Polymer engineered forms of colony stimulating factor-1

Abstract

Conjugates of a CSF-1 moiety and one or more water soluble polymers are provided. Typically, the water soluble polymer is a poly(ethylene glycol) or a derivative thereof. Also provided are compositions comprising conjugates, methods of making conjugates, methods of using the conjugates or compositions in therapeutic treatments, methods of administering conjugates or compositions comprising the conjugates to a patient, and kits comprising conjugates.

Description

POLYMER ENGINEERED FORMS OF COLONY STIMULATING FACTOR- 1CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. 119(e) to U.S. Provisional Patent Application No. 63 / 721,171, filed on November 15, 2024; and to U.S. Provisional Patent Application No. 63 / 717,170, filed November 6, 2024; and to U.S. Provisional Patent Application No. 63 / 696,286, filed September 18, 2024, the disclosures of which are incorporated herein by reference in their entireties.REFERENCE TO A SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (SHE0597_PCT_seqlist.ST26.xml; Size: 7000 bytes; and Date of Creation: September 15, 2025) is herein incorporated by reference in its entirety.FIELD

[0003] The present disclosure relates generally to conjugates comprising a colony stimulating factor 1 (CSF-1) moiety (i.e., a moiety having CSF-1 activity) and a polymer. In addition, the disclosure relates to (among other things) compositions comprising the conjugates, combinations comprising the conjugates, methods for synthesizing the conjugates, and methods for using the conjugates.BACKGROUND

[0004] The current standards of care for chronic inflammatory disease therapies are designed to suppress inflammation and are not optimized for inflammation resolution and / or restoration of tissue homeostasis and function. In general, chronic inflammation or chronic inflammatory disease involves a prolonged inflammatory state often lasting weeks, months or years. This unmet need requires a new class of therapeutics that provide and / or complement current antiinflammatory treatments to enable one or more of resolution of inflammation, tissue repair, extended remission duration, increased remission rates, and / or potentially curative responses.

[0005] Monocytes and tissue macrophages are essential cell types to maintain tissue homeostasis and trigger tissue repair after tissue injuiy for re-establishment of homeostasis. The cytokineCSF-1 , also known as macrophage colony stimulating factor or M-CSF, has a central role in maintaining and differentiating myeloid cell types including tissue macrophage populations as well as in activating macrophages to respond to tissue injury. Through interaction via the CSF-1 receptor, the CSF-1 cytokine activates several signaling pathways directing macrophage functional activation in synergy with other extracellular signals leading to diverse responses from inflammation induction to tissue repair and tissue-specific homeostatic functions. Reporter mice expressing fluorescent proteins under the control of CSF-1 receptor gene promoter in the native CSF-1 R gene locus indicate that tissue macrophages express higher levels of CSF1-R compared to peripheral blood monocytes (Hawley et al., J Immunol, 200(5):2209-2223, 2018). CSF-1 receptor agonists with low receptor affinity may therefore favor selective activation of tissue macrophages to elicit therapeutically beneficial functions like stimulating wound healing and anti-inflammatory activity. PI3K / Akt signaling pathway activation in macrophages has been associated with transition from local inflammation and tissue injury to tissue repair and homeostasis re-establishment in multiple tissues.

[0006] Recombinant human CSF-1 (rhCSF-1) was first produced at Cetus Corporation (later Chiron Corporation) in 1989 (Halenbeck et al., Biotechnology, 7:710-715, 1989). This rhCSF-1 is a 218-amino-acid polypeptide that closely resembles secreted native CSF-1 recovered from human urine. CSF-1 was investigated clinically in the treatment of presbyopia (age related loss in near vision) and in bone marrow transplantation patients whose innate immune system has not been fully restored, and consequently suffer from recurrent fungal and bacterial infections due to the lack of myeloid cells.

[0007] Post-translational modifications of native CSF-1 are extensive, including glycosylation and chondroitin sulfate addition, but are not required for the activity of CSF-1 in vitro. Deletion studies on recombinant CSF-1 have shown that in vitro bioactivity is fully retained in analogs consisting of the first 150 amino acids of mature CSF-lα, a region containing the core of CSF-1 that is common to all of the splice variants. The three-dimensional structure of this region (amino acids 4-154 of CSF-la) has been determined and shows to contain a unique four- helical structure that defines a family of hormones with which CSF-1 shares little significant amino acid sequence identity.

[0008] Administration of an exogenous CSF-1 or a CSF-l-Fc fusion cytokine has been investigated and has been shown to improve tissue damage recovery in certain pre-clinical injurymodels (e.g liver fibrosis, CNS demyelination, renal tubular injury) However, administration of these compounds was also found to induce systemic monocytosis and inflammatory monocyte tissue infiltration (Alikhan, et al.. Am J Pathol, 179(3): 1243-1256, 2001. doi:10.1016 / j. ajpath.2011.05.037; Laflamme et al., Front. Cell. Neurosci., 12:178, 2018. doi: 10.3389 / fncel.2018.00178; Keshvari et al„ Dis Model Meeh., 15(4):dmm049389, 2022. doi: 10.1242 / dmm.049387; Sauter et al., Am J Physiol Gastrointest Liver Physiol, 311 :G533-G547, 2016. doi: 10.1152 / ajpgi.OOl 16.2016).

[0009] Notwithstanding the foregoing approaches, however, there remains a need for new CSF-1 compounds having improved characteristics and profiles. Described herein are polymer conjugated CSF-1 and variants with favorable pharmacokinetic-pharmacodynamic (PKPD) properties that may be used for immunotherapies such as acute or chronic inflammation and tissue injury conditions including fibrotic diseases and neuronal tissue injury.SUMMARY

[0010] In a first aspect, provided is a conjugate comprising a CSF-1 moiety covalently attached to a water-soluble polymer. In some preferred embodiments, the water-soluble polymer is a branched water-soluble polymer. In embodiments, the water-soluble polymer is selected from the group consisting of poly(alkylene oxide), poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline, and poly(acryloylmorpholine). In some preferred embodiments, the water- soluble polymer is a poly(alkylene oxide) polymer. In one particularly preferred embodiment, the poly(alkylene oxide) polymer is a poly(ethylene glycol) polymer. In a preferred embodiment, the water-soluble polymer is a branched poly(alkylene oxide) polymer. In a more preferred embodiment, the water-soluble polymer is a branched poly(ethylene glycol) polymer. In further embodiments, the poly(ethylene glycol) polymer is terminally capped with an end-capping moiety selected from the group consisting of hydroxy, alkoxy, substituted alkoxy, alkenoxy, substituted alkenoxy, alkynoxy, substituted alkynoxy, aryloxy and substituted aryloxy. In one particular embodiment, the poly(ethylene glycol) polymer is terminally capped with methoxy.

[0011] In embodiments, the water-soluble polymer has a weight-average molecular weight in a range of from about 500 daltons to about 100,000 daltons. In further embodiments, the water-soluble polymer has a total weight-average molecular weight in the range of from 5,000daltons, or greater than, to about 150,000 daltons. In even further embodiments, the water- soluble polymer has a total weight-average molecular weight in the range of from about 6,000 daltons to about 100,000 daltons. In additional embodiments, the water-soluble polymer has a total weight-average molecular weight in the range of from about 15,000 daltons to about 85,000 daltons. In some embodiments where the water-soluble polymer is a poly(ethylene glycol) polymer, the poly( ethylene glycol) polymer has a total weight-average molecular weight in the range of from about 20,000 daltons to about 85,000 daltons. In further embodiments, the poly(ethylene glycol) polymer has a total weight average molecular weight in the range of from about 20,000 daltons to about 60,000 daltons. In additional embodiments, the poly(ethylene glycol) polymer has a total weight average molecular weight in the range of from about 10,000 daltons to about 60,000 daltons. In other embodiments, the polyethylene glycol) polymer has a total weight average molecular weight in the range from about 10,000 daltons to about 40,000 daltons. In one particular embodiment, the poly(ethylene glycol) polymer has a total weight average molecular weight of about 40,000 daltons.

[0012] In additional embodiments, one, two or three water-soluble polymers are attached to the CSF-1 moiety. In a preferred embodiment, one water-soluble polymer is attached to the CSF-1 moiety.

[0013] In some further embodiments, the conjugate comprises the structure:wherein each (n) is independently an integer having a value of from 3 to 4000; X2 when present, is a spacer moiety comprised of one or more atoms; (b) is 2 through 6; (c) is 2 through 6; R2, in each occurrence, is independently H or lower alkyl; and CSF-1 is a CSF-1 moiety. In further embodiments, the conjugates comprise the structure:. In even further embodiments, the water-soluble polymer comprises the structure:wherein each (n) is independently an integer having a value of from 3 to 4000.

[0014] In some embodiments, the CSF-1 moiety is selected from the group consisting of a human CSF-1, and biologically active fragments, deletion variants, substitution variants or addition variants of any of the foregoing. In further embodiments, the CSF-1 moiety is recombinantly derived. In one preferred embodiment the CSF-1 moiety is a mutein, which may be a Hl 5A mutation.

[0015] In a second aspect, a pharmaceutical composition is provided. The composition comprises at least a conjugate comprising a CSF-1 moiety covalently attached to a water-soluble polymer as described above and herein and a pharmaceutically acceptable excipient.

[0016] In a third aspect, provided herein is a combination comprising the conjugate or composition as described above and herein, and at least one anti-inflammatory compound.

[0017] In a fourth aspect, provided is a method of treating a condition that is responsive to treatment with CSF-1 by administering to a subject having the condition a therapeutically effective dose of a conjugate as described above and herein. In some embodiments, the condition is selected from a disease characterized by a deficiency in CSF-1 or deficiency in CSF- 1 receptor function, an inflammatory disease, an autoimmune disease, or a fibrotic disease. In a particular embodiment, the inflammatory disease is characterized by chronic inflammation. In another particular embodiment, the inflammatory disease is characterized by acute inflammation. In some embodiments, the disease is selected from systemic lupus erythematosus (SLE), ulcerative colitis, Crohn’s disease, rheumatoid arthritis, psoriatic arthritis, plaque psoriasis, juvenile idiopathic arthritis, atopic dermatitis, systemic sclerosis, ankylosing spondylitis, graft versus host disease (GVHD), Hidradenitis suppurativa, uveitis, polymyositis, organ-specific autoimmune diseases including type 1 diabetes, Addison’s disease, Hashimoto thyroiditis, Graves’ disease, Sjogren’s syndrome, Chronic obstructive pulmonary disease (COPD), vitiligo, pernicious anemia, glomerulonephritis, myasthenia gravis, Goodpasture’s syndrome, autoimmune hemolytic anemia, idiopathic thrombocytopenia purpura, allergic inflammation including peanut allergy, alopecia, inflammatory bowel disease (IBD), multiple sclerosis,autoimmune hepatitis, hepatic fibrosis, and pulmonary fibrosis. In some preferred embodiments, the method further comprises further administering at least one anti-inflammatory compound. In these embodiment, the at least one anti-inflammatory compound may be selected from a tumor necrosis factor-alpha inhibitor, a tyrosine kinase 2 inhibitor, a Janus kinase inhibitor, an antibody that targets interleukin-4 receptor-alpha, an antibody that targets interleukin- 13, an antibody that targets interleukin-23, and / or a corticosteroid.

[0018] In some preferred embodiments, at least one anti-inflammatory compound is administered concurrently or in a staggered treatment timing either preceding or following the treatment with CSF-1 polymer conjugates as described herein.BRIEF DESCRIPTION OF THE FIGURES

[0019] FIG. I provides the DNA sequence of native human mature CSF-1 (hCSF-1) (FIG. 1A, SEQ ID NO:1); the amino acid sequence of native human mature CSF-1 (hCSF-1) protein (FIG. IB, SEQ ID NO:2); the DNA sequence of the H15A mutein of hCSF-1 (FIG. 1C, SEQ ID NO:3); the H15A mutein of hCSF-1 with an included N-terminal methionine for translation initiation (FIG. ID, SEQ ID NO4), where the numbering starts with El as the first M is cleaved; the H15A mutein of hCSF-1 (FIG. IE, SEQ ID NO:5).

[0020] FIG. 2 is a graph showing a reverse phase-HPLC chromatogram analysis showing amounts of native CSF-1, monoPEGylated CSF-1 (1PEG), di-PEGylated CSF-1 (2PEG), and tri- PEGylated CSF-1 (3PEG) obtained using an exemplary method of preparing CSF-1 conjugates as detailed in Example 3.

[0021] FIG. 3 is a graph showing the plasma concentration (ng / mL, mean±SD) of 5 mg / kg CSF- 1 (o), 5 mg / kg Compound 5 (▼), 5 mg / kg Compound 7 (A), or 5 mg / kg Compound 2 (■) after intravenous injection in mice for 0 to 240 hours post injection as detailed in Example 6.

[0022] FIG. 4 is a graph showing the plasma concentration (ng / mL, mean±SD) of 3 mg / kg Compound 1 (■), 1 mg / kg Compound 1 (□), 3 mg / kg Compound 2 (A), or 1 mg / kg Compound 2 (A) after intravenous injection in mice for 0 to 240 hours post injection as detailed in Example 6.

[0023] FIGS. 5A-5B are graphs showing the change in paw volume (mL, from pre) at 14, 21, or 28 days after the first immunization as detailed in Example 12. FIG. 5A is a graph showing the change in paw volume at 14 days, 21 days, and 28 days after first immunization for the normal treatment group (non-immunized) (•), and for rats administered a CIA control (■), etanerceptCompound 2 (▼), or a combination of Compound 2 and etanercept (o). (***P<0.001,Student’s t-test, ###P<0.001, Wilcoxon test). FIG. 5B is a graph showing the change in paw volume (mL, from pre) at 14 days, 21 days, and 28 days after first immunization for the normal treatment group (non-immunized) (•), and for rats administered a CIA control (■), etanercept (▲), Compound 1 (♦), or a combination of Compound 1 and etanercept (□). (***P<0.001, Student’s t-test, ###P<0.001, Wilcoxon test). FIG. 5C is a graph of the area under the curve (AUC) for the change in paw volume (mean ± SEM) for (from left to right) the normal treatment group (non-immunized), rats administered a CIA control, etanercept, Compound 1, or a combination of Compound 1 and etanercept as detailed in Example 12. (***P<0.001, **P<0.0024, 1-way ANOVA).

[0024] FIG. 6 is a graph showing the body weight change (g) from Day 14 to Day 28 after first immunization for the normal treatment group (non-immunized) (***P<0.001, Student’s t-test), and for rats administered a CIA control, etanercept, Compound 2, or a combination of Compound 2 and etanercept as detailed in Example 12.

[0025] FIG. 7 is a graph showing the body weight change (g) from Day 14 to Day 28 after first immunization for the normal treatment group (non-immunized) (***P<0.001, Student’s t-test), and for rats administered a CIA control, etanercept, Compound 1, or a combination of Compound 1 and etanercept as detailed in Example 12.

[0026] FIG. 8A is a graph of synovial tissue weight (mg) for the normal treatment group (nonimmunized) (*P<0.05, Student’s t-test), and for rats administered a CIA control, etanercept, Compound 2, or a combination of Compound 2 and etanercept in a rat collagen-induced arthritis model as detailed in Example 12. FIG. 8B is a graph of synovial tissue weight (mg) for the normal treatment group (non-immunized) (*P<0.05, Student’s t-test), and for rats administered a CIA control, etanercept, Compound 1, or a combination of Compound 1 and etanercept in a rat collagen-induced arthritis model as detailed in Example 12. FIG. 8C is a graph of the monocyte cell count in peripheral blood for the normal treatmentgroup (non-immunized), and for rats administered a CIA control, etanercept, Compound 1, or a combination of Compound 1 and etanercept in a rat collagen-induced arthritis model as detailed in Example 12.

[0027] FIG. 8D is a graph of the al AGP plasma concentration (pg / pl) (mean ± SEM) for the normal treatment group (non-immunized), and for rats administered a CIA control, etanercept,Compound 1 , or a combination of Compound 1 and etanercept in a rat collagen-induced arthritis model as detailed in Example 12.

[0028] FIGS. 9A-9D are graphs showing the percent positive phospho-extracellular signal- regulated kinase (pERK) (% Positive pERK) inmonocytes (FIG. 9 A),monocytes (FIG. 9B),(FIG. 9C), andneutrophils (FIG. 9D) for specified concentrations of CSF-1 (•), Compound 1 (A), Compound 2 (■), or a vehicle ( ▼ ), as a test agent [TA] as detailed in Example 7. FIGS. 9E-9H are graphs showing the percent positive phospho-Akt kinase (pAkt) (% Positive pAkt) in Ly6Ch,ghmonocytes (FIG. 9E),(FIG. 9F),monocytes (FIG. 9G), andneutrophils (FIG. 9H) for specified concentrations of CSF-1 (•), Compound 1 (A), Compound 2 (■), or a vehicle (▼), as a test agent [TA] as detailed in Example 7.

[0029] FIGS. 10A-10D are graphs showing the percent positive pERK (% Positive pERK) inmonocytes (FIG. 10 A),(FIG. 10B),(FIG. 10C), and Ly6G:neutrophils (FIG. 10D) at 4 hours and on each of Days 1, 2, and 3 for 0.3 mg / kg Compound 2 (• indicated with arrow, green), 0.1 mg / kg Compound 2 (■), 0.03 mg / kg Compound 2 (A), 1 mg / kg Compound 1 (▼), 0.3 mg / kg Compound 1 (♦), 0.1 mg / kg Compound 1 (• orange), or a vehicle (□) as detailed in Example 8. FIGS. 10E-10H are graphs showing the percent positive pAkt (% Positive pAkt) in (FIG. 10E),monocytes (FIG. 10F),monocytes (FIG. 10G), and Ly6G neutrophils (FIG. 10H) at 4 hours and on each of Days 1, 2, and 3 for 0.3 mg / kg Compound 2 (• indicated with arrow, green), 0.1 mg / kg Compound 2 (■), 0.03 mg / kg Compound 2 (A), 1 mg / kg Compound 1 (▼), 0.3 mg / kg Compound 1 (♦), 0.1 mg / kg Compound 1 (• orange), or a vehicle (□) as detailed in Example 8.

[0030] FIGSs 11 A-l 1C are graphs illustrating Ki67 expression (expressed as a percentage) (%Ki67+) inmonocytes in blood (FIG. 11 A), inin blood (FIG. 11B),monocytes in blood (FIG. 11C) at 2 or 6 days post administration for each of a vehicle, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 10 mg / kg or 30 mg / kg of Compound 1, or 3 mg / kg of Compound 2 as detailed in Example 9.

[0031] FIGS. 12A-12B are graphs illustrating Ki67 expression (%Ki67+) in Kupffer cells in liver (FIG. 12A) or in monocyte derived macrophages (FIG. 12B) at 2 or 6 days postadministration for each of a vehicle, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 10 mg / kg or 30 mg / kg of Compound 1, or 3 mg / kg of Compound 2 as detailed in Example 9.

[0032] FIGS. 13 A-13C are graphs showing the cell count in blood of monocyte subsets(FIG. 13A),(FIG. 13B) or(FIG. 13C) at 2, 3 or 6 days post administration of 3 mg / kg Compound 2 (•), 1 mg / kg Compound 2 (■), 0.3 mg / kg Compound 2 (A), 3 mg / kg Compound 1 (o), 1 mg / kg Compound 1 (□), 0.3 mg / kg Compound 1 (A), or native CSF-1 (♦) as detailed in Example 9.

