Bifunctional CD206-binding molecules and methods of use

Abstract

Aspects of the present disclosure include bifunctional molecules. The bifunctional molecules comprise a first moiety comprising a CD206 ligand that binds CD206 on the surface of a cell and induces CD206-mediated endocytosis. The bifunctional molecules further comprise a second moiety that specifically binds a molecule on the surface of the cell or an extracellular molecule. In certain embodiments, the CD206 ligand comprises a monosaccharide, disaccharide, or trisaccharide comprising mannose, fucose, a sulfated glycan, or any combination thereof. In some instances, the cell is a tumor-associated macrophage (TAM) and the second moiety is an immune checkpoint molecule, e.g., SIRPα or other immune checkpoint molecule of interest. According to some embodiments, binding of the second moiety to the immune checkpoint molecule does not block activity of the immune checkpoint molecule. Also provided are methods of using the bifunctional molecules of the present disclosure, e.g., in therapy.

Description

[0001] BIFUNCTIONAL CD206-BINDING MOLECULES AND METHODS OF USE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 546,720, filed October 31 , 2023, which application is incorporated herein by reference in its entirety.

[0004] STATEMENT OF GOVERNMENT SUPPORT

[0005] This invention was made with Government support under contracts GM058867 and GM143843 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0006] INTRODUCTION

[0007] Immune suppression in the tumor microenvironment (TME) represents a major barrier to maximizing the clinical potential of immunotherapies. The TME is complex with diverse populations of non-tumor stromal cells that impact tumor immune evasion, response to immunotherapy, and patient survival. In addition to cytotoxic lymphocytes (CTLs) and natural killer cells (NKs) that are generally considered effective anti-tumor immune cells, the TME contains a range of other cell types that are involved in the crosstalk with anti-tumor immune cells, including cancer-associated fibroblasts (CAFs), endothelial cells (ECs), and tumor- associated macrophages (TAMs).

[0008] Macrophages are involved in various processes in both homeostasis and disease. With effector functions such as phagocytosis, antigen presentation, and the plasticity to secrete a wide variety of signaling molecules. TAMs subvert local immune surveillance in the TME because they can directly reduce the activities of T cells and NKs by expressing cell surface proteins or by releasing soluble factors that display immunosuppressive functions (for example, arginase 1 (ARG1 ), indoleamine 2,3-dioxygenase (IDO), IL-10, programmed death ligand 1 (PD-L1 ), and TGF-|3) or indirectly suppress T cell activities through recruitment of other immune suppressive cells such as regulatory T cells (Tregs).

[0009] In checkpoint inhibitor therapy, TAMs can suppress effector T cells directly via the expression of various checkpoint molecules and immunosuppressive cytokines and indirectly through crosstalk with Tregs and hijacking of anti-PD-1 antibodies. Given that TAMs have a profound impact on tumor immunotherapies, there is interest in the therapeutic targeting of TAMs to enhance immune checkpoint inhibitor (ICI)-based immunotherapies. The different approaches that have been explored for targeting TAMs include: (1 ) eliminating TAMs already present in the TME; (2) inhibition of monocyte recruitment; and (3) reprogramming of TAMs. These strategies have been investigated in preclinical models, and some have been translated into the clinical setting as adjuvant to immunotherapy. In homeostasis, normal cells can avoid self-elimination by phagocytes through the expression of anti-phagocytosis molecules, which are therefore called “phagocytosis checkpoints.” However, many studies have shown that tumor cells depend even more on phagocytosis checkpoints to evade immune surveillance. Therefore, identification and intervention with phagocytosis checkpoints might provide a new approach for restoring the phagocytic capacity of TAMs to eliminate tumor cells.

[0010] Signal regulatory protein alpha (SIRPa) is an ITIM-bearing inhibitory receptor expressed on myeloid cells, including macrophages. SIRPa recognizes CD47, which acts as a “don’t eat me” signal, is found to be overexpressed in tumor cells and its expression correlates with patients’ poor survival. Macrophage phagocytosis of tumor cells has been restored by treatment with CD47-blocking antibodies, and this macrophage-mediated phagocytosis was further enhanced in the presence of chemotherapeutic drugs, suggesting that patients with lower CD47 expression were more likely to benefit from adjuvant TACE treatment. Interfering with the CD47-SIRPa interaction promotes phagocytosis in TAMs.

[0011] The bridging between innate and adaptive immune cells provides the rationale for combining phagocytosis checkpoint inhibitors with current ICI-based immunotherapies that boost the adaptive immune response. The potential for such combinations was initially observed when anti-CD47 therapy was shown to have a synergistic effect with PD-L1 inhibitor in a mouse model bearing the B16F10 melanoma. Similarly, a bispecific antibody targeting PD-L1 on tumor cells and SIRPa on APCs showed a more significant anti-tumor effect against murine colon cancer compared with either anti-PD-L1 or anti-SIRPa monotherapy. Overall, these preclinical results along with earlier observations in ICIs confirm the notion that the conventional boundary between innate and adaptive immune checkpoints is becoming blurred, because more of these checkpoints have been found to function at both the innate and adaptive levels.

[0012] SUMMARY

[0013] Aspects of the present disclosure include bifunctional molecules. The bifunctional molecules comprise a first moiety comprising a CD206 ligand that binds CD206 on the surface of a cell and induces CD206-mediated endocytosis. The bifunctional molecules further comprise a second moiety that specifically binds a molecule on the surface of the cell or an extracellular molecule. In certain embodiments, the CD206 ligand comprises a monosaccharide, disaccharide, or trisaccharide comprising mannose, fucose, a sulfated glycan, or any combination thereof. In some instances, the cell is a tumor-associated macrophage (TAM) and the second moiety is an immune checkpoint molecule, e.g., SIRPa or other immune checkpoint molecule of interest. According to some embodiments, binding of the second moiety to the immune checkpoint molecule does not block activity of the immune checkpoint molecule. Also provided are methods of using the bifunctional molecules to remove a molecule from the surface of CD206-expressing cells in a subject in need thereof. BRIEF DESCRIPTION OF THE FIGURES

[0014] FIG. 1A-1 H: A.) CD206 is upregulated on the surface of M2-polarized TAMs and binds multivalent mannose, fucose, and glucosamine via its C-type lectin domains (CTLDs) and sulfated galactose and galactosamine via its N-terminal cysteine rich domain. B.) Two-step protocol for generating Tumor-Immune Cell Targeting Chimeras (TICTACs), where the carbohydrate ligand is represented by a gray sphere. C.) Murine macrophages (RAW264.7) can be polarized to M2 upon treatment with IL-4, leading to increased expression of CD206. D.) Tris- dendron scaffold 1 enables CD206-targeting with mannose, fucose, and 3-SO4galactose ligands 1a-c. E.) TICTAC-mediated internalization of fluorescent rabbit IgG into M2-polarized macrophages. F.) Fold change in mean fluorescence intensity (MFI) relative to the control for M2 polarized RAW264.7 cells incubated at 37 °C for 3 h with 25 nM rabbit lgG-488 and 25 nM goat- anti-rabbit or various goat-anti-rabbit-TICTACs. MFI was determined by live cell flow cytometry. G.) MFI relative to control for M2 polarized murine macrophage cell line BMA3.1 A7 and microglia cell line BV-2. H.) Knocking out CD206 ablates 1 b and 1c-mediated uptake in RAW264.7 cells.

[0015] FIG. 2A-2F: A.) Tris-fucose ligand was independently synthesized and conjugated to a 647 dye to generate tris-Fuc-647 2. B.) Changes in 647 MFI in M2-polarized RAW264.7 cells upon treatment with unconjugated 647 dye or 2 at varying concentrations for 2 h at 37 °C. C.) Confocal microscopy imaging of M2-polarized RAW264.7 cells treated for 2 h with 2 or unconjugated 647 dye. D.) Monocytes were isolated from human blood and differentiated into macrophages either in the absence of presence of IL-4 (20 ng / mL) and IL-10 (50 ng / mL). CD206 is upregulated in M2-polarized macrophages. E.) Changes in 647 MFI in M2-polarized human macrophages upon treatment with unconjugated 647 dye or 2 at varying concentrations for 2 h at 37 °C. F.) Knocking out CD206 ablates uptake of 2 in RAW264.7 cells.

[0016] FIG. 3A-3F: A.) Removal of cell-surface CD54 mediated by TICTACs. B.)

[0017] Downregulation of cell-surface CD54 in M2-polarized RAW264.7 and BMA3.1 A7 cells determined by live cell flow cytometry following 24 h of treatment with 25 nM unconjugated anti- CD54 antibody or conjugates. C.) Visualization of cell-surface CD54 by confocal microscopy after 25 nM TICTAC treatments for 24 h. D.) Dose-response curve for cell-surface CD54 removal in M2-polarized RAW264.7 cells following treatment with unconjugated antibody or 3b at 0.1 nM, 1 nM, 5 nM, 10 nM, and 100 nM for 24 h. E.) Time-course of CD54 downregulation in M2-polarized RAW264.7 cells incubated with 25 nM unconjugated antibody or conjugates at 3 h, 8 h, 24 h, and 48 h. F.) Downregulation of cell-surface CD54 in RAW264.7 and J774A.1 cells that have been polarized with IL-4 (25 ng / mL) or not following treatment with 25 nM unconjugated antibody or 3b.

[0018] FIG. 4A-4F: A.) Left: Canonical checkpoint inhibitor systemically blocks ICP regardless of whether the interacting cell is healthy or cancerous; Flight: Non-blocking TICTACs have no function-ablating activity in healthy tissue but can remove ICPs in CD206high TAMs. B.) Downregulation of cell-surface SIRPa in M2-polarized RAW264.7 and J774A.1 cells determined by live cell flow cytometry following 24 h of treatment with 25 nM unconjugated anti-SIRPa antibody or conjugates (4b, 4c). C.) Dose-response curve for cell-surface SIRPa removal in M2- polarized RAW264.7 cells following treatment with unconjugated antibody or 4b at 0.1 nM, 1 nM, 10 nM, and 100 nM for 24 h. D.) TICTACs generated from multiple antibody clones targeting different epitopes on SIRPa downregulate cell-surface SIRPa in M2-polarized RAW264.7 cells following treatment at 25 nM for 24 h. E.) Orthogonality of SIRPa antibody to CD47 was probed by live cell flow cytometry using a fluorescently labeled CD47-Fc chimera. F.) CD47-binding to M2-polarized RAW264.7 cells was determined using a fluorescently labeled CD47-Fc chimera following treatment with non-blocking clone P84 or its tris-fucose conjugate at 25 nM for 24 h.

[0019] FIG. 5A-5B: A.) Non-blocking TICTAC against a soluble target would have no effect on tissue with low CD206 expression (top), but would internalize and eliminate soluble targets in the presence of TAMs (bottom). B.) Fold change in 488 MFI relative to the control for M2 polarized RAW264.7 cells incubated at 37 °C for 3 h with 25 nM 488-labeled IL-4 and 25 nM anti-IL4 antibody or various anti-IL-4 TICTACs 5b and 5c. MFI was determined by live cell flow cytometry.

[0020] FIG. 6A-6I: Anti-SIRPa TICTACs degrade immune checkpoint protein SIPRa regardless of their ability to block CD47. 6A, Non-blocking TICTACs can degrade ICPs in CD206hiTAMs (left) but have no function-ablating activity in healthy tissue (right). 6B, While antibody clone 119 blocks the CD47-binding site, antibodies 136 and 3 bind a distal, non-blocking site. 6C, Tris-Fuc and 3-SO4-Gal TICTACs generated from blocking clone 1 19 (5a, 5b) induce degradation of SIRPa in human M2-polarized macrophages following treatment at 25 nM for 24 h. 6D, Tris-Fuc TICTACs 6a and 7a generated from non-blocking clones 136 and 3 respectively induce degradation of SIRPa in human M2-polarized macrophages following treatment at 25 nM for 24 h. 6E, Fold change in the abundance of 5,091 proteins detected by quantitative proteomics analysis after 24 h treatment with either unconjugated LALAPG-1 19 or 5a. The no treatment sample represents a PBS control. SIRPa (highlighted in fuscia) is downregulated in 5a-treated macrophages relative to unconjugated 1 19. An adjusted P-value (Padj) threshold of 0.05 and a Iog2 fold change threshold of 1 .0 were set to identify significant changes to protein abundance. Data are the mean of three independent donors. 6F, Co-culture of human macrophages with pHrodo red-labeled Raji cells in the presence of rituximab elicits ADCP, leading to red fluorescence. 6G, Phase contrast and fluorescence microscopy images of human macrophages / Raji co-cultures treated with rituximab at 5 pg / mL. Macrophages were pre-treated with 25 nM unconjugated LALAPG-136 or 6a for 24 h prior to co-culture. Images are representative of 3 independent experiments from a single donor. Scale bars represent 400 pm. 6H, Phagocytosis of Raji cells by M2-polarized human macrophages treated with LALAPG-136 or 6a in the presence or absence of rituximab. Data are mean of 3 independent experiments from a single donor, and error bars represent the SD. P values were determined by unpaired two- tailed t-tests. I, Phagocytosis of Raji cells by M2-polarized human macrophages treated with LALAPG-136 or 6a in the presence or absence of rituximab. Data are mean of 5 independent donors, with each point representing mean of 3 independent experiments per donor. P values were determined by paired two-tailed t-tests. For 6C, 6D, 6H, and 61, statistical significance was defined as P<0.05, and the asterisks * indicates P<0.1 , ** indicates P<0.01 , *** indicates P<0.001 , and **** indicates P<0.0001.

[0021] DETAILED DESCRIPTION

[0022] Before the bifunctional molecules and methods of the present disclosure are described in greater detail, it is to be understood that the bifunctional molecules and methods are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the bifunctional molecules and methods will be limited only by the appended claims.

[0023] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the bifunctional molecules and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the bifunctional molecules and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the bifunctional molecules and methods.

[0024] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the bifunctional molecules and methods belong. Although any bifunctional molecules and methods similar or equivalent to those described herein can also be used in the practice or testing of the bifunctional molecules and methods, representative illustrative bifunctional molecules and methods are now described.

[0026] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and / or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present bifunctional molecules and methods are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed.

[0027] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0028] It is appreciated that certain features of the bifunctional molecules and methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the bifunctional molecules and methods, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present bifunctional molecules and methods and are disclosed herein just as if each and every such subcombination was individually and explicitly disclosed herein.

[0029] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0030] BIFUNCTIONAL MOLECULES

[0031] Aspects of the present disclosure include bifunctional molecules. In some embodiments, the bifunctional molecules comprise a first moiety comprising a CD206 ligand that binds CD206 on the surface of a cell and induces CD206-mediated endocytosis, and a second moiety stably associated with the first moiety. The second moiety specifically binds a molecule on the surface of the cell or an extracellular molecule. The bifunctional molecules of the present disclosure are based in part on the unpredictable demonstration herein that by engaging CD206, which constitutively recycles between the plasma membrane and early endosomes, the bifunctional molecules are able to facilitate robust removal of molecules on the surface of CD206-expressing cells (non-limiting examples of which include CD206h'9htumor associated macrophages (TAMs)) as well as molecules extracellular to such cells. In some instances, the cell surface molecule is an immune checkpoint molecule (e.g., SIRPa, PD-L1 , or the like) such that, in the exemplary context of TAMs, the bifunctional molecules enable / enhance the anti-tumor phagocytic activity of TAMs in the tumor microenvironment (TME). In some instances, the second moiety binds an extracellular molecule in the TME (e.g., an immunosuppressive cytokine), such that the bifunctional molecules would have anti-tumorigenic effects upon removal of such extracellular molecules from the TME. In certain embodiments, in addition to removal of the cell surface molecule or extracellular molecule from the cell surface or extracellular space, respectively, the cell surface molecule or extracellular molecule is degraded upon internalization into the CD206- expressing cell. Details regarding embodiments of the bifunctional molecules of the present disclosure will now be described.

