CD206-targeted drug delivery vehicles carrying novel bisphosphonate drug payloads via degradable linkers

Abstract

Provided are novel compounds, as well as pharmaceutical compositions, methods of synthesis, and methods of use, that include a polymeric carbohydrate backbone, one or more mannose-binding C-type lectin receptor targeting moieties, and a nitrogen-containing bisphosphonate compound attached to the backbone via thiol-maleimide conjugation. The thiol-maleimide conjugation of the bisphosphonate to the polymeric carbohydrate backbone provides methods of use of the compounds and compositions thereof to release a therapeutic payload upon internalization into mannose-binding C-type lectin receptor expressing cells, such as tumor-associated macrophages (TAMs), for the treatment of various diseases, such as cancer, autoimmune diseases, and inflammatory diseases.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 344,134, filed May 20, 2022, entitled "CD206 Targeted Drug Delivery Vehicles Carrying Novel Bisphosphonate Drug Payloads Via a Degradable Linker," and U.S. Provisional Application No. 63 / 352,324, filed June 15, 2022, entitled "CD206 Targeted Drug Delivery Vehicles Carrying Novel Bisphosphonate Drug Payloads Via a Degradable Linker," which are hereby incorporated by reference in their entireties for all purposes.

[0002] The present disclosure relates to therapeutic constructs, methods of preparation and use thereof, which release a therapeutic payload upon internalization into mannose-binding C-type lectin receptor expressing cells, such as tumor-associated macrophages (TAMs), and shift the phenotype towards a pro-inflammatory state. [Background technology]

[0003] Cancer is the second leading cause of death in the United States, with nearly one in four deaths due to cancer. Cancer is characterized by unregulated proliferation and cell division of cancer cells. However, cancer benefits greatly from a chronic maladaptive immune response against the tumor, and macrophages are key mediators of that maladaptive response. Macrophages are a common cell type in all tissues of the body and are a key component of innate immunity. Macrophages also contribute significantly to the maintenance of tissue homeostasis and wound repair. In general, macrophages respond to various stimuli in their local microenvironment by altering the expression patterns of numerous, potentially hundreds of, genes. Macrophages with such altered phenotypes are called activated macrophages. Depending on which stimuli the macrophages respond to, a wide range of activated phenotypic states can be achieved. Among the genes that are differentially expressed upon macrophage activation are cell surface markers (e.g., macrophage mannose receptor, CD206, etc.) and various cytokines, enzymatic pathways leading to the generation of reactive oxygen species (ROS), and other signaling molecules that can regulate the behavior of other components of the immune system, such as T lymphocytes (T cells).

[0004] The extensive literature on macrophage activation reports experimental findings that indicate that a vast number of activation phenotypes exist and that the expression of one or a few genes alone cannot accurately identify any one particular phenotype. However, these activation phenotypes can be characterized for their overall immune status and placed on a continuum, with a highly pro-inflammatory phenotype at one end and an immunosuppressive and wound healing phenotype at the other end of the continuum. Traditionally, as noted in the historical macrophage phenotype literature, activated macrophages have been classified into two phenotypes: (1) a classical activation type called M1, which is highly pro-inflammatory, and (2) an alternative activation type called M2, which is immunosuppressive and promotes wound healing. It is now understood that a strict dichotomous classification of activated macrophage phenotypes is too simplistic and does not represent the true plasticity of macrophage responses to cues from the microenvironment; however, the concept that activated macrophages can influence local immune responses by being either pro-inflammatory (M1-like) or immunosuppressive (M2-like) remains useful in explaining the role of macrophages in various pathological conditions.

[0005] Tumor-associated macrophages (TAMs) are abundant in tumors and are critical contributors to maladaptive immune responses associated with cancer and other conditions. TAMs are the most numerous immune cells that infiltrate tumors and can comprise approximately 5%->30% of all cells within a tumor. Although both M1-like and M2-like TAMs are known, the majority of TAMs present in or near established tumors are immunosuppressive, i.e., M2-like activated macrophages.

[0006] Bisphosphonate drugs have been administered to cancer patients with bone metastases for many years. In healthy individuals, the structural components of bone are constantly turned over in a process of continuous regeneration, and this bone turnover mainly involves two cell types: (1) osteoclasts, which break down bone, and (2) osteoblasts, which continuously form new bone. Because osteoclasts are a type of macrophage, treatment with bisphosphonates may reduce the number of TAMs in bone metastases and, more importantly, change the phenotype of the remaining TAMs to be more M1-like. There is also some evidence that treatment with bisphosphonate drugs can slow the growth of bone metastases, which is hypothesized to be by depriving the tumor of the tumor-promoting effects and support provided by M2-like TAMs.

[0007] However, there are several challenges to using bisphosphonates as cancer immunotherapy targeting TAMs: (1) inefficient cell penetration due to the inability of these molecules to diffuse across cellular lipid membranes due to the highly charged properties of bisphosphonates; (2) rapid localization to bone; and (3) off-target toxicity. Historically, to overcome these limitations, liposomal constructs carrying various bisphosphonates were used to decrease bone localization and increase delivery of payload to TAMs. Several studies in animal models of cancer utilized liposomal constructs carrying a payload of the bisphosphonate drug, clodronate. Clodronate is a non-nitrogen-containing bisphosphonate drug. Clodronate induces apoptosis of macrophages (including TAMs and osteoclasts), but has a lesser ability to alter macrophage phenotype compared to nitrogen-containing bisphosphonates such as zoledronic acid. Although clodronate-containing liposomes reduced the number of TAMs, their potential for tumor therapy was limited.

[0008] Recent studies have evaluated liposomal constructs carrying a payload of zoledronic acid. Some of these studies evaluated targeted delivery of zoledronic acid to TAMs. At least two of these studies attempted targeted delivery of zoledronic acid to TAMs. In one study, liposomes with a zoledronic acid payload were modified to present sialic acid at their exosite. This modification targeted the liposomes to Siglec (sialic acid-binding immunoglobulin-type lectin) receptors on TAMs and other Siglec-expressing cells. This construct inhibited tumor growth in a mouse model. In another study, calcium / zoledronic acid nanoparticles were encapsulated in a modified lipid membrane presenting mannose and biotin. The mannose enabled binding to CD206 on TAMs, and the biotin enabled binding to the biotin receptor on cancer cells. This dual-targeting construct reduced tumor growth in an A549 mouse model of lung cancer, but because this construct is capable of killing both cancer cells and TAMs, it remains unclear whether the observed reduction in tumor growth is due to an anti-tumor cell effect or a TAM-directed effect.

[0009] A further problem with using liposomes or related constructs (TAM targeted or not) to avoid bone localization and deliver bisphosphonates to TAMs is that all liposomal constructs are relatively large compared to the mannosylated amine dextran (MAD) constructs described in this disclosure, which pose challenges with regard to tumor penetration and localization to TAMs.

[0010] Thus, there remains a need for compositions and methods that induce a phenotypic change from M2-like TAMs to M1-like TAMs to treat cancer with greater efficacy and less toxicity. Other objects, advantages and features of the present disclosure will become apparent from the following specification taken in conjunction with the accompanying drawings. Summary of the Invention

[0011] Provided herein are compounds and compositions comprising a carbohydrate polymer backbone and a bisphosphonate compound attached thereto, as well as methods for preparing such compounds and methods of using such compounds for the treatment of disease.

[0012] In Example 1, the compound comprises a polymeric carbohydrate backbone, one or more mannose-binding C-type lectin receptor targeting moieties, and a nitrogen-containing bisphosphonate compound attached to the polymeric carbohydrate backbone via thiol-maleimide conjugation.

[0013] Example 2 relates to a compound of Example 1, wherein the compound comprises a subunit shown in formula (I):

[0014] [ka]

[0015] During the ceremony, Each X is independently H, L 1 -A-Z, or L 2 -R, where each X is bonded to an OH group, L 1 and L 2 each of which is independently an amine-terminated chain; each A independently comprises a substituted or unsubstituted maleimide moiety; each Z independently comprises or is absent a bisphosphonate compound modified with a hydrazone moiety; each R independently comprises a mannose-binding C-type lectin receptor targeting moiety or H; n is an integer greater than 0, and each unit of n may be the same or different; At least one A is a substituted maleimide moiety and a thiol-maleimide conjugation is between A and Z.

[0016] Example 3 relates to the compound of Example 2, wherein at least one X is L 1 -AZ, where at least one X is L 2-R, where R comprises a mannose-binding C-type lectin receptor targeting moiety.

[0017] Example 4 relates to any one of the compounds of Examples 1-3, wherein the molecular weight of the polymeric carbohydrate backbone is from about 1 kD to about 50 kD. Example 5 relates to any one of the compounds of Examples 1-4, wherein the mannose-binding C-type lectin receptor targeting moiety comprises a mannosyl-linked aglycone moiety, mannose, a high mannose glycan or mannose oligosaccharide, fucose, N-acetylglucosamine, a peptide, galactose, or a combination thereof.

[0018] Example 6 relates to any one of the compounds of Examples 2 to 5, wherein at least one L 1 Ha-(CH 2 ) p S(CH 2 ) q It contains -NH-, and p and q are integers of 0 to 5.

[0019] Example 7 relates to any one of the compounds of Examples 2 to 5, wherein at least one L 2 Ha-(CH 2 ) p S(CH 2 ) q It contains -NH-, and p and q are integers of 0 to 5.

[0020] Example 8 relates to any one of Examples 2-5, where the bisphosphonate compound is substituted with a carbonyl functionality before being modified to a hydrazone moiety.

[0021] Example 9 relates to any one of the compounds of Examples 2-5, wherein the hydrazone moiety comprises an acylhydrazone. Example 10 relates to the compound of Example 9, where the hydrazone moiety has the following structure:

[0022] [ka]

[0023] Example 11 relates to the compound of Example 10, wherein R 2 -R 4 -SH, R 4 is a substituted or unsubstituted linear or branched C 1 ~C 12 It may be an alkyl, alkenyl, alkynyl, or aromatic group.

[0024] Example 12 relates to the compound of Example 11, wherein R 2 Ha-(CH 2 ) 2 Includes SH. In Example 13, the pharmaceutical composition comprises a compound according to any one of Examples 1-12 and a pharma- ceutically acceptable carrier.

[0025] Example 14 relates to the composition of Example 13, wherein the compound comprises a subunit shown in formula (I):

[0026] [ka]

[0027] During the ceremony, Each X is independently H, L 1 -A-Z, or L 2 -R, where each X is bonded to an OH group, L 1 and L 2 each of which is independently an amine-terminated chain; each A independently comprises a substituted or unsubstituted maleimide moiety; each Z independently comprises or is absent a bisphosphonate compound modified with a hydrazone moiety; each R independently comprises a mannose-binding C-type lectin receptor targeting moiety or H; n is an integer greater than 0, each unit of n can be the same or different, at least one A is a substituted maleimide moiety, and a thiol-maleimide conjugation is between A and Z.

[0028] In Example 15, a bisphosphonate compound according to formula (II) is provided:

[0029] [ka]

[0030] During the ceremony, R 1 are each independently H, a positively charged counterion, or a substituted or unsubstituted straight-chain or branched C 1 ~C 6 an alkyl group or an acyloxyalkyl group, X 1 H, hydroxyl, C 1 ~C 6 Alkyl, or OC 1 ~C 6 an alkyl group, Y does not exist or C 1 ~C 6 an alkyl group, or a heteroatom, W is a linear or branched C 1 ~C 12 An alkyl, alkenyl, alkynyl, aromatic, or heteroaromatic group, W is substituted with a carbonyl group.

[0031] Example 16 relates to compounds of example 15, wherein W is an aromatic or heteroaromatic group selected from the group consisting of phenyl, pyridine, pyrimidine, pyridazine, pyrazine, triazine, pyrazole, imidazole, triazole, tetrazole, thiazole, isothiazole, oxazole and isoxazole, or a pharmaceutically acceptable salt thereof.

[0032] Example 17 relates to compounds of Examples 15 or 16, wherein W is C 1 ~C 8 It may be further substituted with an alkyl group, an aromatic group, or a heteroaromatic group. Example 18 relates to any one of the compounds of Examples 15 to 17, wherein W is thiazole, the carbonyl group is a ketone group, and W is C 1 Further substituted with an alkyl group, Y is NH, and X 1 is H.

[0033] Example 19 relates to any one of the compounds of Examples 15 to 17, wherein W is imidazole, the carbonyl group is a ketone group, Y is absent, and X 1 is C 1 It is an alkyl group.

[0034] Example 20 relates to any one of the compounds of Examples 15-17, wherein the compound has the following formula (III):

[0035] [ka]

[0036] Example 21 relates to any one of the compounds of Examples 15-17, wherein the compound has the following formula (IV):

[0037] [ka]

[0038] Example 22 relates to any one of the compounds of Examples 15-21, where the carbonyl group is coupled with an acyl hydrazide to form a bisphosphonate compound modified with a hydrazone moiety.

[0039] Example 23 relates to the compound of Example 22, where the acyl hydrazide has the structure: NH 2-NH-C(O)-R 2 . Example 24 relates to compounds of Examples 22 or 23, where the hydrazone moiety has the structure:

[0040] [ka]

[0041] Example 25 relates to the compound of Example 23 or 24, wherein R 2 -R 4 -SH, R 4 is a substituted or unsubstituted linear or branched C 1 ~C 12 It may be an alkyl, alkenyl, alkynyl, or aromatic group.

[0042] Example 26 relates to the compound of Example 25, wherein R 2 Ha-(CH 2 ) 2 Includes SH. Example 27 relates to the compound of Example 22, wherein the compound has the following formula (V):

[0043] [ka]

[0044] In the formula, R 2 -R 4 -SH, R 4 is a substituted or unsubstituted linear or branched C 1 ~C 12 It may be an alkyl, alkenyl, alkynyl, or aromatic group. Example 28 relates to the compound described in Example 27, wherein R 2 Ha-(CH 2 ) 2 Includes SH.

[0045] Example 29 relates to a compound described in Example 22, wherein the compound has the following formula (VI):

[0046] [ka]

[0047] In the formula, R 2 -R 4 -SH, R 4 is a substituted or unsubstituted linear or branched C 1 ~C 12 It may be an alkyl, alkenyl, alkynyl, or aromatic group. Example 30 relates to the compound described in Example 29, wherein R 2 Ha-(CH 2 ) 2 Includes SH.

[0048] In Example 31, a method for preparing a compound described in Example 1 or 2 includes the steps of: (a) synthesizing a dextran backbone having one or more amine-terminated chains attached thereto; (b) synthesizing a nitrogen-containing bisphosphonate compound comprising a carbonyl functional group; (c) reacting the carbonyl functional group with an acyl hydrazide to form a nitrogen-containing bisphosphonate compound modified with a hydrazone moiety; (d) modifying the one or more amine-terminated chains with a maleimide moiety; and (e) replacing the one or more maleimide moieties from step (d) with the hydrazone moiety-modified bisphosphonate compound from step (c) via thiol-maleimide conjugation.

[0049] Example 32 relates to a method according to Example 31, wherein step (d) can be performed before step (b), after step (b), before step (c), or after step (c).