[0033] FIGS. 14A-14C are graphs showing the macrophage subsets and monocytes cell count in liver for Kupffer cells (FIG. 14A), monocyte derived macrophages (FIG. 14B), or monocytes (FIG. 14C) at 2, 3 or 6 days post administration of 3 mg / kg Compound 2 (•), 1 mg / kg Compound 2 (■), 0.3 mg / kg Compound 2 (A), 3 mg / kg Compound 1 (o), 1 mg / kg Compound 1 (□), 0.3 mg / kg Compound 1 (A), or native CSF-1 (♦) as detailed in Example 9.

[0034] FIG. 15 is a graph showing activation of metalloprotease sensor (%MMPsense+- cells) in Kupffer cells at 2, 3, or 6 days after administration of 3 mg / kg Compound ! (▼), ! mg / kg Compound 1 (♦), 0.3 mg / kg Compound 1 (•, dashed line), 3 mg / kg Compound 2 (e, solid line), 1 mg / kg Compound 2 (■), 0.3 mg / kg Compound 2 (A), or a vehicle (o) to mice as detailed in Example 10.

[0035] FIG. 16 is a graph showing induction of IL-4Ra (%IL-4Ra+) in Kupffer cells at 2, 3, or 6 days after administration of 3 mg / kg Compound 1 (o, dashed line), 1 mg / kg Compound 1 (□), 0.3 mg / kg Compound 1 (A), 3 mg / kg Compound 2 (•), 1 mg / kg Compound 2 (■), 0.3 mg / kg Compound 2 (A), or a vehicle (o, solid line) to mice as detailed in Example 10.

[0036] FIG. 17 is a graph showing induction of IL-lORa (%IL-10Ra+) in Kupffer cells at 2, 6, or 10 days after administration of 3 mg / kg Compound 1 (A), 1 mg / kg Compound 2 (■) or a vehicle (•) to mice as detailed in Example 10. The vehicle was tested only at Day 2 after administration.

[0037] FIG. 18 is a graph showing expression of the efferocytosis receptor MerTK (%MerTK+) in Kupffer cells at 2, 3, or 6 days after administration of 3 mg / kg Compound 1 (▼), 1 mg / kg Compound 1 (♦), 0.3 mg / kg Compound 1 (•, dashed line), 3 mg / kg Compound 21 mg / kg Compound 2 (■), 0.3 mg / kg Compound 2 (A), or naive mice (o, solid line) to mice as detailed in Example 10.

[0038] FIG. 19 is a graph showing the effect of Compoundor Compound 2 (▼) on the DSS colitis disease score (DAI score) as compared to no disease control (o) or a DSS vehicle (•)at 5, 9, 14, 17, or 21 days past administration in a dextran sulfate sodium (DSS)-induced colitis mouse model as detailed in Example 11.

[0039] FIG. 20 is a graph showing the effect of a vehicle, Compound 2, or Compound 1 on the DAI score (ACC) during the inflammation resolution phase (days 14-20 after administration) for a vehicle, Compound 2 or Compound 1 in aDSS-induced colitis mouse model as detailed in Example 11.DETAILED DESCRIPTION

[0040] Before describing one or more embodiments in detail, it is to be understood that the present disclosure is not intended to be limited to the particular polymers, synthetic techniques, CSF-1 moieties, assays, and the like, as such may vary as would be understood by one having ordinary skill in the art to which this disclosure applies.

[0041] In describing and claiming certain features of this disclosure, the following terminology will be used in accordance with the definitions described below unless indicated otherwise.

[0042] It must be noted that, as used in this specification and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polymer" includes a single polymer as well as two or more of the same or different polymers, reference to "a pharmaceutically acceptable excipient" refers to a single pharmaceutically acceptable excipient as well as two or more of the same or different pharmaceutically acceptable excipients, and the like.

[0043] The term “colony stimulating factor-1” or “CSF-1”, as used herein, refers to a polypeptide or protein having human CSF-1 activity. The CSF-1 as referenced for use herein may be derived from the natural / wild type protein or may be a “CSF-1 mutein” modified from the natural protein or polypeptide deliberately, as for example, by site directed mutagenesis to include at least one amino acid modification, insertion or substitution. An exemplary CSF-1 mutein comprises an amino acid sequence corresponding to SEQ ID NOs:4-5, as well as any protein or polypeptide substantially homologous thereto. In embodiments, exemplary CSF-1 muteins comprise an amino acid sequence having at least 90%, 95%, or 99% homology to at least one of SEQ ID NOs:2 and 4-5. Further, “CSF-1” as used herein may refer to either of CSF- 1 in dimer form or as a monomer unless otherwise apparent by context. The terms “CSF-1” and “M-CSF” are used interchangeably herein unless otherwise apparent by context. In addition, theterm “CSF-1” encompasses both the CSF-1 polypeptide or protein prior to conjugation as well as the CSF-1 polypeptide or protein following conjugation. As will be explained in further detail below, one of ordinary skill in the art can determine whether any given protein or polypeptide has CSF-1 activity. Exemplary, but non-limiting, methods of determining whether a protein or polypeptide has CSF-1 activity include using cell receptor signaling markers (e.g. pAkt, pERK, and pS6) as described at least in Examples 5, 7, and 8. It will also be understood that when CSF- 1 is attached to a water-soluble polymer such as a polyethylene glycol moiety, CSF-1 is slightly altered due to tire presence of one or more covalent bonds associated with linkage to the polymer(s). Reference to a CSF-1 conjugate as described herein is meant to encompass pharmaceutically acceptable salt formulations thereof. The term includes naturally, recombinantly and synthetically produced CSF-1.

[0044] A “CSF-1 mutein” refers to a CSF-1 polypeptide or protein having one or more amino acid modifications, insertions and / or substitutions. An insertion refers to insertion of an amino acid between two naturally occurring amino acids. A substitution refers to substitution of an amino acid for a naturally occurring amino acid. The position of substitution is typically designated by the original amino acid, the position, and the substituted amino acid. Thus, in an exemplary embodiment, the alanine substitution Hl 5A represents substitution of an alanine for the histidine at position 15 of a specifically referenced sequence.

[0045] The term "CSF-1 moiety," as used herein, refers to a peptide or protein moiety having human CSF-1 activity.

[0046] "Alkyl" refers to a hydrocarbon chain, typically ranging from about 1 to 15 atoms in length. Such hydrocarbon chains are preferably but not necessarily saturated and may be branched or straight chain, although typically a straight chain is preferred. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 3 methylpentyl, and the like. "Lower alkyl" as used herein refers to an alkyl group containing from 1 to 6 carbon atoms, and may be straight chain or branched, as exemplified by methyl, ethyl, n-butyl, i-butyl, and t-butyl.

[0047] The terms "substantially homologous" or “substantially identical” mean that a particular subject sequence, for example, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions. For purposes herein, a sequence having greater than 95 percent homology (identity) and equivalent expression characteristics to a given sequence is considered to be substantially homologous (identical). For purposes of determining homology,truncation of the mature sequence should be disregarded. Exemplary CSF-1 peptides or proteins for use herein include those sequences that are substantially homologous to SEQ ID N0s:2 and 4-5. It will be appreciated in the instance of a CSF-1 mutein as described herein, substantially homologous sequences include the alanine mutation as recited herein.

[0048] The term "fragment" means any protein or polypeptide having the amino acid sequence of a portion or fragment of a CSF-1 protein or polypeptide, and having the biological activity, or substantially the biological activity, of CSF-1. Fragments include proteins or polypeptides produced by proteolytic degradation of a CSF-1 moiety as well as proteins or polypeptides produced by chemical synthesis by methods routine in the art.

[0049] "Water-soluble, non-peptidic polymer" or “water-soluble polymer” refers to a polymer that is at least 35% (by weight) soluble, preferably greater than 70% (by weight), and more preferably greater than 95% (by weight) soluble, in water at room temperature. Typically, an unfiltered aqueous preparation of a "water-soluble" polymer transmits at least 75%, more preferably at least 95%, of the amount of light transmitted by the same solution after filtering. It is most preferred, however, that the water-soluble polymer is at least 95% (by weight) soluble in water or completely soluble in water. With respect to being "non-peptidic," a polymer is non- peptidic when it contains less than 35% (by weight) of amino acid residues An exemplary water-soluble / water-soluble, non-peptidic polymer is a poly(alkylene oxide) such as poly(ethylene glycol).

[0050] "PEG," "polyethylene glycol" and "poly(ethylene glycol)" as used herein, are interchangeable and are meant to encompass any water-soluble poly(ethylene oxide). Unless otherwise indicated, a "PEG polymer" or a polyethylene glycol is one in which substantially all (preferably all) monomeric subunits are ethylene oxide subunits, though, the polymer may contain distinct end capping moieties or functional groups, e.g., for conjugation. Typically, PEGs for use as described herein comprise the following structure:where the variable (n) may range from an average value of from about is 2 to 4000, from about 5 to about 2,000, or from about 45 to about 1818, or from about 113 to about 1818. It will be appreciated that the variable (n) may be selected such that the PEG has a specific molecular weight. For clarity, in one example, the variable (n) is selected such that the PEG has a weight average weight of 40kDa. As used herein, PEG also includesand depending upon whether or not the terminal oxygens have been displaced. Itwill be appreciated that where the PEG polymer is branched, the variable (n) for each polymer chain may independently fall within one of the ranges described above or elsewhere herein. Exemplary or preferred PEG-comprising molecules may however comprise one or more particular PEG architectures and / or linkers, and / or molecular weight ranges. Throughout the specification and claims, it should be remembered that the term "PEG" includes structures having various terminal or "end capping" groups. The term "PEG" also means a polymer that contains a majority, that is to say, greater than 50%, of repeatingsubunits. With respect to specific forms, the PEG can take any number of a variety of molecular weights, as well as structures or geometries such as "branched," "linear," "forked," "multifunctional," and the like, to be described in greater detail below. Where specific reference is made to PEG hereafter as the water-soluble, non-peptidic polymer, it will be understood that the disclosure relates generally to any water-soluble, non-peptidic polymer or poly(alkylene oxide) with poly(ethylene glycol) being preferred.

[0051] The terms "end-capped" and "terminally capped" are interchangeably used herein to refer to a terminal or endpoint of a polymer having an end capping moiety. Typically, although not necessarily, the end capping moiety comprises a hydroxy or Cl -20 alkoxy group, more preferably a Cl-10 alkoxy group, and still more preferably a Cl-5 alkoxy group. Thus, examples of end-capping moieties include alkoxy (e.g., methoxy, ethoxy and benzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and the like. In preferred embodiments, the end capping moiety includes a methoxy. It must be remembered that the end-capping moiety may include one or more atoms of the terminal monomer in the polymer (e.g., the end capping moiety "methoxy" inIn addition, saturated, unsaturated, substituted and unsubstituted forms of each of the foregoing are envisioned Moreover, the end capping group can also be a silane. The end-capping group can also advantageously comprise a detectable label. When the polymer has an end-capping group comprising a detectable label, the amount and / or location of the polymer and / or the moiety (e g., active agent) to which the polymer is coupled can be determined by using a suitable detector. Such labels include, without limitation, fluorescers, chemiluminescers, moieties used in enzyme labeling, colorimetric moieties (e g., dyes), metal ions, radioactive moieties, and the like. Suitable detectors include photometers, films, spectrometers, and the like. The end capping group can also advantageously comprise a phospholipid. When the polymer has an end-capping group comprising a phospholipid, unique properties are imparted to thepolymer and the resulting conjugate. Exemplary phospholipids include, without limitation, those selected from the class of phospholipids called phosphatidylcholines. Specific phospholipids include, without limitation, those selected from the group consisting of di lauroylphosphatidylcholine, dioleylphosphatidylcholine, dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine, behenoylphosphatidylcholine, arachidoylphosphatidylcholine, and lecithin.

[0052] Molecular weight in the context of a water-soluble polymer, such as PEG, can be expressed as either a number average molecular weight or a weight average molecular weight. Unless otherwise indicated, all references to molecular weight herein refer to the weight average molecular weight. Both molecular weight determinations, number average and weight average, can be measured using gel permeation chromatography or other liquid chromatography techniques (e.g. gel filtration chromatography). The most commonly employed methods are gel permeation chromatography and gel filtration chromatography. Other methods for determining molecular weight include end-group analysis or the measurement of colligative properties (e g., freezing-point depression, boiling-point elevation, or osmotic pressure) to determine number average molecular weight or the use of light scattering techniques, ultracentrifugation, MALDI TOP, or viscometry to determine weight average molecular weight. PEG polymers are typically polydisperse (i.e., the number average molecular weight and the weight average molecular weight of the polymers are not equal), possessing low polydispersity values of preferably less than about 1.2, more preferably less than about 1.15, still more preferably less than about 1.10, yet still more preferably less than about 1.05, and most preferably less than about 1.03.

[0053] “Branched,” in reference to the geometry or overall structure of a polymer, refers to a polymer having two or more polymer “arms” or “chains” extending from a branch point. In some preferred embodiments, a branched polymer such as a branched polyethylene glycol possesses two polymer “arms” or “chains” extending from a branch point. Examples of branched polymers are those having two polymer arms comprised of polymer chains having the same structure (for example, comprised of the same monomer subunits), and / or comprised of polymer arms having the same average molecular weight.

[0054] A “releasable linkage” is a relatively labile bond that cleaves under physiological conditions, wherein the cleavage may occur by way of any of a number of different mechanisms. One type of exemplary releasable linkage is a hydrolyzable bond, that is, one that cleaves uponreaction with water (z.e., is hydrolyzed), e.g., under physiological conditions, such as for example, hydrolysis of an ester bond or of a succinimide ring thereby resulting in ring opening. The tendency of a bond to hydrolyze in water may depend not only on the general type of linkage connecting two atoms but also on the substituents attached to these atoms. Exemplary hydrolytically unstable or weak linkages may include but are not limited to carboxylate ester linkages, phosphate ester linkages, anhydride linkages, acetal linkages, ketal linkages, acyloxyalkyl ether linkages, imine linkages, orthoester linkages, peptide linkages, oligonucleotide linkages, thioester linkages, and carbonate linkages. Releasable linkages also include enzymatically releasable linkages, where an "enzymatically releasable linkage" means a linkage that is subject to cleavage by one or more enzymes. Additional types of release mechanisms include but are not limited to 1,6-benzyl elimination, ^-elimination, and the like. While certain bonds may be considered to be stable or releasable, such characterization should be considered within the overall structure of a molecule or structural entity. In certain instances, a polymer conjugate containing a releasable bond may be referred to as a prodrug, wherein upon cleavage of a releasable bond in vivo (i.e., under physiological conditions), the parent drug is released (or may be eventually released, depending upon the number of polymeric moieties releasably attached to an active agent). A covalent “releasable” linkage, for example, in the context of a water-soluble polymer such as polyethylene glycol that is covalently attached to an active moiety such as CSF-1, is one that cleaves under physiological conditions to thereby release or detach a water-soluble polymer from the active moiety, or to detach an active moiety from a water-soluble polymer.

[0055] A “stable” linkage or bond refers to a chemical bond that is substantially stable in water (e.g., under physiological conditions), that is to say, does not undergo hydrolysis under physiological conditions to any appreciable extent over an extended period of time. Examples of hydrolytically stable linkages generally include but are not limited to the following: carbon-carbon bonds (e.g., in aliphatic chains), ether linkages, amide linkages, carbamate linkages, amine linkages, and the like as well as combinations thereof. Generally, a stable linkage is one that exhibits a rate of hydrolysis of less than about 1-2% per day under physiological conditions. Hydrolysis rates of representative chemical bonds can be found in most standard chemistry textbooks. Further, one of ordinary skill in the art can determine whether a given linkage is stable or releasable in a given context by, for example, placing alinkage-containing molecule of interest under conditions of interest (e.g., under physiological conditions) and testing for evidence of release over a suitable time period.

[0056] The terms "spacer moiety," "linkage" and "linker" are used interchangeably and are used herein to refer to a bond or an atom or a collection of atoms optionally used to link interconnecting moieties such as a terminus of a polymer segment and a CSF-1 moiety. The spacer moiety may be hydrolytically stable or may include a physiologically hydrolyzable or enzymatically degradable linkage. Unless the context clearly dictates otherwise, a spacer moiety optionally exists between any two elements of a compound (e.g., the provided conjugates comprising a residue of CSF-1 moiety and water-soluble polymer can be attached directly or indirectly through a spacer moiety).

[0057] As used herein in reference to treatment, the terms “treatment,” “treat,” and “treating” are meant to include the full spectrum of intervention for the condition from which the subject is suffering, such as administration of the combination to alleviate, slow, stop, or reverse one or more symptoms of the condition. Treatment can include, for example, a decrease in the severity of a symptom, the number of symptoms, or frequency of relapse. In some circumstances, treatment in accordance with the instant disclosure leads to an improved prognosis. “Complete response” refers to an absence of clinically detectable disease. “Partial response” refers to at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% decrease in all measurable disease. The term “treatment” contemplates both a complete and a partial response.

[0058] The term "patient," or “subject” as used herein refers to a living organism suffering from or prone to a condition that can be prevented or treated by administration of a compound, composition, or combination as provided herein. Subjects or patients include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and preferably are human.

[0059] “Pharmacologically effective amount,” “physiologically effective amount,” and “therapeutically effective amount” are used interchangeably herein to mean the amount of an active agent, such as, for example, a CSF-1 polymer conjugate, that is needed to provide a desired level of active agent and / or conjugate in the bloodstream or in the target tissue. The precise amount may depend upon numerous factors, e.g., the particular active agent, the components and physical characteristics of the composition, intended patient population, patient considerations, and may readily be determined by one skilled in the art, based upon theinformation provided herein and available in the relevant literature. For example, a therapeutically effective amount of a compound, or a combination of one or more compounds, when administered (either sequentially or concomitantly) is an amount that elicits a desired biological or medicinal response, e g., eliminates, slows or arrests the progression of a disease in a subject. The term also applies to a dose of the compounds that will induce a particular desired response in target cells, e.g., when administered in combination, to provide in a beneficial effect. In certain embodiments, the combined effect is additive, while in certain other embodiments, the combined effect is synergistic.

[0060] “Pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” refers to a component, other than the pharmacologically active agent, that may be included in the compositions described herein and causes no significant adverse toxicological effects to a subject.

[0061] “Combination therapy” or “in combination with” refers to the use of more than one therapeutic agent to treat a particular disorder or condition. By “in combination with,” it is not intended to imply that the therapeutic agents must be administered at the same time and / or formulated for delivery together, although these methods of delivery are within the scope of this disclosure. A therapeutic agent can be administered concurrently with, prior to, or subsequent to, one or more other additional agents on the same or different days. Therapeutic agents that are administered concurrently may be administered in the same formulation or in different formulations during the same treatment session. In embodiments, therapeutic agents administered concurrently are administered separately but within about one hour, about 30 minutes, about 15 minutes, about 10 minutes, about 5 minutes or less from each other. The therapeutic agents in a combination therapy can also be administered on an alternating dosing schedule, with or without a resting period (e.g., no therapeutic agent is administered on certain days of the schedule). The administration of a therapeutic agent “in combination with” another therapeutic agent includes, but is not limited to, sequential administration and concomitant administration of the two or more agents. In general, each therapeutic agent is administered at a dose and / or on a time schedule determined for that particular agent.