[0032] The bifunctional molecules comprise a first moiety comprising a CD206 ligand. CD206 (UniProt Accession No. P22897 (human), No. Q61830 (mouse)), is a member of the C-type lectin (CLEC) family and encompasses a nearly 175 kDa type I transmembrane protein composed of three types of domains. The N-terminal domain is an R-type carbohydrate-recognition domain (CRD) that binds to non-reducing sulfo-N-acetyl-D-galactosamine moieties. The neighboring fibronectin type II domain binds to collagen fragments. This is followed by eight C-type lectin-like domains (CTLDs), a transmembrane region and a short cytosolic region. Of the eight CLTDs, carbohydrate-recognition domain 4 (CRD4) and carbohydrate-recognition domain 5 (CRD5) account for most of the binding activity, and CRD4 is the only C-type lectin domain capable of independently binding glycans. CRD4 is known to bind terminal mannose, fucose, GIcNAc, and glucose residues.

[0033] As used herein, a “CD206 ligand” is a molecule that binds to CD206 and induces CD206- mediated endocytosis. A CD206 ligand may be natural or synthetic. In certain embodiments, the CD206 ligand comprises a carbohydrate. The carbohydrate may be a naturally occurring carbohydrate, a modified carbohydrate, or a carbohydrate derivative. According to some embodiments, when the CD206 ligand comprises a carbohydrate, the CD206 ligand comprises mannose, fucose, a sulfated glycan, or any combination thereof. For example, the CD206 ligand may comprise a monosaccharide, disaccharide, or trisaccharide comprising mannose, fucose, a sulfated glycan, or any combination thereof. Sulfated glycans of interest include, but are not limited to, 3-SO4galactose, 4-SO4galactose, or the like.

[0034] In some instances, the CD206 ligand is tris-fucose (tris-Fuc) or tris-3-SO4galactose (tris-

[0035] 3-SO4-Gal). According to some embodiments, the CD206 ligand is selected from 1a, 1 b, and 1c as shown in FIG. 1 .

[0036] Additional CD206 ligands that may be employed include, but are not limited to, N-acetyl glucosamine (GIcNAc, see e.g., Stahl, P. D. Am. J. Respir. Cell. Mol. Biol. 1990, 2 (4), 317-318),

[0037] 4-SO4-N-Acetyl galactosamine (4-SO4-GalNAc, see e.g., Fiete et al. PNAS, 1998, 95 (5), 2089- 2093), 3-SO4-N-Acetyl galactosamine (3-SO4-GalNAc, see e.g., Martinez-Pomares et al. Laboratory Investigation, 2005, 85, 1238-1249), 2a-mannobiose, 3,6-di-O-(a-D- mannopyranosyl)-a-D-mannopyranoside, 3-fucosyllactose, or any combination thereof.

[0038] The first moiety may comprise a single CD206 ligand, or alternatively, may be multivalent by comprising two or more CD206 ligands, e.g., 2, 3, 4, 5, or more CD206 ligands. The two or more CD206 ligands may be the same ligands (e.g., two or more of any one of the CD206 ligands described elsewhere herein), or the two or more CD206 ligands may comprise at least two different CD206 ligands (e.g., two or more different CD206 ligands independently selected from any of the CD206 ligands described elsewhere herein).

[0039] In some instances, the CD206 ligand comprises a protein. The terms “protein”, “polypeptide”, and “peptide” are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The amino acids may include the 20 “standard” genetically encodable amino acids, amino acid analogs, or a combination thereof.

[0040] According to some embodiments, when the CD206 ligand comprises a protein, the CD206 ligand is an anti-CD206 antibody. By “antibody” is meant an antibody or immunoglobulin of any isotype (e.g., IgG (e.g., lgG1 , lgG2, lgG3, or lgG4), IgE, IgD, IgA, IgM, etc.), whole antibodies (e.g., antibodies composed of a tetramer which in turn is composed of two dimers of a heavy and light chain polypeptide); single chain antibodies (e.g., scFv); fragments of antibodies (e.g., fragments of whole or single chain antibodies) which retain specific binding to CD206, including, but not limited to single chain Fv (scFv), Fab, (Fab’)2, (scFv’)2, and diabodies; chimeric antibodies; monoclonal antibodies, human antibodies, humanized antibodies (e.g., humanized whole antibodies, humanized half antibodies, or humanized antibody fragments, e.g., humanized scFv); and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. In certain embodiments, the antibody is selected from an IgG, single chain Fv (scFv), Fab, (Fab)2, (scFv’)2, or a single variable domain located on a heavy chain (VHH). According to some embodiments, the antibody is a VHH (sometimes referred to herein and elsewhere as a “nanobody”).

[0041] In certain embodiments, the CD206 ligand comprises an aptamer. By “aptamer” is meant a nucleic acid (e.g., an oligonucleotide) that has a specific binding affinity for the target molecule. Aptamers exhibit certain desirable properties for targeted delivery and engagement of CD206, such as ease of selection and synthesis, high binding affinity and specificity, low immunogenicity, and versatile synthetic accessibility. Aptamers that bind to cell surface molecules are known and include, e.g., TTA1 (a tumor targeting aptamer to the extracellular matrix protein tenascin-C). Aptamers that find use in the context of the present disclosure include those described in Zhu et al. (2015) ChemMedChem 10(1 ):39-45; Sun et al. (2014) Mol. The Nucleic Acids 3:e182; and Zhang et al. (201 1 ) Curr. Med. Chem. 18(27):4185-4194. Protein and aptamer CD206 ligands may be designed (and / or screened) for binding to one or more selected domains of CD206. In some instances, the CD206 ligand binds to the N- terminal R-type carbohydrate-recognition domain (CRD) of CD206. On other embodiments, the CD206 ligand binds to C-type CRD4, C-type CRD5, or both, of CD206.

[0042] As summarized above, the second moiety specifically binds a molecule on the surface of the CD206-expressing cell or an extracellular molecule. Non-limiting examples of second moieties that may be employed in the bifunctional molecules of the present disclosure include small molecules, antibodies, aptamers, or any other moieties suitable for binding the cell surface molecule or extracellular molecule. By “small molecule” compound is meant a compound having a molecular weight of 1000 atomic mass units (amu) or less. In some embodiments, the small molecule is 900 amu or less, 750 amu or less, 500 amu or less, 400 amu or less, 300 amu or less, or 200 amu or less. In some instances, the small molecule is not made of repeating molecular units such as are present in a polymer.

[0043] In certain embodiments, when the CD206 ligand and / or second moiety is an antibody, one or more amino acid modifications may be introduced into the Fc region of the antibody, thereby generating an Fc region variant. An Fc region variant may, for example, comprise a human Fc region sequence (e.g., a human lgG1 , lgG2, lgG3 or lgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions (e.g., an lgG4 isotype including the S228P mutation).

[0044] In certain embodiments, the Fc region is mutated to increase its affinity to FcRn at pH 6.0 and consequently extend the antibody half-life. Antibodies with enhanced affinity to FcRn include those with substitution of one or more of Fc region residues 252, 253, 254, 256, 428, 434, including the so called YTE mutation with substitution M252Y / S254T / T256E (Dall’ Acqua et al, J Immunol. 169:5171 -5180 (2002)) or LS mutation M428L / N434S (Zalevsky et al, Nat Biotechnol. 28(2): 157-159 (2010)).

[0045] In certain embodiments, the CD206 ligand and / or second moiety may comprise an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody in vivo is important yet certain effector functions (such as complement activation, ADCC, and ADCP) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC / ADCP activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity) but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes and microglia express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991 ). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat’IAcad. Sci. USA 82:1499- 1502 (1985); 5,821 ,337 (see Bruggemann, M. et al., J. Exp.

[0046] Med. 166:1351 -1361 (1987)).

[0047] Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 234, 235, 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591 -6604 (2001 )). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581 ) or the so-called “DANG” Fc mutant with substitution of residues 265 to alanine and 297 to Glycine. Alternatively, antibodies with reduced effector function include those with substitution of one or more of Fc region residues 234, 235 and 329, so-called “PG-LALA” (or “LALAPG”) Fc mutant with substitution of residues 234 and 235 to alanine and 329 to glycine (Lo, M. et al., Journal of Biochemistry, 292, 3900-3908). Other known mutations at position 234, 235 and 321 , the so- called TM mutant containing mutations L234F / L235E / P331 S in the CH2 domain, can be used (Oganesyan et al. Acta Cryst. D64, 700-704. (2008)). Antibodies from the human lgG4 isotype include mutations S228P / L235E to stabilize the hinge and to reduce FcR binding (Schlothauer et al, PEDS, 29 (10):457-466).

[0048] Other Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311 , 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (US Patent No. 7,371 ,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821.

[0049] As used herein, a first molecule “specifically binds” or “preferentially binds” to a target if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances, e.g., in a sample and / or in vivo. In certain embodiments, the second moiety “specifically binds” the target molecule if it binds to or associates with the target molecule with an affinity or Ka (that is, an association rate constant of a particular binding interaction with units of 1 / M) of, for example, greater than or equal to about 104M1. Alternatively, affinity may be defined as an equilibrium dissociation constant (KD) of a particular binding interaction with units of M (e.g., 102M to 1013M, or less). In certain aspects, specific binding means the second moiety binds to the target molecule with a KD of less than or equal to about 105M, less than or equal to about 106M, less than or equal to about 107M, less than or equal to about 108M, or less than or equal to about 109M, 1010M, 1011M, or 1012M or less. The binding affinity of the second moiety for the target molecule can be readily determined using conventional techniques, e.g., by competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, by using surface plasmon resonance (SPR) technology (e.g., the BIAcore 2000 or BIAcore T200 instrument, using general procedures outlined by the manufacturer); by radioimmunoassay; or the like. According to some embodiments, the second moiety specifically binds a molecule on the surface of the CD206-expressing cell. The second moiety may be selected to specifically bind any cell surface molecule of interest. In certain embodiments, the molecule on the surface of the CD206-expressing cell is a cell surface receptor. For example, the second moiety may be selected to bind a cell surface receptor selected from a stem cell receptor, an immune cell receptor, a growth factor receptor, a cytokine receptor, a hormone receptor, a receptor tyrosine kinase, an immune receptor such as CD28, CD80, ICOS, CTLA4, PD1 , PD-L1 , BTLA, HVEM, CD27, 4-1 BB, 4-1 BBL, 0X40, OX40L, DR3, GITR, CD30, SLAM, CD2, 2B4, TIM1 , TIM2, TIM3, TIGIT, CD226, CD160, LAG3, LAIR1 , B7-1 , B7-H1 , and B7-H3, a type I cytokine receptor such as lnterleukin-1 receptor, lnterleukin-2 receptor, lnterleukin-3 receptor, lnterleukin-4 receptor, lnterleukin-5 receptor, lnterleukin-6 receptor, lnterleukin-7 receptor, lnterleukin-9 receptor, Interleukin-11 receptor, Interleukin-12 receptor, Interleukin-13 receptor, Interleukin-15 receptor, Interleukin-18 receptor, Interleukin-21 receptor, Interleukin-23 receptor, Interleukin-27 receptor, Erythropoietin receptor, GM-CSF receptor, G-CSF receptor, Growth hormone receptor, Prolactin receptor, Leptin receptor, Oncostatin M receptor, Leukemia inhibitory factor, a type II cytokine receptor such as interferon-alpha / beta receptor, interferon-gamma receptor, Interferon type III receptor, Interleukin-10 receptor, Interleukin-20 receptor, Interleukin-22 receptor, Interleukin- 28 receptor, a receptor in the tumor necrosis factor receptor superfamily such as Tumor necrosis factor receptor 2 (1 B), Tumor necrosis factor receptor 1 , Lymphotoxin beta receptor, 0X40, CD40, Fas receptor, Decoy receptor 3, CD27, CD30, 4-1 BB, Decoy receptor 2, Decoy receptor 1 , Death receptor 5, Death receptor 4, RANK, Osteoprotegerin, TWEAK receptor, TACI, BAFF receptor, Herpesvirus entry mediator, Nerve growth factor receptor, B-cell maturation antigen, Glucocorticoid-induced TNFR-related, TROY, Death receptor 6, Death receptor 3, Ectodysplasin A2 receptor, a chemokine receptor such as CCR1 , CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1 , CXCR2, CXCR3, CXCR4, CXCR5, CXCR6 , CX3CR1 , XCR1 , ACKR1 , ACKR2, ACKR3 , ACKR4, CCRL2, a receptor in the epidermal growth factor receptor (EGFR) family, a receptor in the fibroblast growth factor receptor (FGFR) family, a receptor in the vascular endothelial growth factor receptor (VEGFR) family, a receptor in the rearranged during transfection (RET) receptor family, a receptor in the Eph receptor family, a receptor that can induce cell differentiation (e.g., a Notch receptor), a cell adhesion molecule (CAM), an adhesion receptor such as integrin receptor, cadherin, selectin, and a receptor in the discoidin domain receptor (DDR) family, transforming growth factor beta receptor 1 , and transforming growth factor beta receptor 2.

[0050] In certain embodiments, the molecule on the surface of the CD206-expressing cell (e.g., a CD206highTAM) is an immune checkpoint molecule. Immune checkpoint molecules to which the second moiety may specifically bind include, but are not limited to, SIRPa, PD-L1 , PD-L2, CD80, CD86, B7-H4, VISTA, PD-1 , Siglec 10, CSF-1 R, I L-4R / I L-13R, MARCO, and LILRB-1 . For example, in some embodiments, the immune checkpoint molecule is SIRPa. The phagocytic check point protein CD47 inhibits the phagocytic activity of macrophages through binding to SIRPa expressed on macrophages.

[0051] According to some embodiments, the second moiety specifically binds an extracellular molecule. By “extracellular molecule” is meant a molecule which is extracellular with respect to the CD206-expressing cell. In some instances, the extracellular molecule is a soluble ligand. Nonlimiting examples of soluble ligands to which the second moiety may bind include growth factors, cytokines (e.g., immunosuppressive cytokines), chemokines (e.g., immunosuppressive chemokines), hormones, and the like. When the second moiety specifically binds a growth factor, in some embodiments, the growth factor is epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor-a (TGF-a), transforming growth factor-p (TGF-P), platelet- derived growth factor (PDGF), insulin-like growth factor (IGF), or vascular endothelial growth factor (VEGF).

[0052] According to some embodiments, the second moiety specifically binds a cytokine. Cytokines are intercellular signaling molecules that aid cell to cell communication in immune responses and stimulate the movement of cells towards sites of inflammation, infection, and trauma. The downstream effect of a particular cytokine occurs through its high-affinity binding of its receptor expressed on the surface of a target cell. This action may occur in an autocrine (acts on the same cell), paracrine (acts on nearby cells) or endocrine (acts on distant cells) manner. Receptor engagement triggers intracellular signaling cascades leading to altered gene expression in the target cell, leading to a biological effect. Cytokines can be divided into several categories including the interleukins (ILs), transforming growth factors (TGFs), interferons (IFNs), colony-stimulating-factors (CSFs), tumor necrosis factors (TNFs), and chemokines - any of which may be bound by a second moiety of a bifunctional molecule of the present disclosure.

[0053] In some instances, the second moiety specifically binds an interleukin. Interleukins (ILs) are a group of cytokines that are expressed and secreted by white blood cells (leukocytes) as well as some other body cells. Interleukins and associated cytokines serve as the means of communication for innate and adaptive immune cells as well as non-immune cells and tissues. All IL-1 family members share a conserved beta-trefoil structure and bind to members of the IL-

[0054] I receptor (IL-1 R) family. Members of the IL-1 R family contain extracellular Ig-like domains and mediate signaling through an intracellular Toll / IL-1 R (TIR) domain.

