) prompted us to investigate whether T cell-independent innate mechanisms of RP-182, such as cancer cell phagocytosis, might contribute to RP-182's antitumor activity. Therefore, we measured the phagocytosis of several different human and mouse cancer cell lines labeled with the green fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) in M2 BMDMs treated with RP-182. RP-182 significantly increased cancer cell phagocytosis (measured by infiltrating CSFE-positive cells; phagocytic index) of several mouse and human cancer cell lines by 28.2 to 46.6% after 2 hours of treatment with RP-182 (Figure 94A-B). Cancer cell phagocytosis was observed exclusively in the CD86-positive M1 population at baseline and increased after exposure to RP-182 (Figure 95). Although the increased cancer cell phagocytosis by M1 macrophages is similar to the fraction of reprogrammed CD86-positive M1 cells, we cannot exclude that a decrease in inhibitory M2 cues releasing inhibition of M1 function is partially responsible for the observed increased cancer cell phagocytosis of RP-182.

[0255] To demonstrate that RP-182's innate mechanism of action, induced phagocytosis of cancer cells, is involved in its antitumor activity, KPC, MDA-MB231 breast, and C4-2 prostate tumors lack mature T lymphocytes and are unable to mount cell-mediated antitumor immune responses, but are rich in B lymphocytes. RP-182 monotherapy reduced tumor growth across these tumor models, improved antitumor activity in standard gemcitabine models, and inhibited metastatic dissemination in the MDA-MB231 model. H&E review of RP-182-treated tumors showed significant reductions in macrophage activity (Figures 96 and 97). Upon treatment with PR-182, TAMs were able to differentiate into phagocytic cells and phagocytosis of cancer cells. They lose their eosinophilic cytoplasm and show increased hematoxylin uptake along with numerous intracellular inclusions of nuclear material or cellular debris. Tissue sections examined by electron microscopy revealed multiple complete inclusions of cancer cells in TAMs, partial phagocytosis of cancer cells, and a lack of eosinophilic cytoplasm compared with vehicle-treated tumors. We also demonstrated the attachment of activated macrophages to cancer cells (Figure 98).

[0256] Thus, RP-182 can respond to immune checkpoint blockade via CD206. It is unknown to date how adaptive and innate immune cell function is enhanced in tumors.

[0257] Consideration Tumor-associated macrophages are well-positioned to recognize and successfully infiltrate diseased tissue and mount profound antitumor immune responses. Unfortunately, many tumors are able to alter the behavior of these cells and synchronize them to support vascularization, tumor growth, invasion, and metastasis.

[0258] RP-182 selectively killed these problematic macrophages, initiating an apoptotic process that depleted them at tumor sites. Evaluation of the biological effects of RP-182 within M2 macrophages and other immune cells showed that this peptide also altered the function of these macrophages, shifting them from an immunosuppressive state to a proinflammatory, phagocytic phenotype capable of mediating immune antitumor activity. Activation of phagocytosis, autophagy, and NF-κB signaling within RP-182-responsive macrophages resulted in the upregulation of M1 markers. This rapidly leads to the downregulation of M2 markers. These phenotypic changes are accompanied by improved phagocytosis, particularly a reduction in their overall immunosuppressive properties. Both loss of MRC1 / CD206 expression and reprogramming of cells that escape apoptosis, along with induction of cell death in MRC1 / CD206-bearing macrophages, robustly shifts TAM populations toward an M1 phenotype upon prolonged exposure to RP-182, restoring immune surveillance in the tumor microenvironment. The mannose receptor MRC1 / CD206 is expressed on the cell surface and is closely associated with its primary function of phagocytosis of pathogenic microorganisms through recognition of its surface, mannose-containing glycoproteins (MGPs), as well as its scavenger function at sites of tissue damage, particularly collagen.

[0259] RP-182 activates mannose receptor MRC1 / CD206 phagocytosis and autophagy in M2-like macrophages, which reverts these cells to an anti-tumor M1-like phenotype with increased M1 cytokine production and the ability to phagocytose cancer cells. In addition, RP-182 inhibits TNFα cytokines. Autocrine positive signaling induces apoptosis via cleaved caspase 8 Through a feed-forward loop, CD206-positive alveolar macrophage extravasation induces and promotes the depletion of this population, further shifting the balance toward a pro-inflammatory, anti-tumor M1 phenotype. RP-182 was tested in a characterized pulmonary fibrosis model. The observed reduction in collagen deposition and fibrosis associated with the therapeutic efficacy of RP-182 in this inflammatory model appears to be consistent with the known antifibrotic activity of activated M2 macrophages.

[0260] Dynamically, RP-182-induced phagocytosis and autophagy in CD206-positive M2-like macrophages. Induction of apoptosis, followed by induction of apoptosis, M2-like macrophages in the TAM population This was followed by a decrease in TAM expression and an increase in CD8 cytotoxic T cell infiltration and function. This mutation was also associated with a low EMT-like cancer phenotype in RP-182-treated tumors. Of note, the expansion of the M1 population transformed from M2-like cells was observed in vitro and in vivo. Both improved innate antitumor immunity through increased phagocytosis of cancer cells. Results from studies in CD206-low PDX models and CD206- / - knockout allografts suggest that CD206 levels could be used as a future biomarker for this approach.

[0261] In summary, the results presented herein demonstrate that biophysical similarities beyond primary amino acid sequence alignments exist between previously known HDPs and regulators of the innate immune system. RP-182 demonstrates that we can detect unrelated homologies across the HDP and use these motifs to design effective therapeutics. It is a decameric synthetic HDP derived from a mediator involved in immune cloning and innate immune processes. RP-182-induced conformational switching of the mannose receptor MRC1 / CD 206 is a tumor-specific Reprogramming M2-like TAMs in the stromal layer promotes intratumoral innate and adaptive antitumor immunity and tumor Improve control.

[0262] material and method Peptides, cell lines, and chemical sources Peptides were obtained from Poly Peptide Laboratories (San Diego, CA). Peptides included RP-182, KFRKAFKRFF, RP-832C, RWKFGGFKWR, RP-185, FFKKFFKFK, AVP, EKLSAFRNFF, LL37F1, FFRKSKEKIG, and RP-426 KARKAAKRAF. PANC-1 (CRL-1469), HPAF-II (CRL-1997), and LNCaP cells (CRL-1740) were purchased from the American Type Culture Collection (ATCC; Manassas, VA), and primary mouse KP16 and KP20 cells were transfected with 1000 ribosomal RNA (RNA)-derived ... The KPC and KPC pancreatic cancer cell lines were derived from fresh tumor tissue, and the primary-derived low-passage melanoma line 2183 By SNP genotyping using Illumina MiSeq sequencing according to AACR practices. Cells were authenticated and confirmed to be mycoplasma-free. Mesenchymal stem cells, human fibroblasts, and endothelial cells were purchased from Cellular Dynamics International Inc., and DC2.4 murine dendritic cells were purchased from Millipore Sigma. Cells were cultured according to the supplier's instructions or The cells were maintained in PRIMI 1640 medium containing 10% (v / v) FBS and incubated at 37°C in a 5.0% CO2 atmosphere. Small molecule inhibitors were purchased from Selleckchem Inc. (Houston, TX) and included activated B cell (NF-kB) inhibitors JSH-23 (Cat# S7351), QNZ (EVP4593) (Cat# S4902), mitogen-activated protein kinase (MAPK) inhibitor selumetinib (AZD6244) (Cat# S1008), and Ras-associated C3 botulinum toxin substrate 1 (Rac1) GTPase inhibitor NSC 23766 (Cat# S803). 1), and ZCL278 (Cat#S7293), a cell division control protein 42 homolog (CDC42) inhibitor. Contains nuclear factor "kappa-light-chain-enhancer".