[0050] Example 33 relates to the method of example 31 or 32, wherein in formula (I), at least one X is L 1-AZ, where at least one X is L 2 -R, where R comprises a mannose-binding C-type lectin receptor targeting moiety.

[0051] Example 34 relates to the method of any one of Examples 31-33, wherein the molecular weight of the polymeric carbohydrate backbone is from about 1 kD to about 50 kD. Example 35 relates to the method of any one of Examples 31 to 34, wherein the mannose-binding C-type lectin receptor targeting moiety comprises a mannosyl-linked aglycone moiety, mannose, a high mannose glycan or mannose oligosaccharide, fucose, N-acetylglucosamine, a peptide, galactose, or a combination thereof.

[0052] Example 36 relates to the method according to any one of Examples 31 to 35, wherein in Formula (I), at least one L 1 Ha-(CH 2 ) p S(CH 2 ) q It contains -NH-, and p and q are integers of 0 to 5.

[0053] Example 37 relates to the method according to any one of Examples 31 to 35, wherein in formula (I), at least one L 2 Ha-(CH 2 ) p S(CH 2 ) q It contains -NH-, and p and q are integers of 0 to 5.

[0054] Example 38 relates to the method of any one of Examples 31-37, wherein the hydrazone moiety has the structure:

[0055] [ka]

[0056] Example 39 relates to the method described in Example 38, wherein R 2 -R 4 -SH, R 4 is a substituted or unsubstituted linear or branched C 1 ~C 12 It may be an alkyl, alkenyl, alkynyl, or aromatic group.

[0057] Example 40 relates to the method described in Example 39, wherein R 2 Ha-(CH 2 ) 2 Includes SH. In Example 41, a method of repolarizing tumor-associated macrophages (TAMs) from an immunosuppressive (M2-like) phenotype to a pro-inflammatory (M1-like) phenotype comprises administering to a subject in need thereof an effective amount of a compound comprising a polymeric carbohydrate backbone, one or more mannose-binding C-type lectin receptor targeting moieties, and a nitrogen-containing bisphosphonate compound attached to the polymeric carbohydrate backbone via thiol-maleimide conjugation.

[0058] Example 42 relates to the method described in Example 41, wherein the compound comprises a subunit provided by formula (I):

[0059] [ka]

[0060] During the ceremony, Each X is independently H, L 1 -A-Z, or L 2 -R, where each X is bonded to an OH group, L 1 and L 2 each of which is independently an amine-terminated chain; each A independently comprises a substituted or unsubstituted maleimide moiety; each Z independently comprises or is absent a bisphosphonate compound modified with a hydrazone moiety; each R independently comprises a mannose-binding C-type lectin receptor targeting moiety or H; n is an integer greater than 0, and each unit of n may be the same or different; At least one A is a substituted maleimide moiety and a thiol-maleimide conjugation is between A and Z.

[0061] Example 43 relates to a method according to Example 41 or 42, wherein the compound is administered in combination with at least one other therapy or treatment, and the at least one other therapy or treatment is chemotherapy, radiation therapy, or immunotherapy.

[0062] Example 44 relates to the method of any one of Examples 41-43, wherein the bisphosphonate compound is released from the polymeric carbohydrate backbone at a pH of less than about 5.5.

[0063] In Example 45, a method of treating a disease comprises administering to a subject in need of treatment an effective amount of a compound described in any one of Examples 1-12, wherein the disease is cancer, an autoimmune disease, or an inflammatory disease.

[0064] Example 46 relates to a method according to Example 45, wherein the compound is administered in combination with at least one other therapy or treatment, and the at least one other therapy or treatment is chemotherapy, radiation therapy, or immunotherapy.

[0065] Example 47 relates to the method according to Example 45 or 46, wherein the disease is cancer. While multiple embodiments are disclosed, still other embodiments of the present disclosure will become apparent to those skilled in the art from the following detailed description, which shows and describes illustrative embodiments of the present disclosure. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature and not restrictive. [Brief description of the drawings]

[0066] [Figure 1] FIG. 1 shows the synthesis of BIS-1 and BIS-2 from tetraethylvinylidene bisphosphonate and nitrogen heterocycles. [Diagram 2] Figure 1 shows the preparation of hydrazone bonds to BIS-1 and BIS-2. Three linkers were evaluated for different conjugation modes to the MAD scaffold. The linker terminated with a free thiol group was successfully combined with the maleimide-modified MAD scaffold to conjugate bisphosphonates. [Diagram 3] FIG. 1 shows the conjugation of BIS-1 and BIS-2 bearing a hydrazone linker and a terminal thiol group to MAD modified to present a terminal maleimide moiety. [Figure 4] 1 is a graph showing the release of BIS-1 from MAD-BIS-1 over time at pH 4.65. [Diagram 5] Graph showing how various treatments with either MAN-BIS-1 or free BIS-1 affected the expression levels of the eight surface markers evaluated (CD206, CD163, CD80, CD86, MHC1, MHC2, SIRPα, and PD1). All experiments were performed in triplicate using macrophages from three human donors. [Figure 6] Graph showing how various treatments with either MAN-BIS-2 (10 kDa) or free zoledronic acid affected the expression levels of the eight surface markers evaluated (CD206, CD163, CD80, CD86, MHC1, MHC2, SIRPα, and PD1). All experiments were performed in triplicate using macrophages from three human donors. [Figure 7] Graph comparing the effect of MAD-BIS-1 to MAD-BIS-2 on the expression of eight surface markers evaluated (CD206, CD163, CD80, CD86, MHC1, MHC2, SIRPα, and PD1). All experiments were performed in triplicate using macrophages from three human donors. [Figure 8]Graph showing how various treatments with either MAN-BIS-2 (3.5 kDa) or free BIS-2 altered inflammatory marker expression, including expression of eight surface markers evaluated (CD206, CD163, CD80, CD86, MHC1, MHC2, SIRPα, and PD1). All experiments were performed in triplicate using macrophages from three human donors. [Figure 9] Graph showing how various treatments with either MAN-BIS-2 (10 kDa) or MAN-BIS-2 (3.5 kDa) altered inflammatory marker expression, including expression of eight surface markers evaluated (CD206, CD163, CD80, CD86, MHC1, MHC2, SIRPα, and PD1). All experiments were performed in triplicate using macrophages from three human donors. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0067] Various embodiments of the present disclosure will be described in detail with reference to the drawings. Reference to various embodiments is not intended to limit the scope of the present disclosure. The figures shown in this specification are presented for illustrative purposes of the present disclosure, rather than to limit the various embodiments according to the present disclosure.

[0068] The embodiments of the present disclosure are not limited to specific compounds, compositions, and methods, which may vary and are understood by those skilled in the art. Furthermore, it is to be understood that all terms used herein are only for the purpose of describing specific embodiments, and are not intended to be limiting in any manner or scope. For example, as used in this specification and the appended claims, the singular forms "a", "an", and "the" can include plural referents unless the content clearly dictates otherwise. Furthermore, all units, prefixes, and symbols can be denoted in SI accepted form.

[0069] Numerical ranges recited herein are inclusive of the numbers defining the range and include each integer within the defined range. Throughout this disclosure, various aspects of the disclosure are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Thus, the description of a range should be considered to have specifically disclosed all possible subranges, fractions and individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within that range (e.g., 1, 2, 3, 4, 5, and 6), and decimals and fractions (e.g., 1.2, 3.8, 1 and 1 / 2, and 4 and 3 / 4). This applies regardless of the breadth of the range.

[0070] In order to make the present disclosure easier to understand, certain terms are first defined.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the embodiments of the present disclosure pertain.Many methods and materials similar to, modified or equivalent to those described herein can be used to implement the embodiments of the present disclosure without undue experimentation, and preferred materials and methods are described herein.In describing and claiming the embodiments of the present disclosure, the following terms are used according to the definitions set forth below.

[0071] As used herein, the term "about" refers to the variation in quantity that may occur, for example, due to common measuring techniques and equipment, with respect to any quantifiable variable (including, but not limited to, mass, volume, and time). Furthermore, given the solid and liquid handling procedures used in the real world, differences in the manufacture, source, or purity of ingredients used in making compositions or carrying out methods, etc., may result in certain inadvertent errors and variations. The term "about" also encompasses these variations. Whether or not modified by the term "about", the claims include the equivalent of the quantity.

[0072] The methods and compositions of the present disclosure may comprise, consist essentially of, or consist of the components and ingredients of the present disclosure as well as other components described herein. As used herein, "consisting essentially of" means that the methods, systems, devices, and compositions may include additional steps, components, or ingredients, but only if such additional steps, components, or ingredients do not materially alter the basic and novel characteristics of the claimed methods, systems, devices, and compositions.

[0073] The terms "actives" or "percent actives" or "percent actives by weight" or "actives concentration" are used interchangeably herein and refer to the concentration of ingredients involved in cleaning expressed as a percentage minus inactive ingredients such as water or salt. They may also be given as a percentage in parentheses, such as "chemical (10%)."

[0074] As used herein, the term "alkyl" or "alkyl group" refers to a saturated hydrocarbon having one or more carbon atoms, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), cyclic alkyl groups (or "cycloalkyl" or "alicyclic" or "carbocyclic" groups) (e.g., cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.), branched-chain alkyl groups (e.g., isopropyl, tert-butyl, sec-butyl, isobutyl, etc.), and alkyl substituted alkyl groups (e.g., alkyl substituted cycloalkyl groups and cycloalkyl substituted alkyl groups).

[0075] Unless otherwise specified, the term "alkyl" includes both "unsubstituted alkyls" and "substituted alkyls." As used herein, the term "substituted alkyl" refers to alkyl groups having substituents replacing one or more hydrogens on one or more carbons of the hydrocarbon backbone. Such substituents may include, for example, alkenyl, alkynyl, halogeno, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonate, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclic, alkylaryl, or aromatic (including heteroaromatic) groups.

[0076] In some embodiments, the substituted alkyl can include a heterocyclic group. As used herein, the term "heterocyclic group" includes closed ring structures similar to carbocyclic groups, in which one or more of the carbon atoms in the ring is an element other than carbon, such as nitrogen, sulfur, or oxygen. Heterocyclic groups can be saturated or unsaturated. Exemplary heterocyclic groups include, but are not limited to, aziridine, ethylene oxide (epoxide, oxirane), thiirane (episulfide), dioxirane, azetidine, oxetane, thietane, dioxetane, dithietane, dithiete, azolidine, pyrrolidine, pyrroline, oxolane, dihydrofuran, and furan.

[0077] As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Exemplary substituents include, for example, those described below. For appropriate organic compounds, the permissible substituents can be one or more and can be the same or different. For purposes of this disclosure, heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valences of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. The terms "substituted" or "substituted with" also include the implicit proviso that such substitution is in accordance with the permissible valences of the substituted atom and substituent and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation by rearrangement, cyclization, elimination, and the like. It is also contemplated that in certain embodiments, unless expressly stated to the contrary, individual substituents may be further optionally substituted (i.e., further substituted or unsubstituted).

[0078] As described herein, the compounds of the present invention may include "optionally substituted" moieties. In general, the term "substituted", whether preceded by the term "optionally" or not, means that one or more hydrogens of the specified moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when multiple positions in any given structure may be substituted with multiple substituents selected from a specified group, the substituents may be the same or different at all positions. Combinations of substituents envisioned by the present invention are preferably those that result in the formation of stable or chemically feasible compounds. It is also contemplated that in certain embodiments, unless expressly indicated to the contrary, individual substituents may be further optionally substituted (i.e., further substituted or not substituted).

[0079] In defining various terms, 1 ","A 2 ","A 3 ","A 4 ", "X 1 ", "X 2 ","Y 1 ","Y 2 " and the like are used herein as generic symbols to represent various specific substituents. These symbols are not limited to those disclosed herein, but may be any substituent, and in some cases may be defined as a specific substituent, and in other cases may be defined as some other substituent.

[0080] "R 1 ", "R 2 ", "R 3 ", "R n " (where n is an integer), as used herein, can independently have one or more of the groups listed above. For example, R 1 When is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group may be optionally substituted with a hydroxyl group, an alkoxy group, an alkyl group, a halide, etc. Depending on the group selected, the first group may be incorporated within the second group, or alternatively, the first group may be pendant (i.e., attached) to the second group. For example, in the phrase "an alkyl group comprising an amino group," the amino group may be incorporated within the backbone of the alkyl group. Alternatively, the amino group may be attached to the backbone of the alkyl group. The nature of the group(s) selected will determine whether the first group is embedded or attached to the second group.

[0081] Certain of the materials, compounds, compositions, and components disclosed herein are commercially available or can be readily synthesized using techniques generally known to those of skill in the art. For example, the starting materials and reagents used in preparing the disclosed compounds and compositions are available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, NJ), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.), or are generally described in such publications as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991), Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March's Advanced Organic Chemistry (John Wiley and Sons, 4th Edition), and Larock's Comprehensive Organic Transformations (VCH Publishers, 1997). They can be prepared according to procedures described in references such as (E. G., Inc., 1989) and by methods known to those skilled in the art.

[0082] Disclosed are the components used to prepare the compositions of the present disclosure, as well as the compositions themselves used within the methods disclosed herein. These and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that specific references to each of the various individual and collective combinations and permutations of these compounds cannot be explicitly disclosed, but each is specifically contemplated and described herein. For example, when a particular compound is disclosed and discussed, and a number of modifications that can be made to a number of molecules that include that compound are discussed, all combinations and permutations of that compound and possible modifications are specifically contemplated, unless specifically indicated to the contrary.

[0083] Thus, when classes of molecules A, B, and C, and classes of molecules D, E, and F, and an example of a combination molecule A-D are disclosed, each is individually and collectively contemplated even if each is not individually set forth, meaning that combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered to be disclosed. Similarly, any subset or combination of these is also disclosed. Thus, for example, subgroups A-E, B-F, and C-E are considered to be disclosed. This concept applies to all aspects of this application, including but not limited to steps in the methods of preparing and using the compositions of the invention. Thus, when there are various additional steps that can be performed, it is understood that each of these additional steps can be performed in any particular embodiment or combination of embodiments of the method of the invention.

[0084] As used herein, the term "pharmaceutical acceptable carrier" or "carrier" refers to sterile aqueous or non-aqueous solutions, colloids, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions immediately prior to use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc. The absorption of injectable pharmaceutical forms can be prolonged by including agents such as aluminum monostearate and gelatin which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides). Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. Injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium immediately prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

[0085] The term "polarization" is used herein to indicate the phenotypic and functional characteristics of macrophages. The phenotype can be defined through the surface markers expressed by the macrophages. The functionality can be defined, for example, based on the nature and amount of chemokines and / or cytokines expressed, and in particular secreted, by the macrophages. Indeed, macrophages exhibit different phenotypic and functional characteristics depending on their status (pro-inflammatory (M1 type) or anti-inflammatory (M2 type) macrophages). M2 type macrophages can be characterized by the expression of surface markers such as CD206, CD11b, PD-L1, and CD200R, and the subsequent secretion of cytokines such as CCL17. M1 type macrophages can be defined by the expression of surface markers such as CD86 and CCR7, and the secretion of cytokines such as IL-6, TNF-a, and IL12p40. In the context of the present disclosure, the term "repolarization" is used herein to refer to inducing the phenotype of an M1 macrophage population to change to M1-type macrophages.