[0055] According to some embodiments, the second moiety specifically binds a member of the four-helix bundle cytokine superfamily. This superfamily is subdivided into the class I and class

[0056] II cytokine receptor families. Ligands for the class I cytokine receptor family include short-chain and long-chain helical cytokines. The short-chain helical cytokine family includes members of the common gamma-chain and common beta-chain families of cytokines. The common beta-chain and common gamma-chain cytokine families include cytokines such as IL-2, IL-3, IL-4, IL-5, IL- 7, IL-9, IL-15, IL-21 , and GM-CSF. Members of the common beta-chain family signal through heterodimeric receptor complexes that contain the common beta-chain subunit, while members of the common gamma-chain family signal through heterodimeric or heterotrimeric receptor complexes that contain the common gamma-chain subunit. The common y chain (yc) family consists of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 and was named for binding of these factors to the yc receptor (CD132). They act mainly as growth and proliferation factors for progenitors and mature cells and also have roles in lineage-specific cell differentiation.

[0057] In certain embodiments, the second moiety specifically binds a member of the class II cytokine receptor family. Ligands for the class II cytokine receptor family include the IL-10 family cytokines, along with the type I, type II, and type III interferons. Members of the IL-10 family cytokines have structural similarities and signal through heterodimeric receptor complexes with common subunits. Members of the type I, II, and III interferon families include IFN-alpha, IFN- beta, IFN-omega, IFN-epsilon, IFN-kappa, IFN-gamma, IL-28A, IL-28B, IL-29, and IFN-lambda4. These cytokines primarily have anti-viral, anti-proliferative, and immunoregulatory effects.

[0058] In some instances, the second moiety specifically binds a member of the IL-17 family of cytokines which belong to the cysteine-knot superfamily and bind to members of the IL-17 receptor family. They are primarily involved in promoting pro-inflammatory immune responses.

[0059] In certain embodiments, the second moiety specifically binds a member of the tumor necrosis factor (TNF) superfamily. Members of the TNF superfamily form homotrimers or in some cases, heterotrimers, and share a common extracellular domain known as the TNF homology domain (THD). Cytokines in the TNF superfamily bind to oligomeric, type I or type II transmembrane proteins that have multiple extracellular cysteine-rich domains. Many members of the TNF superfamily regulate apoptosis and / or immune cell functions, such as T cell costimulation, natural killer cell activation, and B cell homeostasis. Additionally, they can regulate cell type-specific responses and can play a significant role in regulating the pathogenesis of certain diseases, including chronic inflammation, cancer, and autoimmune diseases.

[0060] In some instances, the second moiety specifically binds a cytokine of the four short-chain helix bundle cytokines that signal through class III receptor tyrosine kinases, including M-CSF, SCF, Fit- 3 Ligand, and IL-34. The receptors for these cytokines contain extracellular Ig-like domains, similar to the IL-1 R family, but they have cytoplasmic domains with tyrosine kinase activity. The long-chain helical cytokine family includes the IL-6 family cytokines, G-CSF, erythropoietin, thrombopoietin, growth hormone, prolactin, and leptin.

[0061] According to some embodiments, the second moiety specifically binds a chemokine. The four subfamilies of chemokines are the C, CX3C, CC, and CXC subfamilies, and are based on the number and spacing of conserved cysteine residues located in the amino terminus. Chemokines bind to conventional G protein-coupled seven transmembrane receptors, with some promiscuity, and have a key role in regulating cell migration during development, and under homeostatic and inflammatory conditions. In certain embodiments, the second moiety specifically binds cardiotrophin-like cytokine factor 1 (CLCF1 ). According to some embodiments, the second moiety specifically binds leukemia inhibitory factor (LIF).

[0062] Regardless of whether the second moiety specifically binds a cell surface molecule or an extracellular molecule, in certain embodiments, binding of the second moiety does not block activity of the molecule to which it binds. In this way, cell surface or extracellular molecules of CD206-expressing cells may be selectively removed while mitigating any off-target effects. By way of example, when the second moiety specifically binds an immune checkpoint molecule (e.g., SIRPa, PD-L1 , PD-L2, CD80, CD86, B7-H4, VISTA, PD-1 , Siglec 10, CSF-1 R, IL-4R / IL-13R, MARCO, or LILRB-1 ), in some instances, binding of the second moiety does not block activity of the immune checkpoint molecule. Non-blocking anti-checkpoint molecule antibodies are known and available to those in the art. Non-blocking anti-SIRPa antibodies (i.e., anti-SIRPa antibodies which do not block binding of SIRPa to CD47) include, e.g., those described in Yanagita, T. et al. JCI Insight, 2017, 2 (1 ): e89140 (antibody P84); clone 3 (PDB 6NMT) and clone 136 (PDB 6NMS) developed by ALX Oncology (Sim et al. mAbs, 2019, 1 1 (6), 1036-1052); and others. Nonblocking anti-PD-L1 antibodies include, e.g., RRID# AB_2860078, RRID# AB_2860079, and single domain antibody Nb-109 (Lv et al. J. Nuc. Med. 2020, 61 (1 ), 1 17-122). Non-blocking anti- IL4R antibodies include Clone S69 (Zurawski et al. J. Biol. Chem. 1995, 270 (23), 13869-13878). Non-blocking VISTA antibodies include mAbl described in US11603406B2 to Johnston et al. Non-blocking Siglec 10 antibodies include S10-D described in US1 1083785B2 to Cornen et al. Non-blocking anti-PD-1 antibodies include Clone RMP1 -30 and NB01 (humanized 135C12) (Fenwick et al. J. Exp. Med. 2019, 216 (7), 1525-1541 .). Non-blocking (for CTLA-4 binding) anti- CD80 antibodies include Galiximab (I DEC-114) (Smith et al. Leuk Lymphoma, 2013, 54 (7), 1405-1410.).

[0063] In certain embodiments, in addition to removal of the cell surface molecule or extracellular molecule from the cell surface or extracellular space, respectively, the cell surface molecule or extracellular molecule is degraded upon internalization into the CD206-expressing cell. Proof-of- concept of such degradation upon internalization is demonstrated in Example 6 below.

[0064] According to the bifunctional molecules of the present disclosure, the second moiety is stably associated with the first moiety. By “stably associated” is meant a physical association between two entities in which the mean half-life of association is one day or more in phosphate buffered saline (PBS) at 4°C. In some embodiments, the physical association between the two entities has a mean half-life of one day or more, one week or more, one month or more, including six months or more, e.g., 1 year or more, in PBS at 4°C. According to some embodiments, the stable association arises from a covalent bond between the two entities, a non-covalent bond between the two entities (e.g., an ionic or metallic bond), or other forms of chemical attraction, such as hydrogen bonding, Van der Waals forces, and the like. According to some embodiments, the first moiety is stably associated with the second moiety via conjugation. The term “conjugation” or “conjugated” generally refers to a chemical linkage, either covalent or non-covalent, usually covalent, that proximally associates one molecule of interest with a second molecule of interest. In certain embodiments, the first moiety is conjugated to the second moiety via a linker. If present, the linker molecule(s) may be of sufficient length to permit the first moiety and second moiety to allow some flexible movement between the first moiety and second moiety. Linker molecules may be, e.g., about 6-50 atoms long. Linker molecules may also be, e.g., aryl acetylene, ethylene glycol oligomers containing 2- 10 monomer units, diamines, diacids, amino acids, or combinations thereof.

[0065] Where the linkers are peptides, the linkers can be of any suitable length, such as from 1 amino acid (e.g., Gly) to 20 or more amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1 , 2, 3, 4, 5, 6, or 7 amino acids in length.

[0066] Flexible linkers include glycine polymers (G)n, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers may be used where relatively unstructured amino acids are of interest and may serve as a neutral tether between components. The ordinarily skilled artisan will recognize that design of conjugates can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer a less flexible structure.

[0067] According to some embodiments, the first moiety is conjugated to the second moiety via a non-cleavable linker. Non-cleavable linkers of interest include, but are not limited to, thioether linkers. An example of a thioether linker that may be employed includes a succinimidyl 4-(N- maleimidomethyl)cyclohexane-1 -carboxylate (SMCC) linker.

[0068] In certain embodiments, the first moiety is conjugated to the second moiety via a cleavable linker. According to some embodiments, the linker is a chemically-labile linker, such as an acid- cleavable linker that is stable at neutral pH (bloodstream pH 7.3-7.5) but undergoes hydrolysis upon internalization into the mildly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0) of a CD206-expressing cell. Chemically-labile linkers include, but are not limited to, hydrazonebased linkers, oxime-based linkers, carbonate-based linkers, ester-based linkers, etc. In certain embodiments, the linker is an enzyme-labile linker, such as an enzyme-labile linker that is stable in the bloodstream but undergoes enzymatic cleavage upon internalization into a CD206- expressing cell. Enzyme-labile linkers include, but are not limited to, linkers that include peptidic bonds, e.g., dipeptide-based linkers such as valine-citrulline (VC) linkers, such as a maleimidocaproyl-valine-citruline-p-aminobenzyl (MC-vc-PAB) linker, a valyl-alanyl-para- aminobenzyloxy (Val-Ala-PAB) linker, and the like. Chemically-labile linkers, enzyme-labile, and non-cleavable linkers are known and described in detail, e.g., in Ducry & Stump (2010) Bioconjugate Chem. 21 :5-13; Nolting, B. (2013) Methods Mol Biol. 1045:71 -100; Tsuchikama and An (2018) Protein & Ce / / 9(1 ):33-46; and elsewhere.

[0069] Numerous strategies are available for linking the first moiety and second moiety directly, or indirectly via a linker. For example, the first moiety may be derivatized by covalently attaching a linker to the first moiety, where the linker has a functional group capable of reacting with a “chemical handle” on the second moiety. Also by way of example, the second moiety may be derivatized by covalently attaching a linker to the second moiety, where the linker has a functional group capable of reacting with a “chemical handle” on the first moiety. The functional group on the linker may vary and may be selected based on compatibility with the chemical handle on the first moiety or second moiety. According to one embodiment, the chemical handle is provided by incorporation of an unnatural amino acid having the chemical handle into the first moiety or second moiety. Unnatural amino acids which find use for preparing the bifunctional molecule of the present disclosure include those having a functional group selected from an azide, alkyne, alkene, amino-oxy, hydrazine, aldehyde (e.g., formylglycine, e.g., SMARTag™ technology from Catalent Pharma Solutions), nitrone, nitrile oxide, cyclopropene, norbornene, iso-cyanide, aryl halide, and boronic acid functional group. Unnatural amino acids which may be incorporated into a first moiety or second moiety of a bifunctional molecule of the present disclosure, which unnatural amino acid may be selected to provide a functional group of interest, are known and described in, e.g., Maza et al. (2015) Bioconjug. Chem. 26(9):1884-9; Patterson et al. (2014) ACS Chem. Biol. 9:592-605; Adumeau et al. (2016) Moi. Imaging Biol. (2) :153-65; and elsewhere. An unnatural amino acid may be incorporated into a first moiety or second moiety via chemical synthesis or recombinant approaches, e.g., using a suitable orthogonal amino acyl tRNA synthetase-tRNA pair for incorporation of the unnatural amino acid during translation of a first moiety or second moiety in a host cell.

[0070] The functional group of an unnatural amino acid present in the first moiety or second moiety may be an azide, alkyne, alkene, amino-oxy, hydrazine, aldehyde, anisaldehyde, nitrone, nitrile oxide, cyclopropene, norbornene, iso-cyanide, aryl halide, boronic acid, diazo, tetrazine, tetrazole, quadricyclane, iodobenzene, or other suitable functional group, and the functional group on the linker is selected to react with the functional group of the unnatural amino acid (or vice versa). As just one example, an azide-bearing unnatural amino acid (e.g., 5-azido-L- norvaline, or the like) may be incorporated into the first moiety or second moiety and the linker portion of a linker-agent moiety may include an alkyne functional group, such that the first moiety or second moiety and linker-agent moiety are covalently conjugated via azide-alkyne cycloaddition. Conjugation may be carried out using, e.g., a copper-catalyzed azide-alkyne cycloaddition reaction.

[0071] In certain embodiments, the chemical handle on the first moiety or second moiety does not involve an unnatural amino acid. A first moiety or second moiety containing no unnatural amino acids may be conjugated by utilizing, e.g., nucleophilic functional groups of the first moiety or second moiety (such as the N-terminal amine or the primary amine of lysine, or any other nucleophilic amino acid residue) as a nucleophile in a substitution reaction with a moiety bearing a reactive leaving group or other electrophilic group. An example would be to prepare a first moiety-linker moiety bearing an N-hydroxysuccinimidyl (NHS) ester and allow it to react with the second moiety under aqueous conditions at elevated pH (~10) or in polar organic solvents such as DMSO with an added non-nucleophilic base, such as N,N-diisopropylethylamine.

[0072] It will be appreciated that the particular approach for attaching a linker, first moiety and / or second moiety to each other may vary depending upon the particular linker, first moiety and / or second moiety and functional groups selected and employed for conjugating the various components to each other.

[0073] According to some embodiments, the linker comprises polyethylene glycol (PEG), alkyl, or peptide spacer groups in a linear or branched orientation. In some instances, the linker comprises:

[0074] In certain embodiments, when the first moiety is proteinaceous (e.g., a CD206 binding protein (e.g., anti-CD206 antibody), or the like) and the second moiety is proteinaceous (e.g., a protein (e.g., antibody) that specifically binds the cell surface or extracellular molecule), the second moiety may be stably associated with the first moiety via fusion of the second moiety and the first moiety. In other words, the second moiety may be part of a fusion protein comprising the second moiety fused directly or indirectly to the first moiety. According to some embodiments, the second moiety is fused indirectly via a linker to the first moiety. Non-limiting examples of linkers that may be employed include glycine-serine linkers, etc.

[0075] Also provided by the present disclosure are bifunctional molecules comprising a first moiety comprising a CD206 ligand that binds CD206 on the surface of a cell and induces CD206- mediated endocytosis, and a cargo moiety stably associated with the first moiety. Such bifunctional molecules find use, e.g., for targeted delivery of the cargo moiety to CD206- expressing cells. The first moiety may be as described elsewhere herein in the context of the bifunctional molecules comprising first a second moieties. The stable association (e.g., conjugation or fusion) of the first moiety and cargo moiety may be as described elsewhere herein in the context of the bifunctional molecules comprising first a second moieties.

[0076] A wide variety of cargo moieties may be conjugated or fused to the first moiety for delivery to CD206-expressing cells. In some embodiments, the cargo moiety is a therapeutic agent, e.g., a chemotherapeutic agent. Therapeutic agents of interest include agents capable of affecting the viability and / or function of a CD206-expressing cell (e.g., TAM). When the function of the CD206-expressing cells / tissue is pathological, an agent that reduces the function of the CD206- expressing cells / tissue may be employed. In certain aspects, a conjugate of the present disclosure includes an agent that reduces the function of CD206-expressing cells by inhibiting cell proliferation and / or killing the CD206-expressing cells. Such agents may vary and include cytostatic agents and cytotoxic agents, e.g., an agent capable of killing a target cell with or without being internalized into a target cell.

[0077] According to some embodiments, the cargo moiety comprises a cytotoxic agent selected from a calicheamicin, a duocarmycin, a pyrrolobenzodiazepines (PBDs), a camptothecins, a daunorubicin, a doxorubicin, an auristatin, a maytansinoid, or a radioisotope. In some instances, the cargo moiety comprises a cytotoxic agent selected from an enediyne, a lexitropsin, a duocarmycin, a taxane, a puromycin, a dolastatin, a maytansinoid, and a vinca alkaloid. In some embodiments, the cytotoxic agent is paclitaxel, docetaxel, CC-1065, CPT-11 (SN-38), topotecan, doxorubicin, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretastatin, calicheamicin, maytansine, maytansine DM1 , maytansine DM4, DM-1 , an auristatin or other dolastatin derivatives, such as auristatin E or auristatin F, AEB (AEB- 071 ), AEVB (5-benzoylvaleric acid-AE ester), AEFP (antibody-endostatin fusion protein), MMAE (monomethylauristatin E), MMAF (monomethylauristatin F), pyrrolobenzodiazepines (PBDs), eleutherobin, netropsin, or any combination thereof.