[0062] "Substantially" or "essentially" means nearly totally or completely, for instance, 95% or greater of a given quantity.

[0063] Similarly, “about” or “approximately” as used herein means within plus or minus 5% of a given quantity.

[0064] "Optional" or "optionally" means that the subsequently described circumstance may, but need not necessarily, occur so that the description includes instances where the circumstance occurs and instances where it does not. "Optional" or "optionally" also means that variables or components described may, but need not necessarily, be present so that the description includes instances where the variables or components are present and instances where they are not present.

[0065] Amino acid residues in peptides are abbreviated as follows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine isMethionine is Met or M; Valine is Vai or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gin or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg or R, and Glycine is Gly or G.

[0066] An exemplary conjugate, active moiety, or other suitably applicable chemical moiety as described herein is meant to encompass, where applicable, analogues, isomers, polymorphs, solvates, and pharmaceutically acceptable salt forms thereof.

[0067] In the context of the present disclosure, it should be recognized that the definition of a variable provided with respect to one structure or formula is applicable to the same variable repeated in a different structure, unless the context dictates otherwise.Overview

[0068] As noted above, current standards of care for chronic inflammatory disease therapies are not optimized for inflammation resolution and restoration of tissue homeostasis and function. This unmet need requires a new class of therapeutics that provide and / or complement current anti-inflammatoiy treatments to enable one or more of resolution of inflammation, tissue repair, extended remission duration, increased remission rates, and / or potentially curative responses. Stimulating inflammation resolution and tissue repair by tissue resident immune cells may increase tissue function recoveiy and / or increase remission rates and / or duration. Macrophages are key drivers of tissue homeostasis and repair after inflammatory insult. Therapies inducingtissue macrophages expansion, reprogramming, and / or activation may synergize with existing cytokine blockade agents to enhance inflammation resolution and tissue function renewal.

[0069] To that end, described herein are novel colony stimulating factor- 1 receptor (CSF-1R) agonists optimized for sustained receptor activation and / or tissue macrophage reprogramming to promote expression of inflammation resolution and / or tissue remodeling functions. The present CSF-1R agonists provide activation of tissue macrophages that is decoupled from pro- inflammatory monocyte tissue infiltration induction that may be a beneficial property' for the treatment of acute or chronic inflammatory conditions and / or autoimmune diseases including fibrotic disease.

[0070] The instant disclosure is directed to, among other things, providing conjugates, combinations, compositions, methods, and kits relating to the treatment of autoimmune diseases, inflammatory conditions, and / or tissue repair comprising a colony stimulating factor- 1 receptor (CSF-1R) agonist, alone or in combination with an inflammatory drug. Such conjugates, combinations, compositions and methods will ideally possess several advantageous and unpredictable features such as, for example at least one, if not more, of the following: improved pharmacokinetic properties as compared to CSF-1 enabling sustained receptor stimulation via modified receptor affinity; limiting inflammatory monocytes tissue infiltration while inducing gene expression programs in tissue macrophages that characterize inflammation resolution and tissue repair functions; limiting induction of monocytosis; and / or selectively modulate resolution processes of inflammation. Surprisingly, the Applicants have arrived at a CSF-1R agonist that possesses a unique combination of advantageous properties, to be described in greater detail below and illustrated in the supporting examples.

[0071] Inflammation resolution and tissue repair inducing agents as described herein also have the potential to synergize with existing anti-inflammatory therapeutics that are designed to suppress chronic inflammation but are not optimized to trigger resolution of inflammation and drive tissue repair that would result in curative therapies. Tissue macrophages are a key cell type to maintain tissue homeostasis by participating in inflammation resolution and tissue repair in inflammatory responses to tissue damage by pathogens and environmental challenges. Pharmacological approaches that could induce and sustain macrophage functions that enhance resolution of inflammatory responses and drive tissue remodeling could enhance therapies of chronic inflammatory diseases to enable curative therapies.

[0072] Macrophage numbers and functional state are controlled by local tissue environment and systemic signals. Cytokines are one major class of cell signaling mediators that dictate macrophage function in tissues. CSF-1 is a central cytokine to all macrophages controlling multiple key functions including survival, proliferation and functional differentiation by signaling via CSF-1 receptor that expressed on all macrophages. Exogenous CSF-1 administration can induce tissue repair stimulating functions in tissue macrophages in multiple tissue injury and chronic inflammation models (Menke et al., J Clin Invest., 119(8):2330-2342, 2009, doi.10.1172 / JCI39087; Alikhan Am J Pathol, 179(3): 1243-1256, 2011. doi:10.1016 / j.ajpath.2011.05.037, 2011; Laflamme et al., Front. Cell. Neurosci., 12: 178, 2018, doi: 10.3389 / fncel.2018.00178; Keshvari, et al., Dis Model Meeh., 15(4):dmm049389, 2022, doi: 10.1242 / dmm.049387).

[0073] CSF-1 use as therapeutic agent has significant limitations due to 1) rapid homeostatic clearance mechanism via receptor mediated endocytosis and degradation, 2) the need for sustained stimulation to trigger expansion of cell numbers and 3) pleiotropic effects stimulating both tissue resident macrophages and also proinflammatory peripheral blood monocytes and their rapid tissue infiltration that can sustain chronic inflammation (Tushinski and Stanley, J Cell Physiol 122(2):221-228, 1985; Hume et al., J Immunol, 141(10):3405-3409, 1988; McCabe et al., Toxicological Sciences, 43:61-67 , 1998, Hume and MacDonald, Blood, 119(8): 1810-1820, 2011 Epub. doi: 10.1182 / blood-2011-09-379214).Colony Stimulating Factor- 1 Receptor Agonists and Related Compositions

[0074] Described herein are chemically and optionally genetically modified CSF-1 derived agonists of CSF-1R that overcome the above limitations by providing one or more of 1) improved pharmacokinetic properties enabling sustained receptor stimulation via modified receptor affinity and pharmacodynamic responses in macrophages in vivo, 2) inducing gene expression programs in tissue macrophages that characterize inflammation resolution and tissue repair functions, 3) limit inflammatory monocytes tissue infiltration from peripheral blood when activating blood monocytes and tissue macrophages, 4) reduced target mediated clearance, and / or 5) sustained target engagement and proliferation and expansion of tissue resident macrophages with minimal off-target effects of monocyte infiltration and production of monocyte-derived macrophages. As used herein, reference to a CSF-1 conjugate applies to otherCSF-1R agonists as described herein unless the context clearly dictates otherwise. Polymer conjugated CSF-1 variants that show preferential and sustained Akt kinase activation may bias functional activation of macrophages to trigger reduction of tissue inflammation and enhance tissue repair with therapeutic effect.

[0075] The novel polymer conjugated CSF-1 and / or polymer conjugated CSF-1 mutein conjugates described herein have at least the following advantages. Altered intracellular cell signaling pathways activation profile in response to receptor agonism: Akt kinase activation signaling pathway is more sensitive to agonist concentrations compared to ERK kinase activation signaling pathway. Tissue macrophages activation is achieved without tissue infiltration of blood monocytes. Agonist half-life in circulation is significantly further increased compared to unconjugated CSF-1.

[0076] Colony-stimulating factor- 1 (CSF-1), also known as macrophage colony stimulating factor (M-CSF), has traditionally been reviewed as a growth / differentiation factor for monocytes, macrophages, and some female-specific tumors. CSF-1 activates the colony-stimulating factor- 1 receptor (CSF-1R), which is mainly expressed in myeloid cells such as monocytes, macrophages, glial cells, and osteoclasts. As a result of alternative mRNA splicing and post-translational processing, several forms of CSF-1 protein are produced, a secreted glycoprotein, a longer secreted form containing proteoglycan, and a short membrane-bound isoform. These different forms of CSF-1 all initiate cell signaling in cells bearing the CSF-1 receptor, also called c-fms. CSF-1 has important roles in bone physiology, the intestinal tract, cancer metastases to the bone, macrophage-mediated tumor cell killing and tumor immunity.

[0077] Native CSF-1 has been isolated from human urine and was shown to be a secreted, homodimeric glycoprotein. The two CSF-1 subunits each contain nine cystines and are linked together by one or more interchain disulfide bonds.

[0078] Three human CSF-1 cDNA clones, each encoding an CSF-1 polypeptide of a different length (a, 256 amino acids; P, 554 amino acids; and y, 438 amino acids), have been isolated from cells expressing the single CSF-1 gene.

[0079] Example 1 describes an exemplary method of preparing a recombinant human CSF-1. In one or more embodiments, CSF-1 can be prepared according to a method as described in Example 1.

[0080] In one embodiment, the CSF-1 is a CSF-1 mutein. In a preferred embodiment, the CSF-1 includes at least one amino acid substitution. An exemplary substitution includes, but is not limited to, histidine at position 15 to alanine (H15A) as shown in SEQ ID NOs:4-5. Example 2 describes preparation of a CSF-1 mutein having a substitution at Hl 5 A. In one or more embodiments, CSF-1 muteins can be prepared according to a method as described in Example 2. Further suitable substitutions may be identified by reference to the structure of the CSF-1 protein dimer.

[0081] In embodiments, the CSF-1 mutein comprises a single mutation or more than one mutation that affect binding of CSF-1 to the CSF-1R. In particular, the mutein comprises one or more amino acid introduction and / or substitution within or near the CSF-1R binding site of the CSF-1 protein. As used herein, “near” with reference to the position of the mutation means that the introduction or substitution at least partially inhibits binding of the CSF-1 mutein to the CSF- 1R. In embodiments, “partially inhibits” refers to an inhibition in binding of the CSF-1 mutein to the CSF-1R of at least or about 5%, 10%, 15%, 20%, 25%, 50%, or more as compared to binding of unmodified CSF-1 to the CSF-1 R. In some embodiments, the mutation is located within 1-10 amino acids from either end of the CSF-1R binding domain. In some exemplary embodiments, the mutation is located within about 1-5 amino acids, or about 5-10 amino acids from either end of the CSF-1R binding domain, or the mutation is located within at least one amino acid from either end of the binding domain. It will be appreciated that the mutation may be introduced within the CSF-1R binding domain. An exemplary substitution includes, but is not limited to, histidine at position 15 to alanine (H15A) of SEQ ID NO:2. Example 2 describes an exemplary method of preparing a recombinant CSF-1 mutein having a substitution at H15A. In one or more embodiments, CSF-1 muteins can be prepared according to a method as described in Example 2. In embodiments, suitable mutations include substitutions at H9, Hl 5, Q20, and V78, or combinations thereof. Suitable substitutions may further be made as identified by Taylor, et al (J. Biol. Chem., 269(49):31171-31177, 1994). Further suitable substitutions may be identified by reference to the crystal structure of the CSF-1 protein dimer in complex with CSF-1R, which has been determined (see Chen et al., PNAS, 105(47): 1827-18272, 2008) in that amino acids on the CSF-1 protein that are involved in binding to the CSF-1R are suitable for mutation such as substitution.

[0082] The CSF-1 moiety can be derived from non-recombinant methods and from recombinant methods, and the disclosure is not limited in this regard. In addition, the CSF-1 moiety can be derived from human sources, animal sources (including insects), fungi sources (including yeasts), and plant sources.

[0083] The CSF-1 protein or CSF-1 protein mutein can be expressed in bacterial (e.g., E. coli, see, for example, Fischer et al., Biotechnol. Appl. Biochem., 21 (3):295-311, 1995), mammalian (see, for example, Kronman et al., Gene, 121 :295-304, 1992), yeast (e.g., Pichia pastoris, see, for example, Morel et al., Biochem. J., 328(1): 121-129, 1997), and plant (see, for example, Mor et al., Biotechnol. Bioeng., 75(3):259-266, 2001) expression systems. The expression can occur via exogenous expression (when the host cell naturally contains the desired genetic coding) or via endogenous expression.

[0084] Although recombinant based methods for preparing proteins can differ, recombinant methods typically involve constructing the nucleic acid encoding the desired polypeptide or fragment, cloning the nucleic acid into an expression vector, transforming a host cell (e.g., plant, bacteria, yeast, transgenic animal cell, or mammalian cell such as Chinese hamster ovary (CHO) cell or baby hamster kidney cell), and expressing the nucleic acid to produce the desired polypeptide or fragment. Methods for producing and expressing recombinant polypeptides in vitro and in prokaryotic and eukaryotic host cells may be used as known to those of ordinary skill in the art. A method for producing and expressing a recombinant CSF-1 is described in U.S. Patent No. 5,470,569. In addition, exemplary recombinant methods of preparing CSF-1 are described in Examples 1 and 2.

[0085] Depending on the system used to express CSF-1, the protein can be unglycosylated or glycosylated and either may be used. In one or more embodiments, the CSF-1 mutein is unglycosylated.

[0086] For any given CSF-1 protein or conjugate, it is possible to determine whether that protein, mutein or conjugate has CSF-1 activity. Various methods for determining in vitro CSF- 1 activity are described in the art. An exemplary approach is based on cell receptor signaling as measured by induction of phosphorylation of endoplasmic reticulum kinase (ERK) (pERK) or Akt (also known as protein kinase B) (pAkt) as described in Example 7.

[0087] Assays for use in connection with measuring the activity of a CSF-1 moiety can also be used to measure the activity of the compounds described herein. See, for example, the supporting examples provided herein.CSF-1 Polymer Conjugates

[0088] As described above, conjugates as described herein comprise at least one water soluble polymer covalently attached (either directly or through a spacer moiety) to a CSF-1 moiety. Typically, for any given conjugate, there will be one to three water soluble polymers covalently attached to a CSF-1 moiety (wherein for each water soluble polymer, the water soluble polymer can be attached either directly to the CSF-1 moiety or through a spacer moiety). In some embodiments, the conjugate may have 1, 2, 3, or more water soluble polymers individually attached to a CSF-1 moiety (again, with respect to each water soluble polymer, attached directly or through a spacer moiety). Preferably, the conjugate includes 1 or 2 water soluble polymers individually attached to CSF-1 moiety. More preferably, the conjugate includes 1 water soluble polymer individually attached to a CSF-1 moiety.

[0089] For compositions described here, preferably at least about 50-95 or even 50-99 mole percent (mol %) of the conjugates comprised within die composition are monoPEGylated. In some embodiments, at least about 50 mol %, at least about 60 mol %, at least about 70 mol %, at least about 75 mol %, at least about 80 mol %, at least about 90 mol %, at least about 95 mol %, or at least about 99 mol % of the conjugates comprised within the composition are monoPEGylated.

[0090] In some instances, the CSF-1 moiety will be in a "monomer" form, wherein a single expression of the corresponding peptide is organized into a discrete unit. In other instances, the CSF-1 moiety will be in the form of a "dimer" (e.g., a dimer of recombinant human CSF-1) wherein two monomer forms of the protein are associated (e.g., by disulfide bonding) to each other. For example, in the context of a dimer of recombinant human CSF-1, the dimer may be in the form of two monomers associated to each other by a disulfide bond. A preferred CSF-1 conjugate comprises two branched water-soluble, non-peptidic polymers (e.g. polyethylene glycol or PEG) covalently attached to a CSF-1 homodimer with one water-soluble, non-peptidic polymer covalently attached to each CSF-1 monomer of the homodimer. The CSF-1 conjugates for use herein are preferably but not necessarily in the form of a homodimer where eachmonomer comprises a single branched water-soluble, non-peptidic polymer (e.g. polyethylene glycol or PEG) covalently attached thereto. It will be appreciated, however, that in some instances, only one monomer of the homodimer may be PEGylated. It will further be appreciated that the CSF-1 conjugates may also be in the form of a heterodimer where each monomer of the dimer is a CSF-1 protein as described herein and comprises a single, branched water-soluble, non-peptidic polymer (e.g. polyethylene glycol or PEG) and where each monomer utilizes a different PEG and / or linkage. Conjugate compositions as described herein may comprise all or substantially all CSF-1 homodimers, all or substantially all CSF-1 heterodimers, or a mixture of homodimers and heterodimers. Preferred compositions are those comprising at least about 75 mol % of CSF-1 conjugates that are homodimers. Where the CSF-1 is present as a dimer, in preferred embodiments, each CSF-monomer of the dimer is monoPEGylated (includes one water soluble polymer covalently attached to each CSF-1 monomer). It will be appreciated that for each CSF-1 monomer of the dimer, the water-soluble polymer may be attached at a different site. In preferred embodiments, the water-soluble polymer is attached at the N-terminus for each CSF-1 monomer of the dimer.

[0091] As previously discussed, each conjugate comprises a CSF-1 moiety covalently attached to at least one water-soluble polymer. With respect to the water-soluble polymer, the water-soluble polymer is nonpeptidic, nontoxic, non-naturally occurring and biocompatible. With respect to biocompatibility, a substance is considered biocompatible if the beneficial effects associated with use of the substance alone or with another substance (e.g., an active agent such as an CSF-1 moiety) in connection with living tissues (e.g., administration to a patient) outweighs any deleterious effects as evaluated by a clinician, e.g., a physician. With respect to non-immunogenicity, a substance is considered non-immunogenic if the intended use of the substance in vivo does not produce an undesired immune response (e.g., the formation of antibodies) or, if an immune response is produced, that such a response is not deemed clinically significant or important as evaluated by a clinician. It is particularly preferred that the nonpeptidic water-soluble polymer is biocompatible and non-immunogenic.

[0092] The water-soluble polymer is not limited to a particular structure and can be linear (e.g., an end capped, e.g., alkoxy PEG or a bifunctional PEG), branched or multi-armed (e.g., forked PEG or PEG attached to a polyol core), a dendritic (or star) architecture, each with or without one or more degradable linkages. Moreover, the internal structure of the water-soluble polymercan be organized in any number of different repeat patterns and can be selected from the group consisting of homopolymer, alternating copolymer, random copolymer, block copolymer, alternating tripolymer, random tripolymer, and block tripolymer.

[0093] The particular linkage between the CSF-1 moiety and the water soluble polymer (or the spacer moiety that is attached to the water soluble polymer) depends on a number of factors. Such factors include, for example, the particular linkage chemistry employed, the particular CSF-1 moiety, the available functional groups within the CSF-1 moiety (either for attachment to a polymer or conversion to a suitable attachment site), the possible presence of additional reactive functional groups within the CSF-1 moiety, and the like.

[0094] Typically, activated PEG and other activated water-soluble polymers (i.e., polymeric reagents) are activated with a suitable activating group appropriate for coupling to a desired site on the CSF-1 moiety. Thus, a polymeric reagent will possess a reactive group for reaction with the CSF-1 moiety. Representative polymeric reagents and methods for conjugating these polymers to an active moiety are known in the art and further described in Zalipsky, S., et al., "Use of Functionalized Poly (Ethylene Glycols) for Modification of Polypeptides" in Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, J. M. Harris, Plenus Press, New York (1992), and in Zalipsky, Advanced Drug Reviews 16:157-182. 1995. Exemplary activating groups suitable for coupling to a CSF-1 moiety include hydroxyl, maleimide, ester, acetal, ketal, amine, carboxyl, aldehyde, aldehyde hydrate, ketone, vinyl ketone, thione, thiol, vinyl sulfone, or hydrazine, among others.