[0086] As used herein, the term "cancer" refers to cells that have the ability to grow autonomously. Examples of such cells include cells that have an abnormal state or condition characterized by rapidly proliferating cell proliferation. The term is meant to include cancerous growths (e.g., tumors), oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. It also includes malignant tumors of various organ systems, such as the respiratory, cardiovascular, renal, reproductive, hematological, nervous, hepatic, gastrointestinal, and endocrine systems, as well as adenocarcinomas, including most colon, renal cell, prostate, and / or testicular, non-small cell lung, small intestine, and esophageal cancers. "Naturally occurring" cancers include any cancer that is not experimentally induced by implanting cancer cells in a subject, including, for example, spontaneous cancers, cancers caused by exposure of a patient to a carcinogen(s), cancers resulting from the insertion of a genetically engineered cancer gene or knockout of a tumor suppressor gene, and cancers caused by infectious diseases, such as viral infections. The term "carcinoma" is art-recognized and refers to a malignant tumor of epithelial or endocrine tissue. In some embodiments, the methods can be used to treat a subject having an epithelial cancer, e.g., a solid tumor of epithelial origin, e.g., lung, breast, ovarian, prostate, renal, pancreatic, or colon cancer.

[0087] As used herein, the term "subject" refers to the target of administration, e.g., an animal. Thus, the subject of the methods disclosed herein can be a vertebrate, such as a mammal, fish, bird, reptile, or amphibian. Alternatively, the subject of the methods disclosed herein can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent. The term does not indicate a particular age or sex. Thus, adult and neonatal subjects, as well as fetuses (whether male or female), are intended to be encompassed. In one aspect, the subject is a mammal. A patient refers to a subject suffering from a disease or disorder. The term "patient" includes human and veterinary subjects. In some aspects of the disclosed methods, the subject has been diagnosed as in need of treatment for one or more cancer disorders prior to the administration step.

[0088] As used herein, the term "synergistic" means that the effect achieved by the methods and combinations of the present invention is greater than the sum of the effects obtained by using the compounds, compositions, treatments and / or methods, or pharma- ceutically acceptable salts thereof, separately. Advantageously, such a synergistic effect provides greater efficacy at the same dose and / or prevents or slows the build-up of multidrug resistance.

[0089] As used herein, the term "treatment" refers to the medical management of a patient with the goal of curing, ameliorating, stabilizing, or preventing a disease, condition, or disorder. The term includes active treatment, i.e., treatment specifically aimed at improving the disease, condition, or disorder, and also includes causal treatment, i.e., treatment aimed at eliminating the cause of the associated disease, condition, or disorder. In addition, the term includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure the disease, condition, or disorder, preventive treatment, i.e., treatment aimed at minimizing or partially or completely inhibiting the onset of the associated disease, condition, or disorder, and supportive treatment, i.e., treatment used to complement another specific therapy aimed at improving the associated disease, condition, or disorder. In various embodiments, the term encompasses any treatment for a subject, including a mammal (e.g., a human), and includes (i) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed as having it, (ii) inhibiting the disease, i.e., halting the progression of the disease, or (iii) relieving the disease, i.e., causing regression of the disease.

[0090] As used herein, the term "prevent" or "preventing" refers to hindering, avoiding, eliminating, forestalling, obstructing, or impeding something from happening, especially by prior action. Where "reduce," "inhibit," or "prevent" is used herein, it is understood that the use of the other two words is expressly disclosed unless specifically indicated otherwise.

[0091] As used herein, the term "diagnosed" means that one has undergone a physical examination by a skilled artisan, e.g., a physician, and has been found to have a condition that can be diagnosed or treated by a compound, composition, or method disclosed herein. For example, "diagnosed with cancer" means that one has undergone a physical examination by a skilled artisan, e.g., a physician, and has been found to have a condition that can be diagnosed or treated by a compound or composition that can reduce tumor size or slow tumor growth rate. A subject having cancer, tumor, or at least one cancer or tumor cell can be identified using methods known in the art. For example, contrast-enhanced MRI or CT can be used to determine the anatomical location, overall size, and / or cellular composition of the cancer cells or tumor. Additional methods for identifying cancer cells can include, but are not limited to, ultrasound, bone scans, surgical biopsies, and biological markers (e.g., serum protein levels and gene expression profiles). An imaging solution containing the cell sensitizing composition of the present invention can be used, for example, in combination with MRI or CT to identify cancer cells.

[0092] As used herein, the terms "administering" and "administration" refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, intranasal administration, topical administration, intravaginal administration, intraocular administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration (including injection, such as intravenous administration, intraarterial administration, administration to a specific organ by infiltration, intramuscular administration, intratumoral administration, and subcutaneous administration). Administration can be continuous or intermittent. In various embodiments, the preparation can be administered therapeutically, i.e., administered to treat an existing disease or condition. In further various embodiments, the preparation can be administered prophylactically, i.e., administered for the prevention of a disease or condition.

[0093] As used herein, the terms "effective amount" and "effective amount" refer to an amount sufficient to achieve a desired outcome or affect an undesired condition. For example, a "therapeutically effective amount" refers to an amount sufficient to achieve a desired therapeutic outcome or affect an undesired condition, but generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will vary depending on a variety of factors, including the disorder being treated and the severity of the disorder, the particular composition used, the patient's age, weight, general health, sex and diet, time of administration, route of administration, excretion rate of the particular compound used, duration of treatment, drugs used in combination or concomitantly with the particular compound used, and similar factors well known in the medical arts. For example, it is well within the skill of the art to begin administering a compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for administration purposes. As a result, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. In the event of any contraindications, the dosage can be adjusted by the individual physician. Dosages vary and can be administered in one or more doses per day for one or several days. Guidance can be found in the literature for appropriate dosages for a given class of pharmaceutical. In further various embodiments, the preparations can be administered in a "prophylactically effective amount", i.e., an amount effective for the prevention of a disease or condition.

[0094] Effective doses can be estimated initially from in vitro assays. For example, initial doses for use in animals may be determined based on the IC 50The pharmaceutical composition can be formulated to achieve or exceed the above. It is well within the ability of one skilled in the art to calculate the dosage to achieve such circulating blood or serum concentration, taking into account the bioavailability of a particular active agent. For guidance, the reader is referred to Fingl & Woodbury, "General Principles", In: Goodman and Gilman's The Pharmaceutical Basis of Therapeutics, Chapter 1, 1-46, latest edition, Pergamagon Press, which is incorporated herein by reference in its entirety, and references cited therein.

[0095] The phrase "anti-cancer composition" can include compositions that exert anti-tumor, chemotherapeutic, anti-viral, anti-mitotic, anti-tumorigenic, anti-angiogenic, anti-metastatic, and / or immunotherapeutic effects, e.g., compositions that prevent the development, maturation, or spread of tumor cells directly on tumor cells, e.g., by a cytostatic or cytocidal effect, rather than indirectly through mechanisms such as biological response modification. There are numerous anti-proliferative agents available in commercial use, clinical evaluation, and preclinical development that can be included in the present application through combination drug chemotherapy. For ease of discussion, antiproliferative agents are divided into the following classes, subtypes, and species: ACE inhibitors, alkylating agents, angiogenesis inhibitors, angiostatin, anthracycline / DNA intercalators, anticancer antibiotics or antibiotic-type agents, antimetabolites, antimetabolites, anti-metastatic compounds, asparaginase, bisphosphonates, cGMP phosphodiesterase inhibitors, calcium carbonate, cyclooxygenase-2 inhibitors, DHA derivatives, DNA topoisomerase, endostatin, epipodophyllotoxins, genistein, hormonal anticancer agents, hydrophilic bile acids (URSO), immunomodulatory or immunological agents, integrin antagonists, interferon antagonists or agents, MMP inhibitors, miscellaneous antitumor agents, monoclonal antibodies, nitrosoureas, NSAIDs, ornithine decarboxylase inhibitors, pBATT, radio / chemosensitizers / protectants, retinoids, selective inhibitors of endothelial cell proliferation and migration, selenium, stromelysin inhibitors, taxanes, vaccines, and vinca alkaloids. The major categories into which some antiproliferative agents fall include antimetabolites, alkylating agents, antibiotic-type agents, hormonal anticancer agents, immunological agents, interferon-type agents, and various antitumor agent categories. Some antiproliferative agents act through multiple or unknown mechanisms and therefore can be classified into multiple categories.

[0096] "Weight percent," "wt.%," "wt-%," "percent by weight," "% by weight," and variations thereof, as used herein, refer to the concentration of a substance calculated by dividing the weight of that substance by the total weight of the composition and multiplying by 100.

[0097] The present disclosure describes compounds, compositions, and methods useful for changing or transitioning the phenotype of activated macrophages from an immunosuppressive phenotype to a pro-inflammatory phenotype (referred to herein as "repolarization"). The ability to change or transition the phenotype of activated macrophages from immunosuppressive to pro-inflammatory constitutes a treatment for cancer, various infectious diseases, and other disease conditions. The present disclosure further describes drug delivery vehicles and methods of use that allow targeted delivery of bisphosphonates and related therapeutics to TAMs for the purpose of repolarizing TAMs. Targeted delivery to TAMs provides a greater amount of bisphosphonates and related therapeutics to TAMs, increasing the effectiveness of the phenotype change while limiting potentially toxic exposure to off-target cells and tissues. Use of the disclosed compounds, compositions, and methods can induce the phenotype of M2-like (immunosuppressive) activated macrophages to switch to an M1-like (pro-inflammatory) activated phenotype.

[0098] Compounds and Compositions In certain embodiments, the compounds disclosed herein use carrier constructs that include polymeric carbohydrate backbones conjugated with mannose-binding C-type lectin receptor targeting moieties (e.g., mannose, fucose, N-acetylglucosamine) to deliver one or more active therapeutic agents. Examples of such constructs include mannosylated amine dextran (MAD), which includes a dextran backbone, with mannose molecules conjugated to the glucose residues of the backbone and active pharmaceutical ingredients conjugated to the glucose residues of the backbone. Tilmanocept is a specific example of MAD. Tilmanocept derivatives, which are tilmanocept without DTPA conjugation, are a further example of MAD.

[0099] MADs are synthetic molecules purposefully designed to be high affinity ligands for mannose-binding C-type lectin receptors, such as CD206. MADs are described in U.S. Pat. No. 6,409,990, which is incorporated herein by reference in its entirety. Thus, the backbone comprises multiple glucose moieties (i.e., residues or subunits) linked primarily by α-1,6 glycosidic bonds. Other bonds, such as α-1,4 and / or α-1,3 bonds, may also be present. In some embodiments, not all backbone moieties are substituted. In some embodiments, one or more amine-terminated chains are attached to the backbone. In further embodiments, mannose-binding C-type lectin receptor targeting moieties are attached to one or more amine-terminated chains. In certain embodiments, mannose-binding C-type lectin targeting moieties are attached to about 15% to about 70%, about 17% to about 65%, or about 20% to about 60% of the glucose residues via amine-terminated chains. In further embodiments, the mannose-binding C-type lectin targeting moiety is attached to up to about 60%, up to about 70%, up to about 80%, up to about 90%, or up to about 100% of the glucose residues via amine-terminated chains. In certain aspects, the percentage may vary depending on the size of the dextran backbone. In further embodiments, one or more therapeutic agents are attached to the glucose residues via amine-terminated chains and hydrazone linkers linked to the backbone using thiol-maleimide conjugation, as described in more detail below. In certain embodiments, the therapeutic agent is attached to about 1% to about 25%, about 2% to about 20%, or about 5% to about 15% of the glucose residues via amine-terminated chains and hydrazone linkers linked to the backbone via thiol-maleimide conjugation, as described herein.

[0100] The size of the MAD can be varied by altering the size of the initial dextran from which the MAD construct is assembled. In some embodiments, the dextran-based portion is about 50-100 kD. The dextran-based portion may be at least about 50 kD, at least about 60 kD, at least about 70 kD, at least about 80 kD, or at least about 90 kD. The dextran-based portion may be less than about 100 kD, less than about 90 kD, less than about 80 kD, less than about 70 kD, or less than about 60 kD. Alternatively, in some embodiments, the MW of the dextran backbone is about 1 kD to about 50 kD, while in other embodiments, the MW of the dextran backbone is about 5 kD to about 25 kD. In yet other embodiments, the MW of the dextran backbone is about 8 kD to about 15 kD, e.g., about 10 kDa. Meanwhile, in other embodiments, the MW of the dextran backbone is about 1 kD to about 5 kDa, e.g., about 3 kDa. As described in Bartels et al (Mol Imaging Biol. 2023 Mar 7. doi:10.1007 / s11307-023-01809-6. Online ahead of print), the MAD-based constructs have an initial dextran (on which the MAD-based constructs are constructed or assembled) and vary in size between 3.5 kDa and 150 kDa, all of which bind strongly to CD206.

[0101] In contrast to larger liposomal constructs, which may be about 84 nm or greater in diameter, the molecular weight of the disclosed MAD constructs, in some embodiments, may have a diameter of about 15 nm or less, about 12 nm or less, about 10 nm or less, about 7 nm or less, or about 5 nm or less. Advantageously, the smaller size of the disclosed constructs allows for greater tumor penetration and localization to TAMs than was possible with previously used liposomal constructs.

[0102] According to certain embodiments, and as further described throughout this disclosure, the one or more mannose-binding C-type lectin receptor targeting moieties and the one or more therapeutic agents are each independently attached to the dextran-based backbone via a tether. As described in more detail below, there may be one or more additional moieties between the tether and the mannose-binding C-type lectin receptor targeting moiety or the therapeutic agent. In further embodiments, the tether is not attached to the mannose-binding C-type lectin receptor targeting moiety or the therapeutic agent, but instead is provided as a stand-alone tether attached to the dextran-based backbone. The tether may be attached to about 50% to about 100% of the backbone moiety, or about 70% to about 90% of the backbone moiety. The tethers may be the same or different. In some embodiments, the tethers are amine-terminated tethers. In some embodiments, the tethers are of the formula -(CH 2 ) p S(CH 2 ) q In a further embodiment, the chain may comprise -NH-, where p and q are integers from 0 to 5. In a further embodiment, the chain may comprise the formula -(CH 2 ) 3 S(CH 2 ) 2 In embodiments where the chains are not attached to a mannose-binding C-type lectin receptor targeting moiety or therapeutic agent, the chains have the formula -(CH 2 ) p S(CH 2 ) q -NH 2 where p and q are integers from 0 to 5.

[0103] In some embodiments, the chain may be a chain of about 1 to about 20 member atoms selected from carbon, oxygen, sulfur, nitrogen, and phosphorus. The chain may be a straight or branched chain. The chain may also be a group selected from, but not limited to, a halo group, a perfluoroalkyl group, a perfluoroalkoxy group, a C 1-4 Alkyl groups such as alkyl, C 1-4 Alkenyl groups such as alkenyl, C 1-4Alkynyl groups such as alkynyl, hydroxy groups, oxo groups, mercapto groups, alkylthio groups, alkoxy groups, nitro groups, azidoalkyl groups, aryl or heteroaryl groups, aryloxy or heteroaryloxy groups, aralkyl or heteroaralkyl groups, aralkoxy or heteroaralkoxy groups, HO-(C=O)- groups, heterocyclic groups, cycloalkyl groups, amino groups, alkyl- and dialkylamino groups, carbamoyl groups, alkylcarbonyl groups, alkylcarbonyloxy groups, alkoxycarbonyl groups, alkylaminocarbonyl groups, dialkylaminocarbonyl groups, arylcarbonyl groups, aryloxycarbonyl groups, alkylsulfonyl groups, arylsulfonyl groups, -NH-NH 2 The mannose-binding C-type lectin targeting moiety may be conjugated to the amine group of the amine-terminated chain via an amidine and / or amide linker.

