Humoral immune activation agents for the treatment of humoral immunosuppressive conditions, cancer and infectious diseases, and humoral immunosuppressive agents for the treatment of inflammatory diseases

Abstract

Compositions, methods, and kits of low molecular weight agents can enhance humoral and immune effector cell responses by binding to and stimulating Fc-γ-activated receptors, and improving immune effector cell and / or antibody-based efficacy against pathogenic microorganisms, as well as virus-infected and dysregulated cells. These can be used for the treatment of infectious diseases, cancer, and other humoral immunosuppressive diseases. Additionally, non-stimulatory analogs that can block Fc-γ-activated receptors can be used to inhibit activation of immune effector cells and downstream inflammatory mediators.

Description

Technical Field

[0001] Technical Field of the Invention The present invention relates to the field of humoral immunity and regulation of immune effector cells. In particular, it relates to compositions, methods, and kits of low molecular weight agents that bind to and stimulate Fc-γ-activating receptors, thereby enhancing the humoral immune response. Stimulation of Fc-γ-activating receptors improves immune effector cell-mediated and antibody-mediated responses against pathogenic microorganisms, virus-infected cells, and dysregulated cells. This low molecular weight agent can be used for the treatment of infectious diseases, cancer, and other humoral immune suppression diseases. Furthermore, non-stimulatory analogs that bind to and inhibit the activation of CD16a can be used for the treatment of inflammatory diseases mediated by CD16a-expressing cells.

Background Art

[0002] Background of the Invention Humoral immunity is a major mechanism by which vertebrate host organisms monitor and defend against infectious pathogens and dysregulated host cells. In cancer treatment, the development of monoclonal antibodies (mAbs) targeting cancer-specific cell surface antigens has been successful and commercially approved. Some of these mAbs have been reported to exert tumor-killing effects through humoral-mediated antibody-dependent cell cytotoxicity (ADCC) and / or antibody-dependent cell phagocytosis (ADCP) via engagement with Fc-γ-activating receptors such as CD16a (FCGRIIIA), CD32a (FCGRIIA), CD64 (FCGRIA) on immune effector cells (NK cells, macrophages, neutrophils, monocytes, dendritic cells, etc.) in addition to complement-dependent cytotoxicity (CDC) (DiLillo DJ, Ravetech JV, Cancer Immunol Res 3:704-713, 2015; Ruck T, et al. Int J Mol Sci. 16:16414-16439, 2015; Pelaia C, et al. Biomed Res Int 4839230:1-9, 2018; VanDerMeid KR, et al. Cancer Immunol Res 6:1150-1160, 2018). In recent years, multiple researchers have reported that tumors produce factors that suppress the humoral immune pathway, which inhibits the anti-tumor effect of mAbs by the mechanisms of ADCC, ADCP, and CDC (Vergote I, et al. J Clin Oncol 34:2271-2278; Kline JB, et al. J Clin Oncol 5:15, 2018; Wang W et al. Cytogenet Genome Res 152:169-179, 2017; Kline JB et al. Eur J Immunol. 48:1872-1882, 2018). The humoral immune responses of ADCC, ADCP, and CDC are controlled by the regulation of the binding of antibodies to cell surface antigens, and the antibodies are arranged on antigen epitopes at specific proximity distances on the cell surface.When this configuration is optimal, antibodies bound to the cell surface engage Fc-γ-activating receptors on natural killer (NK) cells or dendritic cells / myeloid cells / monocytes (all cells involved in ADCC or ADCP are herein referred to as "immune effector cells") to initiate ADCC or ADCP, and also engage the C1q complement initiating protein to kill antibody-bound cells via the classical complement pathway (Reuschenbach M, et al. Cancer Immunol Immunother 58:1535-1544, 2009). These effects have been observed with several therapeutic antibodies, including but not limited to rituximab, trastuzumab, cetuximab, pertuzumab, alemtuzumab, daratumumab, etc. (Zhou X, et al. Oncologist 13:954-966, 2008; Hsu YF, et al. Mol Cancer 9:1-8, 2010; Spiridon CI, et al. Clin Cancer Res 8:1720-1730, 2002; Luo C, et al. Sci Rep 7:46347, 2017; Sanchez L, et al. J Hematol Oncol 9:51-59, 2016). Similarly, humoral effects have been observed in the patient's own immune response to dysregulated cells and in the response to vaccines. Antibodies with anti-proliferative activity, in addition to immune-mediated killing activity, have been observed in patients with slowly progressive diseases (Staff C, et al. J Clin Immunol 32:855-865; Branden S, et al. Cancer Res 63:7995-8005, 2003).However, although many patients suffering from cancer or viral diseases have been found to produce autoantibodies against tumor antigens or viral antigens, their presence is often insufficient to eradicate diseased cells, probably due to overall low levels of autoimmunity-inducing antibodies, suboptimal epitope binding, exhaustion of immune effector cells, and / or humoral immunosuppressive mechanisms by pathogens, virus-infected cells, or dysregulated cells (Bi J and Tian Z. Frontiers Immunol 8:1-10, 2017).