[0263] Synthesis of peptide analogues Biotinylated RP-182 analog (NCGC-00510434) and biotinylated RP-426 containing diazine Phosphorus was synthesized by RS Synthesis LLC (Louisville, KY) and was >95% pure. The Fmoc-diazirine-containing phenylalanine analogs were prepared as shown in Figure 15A-B. Separation of the enantiomers was performed on a chiral column (Chiralpac IB 4.6 x 250 mm, 100% EOH; 1 ml / min). Biotin was added to the phenylalanine analogs using polyethylene glycol (PEG) or hydrocarbons. The lysine residues were introduced onto the side chains of the lysine residues linked to either the hydroxyl group or the hydroxyl group of the lysine residues (Figures 15A-B). (See

[0264] Recombinant human and mouse MRC1 / CD206 Recombinant human CD206 was purchased from R&D Systems (Cat# 2534-MR / CF). Recombinant mouse CD206 was produced by the Protein Expression Laboratory (FNLCR, Frederick, MD). Briefly, a mouse CD206 (NM_008625.2) cDNA fragment encoding the 23 to 1387 sequences was optimized for human codon usage and produced in the pDEST vector with an N-terminal bee melittin signal peptide and a C-terminal 6xHis tag. The protein was transiently expressed in Expi293E cells and purified from supernatants collected 72 hours posttransfection using nickel affinity chromatography. CD206 was eluted in 20 mM HEPES (pH 7.2), 300 mM NaCl, 250 mM imidazole, and dialyzed into PBS (pH 7.4). For electron microscopy experiments, mouse CD206 was further purified by size exclusion chromatography and eluted in PBS (pH 7.4).

[0265] Biophysical homology screening To screen for phylogenetically conserved homology beyond primary amino acid structure, we used the design heuristic Molly Font (Figure 1). The captured chemical properties of the amino acids include the amino acid volume (measured in cubic angstroms (radius of gyration) in H2O) coded by the circle size, and the The most hydrophobic amino acids have the strongest hydrophobicity. The less hydrophobic the molecules, the more cyan they are. ). The most hydrophilic amino acids have the deepest magenta color, and a graduated scale of magenta intensity is used for less hydrophilic amino acids. Implicit in this scheme is that exchanges within a particular queue, i.e., among hydrophobic or hydrophilic amino acids with very similar properties, are more likely to occur due to the specific structural constraints imposed on each particular protein to preserve its functionality (generating the variability observed in proteins with similar functions from evolutionarily distant organisms). Representative glyphs included as mnemonic symbols characterize additional chemical properties of the amino acids, including the charge of the amino acid (a "+" or "-" sign) incorporated within those glyphs and the dissociation constant of their ionizable protons, which is coded by the thickness of the glyph. The numerical value is the energy (in kcal / mol) required to move the amino acid side chain from the inside of the lipid bilayer to the outer aqueous environment (32).

[0266] In silico docking Protein-protein interactions between α-helices derived from identified biophysically homologous sequences and putative target receptors were assessed by in silico docking using the ClusPro® server (Boston University, MA), which performs direct docking in three computational steps: (1) rigid-body docking by sampling billions of conformations; (2) root-mean-square deviation (RMSD)-based clustering of the 1,000 lowest-energy structures generated to find the largest cluster representing the most likely model of the complex; and (3) refinement of selected structures using energy minimization. Docking with each energy parameter set resulted in 10 models defined by the centers of the most populated clusters of the lowest-energy docked structures. Consideration of the centers of the largest clusters of these low-energy structures, rather than simply low-energy structures, is unique to ClusPro® and implicitly accounts for some of the entropic effects that align cluster populations to cluster probabilities under natural assumptions. Structures were compared by ranking the binding coefficients, which combine cluster probabilities and binding energies.

[0267] MRC1 / CD206 modeling The protein sequence of human MR1 / CD206 (1,456 aa) was obtained from UniProt (UniProt ID P22897-1 NCBI The CD206 fragment was derived from the CD206 protein (IDNP_002429.1) and contains two N-terminal domains (ricin-B type lectin and fibronectin type II), followed by eight C-type lectin domains (numbered 1 to 8), a transmembrane domain (TM), and a cytoplasmic domain. A 3D model of CD206 was generated using I-TASSER (https: / / zhanglab.ccmb.med.umich.edu / I-TASSER / ), an Iterative Threading and ASSEmbly Refinement software. I-TASSER utilizes a hierarchical approach that identifies 3D templates from the RCSB-PDB (http: / / www.rcsb.org) using a multithreading approach. The full-length model is finally constructed by iterative template fragment assembly simulations.

[0268] To aid in modeling, cysteine ​​residue positions involved in disulfide bonds were extracted from UniProt (www.uniprot.org), and this list was used as distance restraints during I-TASSER modeling. The top-threading RCSB-PDB template IDs identified by I-TASSER were 5ao5, 3jav, 5ao6, and 4igl. The normalized B-factor values ​​of the models fluctuated around zero, indicating acceptable local accuracy of the models. Model confidence was measured by C-score, which ranged from -5 to 2, with higher values ​​indicating higher confidence. The C-scores of the top four models were -0.35, -1.93, -2.89, and -2.97. The top four models were analyzed, and the top I-TASSER models were identified as possible structural folds for CD206 based on comparison of small-angle X-ray scattering (SAXS) and I-TASSER-predicted secondary structure confidence and C-score.

[0269] Small-angle X-ray scattering (SAXS) data collection and analysis SAXS data were collected at the 12ID-B beamline of the Advanced Photon Source (APS) at Argonne National Laboratory (Lemont, Illinois). The photon energy was 13.3 keV, and the sample-to-detector distance was 2 m, achieving a q range of 0.005 < q < 0.90 A-1, where q = (4π / λ) sinθ, where 2θ is the scattering angle. A series of concentration measurements of CD206 in a buffer solution containing 50 mM Hepes, 100 mM NaCl, and 1 mM DTT were performed, and the data were extrapolated to infinite dilution to remove scattering contributions due to particle-particle interactions (concentration effect). To minimize radiation damage and obtain optimal signal-to-noise ratios, exposure times ranged from 0.75 to 1 second. Thirty 2D image frames were recorded for the sample solution and its matching buffer using a flow cell. The 2D images were reduced to 1D scattering profiles and averaged at the beamline using a Matlab software package.

[0270] A MatLab script developed by the 12-ID-B beamline was used to perform background processing. Band subtraction and intensity extrapolation to infinite dilution were performed. The radius of gyration (Rg) was calculated in the range of qRg<1.3. The R(r) was generated from a Guinier plot. For comparison, Rg was calculated in real and reciprocal space using the program GNOM (https: / / www.embl-hamburg.de / biosaxs / manuals / gnom.html). The pair-distance distribution function P(r) and maximum dimension (Dmax) were also calculated using GNOM. Molecular weights were estimated using two methods based on the Porod volume, Vorod, and correlation volume Vc. Based on an in silico model of the CD206 monomer derived from iTASSER, fits to the experimental SAXS data of the CD206 dimer were calculated using the program CORAL.