[0104] According to certain embodiments, the MAD constructs disclosed herein include at least one mannose-binding C-type lectin receptor targeting moiety. In further embodiments, the MAD constructs disclosed herein include at least one targeting ligand. CD206 is a C-type lectin receptor expressed on macrophages, dendritic cells, and mesangial cells. CD206 binds to molecules that display multiple terminal mannose moieties. Without being limited to any particular mechanism or theory, upon binding of CD206 to a ligand, the receptor / ligand complex is internalized by receptor-mediated endocytosis into endosomes, which are naturally acidified to a pH of 4-5.5. At this low pH, CD206 releases its ligand and recirculates to the cell surface. In some aspects, the inclusion of a mannose-binding C-type lectin receptor targeting moiety within a MAD construct provides a number of advantages in delivering therapeutic agents to targets such as macrophages. According to some embodiments, the mannose-binding C-type lectin receptor targeting moiety comprises mannose, high mannose glycan or mannose oligosaccharide, fucose, or N-acetylglucosamine, peptide, or galactose. In further embodiments, the mannose-binding C-type lectin receptor targeting moiety is linked to an amine-terminated chain via an electrophilic imidate (also referred to herein as a "mannosyl-linked aglycon moiety") appended to the mannose, as described in U.S. Pat. No. 6,409,990 (herein incorporated by reference in its entirety). Thus, in some embodiments, the mannose-binding C-type lectin receptor targeting moiety comprises a mannosyl-linked aglycon moiety, mannose, high mannose glycan or mannose oligosaccharide, fucose, N-acetylglucosamine, peptide, galactose, or a combination thereof. In further embodiments, the mannose-binding C-type lectin receptor targeting moiety comprises mannose. In other embodiments, at least one targeting ligand may be sialic acid.

[0105] According to some embodiments, the MAD constructs described herein may optionally include a maleimide moiety. In some aspects, the maleimide moiety is attached to the carbohydrate-based backbone via a tether as described herein. In further aspects, the tether is an amine-terminated tether. The maleimide moiety may include any compound that includes a maleimide. In some embodiments, the maleimide moiety is terminated with a maleimide group. In embodiments, the maleimide moiety is C 1-12 In some embodiments, the terminal maleimide group in the maleimide moiety may be substituted or unsubstituted. In some aspects, the unsubstituted maleimide moiety has the formula -C(O)(CH 2 ) 2 [H 2 C 2 (CO) 2 In a further aspect, the maleimide moiety may be substituted. In some embodiments, the substituted maleimide moiety has the formula -C(O)(CH 2 ) 2 [H 3 C 2 (CO) 2 N]-. In some embodiments, when the maleimide moiety is substituted, the maleimide moiety is substituted with a compound having a terminal thiol group, thereby forming a thiol-maleimide conjugation. According to certain embodiments, and as discussed in more detail below, a therapeutic agent may be attached to the maleimide moiety via a thiol-maleimide conjugation.

[0106] In some embodiments, one or more therapeutic agents are attached to the MAD construct. In certain embodiments, the therapeutic agent is a bisphosphonate. Suitable bisphosphonates include nitrogen-containing bisphosphonates. Bisphosphonates bind calcium and, as a result, accumulate in bone. Bone is a complex organ with many functions, and in healthy individuals, the structural components of bone are constantly turned over in a process of continuous regeneration. This bone turnover primarily involves two cell types: (1) osteoclasts, which break down bone, and (2) osteoblasts, which continually form new bone. Strong, healthy bone is maintained as a dynamic equilibrium between the actions of osteoclasts and osteoblasts. In pathological conditions, such as osteoporosis or bone metastatic cancer, this equilibrium is disrupted and bone is broken down faster than it is replaced, resulting in weaker bones and more susceptible to fracture. In patients administered bisphosphonate drugs, osteoclasts take up the drug as they break down bone. As a result, osteoclasts that receive bisphosphonates are induced to change their phenotype to degrade bone less efficiently or to die themselves by apoptosis. With reduced osteoclast activity, osteoblasts can form new bone faster than bone is degraded, resulting in stronger bones that resist fracture. Bisphosphonate drugs have been used successfully to treat patients with osteoporosis for decades. Bisphosphonate treatment reduces bone pain associated with metastasis and reduces the incidence of metastasis-related fractures.

[0107] Without being limited to any particular theory or mechanism, bisphosphonates may be able to address two TAM-targeted cancer treatment strategies, including killing or eliminating TAMs and inducing TAMs to switch their phenotype from M2-like to M1-like, making them suitable therapeutic agents for the compositions and methods disclosed herein.

[0108] Bisphosphonates are a class of drugs that have the general structure shown below.

[0109] [ka]

[0110] In this structure, two phosphonate groups are attached to the central carbon atom. The other two possible attachments to the central carbon are R 1 and R 2 A variety of other chemical moieties may be attached, shown as groups. R 1 and R 2 can be different chemical moieties, ranging in complexity from simple hydrogen (H), chlorine (Cl), or hydroxyl (OH) groups to more complex organic structures. In many pharmaceutical bisphosphonate drugs currently on the market, one of the R groups is a complex organic molecule containing one or more nitrogen atoms. Nitrogen-containing (containing nitrogen) bisphosphonate drugs tend to be more pharmacologically active than non-nitrogen-containing bisphosphonate drugs. In some aspects, one of the most pharmacologically active FDA-approved bisphosphonate drugs is zoledronic acid (zoledronate), a nitrogen-containing bisphosphonate. In embodiments, the present disclosure provides novel bisphosphonate drugs that contain a chemical moiety not found in zoledronic acid, which allows the novel bisphosphonate drug to be attached to a MAD construct via a degradable linker, which is described in further detail herein.

[0111] In embodiments, compounds are provided that include a polymeric carbohydrate backbone, one or more mannose-binding C-type lectin receptor targeting moieties, and a bisphosphonate compound attached to the polymeric carbohydrate backbone via thiol-maleimide conjugation as described herein. In some embodiments, the compounds may include a subunit as shown in formula (I):

[0112] [ka]

[0113] During the ceremony, Each X is independently H, L 1 -A-Z, or L 2 -R, where each X is bonded to an OH group, L 1 and L 2 each of which is independently an amine-terminated chain; each A independently comprises a substituted or unsubstituted maleimide moiety; each Z independently comprises or is absent a bisphosphonate compound modified with a hydrazone moiety; each R independently comprises a mannose-binding C-type lectin receptor targeting moiety or H; n is an integer greater than 0, and each unit of n may be the same or different.

[0114] In embodiments, at least one A is a substituted maleimide moiety and a thiol-maleimide conjugation is between A and Z. In a further embodiment, at least one X is L 1 In a further embodiment, at least one X is L 2 -R, wherein R comprises a mannose-binding C-type lectin receptor targeting moiety. As described herein, the mannose-binding C-type lectin receptor targeting moiety comprises a mannosyl-linked aglycone moiety, mannose, a high mannose glycan or mannose oligosaccharide, fucose, N-acetylglucosamine, a peptide, galactose, or a combination thereof.

[0115] As described within the present disclosure, the substituted maleimide moiety and / or the mannose-binding C-type lectin receptor targeting moiety may be attached to the polymer backbone via a tether. The tether may be any tether described throughout the present disclosure. In embodiments, at least one L 1 Ha-(CH 2 ) p S(CH 2 ) q In a further embodiment, at least one L 2 Ha-(CH 2 ) p S(CH 2 ) q where p and q are integers from 0 to 5.

[0116] In some embodiments, n is an integer greater than 0. In other embodiments, n is an integer greater than 1. In further embodiments, n can be an integer from 1 to about 50, from about 5 to about 40, or from about 5 to about 30. As will be appreciated by one of skill in the art, each unit of n can be the same or different. Each X can independently be selected from H, L, 1 -A-Z, or L 2 -R, so each unit of n can be H, L 1 -A-Z, or L 2 -R.

[0117] Bisphosphonate compounds suitable for attachment to a polymeric carbohydrate backbone via thiol-maleimide conjugation are further provided below. In some embodiments, the bisphosphonate compounds themselves are provided. In one aspect, the bisphosphonate compounds may include compounds according to formula (II):

[0118] [ka]

[0119] During the ceremony, R 1 are each independently H, a positively charged counterion, or a substituted or unsubstituted straight-chain or branched C 1 ~C 6 an alkyl group or an acyloxyalkyl group, X 1 H, hydroxyl, C 1 ~C 6 Alkyl, or OC 1 ~C 6 an alkyl group, Y does not exist or C 1 ~C 6 an alkyl group, or a heteroatom, W is an unsubstituted or substituted linear or branched C 1 ~C 12 It is an alkyl, alkenyl, alkynyl, aromatic, or heteroaromatic group.

[0120] In certain embodiments, the positively charged counterion can include, but is not limited to, alkali metal, alkaline earth metal, quaternary amine or ammonium, or any other positively charged counterion.For example, the positively charged counterion can include sodium, potassium, lithium, rubidium, cesium, francium, calcium, magnesium, barium, beryllium, strontium, or radium.In further examples, the positively charged ion can include sodium, potassium, lithium, calcium, or magnesium.As will be understood by those skilled in the art, additional positively charged counterions can be further considered.

[0121] In some embodiments, the acyloxyalkyl group may include, but is not limited to, pivaloyloxymethyl (POM), ethoxycarbonyloxyethyl, or acetoxymethyl (AM). In some embodiments, the acyloxyalkyl group may include pivaloyloxymethyl.

[0122] In certain embodiments, W is a substituted linear or branched C 1 ~C 12 In some embodiments, W is an alkyl, alkenyl, alkynyl, aromatic, or heteroaromatic group, where W is substituted with a carbonyl group. In some embodiments, the carbonyl group is selected from the group consisting of an aldehyde, a ketone, and an enone. In further embodiments, the carbonyl group is a ketone. In some embodiments, W is C 1 ~C 8 It may be further substituted with an alkyl group, a halogen, an aromatic group, or a heteroaromatic group.

[0123] In certain embodiments, W is an aromatic or heteroaromatic group. In some embodiments, the aromatic or heteroaromatic group is selected from the group consisting of phenyl, pyridine, pyrimidine, pyridazine, pyrazine, triazine, pyrazole, imidazole, triazole, tetrazole, thiazole, isothiazole, oxazole, and isoxazole, or a pharma- ceutically acceptable salt thereof. In some embodiments, the aromatic or heteroaromatic group is thiazole or imidazole.

[0124] In some embodiments, W is thiazole, the carbonyl group is a ketone group, and W further comprises C 1 is substituted with an alkyl group, Y is NH, and X 1 is H. In a further embodiment, W is imidazole, the carbonyl group is a ketone group, Y is absent and X 1 is C 1 It is an alkyl group.

[0125] In certain embodiments, the bisphosphonate compound may have the following formula (III):

[0126] [ka]

[0127] In other embodiments, the bisphosphonate compound may have the following formula (IV):

[0128] [ka]

[0129] In certain embodiments, bisphosphonate compounds of formula (II) are provided, where W is substituted with a carbonyl group. In further aspects, the carbonyl group is coupled with an acyl hydrazide to form a bisphosphonate compound modified with a hydrazone moiety. In some embodiments, the acyl hydrazide has the following structure: NH 2 -NH-C(O)-R 2In certain embodiments, R 2 -R 4 -SH, R 4 is a substituted or unsubstituted linear or branched C 1 ~C 12 In an exemplary embodiment, R is an alkyl, alkenyl, alkynyl, or aromatic group. 2 Ha-(CH 2 ) 2 SH. Advantageously, in some embodiments, the hydrazone moiety comprises a terminal thiol group. In aspects, when the acyl hydrazide is bonded to the carbonyl group of formula (II), a hydrazone moiety-modified bisphosphonate compound is formed. In these embodiments, the hydrazone moiety has the structure:

[0130] [ka]

[0131] In the formula, R 2 is the -R of the acyl hydrazide 4 In embodiments, the hydrazone moiety modified bisphosphonate compound has the following formula (V):

[0132] [ka]

[0133] In the formula, R 2 -R 4 -SH, R 4 is a substituted or unsubstituted linear or branched C 1 ~C 12 In an exemplary embodiment, R is an alkyl, alkenyl, alkynyl, or aromatic group. 2 Ha-(CH 2 ) 2 Includes SH.

[0134] In another embodiment, the bisphosphonate compound modified with a hydrazone moiety has the following formula (VI):

[0135] [ka]

[0136] In the formula, R 2 -R 4 -SH, R 4 is a substituted or unsubstituted linear or branched C 1 ~C 12 In an exemplary embodiment, R is an alkyl, alkenyl, alkynyl, or aromatic group. 2 Ha-(CH 2 ) 2 Includes SH.

[0137] In further embodiments, the bisphosphonate compounds disclosed herein can be conjugated to the disclosed MAD constructs. In some aspects, the bisphosphonate compounds are modified with hydrazone moieties before being conjugated to the dextran-based backbone. In further aspects, the bisphosphonate compounds are substituted with carbonyl functional groups before being modified to the hydrazone moieties. In certain embodiments, in which the bisphosphonate compounds modified with hydrazone moieties are conjugated to the MAD constructs, the dextran-based backbone already comprises one or more chains conjugated to maleimide moieties as disclosed herein. In certain aspects, the hydrazone moiety of the bisphosphonate compound comprises a terminal thiol group. This terminal thiol group allows the bisphosphonate compound to be conjugated to the dextran-based backbone via thiol-maleimide conjugation. In certain embodiments, the thiol group of the hydrazone moiety is conjugated to the maleimide group of the maleimide moiety. In embodiments, the hydrazone moiety comprises an acylhydrazone. Without being limited to any particular theory or mechanism, at neutral pH, the constructs retain their payload (such as a drug payload containing a therapeutic agent) long enough to be delivered to endosomes, and once the constructs are inside acidified endosomes, the drug payload is released.

[0138] According to certain embodiments, the disclosed compounds can include a pharma- ceutically acceptable carrier and a compound disclosed herein or a pharma- ceutically acceptable salt of the compound. The disclosed compounds or a pharma- ceutically acceptable salt of the compound can also be included in a pharmaceutical composition in combination with one or more other therapeutically active compounds. In embodiments, the pharma- ceutically acceptable carrier used can be, for example, solid, liquid, or gas. Examples of solid carriers include lactose, mannitol, microcrystalline cellulose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil, ethanol, propylene glycol, and water. Examples of gaseous carriers include carbon dioxide, nitrogen, and compressed air.