[0003] In cancer (also called deregulated cells here), it has been found that the protein MUC16 / CA125 produced by tumors directly binds to a subset of IgG1, IgG3, and IgM type antibodies to suppress the humoral immune response. As a result, the Fc region is disrupted, and the involvement of the Fc region with Fc-γ-activating receptors FCGR2A (also called FCGRIIA and CD32a) and / or FCGR3A (also called FCGRIIIA and CD16a) on immune effector cells, or the effect of the involvement of C1q protein in CDC, is reduced (Pantankar MS, et. al. Gyncol Oncol 99:704-713, 2005; Kline JB, et al. OncoTarget8:52045-52060, 2017; Kline JB, et al. J. Clin. Oncol. 5:15, 2018; Wang W, et al. Cytogenet Genome Res 152:169-179, 2017; Kline JB, et al. Eur J Immunol. 48:1872-1882, 2018; Vergote I, et al. J Clin Oncol 34:2271-2278, 2016; Nicolaides NC, et al. Cancer Biol Ther 13:1-22, 2018; Prochazka V, et al. Int J Hematol 96:58-64, 2012; Grasso L. Oncology Letters 23:2, 2022). This effect is not observed with the high-affinity FCGR1 (also called CD64) Fc-γ-receptor (K D =0.5 nM), suggesting that this effect is partly due to the low-affinity binding of CD16a allotypes 158F (1059 nM) and 158V (415 nM), and CD32a allotypes 131R (218 nM) and 131H (39 nM) (Kline JB, et al. J. Clin. Oncol. 5:15, 2018; Nordstrom JL, et al. Breast Cancer Res 13:R123, 2011).

[0004] Many of the above findings regarding humoral immunosuppression in cancer point to the importance of NK cell and myeloid cell activation and antibody binding for effective killing. In several reports, it has been shown that depletion of NK cells reduces the therapeutic effect of antibody-mediated anti-cancer cell responses (Waldhauer I, Steinle A. Oncogene 27:5932-5943, 2008). Based on these observations, several approaches for utilizing NK cells in cancer immunotherapy have been pursued in experimental models and clinical trials (Vivier E, et al. Nat Rev Immunol 12:239-252, 2012; Shimasaki N, et. al. Nat Rev Drug Dis 19:200-218, 2020). These approaches aim to utilize the ADCC and antibody-independent cell cytotoxicity (AICC) killing mechanisms by NK cells against host target cells lacking major histocompatibility complex class 1 (MHC1) proteins due to viral infection or dysregulation (Bachanova Vand Miller JS. Crit Rev Oncol 19:133-141, 2014; Paul S and Lal G. Front Immunol 8:1124, 2017). Furthermore, NK cell therapy has also been pursued for the treatment of virus-infected patients to eradicate virus-infected host cells that are the downstream source of virus reactivation (Traylen CM, et. al. Future Virol 6:451-463, 2011, Larkin J, et. al. J Interferon Cytokine Res 26:12, 2006). The main drawback of directly utilizing NK cells or through antibody-mediated therapeutic responses is that after administration to patients, a sufficient number of activated cells capable of attacking virus-infected cells and dysregulated cells cannot be maintained.Several attempts to improve NK cell activation by pre-treatment or combination therapy with cytokine agonists such as IL-15 or stimulants that inhibit the negative regulatory pathways of NK cells such as NKG2A have yielded mixed results (Knudsen KM et al. Exp Opinion Biol Ther 20:7705-709, 2020; Miller JS et al. Nature Med 28:392-400, 2022; Creelan BC and Antonia SJ. Nature Rev Clin Oncol 16:277-278, 2019). The suboptimal activity may be due to off-target effects leading to host toxicity or limited stimulation in vivo (Guo, J et al. Cell Res 31:1190-1198, 2021). Considering these drawbacks, it is necessary to identify agents that can preferentially or specifically activate and maintain the killing activity of NK cells and other immune effector cells against virus-infected cells and dysregulated cells.