[0078] According to some embodiments, the cargo moiety comprises a toxin, such as a protein toxin selected from hemiasterlin and hemiasterlin analogs such as HTI-286 (e.g., see USPN 7,579,323; WO 2004 / 026293; and USPN 8,129,407, the full disclosures of which are incorporated herein by reference), abrin, brucine, cicutoxin, diphtheria toxin, batrachotoxin, botulism toxin, shiga toxin, endotoxin, Pseudomonas exotoxin, Pseudomonas endotoxin, tetanus toxin, pertussis toxin, anthrax toxin, cholera toxin, falcarinol, fumonisin Bl, fumonisin B2, afla toxin, maurotoxin, agitoxin, charybdotoxin, margatoxin, slotoxin, scyllatoxin, hefutoxin, calciseptine, taicatoxin, calcicludine, geldanamycin, gelonin, lotaustralin, ocratoxin A, patulin, ricin, strychnine, trichothecene, zearlenone, and tetradotoxin. Enzymatically active toxins and fragments thereof which may be employed include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin , restrictocin, phenomycin, enomycin and the tricothecenes.

[0079] In certain embodiments, the cargo moiety comprises a radiation-sensitizing agent. As used herein, a “radiation-sensitizing agent” is an agent that enhances the ability of radiation to kill CD206-expressing cells, e.g., CD206-expressing TAMs. Non-limiting examples of radiationsensitizing agents that may be conjugated to the CD206 ligand include cisplatin, 5-fluorouracil (5-FU), AZD7762, selumetinib, and the like. In certain embodiments, the cargo moiety comprises a radioisotope, e.g., useful for therapy and / or detection (e.g., imaging). Non-limiting examples of radioisotopes that may be conjugated to the CD206 ligand include but are not limited to225Ac,111Ag,114Ag,71As,72As,77As,

[0080] 169Yb,175Yb,133X, and89Zr. As will be appreciated with the benefit of the present disclosure, the bifunctional molecules of the present disclosure find use, e.g., in radioligand therapy (RLT).

[0081] In certain embodiments, a radioisotope is conjugated to the CD206 ligand via a chelator, for example, a bifunctional chelator. A bifunctional chelator may contain a metal chelating moiety that binds the radioisotope in a stable coordination complex and a reactive functional group that is covalently linked to the CD206 ligand, so that the radioisotope may be properly directed to the desirable molecular target in vivo. Non-limiting examples of bifunctional chelators that may be employed to conjugate a CD206 ligand to a radioisotope include p-SCN-Bn-DOTA and p-SCN- Bn-deferoxamine. Additional examples of bifunctional chelators that may be employed include those described in Price & Orvig (2014) Chem. Soc. Rev. 43:260; and Brechbiel (2008) Q J Nuci Med Mol Imaging 52(2) :166-173.

[0082] According to some embodiments, the radioisotope is a therapeutic radioisotope. In certain embodiments, the radioisotope is an alpha emitting radioisotope, e.g,225Ac,211At,212Bi / 212Pb,213Bi,223Ra, or227Th. In other embodiments, the radioisotope is a beta minus emitting radioisotope, e.g.,32P,33P,67Cu,90Y,1311 or177Lu.

[0083] According to some embodiments, the cargo moiety comprises a labeling agent. By “labeling agent” (or “detectable label”) is meant the agent detectably labels the CD206 ligand, such that the CD206 ligand may be detected in an application of interest (e.g., in vitro and / or in vivo research and / or clinical applications, e.g., in vivo imaging). Detectable labels of interest include radioisotopes (e.g., gamma or positron emitters), enzymes that generate a detectable product (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, etc.), fluorescent proteins, paramagnetic atoms, and the like. In certain aspects, the antibody or fusion protein is conjugated to a specific binding partner of detectable label, e.g., conjugated to biotin such that detection may occur via a detectable label that includes avidin / streptavidin.

[0084] In certain embodiments, the cargo moiety comprises a labeling agent that finds use in in vivo imaging, such as near-infrared (NIR) optical imaging, single-photon emission computed tomography (SPECT) ± CT imaging, positron emission tomography (PET) ± CT imaging, nuclear magnetic resonance (NMR) spectroscopy, or the like. Labeling agents that find use in such applications include, but are not limited to, fluorescent labels, radioisotopes, and the like. In some instances, the labeling agent is a multi-modal in vivo imaging agent that permits in vivo imaging using two or more imaging approaches (e.g., see Thorp-Greenwood and Coogan (201 1 ) Dalton Trans. 40:6129-6143).

[0085] In certain embodiments, the cargo moiety comprises an in vivo imaging agent that finds use in near-infrared (NIR) imaging applications. Such agents include, but are not limited to, a Kodak X-SIGHT dye, Pz 247, DyLight 750 and 800 Fluors, Cy 5.5 and 7 Fluors, Alexa Fluor 680 and 750 Dyes, IRDye 680 and 800CW Fluors. According to some embodiments, the labeling agent is an in vivo imaging agent that finds use in SPECT imaging applications, non-limiting examples of which include99mTc,111ln,123l,201TI, and133Xe. In certain embodiments, the labeling agent is an in vivo imaging agent that finds use in PET imaging applications, e.g.,11C,13N,15O,18F,64Cu,62Cu,124l,76Br,82Rb, “Ga, or the like.

[0086] In certain embodiments, the cargo moiety comprises a nucleic acid. The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to describe a polymer of any length, e.g., greater than about 2 bases, greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, greater than 10,000 bases, greater than 100,000 bases, greater than about 1 ,000,000, up to about 101° or more bases composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, and may be produced enzymatically or synthetically (e.g., PNA as described in U.S. Patent No. 5,948,902 and the references cited therein) which can hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids, e.g., can participate in Watson-Crick base pairing interactions. Naturally-occurring nucleotides include guanine, cytosine, adenine, thymine, uracil (G, C, A, T and U respectively). DNA and RNA have a deoxyribose and ribose sugar backbone, respectively, whereas PNA’s backbone is composed of repeating N-(2- aminoethyl)-glycine units linked by peptide bonds. In PNA various purine and pyrimidine bases are linked to the backbone by methylenecarbonyl bonds. A locked nucleic acid (LNA), often referred to as inaccessible RNA, is a modified RNA nucleotide. The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon. The bridge “locks” the ribose in the 3'-endo (North) conformation, which is often found in the A-form duplexes. LNA nucleotides can be mixed with DNA or RNA residues in the oligonucleotide whenever desired. The term “unstructured nucleic acid,” or “UNA,” is a nucleic acid containing non-natural nucleotides that bind to each other with reduced stability. For example, an unstructured nucleic acid may contain a G' residue and a C residue, where these residues correspond to non-naturally occurring forms, i.e., analogs, of G and C that base pair with each other with reduced stability, but retain an ability to base pair with naturally occurring C and G residues, respectively. Unstructured nucleic acid is described in US20050233340, which is incorporated by reference herein for disclosure of UNA.

[0087] According to some embodiments, when the cargo moiety comprises a nucleic acid, the nucleic acid is an RNAi construct, an antisense oligonucleotide (ASO), or an mRNA. RNAi constructs of interest include, but are not limited to, siRNA constructs. siRNAs and their use in therapy are known and described, e.g., in Hu et al. (2020) Sig Transduct Target Ther 5, 101 .

[0088] In some embodiments, the cargo moiety comprises an RNAi construct or ASO targeting an mRNA encoding an immune checkpoint molecule (e.g., SIRPa, PD-L1 , PD-L2, CD80, CD86, B7-H4, VISTA, PD-1 , Siglec 10, CSF-1 R, IL-4R / IL-13R, MARCO, or LILRB-1 ) or an immunosuppressive cytokine.

[0089] In certain embodiments, the cargo moiety comprises a protein. Non-limiting examples of proteins which may be conjugated or fused to the CD206 ligand include enzymes and binding proteins. Binding proteins of interest include, but are not limited to, antibodies.

[0090] COMPOSITIONS

[0091] Aspects of the present disclosure further include compositions. A composition of the present disclosure comprises a bifunctional molecule of the present disclosure. For example, the bifunctional molecule may be any of the bifunctional molecules described in the Bifunctional Molecules section hereinabove or in the Experimental section below, which descriptions are incorporated but not reiterated herein for purposes of brevity.

[0092] In certain embodiments, a composition of the present disclosure includes the bifunctional molecule present in a liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffered solution, or the like. One or more additives such as a salt (e.g., NaCI, MgCI2, KCI, MgSO4), a buffering agent (a Tris buffer, N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N- tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), a solubilizing agent, a detergent (e.g., a non-ionic detergent such as Tween-20, etc.), a nuclease inhibitor, a protease inhibitor, glycerol, a chelating agent, and the like may be present in such compositions.

[0093] Aspects of the present disclosure include pharmaceutical compositions. In some embodiments, a pharmaceutical composition of the present disclosure includes a bifunctional molecule of the present disclosure, and a pharmaceutically acceptable carrier.

[0094] The bifunctional molecule can be incorporated into a variety of formulations for therapeutic administration. More particularly, the bifunctional molecules can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable excipients or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, injections, inhalants and aerosols.

[0095] Formulations of the bifunctional molecules for administration to an individual (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration.

[0096] In pharmaceutical dosage forms, the bifunctional molecules can be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and carriers / excipients are merely examples and are in no way limiting.

[0097] For oral preparations, the bifunctional molecules can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

[0098] The bifunctional molecules can be formulated for parenteral (e.g., intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration. In certain embodiments, the bifunctional molecules are formulated for injection by dissolving, suspending or emulsifying the bifunctional molecules in an aqueous or non-aqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

[0099] Pharmaceutical compositions that include the bifunctional molecules may be prepared by mixing the bifunctional molecules having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and / or tonicity agents. Acceptable carriers, excipients and / or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or nonionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG). The pharmaceutical composition may be in a liquid form, a lyophilized form or a liquid form reconstituted from a lyophilized form, wherein the lyophilized preparation is to be reconstituted with a sterile solution prior to administration. The standard procedure for reconstituting a lyophilized composition is to add back a volume of pure water (typically equivalent to the volume removed during lyophilization); however solutions comprising antibacterial agents may be used for the production of pharmaceutical compositions for parenteral administration.

[0100] An aqueous formulation of the bifunctional molecules may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.

[0101] A tonicity agent may be included to modulate the tonicity of the formulation. Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term "isotonic" denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM.

[0102] A surfactant may also be added to the formulation to reduce aggregation and / or minimize the formation of particulates in the formulation and / or reduce adsorption. Example surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylene sorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylenepolypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable Polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Example concentrations of surfactant may range from about 0.001% to about 1% w / v.

[0103] A lyoprotectant may also be added in order to protect the bifunctional molecules against destabilizing conditions during a lyophilization process. For example, known lyoprotectants include sugars (including glucose and sucrose); polyols (including mannitol, sorbitol and glycerol); and amino acids (including alanine, glycine and glutamic acid). Lyoprotectants can be included, e.g., in an amount of about 10 mM to 500 nM.

[0104] In some embodiments, the pharmaceutical composition includes the bifunctional molecule, and one or more of the above-identified components (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% weight / volume (w / v).

[0105] METHODS OF USE

[0106] Aspects of the present disclosure further include methods of using the bifunctional molecules of the present disclosure. For example, in certain embodiments, provided are methods of removing a molecule from the surface of CD206-expressing cells or an extracellular molecule in a subject in need thereof. Such methods comprise administering a bifunctional molecule of the present disclosure to the subject in an amount effective to remove the molecule from the surface of the CD206-expressing cells or from the extracellular space of the CD206-expressing cells. CD206-expressing cells of interest include, but are not limited to, macrophages, dendritic cells, sinusoidal endothelial cells, and the like.

[0107] In certain embodiments, the subject has cancer. The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. The methods of the present disclosure may be employed for the treatment of a large variety of cancers. Examples of cancers that may be treated using the methods include, but are not limited to, a carcinoma, a sarcoma, a myeloma, a leukemia, a lymphoma, or mixed type. More particular examples of such cancers include renal cancer; kidney cancer; glioblastoma multiforme; metastatic breast cancer; breast carcinoma; breast sarcoma; neurofibroma; neurofibromatosis; pediatric tumors; neuroblastoma; malignant melanoma; carcinomas of the epidermis; leukemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as but not limited to smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone cancer and connective tissue sarcomas such as but not limited to bone sarcoma, myeloma bone disease, multiple myeloma, cholesteatoma-induced bone osteosarcoma, Paget's disease of bone, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft- tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangio sarcoma, neurilemmoma, rhabdomyosarcoma, and synovial sarcoma; brain tumors such as but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, and primary brain lymphoma; breast cancer including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease (including juvenile Paget's disease) and inflammatory breast cancer; adrenal cancer such as but not limited to pheochromocytoma and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer such as but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancers such as but limited to Cushing's disease, prolactinsecreting tumor, acromegaly, and diabetes insipius; eye cancers such as but not limited to ocular melanoma such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers such as but not limited to, squamous cell carcinoma, and adenocarcinoma; uterine cancers such as but not limited to endometrial carcinoma and uterine sarcoma; ovarian cancers such as but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; cervical carcinoma; esophageal cancers such as but not limited to, squamous cancer, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; stomach cancers such as but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; colorectal cancer, KRAS mutated colorectal cancer; colon carcinoma; rectal cancers; liver cancers such as but not limited to hepatocellular carcinoma and hepatoblastoma, gallbladder cancers such as adenocarcinoma; cholangiocarcinomas such as but not limited to papillary, nodular, and diffuse; lung cancers such as KRAS-mutated non-small cell lung cancer, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinoma and small-cell lung cancer; lung carcinoma; testicular cancers such as but not limited to germinal tumor, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers such as but not limited to, androgen-independent prostate cancer, androgen-dependent prostate cancer, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers such as but not limited to squamous cell carcinoma; basal cancers; salivary gland cancers such as but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; pharynx cancers such as but not limited to squamous cell cancer, and verrucous; skin cancers such as but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma (ALM); kidney cancers such as but not limited to renal cell cancer, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and / or ureter); renal carcinoma; Wilms' tumor; and bladder cancers such as but not limited to transitional cell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. In some embodiments, the cancer is myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, or papillary adenocarcinomas.

[0108] According to some embodiments, the subject has cancer characterized by a solid tumor, and wherein the CD206-expressing cells are CD206hi9htumor-associated macrophages (TAMs). The immune cells in the tumor microenvironment (TME) are not only powerful regulators of immunosuppression and tumorigenesis, but also represent a dominant cell type, with tumor- associated macrophages (TAMs) comprising up to 50% of total cell mass in solid tumors.

[0109] In some instances, when the CD206-expressing cells are TAMs (e.g., CD206h'9hTAMs), the second moiety specifically binds an immune checkpoint molecule, e.g., SIRPa, PD-L1 , PD- L2, CD80, CD86, B7-H4, VISTA, PD-1 , Siglec 10, CSF-1 R, IL-4R / IL-13R, MARCO, or LILRB-1. Immunotherapies such as immune checkpoint inhibitors (ICIs) derive their efficacy from this cancer-immune cell interface, however, auto-immune related adverse events resulting from systemic administration remain a significant challenge. The bifunctional molecules of the present disclosure address the need for potent, yet highly tumor-specific immunotherapies, via their capability of selectively removing immune checkpoint (ICP) molecules from the surface of TAMs.

[0110] In certain embodiments, the immune checkpoint molecule is SIRPa. According to such embodiments, SIRPa molecules are removed from the surface of TAMs, thereby reducing antiphagocytosis (CD47) signaling to the TAMs, in turn restoring or enhancing the phagocytic capacity of TAMs to eliminate tumor cells.