[0095] In one or more embodiments, the linkage between the CSF-1 moiety and the polymer (or the spacer moiety that is attached to the polymer) is a hydrolytically stable linkage, such as an amide, urethane (also known as carbamate), amine, thioether (also known as sulfide), or urea (also known as carbamide). In one or more embodiments, the linkage does not result from reaction of the polymeric reagent bearing a functional group with the CSF-1 moiety, wherein the functional group is selected from the group consisting of triazine, hydrazine, hydrazide, aldehyde, semicarbazide, maleimide, vinylsulfone, phenylglyoxal, isocyanate, isothiocyanate, amine and tresyl functional group with the CSF-1 moiety.

[0096] In one or more embodiments, the linkage between the CSF-1 moiety and the water soluble polymer (or the spacer moiety that is attached to the water soluble polymer) is a degradable linkage. In this way, the linkage linking the CSF-1 moiety is "degradable." That is,the water soluble polymer (and the spacer moiety, when present) cleaves (either through hydrolysis, enzymatic processes, or otherwise), thereby resulting in the native or an unconjugated CSF-1 moiety. Preferably, degradable linkages result in the water soluble polymer (and any spacer moiety) detaching from the CSF-1 moiety in vivo without leaving any fragment of the water soluble polymer (and any spacer moiety). Exemplary degradable linkages include carbonate, carboxylate ester, phosphate ester, thiolester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines, and orthoesters. Such linkages can be readily prepared by appropriate modification of either the CSF-1 moiety (e.g., the carboxyl group C terminus of the protein or a side chain hydroxyl group of an amino acid such as serine or threonine contained within the protein) and / or the polymeric reagent using coupling methods commonly employed in the art. Most preferred releasable linkages, however, are hydrolyzable linkages that are readily formed by reaction of a suitably activated polymer with a non-modified functional group contained within the CSF-1 moiety.

[0097] In some preferred embodiments, CSF-1 conjugates are prepared by reaction of the CSF-1 protein with an amine-selective or amine-specific water-soluble, non-peptidic polymer reagent, e.g. an amine-selective or amine-specific PEGylation reagent, to provide a CSF-1 protein with a PEG moiety covalently attached thereto. Exemplary polymer reagents, CSF-1 proteins and muteins, and reaction conditions for preparing the subject conjugates are described in Example 3, and such are suitable or may be readily adapted in light of the teachings provided herein, when considered along with knowledge commonly available in the chemical and polymer arts, for forming CSF-1 conjugates having one or more of the advantageous features described herein. Conjugates can be formed using amine-selective polymeric reagents in a number of ways and the invention is not limited in this regard.

[0098] One exemplary polymer reagent is a mPEG ButyrALD reagent. The aldehyde group of mPEG ButyrALD reacts with primary amines and covalently bonds to them via a secondary amine linkage upon reduction by a reducing reagent such as sodium cyanoborohydride. Selectivity for which amine(s) becomes attached with the polymer can be modulated by adjusting the pH of the conjugation conditions. Relatively low pH conditions (e.g., around a pH of 5.5) will direct conjugation toward the N terminus. At relatively neutral pH conditions (e.g., around 7.5 and slightly above), covalent attachment becomes more frequent at other locations (i.e., at the amine side chains of lysine residues contained within the protein). Adjusting the pHof the conjugation conditions will allow some degree of control as to which locations conjugation occurs, thereby having a better ability to arrive at the desired positional isomers. In a preferred embodiment, the polymer is attached, or predominantly attached, at the N-terminus of the CSF-1. For compositions described here, preferably the polymer is attached at the N- terminus of the CSF-1 for at least about 50-95% of the conjugates comprised within the composition. In some embodiments, the polymer is attached at the N-terminus of the CSF-1 for at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% of the conjugates comprised within die composition.

[0099] In preferred embodiments, the water-soluble, non-peptidic polymer is a poly(alkylene oxide) such as a poly(alkylene glycol). A preferred poly(alkylene glycol) is polyethylene glycol). The polymer is not limited to a particular structure and can be linear or branched. When the water-soluble, non-peptidic polymer is a poly(ethylene glycol), the polymer will comprise a number ofmonomers ormonomers, depending on how the poly(ethylene glycol) is defined. As used throughout the description, the number of repeat units is identified by the subscript “n" inIn one or more embodiments, n is an integer having a value that corresponds to a poly(ethylene glycol) polymer having a weight average molecular weight selected from the group consisting of about 2,000 daltons (where n is -45), about 5,000 daltons (where n is -113), or about 10,000 daltons (where n is -227), or about 15,000 daltons (where n is -340), or about 20,000 daltons (where n is -454), or about 25,000 daltons (where n is -568), or about 30,000 daltons (where n is -681), or about 40,000 daltons (where n is -909), or about 50,000 daltons (where n is -1136) or about 60,000 daltons (where n is -1364), or about 80,000 daltons (where n is -1818), or greater. In embodiments, n is an integer having a value selected from about 3 to about 4000. In some embodiments, the water- soluble polymer has a weight average molecular weight in a range of from about 500 daltons to about 100,000 daltons. In some embodiments, the water-soluble polymer has a weight average molecular weight in a range of from about 20,000 daltons to about 85,000 daltons. In further embodiments, the water-soluble polymer has a weight average molecular weight in a range of from about 20,000 daltons to about 60,000 daltons. For any given polymer in which the molecular weight is known, it is possible to determine the number of repeating units (i.e., “n”) bydividing the total weight-average molecular weight of the polymer by the molecular weight of the repeating monomer unit.

[0100] Further exemplary weight average molecular weights for the polyethylene glycol portion of the conjugate, that is for each CSF-1 monomer, in addition to the foregoing, include about 10,000 daltons, about 11,000 daltons, about 12,000 daltons, about 13,000 daltons, about 14,000 daltons, about 20,000 daltons, about 22,500 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 55,000 daltons, about 65,000 daltons, about 70,000 daltons, about 75,000 daltons, or about 80,000 daltons. In some embodiments, the weight-average molecular weight of the polyethylene glycol portion of each CSF-1 monomer of the conjugate is about 10,000 to about 80,000 daltons.

[0101] Where the CSF-1 is attached to a branched PEG polymer, it will be appreciated that each arm of the branched polymer may have the same or different weight-average molecular weight. Thus, the weight average molecular weight of each arm may be the same or may be different between the arms. In preferred embodiments, each arm of the branched polymer has the same weight-average molecular weight.

[0102] In some embodiments, the CSF-1 conjugate has the structure of Formula I:wherein CSF-1 is a CSF-1 moiety, each (n) is independently an integer having a value of from 3 to 4000; X2 when present, is a spacer moiety comprised of one or more atoms; (b) is 2 through 6; (c) is 2 through 6; and R2, in each occurrence, is independently H or lower alkyl. It will be appreciated that the above formula may apply to each CSF-1 monomer of a dimer.

[0103] In some embodiments related to one or more of the foregoing, the value of n ranges from 5-2000. In some other embodiments, the value of n ranges from 10-1000. In yet some additional embodiments, the value of n ranges from 10-750. In some embodiments the value of n ranges from 10-500, or from 20-250. In one preferred embodiment, the value of each n is independently about 226, about 227, or about 454.

[0104] The value of n in the embodiments provided herein can vary independently at each occurrence. In one or more embodiments described herein, the value of n in each of thepolyethylene glycol arms of the branched polymer is substantially the same. In some further embodiments, the value of n in each of the polymer arms comprising the branched polymer ranges from about 170 to 285. In yet some further embodiments, the value of n in each of the polymer arms comprising the branched polymer ranges from about 204 to about 250. In one or more particular embodiments, the value of n in each of the polymer arms comprising the branched polymer is about 226 or about 227. In some further embodiments, the value of n in each of the polymer arms ranges from about 227 to about 909. In other embodiments, the value of n in each of the polymer arms ranges from about 227 to about 454.Methods of Use

[0105] In a further aspect, provided is a method for treating a subject afflicted with a disease or indication treatable by administration of CSF-1, such as a condition characterized by a deficiency in CSF-1, or an inflammatory indication. The CSF-1 conjugates may be used as first line treatment, second line treatment, or third line treatment as appropriate and described herein. The method comprises administering, alone or in combination with a further therapeutic agent or procedure, a CSF-1 conjugate as described herein. See, for example, the results described in the accompanying examples herein. It will be appreciated that a combination combining the CSF-1 conjugates described herein may be administered either concurrently or in a staggered regimen where generally the anti-inflammatory treatment is administered first to suppress and / or reduce inflammation. The CSF-1 conjugate as described herein is administered thereafter in a combination or as a single agent e.g. to promote inflammation resolution repair. In embodiments, the presently described conjugates, combinations, compositions, and kits are contemplated for use in the treatment of autoimmune diseases, inflammatory diseases, and / or other conditions responsive to CSF-1 therapy. In embodiments, the presently described conjugates, combinations, compositions, and kits are contemplated to support resolving inflammation and / or to repair damage caused by inflammation.

[0106] The immune system is the body’s main line of defense against invasion by infectious organisms. In a normally functioning immune system, an immune response does not occur against self-antigens; this is referred to as self-tolerance. Autoimmune disease occurs when body tissues are attacked by the body’s own immune system due to a loss of tolerance to selfantigens (Dejaco, C., et al., Immunology, 117(3): 289-300, 2006). In subjects having anautoimmune disease, body tissues are destroyed by antigen-specific cytotoxic T cells or autoantibodies, where the accompanying inflammation can cause functional disability and, in some cases, death. Autoimmune diseases are a heterogeneous collection of diseases with a wide spectrum of symptoms that affect approximately six percent of the population (Siatskas, C., et al., Curr Gene Ther., 6(l):45-58, 2006). While the clinical features of autoimmune diseases are very different, immune-mediated mechanisms are associated with the generation of an adaptive immune response toward the target antigen (Kuby, J. (1994). Autoimmunity. Immunology, 2nd ed., p 445-467. WH Freeman and Company, New York).

[0107] As described above, CSF-1 is known to have a central role in maintaining and differentiating myeloid cell types including tissue macrophage populations and activating macrophages. Further, macrophages control the development and proliferation of the liver. Use of a biologically active fragment of CSF-1 has been proposed for use in enhancing liver regeneration, restoring liver function, and / or modulating liver homeostasis (U.S. Patent Publication No. 2018 / 0112193). Use of a CSF-l-fusion protein was investigated and shown to have potential for treating chronic liver disease using a mouse hepatocyte proliferation model (Keshvari, et al., Dis Model Meeh, 15(4):dmm049387, published online, 2022). Fibrosis is a physiological response to acute and chronic tissue injury and is the excessive accumulation of extracellular matrix proteins including collagen (e.g. scar tissue). In some embodiments, the present compounds are contemplated for use in treating hepatic disorders such as the prevention and / or treatment of acute or chronic liver disease and including use as a supportive therapy following liver resection or liver transplant by promoting recruitment and maturation of macrophages in the liver. In other embodiments, the present compounds are contemplated for use in treating and / or resolving liver fibrosis.

[0108] In some embodiments, the CSF-1 conjugates are contemplated for providing inflammation resolution and tissue repair after inflammation. As described in Example 10, CSF- 1 conjugates as described herein were effective to induce MMP activity in Kupffer cells. Administration of the CSF-1 conjugates induced IL-4Ra, which was sustained for at least 6 days for the CSF-1 mutein conjugate. Administration of the CSF-1 conjugates induced IL-lORa, which was sustained after administration of both conjugates. Administration of the CSF-1 conjugates induced expression of MerTK in Kupffer cells as shown by the percentage of MerTK+ cells in Fig. 18.

[0109] Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) involves impairment of CSF-1 receptor activity due to mutations in the gene, which is characterized by development of neurodevelopment degenerative disease. A mouse model of this disease has been described which involves introducing a human patient mutation into the mouse CSF-1 receptor gene (Stables et al.. Development, 149(8): 1-16, 2022. doi: 10.1242 / dev.200237). These mice were injected with CSFl-Fc fusion protein to increase CSF-1 signaling resulting in restoration or enhancement of a number of biomarkers that are affected by the disease mutation (see Stables Fig. 8). In some embodiments, the present compounds are contemplated for use in treating adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP).

[0110] The present compounds and related compositions, provided herein, are useful for (among other things) treating autoimmune diseases and disorders. In some embodiments, the present compounds are administered as a combination treatment with one or more compounds or therapies that control and / or reduce inflammation such as through immune suppression. In particular, the present compounds are useful as a combination treatment for autoimmune and inflammatory diseases.

[0111] Exemplary autoimmune or inflammatory diseases that can be treated by administration of the compounds as described herein include systemic lupus erythematosus (SLE), ulcerative colitis, Crohn’s disease, rheumatoid arthritis, psoriatic arthritis, plaque psoriasisjuvenile idiopathic arthritis, atopic dermatitis, systemic sclerosis, ankylosing spondylitis, graft versus host disease (GVHD), Hidradenitis suppurativa, uveitis, polymyositis, organ-specific autoimmune diseases include type 1 diabetes, Addison’s disease, Hashimoto thyroiditis, Graves’ disease, Sjogren’s syndrome, Chronic obstructive pulmonary disease (COPD), vitiligo, pernicious anemia, glomerulonephritis, myasthenia gravis, Goodpasture’s syndrome, autoimmune hemolytic anemia, idiopathic thrombocytopenia purpura, allergic inflammation including peanut allergy, alopecia, inflammatory bowel disease (IBD), multiple sclerosis, autoimmune hepatitis, hepatic fibrosis, and pulmonary fibrosis.

[0112] In some embodiments, the compounds described herein are administered in combination with one or more compounds that control inflammation by targeting inflammatory cytokines such as tumor necrosis factor-alpha (TNFa) or downstream mediators of cytokine signaling such as tyrosine kinase 2 (TYK2).

[0113] In embodiments, the compounds described herein are administered in combination with one or more Janus kinase (JAK) inhibitors including, without limitation, abrocitinib (CIBINQO®), tofacitinib (XELJANZ®), baricitinib (OLUMIANT®), or upadacitinib (RINVOQ®). In particular embodiments, the compounds described herein are administered in combination with a JAK inhibitor for treatment of a chronic inflammatory disorder or autoimmune disease. In one particular embodiment, the compounds described herein are administered in combination with a JAK inhibitor for treatment of rheumatoid arthritis (RA).

[0114] In embodiments, the compounds described herein are administered in combination with one or more tyrosine kinase 2 (TYK2) inhibitors including, without limitation, deucravacitinib (SOTYKTU®), brepocitinib, or ropsacitinib. In particular embodiments, the compounds described herein are administered in combination with a TYK2 inhibitor for treatment of psoriasis and / or psoriatic arthritis.

[0115] In embodiments, the compounds described herein are administered in combination with one or more tumor necrosis factor-alpha (TNFa) inhibitors including, without limitation, etanercept (ENBREL®), infliximab (REMICADE®), adalimumab (HUMIRA®), certolizumab pegol (CIMZIA®), or golimumab (SIMPONI®). In some embodiments, a combination of a TNFa inhibitor and compounds as described herein are useful for treating ankylosing spondylitis, Crohn's disease, ulcerative colitis, rheumatoid arthritis, psoriatic arthritis, psoriasisjuvenile idiopathic arthritis, Hidradenitis suppurativa, and uveitis.

[0116] As described in Example 12, administration of compounds in a rat collagen- induced arthritis model as described herein in combination with an anti-inflammatory TNF inhibitor (etanercept) was effective to decrease initial inflammation, as measured by a decrease in paw volume (see, e.g., FIG. 5B), and to provide an inflammation resolution effect. In embodiments, the present compounds are contemplated for use with an anti-inflammatory compound as described herein in the treatment of rheumatoid arthritis.

[0117] In embodiments, the compounds described herein are administered in combination with an antagonist or antibody that targets (e.g. inhibits) one or more of interleukin- 4 receptor-alpha (IL-4Ra), interleukin- 13 (IL-13), interleukin-23 (IL-23), or IL-4Ra and IL-13 dual blockade. One suitable antibody that targets IL-13 is lebrikizumab (Ebglyss™, Eli Lilly), which is approved for treatment of atopic dermatitis. Suitable antibodies that target IL-23 include guselkumab, which has been investigated for treating inflammatory bowel disease (IBD)mirikizumab-mrkz (Omvoh™, Eli Lilly), which is approved for treating ulcerative colitis, and risankizumab-rzaa (Skyrizi®, Abbvie), which is approved for treating plaque psoriasis, psoriatic arthritis, Crohn’s disease, and ulcerative colitis. A further suitable antibody that inhibits IL-4 and IL-13 signaling is dupilumab (DUPIXENT®), which is approved for treatment of atopic dermatitis and asthma, among others.

[0118] In embodiments, the compounds described herein are administered in combination with one or more corticosteroids including, without limitation, cyclophosphamide, azathioprine, prednisolone, methylprednisolone, and budesonide. In some embodiments, a combination of a steroid and compounds as described herein are useful for treating autoimmune disease including systemic lupus erythematosus (SLE), autoimmune hepatitis, Crohn’s disease as well as chronic inflammatory diseases.

[0119] In embodiments, the compounds described herein are administered in combination with one or more compounds including, without limitation, cyclophosphamide, azathioprine, and methotrexate.

[0120] In embodiments, the compounds described herein are administered in combination with an anti-fibrotic agent. In some embodiments, the anti-fibrotic agent is selected from nintedanib (Ofev®), pirfenidone (Esbriet®), or saracatinib for treatment of pulmonary fibrosis. In some embodiments, the anti-fibrotic agent is selected from resmetirom (Rezdiffra™), cenicriviroc, or elafibranor (IQIRVO®) for treatment of hepatic fibrosis and / or nonalcoholic steatohepatitis (NASH). In particular embodiments, the compounds described herein are administered in combination with an anti-fibrotic agent to reduce, slow the progression of or eliminate fibrosis. In particular embodiments, the present compounds are contemplated for use with an anti-fibrotic agent as described herein in the treatment of hepatic fibrosis or idiopathic pulmonary fibrosis (IFF).

[0121] In a further aspect, provided herein is a method for promoting and / or maintaining homeostasis and / or tissue repair when combined with one or more anti-inflammatory compounds for treating chronic inflammation or an autoimmune disease. In this embodiment, administration of the presently disclosed compounds is effective to resolve inflammation and promote immune homeostasis as well as to repair tissue damage caused or exacerbated by inflammation.

[0122] As described in Example 11, CSF-1R agonists in accord with the present disclosure (Compound 1 and Compound 2) were effective to accelerate a decrease in diseaseactivity index (DAI) score in a DSS-induced colitis mouse model as indicated by a lower AUC during the last week of the study (see FIG. 19). These results indicate CSF-1R agonists as described herein provide a specific effect in the tissue recovery phase of the inflammatory phase of colitis.