[0139] Any convenient pharmaceutical medium can be used when preparing the composition into a dosage form. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like can be used to form oral liquid preparations such as suspensions, elixirs, and solutions, while carriers such as starch, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrants, and the like can be used to form oral solid preparations such as powders, capsules, and tablets. Because of ease of administration, tablets and capsules are the preferred oral dosage units where solid pharmaceutical carriers are used. If necessary, tablets can be coated by standard aqueous or non-aqueous techniques. In further aspects, aerosol carriers, propellants, liquid carriers, and gas carriers such as sugars, including glucose, fructose, mannitol, sucrose, lactose, and cellulose, can be utilized to form a formulation suitable for inhalation. In certain aspects, the composition is administered intravenously, intraperitoneally, intramuscularly, orally, subcutaneously, intratumorally, or transdermally. In a preferred embodiment, the composition is administered intravenously.

[0140] In certain embodiments, the compound is administered in a therapeutically effective amount. In further embodiments, the compound is administered in a prophylactically effective amount. The molecular weights referred to herein, as well as the number and degree of conjugation of receptor substrates, chains, and therapeutic moieties attached to the dextran backbone, refer to the average amount of a given amount of carrier molecule, since some variation occurs depending on the synthetic technique.

[0141] method In living humans and animals, activated macrophages with a mixed activation phenotype, with characteristics of both M1-like and M2-like phenotypic states, have been observed. Examples of stimuli that can induce an M1-like phenotype in macrophages include tumor necrosis factor (TNF), interferon gamma (INFγ), and Toll-like receptor (TLR) agonists such as lipopolysaccharide (LPS). Examples of stimuli that can induce an M2-like phenotype in macrophages include interleukin 4 (IL4), interleukin 13 (IL13), and transforming growth factor beta (TGFβ). There are many other hormones, cytokines, chemokines, and environmental factors that can affect the phenotype of macrophages.

[0142] Examples of cell surface markers and secreted proteins whose expression levels frequently change in M1-like and M2-like activated macrophages are shown in Table 1. In addition, two immune checkpoint receptors, PD-1 and SIRPα, can be expressed on the surface of macrophages. When these immune checkpoint receptors bind to their ligands, PD-L1 and CD47, respectively, signals are generated that suppress the phagocytic activity of macrophages. M1-like macrophages are expected to attack and phagocytose recognized pathogens or tumor cells. However, if M1-like macrophages express PD-1 and / or SIRPα bound to their ligands, phagocytosis will be suppressed.

[0143] [Table 1]

[0144] Under the influence of M2-like TAMs, the tumor immune microenvironment becomes tumor-promoting and immunosuppressive, suppressing the antitumor activity of other immune cells, such as lymphocytes. The tumor-promoting and immunosuppressive activities of M2-like TAMs reduce the efficacy of anticancer therapies, perhaps most notably, anticancer immunotherapy. For these reasons, TAMs are important therapeutic targets in cancer. Cancer treatment strategies targeting TAMs include (1) killing or eliminating TAMs, (2) blocking the recruitment of TAMs to tumors, or (3) forcing TAMs to switch their phenotype from M2-like to M1-like. This third strategy is sometimes called TAM repolarization or TAM reeducation. M1-like TAMs attack tumor cells and stimulate other types of immune cells, such as lymphocytes, to do the same.

[0145] In certain embodiments, methods are disclosed for repolarizing TAMs from an immunosuppressive (M2-like) phenotype to a pro-inflammatory (M1-like) phenotype. In certain aspects, MAD constructs carrying therapeutic agents, such as the disclosed bisphosphonate compounds, are attached via a pH-sensitive hydrazone and linked to a dextran-based backbone using thiol-maleimide conjugation. In certain aspects, at neutral pH, the constructs retain their payload (i.e., therapeutic agent) for a sufficient time to deliver it to a mannose-binding C-type lectin receptor (e.g., CD206, etc.). Upon binding of the mannose-binding C-type lectin receptor to the construct, the receptor / ligand complex is internalized by receptor-mediated endocytosis into an endosome, which naturally becomes acidified to a pH of about 4-5.5. Once inside the acidified endosome, the drug payload is released. In some aspects, the bisphosphonate-bearing MADs are less toxic to human macrophages, yet have a superior ability to change their phenotype to become more pro-inflammatory and anti-tumor. Importantly, they also induce a highly significant decrease in the expression of SIRPα (the "don't eat me" receptor for CD47), which is frequently expressed on cancer cells. Without being limited to any particular theory or mechanism, the combination of the ability to transition to a pro-inflammatory phenotype with reduced expression of SIPRα suggests that TAMs exposed to the disclosed constructs actively attack and phagocytose cancer cells and stimulate other immune cells, such as lymphocytes, to adopt an anti-tumor phenotype.

[0146] In certain embodiments, the method of repolarizing TAM comprises administering to a subject in need thereof an effective amount of a compound disclosed herein.In further embodiments, the method comprises administering a compound comprising a dextran backbone, one or more mannose-binding C-type lectin receptor targeting moieties, and a bisphosphonate compound bound to the dextran backbone via thiol-maleimide conjugation.In further embodiments, the compound comprises a subunit provided by formula (I) disclosed herein.

[0147] In certain embodiments, the compound is administered in a therapeutically effective amount. In further embodiments, the compound is administered in a prophylactically effective amount. In a further aspect, the method further comprises administering the compound intravenously, intraperitoneally, intramuscularly, orally, subcutaneously, intraocularly, intratumoral injection, or transdermally, or delivering the compound directly to the tumor organ by an invasive technique.

[0148] In a further embodiment, the method further comprises administering the composition in combination with at least one other treatment or therapy.In a further embodiment, the other treatment or therapy comprises the simultaneous administration of an anti-cancer agent.In a further embodiment, the other treatment or therapy is chemotherapy.In certain embodiments, the compound is administered alone or in combination with other chemical-based therapeutic agents or radiation therapy or hyperthermia therapy or physical therapy or diet therapy.

[0149] According to further embodiments, the at least one other treatment or therapy is an immunotherapy, e.g., administration of an immunomodulatory agent. According to certain embodiments, the at least one other treatment or therapy is an anti-CTLA4 immunotherapy. In certain embodiments, the immunomodulatory agent is an immunostimulant. In some embodiments, the immunomodulatory agent is a glucocorticoid, hydrocortisone (cortisol), cortisone acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, deoxycorticosterone acetate (doca), aldosterone, non-glucocorticoid steroids, cytostatic agents, alkylating agents, nitrogen mustard (cyclophosphamide), nitrosoureas, platinized compounds, antimetabolites, purine analogs, azathioprine, mercaptopurine, mycophenolic acid, pyrimidine synthesis inhibitors, leflunomide, teriflunomide, folic acid analogs, methotrexate, cytotoxic antibiotics, dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin, antibodies or fusions thereof, antithymocyte globulin, antilymphocyte globulin, anti-IL-2 receptor antibodies, anti-CD3 antibodies, OKT3 (muromonab), otelixizumab, teplizumab, visilani uzumab, anti-CD4 antibody, clenoliximab, keliximab, zanolimumab, anti-CD11a antibody, efalizumab, anti-CD18 antibody, erlizumab, rovelizumab, anti-CD20 antibody, afutuzumab, ocrelizumab, ofatumumab, pascolizumab, rituximab, anti-CD23 antibody, lumiliximab, anti-CD40 antibody, teneliximab, toralizumab, anti-CD40L antibody, ruplizumab, anti-CD62L antibody, acelizumab, anti-CD80 antibody, galiximab, anti-CD147 antibody, gavilimo Mabs, B-lymphocyte stimulator (BLyS) inhibitor antibodies, belimumab, anti-PD1 (anti-programmed cell death protein 1, or anti-CD279) antibodies or antibody fragments, anti-PD-L1 (anti-programmed death-ligand 1, anti-B7 homolog 1 (B7-H1), or anti-CD274) antibodies or antibody fragments, anti-CTLA4 (anti-cytotoxic T-lymphocyte-associated protein 4, or anti-CD152) antibodies or antibody fragments, CTLA4-Ig fusion proteins, abatacept, belatacept, ipilimumab, tremelimumab,Anti-eotaxin 1 antibody, bertilimumab, anti-α4 integrin antibody, natalizumab, anti-IL-6R antibody, tocilizumab, anti-LFA-1 antibody, odulimomab, anti-CD25 antibody, basiliximab, daclizumab, inolimomab, anti-CD5 antibody, zolimomab, anti-CD2 antibody, siplizumab, nerelimomab, faralimomab, atlizumab, atolimumab, cedelizumab, dorlimomab alitoxin, dorlixizumab, fontolizumab, gantenerumab, gomilikimab, lebrilizumab, maslimomab, morolim Mab, pexelizumab, reslizumab, rovelizumab, talizumab, terimomab alitox, bapaliximab, beparimomab, aflibercept, alefacept, rilonacept, immunophilin modulator, rapamycin, calcineurin inhibitor, tacrolimus, cyclosporine, pimecrolimus, avetimus, gusperimus, ridaforolimus, everolimus, temsirolimus, zotarolimus, TNF inhibitor, infliximab, adalimumab, certolizumab pegol, golimumab, etanercept, thalidomide, lenalid Mydo, pentoxifylline, bupropion, curcumin, catechin, IL-1 receptor antagonist, anakinra, anti-IL-5 antibody, mepolizumab, IgE inhibitor, omalizumab, talizumab, IL12 inhibitor, IL23 inhibitor, ustekinumab, opioid, IMPDH inhibitor, mycophenolic acid, myriocin, fingolimod, NF-κB inhibitor, raloxifene, drotrecogin alfa, denosumab, NF-κB signaling cascade inhibitor, disulfiram, olmesartan, dithiocarbamate, proteasome inhibitor, bortezomib, M G132, Pro1, NPI-0052, curcumin, genistein, resveratrol, parthenolide, thalidomide, lenalidomide, flavopiridol, nonsteroidal anti-inflammatory drugs (NSAIDs), arsenic trioxide, dehydroxymethylepoxyquinomycin (DHMEQ), 13C(indole-3-carbinol) / DIM(di-indolemethane)(I3C / DIM), Bay11-7082, luteolin, cell-penetrating peptide SN-50, IκBα super-repressor overexpression, NFκB decoy oligodeoxynucleotide (ODN),or any derivative or analogue thereof.

[0150] In an exemplary embodiment, the combined administration of the compound and at least one treatment or therapy is synergistically or additively effective compared to either administered alone. According to certain embodiments, administering the compounds disclosed herein in combination with another therapy or treatment reduces toxicity compared to administering the other therapy or treatment alone.In further embodiments, co-administration of the compounds disclosed herein with the other therapy or treatment produces synergistic effects.In further embodiments, co-administration of the compounds disclosed herein reduces the effective dosage of the other therapy or treatment.

[0151] The methods provided herein can be performed in an adjuvant setting. In some embodiments, the methods are performed in a neoadjuvant setting, i.e., the methods can be performed before a primary / definitive therapy. In some embodiments, the methods are used to treat individuals who have previously been treated. Any of the treatment methods provided herein can be used to treat individuals who have not previously been treated. In some embodiments, the methods are used as a first-line therapy. In some embodiments, the methods are used as a second-line or subsequent therapy.

[0152] In further embodiments, methods of treating a disease are provided. In some embodiments, the methods comprise administering to a subject in need of treatment an effective amount of a compound disclosed herein. In some aspects, the disease is cancer, an autoimmune disease, or an inflammatory disease.

[0153] According to other embodiments, the subject has been diagnosed with melanoma, breast cancer, lung cancer, pancreatic cancer, renal cancer, ovarian cancer, prostate cancer or cervical cancer, glioblastoma or colorectal cancer, brain and spinal cord cancer, head and neck cancer, thymoma, mesothelioma, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, bile duct cancer, bladder cancer, testicular cancer, germ cell tumors, ovarian cancer, cervical cancer, endometrial cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, or any combination thereof.

[0154] In certain embodiments, the method further comprises administering the composition as a bolus and / or at regular intervals. In certain embodiments, the disclosed method further comprises administering the composition intravenously, intraperitoneally, intramuscularly, orally, subcutaneously, intratumorally, or transdermally.

[0155] According to certain further embodiments, the method further comprises diagnosing the subject with cancer. In a further aspect, the subject is diagnosed with cancer prior to administration of the composition. According to a further aspect, the method further comprises evaluating the effectiveness of the composition. In a further aspect, evaluating the effectiveness of the composition comprises measuring a tumor size prior to administration of the composition and measuring a tumor size after administration of the compound. In a further aspect, evaluating the effectiveness of the composition is performed at regular intervals. According to certain aspects, the disclosed method further comprises optionally adjusting at least one aspect of the method. In a further aspect, adjusting at least one aspect of the method comprises changing the dosage of the composition, the frequency of administration of the composition, or the route of administration of the compound.

[0156] According to certain alternative embodiments, the subject has been diagnosed with a disease associated with elevated levels of CD206+ macrophages, dendritic cells, and / or myeloid-derived suppressor cells (MDSCs), or in which such CD206+ cell types contribute to the pathobiology of the disease, including, but not limited to, acquired immune deficiency syndrome (AIDS), acute disseminated encephalomyelitis (ADEM), Addison's disease, agammaglobulinemia, allergic diseases, alopecia areata, Alzheimer's disease, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, antisynthetase syndrome, arterial plaque disorders, asthma, atherosclerosis, cardiovascular disease, atopic allergy, atopic dermatitis, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune inflammatory bowel disease ... Infectious hepatitis, autoimmune hypothyroidism, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, Balo disease / Balo concentric sclerosis, Behcet's disease, Berger's disease, Bickerstaff encephalitis, Blau syndrome, bullous pemphigoid, Castleman disease, celiac disease, Chagas disease, chronic inflammatory demyelinating multiple myelopathy chronic recurrent multiple osteomyelitis, chronic obstructive pulmonary disease, chronic venous stasis ulcer, Churg-Strauss syndrome, cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, complement component 2 deficiency, contact dermatitis, cranial arteritis, CREST syndrome, Crohn's disease, Cushing's syndrome, cutaneous leukocytoclastic vasculitis, Degos disease, Derkam's disease, dermatitis herpetiformis, dermatomyositis, type 1 diabetes, type 2 diabetes, diffuse cutaneous systemic sclerosis, Dressler's syndrome, drug-induced lupus, discoid lupus erythematosus, eczema, emphysema, fetal Endometriosis, Enthesitis-related arthritis, Eosinophilic fasciitis, Eosinophilic gastroenteritis, Eosinophilic pneumonia, Epidermolysis bullosa acquisita, Erythema nodosum, Erythroblastosis fetalis, Essential mixed cryoglobulinemia, Evans syndrome, Fibrodysplasia ossificans progressiva, Fibrosing alveolitis (or idiopathic pulmonary fibrosis), Gastritis, Gastrointestinal pemphigoid, Gaucher disease, Glomerulonephritis, Goodpasture syndrome, Graves' disease, Guillain-Barre syndrome (GBS), Hashimoto's encephalopathy, Hashimoto's thyroiditis, Heart disease, Henoch-Schönlein purpura, Herpes gestationis (also known as pemphigoid gestationis),Hidradenitis suppurativa, HIV infection, Hughes-Stovin syndrome, hypogammaglobulinemia, infections (including bacterial, viral, parasitic, and helminthic infections), idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA nephropathy, inclusion body myositis, inflammatory arthritis, inflammatory bowel disease, inflammatory dementia, interstitial cystitis, interstitial pneumonia, juvenile idiopathic arthritis (aka juvenile rheumatoid arthritis), Kawasaki disease, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear IgA disease (LAD), lupoid hepatitis (aka autoimmune hepatitis), lupus erythematosus, Lymphomatoid granulomatosis, Majeed syndrome, malignancies including cancer (e.g., sarcoma, Kaposi's sarcoma, lymphoma, leukemia, carcinoma, and melanoma), Meniere's disease, microscopic polyangiitis, Miller Fisher syndrome, mixed connective tissue disease, morphea, Mouka-Haberman disease (also known as pityriasis lichenoidis acute), multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (also known as Devic's disease), neuromyotonia, ocular cicatricial pemphigoid, opsoclonus-myoclonus syndrome, Ord's thyroiditis, relapsing rheumatism, PANDAS (pediatric autoimmune neuropathies associated with streptococcus) psychiatric disorders), paraneoplastic cerebellar degeneration, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Parsonage-Turner syndrome, peripheral uveitis, pemphigus vulgaris, peripheral arterial disease, pernicious anemia, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pyoderma gangrenosum, pure red cell aplasia, Rasmussen's encephalitis, Raynaud's phenomenon, relapsing polychondritis, Reiter's syndrome, restenosis, restless legs syndrome, retroperitoneal fibrosis, rheumatoid arthritis rheumatic fever, sarcoidosis, schizophrenia, Schmidt syndrome, Schnitzler syndrome, scleritis, scleroderma, sepsis, acute respiratory distress syndrome (ARDS), serum sickness, Sjogren's syndrome, spondyloarthropathy, Still's disease (adult onset), stiff-person syndrome, stroke, subacute bacterial endocarditis (SBE), Susac syndrome, Sweet syndrome, Sydenham chorea, sympathetic ophthalmia, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis (also known as "giant cell arteritis"), thrombocytopenia, Tolosa-Hunt syndrome, transplant (e.g., heart / lung transplant) rejection, transverse myelitis, tuberculosis,These include, but are not limited to, ulcerative colitis, undifferentiated connective tissue disease, undifferentiated spondyloarthropathy, urticarial vasculitis, vasculitis, vitiligo, and Wegener's granulomatosis.