[0111] Accordingly, in some embodiments, provided are methods of restoring or enhancing the phagocytic capacity of TAMs (e.g., CD206h'9hTAMs) to eliminate tumor cells in a subject in need thereof. In some instances, such methods comprise administering a bifunctional molecule of the present disclosure to the subject, where the second moiety binds an immune checkpoint molecule (e.g., SIRPa, PD-L1 , PD-L2, CD80, CD86, B7-H4, VISTA, PD-1 , sialic-acid binding immunoglobulin-like lectin-10 (Siglec 10), CSF-1 R, IL-4R / IL-13R, MARCO, or LILRB-1 ), and where the administration results in removal of the immune checkpoint molecule from the surface of the TAMs, e.g., in the TME. In certain embodiments, binding of the second moiety to the immune checkpoint molecule does not block the activity of the immune checkpoint molecule. For example, the second moiety may specifically bind SIRPa without blocking binding of SIRPa to CD47. Also provided are methods of delivering a cargo moiety into CD206-expressing cells of a subject in need thereof, the method comprising contacting the CD206-expressing cells with a bifunctional molecule of the present disclosure comprising the first moiety stably associated with a cargo moiety. Cargo moieties of interest include, but are not limited to, cytotoxic agents, nucleic acids (e.g., RNAi constructs, ASOs, mRNAs, or the like), proteins (e.g., enzymes, binding proteins such as antibodies, transcription factors, or the like), etc. In certain embodiments, the CD206-expressing cells are macrophages, dendritic cells, or sinusoidal endothelial cells. According to some embodiments, the subject has cancer characterized by a solid tumor, and wherein the CD206-expressing cells are CD206h'9htumor-associated macrophages (TAMs).

[0112] The bifunctional molecules of the present disclosure may be administered via a route of administration selected from oral (e.g., in tablet form, capsule form, liquid form, or the like), parenteral (e.g., by intravenous, intra-arterial, subcutaneous, intramuscular, epidural injection), topical, intra-nasal, intra-tumoral administration, or intraperitoneal (IP) administration.

[0113] The bifunctional molecules of the present disclosure may be administered (e.g., in a pharmaceutical composition) in a therapeutically effective amount. By “therapeutically effective amount” is meant a dosage sufficient to produce a desired result, e.g., an amount sufficient to effect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of a cancer, as compared to a control. With respect to cancer, in some embodiments, the therapeutically effective amount is sufficient to slow the growth of a tumor, reduce the size of a tumor, and / or the like. An effective amount can be administered in one or more administrations.

[0114] In some embodiments, the methods are for treatment of a subject in need thereof. By treatment is meant at least an amelioration of one or more symptoms associated with a condition of the subject (e.g., cancer), where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the condition being treated. As such, treatment also includes situations where the condition, or at least one or more symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the individual no longer suffers from the condition, or at least the symptoms that characterize the condition.

[0115] A bifunctional molecule of the present disclosure may be administered to the subject alone or in combination with a second agent. Second agents of interest include, but are not limited to, agents approved by the United States Food and Drug Administration and / or the European Medicines Agency (EMA) for use in treating cancer. In some embodiments, the second agent is an immune checkpoint inhibitor. Immune checkpoint inhibitors of interest include, but are not limited to, a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, a programmed cell death-1 (PD-1) inhibitor, a programmed cell death ligand-1 (PD-L1 ) inhibitor, a lymphocyte activation gene-3 (LAG-3) inhibitor, a T-cell immunoglobulin domain and mucin domain 3 (TIM- 3) inhibitor, an indoleamine (2,3)-dioxygenase (IDO) inhibitor, a T cell immunoreceptor with Ig and ITIM domains (TIGIT) inhibitor, a V-domain Ig suppressor of T cell activation (VISTA) inhibitor, a B7-H3 inhibitor, and any combination thereof.

[0116] When a bifunctional molecule of the present disclosure is administered with a second agent (e.g., immune checkpoint inhibitor or other second agent of interest), the bifunctional molecule and the second agent may be administered to the subject according to any suitable administration regimen. According to certain embodiments, the bifunctional molecule and the second agent are administered according to a dosing regimen approved for individual use. In some embodiments, the administration of the bifunctional molecule permits the second agent to be administered according to a dosing regimen that involves one or more lower and / or less frequent doses, and / or a reduced number of cycles as compared with that utilized when the second agent is administered without administration of the bifunctional molecule. In certain aspects, the administration of the second agent permits the bifunctional molecule to be administered according to a dosing regimen that involves one or more lower and / or less frequent doses, and / or a reduced number of cycles as compared with that utilized when the bifunctional molecule is administered without administration of the second agent.

[0117] In some embodiments, one or more doses of the bifunctional molecule and the second agent are administered concurrently to the subject. By “concurrently” is meant the bifunctional molecule and the second agent are either present in the same pharmaceutical composition, or the bifunctional molecule and the second agent are administered as separate pharmaceutical compositions within 1 hour or less, 30 minutes or less, or 15 minutes or less.

[0118] In some embodiments, one or more doses of the bifunctional molecule and the second agent are administered sequentially to the subject.

[0119] In some embodiments, the bifunctional molecule and the second agent are administered to the subject in different compositions and / or at different times. For example, the bifunctional molecule may be administered prior to administration of the second agent, e.g., in a particular cycle. Alternatively, the second agent may be administered prior to administration of the bifunctional molecule, e.g., in a particular cycle. The second agent to be administered may be administered a period of time that starts at least 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or up to 5 days or more after the administration of the first agent to be administered.

[0120] In one example, the second agent is administered to the subject for a desirable period of time prior to administration of the bifunctional molecule. In certain aspects, when the subject has cancer, such a regimen “primes” the cancer cells to potentiate the anti-cancer effect of the bifunctional molecule. Such a period of time separating a step of administering the second agent from a step of administering the bifunctional molecule is of sufficient length to permit priming of the cancer cells, desirably so that the anti-cancer effect of the bifunctional molecule is increased. In some embodiments, administration of one agent is specifically timed relative to administration of the other agent. For example, in some embodiments, the bifunctional molecule is administered so that a particular effect is observed (or expected to be observed, for example based on population studies showing a correlation between a given dosing regimen and the particular effect of interest).

[0121] In certain aspects, desired relative dosing regimens for agents administered in combination may be assessed or determined empirically, for example using ex vivo, in vivo and / or in vitro models; in some embodiments, such assessment or empirical determination is made in vivo, in a patient population (e.g., so that a correlation is established), or alternatively in a particular subject of interest.

[0122] In some embodiments, the bifunctional molecule and the second agent are administered according to an intermittent dosing regimen including at least two cycles. Where two or more agents are administered in combination, and each by such an intermittent, cycling, regimen, individual doses of different agents may be interdigitated with one another. In certain aspects, one or more doses of a second agent is administered a period of time after a dose of the first agent. In some embodiments, each dose of the second agent is administered a period of time after a dose of the first agent. In certain aspects, each dose of the first agent is followed after a period of time by a dose of the second agent. In some embodiments, two or more doses of the first agent are administered between at least one pair of doses of the second agent; in certain aspects, two or more doses of the second agent are administered between at least one pair of doses of the first agent. In some embodiments, different doses of the same agent are separated by a common interval of time; in some embodiments, the interval of time between different doses of the same agent varies. In certain aspects, different doses of the bifunctional molecule and the second agent are separated from one another by a common interval of time; in some embodiments, different doses of the different agents are separated from one another by different intervals of time.

[0123] One exemplary protocol for interdigitating two intermittent, cycled dosing regimens may include: (a) a first dosing period during which a therapeutically effective amount the bifunctional molecule is administered to the subject; (b) a first resting period; (c) a second dosing period during which a therapeutically effective amount of the second agent is administered to the subject; and (d) a second resting period. A second exemplary protocol for interdigitating two intermittent, cycled dosing regimens may include: (a) a first dosing period during which a therapeutically effective amount the second agent is administered to the subject; (b) a first resting period; (c) a second dosing period during which a therapeutically effective amount of the bifunctional molecule is administered to the subject; and (d) a second resting period.

[0124] In some embodiments, the first resting period and second resting period may correspond to an identical number of hours or days. Alternatively, in some embodiments, the first resting period and second resting period are different, with either the first resting period being longer than the second one or, vice versa. In some embodiments, each of the resting periods corresponds to 120 hours, 96 hours, 72 hours, 48 hours, 24 hours, 12 hours, 6 hours, 30 hours, 1 hour, or less. In some embodiments, if the second resting period is longer than the first resting period, it can be defined as a number of days or weeks rather than hours (for instance 1 day, 3 days, 5 days, 1 week, 2, weeks, 4 weeks or more).

[0125] If the first resting period’s length is determined by existence or development of a particular biological or therapeutic event, then the second resting period’s length may be determined on the basis of different factors, separately or in combination. Exemplary such factors may include type and / or stage of a cancer against which the therapy is administered; properties (e.g., pharmacokinetic properties) of the bifunctional molecule, and / or one or more features of the patient’s response to therapy with the bifunctional molecule. In some embodiments, length of one or both resting periods may be adjusted in light of pharmacokinetic properties (e.g., as assessed via plasma concentration levels) of one or the other of the administered agents. For example, a relevant resting period might be deemed to be completed when plasma concentration of the relevant agent is below a pre-determined level, optionally upon evaluation or other consideration of one or more features of the subject’s response.

[0126] In certain aspects, the number of cycles for which a particular agent is administered may be determined empirically. Also, in some embodiments, the precise regimen followed (e.g., number of doses, spacing of doses (e.g., relative to each other or to another event such as administration of another therapy), amount of doses, etc.) may be different for one or more cycles as compared with one or more other cycles.

[0127] The bifunctional molecule and the second agent may be administered together or independently via any suitable route of administration. The bifunctional molecule and the second agent may be administered via a route of administration independently selected from oral, parenteral (e.g., by intravenous, intra-arterial, subcutaneous, intramuscular, or epidural injection), topical, or intra-nasal administration. According to certain embodiments, the bifunctional molecule and the second agent are both administered orally (e.g., in tablet form, capsule form, liquid form, or the like) either concurrently (in the same pharmaceutical composition or separate pharmaceutical compositions) or sequentially.

[0128] KITS

[0129] Aspects of the present disclosure further include kits. In certain embodiments, the kits find use in practicing the methods of the present disclosure, e.g., methods of removing a molecule from the surface of CD206-expressing cells or an extracellular molecule in a subject in need thereof.

[0130] Accordingly, in certain embodiments, a kit of the present disclosure comprises any of the bifunctional molecules of the present disclosure (e.g., formulated for administration to a subject), and optionally, instructions for administering the bifunctional molecule to a subject in need thereof. As will be appreciated, the kits of the present disclosure may include any of the bifunctional molecules having any of the features (e.g., any of the first moieties, any of the second moieties, etc.) described above in the section relating to the bifunctional molecules of the present disclosure, which are not reiterated herein for purposes of brevity.

[0131] The kits of the present disclosure may include a quantity of the bifunctional molecule, present in unit dosages, e.g., ampoules, or a multi-dosage format. As such, in certain embodiments, the kits may include one or more (e.g., two or more) unit dosages (e.g., ampoules) of a bifunctional molecule of the present disclosure. The term “unit dosage”, as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the bifunctional molecule calculated in an amount sufficient to produce the desired effect. The amount of the unit dosage depends on various factors, such as the bifunctional molecule employed, the effect to be achieved, and the pharmacodynamics associated with the bifunctional molecule, in the subject. In yet other embodiments, the kits may include a single multi dosage amount of the bifunctional molecule.

[0132] The instructions (e.g., instructions for use (I FU)) included in the kits may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., portable flash drive, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet) are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, the means for obtaining the instructions is recorded on a suitable substrate.

[0133] For purposes of completeness, non-limiting aspects and embodiments of the present disclosure are further disclosed in the following numbered clauses.

[0134] 1 . A bifunctional molecule comprising: a first moiety comprising a CD206 ligand that binds CD206 on the surface of a cell and induces CD206-mediated endocytosis; and a second moiety stably associated with the first moiety, wherein the second moiety specifically binds a molecule on the surface of the cell or an extracellular molecule.

[0135] 2. The bifunctional molecule of clause 1 , wherein the CD206 ligand comprises a carbohydrate.

[0136] 3. The bifunctional molecule of clause 2, wherein the CD206 ligand comprises mannose, fucose, a sulfated glycan, or any combination thereof. 4. The bifunctional molecule of clause 3, wherein the CD206 ligand comprises a monosaccharide, disaccharide, or trisaccharide comprising mannose, fucose, a sulfated glycan, or any combination thereof.

[0137] 5. The bifunctional molecule of clause 3 or 4, wherein the sulfated glycan is 3-SO4galactose or 4-SO4galactose.

[0138] 6. The bifunctional molecule of any one of clauses 2-5, wherein the CD206 ligand is trisfucose.

[0139] 7. The bifunctional molecule of any one of clauses 2-5, wherein the CD206 ligand is tris-3- SO4galactose.

[0140] 8. The bifunctional molecule of clause 2, wherein the CD206 ligand comprises N-acetyl glucosamine (GIcNAc), 4-SO4-N-Acetyl galactosamine (4-SO4-GalNAc), 3-SO4-N-Acetyl galactosamine (3-SO4-GalNAc), 2a-mannobiose, 3,6-di-O-(a-D-mannopyranosyl)-a-D- mannopyranoside, 3-fucosyllactose, or any combination thereof.

[0141] 9. The bifunctional molecule of clause 1 , wherein the CD206 ligand comprises a protein.

[0142] 10. The bifunctional molecule of clause 9, wherein the CD206 ligand is an anti-CD206 antibody.

[0143] 11 . The bifunctional molecule of clause 1 , wherein the CD206 ligand comprises an aptamer.

[0144] 12. The bifunctional molecule of any one of clauses 9-11 , wherein the CD206 ligand binds to the N-terminal R-type carbohydrate-recognition domain (CRD) of CD206.

[0145] 13. The bifunctional molecule of any one of clauses 9-11 , wherein the CD206 ligand binds to C-type CRD4, C-type CRD5, or both, of CD206.

[0146] 14. The bifunctional molecule of any one of clauses 1-13, wherein the second moiety is a small molecule, an antibody, or an aptamer.

[0147] 15. The bifunctional molecule of clause 14, wherein the second moiety is an antibody.

[0148] 16. The bifunctional molecule of any one of clauses 1-15, wherein the cell is a tumor- associated macrophage (TAM), a dendritic cell, or a sinusoidal endothelial cell.

[0149] 17. The bifunctional molecule of clause 16, wherein the cell is a CD206h'9htumor-associated macrophage (TAM).

[0150] 18. The bifunctional molecule of clause 17, wherein the second moiety specifically binds an immune checkpoint molecule.

[0151] 19. The bifunctional molecule of clause 18, wherein the immune checkpoint molecule is SIRPa, PD-L1 , PD-L2, CD80, CD86, B7-H4, VISTA, PD-1 , Siglec 10, CSF-1 R, IL-4R / IL-13R, MARCO, or LILRB-1.

[0152] 20. The bifunctional molecule of clause 19, wherein the immune checkpoint molecule is SIRPa. 21 . The bifunctional molecule of any one of clauses 18-20, wherein binding of the second moiety to the immune checkpoint molecule does not block activity of the immune checkpoint molecule.

[0153] 22. The bifunctional molecule of any one of clauses 1 -17, wherein the second moiety specifically binds an extracellular molecule.

[0154] 23. The bifunctional molecule of clause 22, wherein the extracellular molecule is a cytokine.

[0155] 24. The bifunctional molecule of clause 22 or 23, wherein binding of the second moiety to the extracellular molecule does not block activity of the extracellular molecule.

[0156] 25. The bifunctional molecule of any one of clauses 1-24, wherein the second moiety is stably associated with the first moiety via conjugation.

[0157] 26. The bifunctional molecule of clause 25, wherein the first moiety and second moiety are conjugated via a linker.

[0158] 27. The bifunctional molecule of clause 26, wherein the linker comprises polyethylene glycol (PEG), alkyl, or peptide spacer groups in a linear or branched orientation.

[0159] 28. The bifunctional molecule of clause 26, wherein the linker comprises:

[0160] 29. The bifunctional molecule of any one of clauses 1 -21 , wherein the second moiety is stably associated with the first moiety via fusion of a protein domain of the second moiety with a protein domain of the first moiety.