[0123] In an additional aspect, provided is a method for reducing binding affinity of the CSF-1 compound to CSF-1R. As described in Example 4, CSF-1 conjugates as described herein exhibited a reduced receptor affinity (binding) to the CSF-1 R as compared to unmodified native CSF-1 as measured by SPR. Reduced receptor affinity provides one or more of the therapeutically desirable benefits including, but not limited to, improved pharmacokinetic properties and sustained receptor stimulation via the modified receptor affinity. In some embodiments, the instant CSF-1 conjugates exhibit at least about a 100-fold to at least about a 1000-fold reduction in CSF-1 receptor affinity as compared to unmodified CSF-1. In some embodiments, the CSF-1 conjugates described herein exhibit about a 100-fold, about 125-fold, about 150-fold, about 175-fold, about 200-fold, about 250-fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, about 750-fold, about 800-fold, about 900-fold, or at least about 1000-fold or greater reduction in receptor affinity to the CSF-1R as compared to unmodified CSF-1

[0124] In addition to reduction in receptor affinity to the CSF-1R as compared to unmodified CSF-1, the CSF-1 conjugates as described herein exhibit reduced potency as compared to unconjugated CSF-1. As described in Example 5, CSF-1 conjugates had significantly reduced potency in human whole blood monocytes as measured by pERK, pAkt and pS6. Further evidence is provided in Example 7 where the CSF-1 conjugates exhibited significantly reduced potency as compared to unconjugated CSF-1 as measured by pERK and pAkt in mouse blood monocytes and neutrophils. In both of these examples, the CSF-1 mutein conjugate had the greatest loss of potency as compared to unconjugated CSF-1 or other CSF-1 conjugates.

[0125] In some preferred embodiments, the CSF-1 conjugate is administered at a therapeutically effective dose. A therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the CSF-1 conjugate in order to determine an amount that produces a clinically desired endpoint as described herein. Generally, a therapeutically effective amount will range from about 0.1 pg / m2to about 1000pg / m2per administration, or about 25 pg / m2to about 1000 pg / m2per administration, inclusive In further embodiments, the therapeutically effective amount ranges from about 0.1 pg / m2to about 500 pg / m2, about 0.1 pg / m2to about 250 pg / m2, about 0.1 pg / m2to about 100 pg / m2, about 0.1 pg / m2to about 50 pg / m2, about 0.1 pg / m2to about 25 pg / m2, about 0.1 pg / m2to about 10 pg / m2, about 0.1 pg / m2to 1 pg / m2, about 1 pg / m2to about 500 pg / m2, about 1 pg / m2to about 250 pg / m2, about 1 pg / m2to about 100 pg / m2, about 1 pg / m2to about 50 pg / m2, about 1 pg / m2to about 25 pg / m2, about 1 pg / m2to about 10 pg / m2, about 10 pg / m2to about 500 pg / m2, about 10 pg / m2to about 250 pg / m2, about 10 pg / m2to about 100 pg / m2, about 10 pg / m2to about 50 pg / m2, about 10 pg / m2to about 25 pg / m2, about 25 pg / m2to about 100 pg / m2, from about 25 pg / m2to about 75 pg / m2, from about 25 pg / m2to about 50 pg / m2, from about 25 pg / m2to about 100 pg / m2, from about 25 pg / m2to about 150 pg / m2, from about 25 pg / m2to about 250 pg / m2, from about 25 pg / m2to about 500 pg / m2, from about 25 pg / m2to about 750 pg / m2, from about 25 pg / m2to about 1000 pg / m2, from about 50 pg / m2to about 150 pg / m2, from about 50 pg / m2to about 100 pg / m2, from about 50 pg / m2to about 75 pg / m2, from about 50 pg / m2to about 100 pg / m2, from about 50 pg / m2to about 150 pg / m2, from about 50 pg / m2to about 250 pg / m2, from about 50 pg / m2to about 500 pg / m2, from about 50 pg / m2to about 750 pg / m2, from about 50 pg / m2to about 1000 pg / m2, from about 75 pg / m2to about 150 pg / m2, from about 75 pg / m2to about 1000 pg / m2, from about 100 pg / m2to about 1000 pg / m2, from about 200 pg / m2to about 1000 pg / m2, from about 300 pg / m2to about 1000 pg / m2, from about 400 pg / m2to about 1000 pg / m2, from about 500 pg / m2to about 1000 pg / m2, from about 600 pg / m2to about 1000 pg / m2, from about 700 pg / m2to about 1000 pg / m2, from about 800 pg / m2to about 1000 pg / m2, or from about 900 pg / m2to about 1000 pg / m2per administration. In some particular, but not limiting embodiments, the CSF-1 conjugate is administered at a dose of about 0.1 pg / m2, 1.0 pg / m2, 19 pg / m2, 25 pg / m2, 50 pg / m2, 75 pg / m2, 100 pg / m2, 150 pg / m2, 200 pg / m2, 250 pg / m2, 300 pg / m2, 400 pg / m2, 500 pg / m2, 600 pg / m2, 700 pg / m2, 800 pg / m2, 900 pg / m2, or 1000 pg / m2per administration. In other embodiments, a therapeutically effective amount ranges from about 1.0 pg / kg to about 10 pg / kg per administration. In additional embodiments, the therapeutically effective amount ranges from about 1 .0 pg / kg to about 5 pg / kg, from about 1 0 pg / kg to about 2 pg / kg, from about 1.0 pg / kg to about 1.5 pg / kg, from about 1.5 pg / kg to about 10 pg / kg, from about 1.5 pg / kg to about 5 pg / kg, or from about from about 5.0 pg / kg to about 10 pg / kg per administration. In some specific, but not limiting embodiments, a therapeutically effectiveamount is aboutper administration. In some further, but not limiting embodiments, a therapeutically effective amount is about 0.1 gg, about 1.0 gg, about 10 gg, about 25 gg, about 50 gg, about 75 gg, about 100 gg, about 200 gg, about 250 gg, about 500 gg, about 600 gg, about 700 gg, about 750 gg, about 800 gg, about 900 gg, or about 1000 gg per administration. It will be appreciated that the therapeutically effective amount of the conjugate may be any dose as approved by a governmental regulatory agency for a CSF-1 protein, peptide, or fragment thereof. With reference to the doses referenced in the examples herein, one of ordinary skill in the art could convert the animal doses (e g. mouse) to a corresponding dose in humans using conversions as known in the art (e g. Nair et al, J Basic and Clin. Pharmacy, 7:27-31, 2016).

[0126] It will be appreciated that the doses for the CSF-1 conjugate as described above may refer to either of the compound or the CSF-1 protein equivalent. In preferred embodiments, the doses refer to the CSF-1 protein equivalents.

[0127] It will be appreciated that the actual dose of the conjugate to be administered will vary depending upon the age, weight, body surface area, and general condition of the subject as well as the severity of the condition being treated, the judgment of the health care professional, and conjugate being administered. Therapeutically effective amounts are known to those skilled in the art and / or are described in the pertinent reference texts and literature.

[0128] The dose of the conjugate (preferably provided as part of a pharmaceutical composition or preparation) can be administered in a variety of dosing schedules depending on the judgment of the clinician, needs of the patient, and so forth. The specific dosing schedule will be known by those of ordinary skill in the art or can be determined experimentally using routine methods. Exemplary dosing schedules include, without limitation, administration once daily, three times weekly, twice weekly, once weekly (qlw), twice monthly (e.g. q / 14 days), once monthly (e.g. q / 30 days or 31 days, q / 28 days or q / 21 days), and any combination thereof. It will be appreciated that the dosing schedule may be adjusted as needed, e.g. administration once weekly for a period of time and then adjusted to a shorter or longer schedule as needed. Once a desired clinical endpoint has been achieved, dosing of the composition is halted or reduced. In some embodiments, the unit dose of any given conjugate may be administered once to provide sustained effect. A given dose can be periodically administered up until, for example, the clinician determines an appropriate endpoint (e.g., cure, regression, partial regression,remission, and so forth) is achieved. As described in Example 6, polymer conjugation dramatically reduced the clearance rate for all CSF-1 polymer conjugates. As described in Example 8, administration of the CSF-1 conjugate provided sustained pERK activation in monocytes in mice. Accordingly, the CSF-1 conjugates as described herein may provide sustained pharmacodynamic (PD) activity from administration of a single dose.

[0129] It will be appreciated that when the CSF-1 conjugate is administered as part of a combination therapy, the frequency of administration for the CSF-1 conjugate and the additional therapeutic agent(s) may be the same or different and one of ordinary skill in the art would be able to determine an appropriate frequency for each component of the combination therapy. In addition, as some anti-inflammatory compounds are either in advanced clinical testing or commercially available, it is also possible to refer to the literature to obtain an appropriate frequency of administration (keeping in mind that some adjustment may be necessary in view of the combined effects of the treatment regimen). In one preferred embodiment, an antiinflammatory compound is administered before, during or after early or peak inflammatory disease phases in order to control or reduce inflammation. Thereafter, which may be concurrently at least in part, the CSF-1 conjugate is administered to effect inflammation resolution and tissue repair. In other words, the anti-inflammatory agent and the CSF-1 conjugate may be administered sequentially to control / reduce inflammation and promote homeostasis / resolve inflammation / promote tissue repair / extend the rate and / or duration of remission.

[0130] The CSF-1 conjugate may be administered by any suitable means as known in the art. In some embodiments, the CSF-1 conjugate may be administered parenterally which includes subcutaneous, intravenous, intra-arterial, intraperitoneal, intracardiac, intrathecal, and intramuscular injection, as well as infusion injections. Suitable formulation types for parenteral administration include ready-for-inj ection solutions, dry powders for combination with a solvent prior to use, suspensions ready for injection, dry insoluble compositions for combination with a vehicle prior to use, and emulsions and liquid concentrates for dilution prior to administration, among others. In some particular embodiments, the CSF-1 conjugate is provided in a formulation suitable for intravenous administration and is administered intravenously. In some other embodiments, the CSF-1 conjugate is provided in a formulation suitable for subcutaneousadministration and is administered subcutaneously. Other modes of administration are also contemplated, such as pulmonary, nasal, buccal, rectal, sublingual, and transdermal.EXPERIMENTAL

[0131] It is to be understood that the foregoing description as well as the examples that follow are intended to illustrate and not limit the scope of the methods, combinations, kits, etc. provided herein. Other aspects, advantages and modifications will be apparent to those skilled in the art to which this disclosure pertains.

[0132] In the following examples, efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.) but some experimental error and deviation should be taken into account. Unless indicated otherwise, temperature is in degrees C and pressure is at or near atmospheric pressure at sea level. Each of the following examples is considered to be instructive to one of ordinary skill in the art for carrying out one or more of the embodiments described herein.Materials and MethodsSDS-PAGE Analysis

[0133] Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using Invitrogen gel electrophoresis system (XCell SureLock Mini-Cell). Samples were mixed with sample buffer. Then, the prepared samples were loaded onto a NuPAGE Novex precast gel and run for approximately thirty minutes.RP-HPLC Analysis

[0134] Reversed-phase chromatography (RP-HPLC) analysis was performed on an Agilent 1200 HPLC system (Agilent). Samples were analyzed using a Poroshell® 300SB-C3 column (2.1 x 75 mm, Agilent) at 60°C. The mobile phases are 0.1%TFA / H2O (A) and 0.1%TFA / CH3CN (B). The flow rate for the column was 0.5 ml / min. Eluted protein and PEG- protein conjugates were detected using UV at 215nm or 280nm.EXAMPLE 1Preparation of CSF-1

[0135] A rhCSF-1 fragment (SEQ ID NO:2) with a 159 amino acid sequence is available from commercially available sources (e g. PeproTech). Initially an N-terminal methionine was included for translation (at position -1).

[0136] Production: rhCSF-1 was constructed into a plasmid by Atum (Newark, CA).

[0137] Expression: Plasmids containing the rhCSF-1 expression cassette was transformed into chemically competent BL21(DE3) £ coti cells (New England Biolabs). Transformed cells were grown on LB agar plates with ampicillin for selection.

[0138] 2mL of the transformed colonies from the transformed plate (2mL LB / AMP,medium was inoculated with glycerol stock. The inoculated 2mL culture media was incubated at 37°C at 220 rpm for 7 hours. The OD600 of the cell culture was reached around 0.51.

[0139] A 250mL shake flask with 50mL of the overnight culture medium LB / AMP(100was inoculated withpre-culture. The inoculated 50mL overnight culture was incubated at 37°C at 220 rpm overnight. The OD600 of cell culture was reached around 3.5.

[0140] A 2.8L shake flask withwas inoculated with 10 mL of overnight culture and incubated at 37 °C at 220 rpm for around 3.5 hours till OD600 reaching 0.7 - 0.9, then ImM isopropyl-D-thiogalactopyranoside (IPTG) (final) was added for induction and further incubated for 3 hours. The final OD600 reached around 2.10. A total of 6 shake flask fermentations were performed in parallel. The expression levels of rhCSF-1 was determined by SDS-PAGE gel analysis.

[0141] Cells were harvested by centrifugation at 9000 xg for 30 minutes. A total 18.68g of wet cell paste was obtained from the 6 shake flask fermentations.

[0142] The CSF-1 protein was expressed as inclusion bodies (IBs) in the bacterial cells. The cell paste was washed once with cell lysis buffer (50mM Tris, 5mM EDTA, pH 8.0) at a ratio of 20mL buffer / g wet cell paste by sufficient mixing using a homogenizer. The cell suspension was centrifuged at 9000 xg at 4°C for 30 minutes. The resulting pellet was collected. The washed cell paste was re-suspended in cell lysis buffer at a ratio of lOmL buffer / g wet cell paste and mixed well by homogenizer.

[0143] The resuspended cells were then lysed using an M-l 10P microfluidizer (Microfluidics) at 15,000 psi for three passes. After cell lysis, the lysis broth was centrifuged at 15,000 xg at 4°C for 1.5 hours to obtain a crude IB pellet.

[0144] The crude IB pellet was then washed with three different buffers: (1stwash) 50 mM Tris, 5 mM EDTA, 2% (w / v) Triton X-100, and 1% (w / v) Tween 20, pH 8.0, (2ndwash) 50 mM Tris, 5 mM EDTA, and IM sodium chloride, pH 8.0, and (3rdwash) 50 mM Tris, 5 mM EDTA, and 0.02M sodium chloride, pH 8.0. For each wash, the IBs were resuspended in the respective buffer at a ratio of 20mL buffer per gram of IBs and mixed well by homogenizer. The resuspended IBs were then harvested by centrifugating at 15,000 xg at 4°C for 1.5 hours.

[0145] Protein Refolding and Purification: Refolding of the rhCSF-1 was performed through IB dissolving and reduction clarification, and step dilution refolding. The washed IBs were dissolved in an IB dissolving buffer (8M Urea, 2mM Tris, 0.55mM EDTA, pH 8.5) at a ratio of 10-30mL buffer / g IB with gentle mixing at 70 RPM at room temperature for 1.0 hours. A fresh prepared IM DTT in water solution was added to bring the final DTT concentration to 5mM. The solution was incubated in the cold room overnight for complete dissolving and reduction.

[0146] After dissolving and reduction, the solution was centrifuged at 20,000 xg at 4 °C for 2 hours and the supernatant was collected. The protein concentration of the solution was determined by Nanodrop A280 using distinction coefficient of 0.64 (rhM-CSF) and 1 :5 dilution ratio.

[0147] The dissolved and reduced IB solution was diluted further with IB dissolving buffer (8M Urea, 2mM Tris, 0.5mM EDTA, pH 8.50) to a protein concentration of around 1.5- 2.0 mg / mL Fresh prepared IM DTT in water solution was added to bring the final DTT concentration to 5 mM. The IB solution was incubated in 4 °C cold room for reduction.

[0148] A refolding buffer (50mM Tris, 5mM EDTA, 20mM NaCl, 2mM GSH, ImM GSSG, pH 8.5) was prepared according to the dilution ratio of 11 -12 of refolding buffer / IB solutionThe refolding buffer was pre-chilled in a cold room (4°C). Reduced and cooled IB solution (300mL) was slowly added to the pre-chilled refolding buffer using a pipette with vigorous stirring (300-340 rpm). After adding, the stirring was slowed to 60 rpm. The solution was loose caped and incubated in the cold room for 4-5 days.

[0149] The refolded rh-CSF-1 was purified using Q Sepharose HP column purification and Phenyl Sepharose HP column chromatography.EXAMPLE 2Design and Preparation of CSF-1 Muteins

[0150] The mutein prepared was derived from the amino acid sequence of a hCSF-1 (SEQ ID NO:2) available from commercially available sources (e.g. PeproTech). Initially an N- terminal methionine was included for translation (at position -1) (SEQ ID NO.4).

[0151] To generate the CSF-1 H15A sequence, the histidine at position 15 of the CSF-1 sequence was replaced with an alanine (SEQ ID NO:5). The amino acid sequence of the mutein prepared is shown below with the alanine residue highlighted in bold and underlined: hCSF-1 H15A:MEEVSEYCSHMIGSGALQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAF LLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYET PLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQGHERQSEGS (SEQ ID NO: 4)

[0152] Mutein Production: The nucleotide sequences encoding for the mutein polypeptides were generated using GeneGPS® technology (Atum, Newark, CA). The mutein nucleotide sequences were then inserted into the pD454-SR plasmid (Atum). The utilized plasmid conferred ampicillin resistance.

[0153] Mutein Expression: Plasmids containing the rhCSF-1 H15A expression cassette was transformed into chemically competent BL21(DE3) E. colt cells (New England Biolabs). Transformed cells were grown on LB agar plates with ampicillin for selection.

[0154] Transformed colonies from the transformed plate were used to inoculate a small 2mL culture in 2xYT media (Teknova) with lOOpg / mL of ampicillin. The 2mL cultures were grown at 37°C and shaken at 220 rpm for 7 hours and OD600 around 0.6 Glycerol stocks were generated by adding 80% v / v glycerol to the cultures in a 1 : 1 volume ratio (final concentration of 40% v / v glycerol). This glycerol stock was used for expression.

[0155] To initiate a production culture in shake flasks, a 250 mL shake flask with 50 mL of 2xYT medium and 100 μg / mL of ampicillin was inoculated with the glycerol stock. The inoculated 50 mL culture was incubated at 30 °C and shaken at 220 rpm for 14 hours.

[0156] After 14 hours, the overnight culture reached OD600 of around 1.0. The expression culture was started in a 2.8L shake flask with IL of 2xYT medium and 100 pg / mL of ampicillin. The IL expression cultures were inoculated using 10mL of the overnight pre-culture. The expression culture was incubated at 37°C and shaken at 220 rpm for around 3.5 hours until OD600 reached 0.7-1.0, then IPTG was added to final concentration of ImM for induction of expression. A 500pl sample was taken and centrifuged before induction as a starting timepoint (TOhr).

[0157] 3 hours after induction another 500μl sample was taken as a final time point(T3hr), and OD600 was recorded to be between 2.0 and 3.0. Cells were harvested at 9,000xg for 30min.

[0158] For each culture, the starting and final time point samples were resuspended in milli-q water to normalize the calculated OD600 to 20. Samples were combined with 6 / zl of sample buffer (InvitrogenNuPAGE™LDS Sample Buffer 4X), 4 / 4 of reducing agent (Invitrogen NuPAGE™ Sample Reducing Agent 10X), and 18 / 4 of resuspended sample. The expected MW of the expressed protein in a reducing gel is 18kDa.

[0159] The expression levels of rhCSF-1 was determined by SDS-PAGE gel analysis.