[0157] Further, the present disclosure provides a method for preparing the compound.In certain embodiments, the method for preparing the compound according to formula (I) may include the steps of: (a) synthesizing a dextran backbone with one or more amine-terminated chains; (b) synthesizing a bisphosphonate compound with a carbonyl functional group; (c) reacting the carbonyl functional group with an acyl hydrazide to form a bisphosphonate compound modified with a hydrazone moiety; (d) modifying one or more amine-terminated chains with a maleimide moiety; and (e) replacing one or more maleimide moieties from step (d) with the bisphosphonate compound modified with a hydrazone moiety from step (c) via thiol-maleimide conjugation.

[0158] In one aspect, steps (a)-(e) need not be performed in exactly the same order. In further aspects, additional steps may be performed between each of steps (a)-(e). In some embodiments, step (d) may be performed before step (b), after step (b), before step (c), or after step (c). In further embodiments, step (a) is performed before any of steps (b)-(e). In further embodiments, step (e) is performed after any of steps (a)-(d).

[0159] In certain embodiments, the bisphosphonate compound is prepared using hydrazone before being attached to the MAD construct. In a further aspect, the bisphosphonate compound is substituted with a carbonyl functional group before being modified to the hydrazone moiety. In some embodiments, the MAD construct is prepared with at least one chain attached to a maleimide moiety before adding the bisphosphonate compound. In an exemplary embodiment, the bisphosphonate compound modified with a hydrazone moiety is attached to the MAD construct via thiol-maleimide conjugation.

[0160] In some embodiments, the mannose-binding C-type lectin receptor targeting moiety is further attached to one or more amine-terminated chains. In certain embodiments, the mannose-binding C-type lectin targeting moiety is attached to about 15% to about 70%, about 17% to about 65%, or about 20% to about 60% of the glucose residues via amine-terminated chains. In further embodiments, the mannose-binding C-type lectin targeting moiety is attached to up to about 60%, up to about 70%, up to about 80%, up to about 90%, or up to about 100% of the glucose residues via amine-terminated chains. In certain aspects, the percentage may vary depending on the size of the dextran backbone.

[0161] Further discussion regarding how to prepare the compounds can be found in the non-limiting examples provided herein. All publications and patent applications in this specification are indicative of the level of ordinary skill in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. EXAMPLES

[0162] The embodiments of the present disclosure are further defined in the following non-limiting examples. It should be understood that these examples, while showing specific embodiments of the present disclosure, are given by way of illustration only. From the above discussion and these examples, those skilled in the art can ascertain the essential features of the present disclosure, and can make various changes and modifications to the embodiments of the present disclosure to adapt them to various applications and conditions without departing from the spirit and scope thereof. Thus, in addition to those shown and described herein, various modifications of the embodiments of the present disclosure will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0163] As a drug delivery construct targeting CD206-expressing cells, mannosylated amine dextran (MAD) bearing a bisphosphonate payload (MAD-BIS) was synthesized in three parts: (1) synthesis of the MAD backbone, (2) synthesis of the bisphosphonate drug payload, and (3) modification of the MAD backbone and bisphosphonate drug payload to enable conjugation with a pH-sensitive hydrazone linker using thiol-maleimide conjugation. After MAD-BIS synthesis was completed, the MAN-BIS construct was evaluated to determine its ability to release the bisphosphonate payload at physiological temperature (approximately 37° C.) and under the mildly acidic conditions found within endosomes.

[0164] Example 1: Synthesis of MAD-BIS-1 and MAD-BIS-2 drug delivery constructs Synthesis of MAD backbone: Starting with 10 kDa (Mw) dextran, the MAD backbone was synthesized as described in U.S. Patent No. 6,409,990 (previously incorporated by reference in its entirety) for mannosylated amine DTPA dextran, except that the conjugation of the chelator (i.e., DTPA) was omitted. The resulting construct had a combination of (1) a glucose moiety modified by attachment of an amine-terminated chain to which a mannose moiety was conjugated, (2) a glucose moiety having an amine-terminated chain to which no mannose moiety was conjugated (i.e., a free amine-terminated chain), and (3) an unmodified glucose moiety having neither an amine-terminated chain nor a conjugated mannose. The resulting construct had the following structure:

[0165] [ka]

[0166] The free amine-terminated chains can later be utilized as attachment points for drug payloads via degradable linkers. In the above structure, for simplicity, the amine-terminated chains are shown attached to the C2 hydroxyl groups of the glucose moieties of the dextran polymer, but these chains may be distributed among any of the hydroxyl groups of the MAD backbone. In this example, the MAD backbone had about 36 amine-terminated chains with an average of 22 mannose moieties attached, leaving about 14 free amines. Multiple mannose moieties were required to allow high affinity binding to CD206. The MAD backbone also had an average of about 16 glucose moieties that were not modified by the addition of amine-terminated chains. This MAD backbone construct is provided by way of example only. MAD backbone suitable for drug delivery of bisphosphonate payloads to CD206-expressing cells can be constructed using initial dextran polymers with average molecular weights (Mw) ranging from 1 kDa to over 150 kDa. Varying the initial Mw of the starting dextran will determine the final size (Mw) of the final drug delivery construct. Drug delivery vehicles of different Mw have different biodistributions when administered to either animal or human subjects. These differences in biodistribution may allow the optimization of drug delivery vehicles for different pharmacological applications. In addition, the number of amine-terminated chains and / or the number of conjugated mannose moieties can be intentionally changed, theoretically ranging from 0 to the number of hydroxyl groups on the glucose moiety of the initial dextran. Changing the number of free amines and mannose moieties also changes the biodistribution and CD206 receptor affinity, which may allow the optimization of drug delivery vehicles for different pharmacological applications.

[0167] Synthesis of bisphosphonate drug payloads (BIS-1 and BIS-2): To form a hydrazone bond with desirable hydrolysis kinetics, one coupling partner (either the drug or the dextran backbone) bears a carbonyl moiety and the other bears a hydrazide moiety. Because commercially available bisphosphonates (including zoledronate) do not contain either of these groups, bisphosphonates BIS-1 and BIS-2, which bear a ketone functional group on the aromatic ring, were prepared as payloads for MAD-BIS CD206-targeted drug delivery vehicles. All reagents used in the synthesis of these new bisphosphonates were purchased from commercial suppliers. As shown in Figure 1, both syntheses were initiated by the addition of an arylamine to tetraethylvinylidene bisphosphate. For the first bisphosphonate (BIS-1), 2-amino-4-methyl-5-acetylthiazole was mixed with tetraethylvinylidene bisphosphonate in N,N-dimethylformamide (DMF) and heated at 70°C with constant stirring for 15 hours. The contents were cooled and consumption of the starting vinylidene was confirmed by thin layer chromatography (5% MeOH / DCM, KMnO4 stain). The reaction slurry was then dry loaded onto 20 g of silica. Column purification with a 2-10% methanol-dichloromethane gradient afforded the desired protected adduct as a clear oil (3.24 g, 85%).

[0168] To remove the ethylphosphonate protection, 0.973 g (2.13 mmol) of the purified oil was dissolved in 16 ml of anhydrous acetonitrile (ACN) under nitrogen and 8 equivalents (2.25 ml, 17 mmol) of trimethylsilyl bromide (TMSBr) was added dropwise. The reaction was confirmed to be complete by LC-MS at 4 h and the solution was concentrated in vacuo. Precipitation with water and acetone afforded compound BIS-1, 2-(4-methyl-5-acetylthiazol-2-yl-amino)ethylidene 1,1-bisphosphonic acid as an off-white powder (606 mg, 83%).

[0169] The synthesis of BIS-2, 2-(4-acetylimidazole)ethylidene-1-methyl-1,1-bisphosphonic acid, is further illustrated in Figure 1. BIS-2 was prepared by mixing 1 g (3.3 mmol) of vinylidene bisphosphonate and 0.55 g (5.0 mmol) of 4-acetylimidazole with 20 ml of anhydrous tetrahydrofuran (THF) in a pressure bottle. The bottle was sealed and heated at 75 °C with stirring for 3 h. The contents were cooled and acceptable reaction progress was confirmed by ESI+LC-MS. The reaction was filtered to remove excess imidazole and concentrated in vacuo. The crude material (1.13 g, HPLC purity approx. 70%) was not stable to purification, instead the starting 4-acetylimidazole and vinylidene were removed. Therefore, it was necessary to add a non-hydrogen functionality at the alpha position. Therefore, prior to purification, the crude intermediate was reconstituted in 35 ml of anhydrous DMF, cooled to -40°C, and 88 mg of 60% sodium hydride (NaH) dispersion in oil (53 mg, 2.2 mmol) was added. After stirring for approximately 10 minutes, 0.14 ml (2.2 mmol) of methyl iodide (MeI) was added, stirred for 15 minutes, and equilibrated to ambient temperature for 1 hour. The crude product was stable to purification by silica column chromatography using 5% methanol in dichloromethane to give 154 mg (11% overall yield) of the pure methylated protected bisphosphonate as a pale yellow oil.

[0170] The isolated yield of the previously described BIS-2 synthesis was affected by the formation of by-products during the methylation reaction. Below is an alternative synthesis in which the imidazole ketone is protected as a ketal (1,3-dioxolane, 3) prior to addition to the vinylidene.

[0171] To prepare N-pivaloyloxymethyl (POM) protected 4-acetylimidazole (1), 5.0 g (45.4 mmol) of 4-acetylimidazole was suspended in 100 ml of anhydrous THF and cooled on an ice bath. 1.2 eq. of NaH (1.31 g, 54.5 mmol, 2.18 g of 60% dispersion in oil) was added and the suspension was stirred for 40 min under nitrogen atmosphere. 1.2 eq. (54.5 mmol, 7.85 ml) of chloromethyl pivalate was added and stirred for 30 min on an ice bath and the reaction was continued at ambient temperature overnight. The contents of the flask were diluted with ethyl acetate and saturated sodium bicarbonate, the organics were washed with brine, dried over anhydrous sodium sulfate and concentrated to give an off-white solid. Single crystallization from boiling ethyl acetate and hexanes gave 9.04 g (89%) of POM protected acetylimidazole (1) as white needles.

[0172] 4.88 g (21.8 mmol) of POM-protected acetylimidazole (1) was mixed under nitrogen with 70 ml of freshly distilled ethylene glycol and 25 ml of anhydrous dichloromethane (DCM). Eight equivalents (0.174 mol, 22 ml) of trimethylsilyl chloride were added over several minutes and the opaque solution was stirred overnight at ambient temperature under nitrogen. The reaction was concentrated in vacuo and placed on high vacuum to remove excess reagents, then diluted with saturated sodium bicarbonate and the product extracted with DCM. The organics were washed and dried with saturated brine and anhydrous sodium sulfate, filtered and concentrated to give 3.42 g of an off-white solid. Crystallization from warm ethyl acetate and hexanes gave 2.44 g (42%) of the fully protected ketal product (2) as a white powder.

[0173] 2.28 g (8.5 mmol) of the fully protected imidazole (2) was dissolved in 45 ml of 7 M ammonia in methanol, the flask was sealed, and the POM was removed by stirring at room temperature overnight. The reaction was completed by high sensitivity ESI+LC-MS and concentrated in vacuum twice with methanol without a water bath. The solid residue was dissolved in a small amount of methanol over a 50°C water bath and equilibrated to ambient temperature with stirring while exposed to air and unsealed. The flask was sealed and placed in a -20°C freezer for further crystallization. After filtration and placing under high vacuum, 756 mg (58% yield) of 5-(2-methyl-1,3-dioxolan-2-yl)-1H imidazole (3) was recovered as a bright white powder (calculated mass 154.07, found ESI+LC-MS [M+1] 155.09).

[0174] Under nitrogen atmosphere, 55 mg (0.36 mmol) of 5-(2-methyl-1,3-dioxolan-2-yl)-1H-imidazole (3) in 1 ml of anhydrous THF was added with 106 mg (0.35 mmol) of tetraethylvinylidene-1,1-bisphosphonate in 1 ml of anhydrous THF. After stirring at room temperature for 2 hours, a satisfactory addition product was formed by sensitive ESI+LC-MS. The flask was cooled on an ice bath and charged with 1.05 eq (9 mg, 0.37 mmol, 15 mg of 60% dispersion in oil) of NaH and stirred for 15 minutes. Next, 1.5 eq (0.53 mmol, 33 uL) of iodomethane was added to the reaction mixture, which was removed from the ice bath and stirred overnight. The mixture was diluted with DCM and 0.1 M pH 7 PBS, and the organics were washed with brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. A gradient of 0-10% MeOH in DCM on a 10 g silica column gave 99 mg (59% yield) of the fully protected BIS-2 product as a clear oil (calculated 468.18, found ESI+LC-MS [M+1] 469.28).