[0161] 30. A composition comprising the bifunctional molecule of any one of clauses 1 -29.

[0162] 31 . The composition of clause 30, wherein the composition is formulated for administration to a subject in need thereof.

[0163] 32. The composition of clause 31 , wherein the composition is formulated for parenteral administration to a subject in need thereof.

[0164] 33. The composition of clause 32, wherein the composition is formulated for intravenous administration to a subject in need thereof.

[0165] 34. A method of removing a molecule from the surface of CD206-expressing cells or an extracellular molecule in a subject in need thereof, the method comprising administering the bifunctional molecule of any one of clauses 1-29 to the subject in an amount effective to remove the molecule from the surface of the CD206-expressing cells or from the extracellular space of the CD206-expressing cells.

[0166] 35. The method of clause 34, wherein the CD206-expressing cells are macrophages, dendritic cells, or sinusoidal endothelial cells. 36. The method of clause 34, wherein the subject has cancer characterized by a solid tumor, and wherein the CD206-expressing cells are CD206h'9htumor-associated macrophages (TAMs).

[0167] 37. The method of clause 36, wherein the second moiety specifically binds an immune checkpoint molecule.

[0168] 38. The method of clause 37, wherein the immune checkpoint molecule is SIRPa, PD-L1 , PD-L2, CD80, CD86, B7-H4, VISTA, PD-1 , Siglec 10, CSF-1 R, IL-4R / IL-13R, MARCO, or LILRB-1.

[0169] 39. The method of clause 38, wherein the immune checkpoint molecule is SIRPa.

[0170] 40. The method of any one of clauses 37-39, wherein binding of the second moiety to the immune checkpoint molecule does not block the activity of the immune checkpoint molecule.

[0171] 41 . The method of clause 40, wherein the second moiety specifically binds SIRPa and does not block binding of SIRPa to CD47.

[0172] 42. The method of any one of clauses 36-41 , wherein the solid tumor is a carcinoma, sarcoma, blastoma, or lymphoma.

[0173] 43. A bifunctional molecule comprising: a first moiety comprising a CD206 ligand that binds CD206 on the surface of a cell and induces CD206-mediated endocytosis; and a cargo moiety stably associated with the first moiety.

[0174] 44. The bifunctional molecule of clause 43, wherein the first moiety is as defined in any one of clauses 2-13

[0175] 45. The bifunctional molecule of clause 43 or 44, wherein the cargo moiety is a cytotoxic agent.

[0176] 46. The bifunctional of clause 45, wherein the cytotoxic agent is a calicheamicin, a duocarmycin, a pyrrolobenzodiazepines (PBDs), a camptothecins, a daunorubicin, a doxorubicin, an auristatin, a maytansinoid, or a radioisotope.

[0177] 47. The bifunctional of clause 46, wherein the auristatin is MMAF or MMAE.

[0178] 48. The bifunctional of any one of clauses 43-47, wherein the cargo moiety comprises a nucleic acid.

[0179] 49. The bifunctional of clause 48, wherein the nucleic acid is an RNAi construct, an antisense oligonucleotide (ASO), or an mRNA.

[0180] 50. The bifunctional of clause 49, wherein the RNAi construct is an siRNA construct.

[0181] 51 . The bifunctional of any one of clauses 43-50, wherein the cargo moiety comprises a protein.

[0182] 52. The bifunctional of clause 51 , wherein the protein is an enzyme. 53. The bifunctional of clause 51 , wherein the cargo moiety comprises a binding protein.

[0183] 54. The bifunctional of clause 53, wherein the binding protein is an antibody.

[0184] 55. The bifunctional of clause 53, wherein the binding protein is a transcription factor.

[0185] 56. A method of delivering a cargo moiety into CD206-expressing cells of a subject in need thereof, the method comprising contacting the CD206-expressing cells with the bifunctional molecule of any one of clauses 43-55.

[0186] 57. The method of clause 56, wherein the CD206-expressing cells are macrophages, dendritic cells, or sinusoidal endothelial cells.

[0187] 58. The method of clause 56, wherein the subject has cancer characterized by a solid tumor, and wherein the CD206-expressing cells are CD206h'9htumor-associated macrophages (TAMs).

[0188] The following examples are offered by way of illustration and not by way of limitation.

[0189] EXPERIMENTAL

[0190] Example 1 - Development of bifunctional molecules that target CD206 on M2-polarized macrophages

[0191] Prior approaches for targeting CD206 primarily employ polymeric mannose to invoke avidity effects that would compensate for the low monomeric affinity of mannose.16 21However, the heterogeneous nature of such polymers would render reproducible synthesis and characterization of the resulting antibody-conjugates challenging. The development of alternative ligands that bind CD206 is described in the present example. A recent glycan array study identified fucose as a promising candidate: >60% of the top 20 binders contained fucose, and fucose-containing glycans were structurally simpler than those containing mannose.22Also identified were sulfated glycans such as 3-SC galactose, which bind the N-terminal R-type CRD instead of the CTLDs commonly targeted by mannose (Fig. 1 A).23Therefore, a variety of propargyl-, azide-, or hydroxylamine-containing mannose, fucose, and 3-SO4galactose mono, di, and trisaccharides were prepared. The antibody-carbohydrate conjugates were generated in a two-step sequence, where the antibodies were first non-specifically labeled with linkers displaying alkynes, azides, or aldehydes in varying valencies and orientations, then conjugated to the carbohydrate ligands via Cu-catalyzed click reactions, Cu-free strain promoted reactions, or oxime ligations (Fig. 1 B). Characterization of the antibody-conjugates by MALDI-MS analysis revealed ligand to antibody ratios between 10 and 15.

[0192] Next sought was to identify a suitable in vitro macrophage model system. Phorbol 12- myristate 13-acetate (PMA) differentiated THP-1 or U-937 cells are frequently utilized as human macrophage surrogates, however, polarization with IL-4, IL-10, or IL-13 all failed to induce expression of CD206.24Thus, turned to was the murine macrophage cell line RAW264.7, another extensively used model for macrophage biology.25Addition of 25 ng / mL of IL-4 over 24 hours resulted in robust upregulation of CD206 (M2-polarized) in comparison to unpolarized cells (Fig. 1C). The ability of the antibody-carbohydrate conjugates to internalize extracellular targets via CD206 was examined, leading to the identification of tris-dendron TICTACs 1a-c (Fig. 1 D). M2- polarized RAW264.7 cells were incubated with rabbit lgG-488 and goat-anti-rabbit (control) or goat-anti-rabbit TICTACs 1 for 3 hours, then analyzed by flow cytometry for intracellular 488 fluorescence (Fig. 1 E). Because of the broad expression profile of CD206, cells were co-stained with an anti-CD206 antibody and gated for CD206high and CD206iowpopulations. In the CD206high population, 3-SO4Gal TICTAC 1c resulted in a 6-fold increase in intracellular fluorescence relative to the control, while Fuc TICTAC 1b and Man TICTAC 1a resulted in a 2.6-fold and 2- fold increase respectively (Fig. 1 F). Direct triazole linkage at the anomeric position of mannose (1d) completely ablated internalization. In the CD206iowpopulation, 1a-d did not result in significant increases in intracellular fluorescence. Evaluated next was whether TICTAC-mediated uptake was generalizable across other macrophage cell lines. Internalization of fluorescent rabbit-IgG was observed for M2-polarized BMA3.1 A726and J774A.1 cell lines for TICTACs 1a-c (Fig. 1G left), with 1b and 1c resulting in superior uptake. M2-polarized microglial cell line BV-2 was also subjected to control, TICTACs 1 b, 1c, or a tris-Gal analogue, resulting in internalization of 1 b and 1c, but not tris-Gal (Fig. 1G right). Finally, to confirm the mechanism of uptake, a CD206-knockout RAW264.7 cell line was generated and subjected to M2-polarization conditions before treatment with fluorescent rabbit IgG and control, 1 b, or 1c (Fig. 1 H). Uptake was completely abolished in the knockout cells, while preserved in cells that were treated with Cas9 and a non-targeting sgRNA (mock control).

[0193] Example 2 - Independent Function of a Tris-Fucose Ligand as a Small Molecule Scaffold

[0194] Asked next was whether the conjugated tris-fucose ligand in 1b could function independently as a small molecule scaffold. Tris-Fuc-647 (2) was synthesized, where the tris- fucose ligand was conjugated to a 647-dye in a 1 :1 stoichiometry (Fig. 2A). Polarized RAW264.7 cells were subjected to varying concentrations of the unconjugated dye or 2 for 2 hours, and intracellular fluorescence was analyzed by flow cytometry (Fig. 2B). Uptake was dependent on concentration of 2, and significant differences were observed between 2 and the unconjugated control at concentrations as low as 0.1 nM. This result was confirmed by confocal microscopy, where treatment of 2 resulted in a high 647 signal that concentrated in certain cells over others, mirroring the broad expression profile of CD206 (Fig. 2C). Sought next was to determine whether 2 could induce internalization in primary human macrophages. Upregulation of CD206 was first confirmed upon polarizing the macrophages with IL4 / IL10 over several days (Fig. 2D). M2- polarized and non-polarized macrophages were subjected to the unconjugated dye or 2 at varying concentrations, and intracellular fluorescence was analyzed by flow cytometry (Fig. 2E). In addition to a significant increase in fluorescence for 2 over the unconjugated control, a notable preference for M2-polarized over nonpolarized cells was observed. Finally, CD206-knockout RAW264.7 cells were subjected to 2, which resulted in ablation of uptake, indicating the selectivity of 2 for CD206 (Fig. 2F). Binding of 2 to CD206 was also analyzed by surface plasmon resonance (SPR), which revealed an extremely slow kOff that likely drives internalization even at very low nM concentrations.

[0195] Example 3 - TICTAC-Mediated Removal of Cell-Surface Proteins

[0196] Having demonstrated that TICTACs can efficiently internalize extracellular targets, investigated next was whether they could mediate internalization of membrane proteins in M2- polarized macrophages. For proof-of-concept, CD54 (ICAM-1 ) was chosen as a target due to its robust expression in several macrophage cell lines. TICTACs were constructed using a commercially available primary antibody against CD54 following a similar scheme to 1 b and 1c to generate 3b and 3c (Fig. 3A). RAW264.7 cells were treated with 3b and 3c, followed by measurement of surface levels of CD54 by flow cytometry using an orthogonal detection antibody. TICTAC 3b resulted in >80% removal of surface CD54 in CD206high RAW264.7 cells, whereas TICTAC 3c resulted in >60% removal (Fig. 3B, left). In CD206iOw cells, this effect is greatly attenuated, with 3b and 3c resulting in 30% and 15% removal of surface CD54 respectively. Also evaluated was the activity of 3b in BMA3.1 A7 cells and J774A.1 cell lines. >60% removal of surface CD54 in CD206high BMA3.1 A7 cells was observed, while <30% downregulation was observed in CD206iowcells (Fig. 3B, right). J774A.1 cells also exhibited comparable removal of surface CD54, which was dependent on expression levels of CD206. Visualization of surface CD54 by confocal microscopy following TICTAC treatment showed significantly reduced membrane CD54, consistent with our flow cytometry observations (Fig. 3C). Downregulation of CD54 on CD206high RAW264.7 cells was concentration dependent, where 3b demonstrated a detectable decrease in surface CD54 at concentrations as low as 0.1 nM. Maximum activity of 3b was reached at 5 nM, where the same degree of downregulation was maintained at higher concentrations up to 100 nM with no observable “hook effect” (Fig. 3D). 3b and 3c mediated downregulation of surface CD54 over 48 hours, where >50% removal was observed for 3b after 3 hours, which further increased to >80% removal after 24 hours (Fig. 3E). Downregulation mediated by 3c was markedly slower than that by 3b. Finally, because CD206 expression is dependent on the cytokine IL-4, it was reasoned that the TICTACs must also be dependent on IL-4. Two parallel experiments were set up where RAW264.7 or J774A.1 cells were either polarized with IL-4 or not prior to treatment with 3b. 3b-mediated downregulation of CD54 that was dependent on the presence of IL-4 was observed, demonstrating that TICTACs can be “turned on” with IL-4 (Fig. 3F). Example 4 - TICTACs as a TME-Specific Immune Checkpoint Blockade

[0197] Investigated next was whether TICTACs could provide an alternative form of immune checkpoint blockade that was specific to the tumor microenvironment. In a canonical checkpoint blockade, the antibody systemically blocks the immune checkpoint protein (ICP) and activates immune cells in healthy and cancerous tissues alike, leading to irAEs. It was hypothesized that by generating TICTACs from non-blocking antibodies that bind but do not inhibit the ICP, the ICP could be selectively removed from TAMs while mitigating any off-target effects (Fig. 4A). Targeted in this example was signal regulatory protein alpha (SIRPa), a known engager of the “don’t eat me” signal CD47 and highly expressed on myeloid-derived immune cells.27 31TICTACs 4b and 4c were first generated using a commercially available primary antibody against SIRPa following a similar scheme to 1 b and 1c. M2-polarized RAW264.7 and J774A.1 cells were subjected to 4b and 4c, after which surface SIRPa levels were quantified by flow cytometry using an orthogonal detection antibody. TICTAC treatment resulted in significant decreases in cellsurface SIRPa (>60%) for CD206high cells across both cell lines (Fig. 4B). 4b-mediated downregulation of SIRPa was concentration dependent, reaching a maximum at 100 nM (Fig. 4C). To begin to test the hypothesis regarding non-blocking TICTACs, determined first was whether TICTACs could withstand changes in the binding epitope of the target protein. Trisfucose TICTACs were generated from four different monoclonal antibodies against SIRPa and their activities in M2-polarized RAW264.7 cells were tested. TICTACs derived from all clones reduced surface SI RPa by >40% in CD206high cells (Fig. 4D). Determined next was whether each of the antibodies inhibited CD47 binding by assessing orthogonality with a CD47-Fc chimera by flow cytometry. Only clone P84 was fully orthogonal to CD47, whereas other clones such as 012 resulted in almost complete blockade of CD47 binding (Fig. 4E). Finally, RAW264.7 cells were subjected to P84-derived tris-Fuc TICTAC or the unconjugated P84 antibody, followed by measurement of levels of CD47 binding after treatment. TICTAC treatment resulted in a 50% decrease in CD47 binding relative to the unconjugated control, demonstrating that TICTACs can selectively activate non-blocking ICP antibodies in the presence of CD206.

[0198] Example 5 - TICTAC-Mediated Removal of Extracellular Targets

[0199] Having established the concept of non-blocking TICTACs for membrane targets, investigated next was whether this technology could be applied to the selective clearance of soluble targets within the tumor microenvironment. Cytokines are ubiquitous signaling molecules that regulate a large number of processes, often having differing or even contrasting effects on different cell-types or tissues. Envisioned was a strategy where TICTACs generated from nonblocking antibodies that do not disrupt binding of the cytokine to its receptor could result in tumorspecific clearance mediated by CD206 while having no inhibitory effect elsewhere (Fig. 5A). IL- 4, a driver for M2 polarization in TAMs and immunosuppression within the tumor microenvironment, was chosen as a target as proof-of-concept.32TICTACs 5b and 5c were generated through conjugation of tris-Fuc and tris-3-SO4Gal ligands with a known nonneutralizing anti-l L4 antibody (clone BVD6-24G2).33Polarized RAW264.7 cells were subjected to 488-labeled IL4 and unconjugated anti-l L4 antibody, 5b, or 5c. In CD206high populations, 5b and 5c induced >10-fold increase in intracellular 488 fluorescence relative to the unconjugated antibody, whereas the change in CD206iOw populations was less than 2-fold. (Fig. 5B). These results demonstrate that non-blocking TICTACs can also be employed for tumor-specific removal of soluble targets such as cytokines.