[0160] The CSF-1 muteins were expressed as inclusion bodies (IBs) in the bacterial cells. The cell paste was washed once with cell lysis buffer (50mM Tris, 5mM EDTA, pH 8.0) at a ratio of 20mL buffer / g wet cell paste by sufficient mix using a homogenizer. The cell suspension was centrifuged at 9000 xg at 4°C for 30 minutes. The resulting pellet was collected. The washed cell paste was re-suspended in cell lysis buffer at a ratio of lOmL buffer / g wet cell paste and mixed well by homogenizer.

[0161] The resuspended cells were then lysed using an M-l 10P microfluidizer (Microfluidics). After cell lysis, the lysis broth was centrifuged at 15,000 xg at 4°C for 1.5 hours to obtain 6.96g of crude IB pellet.

[0162] The IBs were then washed with three different buffers:wash) 50 mM Tris, 5 mM EDTA, 2% (w / v) Triton X-100, and 1% (w / v) Tween 20, pH 8.0 to obtain a 6.36g IB pellet, (2ndwash) 50 mM Tris, 5 mM EDTA, and IM sodium chloride, pH 8.0 to obtain a 5 96g IB pellet, and (3rdwash) 50 mM Tris, 5 mM EDTA, and 0.02M sodium chloride, pH 8.0 to obtain a 5.36g pellet. For each wash, the IBs were resuspended in the respective buffer at a ratio of 20mLbuffer per gram of IBs and mixed well by homogenizer. The resuspended TBs were then harvested by centrifugating at 15,000 xg at 4°C for 1.5 hours.

[0163] Mutein Refolding and Purification: Refolding of the rhCSF-1 was performed through IB dissolving and reduction clarification, and step dilution refolding. The washed IBs were dissolved in an IB dissolving buffer (8M Urea, 2mM Tris, 0.5 mM EDTA, pH 8.5) at a ratio of 10-30 mL buffer per gram IB and gently mixed at 70 rpm at room temperature for 1.0 hours. Freshly prepared IM DTT in water solution was added to bring the final DTT concentration to 5 mM. The resuspended mixture was incubated in the cold room overnight for complete dissolving and reduction. After dissolving and reduction, the resuspended mixture was centrifuged at 20,000 xg at 4°C for 2 hours. The supernatant containing the resolubilized protein was collected. The protein concentration of the solution was determined by Nanodrop A280 using distinction coefficient of 0.64 (rh-CSF-1) and 1:5 dilution ratio

[0164] The dissolved and reduced IB solution was diluted further with IB dissolving buffer (8M Urea, 2mM Tris, 0.5mM EDTA, pH 8.50) to a protein concentration of around 1.5- 2.0 mg / mL. Fresh prepared IM DTT in water solution was added to bring the final DTT concentration to 5 mM. The DTT was added to reduce any oxidized cysteines and to maintain a reducing environment for refolding. The IB solution was incubated in 4°C cold room for reduction.

[0165] Prior to use, a refolding buffer (50mM Tris, 5mM EDTA, 20mM NaCl, 2mM GSH, ImM GSSG, pH 8.5) was pre-chilled in a cold room at 4°C. Reduced and cooled IB solution (300mL) was slowly added to the pre-chilled refolding buffer (3500mL) using a pipette with vigorous stirring (300-340 rpm). The solution was incubated in the cold room for 4-5 days with stirring at 60 rpm.

[0166] The refolded rh-CSF-1 was purified using Q Sepharose® HP column purification and Phenyl Sepharose® HP column chromatography (Cytiva).EXAMPLE 3Preparation of di-mPEG2-ru-butyrALD-40kDa-CSF-l-H15ACompound 1

[0167] rhCSF-l-Hl 5 A was prepared as described in Example 2. The purified rhCSF-1-H15A in lOmM sodium phosphate pH 7.4, 150 mM NaCl and 10% mannitol was diluted to 1.0 g / L with MiliQ water and brought to room temperature.

[0168] The branched mPEG reagent di-mPEG2-ru-butyrALD-40kDawas used to prepare rhCSF-l-H15A conjugates, where the weight average molecular weight of the PEG reagent used to prepare the conjugates was about 40,000 daltons.

[0169] In a separate vessel, the PEG solution was prepared by solubilizing the PEG reagent into MiliQ water at 1 mL per 100 mg of PEG reagent at room temperature. The amount of PEG charged is based on a 25 to 1 mol / mol ratio of PEG to CSF-1 mutein.

[0170] PEG is a light powder and is a prone to static adhesion. To ease the process of solubilization of PEG in water, the solution was centrifuged at low RCF (200-300 RCF) proceeded by gentle mixing.

[0171] After PEG dissolution, the CSF-1 solution was brought to pH 5.5 ± 0.1 by charging with 1 M sodium acetate buffer at pH 5 with gentle agitation. The amount of acetate buffer was relative to the volume of the CSF-1 solution once it was adjusted to 1.0 g / L. The ratio was ~ 0.02 ± 0.004 to 1 v / v, IM acetate buffer to reaction solution volume. Small amounts of acetate buffer were charged at the time and the pH was monitored using a pH probe.

[0172] The PEG solution was slowly charged into the CSF-1 solution using a serological pipette and PEG solution was applied onto the walls of the reaction vessel allowing gentlemixing. The PEG CSF-1 reaction mixture was slowly mixed for 30-minutes using a nutator at room temperature.

[0173] Sodium cyanoborohydride powder was reconstituted as a IMsolution in VBSE hood using MiliQ water. IM NaBHjCN solution was charged into the conjugation reaction to a 0.0 IM final concentration.

[0174] The pH of the reaction was confirmed at pH 5.5 using a pH meter. The reaction mixture was moved into a cold room and the reaction continued with gentle mixing on a nutator at 4°C overnight.

[0175] After about 16 hours, a small aliquotfrom the reaction mix was taken for analysis of the ongoing reaction using RP-HPLC.

[0176] Progress of conjugation reaction was monitored with sample injection onto the RP-HPLC column every' hour. Because it is hard to separate Di-PEG-CSF-1 from Tri-PEG-CSF- 1 it is important to limit the amount of Tri-PEG-CSF-1 formed. Preferably, the relative % of Tri- PEG-CSF-1 is kept below 8%. The reaction was run for 26-28 hours before quenching. Di-PEG- CSF-1 is expected to be around 55%.

[0177] To quench, the reaction was charged with l / 10th of its volume of 0.5 M glycine pH 8.0. The reaction mixture was gently mixed on a nutator for 30 minutes at room temperature.Afterwards, the quenched reaction mixture was diluted in MiliQ water (DF 15) and filtered through a 0.22 μm disposable bottle filter. It was stored at 2-8°C for up to 72 hours. The quenched PEGylation reaction mixture was characterized by RP-HPLC with the results shown in Figure 2. The PEGylation reaction yielded 35.81% mono-conjugate (one PEG attached to rhCSF-l-H15A), 54.6% di-conjugate (two PEG attached to rhCSF-l-H15A), and 5.28% triconjugate (three PEG attached to rhCSF-l-H15A).

[0178] Using the same approach, conjugates of rhCSF-1 (wild type) or other CSF-1 mutein conjugate can be prepared with branched mPEG-ru-butyrALD reagents can be prepared. Using the same approach, a CSF-1 conjugate was prepared having the structure:Compound 2.

[0179] Further using this same approach, other CSF- 1 (wild type or mutein) conjugates can be prepared with linear or branched mPEG-ru-butyrALD reagents having other weight average molecular weights, e.g. 20 kDa, 30 kDa, 50 kDa, 60 kDa, etc. as described herein.EXAMPLE 4Polymer Conjugated CSF-l Variants with Reduced Receptor Affinity by SPR Screening

[0180] A surface plasmon resonance (SPR) based screening method was developed to evaluate CSF-1R binding affinity of recombinant CSF-l polymer conjugates to identify low affinity binders. Parameters used for generating conjugates for screening included properties of the polymer (e.g. polymer size and geometry), polymer conjugation chemistry, and CSF-l protein sequence. Biacore® sensor chips (Cytiva) coated with anti-Fc antibody were bound by CSF-IR-Fc fusion protein containing residues Ile20-Glu512 that includes the CSF-l binding domain. Binding affinities of PEG polymer CSF-l conjugates as shown in Table 1 to CSF-IR-Fc were determined by kinetic and steady state binding analysis with the results shown in Table 2. Table 1: PEGylated CSF-l conjugates for screeningTable 2: SPR rate constants (kon, koff) and dissociation constant (KD) screening result for the polymer conjugates described in Table 1

[0181] CSF1-R binding affinity was dramatically reduced as a function of polymer size and geometry, and the conjugate's protein component sequence. Receptor binding was most dramatically impacted (reduced) for the polymer conjugated with the mutagenized CSF-1 variant, Compound 1.EXAMPLE SCell Signaling Potency Evaluation of Low Affinity CSF-1R Binding Conjugates in Human Whole Blood Monocytes

[0182] Three compounds with the lowest CSF-1R binding affinity in the SPR assay described in Example 4 were selected for cell signaling evaluation in CD 14+CD 16- monocytes in human whole blood samples in vitro. CSF-1R activation in myeloid cells triggers MAPK and PI3K-Akt signaling pathways activation. Flow cytometry based phospho-signaling assays to monitor intracellular phosphorylation of extracellular signal-regulated kinase (pERK), Akt kinase (pAkt) and S6 ribosomal protein (pS6) were developed to measure cell signaling potency after 20 minutes (pAkt, pS6) or 30 minute (pERK) incubation with test compounds at 37°C in vitro in CD14+CD16- monocytes. Dose response experiments with 11 -point concentration series with each compound were conducted to determine EC50 and Emax of CD14+CD16- monocytes population fraction that was positive for each of the three phospho-markers (pERK, pAkt or pS6) for each compound and the parental CSF-1 protein fragment with wildtype amino acid sequence. Flow cytometry data were analyzed on FlowJo® software (BD Biosciences). Each phosphomarker dose response percent positivity values were fitted to variable slope four parameter log(agonist) vs. response model in GraphPad Prism® software (GraphPad Software) for EC50 and Emax calculation. The resulting ECJO and Emax best-fit values are shown in Table 3. Table 3: Polymer conjugated CSF-1 conjugates potency in cell signaling in human monocytes

[0183] The potency of all compounds was significantly reduced as compared to unconjugated CSF-1. Loss of potency correlated with conjugated polymer size and geometry.The most dramatic loss of potency was observed for the polymer conjugated mutagenized CSF-1 variant, Compound 1. All compounds in Table 3 showed full agonist activity reaching similar levels of Emax as parental unconjugated CSF-1.EXAMPLE 6Polymer Conjugation and CSF-1 Mutagenesis Effect on CSF-1R AgonistsPharmacokinetics in Mice

[0184] Balb / c (Figure 3) or C57B16 (Figure 4) female mice were intravenously (TV) injected with indicated PEG-CSF1 conjugates at dose levels as indicated in Tables 4 and 5. K2EDTA anticoagulant plasma was collected and each conjugate or unconjugated CSF-1 concentration in plasma was measured at indicated timepoints by a bioanalytical assay that captured unconjugated CSF-1 or PEG-conjugated CSF-1 to MSD plate via anti-human CSF-1 antibody (Figure 3) or via anti-PEG antibody (Figure 4). After diluted plasma sample incubation and washing, the bound unconjugated CSF-1 or PEG-conjugated CSF-1 was detected by sulfotag bound human CSF-1 specific antibody. Test article plasma concentrations were calculated based on CSF-1 standard curve. Plasma concentrations-based calculation of PK parameters in Tables 4 and 5 were calculated in Phoenix WinNonlin® software (Certara).Table 4: Pharmacokinetic parameters in Balb / c miceTable 5: Pharmacokinetic parameters in C57B16 mice00185] Polymer conjugation dramatically improved the PK properties increasing exposure, half-life and reducing clearance rate for all CSF-1 polymer conjugates. These effects were driven by polymer size and the number of PEG polymers conjugated to the CSF-1 dimer (Figure 3, Table 4). Further reduction of receptor affinity was achieved by mutagenesis of the protein component in the polymer conjugate. As seen in Figure 4 and Table 5, use of a CSF-1mutein in the polymer conjugate further increased exposure, half-life and reduced clearance rate and PK nonlinearity when compared to a non-mutated protein polymer conjugate utilizing the same polymer and linker composition (Figure 4 and Table 5).EXAMPLE 7INDUCTION OF ERK AND AKT PHOSPHORYLATION IN MOUSE BLOODMONOCYTES AND NEUTROPHILS BY COMPOUND 1 OR COMPOUND 2 IN VITRO

[0186] Reduction in cell signaling potency of Compound 1 and Compound 2 was evaluated in comparison to CSF-1 in pERK and pAkt induction in monocyte subsets and neutrophils in fresh mouse whole blood in a dose response experiment. Blood samples from c57B16 mice were incubated for 30 minutes with CSF-1, Compound 1, or Compound 2 followed by flow cytometry based detection of phosphorylated ERK and Akt within major blood immune cell populations including classical pro-inflammatorynon-classicalFlow cytometry data was analyzed on Flowjo® software (BD Biosciences). Each phospho-marker dose response percent positivity values were fitted to variable slope four parameter log(agonist) vs response model in GraphPad Prism software for EC50 and Emax calculations. Resulting EC50 and Emax best-fit values are shown in Table 6 formonocytes, Table 7 formonocytes, Table 8 formonocytes and Table 9 for neutrophils.Table 6: Signaling potency and Emax values in mouse peripheral blood monocyte subsets and neutrophilsTable 7:y yTable 8:y yTable 9:y p

[0187] The potency of each of Compound 1 and Compound 2 was significantly reduced as compared to unconjugated CSF-1. Potency loss correlated with conjugated polymer size and geometry. The most dramatic potency loss was observed for Compound 1, which combinespolymer conjugation with a protein sequence that is a mutagenized CSF-1 variant that impacts receptor affinity. Compound 2 maintained full agonist activity without a significant reduction in Emax as compared to the unconjugated CSF-1 protein. Compound 1 lost any observable ability to activate pAkt signaling in neutrophils changing qualitatively the cell signaling pattern compared to CSF-1, which was active on both monocytes and neutrophils. In mouse monocyte subsets Compound 1 top concentration did not reach maximal pERK (Figures 9A-9D) or pAkt (Figures 9E-9H) induction. Approximate EC50 values were estimated assuming comparable Emax values to CSF-1.EXAMPLE 8SUSTAINED TARGET ENGAGEMENT IN VIVO IN MICE BY A SINGLE DOSE OFCOMPOUND 1 OR COMPOUND 2

[0188] Compound 1 and Compound 2 were evaluated in c57Bl mice in peripheral blood myeloid cells ERK and Akt phosphorylation induction in a dose response time course experiment Blood was collected at 4 h, 24h, 48h and 72h after intravenous injection of 1 mg / kg, 0.3 mg / kg, or 0.1 mg / kg of Compound 1, 0.3 mg / kg, 0.1 mg / kg, or 0.03 mg / kg of Compound 2, or a vehicle. Phospho-ERK (pERK) and phospho-Akt (pAkt) levels in classical pro- inflammatorymonocytes, non-classical andmonocytes and Ly6G+neutrophils were measured by flow cytometry in comparison to vehicle treated mice. Flow cytometry data was analyzed in FlowJo® software and percentage changes of pERK and pAkt frequency in target cells were graphed in GraphPad Prism® as shown in Figures 10A-10D for %pERK+ and Figures 10E-10H for %pAkt+.

[0189] Sustained pERK activation in monocytes was observed after administration of Compound 1 or Compound 2 consistent with sustained exposure profiles of both conjugates. Quantitative differences were observed when comparing the conjugates to each other. At comparable starting blood concentration at 0.1 and 0.3 mg / kg dose levels Compound 1 showed saturated pERK activation inmonocytes whereas Compound 2 showed minimal pERK activation. 10-fold higher Compound 2 dose level (1 mg / kg) was necessary to reach comparable maximum pERK level at the 4 hour timepoint correlating with significantly lower potency of Compound 2. These data also indicate that Compound 2 has comparable Emax in pERKactivation to Compound 1 when measured in vivo. Among the monocytes subsets Ly6Chighmonocytes showed highest sensitivity to both conjugates and differences in pERK activation were quantitative in nature consistent with potency difference between both conjugates. Compound 1 sustained elevated pERK longer than Compound 2 showing upregulated pERK inmonocytes at 3 days after treatment whereas Compound 2 dependent pERK activation returned to baseline by Day 2 after treatment. pAkt activation profile in monocytes showed qualitatively different profiles when comparing Compound 2 to Compound 1. At comparable conjugate maximal starting concentrations (0.1 and 0.3 mg / kg dose levels) Compound 2 activation of pAkt was highest at the earliest 4 hour timepoint and was sustained at half-maximal level on Day 3 after treatment. At the same dose levels providing similar initial peak concentration Compound 1 profile of pAkt activation showed delayed maximal activation reaching peak activation at lower maximal activation level relative to Compound 2 on Day 1 after treatment. At comparable dose levels, Compound 2 dependent pAkt upregulation was sustained through Day 3 after treatment whereas Compound 1 dependent pAkt activation in monocytes returned to baseline by Day 3 despite higher plasma concentration as determined by PK measurement (Example 6). In neutrophils either conjugate did not show pERK sustained activation at 4 hour or any of the later timepoints. Compound 2 showed maximal activation at 4 hours after administration that was sustained through Day 3 at 0.1 and 0.3 mg / kg doses. At the same dose levels Compound 1 showed delayed and transient pAkt activation in neutrophils with no activity at the 4 hour timepoint and maximal activity at 24 hours after administration to same level as saturated activity of Compound 2. By Day 3 Compound 1 induced pAkt activation had returned to baseline levels unlike Compound 2 at same dose levels despite expected higher blood concentration than Compound 2.

[0190] These data indicate that reducing CSF-1R agonist affinity and signaling potency translate to sustained multi-day monocytes and neutrophils activation from a single treatment. Compound 1 can elicit differentiated signaling profile compared to Compound 2 conjugate when normalized to same dose levels and comparable Cmax concentration.EXAMPLE 9EFFECT ON BLOOD MONOCYTES AND LIVER MACROPHAGESPROLIFERATION, CELL NUMBER INCREASE AND MONOCYTES TISSUE INFILTRATION AFTER ADMINISTRATION OF COMPOUND 1 OR COMPOUND 2

[0191] A key CSF-1 function is to maintain cell numbers of blood monocytes and tissue macrophages. CSF-1 can trigger peripheral blood monocytes and tissue macrophages proliferation, increase cell numbers for these cells, and increase and induce tissue infiltration of peripheral blood pro-inflammatory monocytes when elevated during inflammation or provided through treatment exogenously.

[0192] C57B16 mice were administered (0.3, 1, 3, 10, and 30 mg / kg) of Compound 1 intravenously and the positivity of the Ki67 proliferation marker was measured by flow cytometry on Day 2 and Day 6 after treatment. Compound 2 was administered 3 mg / kg intravenously for evaluation at 2 days after treatment. The frequency of Ki67 positive cells (%Ki67+) was measured in blood monocyte subsets includingpro-inflammatory monocytes,nonclassical monocytes from peripheral blood. Tissue macrophage subsets Ki67 positive cells frequency was determined from liver tissue includingTIM4+tissue resident macrophages also called Kupffer cells (KC) andTIM4" monocyte derived macrophages (MdM).