[0271] electron microscope Purified recombinant mouse CD206 full-length protein and its complexes with peptides RP-182, RP-185, RP-832C, AVP1, LL37F1, and RP-426 were analyzed by negative-stain electron microscopy. A 3-μL aliquot containing approximately 0.01 mg / mL of sample was applied for 20 seconds to a carbon-coated 200Cu mesh grid that had been glow-discharged at 30 mA for 30 seconds and then negatively stained with 0.7% (w / v) uranyl formate for 40 seconds. Data for unbound CD206 and its complexes with RP-182, RP-426, and RP-832C were collected using an FEI T20 electron microscope operating at 200 kV, resulting in an electron dose of approximately 40 e / A² and a pixel size of 2.19 A at the sample plane with 100,000x magnification. Images were acquired with an Eagle 2kx2k CCD camera (http: / / FEI.com) using a nominal defocus of 1500 nm and SerialEM software (54). Data for the complex containing RP-185, AVP1, and LL37F1 were collected using an FEI Talos electron microscope operating at 200 KV with an electron dose of approximately 40 e / A² at 73,000x magnification, resulting in a pixel size of 1.98 A at the specimen plane. Images were acquired with a Ceta 4kx4k CCD camera (http: / / FEI.com) using a nominal defocus of 1,200 nm and EPU software. For electron microscopy data processing, particles were selected and extracted from the micrographs, and reference-free 2D class averages were obtained using RELION 2.1.0. Microscale thermophoresis and cellular thermal shift assays.

[0272] The binding of RP-182 and RP-426 peptides to purified recombinant MRC1 / CD206 was assessed by microscale thermophoresis (MST) using a label-free approach. Specifically, two-fold serial dilutions of the peptides were prepared in PBS and incubated with an equal volume of 250 nM recombinant human and mouse CD206 in PBS. After 5 min of incubation at room temperature (RT), measurements were performed in standard capillary tubes using a Monolith NT label-free instrument (Nanotemper Technologies) with 40% LED excitation power, 40% IR laser power, 30 s laser ON / OFF, and 5 s laser ON / OFF. KD values ​​were calculated by fitting the T-Jump signals of the thermograph T using MOAffinity analysis software (Nanotemper Technologies).

[0273] Target engagement of the peptides in macrophages was assessed using a cellular thermal shift assay (CETSA) according to the protocol of Jafari et al. with slight modifications. Briefly, a suspension of M2-polarized macrophages was prepared using cell dissociation buffer (Gibco BRL) for 5 min at RT, followed by one wash with DMEM (Gibco, Cat. #11965118). Aliquots of 6 x 10 cells were incubated with 100 μM RP-182, 100 μM RP-426, or an equivalent volume of PBS for 45 min at 37°C. After treatment, cells were collected by centrifugation at 300 x g for 5 min and resuspended in 600 μL of DMEM. A 50 μL aliquot of the cell suspension was heated in 3°C steps over a temperature range of 37 to 64°C for 3 min, cooled at RT for 3 min, and lysed in 10 μL of DMEM containing NP-40 (1% (v / v) final concentration) and Halt protease inhibitor cocktail (ThermoFisher, Cat. #78430) and supported by three freeze-thaw cycles. Samples were centrifuged at 20,000 x g for 20 min at 4°C. The supernatant was subsequently analyzed by Western blot using a 12-230 kDa Peggy Sue separation module (ProteinSimple, Cat. #SM-S001) and a Peggy Sue instrument (ProteinSimple) with the following settings: electrophoresis at 250 volts for 45 min; block for 23 min; primary antibody for 30 min; secondary antibody for 30 min. Quantitative analysis of CD206 levels multiplexed with anti-CD206 antibody at a 1:70 concentration and anti-SOD1 antibody at a 1:300 dilution as an internal control for normalization was performed using Compass software (ProteinSimple, San Jose, CA).

[0274] MRC1 / CD206 fragment analysis by LC-MS / MS To identify the binding domain of MRC1 / CD206 to PR-182, two different approaches were used. First, 5 μg of recombinant human CD206 digested with trypsin (Thermo Scientific, Cat# 90057) was incubated with biotinylated RP-182 (NCGC-00510434) immobilized on magnetic beads (Thermo Scientific, Cat# 65001) or beads alone for 4 hours at RT. After washing the beads three times in PBS containing 100 μM Tween-20 PBS-T, the samples on the beads were eluted, desalted using a C18-ziptip (Millipore, ZTC18S960), and analyzed by LC-MS / MS. Next, a diazirine and biotin-containing RP-182 analog (NCGC-00510434) was crosslinked to full-length recombinant CD206 by photolabeling and then digested with trypsin. Specifically, 50 μg of MRC1 / CD206 protein was incubated with 100 μM NCGC-00510434 or PBS, incubated at room temperature for 5 minutes, photolabeled on ice for 30 minutes, and digested with trypsin. The samples were then incubated with streptavidin magnetic beads for 4 hours at room temperature. The beads were collected and washed three times with PBS-T. The samples on the beads were treated as described above and analyzed by LC-MS / MS.

[0275] LC-MS / MS analysis of the samples was performed using a Thermo Scientific Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer and a Thermo Dionex UltiMate 3000 RSLCnano system. Peptide mixtures from each sample were loaded onto a peptide trap cartridge and eluted onto a reversed-phase PicoFrit column (New Objective, Woburn, MA) using a linear gradient of acetonitrile (3 to 36%) in 0.1% (v / v) formic acid. The peptides were then ionized and sprayed into the mass spectrometer using a Nanospray Flex Ion Source ES 071 (Thermo Scientific) under the following settings: spray voltage 1.8 kV, capillary temperature 250 °C. For peptide identification and protein assembly, data were analyzed using the Thermo Proteome Discoverer 1.4.1 platform (Thermo Scientific, Bremen, Germany). Database searches against the CD206 sequence were performed using the SEQUEST algorithm through the Discoverer 1.4.1 platform. Carbamidomethylation of cysteine ​​was set as a fixed modification, and oxidation and deamidation Q / N-deamidation (+0.98402 Da) were set as dynamic modifications. The minimum peptide length was specified to be 5 amino acids with a maximum false peptide discovery rate of 0.01. The precursor and fragment mass tolerances were set to 15 ppm and 0.05 Da, respectively.

[0276] Proteomic analysis of the CD206 complex by LC-MS / MS 5x10 per treatment to identify proteins involved in downstream signaling induced by RP-182 via CD206 6M2 cells were resuspended in 3 mL of RPMI medium and incubated with 100 μM biotinylated RP-182 or PBS for 30 minutes at 37°C. Cells were pelleted and lysed in 500 μL of Pierce IP Lysis Buffer (Thermo Scientific, Cat# 78440) with protease and phosphatase inhibitors (Thermo Scientific, Cat# 87787) for 15 minutes at 4°C. Then, the cells were cleared by centrifugation at 15,000 x g for 15 minutes at 4°C. The supernatant was transferred to a new tube and incubated with 20 μL of streptavidin magnetic beads (Thermo Scientific, Cat# 65001) for 30 minutes at 4°C. The beads were collected and washed four times with PBS-T. Samples on the beads were separated by SDS-PAGE, reduced with DTT, alkylated with iodoacetamide, and digested with MS-grade trypsin. The digested peptide mixture was concentrated, desalted using a C18 Zip-Tip, reconstituted in 20 μL of 0.1% formic acid, and analyzed by LC-MS / MS as described above.