[0200] Example 6 - Anti-SIRPa TICTACs Degrade Immune Checkpoint Protein SIRPa Regardless of Their Ability to Block CD47

[0201] Assessed in this example was whether TICTACs could provide an alternative form of immune checkpoint blockade specific to M2-polarized macrophages which are known to drive immune suppression in the tumor microenvironment. In a canonical checkpoint blockade, the antibody drug systemically blocks the immune checkpoint protein (ICP) and activates immune cells in healthy and cancerous tissue alike, leading to irAEs. It was hypothesized that by generating TICTACs from non-blocking antibodies that bind but do not inhibit the ICP, the ICP could be selectively removed from TAMs while mitigating off-target effects (Fig. 6A). This example involved targeting signal regulatory protein alpha (SIRPo), which is highly expressed on myeloid-derived immune cells and potently engages the “don’t eat me” ligand CD47. Monoclonal antibodies against both SIRPo and CD47 are currently being evaluated as cancer immune therapies. Blocking antibody clone 119 and non-blocking antibody clones 136 and 3 were recently developed as pan-allelic, high affinity SIRPa binders (Fig. 6B) (Sim, J. et al. Discovery of high affinity, pan-allelic, and pan-mammalian reactive antibodies against the myeloid checkpoint receptor SIRPa. mAbs l l , 1036-1052 (2019)).

[0202] LALAPG-119, LALAPG-136, and LALAPG-3 were generated using the corresponding Fab fragments and human lgG1 containing Fc-silencing mutations L234A / L235A / P329G (‘LALAPG’) to mitigate competing Fc receptor (FcR) binding (Schlothauer, T. et al. Novel human lgG1 and lgG4 Fc-engineered antibodies with completely abolished immune effector functions. Protein Eng. Des. Sei. 29, 457-466 (2016)). TICTACs were assembled following a similar scheme to 1a and 1 b, generating tris-Fuc and tris-3-SO4-Gal functionalized LALAPG-119 (5a and 5b respectively), tris-Fuc functionalized LALAPG-136 (6a), and tris-Fuc functionalized LALAPG- 3 (7a). M2-polarized primary human macrophages were subjected to 25 nM unconjugated LALAPG-119, 5a, or 5b for 24 h, and total SIRPa levels were quantified by Western blot analysis (Fig. 6C). While the unconjugated antibody had no effect on SIRPa levels, TICTAC treatment resulted in almost complete (>90%) degradation of SIRPa across three human donors. Assessed next was whether TICTACs that bind different epitopes of the target protein could also induce selective degradation. M2-polarized macrophages were treated with non-blocking LALAPG-136, 3, or the corresponding tris-Fuc TICTACs 6a and 7a. Upon assessing SIRPa levels by Western blot, non-blocking TICTACs were equally as effective as their blocking counterpart, again resulting in >90% degradation across three independent donors (Fig. 6D). These results demonstrate that TICTACs enable decoupling of receptor binding from the inhibitory effect (via degradation), giving rise to highly TAM-specific activity. Further, high levels of SIRPa degradation (>70% relative to no treatment control) was observed when M2-polarized primary murine BMDMs were treated with 6a (25 nM for 24 h), indicating excellent cross-reactivity in mice.

[0203] Quantitative mass spectrometry-based proteomics was also used to measure proteome- wide changes in human macrophages (across 3 independent donors) treated with LALAPG-119 or 5a (Fig. 6E). While no significant changes were observed upon treatment with LALAPG-1 19, 5a promoted a marked reduction in SIRPa levels. Moreover, TICTACs exert high target precision: downmodulation was highly localized to SIRPa across the entire proteome.

[0204] Determined next was the selectivity of anti-SIRPa TICTAC activity for M2-polarized macrophages over non-polarized macrophages and other immune cells. M2- (CD206hi) and M0- polarized (CD2O610) macrophages were generated from the same donor and treated with LALAPG-119 or 5a. While M2-macrophages again exhibited substantial TICTAC-induced degradation (90% degradation relative to no treatment), this effect was greatly attenuated in M0- macrophages (30% degradation relative to no treatment) which expressed basal levels of CD206. Signal regulatory protein gamma (SIRPy) is another member of the SIRP family that is uniquely expressed by human T-cells. While its function is less well-characterized, its engagement with CD47 on antigen presenting cells (APCs) enhances antigen-specific T-cell proliferation and facilitates endothelial migration of T-cells in vitro. Because many antibodies targeting SIRPa are cross-reactive with SIRPy, assessed was whether TICTACs exhibited any off-target degradation of SIRPy expressed in human T-cells. PBMCs isolated from three independent donors were subjected to LALAPG-1 19, LALAPG-136, or the corresponding TICTACs 5a and 6a. After 24 h, the cells were analyzed by flow cytometry, where T-cells were identified as the CD3+population. Upon quantifying cell-surface SIRPy levels, it was found that TICTACs induced no appreciable downmodulation of SIRPy relative to the unconjugated antibody. These data indicate TICTAC- mediated degradation is highly specific to M2-polarized macrophages, preserving target protein expression on non-polarized macrophages and other immune cells.

[0205] Finally, assessed was whether TICTACs could have a functional anti-tumor effect on M2- polarized macrophage activity. Antibody-dependent cellular phagocytosis (ADCP) and antibodydependent cellular cytotoxicity (ADCC) are tumoricidal processes resulting from co-engagement of FcRs on immune cells and antigens on the cancer cell surface. Because inhibitory checkpoint receptors such as SIRPa transmit “don’t eat me” signals that potently block ADCP, it was hypothesized that TICTAC-mediated degradation of SIRPa would enhance phagocytosis in the presence of therapeutic tumor-targeting antibodies. To test this, M2-polarized human macrophages were first treated with LALAPG-136 or 6a for 48 h, then co-cultured with Raji cells that were labeled with pHrodo red, a pH-sensitive dye that fluoresces red in acidic phagosomes (Fig. 6F). Fluorescence microscopy images were acquired at 1 h intervals, and phagocytosis was quantified as total integrated red fluorescence normalized by cellular confluence in each well (Fig. 6H, G). Maximum phagocytosis was observed after 4 h of co-culture in the presence of rituximab, a CD20-targeting therapeutic antibody. TICTAC-treated macrophages were significantly more phagocytic than either the vehicle or unconjugated LALAPG-136 control, which was consistent across macrophages from 5 donors (Fig. 6I). Taken together, these results show that TICTACs can have a functional effect on antibody effector functions such as ADCP in M2- polarized macrophages.

[0206] Methods

[0207] General Synthetic Chemistry Procedures

[0208] Unless otherwise noted, all reactions were carried out in flame-dried glassware sealed with rubber septa under a nitrogen atmosphere with Teflon-coated magnetic stir bars. Reaction progress was monitored using thin layer chromatography on Millipore Sigma glass-backed TLC plates (250 pm thickness, F-254 indicator) and visualized with 254 nm UV light or stained by submersion in a basic potassium permanganate solution or 5% H2SO4 in methanol solution. Flash column chromatography was performed on a Biotage instrument using pre-packed silica gel columns. Reagents were purchased in reagent grade from commercial suppliers and used as received, unless otherwise described. Anhydrous solvents (dichloromethane, tetrahydrofuran, methanol, and acetonitrile) were purchased as sure-sealed bottles and used as received, unless otherwise described. See Supplemental Methods for detailed synthetic procedures and characterization of all new compounds.

[0209] Amino-PEG4-tris-a-L-fucose (S1)

[0210] To a 1 -dram vial equipped with a magnetic stir bar was added amino-PEG4-tris-alkyne (10 mg, 0.02 mmol, 1 equiv.) and 2-azidoethyl a-L-fucopyranoside (23.3 mg, 0.14 mmol, 5 equiv.) as a solution in 0.5 mL of degassed 1 :1 THF / ddH2O. In a separate one-dram vial, CuSO4(5 mg, 0.02 mmol, 1 equiv.), BTTP (17.22 mg, 0.04 mmol, 2 equiv.), and sodium ascorbate (1 1.9 mg, 0.06 mmol, 3 equiv.) were combined in 0.2 mL of ddH2O, then added to the reaction mixture. The solution was heated to 40 °C and stirred for 16 h. The mixture was purified by preparative HPLC using a 0-30% acetonitrile gradient over 1 1 min, and the product was isolated as a white solid after lyophilization (15.5 mg, 65% yield).

[0211] 1H NMR (500 MHz, MeOD) 5 8.1 1 (s, 3H), 4.76 (d, J = 3.8 Hz, 3H), 4.71 - 4.60 (m, 5H), 4.58 (s, 8H), 4.06 (ddd, J = 1 1 .2, 7.9, 3.6 Hz, 3H), 3.83 (ddd, J = 10.9, 5.6, 3.6 Hz, 3H), 3.78 - 3.61 (m, 22H), 3.59 (s, 4H), 3.58 - 3.55 (m, 3H), 3.41 (q, J = 6.8 Hz, 4H), 3.14 (t, J = 5.1 Hz, 2H), 2.48 (t, J = 6.0 Hz, 2H), 1 .10 (d, J = 6.6 Hz, 9H). Found [M+H+]: 1 183. Tris-fucose-647 (2)

[0212] To a 1 -dram vial equipped with a magnetic stir bar was added S1 (3.62 mg, 0.0031 mmol, 1 equiv.), APDye Fluor 647 acid (3 mg, 0.0032 mmol, 1 equiv.), and 1 -hydroxy-7- azabenzotriazole (0.52 mg, 0.0038 mmol, 1 .2 equiv.) as a solution in 0.2 mL DMF. To this mixture was added 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.73 mg, 0.0038 mmol, 1 .2 equiv.) and diisopropylethylamine (1 .33 piL, 0.0077 mmol, 2.5 equiv.). The mixture was stirred for 24 h at room temperature. The crude mixture was purified by prep-HPLC using 5-95% acetonitrile gradient over 11 min, and the product was isolated as a bright blue solid after lyophilization.

[0213] TICTAC antibody conjugation

[0214] General procedure for antibody linker labeling

[0215] A 1 mg / mL solution of antibody was buffer exchanged into PBS using a 7 KDa Zeba sizeexclusion column. The antibody was reacted with 50 equiv. of NHS-linker (10 mM stock solution in DMSO), and the reaction was incubated overnight at room temperature. The resulting mixture was filtered using a 7 KDa Zeba size-exclusion column to yield the conjugate.

[0216] General procedure for Cu-catalyzed click reaction

[0217] To a 1 mg / mL solution of the antibody-linker conjugate was added 75 equiv. of the carbohydrate ligand (10 mM stock solution in H2O). BTTP-Cu(ll) pre-catalyst was generated by mixing CuSO4 (aqueous solution) and BTTP (DMSO solution) in a 1 :4 molar ratio. 150 pM BTTP- Cu and 3 mM sodium ascorbate (100 mM solution in H2O, prepared immediately prior to addition) were added, and the reaction was briefly vortexed. The reaction mixture was allowed to incubate at room temperature for 1 h and was filtered using a 7 KDa or 40 KDa Zeba size-exclusion column.

[0218] Cell Culture

[0219] All cell lines were purchased from the American Type Culture Collection (ATCC) unless otherwise noted. RAW26.7, J774A.1 , BV-2, and U937 were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 10011 / mL Penicillin, and 100 pg / mL Streptomycin. THP-1 cells were cultured in RPMI supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100U / mL Penicillin, and 100 pg / mL Streptomycin. The CB7BL / 6-derived immortalized bone marrow macrophage cell line BMA3.1 A7 was a generous gift from Dr. Kenneth Rock (University of Massachusetts Medical School), and the cells were maintained as previously described. Cells were cultured in T-75 flasks (Fisher Scientific) and incubated at 37 °C and 5% CO2and tested negative for mycoplasma quarterly using a PCR-based assay.

[0220] Gene Knockout Pool

[0221] CRISPR-Cas9 mediated knockout cell pool of CD206 in RAW264.7 cells were generated by Synthego Corporation (Redwood City, CA). Cells were electroporated with Sp-Cas9 and sgRNAs targeting CD206 using Synthego’s optimized protocol. 48 h post-electroporation, genomic DNA was extracted, PCR amplified, and sequenced using Sanger sequencing to verify editing efficacy. Resulting chromatograms were analyzed using Synthego Inference of CRISPR edits software (ice.synthego.com). Mock control cells were generated by electroporating Sp-Cas9 without sgRNA. Cells were cultured in DMEM supplemented with 10% heat- inactivated fetal bovine serum (FBS), 100U / mL Penicillin, and 100 pg / mL Streptomycin.

[0222] Surface plasmon resonance

[0223] Binding affinity of tris-fucose small molecule was determined by the Biacore 3000 instrument (Cytiva).

[0224] Soluble target uptake assay via flow cytometry

[0225] For uptake experiments, cells were plated (60,000 cells / well in 24-well plates) two days before treatment in complete media supplemented with 25 ng / mL murine IL-4 (Peprotech) for M2-polarization or without any cytokines (M0). Cells were incubated with 250 pL of M2- or M0- media with fluorescent proteins and TICTACs / controls or conjugated / unconjugated dyes for the indicated times (<8 h). For overnight uptake experiments, cells were plated / polarized one day before treatment. After treatment, cells were washed with PBS, then treated with 200 pL of enzyme-free Cell Dissociation Buffer (Gibco) for 10 minutes to dissociate the cells. The resuspended cells were transferred to a pre-chilled 96-well v-bottom plate (Corning) and washed with cold blocking buffer (PBS supplemented with 0.5% BSA and 5 mM EDTA) two times, pelleting by centrifugation at 500g for 3 min at 4 °C between washes. For experiments requiring co-staining for CD206 levels, Fc-receptors were first neutralized by incubating cells on ice for 5- 10 minutes with TruStain FcX (BioLegend) at 1 .0 pg / 1 x 106cells in 100 pL of blocking buffer. The cells were then stained with the fluorescently labeled anti-CD206 antibody in 100 pL of blocking buffer for 30 minutes at 4 °C. Cells were washed two times with blocking buffer, then incubated with Sytox Blue according to the manufacturer’s specifications for 10 min at 4 °C. Flow cytometry was performed on either a MACSQuant Analyzer 10 (Miltenyi Biotec), Novocyte Quanteon (Agilent), or Novocyte Penteon (Agilent), and FlowJo software was used to gate on single cells, live cells, and CD206high / iow cells for analysis.

[0226] Cell-surface protein internalization assay via flow cytometry

[0227] Cells were plated (60,000 cells / well in 24-well plates) one day before treatment in M2- or MO-media. Cells were incubated with 250 pL of M2- or MO-media with 25 nM TICTACs / controls for the indicated amount of time, washed with PBS, and dissociated with Cell Dissociation Buffer for 5-10 minutes. Resuspended cells were transferred to a 96-well v-bottom plate and washed with cold blocking buffer, Fc-neutralized, and incubated with primary antibodies for 30 min at 4 °C. Cells were washed two times with blocking buffer, then incubated with Sytox Blue for 10 min before flow cytometry analysis. Isolation and differentiation of donor macrophages

[0228] LRS chambers were obtained from healthy anonymous blood bank donors. PBMCs were isolated using Ficoll-Paque (GE Healthcare Life Sciences) density gradient separation. Monocytes were isolated by plating ~1 x 108PBMCs in T-25 flasks with serum-free RPMI and incubating for 1 h, followed by 3x rigorous washes with DPBS (PBS +Ca +Mg) to remove nonadherent cells and ensuring that the final wash results in clear supernatant. The media was replaced with IMDM supplemented with 10% Human AB Serum (Gemini), and the cells were allowed to differentiate into macrophages over 7-9 days. To generate M2 macrophages, the media was replaced on day 4 with IMDM supplemented with 10% Human AB Serum, 20 ng / mL IL-4 (Peprotech), 50 ng / mL IL-10, and 50 ng / mL TGF-p. No media changes were conducted for M0 macrophages throughout the differentiation period.