[0193] Treatment with Compound 1 did not change the Ki67+ cell fraction in monocyte subsets in blood when tested up to 30 mg / kg dose level in contrast to treatment with Compound 2 at 3 mg / kg dose, which significantly increased pro-inflammatorymonocytes Ki67 positivity (Figure 11 A) and showed a modest 2-3-fold increase of Ki67+ cells frequency insubsets (Figures 1 IB-11C). Unlike blood monocytes, liver tissue resident macrophages (Kupffer cells) showed dose responsive Ki67 induction in response to treatment with Compound 1 saturating the response at the 3 mg / kg dose level (Figure 12A). A comparable Ki 67 induction of Ki67 positive cells was observed with treatment with Compound 2 at 3 mg / kg. Monocyte derived macrophages showed no additional increase of Ki67 positivity compared to baseline in Compound 1 treatment animals when treated with up to 30 mg / kg doselevel (Figure 12B). In contrast, and similarly to blood monocytes, treatment with Compound 2 at a 3 mg / kg dose level increased Ki67+ cells in the MdM population, to nearly 100% positivity.

[0194] The increase in cell count in blood for monocyte subsets reflected the Ki67 proliferation marker results.monocytes showed a nearly 10-fold increase in mice treated with 3 mg / kg Compound 1 and a dose responsive increase at 0.3 mg / kg and 1 mg / kg dose levels (Figure 13 A). Treatment with Compound 1 at a Ki67 induction-saturating dose level of 3 mg / kg showed minimalpro-inflammatory monocytes increase and a modest below 2- fold increase insubsets (Figures 13B and 13C) that may reflect enhanced survival signaling which is known to require lower CSF-1 activity compared to proliferation induction. Treatment with Compound 1 showed a dose responsive increase in Kupffer cells up to a 2-3-fold increase at the 3 mg / kg dose level that reached maximal Ki67 induction level (Figure 14A). Similarly to the increase obtained for blood monocytes, administration of Compound 2 induced up to 10-fold increase in monocyte derived macrophages (MdM) increase in liver tissue and increased monocytes cell count several fold in the liver (Figures 14B and 14C). MdM are derived fromperipheral blood monocytes that infiltrate into solid tissues after increased CSF-1R stimulation. High bloodinduction explains the substantial MdM increase observed in animals treated with Compound 2.

[0195] In a homeostatic state, monocyte infiltration into liver is low and Kupffer cells represent -90% of macrophage subsets in the liver with small monocyte derived macrophage population. In inflammatory conditions, significant pro-inflammatory monocyte tissue infiltration is commonly observed that can sustain tissue inflammation. Treatment with Compound 2 changed MdM and Kupffer cell ratio to approximately 1 : 1 due to significant infiltration of Ly6Chighmonocytes that differentiated to MdM, peaking at days 2 and 3 after treatment before returning to baseline by day 3 (Figures 14A-14C). Treatment with Compound 1 even to up to 30 mg / kg dose level did not induce significant tissue infiltration of monocytes as shown by monocyte count minimal change in liver tissue (Figures 14A-14C) and accordingly increased minimally MdM levels compared to vehicle treated animals. The Kupffer cell to MdM cell ratio for Compound 1 treated animals was more similar to vehicle treated animals where tissue resident macrophages are the dominant macrophage subset in the liver.EXAMPLE 10INDUCTION OF FUNCTIONAL MARKERS RELATED TO INFLAMMATIONRESOLUTION AND TISSUE REPAIR FUNCTIONS IN LIVER TISSUE RESIDENTMACROPHAGES BY COMPOUND 1 OR COMPOUND 2

[0196] Inflammation resolution and tissue repair supporting macrophages adopt distinct functional gene expression signatures that are characterized by expression of receptors and enzymes that mediate anti-inflammatory polarization, synthesis of inflammation resolution mediating cytokine and lipid mediators as well as removal of dead cells, tissue debris and remodeling of tissue architecture. A subset of functional markers including metalloproteases (MMP) activation, and upregulation of anti-inflammatory tissue repair cytokines IL-4 and IL- 10 receptors (IL-4Ra> IL-lORa) and efferocytosis receptor MerTK was evaluated on liver tissue resident macrophages (Kupffer cells) cells in vivo after administration of Compound 1 or Compound 2.

[0197] C57B16 mice were administered 0.3, 1, or 3 mg / kg of Compound 1 intravenously,0.3, 1, or 3 mg / kg of Compound 2 intravenously, or a vehicle intravenously and the MMP activity, IL-4a induction, IL-2Ra upregulation, and MerTK+ expression in Kupffer cells was measured on Days 2, 3 and 6, or Days 2, 6, and 10 after treatment.

[0198] MMP activity in Kupffer cells (KC) was measured by flow cytometric detection of MMP sensor MMPSense® (ThermoFisher) which is a quenched peptide that is converted to a fluorescent peptide by cleavage of MMPs in vivo when administered 24 hours prior to tissue measurement with the results shown in Figure 15. IL-4Ra, IL-lORa and MerTK levels on Kupffer cells were determined by flow cytometry from liver single cells suspensions with the results shown in Figure 16 (%IL-4Ra+), Figure 17 (%IL-10Ra+), and Figure 18 (%MerTK+). Kupffer cells were defined ascells with standard antibody staining and flow cytometry methods.

[0199] MMP activity in KC was induced by Compound 2 and Compound 1 at tested dose levels of 0.3, 1, and 3 mg / kg. Maximal signal was achieved for the mice administered 1 and 3 mg / kg of Compound 2, which showed initial signal at 2 days after treatment that exceeded Compound 1 induction of MMP activity. However, the inductions provided by Compound 1 treatment were more sustained over time and by day 6 after treatment higher a MMPSense®signal was observed in the 3 mg / kg Compound 1 treated animals as compared to the same dose level of Compound 2 correlating with clearance profiles of both conjugates.

[0200] IL-4Ra induction was significantly more robust with Compound 2 treatment as compared to Compound 1, up to 10-fold increase at the highest 3 mg / kg dose. Induction provided by Compound 1 was saturated at approximately 2-fold higher level as compared to vehicle treatment. Compound 2 induced IL-4Ra upregulation was not sustained and returned to baseline levels by Day 3 after treatment. While Compound 1 induced IL-4Ra upregulation was lower in magnitude, it was sustained longer and maintained elevated levels at Day 6 after treatment at the highest dose level of 3 mg / kg correlating with a sustained exposure profile.

[0201] IL-lORa was induced by both conjugates in a sustained manner in KC. Compound 2 was more effective at IL-lORa induction showing higher levels of induction at 1 mg / kg dose level as compared to 3 mg / kg Compound 1. IL-lORa upregulation was sustained from a single dose of either conjugate for at least 10 days (see Figure 17).

[0202] Efferocytosis receptor MerTK expression in KC was induced by both Compound 2 and Compound 1. MerTK expression and signaling in macrophages promotes proresolving mediators production in cells and suppression of inflammation (Cai et al., 2016, doi: 10.1073 / pnas.1524292113). Compound 2 was more potent than Compound 1 in MerTK upregulation on Day 2 of the dose response showing saturated maximal signal at 0.3 mg / kg dose level. Over time Compound 2 induction of MerTK decayed dose responsively returned to baseline at the lowest 0.3 mg / kg dose level and maintained approximately half-maximal signal at the 1 mg / kg and 3 mg / kg dose levels. In contrast, for the 3 mg / kg dose level of Compound 1, dependent induction at of MerTK reached approximately half-maximal induction of MerTK upregulation compared to Compound 2 but maintained the upregulated expression levels through 6 days after treatment correlating with a reduced clearance profile.EXAMPLE 11IN VIVO ADMINISTRATION OF rCSF-1 POLYMER CONJUGATES IN A DEXTRANSULFATE SODIUM (DSS)-INDUCED COLITIS MOUSE MODEL

[0203] Enhancement of inflammation resolution effect of Compound 1 and Compound 2 was evaluated in the dextran sulfate sodium (DSS)-induced colitis model in c57B16 mice. DSScolitis model at moderate DSS concentrations goes through three phases of disease state including (i) a buildup phase of inflammation and tissue injury described by a rate of disease score increase that culminates in (ii) a peak disease level phase with maximal inflammation and tissue injury. After cessation of DSS administration, a third phase (iii) of the disease includes a decrease in inflammation and repair of accumulated tissue injury that is described by certain rate of disease activity index (DAI) score decrease which is reflective of inflammation resolution and tissue repair that drives a return to homeostasis dissipation of disease symptoms. Compound 2 and Compound 1 were evaluated in this disease model to assess an effect on the rate of disease reduction after DSS administration during the inflammation resolution and tissue repair phase of the disease model.

[0204] Mice were provided 1.75% DSS in drinking water ad libitum for 7 days to induce disease followed by 14 days of regular tap water. No disease control animals received regular water without DSS. Mice were randomized based on disease activity index (DAI) on day 5 into treatment groups of 10 animals each and injected intravenously with vehicle (PBS), Compound 2 (1 mg / kg) or Compound 1 (10 mg / kg) on days 5 and 8 after DSS treatment start. Disease severity was evaluated by DAI score on days 0, 5, 7, 9, 12, 14, 17 and 21. The DAI score was calculated by grading on a scale of 0 to 4 using the following parameters: loss of weight (0: normal, 1: 0 - 5%, 2: 5 - 10%, 3: 10 - 20%, 4: >20%), stool consistency (0: normal, 2: loose stools, 4: watery diarrhea) and occurrence of gross blood in stool (0: negative, 4: positive). The mean DAI score after Compound 1 or Compound 2 treatment did not affect the rate of increase of disease score in the early inflammatory phase of the disease induction. Further, no reduction in peak disease score compared to vehicle treatment (Figure 19) was observed in treated animals. However, treatment with Compound 1 or Compound 2 accelerated a decrease in disease score in the late phase of the disease course that occurs in the model and involves inflammation resolution and tissue repair as indicated by a lower area under the curve (AUC) under the DAI score curve during the last week of study (Day 14-21) (Figure 19 and 20). These results indicate single agent treatment with Compound 1 or Compound 2 provide a specific effect in the tissue recovery phase but not efficacy in the inflammatory phase of the disease.EXAMPLE 12IN VIVO ADMINISTRATION OF rCSF-1 POLYMER CONJUGATES ALONE OR INCOMBINATION WITH AN ANTI-INFLAMMATORY TNF INHIBITOR IN A RATCOLLAGEN-INDUCED ARTHRITIS MODEL

[0205] Studies were conducted to evaluate and compare the inflammation resolution effect of Compound 1 (di-mPEG2-ru-butyrALD-40kDa-CSF-l-H15A) or Compound 2 (dimPEG2-ru-butyrALD-40kDa-CSF-1), each a CSF-1 polymer conjugate, as a monotherapy or in combination with an anti-inflammatory TNFa inhibitor (etanercept, ENBREL®, Amgen) in a rat collagen-induced (CIA) model.

[0206] Rats used were ~6 weeks old female LEW / CrlCrlj. Non-immunized rats (Normal group) and immunized rats were allocated as shown in Table 10.Table 10: Study Treatment groups

[0207] Arthritis was induced by immunization on Day 0 and Day 7 with an emulsion containing bovine type II collagen. Etanercept (EMBREL®) was subcutaneously administered twice a week from Day 14 to Day 28. Compound 1 or Compound 2 was intravenously administered once a week from Day 14 to Day 28. Paw volume was measured on Day 14, Day 21 and Day 28. Body weight was measured every day from Day 14 to Day 28. Data represent the mean±S.E. for 8 animals / group. ***P<0.001 (CIA control group vs. each normal group, Student’s t-test), ###P<0.001 vs. each normal group (CIA control group vs. each normal group, Wilcoxon test).

[0208] The CIA rats were immunized by intradermal injection at four sites on the back and one site on the tail base with 0.5mL of an emulsion of bovine type II collagen and Freund’s incomplete adjuvant (FIA) (3 mg / ml bovine type II collagen solution mixed with an equal volume of FIA) on day 0. Seven days after the first immunization (day 7), the CIA rats were immunized with 0.2 mL of the emulsion intradermally at one site on the tail base. Normal rats were not immunized.

[0209] Paw volume (PV) of the hind paws of each rat was measured one day before the second immunization (day 6) and on Day 14, Day 21 and Day 28 using a plethysomometer (Ugo Basile). Body weight was measured on Day 7 and every day from Day 14 to Day 28. The change in body weight (g) from day 14 to day 28 is shown in Figures 6 and 7. The immunized rats were selected and grouped based on the changes in PV from the day before the second immunization to Day 14 and the body weight on Day 14.

[0210] Vehicle (formulation buffer), Compound 1, or Compound 2 were administered intravenously at 5 mL / kg once a week to Group 1 - Group 7 from Day 14 as detailed in Table 10. Vehicle (saline) or etanercept were administered subcutaneously at 5 mL / kg twice a week with an interval of 3 to 4 days between doses to Group 1- Group 7 from Day 14.

[0211] Figure 5 A shows the change in PV (mL, from pre) for the normal control (•), the CIA control (■), etanercept (A), Compound 2 (▼), or Compound 2 + etanercept (o) combination treatment. Figure 5B shows the change in PV (mL, from pre) for the normal control (•), the CIA control (■), etanercept (A), Compound 1 (♦), or Compound 1 + etanercept (□) combination treatment. Figure 5C shows the area under the curve (AUC) for the change in paw volume (mean ± SEM) for eight animals for the normal control, the CIA control, etanercept, Compound 1, or Compound 1 + etanercept combination treatment (*** p<0.0001, Student’s t-test, ** p=0.0024, 1-way ANOVA, ###P<0.001, Wilcoxon test). Combination treatment showed lowest AUC indicating lowest total level of inflammation through the inflammation resolution phase.

[0212] Changes in paw volume from pre-immunization values in the CIA control group increased significantly on Day 14 compared to the Normal group. The increased paw volume gradually decreased from Day 14 to Day 28, but still was significantly higher than that in the Normal group. Increase of body weight from Day 14 was significantly suppressed in the CIA control group compared to the Normal group on both Day 21 and Day 28.

[0213] Etanercept (10 mg / kg, SC, twice a week) or Compound 1 (2 mg / kg, IV, once a week) was administered from Day 14 to Day 28, and the inflammation resolution effect was evaluated in CIA rats. Monotherapy treatment with Compound 1 as well as combination treatment with etanercept was tolerated with no negative impact on body weight (Figure 7).. Compound 1 also tended to increase synovial tissue weight, but no interaction between etanercept and Compound 1 was observed (Figure 8B).

[0214] In human rheumatoid arthritis patients, the synovial tissue macrophage phenotype is characterized by inflammation resolution markers that drive disease remission. This observation is contrasted with disease progressing infiltrating monocytes derived proinflammatory macrophages.

[0215] Etanercept (10 mg / kg, SC, twice a week) or Compound 2 (0.5 mg / kg, IV, once a week) was administered from Day 14 to Day 28, and the inflammation resolution effect was evaluated in CIA rats. As shown in Figure 5 A, the paw volume in the CIA control group significantly decreased from Day 14 to Day 21 and Day 28. Single treatment of etanercept accelerated the decrease in paw volume on Day 21 that was maintained through Day 28 (Figure 5 A). On the other hand, monotherapy treatment of Compound 2 did not have effect on the decrease in paw volume (Figure 5 A). No significant effect on paw volume was observed from administering the combination of Compound 2 with etanercept (Figure 5 A). However, paw volume for the combined treatment was decreased as compared to etanercept alone on Day 28.

[0216] The number of monocytes in peripheral blood (cell count xlO2cells / pl) was measured on day 28 with the results shown in Figure 8C. The monocyte cell count in peripheral blood showed no increase in the Compound 1 single treatment group or in the Compound 1 andetanercept combination treatment compared to CIA control group indicating no increase in systemic inflammation.

[0217] The αl acid-glycoprotein (al AGP) concentration (pg / mL) in plasma was measured with the results shown in Figure 8D. No increase in al acid-glycoprotein (al AGP) concentration was observed in the Compound 1 single treatment group or in the Compound 1 and etanercept combination treatment compared to CIA control group indicating no increase in systemic inflammation.

[0218] Single treatment of etanercept showed significant effect on the suppression of body weight gain on Day 21, but not on Day 28 (Figure 6). Compound 2 did not appear to affect the suppression of body weight gain by the disease either in single treatment or combination treatment with etanercept (Figure 6). Monotherapy treatment with Compound 2 as well as combination treatment with etanercept was tolerated with no negative impact on body weight (Figure 6). Synovial tissue weight in the CIA control group (Figure 8A) significantly increased compared to the Normal group. Etanercept tended to augment the increase of synovial tissue weight, while Compound 2 or its combination treatment with etanercept tended to reduce the increase in synovial tissue weight (Figure 8A).

[0219] Findings in the study suggest that Compound 1 might be expected to provide tissue repair effect in inflammatory conditions such as rheumatoid arthritis (RA).

[0220] Single treatment of etanercept accelerated the decrease in paw volume on Day 21 . Single treatment of Compound 1 did not show effect on the decrease in paw volume, but Compound 1 had significant combination effect with etanercept accelerating the decrease of paw volume on Day 21 as compared to etanercept single agent treatment. On the other hand, Compound 2 did not show combination effect with etanercept on the decrease in paw volume. These results suggested that Compound 1 would have inflammation resolution effect in combination with anti-TNF α treatment.