[0277] The raw data files were analyzed for mouse protein sequences using Proteome Discoverer 1.4 software (Thermo Scientific, San Jose, CA) based on the SEQUEST algorithm. The peptides were screened against a protein sequence database. Carbamidomethylation of cysteine ​​(+57.021 Da) was the fixed modification, and oxidation / +15.995 Da (M), deamidation / +0.984 Da (N,Q), methyl / +14.016 Da (K,R), acetyl / +42.011 Da (K), phospho / +79.966 Da (S,T,Y), and dimethyl / +28.031 Da (K,R) were set as dynamic modifications. The minimum peptide length was specified to be five amino acids. The precursor mass tolerance was set to 15 ppm, while the fragment mass tolerance was set to 0.05 Da. The maximum false peptide discovery rate was specified as FDR<0.01.

[0278] Mouse and human macrophages Murine monocyte progenitor cells were obtained by draining bone marrow from the femurs of 6- to 8-week-old healthy C57B / L mice. Experiments were performed in accordance with the Animal Care and Use Committee of the National Institutes of Health (ACUC protocol SB-210 -3) Protocols and policies approved by and the Humane Care and Use of Laboratory Animals The study was conducted in accordance with the NIHS policy regarding monocyte isolation (https: / / olaw.nih.gov / home.htm). After 1 week of incubation at 37°C with 5% CO, myeloid precursors were polarized into M1 and M2 macrophages using the respective cytokines (Figure 14A). Human macrophages were isolated using a classical monocyte isolation kit (Cat# 130-117-337 MACS Miltenyi Biotec, San Diego, CA). Peripheral blood mononuclear cells (PBMCs) obtained from anonymized healthy human donors from the NIH, Division of Transfusion Medicine (DTM) (under institutional review board (IRB)-approved NIH protocol 99-C-0168) were depleted of CD14+ cells and polarized to M1 and M2 macrophages using cytokines, as described in Figure 21B-E (57). Recombinant mouse M-CSF (Cat# PMC2044) and IFN-γ recombinant human protein (Cat# PHC4033) were purchased from ThermoFisher Scientific, and recombinant mouse INF-γ (Cat# 485-MI-100), mouse IL-4 (Cat# 404-ML-050), recombinant human IL-4 protein (Cat# 204-IL-050), recombinant human IL-13 protein (Cat# 213-ILB-025), and recombinant human IL-6 protein (Cat# 206-IL-050) were purchased from R&D Systems (Minneapolis, MN). Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) (Cat#8922SC), human macrophage colony-stimulating factor (hM-CSF) (Cat#8929SC) were purchased from Cell Signaling, and lipopolysaccharide from Escherichia coli O111:B4 (Cat#L3012-5MG) was purchased from Sigma Aldrich.

[0279] RNA sequencing (RNASeq) experiments and data analysis Total RNA was harvested from M1- and M2-polarized macrophages treated with 20 μM RP-182 or vehicle for 2 hours and subjected to global RNASeq analysis on an Illumina NextSeq500 sequencer. Reads were trimmed to remove adapter sequences and reads shorter than 25 base pairs (bp) using the Trimmomatic ver. 0.32 tool. Trimmed reads were mapped to the mouse genome mm10 using the STAR aligner. A transcriptome BAM, along with the genome BAM, was generated for use in RSEM quantification (accurate transcript quantification from RNASeq data with or without a reference genome).

[0280] EdgeR (Empirical Analysis of Digital Gene Expression Data; v3.30.09) analysis uses a negative binomial model The EdgeR analysis was performed in R based on the variance and count data. Low-count transcripts were manually excluded in the EdgeR analysis, and only genes with at least 1 count per million were used for further analysis. Normalization coefficients were calculated using the trimmed mean M (TMM) method, and the variance parameter for each gene was estimated as the Cox-Reid common variance method. The GLM (generalized linear M) likelihood ratio test was based on fitting a negative binomial GLM with a Cox-Reid variance estimate to account for known sources of variation. Significant DEGs were detected using a cutoff value of false discovery rate (FDR) < 0.05 and log2 fold change > 1. did.

[0281] Functional GO enrichment and network analysis: Differentially expressed genomic features (p < 0.05; q ≤ 0.05) in M2 macrophages after RP-182 treatment were imported into Cytoscape (v. 3.7.1) to assess functional Gene Ontology enrichment and visualize GO term interaction networks. After import, functions were isolated by up- or down-regulation using the setsApp (v. 2.2.0) plugin. Once isolated, functional analysis and network building were completed using the ClueGO plugin (v. 2.5.4). KEGG pathway (v. 27.02.2019) enrichment was determined using a two-sided (enrichment / depletion) hypergeometric test with the Bonferroni step-down method. Analysis thresholds included an enrichment significance of p ≤ 0.05, at least 5% gene inclusion, and a kappa score threshold of 0.4 or higher. ClueGO uses the kappa score to determine the likelihood of GO term interactions and grouping.

[0282] Pathway Studio® analysis calculated differentially expressed gene sets in RR-182- vs. vehicle-treated M2 BMDMs. DESeq2 analysis (version 1.22.2; in R) yielded 1,224 differentially expressed genes (DEGs) with p ≤ 0.05 and 382 DEGs with FDR-adjusted q ≤ 0.05, which were used for gene ontology analysis in Mouse Genome Informatics (http: / / www.informatics.jax.org / ). The top 25 gene sets were enriched for analysis.

[0283] DEGs generated p-values ​​for enrichment and enrichment scores, both of which were incorporated into ranking metrics. Leading Edge Analysis was used to rank DEGs using Pathway Studio (registered trademark). ) Finder (https: / / www.pathwaystudio.com / ) identified the most common genes across enriched gene sets that remained consistent despite changes in estimated ranking metrics.

[0284] Single-cell RNA sequencing Normal pancreas and pancreatic tumors were harvested from treated and untreated KPC mice, and single-cell suspensions were prepared using a Mouse Tumor Dissociation Kit (#130-096-730, MACS Miltenyi Biotec, San Diego, CA) and a Gentle Macs Agitator (Miltenyi Biotec, San Diego, CA) according to the company's protocol. Using 9,000-12,000 cells, single-cell barcoded cDNA libraries were generated using a 10X Genomics Chromium Controller. Gene expression libraries were generated using a single-cell 5' library and gel bead kit (10X Genomics, Cat. #1000006). The manufacturer's instructions were followed to generate the libraries. The libraries were then sequenced in multiple runs on an Illumina NextSeq500 with V2 chemistry. The sequencing run for the gene expression library was configured as an asymmetric run of 26 cycles + 8 cycles + 57 cycles.