[0005] Cells expressing CD16a are associated with inflammatory diseases and have been shown to be directly involved (Cooper DL, et al. PLOS 7:e28918, 2012). In several related studies examining cell infiltration in rheumatoid arthritis, a correlation between CD16a-expressing cells and disease severity has been revealed (Robinson JI, et al. Ann Rheum Dis 69:1054-1057, 2010). Furthermore, in experimental animal models of inflammatory diseases, it has been shown that CD16a activity directly affects the progression of inflammatory diseases (Ji H, et al. Immunity 16:157-168, 2002; Diaz DS, et al. Eur J Immunol 32:2915-2922, 2002). From these findings, the development of agents that inhibit the activation of CD16a may be useful as therapeutic agents for CD16a-related inflammatory diseases.

Summary of the Invention

[0006] The present invention provides natural and synthetic low molecular weight agents that enhance the binding of IgG antibodies to CD16a-158F Fc receptor (FcR) (SEQ ID NO:1) and CD16a-158V Fc receptor (SEQ ID NO:2), and also stimulate CD16a FcR on immune effector cells for cell activation and downstream target cell killing. The low molecular weight agents of the present invention are related to lanosta-8,24-dien-3β-ol (Compound 1a) and 9β-19-cyclo-24-lanosten-3β-ol (Compound 2a), which are a kind of tetracyclic triterpenoid. These enhance the binding of IgG1 antibodies to CD16a-158F FcR and its high affinity allotype CD16a-158V FcR, and can also activate the CD16a pathway of NK cells and other immune effector cells. This class of therapeutic agents can be used for the treatment of infectious diseases, cancer, and other humoral immune suppression diseases through the activation of CD16a in immune effector cells (see also Nes WD and Heftmann E. Nat Prod 44:377-400, 1981). Alternatively, by changing the use of these scaffolds at specific carbons, CD16a can be bound and its unwanted activation in inflammatory diseases can be blocked.

[0007] Cholestane-based lanosta-8,24-dien-3β-ol (Compounds 1a and 1b), 9β-19-cyclo-24-lanosten-3β-ol (Compounds 2a and 2b), bisnor alcohol-based Compounds 3, 4a and 4b, and analogs containing their three cyclohexyl (A, B, C) and cyclopentyl (D) ring backbone structures as scaffolds can be used to modify CD16a to enhance affinity binding to IgG-type antibodies and / or CD16a receptor activation in the presence or absence of IgG, while other analogs can be used to inhibit the binding and activation of CD16a to IgG. Low molecular weight agents based on the following structures were analyzed and tested. TIFF2025522432000002.tif101134TIFF2025522432000003.tif86147Here, R1 is hydroxyl (OH) or formyl (CH=O), R2 and R3 are methyl (CH3), R4 is methyl or 14α-difluoromethyl (CHF2), R5 and R6 are independently aldehyde (CH3CHO), amine (NH2), dimethylamine ((CH3)2NH), piperidine ((CH2)5NH), isopropylamine ((CH3)2CHNH2), morpholine (O(CH2CH2)2NH), carboxylic acid (C(=O)OH), sulfonamide (CH3SO2NH2), acetamide (CH3CONH2) or (C2H5NO), alcohol (OH), ketone (CH3C(O)CH3), isopropyl methyl ketone (CH3COCH(CH3)2) or methyl isobutyl ketone ((CH3)2CHCH2COCH3), methyl (CH3), 2-methylheptane 2,3-diol (CH3CH(CH2)CH(OH)C(OH)(CH3)2, 2-methylheptane (CH3CH(CH2)3CH(CH3)2, a pegylated amine containing 2 to 12 PEG (polyethylene glycol) units, PEG5 amine, and a biotinylated amine in which a biotin moiety is linked to the amine moiety by PEG2 to 12.