[0221] Embodiments of the present conjugates, compositions, combinations, methods, and kits include, but are not limited to:Embodiment 1: A conjugate comprising a colony stimulating factor- 1 (CSF-1) moiety covalently attached to a water-soluble polymer.Embodiment 2: The conjugate of embodiment 1, wherein the water-soluble polymer is a polymer selected from the group consisting of poly(alkylene oxide), poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline, and poly(acryloylmorpholine).Embodiment 3: The conjugate of the combined or separate embodiments 1-2, wherein the water- soluble polymer is a poly(alkylene oxide) polymer.Embodiment 4: The conjugate of the combined or separate embodiments 1-3, wherein the poly(alkylene oxide) polymer is a poly(ethylene glycol) polymer.Embodiment 5: The conjugate of the combined or separate embodiments 1-4, wherein the water- soluble polymer is a branched water-soluble polymer.Embodiment 6: The conjugate of the combined or separate embodiments 1-5, wherein the branched water-soluble polymer is a branched poly(alkylene oxide) polymer.Embodiment 7: The conjugate of the combined or separate embodiments 1-6, wherein the branched poly(alkylene oxide) polymer is a branched polyethylene glycol (PEG) polymer. Embodiment 8: The conjugate of the combined or separate embodiments 1-7, wherein the branched poly(ethylene glycol) polymer is terminally capped with an end-capping moiety selected from the group consisting of hydroxy, alkoxy, substituted alkoxy, alkenoxy, substituted alkenoxy, alkynoxy, substituted alkynoxy, aryloxy and substituted aryloxy.Embodiment 9: The conjugate of the combined or separate embodiments 1-8, wherein the branched poly(ethylene glycol) polymer is terminally capped with methoxy.Embodiment 10: The conjugate of the combined or separate embodiments 1-9, wherein the water-soluble polymer has a weight-average molecular weight in a range of from about 500 daltons to about 100,000 daltons.Embodiment 11 : The conjugate of the combined or separate embodiments 1-10, wherein the water-soluble polymer has a total weight-average molecular weight in the range of from greater than 5,000 daltons to about 150,000 daltons.Embodiment 12: The conjugate of the combined or separate embodiments 1-11, wherein the water-soluble polymer has a total weight-average molecular weight in the range of from about 6,000 daltons to about 100,000 daltons.Embodiment 13: The conjugate of the combined or separate embodiments 1-12, wherein the water-soluble polymer has a total weight-average molecular weight in the range of from about 15,000 daltons to about 85,000 daltons.Embodiment 14: The conjugate the combined or separate embodiments 1-13, wherein the poly(ethylene glycol) polymer has a total weight-average molecular weight in the range of from about 20,000 Daltons to about 85,000 Daltons.Embodiment 15: The conjugate of the combined or separate embodiments 1-14, wherein the poly(ethylene glycol) polymer has a total weight average molecular weight in the range of from about 20,000 Daltons to about 60,000 Daltons.Embodiment 16: The conjugate of the combined or separate embodiments 1-15, wherein one, two or three water-soluble polymers are attached to the CSF-1 moiety.Embodiment 17: The conjugate of the combined or separate embodiments 1-16, wherein one water-soluble polymer is attached to the CSF-1 moiety.Embodiment 18: The conjugate of the combined or separate embodiments 1-17, comprising the structure:wherein: each (n) is independently an integer having a value of from 3 to 4000;X2when present, is a spacer moiety comprised of one or more atoms;(b) is 2 through 6;(c) is 2 through 6;R2, in each occurrence, is independently H or lower alkyl; and CSF-1 is a CSF-1 moiety.Embodiment 19: The conjugate of the combined or separate embodiments 1-18, comprising the structure:Embodiment 20: The conjugate of the combined or separate embodiments 1-19, wherein the water-soluble polymer comprises the structure:wherein each (n) is independently an integer having a value of from 3 to 4000. Embodiment 21 : The conjugate of the combined or separate embodiments 1-20, wherein the CSF-1 moiety is selected from the group consisting of a human CSF-1, and biologically active fragments, deletion variants, substitution variants or addition variants of any of the foregoing. Embodiment 22: The conjugate of the combined or separate embodiments 1-21, wherein the CSF-1 moiety is recombinantly derived.Embodiment 23 : The conjugate of the combined or separate embodiments 1-22, wherein the CSF-1 moiety is a mutein.Embodiment 24: The conjugate of the combined or separate embodiments 1-23, wherein the mutein has a Hl 5 A mutation.Embodiment 25: A pharmaceutical composition comprising:(i) a conjugate comprising a colony stimulating factor- 1 (CSF-1) moiety covalently attached to a water-soluble polymer; and(ii) a pharmaceutically acceptable excipient.Embodiment 26: The composition of embodiment 25, wherein the water-soluble polymer is selected from the group consisting of poly(alkylene oxide), poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline, and poly(acryloylmorpholine).Embodiment 27: The composition of the combined or separate embodiments 25-26, wherein the water-soluble polymer is a poly(alkylene oxide) polymer.Embodiment 28: The composition of the combined or separate embodiments 25-27, wherein the poly(alkylene oxide) polymer is a pdy(ethylene glycol) (PEG) polymer.Embodiment 29: The composition of the combined or separate embodiments 25-28, wherein the poly(ethylene glycol) polymer is terminally capped with an end-capping moiety selected from the group consisting of hydroxy, alkoxy, substituted alkoxy, alkenoxy, substituted alkenoxy, alkynoxy, substituted alkynoxy, aryloxy and substituted aryloxy.Embodiment 30: The composition of the combined or separate embodiments 25-29, wherein the poly(ethylene glycol) polymer is terminally capped with methoxy.Embodiment 31 : The composition of the combined or separate embodiments 25-30, wherein the water-soluble polymer has a weight-average molecular weight in a range of from about 500 daltons to about 100,000 daltons.Embodiment 32: The composition of the combined or separate embodiments 25-31, wherein the poly(ethylene glycol) has a total weight-average molecular weight in the range of from about 20,000 daltons to about 85,000 daltons.Embodiment 33: The composition of the combined or separate embodiments 25-32, wherein the poly(ethylene glycol has a total weight average molecular weight in the range of from about 20,000 daltons to about 60,000 daltons.Embodiment 34: The composition of the combined or separate embodiments 25-33, wherein the water-soluble polymer is a branched water-soluble polymer.Embodiment 35: The composition of the combined or separate embodiments 25-34, wherein one, two or three water-soluble polymers are attached to the CSF-1 moiety.Embodiment 36: The composition of the combined or separate embodiments 25-35, wherein one water-soluble polymer is attached to the CSF-1 moiety.Embodiment 37: The composition of the combined or separate embodiments 25-36, wherein the conjugate comprising the structure: wherein: each (n) is independently an integer having a value of from 3 to 4000;X2when present, is a spacer moiety comprised of one or more atoms;(b) is 2 through 6;(c) is 2 through 6;R2, in each occurrence, is independently H or lower alkyl; andCSF-1 is a CSF-1 moiety.Embodiment 38: The composition of the combined or separate embodiments 25-37, wherein the conjugate comprising the structure:Embodiment 39: The composition of the combined or separate embodiments 25-38, wherein the branched water-soluble polymer comprises the structure:wherein each (n) is independently an integer having a value of from 3 to 4000.Embodiment 40: The composition of the combined or separate embodiments 25-39, wherein the CSF-1 moiety is selected from the group consisting of a human CSF-1, and biologically active fragments, deletion variants, substitution variants or addition variants of any of the foregoing.Embodiment 41 : The composition of the combined or separate embodiments 25-40, wherein the CSF-1 moiety is recombinantly derived.Embodiment 42: The composition of the combined or separate embodiments 25-41, wherein the CSF-1 moiety is a mutein.Embodiment 43: The composition of the combined or separate embodiments 25-42, wherein the mutein has a Hl 5 A mutation.Embodiment 44: A combination comprising:(a) the conjugate of any one of embodiments 1-24 or the composition of any one of embodiments 25-43; and(b) an anti-inflammatory compound.Embodiment 45: A method of treating a condition that is responsive to treatment with colony stimulating factor- 1 (CSF-1) by administering to a subject having the condition a therapeutically effective dose of a conjugate comprising a colony stimulating factor- 1 (CSF-1) moiety covalently attached to a branched water-soluble polymer.Embodiment 46: The method of embodiment 45, wherein the condition is selected from a disease characterized by a deficiency in CSF-1, an inflammatory disease, an autoimmune disease, or a fibrotic disease.Embodiment 47: The method of the combined or separate embodiments 45-46, wherein the inflammatory disease is characterized by chronic inflammation.Embodiment 48: The method of the combined or separate embodiments 45-47, wherein the disease is selected from systemic lupus erythematosus (SLE), ulcerative colitis, Crohn’s disease, rheumatoid arthritis, psoriatic arthritis, plaque psoriasis, juvenile idiopathic arthritis, atopic dermatitis, systemic sclerosis, ankylosing spondylitis, graft versus host disease (GVHD), Hidradenitis suppurativa, uveitis, polymyositis, organ-specific autoimmune diseases include type 1 diabetes, Addison’s disease, Hashimoto thyroiditis, Graves’ disease, Sjogren’s syndrome, Chronic obstructive pulmonary disease (COPD), vitiligo, pernicious anemia, glomerulonephritis, myasthenia gravis, Goodpasture’s syndrome, autoimmune hemolytic anemia, idiopathic thrombocytopenia purpura, allergic inflammation including peanut allergy, alopecia, inflammatory bowel disease (IBD), multiple sclerosis, autoimmune hepatitis, hepatic fibrosis, and pulmonary fibrosis.Embodiment 49: The method of the combined or separate embodiments 45-48, further comprising administering at least one anti-inflammatory compound.Embodiment 50: The method of the combined or separate embodiments 45-49, wherein the at least one anti-inflammatory compound is selected from a tumor necrosis factor-alpha inhibitor, a tyrosine kinase 2 inhibitor, a Janus kinase inhibitor, an antibody that targets interleukin-4 receptor-alpha, an antibody that targets interleukin-13, an antibody that targets interleukin-23, and a corticosteroid.SEQUENCE LISTINGSEQ ID NO: 1 human CSF-1ATGGAAGAAGTTTCTGAGTACTGTTCACACATGATTGGTTCTGGTCATCTGCAAAGCCTGCAACGTTTGATTGACAGCCAGATGGAAACGTCCTGTCAGATTACCTTTGAGTTCGTCGATCAAGAGCAACTGAAAGATCCGGTTTGCTATCTGAAAAAAGCATTCCTGCTGGTGCAAGACATCATGGAAGATACGATGCGCTTTCGCGACAATACGCCGAACGCCATCGCGATCGTCCAGCTGCAAGAACTGAGCCTGCGTCTGAAGTCGTGCTTCACCAAAGACTATGAAGAACATGATAAGGCTTGCGTGCGTACCTTTTACGAGACTCCGCTGCAGCTGCTGGAAAAAGTGAAAAATGTTTTCAATGAAACCAAGAACCTGTTGGATAAAGACTGGAATATCTTCAGCAAGAACTGCAACAACAGCTTTGCGGAGTGTAGCAGCCAGGGCCACGAGCGTCAGAGCGAANGCTCCTGATAASEQ ID NO:2 human CSF-1MEEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQGHERQSEGSSEQ ID NO:3 CSF-1 muteinATGGAAGAAGTTTCTGAGTACTGTTCACACATGATTGGTTCTGGTGCCCTGCAAAGCCTGCAACGTTTGATTGACAGCCAGATGGAAACGTCCTGTCAGATTACCTTTGAGTTCGTCGATCAAGAGCAACTGAAAGATCCGGTTTGCTATCTGAAAAAAGCATTCCTGCTGGTGCAAGACATCATGGAAGATACGATGCGCTTTCGCGACAATACGCCGAACGCCATCGCGATCGTCCAGCTGCAAGAACTGAGCCTGCGTCTGAAGTCGTGCTTCACCAAAGACTATGAAGAACATGATAAGGCTTGCGTGCGTACCTTTTACGAGACTCCGCTGCAGCTGCTGGAAAAAGTGAAAAATGTTTTCAATGAAACCAAGAACCTGTTGGATAAAGACTGGAATATCTTCAGCAAGAACTGCAACAACAGCTTTGCGGAGTGTAGCAGCCAGGGCCACGAGCGTCAGAGCGAANGCTCCTGATAASEQ ID N0:4 CSF-1 muteinMEEVSEYCSHMIGSGALQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQ DIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVK NVFNETKNLLDKDWNIFSKNCNNSFAECSSQGHERQSEGSSEQ ID N0:5 CSF-1 muteinEEVSEYCSHMIGSGALQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQD IMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKN VFNETKNLLDKDWNIFSKNCNNSFAECSSQGHERQSEGS

Claims

We Claim:

1. A conjugate comprising a colony stimulating factor- 1 (CSF-1) moiety covalently attached to a water-soluble polymer.

2. The conjugate of claim 1, wherein the water-soluble polymer is a polymer selected from the group consisting of poly(alkylene oxide), poly( vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline, and poly(acryloylmorpholine).

3. The conjugate of claim 2, wherein the water-soluble polymer is a poly(alkylene oxide) polymer.

4. The conjugate of claim 3, wherein the poly(alkylene oxide) polymer is a poly(ethylene glycol) polymer.

5. The conjugate of any previous claim, wherein the water-soluble polymer is a branched water-soluble polymer.

6. The conjugate of claim 5, wherein the branched water-soluble polymer is a branched poly(alkylene oxide) polymer.

7. The conjugate of claim 6, wherein the branched poly(alkylene oxide) polymer is a branched polyethylene glycol (PEG) polymer.

8. The conjugate of claim 7, wherein the branched poly(ethylene glycol) polymer is terminally capped with an end-capping moiety selected from the group consisting of hydroxy, alkoxy, substituted alkoxy, alkenoxy, substituted alkenoxy, alkynoxy, substituted alkynoxy, aryloxy and substituted aryloxy.

9. The conjugate of claim 7 or 8, wherein the branched poly(ethylene glycol) polymer is terminally capped with methoxy.

10. The conjugate of any previous claim, wherein the water-soluble polymer has a weight-average molecular weight in a range of from about 500 daltons to about 100,000 daltons.

11. The conjugate of any previous claim, wherein the water-soluble polymer has a total weight-average molecular weight in the range of from greater than 5,000 daltons to about 150,000 daltons.

12. The conjugate of any previous claim, wherein the water-soluble polymer has a total weight-average molecular weight in the range of from about 6,000 daltons to about 100,000 daltons.

13. The conjugate of any previous claim, wherein the water-soluble polymer has a total weight-average molecular weight in the range of from about 15,000 daltons to about 85,000 daltons.

14. The conjugate of any one of claims 4-13, wherein the poly(ethylene glycol) polymer has a total weight-average molecular weight in the range of from about 20,000 daltons to about 85,000 daltons.

15. The conjugate of claim 14, wherein the poly(ethylene glycol) polymer has a total weight average molecular weight in the range of from about 20,000 daltons to about 60,000 daltons.

16. The conjugate of any previous claim, wherein one, two or three water-soluble polymers are attached to the CSF-1 moiety.

17. The conjugate of any one of claims 1-16, wherein one water-soluble polymer is attached to the CSF-1 moiety.

18. The conjugate of any previous claim, comprising the structure:wherein: each (n) is independently an integer having a value of from 3 to 4000;X2when present, is a spacer moiety comprised of one or more atoms;(b) is 2 through 6;(c) is 2 through 6;R2, in each occurrence, is independently H or a lower alkyl; and CSF-1 is the CSF-1 moiety.

19. The conjugate of claim 18, comprising the structure:

20. The conjugate of any previous claim, wherein the water-soluble polymer comprises the structure:wherein each (n) is independently an integer having a value of from 3 to 4000.

21. The conjugate of any previous claim, wherein the CSF-1 moiety is selected from the group consisting of a human CSF-1, and biologically active fragments, deletion variants, substitution variants or addition variants of any of the foregoing.

22. The conjugate of any previous claim, wherein the CSF-1 moiety is recombinantly derived.

23. The conjugate of any previous claim, wherein the CSF-1 moiety is a mutein.

24. The conjugate of claim 23, wherein the mutein has a Hl 5 A mutation.

25. A pharmaceutical composition comprising:(iii) a conjugate comprising a colony stimulating factor- 1 (CSF-1) moiety covalently attached to a water-soluble polymer; and(iv) a pharmaceutically acceptable excipient.

26. The composition of claim 25, wherein the water-soluble polymer is selected from the group consisting of poly(alkylene oxide), poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline, and poly(acryloylmorpholine).

27. The composition of claim 25 or 26, wherein the water-soluble polymer is a poly(alkylene oxide) polymer.

28. The composition of any one of claims 25-27, wherein the poly(alkylene oxide) polymer is a poly(ethylene glycol) (PEG) polymer.

29. The composition of claim 28, wherein the poly(ethylene glycol) polymer is terminally capped with an end-capping moiety selected from the group consisting of hydroxy,alkoxy, substituted alkoxy, alkenoxy, substituted alkenoxy, alkynoxy, substituted alkynoxy, aryloxy and substituted aryloxy.

30. The composition of any one of claims 25-29, wherein the poly(ethylene glycol) polymer is terminally capped with methoxy.

31. The composition of any one of claims 25-30, wherein the water-soluble polymer has a weight-average molecular weight in a range of from about 500 daltons to about 100,000 daltons.

32. The composition of any one of claims 28-30, wherein the poly(ethylene glycol) has a total weight-average molecular weight in the range of from about 20,000 daltons to about 85,000 daltons.

33. The composition of any one of claims 28-30 or 32, wherein the poly(ethylene glycol has a total weight average molecular weight in the range of from about 20,000 daltons to about 60,000 daltons.

34. The composition of any one of claims 25-33, wherein the water-soluble polymer is a branched water-soluble polymer.

35. The composition of any one of claims 25-34, wherein one, two or three water-soluble polymers are attached to the CSF-1 moiety.

36. The composition of any one of claims 25-35, wherein one water-soluble polymer is attached to the CSF-1 moiety.

37. The composition of any one of claims 25-36, wherein the conjugate comprising the structure:wherein: each (n) is independently an integer having a value of from 3 to 4000; X2when present, is a spacer moiety comprised of one or more atoms;(b) is 2 through 6;(c) is 2 through 6;R2, in each occurrence, is independently H or lower alkyl; andCSF-1 is the CSF-1 moiety.

38. The composition of any one of claims 25-37, wherein the conjugate comprising the structure:

39. The composition of any one of claims 25-38, wherein the branched water-soluble polymer comprises the structure:wherein each (n) is independently an integer having a value of from 3 to 4000.

40. The composition of any one of claims 25-39, wherein the CSF-1 moiety is selected from the group consisting of a human CSF-1, and biologically active fragments, deletion variants, substitution variants or addition variants of any of the foregoing.

41. The composition of any one of claims 25-40, wherein the CSF-1 moiety is recombinantly derived.

42. The composition of any one of claims 25-41, wherein the CSF-1 moiety is a mutein.

43. The composition of claim 42, wherein the mutein has a Hl 5 A mutation.

44. A combination comprising:(c) the conjugate of any one of claims 1-24 or the composition of any one of claims 25- 43; and(d) an anti-inflammatory compound.

45. A method of treating a condition that is responsive to treatment with colony stimulating factor- 1 (CSF-1) by administering to a subject having the condition a therapeutically effective dose of a conjugate comprising a colony stimulating factor- 1 (CSF-1) moiety covalently attached to a branched water-soluble polymer.

46. The method of claim 45, wherein the condition is selected from a disease characterized by a deficiency in CSF-1, an inflammatory disease, an autoimmune disease, and / or a fibrotic disease.

47. The method of claim 46, wherein the inflammatory disease is characterized by chronic inflammation.

48. The method of any one of claims 45-47, wherein the disease is selected from systemic lupus erythematosus (SLE), ulcerative colitis, Crohn’s disease, rheumatoid arthritis, psoriatic arthritis, plaque psoriasis, juvenile idiopathic arthritis, atopic dermatitis, systemic sclerosis, ankylosing spondylitis, graft versus host disease (GVHD), Hidradenitis suppurativa, uveitis, polymyositis, organ-specific autoimmune diseases include type 1 diabetes, Addison’s disease, Hashimoto thyroiditis, Graves’ disease, Sjogren’s syndrome, Chronic obstructive pulmonary disease (COPD), vitiligo, pernicious anemia, glomerulonephritis, myasthenia gravis, Goodpasture’s syndrome, autoimmune hemolytic anemia, idiopathic thrombocytopenia purpura, allergic inflammation including peanut allergy, alopecia, inflammatory bowel disease (IBD), multiple sclerosis, autoimmune hepatitis, hepatic fibrosis, and pulmonary fibrosis.

49. The method of any one of claims 45-48, further comprising administering at least one anti-inflammatory compound.

50. The method of claim 49, wherein the at least one anti-inflammatory compound is selected from a tumor necrosis factor-alpha inhibitor, a tyrosine kinase 2 inhibitor, a Janus kinase inhibitor, an antibody that targets interleukin-4 receptor-alpha, an antibody that targets interleukin-13, an antibody that targets interleukin-23, and a corticosteroid.

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