[0285] Single-cell transcriptome For single-cell gene expression libraries, demultiplexing, alignment to the mm10 transcriptome, and gene barcode matrix generation were performed for each dataset using the 10X Genomics Cellranger toolkit (v2.2.0). Library batches were normalized using the CellRanger Aggregate function to aggregate all treated and untreated tumor sample datasets together, and the resulting gene barcode matrix was fed into Seurat (v2.3.4). Each sample in the sample set was preprocessed in Seurat (v2.3.4) by removing genes detected in fewer than three cells and removing cells with fewer than 100 detected genes. Samples were then further processed using the global scaling normalization method LogNormalize, which normalizes each cell's gene expression measurement by total expression, multiplies this number by a scale factor (default 10,000), and log-transforms the result. The number of principal components to use in the analysis was estimated for each sample set using the calcPCA function in URD with the mp.factor parameter set to 2. The total number of significant principal components identified by this algorithm was used as the estimated number of principal components to be used in Seurat. Dimensionality reduction was performed in Seurat via principal component analysis, followed by clustering of the t-SNE visualization using the top significant components. Major clusters were identified by differentially expressed canonical marker genes and subjected to additional rounds of cluster refinement. For CD11b+ and Krt19, CD11c, and Ly6G-negative cells, the marker gene ITGAM was used to identify CD11b cells, and cells co-expressing the Krt19 gene (cytokeratin-19), ITGAX (CD11c), and LY6G were removed. The filtered gene barcode matrix of CD11b+ and Krt19, ITGAX, and LY6G-negative cells was used for cluster refinement and analysis. Differential expression analysis was performed using the EdgeR program. Differentially expressed genes from RP-182-treated M2 BMDMs (adjusted p-value < 0.05) were ranked and a fold-change cutoff (-1 < Log(FC) > 1) was applied.All genes above the fold-change p-value threshold reported by EdgeR were compared with their counterparts from the bulk RNASeq dataset and submitted to the GSEA program, where the FindMarkers function was used to identify marker genes discriminating specific subpopulations. Top markers, ranked by Bonferroni-adjusted p-values, are displayed on a log(10) fold-change color scale normalized across all cells.

[0286] Co-immunoprecipitation experiments M2 macrophages were resuspended in RPMI medium and treated with 100 μM peptide or PBS for 10 minutes at 37°C. Cells were pelleted and lysed in 500 μL of Pierce IP Lysis Buffer (Thermo Scientific, Cat# 87787) containing protease and phosphatase inhibitors (Thermo Scientific, Cat# 78440). To pull down CD206-associated proteins via biotinylated RP-182 or RP-426, 50 μL of streptavidin beads (Thermo Scientific, Cat# 65001) were added to the cleared cell lysate and incubated for 30 minutes at room temperature with gentle mixing. The beads were washed four times with PBS containing 0.05% Tween-20, incubated in PBS with 0.1% SDS, and boiled at 95°C for 5 minutes. The supernatant was then collected from the beads. To verify GRB2-interacting proteins, 25 μg of anti-GRB2 antibody was immobilized onto agarose resin using the Pierce Classic IP Kit (Thermo Scientific, Cat# 26146), and co-immunoprecipitation was performed according to the manufacturer's protocol. Specifically, lysates were incubated with the immobilized antibody overnight at 4°C with gentle rotation. The column was washed three times with PBS containing 0.25% Triton X100 and eluted using the provided elution buffer (Thermo Scientific, Cat# 21027). Samples were visualized by Western blotting using the indicated antibodies. The activated forms of the RhoGTPases Rac1 and CDC42 (GTP-Rac1 and GTP-CDC42) were measured with the RhoA / Rac1 / Cdc42 Combo Activation Assay Kit (Abcam, Cambridge, MA) according to the manufacturer's instructions.

[0287] Immunofluorescence assay Immunocytochemistry was performed using a Zeiss LSM 880 confocal microscope. Precursors were seeded into 8-well chamber slides and polarized into M1 and M2 macrophages. Cells were treated with 20 μM RP-182 for 2 hours at 37°C, then fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.3% Triton for 5 minutes, and blocked with 3% BSA in PBS for 1 hour. After blocking, cells were incubated with the respective primary antibodies (Table 6A-D) for 1 hour at room temperature. Secondary antibody staining was performed for 1 hour at room temperature, followed by washing and the addition of DAPI using mounting medium (H-1200 Vectashield, Burlingame, CA). Images were taken at 63x magnification, and three separate images for each treatment group containing approximately 200 cells were analyzed using ImagePro software (Media Cybernetics, Rockville, MD). The number of automatically counted bright objects (fluorescence of the secondary antibody for a specific protein) was normalized to the number of DAPI-stained nuclei. For relative comparison, the fluorescence ratio of vehicle-treated cells was set to 1.

[0288] Cell viability assay Dose-response curves in terms of cell viability were performed using the Live / Dead Viability Cytotoxicity Kit (#L Macrophages were determined using a 3224 ThermoFisher Scientific, Grand Island, NY. Macrophages were seeded in glass-bottom 96-well plates and polarized into M1 and M2 phases. Cells were treated with different concentrations of RP-182 and the control peptide PR-426, ranging from 0.01 μM to 100 μM, for 48 hours. After drug treatment, a 100 μL mixture of 2 μM calcein-AM and 4 μM ethidium homodimer was added and incubated for 1 hour. Images were captured using a similar method as described for the immunofluorescence assay. 200 cells were manually counted from three different random fields across three technical replicates, and the percentage of viable cells was calculated using GraphPad Prism version 7.0.

[0289] Animal models A colony of transgenic mice was established at the National Cancer Institute (NCI) in Bethesda, Maryland, and all animal experiments were conducted in accordance with the Animal Care and Use Committee (ACUC) of the National Institutes of Health. All animal studies were conducted in accordance with ACUC-approved protocols SB-210 and SB-211. Mice carrying the individual genes Pdx-1-cre, LSL-KrasG12D / +, Trp53R172H / +, and Ink4a(p16) / Arf(p19) floxed / floxed were obtained from the NCI's Mouse Repository, Frederick National Cancer Institute (https: / / frederick.cancer.gov / science / technology / mouserepository) and bred to generate animals with the triple genotypes Pdx-1-cre; LSL-KrasG12D / +; Ink4a(p16) / Arf(p19) floxed / floxed (KP16) or Pdx-1-cre; LSL-KrasG12D / +; LSL-Trp53R172H / + (KPC) (49, 60). B6.129P2-Mrc1tm1Mnz / J mice were obtained from The Jackson Laboratory (JAX stock #007620) ( 61 ). Genotypes were verified using PCR performed by Transnetyx, Inc. (Cordova, TN).

[0290] Human pancreatic cancer tissues for xenografting were obtained from the NCI Patient-Derived Model Repository (PDMR; https: / / pdmr.cancer.gov / ) initiative and subcutaneously implanted into NOD-scid IL2R gamma null (NSG) immunodeficient mice (F generation). After tumors reached 2 cm, they were explanted, cut into equal pieces, and regenerated in another generation (F generation). Therapeutic experiments were performed in F mice.

[0291] Syngeneic mouse models of cancer included the murine CD-26 colon cancer and B16 melanoma models. Approximately 1x10 6CT-26 cells / 100 μL medium were subcutaneously implanted into 6- to 8-week-old BALB / c mice. 3 Once tumor volume was reached, mice began treatment as outlined below. Area (mm 3 ) is calculated as (L × W2) / 2, where L = length (mm) and W = width (mm) are the dimensions of the two-dimensional carrier. The mice were measured during the perioperative period, and total body weight was recorded twice weekly. Two hours after the last injection, the mice were sacrificed, and tumors were excised, weighed, and fixed in formalin. Similarly, 0.5x10 6 Mouse B16 melanoma cells were injected subcutaneously into the flanks of BALB / c animals until tumors grew to approximately 50 to 100 mm 3 Treatment began when the volume reached 0.5x10 6 Human breast MDA-MB23, prostate C4-2, or KPC cells were injected subcutaneously into the flanks of homozygous athymic (nu / J) female nude mice. Tumors grew to 250 mm 3 After reaching 100 mm, KPC tumors were treated for 3 weeks and C4-2 tumors were treated for 4 weeks. 3 After reaching volume, MDA-MB231 tumors were treated for 6 weeks, at which time the draining lymph node basins were removed for H&E staining determination of the local metastatic index (number of lymph nodes involved with cancer per total number of lymph nodes in the basin excised and examined).