[0008] An example of a biotinylated PEG2 amine that can be used as R6 is TIFF2025522432000004.tif34128.

[0009] An example of a PEG5 amine used as R6 is TIFF2025522432000005.tif32128.

[0010] In another aspect of the present invention, the above compounds, and other specific compounds, are used as effective binders to CD16a FcR, and stimulants of immune effector cells expressing such receptors, to enhance the therapeutic effect against pathogens, as well as against virus-infected cells and dysregulated cells including malignant cells, in a patient.

[0011] The present invention also includes a therapeutic pharmaceutical composition for activating immune effector cells in vitro and in vivo in mammals including humans. The pharmaceutical composition contains an effective amount of compound 1a, 1b, 2a, 2b, 3, 4a, or 4b.

[0012] Some of the above compounds are preferred for reasons such as ease of synthesis and / or high efficacy. Preferred compounds include lanosterol 14α-difluoromethyl, lanosterol 24-aldehyde, lanosterol 24-amine, lanosterol 24-dimethylamine, lanosterol 24-piperidine, lanosterol 24-isopropylamine, lanosterol 24-morpholine, lanosterol 24 carboxylic acid, lanosterol 24-sulfonamide, lanosterol 24-acetamide, lanosterol 24-alcohol, lanosterol 24-PEG(2-12), lanosterol 24-biotin, lanosterol 24-PEG5-biotin, lanosterol 24-PEG5-lanosterol, lanosterol 24-PEG5-cycloartenol, lanosterol 24-isopropyl methyl ketone, lanosterol 24-methyl-isobutyl ketone, lanosterol 24-methyl, dihydrolanosterol, and 24,25-dihydroxy lanosterol, etc.

[0013] Other preferred low molecular weight agents that can be used include cycloartenol 24-aldehyde, cycloartenol 24-amine, cycloartenol 24-dimethylamine, cycloartenol 24-piperidine, cycloartenol 24-isopropylamine, cycloartenol 24-morpholine, cycloartenol 24 carboxylic acid, cycloartenol 24-sulfonamide, cycloartenol 24-acetamide, cycloartenol 24-alcohol, cycloartenol 24-PEG(2-12), cycloartenol 24-biotin, cycloartenol 24-PEG5-biotin, cycloartenol 24-PEG5-lanosterol, cycloartenol 24-PEG5-cycloartenol, cycloartenol 24-isopropyl methyl ketone, cycloartenol 24-methyl isobutyl ketone, cycloartenol 24-methyl.

[0014] In other embodiments, the formulation of the above low molecular weight agents and analogs using parenteral and non-parenteral pharmaceutical formulations is included. Such pharmaceutical formulations include, but are not limited to, α-tocopherol, cetyl alcohol, cetearyl alcohol, corn oil monoglyceride, Cremophor EL, enteric acrylic resin, ethanol, ethyl oleate, glycerol, isopropyl palmitate, lauryl lactate, liposomes, mono / diglycerides of caprylic acid / capric acid, oleic acid, PEG, phosphate buffered saline, polysorbate 80, propylene glycol, sorbitan monocrete, sulfonamide, trehalose, and the like.