[0229] Western blot analysis

[0230] Cells were plated (60,000 cells / well in 24-well plates) one day before treatment in M2- or MO-media. Cells were incubated with 250 pL of M2- or MO-media with 25 nM TICTACs / controls for the indicated amount of time, washed with 3x with PBS, and lysed with RIPA buffer supplemented with protease inhibitor cocktail (Roche), 0.1% benzonase (Millipore-Sigma), and phosphatase inhibitor cocktail (Cell Signaling Technologies) on ice for 30 min. The cells were scraped and transferred to 1 .5 mL Eppendorf tubes and centrifuged at 21 ,000g for 15 min at 4 °C. The supernatant was collected, and the concentration was determined using a BSA assay (Pierce). Equal amounts of protein were loaded onto a 4-12% Bis-Tris gel (BioRad) and separated by SDS-PAGE. The gel was transferred to a nitrocellulose membrane, stained with REVERT Total Protein Stain (LI-COR) and blocked with Odyssey Blocking Buffer (PBS) (LI-COR) for 1 h at room temperature. The membrane was then incubated with primary antibody in Blocking Buffer overnight at 4 °C, then washed three times with PBS with 0.1 % Tween-20 (PBS-T). The membrane was incubated with secondary antibody for 1 h at room temperature, washed three times with PBST, and visualized with an Odyssey CLx imager (LI-COR). Image Studio (LI-COR) was used to analyze the image and quantify band intensities.

[0231] Confocal microscopy

[0232] Membrane protein analysis

[0233] Cells were plated (20,000 cells per well in an 8-chamber Labtek plate) 3 days prior to treatment. Cells were incubated with 250 pL of complete growth medium with 25 nM TICTACs or control for the indicated amount of time. Cells were then washed 3x with DPBS (300 pL / wash) and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Cells were washed 2x with PBS and incubated with primary antibody in 300 pL blocking buffer (PBS + 1% BSA) for 1 h at room temperature. After washing with PBS, cells were incubated with Hoescht stain in PBS for 10 min at room temperature. The wells were aspirated, and the chambers were removed according to the manufacturer’s specifications. 15 pL of mounting medium (Vector Laboratories) was added followed by a coverslip. The slides were cured for 30 min at room temperature and imaged with Nikon A1 R confocal microscope using a Plan Fluor x60, 1 ,30-NA oil objective. The following laser settings were used: 405 nm violet laser, 488 nm blue laser, 561 nm green laser, and 639 nm red laser.

[0234] Matrix-assisted laser desorption / ionization-mass spectrometry

[0235] A 1 mg / mL solution of the antibody conjugate was buffer exchanged into ddH2O using a 7 KDa Zeba size-exclusion column to remove excess salts. Samples were prepared by mixing 2 pL of sinapinic acid (SPA) matrix (10 mg / mL in 0.1 % trifluoroacetic acid and 50% acetonitrile) and 2 pL of the antibody sample. The mixture was vortexed, and 1 pL was loaded onto a MALDI stainless steel plate. The sample was dried at room temperature for 15 min, and the MALDI-MS was acquired by AB SCIEX TOF / TOF and a 5800 CovalX High Mass Detector with a mass range of 10,000 - 250,000 Da and a fixed laser intensity of 5,900. Greater than 3 scans were taken per sample, and the spectra was analyzed and averaged using MALDIquant.

[0236] Phagocytosis assays

[0237] On Day 7, M2-polarized human macrophages were washed with PBS and lifted by incubating with 8 mL TrypLE (Thermo Fisher Scientific) for 30 min at 37 °C. The macrophages were pelleted by centrifugation at 400 x c? for 4 min and resuspended in phenol-red free IMDM supplemented with 10% Human AB serum and M2 cytokine cocktail (20 ng / mL IL-4, 50 ng / mL IL-10, and 50 ng / mL TGF-p). 10,000 macrophages were plated in 100 pL of media in a 96-well flat-bottom plate (Corning) and incubated in a humidified incubator for 1 h at 37 °C. TICTACs or unconjugated antibody were added at 25 nM (in 5 pL volume), and the cells were incubated for 48 h at 37 °C. The wells were aspirated, then replaced with 100 pL IncuCyte medium (phenol- red free RPMI + 10% HI FBS). Raji cells were washed 1x with PBS, then incubated with 1 :80,000 diluted pHrodo red succinimidyl ester dye (Thermo Fisher Scientific) in PBS at 37 °C for 30 min on a shaker, washed with 1 x PBS, and resuspended in IncuCyte medium. The labeled Raji cells were added to the macrophages (20,000 cells, 90 pL), followed by 10 pL of 20x rituximab stocks (final concentration of 5 pg / mL). Cells were plated by gentle centrifugation (50 x g, 1 min) then placed in an IncuCyte S3 Live-Cell Analysis System (Sartorius). Two images per well were acquired at 1 h intervals until maximum signal was reached (at least 4 h). Images were analyzed using a threshold of 1 .1 , an edge sensitivity of -45, and areas between 100 and 2000 pm2. The total red object integrated intensity (RCU x pm2 / image) was quantified for each well and normalized by dividing by the total confluence (%). This normalized total integrated intensity value is reported for each treatment condition. Acquisition parameters for IncuCyte images

[0238] IncuCyte S3 Live-Cell Analysis System (Sartorius) was placed within a tissue culture incubator (Thermo Fisher Scientific) maintained at 37 °C with 5% CO2. Images were acquired from a 10x objective lens in phase contrast and from a red fluorescence channel (ex. 585 ± 20, em: 665 ± 40, acquisition time: 400 ms). Unless otherwise specified, cells were analyzed by Top- Hat segmentation with 100 pm radius, edge split on, hole fill: 0 pm2.

[0239] Proteomics sample preparation

[0240] Macrophage samples cultured on 24-well plates were washed three times with cold (4SC) PBS (pH 7.4) to remove cell culture media. Following PBS washes, the macrophage samples were lysed in RIPA buffer containing 25 mM Tris-HCI (pH 7.6), 150 mM NaCI, 1% (m / V) NP-40, 1% (m / V) sodium deoxycholate, 0.1 % (m / V) and sodium dodecyl sulfate (SDS), and 1 x Halt protease inhibitor cocktail (78430, Thermo Fisher Scientific). The cell lysates were then processed using a suspension trapping (S-Trap) microcolumn (Protifi, C02-micro-80, < 100 pg) following vendor protocols as described by Protifi (Fairport, NY, USA) with slight modifications. Briefly, protein samples were first reduced using 20 mM tris(2-carboxyethyl)phosphine (TCEP) at 37°C for 15 min and then alkylated using 40 mM 2-chloroacetamide for 20 min at room temperature. The denatured, non-digested proteins were then acidified using phosphoric acid (85 % w / v) to a final phosphoric acid concentration of 2.5 % w / v before being loaded on the S-trap microcolumn. The bound proteins were washed by repeated centrifugation using 100 mM triethylammonium bicarbonate (TEAB) in 90% methanol to remove MS incompatible buffer salts and detergents. The protein samples were then digested on the S-trap using 50 pL of a 1 :20 enzyme to substrate mass ratio of Trypsin (Promega, V5280) at 37 °C overnight for 16 h. The resulting peptides were then eluted from the S-trap using 40 pL of 50 mM TEAB, followed by 40 pL of 0.2% formic acid (FA), and finally with 40 pL of 50% acetonitrile (ACN). The pooled elution mixtures were then dried using a Labconco CentriVap vacuum concentrator (Kansas City, MO, USA). Dried peptides were resuspended in 12 pL of 0.2% FA, 2% ACN and peptide concentrations were measured by absorbance at 205 nm using a Nanodrop (Thermo Fisher Scientific) prior to LC / MS analysis.

[0241] Statistical analyses

[0242] Statistical analysis was performed in GraphPad Prism (version 9). Two-tailed tests with Welch’s correction were used for all t-tests. For SPR data, association then dissociation models were used to calculate the dissociation constant. For EC5o data, one-site total and nonspecific binding models were used to determine the apparent dissociation constant for specific binding. Statistically significant was defined as p<0.05, and the asterisk * indicates a p<0.05, ** indicates p<0.01 , *** indicates p<0.001 , and **** indicates p<0.0001 . References

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[0276] Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

WHAT IS CLAIMED IS:1 . A bifunctional molecule comprising: a first moiety comprising a CD206 ligand that binds CD206 on the surface of a cell and induces CD206-mediated endocytosis; and a second moiety stably associated with the first moiety, wherein the second moiety specifically binds a molecule on the surface of the cell or an extracellular molecule.

2. The bifunctional molecule of claim 1 , wherein the CD206 ligand comprises a carbohydrate.

3. The bifunctional molecule of claim 2, wherein the CD206 ligand comprises mannose, fucose, a sulfated glycan, or any combination thereof.

4. The bifunctional molecule of claim 3, wherein the CD206 ligand comprises a monosaccharide, disaccharide, or trisaccharide comprising mannose, fucose, a sulfated glycan, or any combination thereof.

5. The bifunctional molecule of claim 3 or 4, wherein the sulfated glycan is 3-SO4galactose or 4-SO4galactose.

6. The bifunctional molecule of any one of claims 2-5, wherein the CD206 ligand is trisfucose.

7. The bifunctional molecule of any one of claims 2-5, wherein the CD206 ligand is tris-3- SO4galactose.

8. The bifunctional molecule of claim 2, wherein the CD206 ligand comprises N-acetyl glucosamine (GIcNAc), 4-SO4-N-Acetyl galactosamine (4-SO4-GalNAc), 3-SO4-N-Acetyl galactosamine (3-SO4-GalNAc), 2a-mannobiose, 3,6-di-O-(a-D-mannopyranosyl)-a-D- mannopyranoside, 3-fucosyllactose, or any combination thereof.

9. The bifunctional molecule of claim 1 , wherein the CD206 ligand comprises a protein.

10. The bifunctional molecule of claim 9, wherein the CD206 ligand is an anti-CD206 antibody.11 . The bifunctional molecule of claim 1 , wherein the CD206 ligand comprises an aptamer.

12. The bifunctional molecule of any one of claims 9-11 , wherein the CD206 ligand binds to the N-terminal R-type carbohydrate-recognition domain (CRD) of CD206.

13. The bifunctional molecule of any one of claims 9-11 , wherein the CD206 ligand binds to C-type CRD4, C-type CRD5, or both, of CD206.

14. The bifunctional molecule of any one of claims 1 -13, wherein the second moiety is a small molecule, an antibody, or an aptamer.

15. The bifunctional molecule of claim 14, wherein the second moiety is an antibody.

16. The bifunctional molecule of any one of claims 1 -15, wherein the cell is a tumor- associated macrophage (TAM), a dendritic cell, or a sinusoidal endothelial cell.

17. The bifunctional molecule of claim 16, wherein the cell is a CD206h'9htumor-associated macrophage (TAM).

18. The bifunctional molecule of claim 17, wherein the second moiety specifically binds an immune checkpoint molecule.

19. The bifunctional molecule of claim 18, wherein the immune checkpoint molecule is SIRPa, PD-L1 , PD-L2, CD80, CD86, B7-H4, VISTA, PD-1 , Siglec 10, CSF-1 R, IL-4R / IL-13R, MARCO, or LILRB-1.

20. The bifunctional molecule of claim 19, wherein the immune checkpoint molecule is SIRPa.21 . The bifunctional molecule of any one of claims 18-20, wherein binding of the second moiety to the immune checkpoint molecule does not block activity of the immune checkpoint molecule.

22. The bifunctional molecule of any one of claims 1 -17, wherein the second moiety specifically binds an extracellular molecule.

23. The bifunctional molecule of claim 22, wherein the extracellular molecule is a cytokine.

24. The bifunctional molecule of claim 22 or 23, wherein binding of the second moiety to the extracellular molecule does not block activity of the extracellular molecule.

25. The bifunctional molecule of any one of claims 1 -24, wherein the second moiety is stably associated with the first moiety via conjugation.

26. The bifunctional molecule of claim 25, wherein the first moiety and second moiety are conjugated via a linker.

27. The bifunctional molecule of claim 26, wherein the linker comprises polyethylene glycol (PEG), alkyl, or peptide spacer groups in a linear or branched orientation.

28. The bifunctional molecule of claim 26, wherein the linker comprises:

29. The bifunctional molecule of any one of claims 1 -21 , wherein the second moiety is stably associated with the first moiety via fusion of a protein domain of the second moiety with a protein domain of the first moiety.

30. A composition comprising the bifunctional molecule of any one of claims 1-29.31 . The composition of claim 30, wherein the composition is formulated for administration to a subject in need thereof.

32. The composition of claim 31 , wherein the composition is formulated for parenteral administration to a subject in need thereof.

33. The composition of claim 32, wherein the composition is formulated for intravenous administration to a subject in need thereof.

34. A method of removing a molecule from the surface of CD206-expressing cells or an extracellular molecule in a subject in need thereof, the method comprising administering the bifunctional molecule of any one of claims 1 -29 to the subject in an amount effective to remove the molecule from the surface of the CD206-expressing cells or from the extracellular space of the CD206-expressing cells.

35. The method of claim 34, wherein the CD206-expressing cells are macrophages, dendritic cells, or sinusoidal endothelial cells.

36. The method of claim 34, wherein the subject has cancer characterized by a solid tumor, and wherein the CD206-expressing cells are CD206hi9htumor-associated macrophages (TAMs).

37. The method of claim 36, wherein the second moiety specifically binds an immune checkpoint molecule.

38. The method of claim 37, wherein the immune checkpoint molecule is SIRPa, PD-L1 , PD-L2, CD80, CD86, B7-H4, VISTA, PD-1 , Siglec 10, CSF-1 R, IL-4R / IL-13R, MARCO, or LILRB-1.

39. The method of claim 38, wherein the immune checkpoint molecule is SIRPa.

40. The method of any one of claims 37-39, wherein binding of the second moiety to the immune checkpoint molecule does not block the activity of the immune checkpoint molecule.41 . The method of claim 40, wherein the second moiety specifically binds SIRPa and does not block binding of SIRPa to CD47.

42. The method of any one of claims 36-41 , wherein the solid tumor is a carcinoma, sarcoma, blastoma, or lymphoma.

43. A bifunctional molecule comprising: a first moiety comprising a CD206 ligand that binds CD206 on the surface of a cell and induces CD206-mediated endocytosis; and a cargo moiety stably associated with the first moiety.

44. The bifunctional molecule of claim 43, wherein the first moiety is as defined in any one of claims 2-1345. The bifunctional molecule of claim 43 or 44, wherein the cargo moiety is a cytotoxic agent.

46. The bifunctional of claim 45, wherein the cytotoxic agent is a calicheamicin, a duocarmycin, a pyrrolobenzodiazepines (PBDs), a camptothecins, a daunorubicin, a doxorubicin, an auristatin, a maytansinoid, or a radioisotope.

47. The bifunctional of claim 46, wherein the auristatin is MMAF or MMAE.

48. The bifunctional of any one of claims 43-47, wherein the cargo moiety comprises a nucleic acid.

49. The bifunctional of claim 48, wherein the nucleic acid is an RNAi construct, an antisense oligonucleotide (ASO), or an mRNA.

50. The bifunctional of claim 49, wherein the RNAi construct is an siRNA construct.51 . The bifunctional of any one of claims 43-50, wherein the cargo moiety comprises a protein.

52. The bifunctional of claim 51 , wherein the protein is an enzyme.

53. The bifunctional of claim 51 , wherein the cargo moiety comprises a binding protein.

54. The bifunctional of claim 53, wherein the binding protein is an antibody.

55. The bifunctional of claim 53, wherein the binding protein is a transcription factor.

56. A method of delivering a cargo moiety into CD206-expressing cells of a subject in need thereof, the method comprising contacting the CD206-expressing cells with the bifunctional molecule of any one of claims 43-55.

57. The method of claim 56, wherein the CD206-expressing cells are macrophages, dendritic cells, or sinusoidal endothelial cells.

58. The method of claim 56, wherein the subject has cancer characterized by a solid tumor, and wherein the CD206-expressing cells are CD206h'9htumor-associated macrophages (TAMs).

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