[0292] Animal Imaging Mice bearing the KP16 and KPC genotypes were imaged weekly by ultrasound from 6 weeks of age. Ultrasound imaging was performed using a 40 mHz transducer and a Vevo700 ultrasound machine (Visualsonics, Toronto, Canada). Mice were anesthetized with isoflurane (Baxter, Deerfield, IL), shaved, and injected intraperitoneally with 1.5 mL of saline (eBioscience, San Jose, CA). B-mode images were recorded to obtain tumor measurements.

[0293] Treatment Protocol KP16 and KPC mice were analyzed by flow cytometry, immune cell pull-down, or immunohistochemistry. Treat for 7 days prior to harvest of tumors used in the assay or until the predetermined study endpoint All animal treatments were initiated after pancreatic tumors of 4 to 5 mm or larger were confirmed by ultrasound examination and individual animals were randomized to treatment groups. Animal survival was measured from the first day of treatment until death. In control and treatment cohorts, animals were allowed to progress under continuous treatment conditions until the study endpoint was reached (determined as 20% weight loss, recognizable signs of illness, general lack of reflexes, abnormal posture, loss of mobility, respiratory distress, or inability to drink or feed). To avoid animal suffering, animals were euthanized in accordance with ACUC animal care guidelines. For experiments in KP16 and KPC mice, saline as vehicle, 20 mg / kg RP-182 (PolyPeptide Group, San Diego, CA), 50 mg / kg gemcitabine (Fresenius Kabi, Lake Zurich, IL), or RP-182 combined with gemcitabine were injected intraperitoneally (IP) in a final volume of 200 μL. RP-182 was injected every other day, and gemcitabine was injected twice weekly. Anti-PD-L1 (Biolegend, Cat. #124329) was administered intraperitoneally at 150 μg per mouse three times weekly. Mice were treated IP twice weekly with 100 μg of anti-CTLA-4 antibody (Bioxcell; 9D9). For CD8 depletion, mice received two doses of 100 μg of anti-mCD8 (8ioxcell, Cat. #BE0061) per mouse on days 1 and 5. Rat isotype control IgG1 (Bioxcell, Cat. #BE0090) was administered at an equivalent dose on the same schedule. Mice bearing CT-26, MDA-MB231, C4-2, and B16 tumors were administered 10 mg / kg of RP-182 daily via IP injection for tumor growth studies. Gemcitabine administration remained unchanged, and the docetaxel dose administered in the C4-2 model was replaced by 2.5 mg / kg of docetaxel daily for 7 days and then discontinued. For intratumoral injections, 50,000 BMDMs pretreated with vehicle or 20 μM RP-182 for 2 hours were injected into 500 m3 or larger KPC tumors grown in C57B / L wild-type mice on days 2, 5, 7, and 9.Prior to injection, M2 BMDMs grown and polarized on T75 flasks were washed twice, lifted, counted, and resuspended in HBSS in an injection volume of less than 50 μL.

[0294] Bleomycin pulmonary fibrosis model Animals were anesthetized for a short period to facilitate placement of intratracheal bleomycin. A single dose of 0.5 mg / kg (1-4 U / mg) of bleomycin in sterile isotonic saline (total volume 50 μL) was administered intratracheally via a 22-gauge plastic cannula to n = 12 BALB / c mice, followed by an equal volume of sterile saline. Saline was administered to the control group of mice (N = 6 mice). Patients were randomized on day 1 to receive 20 mg / kg RP via daily IP injection or vehicle control. Mice were weighed daily, and animal survival was measured from the first day of treatment until death. In the control and PR-182-treated cohorts, animals were allowed to progress under continuous treatment conditions until the study endpoint (determined by the study veterinarian as 20% weight loss, recognizable signs of disease, general lack of reflexes, abnormal posture, loss of mobility, labored breathing, inability to drink or feed, and moribund status with low survival rate). To avoid animal suffering, animals were euthanized in accordance with ACUC animal care guidelines. Only "warm" necropsy specimens (lungs) were used for histological analysis. Lungs were weighed before being fixed in formalin and embedded in paraffin, and stained with H&E, Masson's trichrome, and anti-CD206. Image J was used to quantify the level of fibrosis between the vehicle and RP-182-treated groups.

[0295] Flow cytometry analysis Multicolor flow cytometry analysis was performed after 7 days of treatment with RP-182, gemcitabine, the combination, or vehicle. After euthanasia of the animals, pancreatic tumors were harvested, washed with PBS, and pulverized with a scalpel.

[0296] Tumor lysates were digested using a Mouse Tumor Dissociation Kit (# 130-096-730, MACS Miltenyi Biotec, San Diego, CA) and a Gentle Macs Agitator (Miltenyi Biotec, San Diego, CA) according to the company's protocol. Tumor lysates were passed through a 70 μm filter, washed in PBS, and stained for flow cytometry analysis. BMDMs subjected to flow cytometry were treated with RP-182 and control peptides for 2 or 24 hours at 37°C before staining. Cells were stained with a Live / Dead Fixable Blue Dead Cell Stain Kit (ThermoFisher Scientific) using antibodies conjugated to the fluorophores listed in Tables 6A to D (Antibodies for Flow Cytometry). Stained cells were washed with FACS buffer before sample acquisition on a BD LSRFortessa SORP I flow cytometer (BD Bioscience). Flow cytometry data were analyzed using FlowJo software (TreeStar, Ashland, OR).

[0297] histology Harvested tumors were prepared for histological analysis using standard protocols and 4% paraformaldehyde. In addition to H&E staining, tumors were examined by immunocytochemistry and immunohistochemistry using the antibodies listed in Tables 6A to D (antibodies for tissue staining). A commercially available pancreatic cancer tissue microarray (TMA) containing 80 single cores (70 from adenocarcinoma and 10 from normal pancreas) was purchased from US Biomax, Inc., Maryland, for anti-CD206 staining. Brightfield images (immunostaining) were obtained using an Aperio ScanScope XT (Aperio, California, USA) for whole-slide scanning at 40x magnification. Images were acquired using a 3D scanner (Vista, California) and analyzed using ImageScope Analysis. Quantitative analysis was performed using the membrane algorithm. For immunofluorescence analysis, slides were deparaffinized as follows: two 5-minute xylene immersions, two 5-minute 100% ethanol immersions, and two 5-minute 100% ethanol immersions. Rehydration was performed in 95% ethanol for 5 minutes, 80% ethanol for 5 minutes, 70% ethanol for 5 minutes, and water for 5 minutes. After antigen retrieval after 60-minute incubation at 60°C, slides were blocked with H2O2 peroxidase, washed twice for 30 minutes in PBS blocking solution with FBS or NGS, and incubated with primary antibodies overnight at 4°C. Alexa 488 anti-mouse or Alexa 594 anti-rabbit secondary antibodies were incubated for 30 minutes at 27°C. After 1 minute of DAPI counterstaining, slides were then incubated overnight at 4°C with Alexa 488 anti-mouse or Alexa 594 anti-rabbit secondary antibodies. Coverslips were performed with lycerol mounting medium. Images were collected and analyzed using Zeiss AxioScan imaging.