[0015] In another embodiment of the present invention, CD16a is used in a molecular binding assay to measure the binding affinity of CD16a to a full-length IgG antibody or to an IgG Fc fragment via any ELISA-based or affinity kinetics-based method such as surface plasmon resonance (SPR), Octet™ assay, or other kinetic binding analysis platforms in the presence of compound 1a, 1b, 2a, 2b, 3, 4a, or 4b.

[0016] Another aspect of the present invention is to apply biotinylated compounds 1a, 1b, 2a, 2b, 3, 4a, or 4b to monitor the binding of the biotinylated compounds to the CD16a domain. This can be used to identify important binding domains, improve antibody binding affinity, improve CD16a FcR activation, and / or determine the biological processing of activated FcR in CD16a-expressing cells.

[0017] Another aspect of the present invention is to use compounds 1a, 1b, 2a, 2b, 3, 4a, or 4b to enhance the binding between CD16a and IgG in a molecular assay. Using this, it becomes easier to screen for substances that inhibit the binding between CD16a and IgG Fc. Using such compounds may enhance the binding signal and facilitate the screening of such inhibitory substances.

[0018] Another aspect of the present invention is a kit for enhancing CD16a binding to the IgG Fc domain for in vitro screening. The kit contains compounds 1a, 1b, 2a, 2b, 3, 4a, or 4b and can be used in the binding assay between CD16a and IgG Fc to enhance the binding signal. The enhanced binding signal can be used to test for substances that inhibit the binding between CD16a and IgG Fc. Any method known to those skilled in the art can be used for screening protein-protein antagonists.

[0019] Yet another aspect of the present invention is to bind compounds 1a, 1b, 2a, 2b, 3, 4a, or 4b to an antibody to enhance the antibody's binding to CD16a and improve the therapeutic response mediated by the antibody.

[0020] Yet another aspect of the present invention is to administer compound 1a, 1b, 2a, 2b, 3, 4a, or 4b to a patient with an infectious disease or cancer, regardless of the presence or absence of standard treatment. This compound enhances the patient's humoral immune response (i.e., activation of immune effector cells expressing CD16a), thereby improving the clinical outcome. The compound can be administered orally, transdermally, and / or by injection.

[0021] Another embodiment of the present invention is to use analogs of compound 1a, 1b, 2a, 2b, 3, 4a, and / or 4b that bind to CD16a and inhibit its activation in immune effector cells to suppress inflammation.

[0022] These and other aspects of the present invention will be apparent to those skilled in the art upon reading this specification, and provide methods, compositions, and kits in the art for enhancing CD16a-expressing immune effector cells, including but not limited to NK cells and myeloid cells. These can be used to improve the patient's response to pathogens, viral infections, and dysregulated cells including cancer. The compounds of the present invention can be used as a single agent or in combination with other therapies including antibody-based ones. The antibody can be conjugated to one or more compounds if desired. These compounds may improve the antibody-mediated humoral immune response in immune-suppression related diseases such as cancer and non-neoplastic diseases where antibody therapeutics are used to stimulate the humoral immune response. A second aspect of the present invention is to suppress the activity of immune effector cells that support unnecessary inflammation in the treatment of inflammatory diseases by using compounds that inhibit the activation of CD16a. BRIEF DESCRIPTION OF THE DRAWINGS

[0023]

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Modes for Carrying Out the Invention