[0175] To deprotect the phosphonate group, 0.148 g (0.349 mmol) of the purified oil was dissolved in 2.7 ml of anhydrous acetonitrile (ACN) under nitrogen and 8 equivalents (0.37 ml, 2.8 mmol) of trimethylsilyl bromide (TMSBr) were added dropwise. The reaction was confirmed to be complete by LC-MS at 4 hours and the solution was concentrated in vacuo. For ketal protection, the reaction was diluted with water and lyophilized after completion of phosphonate deprotection. The crude material was purified by C-18 reverse phase chromatography using water as the mobile phase. Pure fractions were combined, frozen, and lyophilized to give BIS-2 as a pale green powder (74.4 mg).

[0176] Modification of the MAD backbone and bisphosphonate drug payload to allow for linkage via a pH-sensitive hydrazone linker: BIS-1 and BIS-2 were further modified to incorporate pH-sensitive hydrazones and functional groups for linkage to MAD, as shown in Figure 2. The hydrazones of BIS-1 and BIS-2 (7-9 and 9') were modified to incorporate functional groups for linkage to the side chain (R 2 ) were successfully prepared by condensation with acyl hydrazides (4-6) that have different functional groups and therefore different chemical strategies for attachment to the MAD backbone. However, subsequent attempts to link the BIS-1 hydrazone azide (7) via the click chemistry method disclosed in US Patent Application No. 63 / 294,996 were unsuccessful. Direct coupling of the BIS-1 hydrazone (8) carboxylic acid, and hydrazone formation by condensation reaction of the carbonyl groups on BIS-1 and BIS-2 with hydrazides attached to the MAD backbone also failed to deliver the desired conjugation products.

[0177] However, conjugation of BIS-1 and BIS-2 hydrazones (9) and (9') to MAD was successful using thiol-maleimide conjugation, as shown in Figure 3. The thiol coupling partner was intentionally placed on the hydrazone (9 / 9') and the maleimide on MAD, because if reversed, unreacted thiols attached to the backbone could crosslink as disulfides after conjugation. As shown in Figure 3, the MAD backbone was first modified by adding a maleimide moiety to the free amine-terminated chain. In the second step, (9) or (9') was placed into an aqueous solution containing maleimide-derivatized MAD.

[0178] To prepare MAD-BIS-1 (13), 90 mg of mannose dextran (MAD) (1H NMR: average 22-mannose, 14 amines, Mw 19,393 g / mol) was dissolved in 3.6 ml of 0.05 M PBS buffer (pH 7.2) and the pH was adjusted to 7.0 with a minimum amount of phosphoric acid and 1 N NaOH. In a separate flask under inert atmosphere, 33 mg (0.195 mmol) of 3-maleimidopropionic acid and 24 mg (0.205 mmol) of N-hydroxysuccinimide were dissolved in 0.28 ml of anhydrous DMF. 39 mg (0.205 mmol) of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC·HCl) was added, the flask was sealed, and the activated mixture was stirred at room temperature for 1 h. The entire DMF solution of 3-maleimidopropionic acid N-hydroxysuccinimide ester (3 equivalents of NHS ester relative to the amine concentration of dextran) was charged to the flask containing mannose dextran. The progress of the reaction was monitored by following the complete loss of amine content in the solution. Upon completion, the reaction was transferred to a 10 kDa MWCO Amicon centrifugal spin filter, diluted to 12 ml with 0.05 M PBS buffer (pH 7.1) and concentrated to a volume of approximately 0.5 ml. Ultrafiltration was repeated for two more cycles and the final retentate containing MAD maleimide (10) was transferred from the spin filter and diluted to 3 ml with 0.05 M PBS buffer (pH 7.1). 58 mg (0.130 mmol) of bisphosphonate hydrazone (9), which is 2 equivalents relative to the maleimide content on (10), was added and stirred at ambient temperature for 1.5 h. The reaction was diluted to 12 ml with distilled water and concentrated to 0.5 ml in a 10 kDa MWCO centrifugal spin filter. The retentate was washed five more times with distilled water, passed through a 0.45 μm syringe filter, frozen and lyophilized to give 76.8 mg of MAD-BIS-1 (13). This construct had an average of 6.3 BIS-1 moieties per MAD as determined by HPLC analysis after complete hydrolysis from the backbone. The synthesis of MAD-BIS-2 (13') followed the same procedure, substituting BIS-2 hydrazone (9'). 53.5 mg of MAD-BIS-2 (13') was prepared (average of 6.0 BIS-2 moieties per dextran).

[0179] An important difference between this chemical method and those disclosed in U.S. Pat. No. 10,806,803 or in the current scientific literature is that in the prior literature, a hydrazine moiety is first attached to the drug delivery vehicle (in this case the MAD backbone) and in a second step, a point of attachment to the drug payload is created as a hydrazone. In certain embodiments of the present disclosure, a hydrazone linker is formed as a derivative of the drug payload, and the drug-hydrazone construct is then attached to the drug delivery vehicle using a different chemical method than described in the prior literature. Furthermore, the entire linker that attaches the drug payload to the amine-terminated chain on the MAD backbone in the final synthesis product is unique. All three hydrazone linkers shown in FIG. 2 contained the same hydrazone moiety, but different side chain alterations (R 2 ) but the results were all R 2 We have demonstrated that the thiol-terminated R 2 Conjugation was successful only between group (6) and MAD maleimide (10). This observation highlights the unexpected results achieved with the constructs of the present disclosure, as only thiol hydrazone (6) was successful. Therefore, the other two R 2 In view of the failure of the group, the success of the thiol hydrazone (6) was surprising.

[0180] Release of bisphosphonates from MAN-BIS-1 at pH 4.65 (37°C): After the synthesis of MAN-BIS-1 was completed, samples were evaluated by HPLC. Examination of the HPLC results revealed that MAN-BIS-1 was free of BIS-1 and BIS-1 with hydrazone linkers. A 1 mg / ml solution of MAN-BIS-1 in 1N HCl was then evaluated after 1 and 2 hours to determine the amount of BIS-1 released. All of the BIS-1 should have been released under these highly acidic conditions. The amount of BIS-1 released under these highly acidic conditions was found to be 9.2% wt / wt or more than 6 BIS-1 moieties per MAD backbone.

[0181] Next, an experiment was performed in which MAD-BIS-1 was dissolved in an aqueous solution at pH 4.65. Samples were analyzed after exposure to pH 4.65 for various times. The amount of BIS-1 released over time was monitored by HPLC. Free BIS-1 was not detected at 5 and 30 minutes, but was observed after 2 hours of incubation at pH 4.65. The release was monitored over 47 hours, as shown in Figure 4. BIS-1 was released gradually over the duration of the experiment. After 47 hours of incubation, 6.4% by weight (>4 drug moieties per dextran chain) of free BIS-1 was detected by HPLC.

[0182] This observation highlights an important feature of the constructs, since they allow for a gradual and sequential release of the drug payload to CD206-expressing cells. This would not have been possible if the drug had not been delivered on the MAD scaffold. If BIS-1 or other bisphosphonates were administered without the MAD delivery construct, it would be rapidly cleared from the blood and localized to the bone. The MAD-BIS construct allows for continuous exposure of the drug to CD206-expressing cells over a relatively long period of time.

[0183] Example 2: Bisphosphonate 1 (MAD-BIS-1) - Evaluation in a human macrophage cell culture assay MAD-BIS-1 was evaluated in a human macrophage cell culture assay. In this assay, human peripheral blood monocytes (hPBMCs) were incubated in 48-well tissue culture plates at a concentration of 500,000 monocytes per well in RPMI + 10% FBS + 1X penicillin / streptomycin / L-glutamine + 50ng / ml GM-CSF (complete medium) for 5 days. During this 5-day incubation, monocytes differentiated into macrophages. GM-CSF induces macrophages to adopt an activated phenotype that is intermediate between the extremes of M1 or M2. After 5 days of incubation, the medium was removed and replaced with complete medium supplemented with various concentrations of MAD-BIS-1 or unbound BIS-1 (free Bis-1). Saline and vehicle (MAD without drug payload, 80μg / ml) were added as alternative supplements to other cultures to serve as negative controls. Macrophage cell cultures were incubated with the supplemented complete medium for 24 hours, after which the supplemented complete medium was removed and replaced with fresh complete medium. Macrophage cultures were then incubated for an additional 3 days. The 3-day incubation period following treatment was performed to allow for the assessment of any permanent changes in macrophage phenotype. After an additional 3-day incubation in fresh complete medium, cells were harvested and assessed by flow cytometry for viability (DAPI-) or expression of macrophage surface markers, either representative of markers well considered to be indicative of either an M1-like or M2-like phenotype, or known immune checkpoint receptors.

[0184] The cell surface markers assessed using antibodies specific for each marker were CD206, CD163, CD80, CD86, MHC1, MHC2, SIRPα, and PD-1. The observed amounts of each surface marker varied considerably between markers and treatment groups, but nearly all live cells expressed detectable amounts of all markers. The marker with the lowest expression in saline and vehicle-treated controls was PD-1. The output of the flow cytometry assay was mean fluorescence intensity (MFI). Macrophages differentiated from monocytes from three separate donors were assessed for all treatments. All experiments were performed in triplicate for macrophages from each donor.

[0185] Results: In all repetitions of this experiment, the majority of macrophages treated with saline controls survived until the end of the experiment (9 days). For cell viability and all surface markers evaluated, the results for drug-free vehicle-treated controls were not statistically different from those observed in the saline (no drug) controls, indicating that no pharmacological activity was observed in the drug-free vehicle in this study. Furthermore, neither MAD-BIS-1 nor free BIS-1 reduced cell viability at any of the concentrations tested.

[0186] Figure 5 provides a summary representation of how various treatments with either MAD-BIS-1 or free BIS-1 affected the expression levels of the eight surface markers evaluated. Values ​​shown are the mean fold change (MFI) in expression compared to the MFI values ​​observed in saline controls. The statistical significance of these changes in expression was measured by Z-test.

[0187] The data presented in Figure 5 show the fold change in expression of the indicated surface markers treated with either 80 μg of MAD-BIS-1 or an equimolar amount of free BIS-1 not bound to the mannosylated amine dextran vehicle. A fold change of 1.0 indicates no change in expression, and the mean expression level (MFI) observed in treated cells was the same as that observed in the saline control. A value of 2.0 means that the mean MFI of treated cells was twice that observed in the saline treated control. Conversely, a value of 0.5 means that the MFI observed in treated cells was half that observed in the saline control.

[0188] Several important observations can be made from the data shown in Figure 5. First, free BIS-1 had little effect on the expression levels of any of the markers tested. This may be because, as a highly charged polar molecule, BIS-1 cannot efficiently cross the cell membrane and interact with effector proteins inside the cell. Conversely, MAD-BIS-1 actively transports BIS-1 into the cell through interaction with CD206 and release of the payload in the mildly acidic conditions of the endosome. MAD-BIS-1 significantly altered the expression of six of the surface markers evaluated. CD206 and CD163, which are frequently associated with an M2-like phenotype, were significantly reduced in expression by more than half. Conversely, CD80 and CD86, which bind to CD28 on T cells and activate T cells, are often considered to be indicative of an M1-like phenotype. Both CD80 and CD86 are significantly increased in expression by treatment with MAD-BIS-1.

[0189] Also important is the observation that MAD-BIS-1 significantly reduced the expression of SIRPα. SIRPα is a receptor for CD47. CD47 is frequently expressed on many types of cells, including many cancer cells. CD47 is called a "don't eat me" signal. When CD47 binds to SIRPα, it inhibits the phagocytic response of macrophages. Reduced expression of SIRPα may indicate a more M1-like pro-inflammatory macrophage phenotype in which macrophages are more likely to phagocytose cancer cells. PD-1 expression is increased by MAD-BIS-1. However, the very low levels of PD-1 expression in saline control-treated macrophages suggests that the small but significant increase in PD-1 expression induced by MAD-BIS-1 may be of limited phenotypic significance.

[0190] Overall, the decreased expression of CD206, CD163, and SIRPα, coupled with increased expression of CD80 and CD86, indicates that treatment with MAD-BIS-1 primed macrophages to adopt a more M1-like pro-inflammatory and anti-tumor phenotype. It is also important to note that the observed changes in macrophage phenotype could not be reproduced by free BIS-1 at any dose. This indicates that BIS-1 activity was dependent on intracellular trafficking mediated by binding of MAD to CD206 and possibly other circulating pattern recognition receptors.

[0191] Example 3: Bisphosphonate 2 (MAD-BIS-2) MAD-BIS-2 was evaluated in a human macrophage culture assay similar to that described in Example 2, with two differences. First, for the changes in cell surface markers selected for presentation, the MAD-BIS-2 concentration selected for comparison was 40 μg / ml, instead of 80 μg / ml for MAN-BIS-1. More importantly, the comparator for MAN-BIS-2 was free zoledronic acid (zoledronate), instead of free BIS-2. Like BIS-1 and BIS-2, zoledronic acid is a nitrogen-containing bisphosphonate. Zoledronic acid is the most pharmacologically active bisphosphonate drug approved by regulatory agencies. In the data shown in Figure 6, the phenotypic changes induced by MAD-BIS-2 were compared to equimolar concentrations of zoledronic acid, rather than free BIS-2.

[0192] As shown in Figure 6, the changes in surface marker expression induced by MAN-BIS-2 were similar to those induced by MAN-BIS-1, with a significant decrease in the expression levels of CD206, CD163, and SIRPα, and an increase in the expression levels of CD80, CD86, and PD-1. One difference was that MAN-BIS-2 induced a slight increase in the expression of MHC2, whereas MAN-BIS-1 induced a slight decrease in the expression of MHC2. A direct comparison of the changes in surface marker expression induced by MAD-BIS-1 (80 μg / ml) and MAD-BIS-2 (40 μg / ml) is shown in Figure 7. The changes in surface marker expression levels induced by MAD-BIS-1 and MAD-BIS-2 were not identical, but were sufficiently similar, suggesting that the two bisphosphonate constructs have a similar, if not identical, pharmacological mechanism of action. As with MAD-BIS-1, MAD-BIS-2 modified the phenotype of human macrophages to be more M1-like, and MAD-BIS-2 was likely more pharmacologically active than MAD-BIS-1.

[0193] As shown in Figure 6, free zoledronic acid was observed to have a similar phenotypic effect on treated macrophages to that observed for MAN-BIS-1 and MAN-BIS-2. Free zoledronic acid reduced the expression of CD206, CD163, and SIRPα, and increased the expression of CD86 and (although not significantly) MHC2. However, although these changes were qualitatively similar to the changes in marker expression seen with treatment with the MAD-BIS-2 construct, the changes observed when macrophages were treated with free zoledronic acid were quantitatively smaller and less statistically significant. The mechanism by which free zoledronate enters macrophages and mediates these modest changes in surface marker expression is unclear, but is likely due to small amounts of drug taken up during pinocytosis. Once inside the cells, zoledronic acid may be more pharmacologically active, but this activity is muted because the highly charged zoledronic acid cannot cross the cell membrane to reach intracellular pharmacological targets. Conversely, BIS-2 may be less pharmacologically active compared to zoledronic acid, but when bound to MAD, it can achieve higher intracellular concentrations through active intracellular trafficking mediated by CD206.