[0298] TCGA gene expression data analysis Genomic data from the TCGA project are available from the National Cancer Institute's Genomic Data Commons ( https: / / gdc.cancer.gov / ). Gene-level gene expression data from RNA-seq experiments for all tumors ( N = 9,452) and pancreatic adenocarcinomas ( N = 125) were included in the analysis and correlated with individual gene expression levels and immune signature-based scoring as previously shown ( 62 , 63 ).

[0299] Isolation of tumor-associated macrophages and bone marrow-derived macrophages (BMDM) and qRT-PCR Tumor digests derived from KP16 and KPC tumors were analyzed using EasySep TMCD11b (clone M1 / 70, BD Biosciences) and Gr1 (clone RB6-8C5, Biolegend) antibodies were used to isolate macrophages from tumor digests derived from KP16 and KPC tumors. After tumor harvest and digestion, Gr-1 positive cells were first removed before isolating CD11b positive macrophages via magnetic cell isolation. Total RNA from TAM isolates and bone marrow-derived macrophages (BMDMs) was extracted using the RNeasy Mini Kit (reference #74104, Qiagen). Bone marrow-derived macrophages were sorted for the CD11b+GR1-F4 / 80+CD206+ population using a fluorescence-activated cell sorting (FACS) FACS sorter and lysates from the same cells used for RT-PCR. cDNA was generated using the Superscript III First - Strand (reference: #18080 -051, Invitrogen) synthesis system for RT-PCR. Following first-strand cDNA synthesis, individual primer master mixes (IL1b-Mm00434228_m1, TNFα-Mm00443258_m1, IL12-Mm01288989_m1, CD40-Mm00441891_m1, CLEC4e-Mm01183703_m1, CD86-Mm00444540, IL10- Mm01288386_m1, IL27-Mm00461162, PDL1-Mm00452054_m1, SIRPa- Mm00455928, Chil3-Mm00657889_m1, MRC1-Mm01339362_m1, actb-Mm02619580_g1; gapdh-Mm99999915_g1 (TaqMan Assays, ThermoFisher Scientific) was added, and RT-PCR reactions were performed and read on a BioRad CFX96 cycler. Target gene expression was calculated using the formula "relative gene expression 2 - (ΔCt)."where ΔCt is the cycle number of the target gene (Cttarget) normalized to the reference / housekeeping gene (Ctreference). Individual qRT-PCR reactions were run in triplicate and graphs were generated using GraphPad Prism.

[0300] ELISpot assay T cell reactivity against cancer cells was assessed in a 20-hour co-culture assay in flat-bottom 96-well PVDF membrane microtiter plates (Cat.# MAIPSWU10, EMD Millipore). CD8a+ T cells were isolated from single-cell suspensions of digested tumor or whole spleen using the EasySep™ Mouse CD8a Positive Selection Kit II (Cat.# 18953, StemCell). 1x10 5 4x10 KP16 cancer cells 4 The positive control contained CD8a+ T cells with PMA / ionomycin, and the negative control contained only CD8a+ T cells. Visualization of immobilized cytokines as "ImmunoSpots" was performed according to the manufacturer's instructions (Cat# 3321-2A, Mabtech). ELISpots were read and quantified on an ImmunoSpot S6 Universal Analyzer (CTL). 4x10 pulldowns were performed. 4 CD8+ T cells were added to 1x105 KP16 cancer cells, and Elispots were then analyzed. In triple co-culture experiments, 4x105 CD8+ T cells isolated from tumor and spleen from tumor-bearing animals via CD11b pulldown after negative selection for Gr-1 were added. 4 TAMs were isolated from the spleens of tumor-bearing mice using 1x10 KP16 cells and 4x10 4 CD8+ T cells were added to the tumor cells. After 20 hours of co-culture, T cell reactivity against the tumor cells was assessed as described above.

[0301] Phagocytosis assay The ability of BMDMs and TAMs to phagocytose cancer cells was analyzed by confocal microscopy and flow cytometry. Cancer cell lines, including KPC, PANC1, HPAF-II (pancreatic cancer), primary melanoma tissue culture line 2183, and LNCaP (prostate cancer), were labeled with CFSE (5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester) dye (#C1157, ThermoFisher Scientific, Grand Island, NY) for 1 hour at 37°C and 5% CO2 according to the manufacturer's instructions. CFSE-labeled cancer cells were added to BMDMs pretreated with RP182 or vehicle (2 hours) and incubated for 6 hours before washing twice to remove excess cancer cells. Images were taken using a Zeiss LSM 880 confocal microscope at 63x magnification using the green and phase contrast channels. For flow cytometry, CFSE-labeled cancer cells were incubated with macrophages pretreated with RP-182 and vehicle (2 h) and cultured in a T75 flask for 4 h. Excess CFSE-labeled cells were washed away, and macrophages were harvested from the T75 flask and analyzed using a BD LSRFortessa SORP I flow cytometer (BD Bioscience). For phagocytosis assays involving beads, the pHrodo™ Red E. coli BioParticles™ Phagocytosis Kit (#A10025, ThermoFisher Scientific, Grand Island, NY) was used. Macrophage polarization, treatment, and flow and immunofluorescence assay analysis were performed in a similar manner as described in previous experiments.

[0302] statistical analysis Data were analyzed using SPSS software version 16 (IBM, Armonk, NY). Data were statistically analyzed. Tumor volume was assessed as the best objective response (BOR), follow-up measurements, and Best recorded response from the start of study treatment compared with either 3) were used to compare the four groups. Continuous data, including tumor volume, gene expression levels, or immune cell population percentages, were analyzed using Student's t-test in GraphPad Prism. Kaplan-Meier curves were compared using the log-rank test. Bars indicate standard error of the mean (SEM) unless otherwise indicated. Calculated p-values ​​are given with numbers and asterisk(es) indicating p<0.05, ** p<0.01, and *** p<0.001.

[0303] Notwithstanding the appended claims, the disclosure set forth herein is also defined by the following appendix. 1. A method for modulating macrophage activity, comprising contacting macrophages with a CD206-binding agent. and modulating macrophage activity. 2. The CD206-binding agent binds to a site selected from the fibronectin II domain of CD206, the C-type lectin carbohydrate recognition domain 3 (CRD3) of CD206, the C-type lectin carbohydrate recognition domain 4 (CRD4) of CD206, and the C-type lectin carbohydrate recognition domain 5 (CRD5) of CD206. , the method described in Appendix 1. 3. The method of claim 1, wherein the CD206-binding agent binds to CD206 with a binding energy of at least -650 / mol. 4. The method of claim 1, wherein the modulated macrophage activity is macrophage polarization. 5. The method of claim 1, wherein the viability of the macrophages is reduced.

[0304] 6. The macrophages are M2 macrophages or tumor-associated macrophages (TAMs). The method according to claim 1, 7. The method of claim 1, wherein the CD206-binding agent inhibits macrophage activity. 8. The method of claim 1, wherein the CD206-binding agent induces apoptosis of the macrophages. How to post. 9. The method of claim 1, wherein the CD206-binding agent stimulates phagocytosis. 10. The method according to any one of appendices 1 to 9, wherein the macrophages are in vitro. Law.