[0194] FIG. 8 shows the results of an experiment similar to that shown in FIG. 6, except that the activity of MAD-BIS-2 was compared to free BIS-2 rather than free zoledronic acid, and the MAD-BIS-2 construct was constructed on a smaller dextran backbone with a starting molecular weight (Mw) of 3.5 kDa compared to the MAD-BIS-2 construct (starting molecular weight (Mw) of 10 kDa) that provided the results shown in FIG. 6. Otherwise, the two MAD-BIS-2 constructs have similar molecular architectures. On a mass basis, the two MAD-BIS-2 constructs carried similar masses of BIS-2 payload. After synthesis was completed, the final molecular weights (Mw) of MAD-BIS-2 (10 kDa dextran) and MAD-BIS-2 (3.5 kD backbone) were approximately 18-22 kDa and 8-11 kDa, respectively. The results, shown in Figure 8, show that the MAD-BIS-2(3.5) construct induced a decrease in the expression of CD206, CD163, and SIRPα, and an increase in the expression of CD80, CD86, and MHC2. Importantly, as with BIS-1, free BIS-2 did not alter the expression levels of any of the surface markers evaluated, indicating that its internalization into cells was also dependent on binding to the MAD drug delivery vehicle.

[0195] Figure 9 shows a direct comparison of surface marker expression results aggregated from the results shown in Figure 6 (MAD-BIS-2, 10 kDa dextran) and Figure 8 (MAD-BIS-2, 3.5 kDa dextran). Figure 9 demonstrates that two MAD-BIS-2 constructs, differing primarily only in the size of the initial dextran backbone and the final molecular weight, induce comparable changes in macrophage surface marker expression (i.e., phenotype) that are statistically nearly identical. The pharmacological effects of MAD-BIS-2 constructs are independent of the size (Mw) of the MAD-BIS-2 constructs or the initial size of their dextran backbone. MAD-BIS-2 constructs constructed on dextran backbones of less than 3.5 kDa, between 3.5 kDa and 10 kDa, or greater than 10 kDa are expected to have similar pharmacological effects as long as they have a sufficient number of mannose moieties to bind potently to CD206. Furthermore, because MAD-BIS-1, MAD-BIS-2, and zoledronic acid are all nitrogen-containing bisphosphonates and all induce a similar pattern of pharmacological effects on macrophage surface marker expression and macrophage phenotype, various other nitrogen-containing bisphosphonate drugs are also expected to induce a similar pattern of pharmacological effects on macrophage surface marker expression and macrophage phenotype when delivered to CD206-expressing cells, such as macrophages, linked to MAD constructs via degradable linkers. Other MAD constructs carrying other nitrogen-containing bisphosphonates may be more or less pharmacologically active compared to MAD-BIS-1 or MAD-BIS-2, but all are expected to induce a similar pattern of changes in surface marker expression, similarly shifting the macrophage phenotype from an M2-like immunosuppressive and tumor-promoting phenotype to a more M1-like anti-tumor phenotype.

[0196] The exemplary mannosylated dextran constructs evaluated have several important properties of note. They are both nitrogen-containing bisphosphonates. Both altered the phenotype of human macrophages to be more M1-like. This indicates that MAD-BIS constructs can be used to tailor the tumor immune microenvironment to be less immunosuppressive and protumor, and more proinflammatory and antitumor. Furthermore, both MAD-BIS constructs significantly reduced the expression of SIRPα, indicating that TAMs treated with MAD-BIS are more likely to phagocytose tumor cells. Furthermore, the pharmacological activity of MAD-BIS constructs is independent of the size of the MAD component of the construct, as demonstrated by experiments with the MAD-BIS-2 construct.

[0197] These results further suggest that MAD-BIS constructs may improve the efficacy of other anti-cancer therapies, such as radiation therapy, cytotoxic therapy, and immunotherapy, by shifting the tumor immune microenvironment to be less immunosuppressive and tumor-promoting.

[0198] An additional advantage of the MAD-BIS construct is that it targets CD206-expressing cells, such as macrophages and dendritic cells, thus reducing off-target toxicity. The MAD-BIS constructs can deliver their payloads internally to targeted CD206-expressing cells, overcoming the challenges associated with the limitations of highly charged polar molecules, such as bisphosphonates, in crossing cell membranes to reach intracellular drug targets. As demonstrated in the Examples, the MAD-BIS constructs have pharmacological activity at least equal to or greater than that of free zoledronic acid, the most active drug in the bisphosphonate drug class.

[0199] The disclosure being thus described, it will be apparent that the same may be modified in many ways. Such variations are not to be regarded as departures from the spirit and scope of the disclosure, and all such modifications are intended to be included within the scope of the following claims.

Claims

1. It is a compound, Polymer carbohydrate backbone, One or more mannose-binding C-type lectin receptor targeting moieties, A compound comprising a nitrogen-containing bisphosphonate compound bonded to the polymer carbohydrate skeleton via a thiol-maleimide conjugation.

2. The compound comprises the subunit shown in formula (I), 【Chemistry 1】 During the ceremony, Each X independently corresponds to H and L １ -A-Z, or L ２ -R, and each X is bonded to an OH group, L １ and L ２ Each of these is independently an amine-terminated chain, Each A independently includes a substituted maleimide moiety or an unsubstituted maleimide moiety. Each Z independently contains or does not contain a bisphosphonate compound modified with a hydrazone moiety. Each R independently comprises the mannose-binding C-type lectin receptor targeting moiety or H. n is an integer greater than 0, and the units of n may be the same or different. The compound according to claim 1, wherein at least one A is the substituted maleimide moiety, and the thiol-maleimide conjugation is between A and Z.

3. At least one X is L １ -A-Z, and at least one X is L ２ The compound according to claim 2, wherein R comprises the mannose-binding C-type lectin receptor targeting moiety.

4. The compound according to claim 1, wherein the molecular weight of the polymer carbohydrate backbone is about 1 kD to about 50 kD.

5. The compound according to claim 1, wherein the mannose-binding C-type lectin receptor targeting moiety comprises a mannosyl-binding aglycone moiety, mannose, high-mannose glycan or mannose oligosaccharide, fucose, N-acetylglucosamine, peptide, galactose, or a combination thereof.

6. At least one L １ is -(CH ２ ), ｐ S(CH ２ ), ｑ -NH-, and p and q are integers from 0 to 5, the compound according to claim 2.

7. at least one L ２ ga- (CH ２ ) ｐ S (CH ２ ) ｑ The compound according to claim 2, comprising -NH-, wherein p and q are integers from 0 to 5.

8. The compound according to claim 2, wherein the bisphosphonate compound is substituted with a carbonyl functional group before being modified with a hydrazone moiety.

9. The compound according to claim 2, wherein the hydrazone portion comprises an acylhydrazone.

10. The compound according to claim 9, wherein the hydrazone portion has the following structure. 【Chemistry 2】

11. R ２ ga-R ４ - Includes SH, R ４ is a substituted or unsubstituted linear or branched C １ ~C １２ The compound according to claim 10, wherein the group is alkyl, alkenyl, alkynyl, or aromatic.

12. R ２ ga- (CH ２ ) ２ The compound according to claim 11, comprising SH.

13. A pharmaceutical composition, A compound according to any one of claims 1 to 12, A pharmaceutical composition comprising a pharmaceutically acceptable carrier.

14. The compound comprises the subunit shown in formula (I), 【Transformation 3】 During the ceremony, Each X independently corresponds to H and L １ -A-Z, or L ２ -R, and each X is bonded to an OH group, L １ and L ２ Each of these is independently an amine-terminated chain, Each A independently includes a substituted maleimide moiety or an unsubstituted maleimide moiety. Each Z independently contains or does not contain a bisphosphonate compound modified with a hydrazone moiety. Each R independently comprises the mannose-binding C-type lectin receptor targeting moiety or H. n is an integer greater than 0, and the units of n may be the same or different. The composition according to claim 13, wherein at least one A is the substituted maleimide moiety, and the thiol-maleimide conjugation is between A and Z.

15. A bisphosphonate compound of formula (II), 【Chemistry 4】 During the ceremony, R １ Each of these is independently H, a positively charged counterion, and a substituted or unsubstituted linear or branched C. １ ~C ６ It is an alkyl group, or an acyloxyalkyl group. X １ H, hydroxyl, C １ ~C ６ Alkyl, or O-C １ ~C ６ It is one of the alkyl groups, Y does not exist, or C １ ~C ６ It is an alkyl group or heteroatom, W is a linear or branched carbon containing at least one nitrogen atom. １ ~C １２ A bisphosphonate compound having an alkyl, alkenyl, alkynyl, aromatic, or heteroaromatic group, where W is substituted with a carbonyl group.

16. The bisphosphonate compound according to claim 15, wherein W is an aromatic or heteroaromatic group, and the aromatic or heteroaromatic group is selected from the group consisting of phenyl, pyridine, pyrimidine, pyridazine, pyrazine, triazine, triazole, pyrazole, imidazole, triazole, tetrazole, thiazole, isothiazole, oxazole, and isoxazole, or pharmaceutically acceptable salts thereof.

17. W is C １ ~C ８ The bisphosphonate compound according to claim 15, further substituted with an alkyl group, an aromatic group, or a heteroaromatic group.

18. W is a thiazole, the carbonyl group is a ketone group, and W is C １ Further substituted with alkyl groups, where Y is NH and X １ A bisphosphonate compound according to any one of claims 15 to 17, wherein is H.

19. W is imidazole, the carbonyl group is a ketone group, Y is absent, X １ C １ A bisphosphonate compound according to any one of claims 15 to 17, wherein the compound is an alkyl group.

20. The bisphosphonate compound according to any one of claims 15 to 17, wherein the bisphosphonate compound has the following formula (III). 【Transformation 5】

21. The bisphosphonate compound according to any one of claims 15 to 17, wherein the bisphosphonate compound has the following formula (IV). 【Transformation 6】

22. The bisphosphonate compound according to any one of claims 15 to 17, wherein the carbonyl group is bonded to an acylhydrazide to form a bisphosphonate compound modified with a hydrazone moiety.

23. The bisphosphonate compound according to claim 22, wherein the acylhydrazide has the following structure: NH ２ -NH-C(O)-R ２

24. The bisphosphonate compound according to claim 22, wherein the hydrazone portion has the following structure. 【Transformation 7】

25. R ２ ga-R ４ - Includes SH, R ４ is a substituted or unsubstituted linear or branched C １ ~C １２ The bisphosphonate compound according to claim 23, wherein the group is alkyl, alkenyl, alkynyl, or aromatic.

26. R ２ ga- (CH ２ ) ２ A bisphosphonate compound according to claim 25, comprising SH.

27. The bisphosphonate compound has the following formula (V): 【Transformation 8】 In the formula, R ２ Ha-R ４ - Includes SH, R ４ is a substituted or unsubstituted linear or branched C １ ~C １２ The bisphosphonate compound according to claim 22, wherein the group is alkyl, alkenyl, alkynyl, or aromatic.

28. R ２ ga- (CH ２ ) ２ A bisphosphonate compound according to claim 27, comprising SH.

29. The bisphosphonate compound has the following formula (VI): 【Chemistry 9】 In the formula, R ２ Ha-R ４ - Includes SH, R ４ is a substituted or unsubstituted linear or branched C １ ~C １２ The bisphosphonate compound according to claim 22, wherein the group is alkyl, alkenyl, alkynyl, or aromatic.

30. R ２ ga- (CH ２ ) ２ A bisphosphonate compound according to claim 29, comprising SH.

31. A method for preparing the compound according to claim 1 or 2, (a) A step of synthesizing a polymer carbohydrate skeleton to which one or more amine terminal chains are attached, Step (b) of synthesizing a nitrogen-containing bisphosphonate compound containing a carbonyl functional group, (c) a step in which the carbonyl functional group is reacted with an acyl hydrazide to form the nitrogen-containing bisphosphonate compound modified with a hydrazone moiety, (d) modifying one or more amine terminal chains with maleimide moieties, A method comprising step (e) substituting one or more maleimide moieties from step (d) with the bisphosphonate compound modified with the hydrazone moiety from step (c) via thiol-maleimide conjugation.

32. The method according to claim 31, wherein step (d) may be performed before step (b), after step (b), before step (c), or after step (c).

33. In equation (I), at least one X is L １ -A-Z, and at least one X is L ２ The method according to claim 31, relating to claim 2, wherein -R comprises the mannose-binding C-type lectin receptor targeting portion.

34. The method according to claim 31, wherein the molecular weight of the polymer carbohydrate backbone is about 1 kD to about 50 kD.

35. The method according to claim 31, wherein the mannose-binding C-type lectin receptor targeting moiety comprises a mannosyl-binding aglycone moiety, mannose, high-mannose glycan or mannose oligosaccharide, fucose, N-acetylglucosamine, peptide, galactose, or a combination thereof.

36. In equation (I), at least one L １ ga- (CH ２ ) ｐ S (CH ２ ) ｑ The method according to claim 31, which references claim 2, wherein the formula includes -NH- and p and q are integers from 0 to 5.

37. In equation (I), at least one L ２ ga- (CH ２ ) ｐ S (CH ２ ) ｑ The method according to claim 31, which references claim 2, wherein the formula includes -NH- and p and q are integers from 0 to 5.

38. The method according to claim 31, wherein the hydrazone portion has the following structure. 【Chemistry 10】

39. R ２ ga-R ４ - Includes SH, R ４ is a substituted or unsubstituted linear or branched C １ ~C １２ The method according to claim 38, wherein the group is alkyl, alkenyl, alkynyl, or aromatic.

40. R ２ is - (CH ２ ) ２ The method according to claim 39, comprising - SH.

41. A pharmaceutical composition for use in repolarizing tumor-associated macrophages (TAMs) from an immunosuppressive (M2-like) phenotype to a pro-inflammatory (M1-like) phenotype, comprising an effective amount of the compound described in claim 1, and for administration to a target.

42. The compound comprises the subunit shown in formula (I), 【Chemistry 11】 During the ceremony, Each X independently corresponds to H and L １ -A-Z, or L ２ -R, and each X is bonded to an OH group, L １ and L ２ Each of these is independently an amine-terminated chain, Each A independently includes a substituted maleimide moiety or an unsubstituted maleimide moiety. Each Z independently contains or does not contain a bisphosphonate compound modified with a hydrazone moiety. Each R independently comprises the mannose-binding C-type lectin receptor targeting moiety or H. n is an integer greater than 0, and the units of n may be the same or different. The pharmaceutical composition according to claim 41, wherein at least one A is the substituted maleimide moiety, and the thiol-maleimide conjugation is between A and Z.

43. The pharmaceutical composition according to claim 41 or 42, wherein the compound is administered in combination with at least one other therapy or treatment, the at least one other therapy or treatment being chemotherapy, radiotherapy, or immunotherapy.

44. The pharmaceutical composition according to claim 41 or 42, wherein the bisphosphonate compound is released from the polymer carbohydrate backbone at a pH of less than approximately 5.

5.

45. A pharmaceutical composition for use in treating a disease, comprising an effective amount of any one of claims 1 to 12, for administration to a subject, A pharmaceutical composition in which the disease is cancer, an autoimmune disease, or an inflammatory disease.

46. The pharmaceutical composition according to claim 45, wherein the compound is administered in combination with at least one other therapy or treatment, the at least one other therapy or treatment being chemotherapy, radiotherapy, or immunotherapy.

47. The pharmaceutical composition according to claim 45, wherein the disease is cancer.

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