[0305] 11. The method according to any one of appendices 1 to 9, wherein the macrophages are in vivo. . 12. The method of any one of claims 1 to 11, wherein the CD206-binding agent is an immunomodulatory peptide. How to post. 13. The immunomodulatory peptide is 5 to 18 amino acid residues in length, 13. The method of claim 12, wherein the domain comprises a striatal region of alternating hydrophilic and hydrophobic modules that adopt an amphipathic conformation under physiological conditions. 14. The method of claim 13, wherein the striatal region comprises: three or more hydrophobic modules, and two or more hydrophilic modules, each comprising at least one cationic residue, wherein the immunomodulatory peptide specifically binds to CD206. 15. The immunomodulatory peptide of claim 12, wherein the immunomodulatory peptide comprises a sequence defined by one of the following formulas: A method according to any one of claims 1 to 14. [J 1a J 1b ]-[X 1a X 1b ]-[J 2a J 2b ]-[X 2a X 2b ]-[J 3a ]-[X 3a ]; and [X 3a ]-[J 3a ]-[X 2b X 2a ]-[J 2b J 2a ]-[X 1b X 1a ]-[J 1b J 1a]; Here, J 1a , J 1b , J 2a , J 2b and J. 3a are each independently selected from phenylalanine, tryptophan, alanine, valine, and glycine; 1a , X 1b , X 2a , X 2b oh Call X 3a are independently lysine, arginine, histidine, aspartic acid, and glutamic acid. The amino acid is selected from the group consisting of glutamine, thiamin, asparagine, and thiamin.

[0306] 16. A method according to claim 15, which satisfies the following: J 1a , J 1b , J 2a , J 2b and J. 3a are phenylalanine, respectively, X 1a , X 1b , X 2a , X 2b and X 3a are each independently selected from lysine and arginine will be done. 17. The method of any one of appendices 12 to 16, wherein the immunomodulatory peptide comprises: a) a sequence selected from: KFRKAFKRFF (RP182); FFRKFAKRFK (RP183); FFKKFFKKFK (RP185); FFKKFFKKFK (RP186); and FFKKFFKKFK (RP233); or b) A sequence having one or two amino acid substitutions relative to the sequence defined in a). 18. The method of claim 17, wherein the immunomodulatory peptide comprises the amino acid sequence KFRKAFKRFF (RP182). 19. The method of claim 17, wherein the immunomodulatory peptide comprises the amino acid sequence FFRKFAKRFK (RP183). 20. The method of claim 17, wherein the immunomodulatory peptide comprises the amino acid sequence FFKKFFKKFK (RP185).

[0307] 21. The method of any one of appendices 12 to 16, wherein the immunomodulatory peptide comprises: a) a peptide sequence selected from: RWKFGGFKWR (RP832C); FKWRGGRWKF (RP837C); FWKRGGRKWF (RP837A); FWKRFV (RP837N); FVRKWR (RP837C1); FAOOFAOOFO (RP850); FWKRFVRKWR (RP837); FWKKFVKKWK (RP841); WWHHWWHHWH (RP847); WWRHWWHRWR (RP848); WWKHWWHKWK (RP849); GDRGIKGHRGF (RP842); LYKKIIKKLL (RP846); FYPDFFKKFF (RP844); FFRKSKEKIG (RP853); FFRHFATHLD (RP845); and EKLSAFRNFF (RP843); or b) A sequence having one or two amino acid substitutions relative to the sequence defined in a). 22.b) One or two amino acid substitutions as defined in the cationic amino acids of the sequence The method of claim 21, consisting of highly conservative substitutions. 23. The method of claim 21, comprising a peptide sequence selected from the following: RWKFGGFKWR(RP832C), FKWRGGRWKF(RP837C) and FWKRGGRKWF(RP837A). 24. Contains a peptide sequence selected from FWKRFV (RP837N) and FVRKWR (RP837C1). , the method described in Appendix 21. 25.Select from FAOOFAOOFO (RP850), FWKRFVRKWR (RP837) and FWKKFVKKWK (RP841). 22. T...

Claims

1. 1. A method for modulating macrophage activity, comprising: contacting a macrophage with a CD206-binding agent to modulate the activity of said macrophage; and A method comprising:

2. the CD206-binding agent binds to a site selected from the fibronectin II domain of CD206, C-type lectin carbohydrate recognition domain 3 (CRD3) of CD206, C-type lectin carbohydrate recognition domain 4 (CRD4) of CD206, and C-type lectin carbohydrate recognition domain 5 (CRD5) of CD206. The method according to claim 1.

3. the CD206-binding agent binds to CD206 with a binding energy of at least -650 kcal / mol. The method of claim 1.

4. 2. The method of claim 1, wherein the modulated macrophage activity is macrophage polarization. method.

5. The method of claim 1 , wherein the viability of the macrophages is reduced.

6. The macrophages are M2 macrophages or tumor-associated macrophages (TAMs). The method of claim 1 .

7. The method of claim 1 , wherein at least one of the CD206-binding agents inhibits macrophage activity, induces apoptosis of the macrophages, and stimulates phagocytosis.

8. The method of any one of claims 1 to 7, wherein the macrophages are in vitro.

9. The method of any one of claims 1 to 7, wherein the macrophage is in vivo.

10. 10. The method of claim 1, wherein the CD206-binding agent is an immunomodulatory peptide. method.

11. The immunomodulatory peptide is 5 to 18 amino acid residues in length, The method of claim 10, wherein the peptide comprises a striatal region of alternating hydrophilic and hydrophobic modules that adopts an amphipathic conformation under physiological conditions.

12. The striatal region is three or more hydrophobic modules, and two or more hydrophilic modules, each containing at least one cationic residue; It includes, the immunomodulatory peptide specifically binds to CD206; The method of claim 11.

13. 10. The immunomodulatory peptide of claim 10, wherein the immunomodulatory peptide comprises a sequence defined by one of the following formulas:

13. The method according to any one of claims 1 to 12. [J 1a J 1b ]-[X 1a X 1b ]-[J 2a J 2b ]-[X 2a X 2b ]-[J 3a ]-[X 3a ]; and [X 3a ]-[J 3a ]-[X 2b X 2a ]-[J 2b J 2a ]-[X 1b X 1a ]-[J 1b J 1a ]; Here, J 1a , J 1b , J 2a , J 2b and J. 3a are each independently selected from phenylalanine, tryptophan, alanine, valine, and glycine; 1a , X 1b , X 2a , X 2b oh Call X 3a are independently lysine, arginine, histidine, aspartic acid, and glutamic acid. The amino acid is selected from the group consisting of glutamine, thiamin, asparagine, and thiamin.

14. J 1a , J 1b , J 2a , J 2b and J. 3a are phenylalanine, respectively, X 1a , X 1b , X 2a , X 2b and X 3a are each independently selected from lysine and arginine will be The method of claim 13.

15. The immunomodulatory peptide a) a sequence selected from: KFRKAFKRFF (RP182); FFRKFAKRFK (RP183); FFKKFFKKFK (RP185); FFKKFFKKFK (RP186); and FFKKFFKKFK (RP233); or b) a sequence having one or two amino acid substitutions relative to the sequence defined in a); Including, The method according to any one of claims 10 to 14.

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