[0024] Detailed Description of the Invention The inventors have identified low molecular weight agents that specifically bind to CD16a Fc-γ-activating receptors across species and lead to the activation of NK cells and other immune effector cells having CD16a in vitro and in vivo. This low molecular weight agent enhances the binding of IgG antibodies to low affinity (158F) and high affinity (158V) CD16a Fc-γ-activating receptors (FcR). This low molecular weight agent exerts its effect by changing the dynamic structure of the FcR, increasing FcR-IgG binding, enhancing CD16a receptor signaling, and as a result, promoting the activation of immune effector cells and the killing of target cells. Here, the use of low molecular weight agents and their active cores that stimulate the activity of FcR and lead to the activation of immune effector cells, enhancement of IgG1 affinity binding, and subsequent killing of target cells will be described (see also Bruhns P et al. 113:3716-3725, 2009). This low molecular weight agent that stimulates FcR activation and enhances IgG binding can be used for the treatment of infectious diseases, cancer, and other humoral immune suppression diseases. Also, the use of compounds (e.g., but not limited to compound 37) that can suppress CD16a activation is taught using a composition that contains an important skeletal scaffold for CD16a binding without having a stimulating domain that can suppress CD16a activation in immune effector cells involved in inflammatory diseases.

[0025] The identified low molecular weight agents can bind to and activate murine and human CD16a Fc receptor-expressing immune effector cells by directly binding to the CD16a Fc-γ-activating receptor (FcR). Furthermore, these compounds also have the ability to enhance the binding of IgG-type antibodies to high-affinity CD16a receptors and low-affinity CD16a receptors, thereby enhancing the antibody-mediated killing mechanism against antigen-expressing target cells (Rugo HS, et al. J Clin Oncol 37:1000, 2019). Alternatively, non-stimulatory compounds provide an opportunity to inhibit the activation of CD16a, thereby suppressing the activation of immune effector cells and unwanted inflammatory responses. Without wishing to be limited by a particular theory or mechanism of action, the applicants believe that active low molecular weight compounds are involved in CD16a FcR on immune effector cells, including macrophages, NK cells, and any other cell subtypes expressing CD16a FcR. These change the dynamic structure of CD16a, alter receptor signaling and the activation of immune effector cells, and result in improved affinity for the IgG Fc domain, either or both of which lead to enhanced killing responses of humoral immune cells against target cells. Furthermore, one or more of these compounds can be administered to a patient to enhance the humoral immune response against dysregulated cells such as infectious pathogens and cancer. Alternatively, these compounds can be modified to bind to CD16a and inhibit its activation, thereby suppressing the activation of immune effector cells and the accompanying downstream inflammatory cascade. This use is useful for suppressing unwanted inflammation associated with inflammatory diseases generally known to those skilled in the art.

[0026] Additional analogs of the above compounds are similarly identified and may further improve in vivo therapeutic activity due to enhanced binding and activation, or blockade and inactivation, of CD16a FcR, as well as pharmacokinetic (PK) and pharmacodynamic (PD) properties. Such enhancements may improve the therapeutic effect and patient tolerance, all of which can be further determined through preclinical and human trials.

[0027] Some of the methods, compositions, and kits can be divided into three categories. In one category, the compounds are developed for use as therapeutic agents. It is well known in the art that the stronger the binding affinity of an antibody to the CD16a Fc receptor, particularly the low-affinity human CD16a-158F FcR allotype, the more improved the therapeutic response to target cells expressing a specific antigen of the antibody (Stavenhagen, JB et al. Cancer Res 67:8882-8890, 2007). Furthermore, it is also known in the art that activated NK cells are effective in killing virus-infected cells and dysregulated cell types (Minetto, P, et al. Front Immunol 10:1-10, 2019; Market M, et al. Front Immunol 11:1-23, 2020). The compounds described herein can activate NK cells and enhance the binding of antibodies to the CD16a Fc receptor. In yet another category, it is known to those skilled in the art that dysregulated inflammatory cells expressing CD16a can cause unwanted inflammation that contributes to various inflammatory diseases (Cooper DL, et al. PLOS7:e28918, 2012). The use of analogs and methods with the compositions described herein enables the development of compounds that can activate immune effector cells and bind to and block the downstream inflammatory cascade. Finally, another category involves using the active compounds as molecular biology reagents that enhance CD16a-IgG antibody binding in ELISA and other binding formats to facilitate the screening of inhibitors and activators of CD16a FcR-IgG antibody binding and subsequent pathway inhibition / activation. These compounds can be used in kits or as single assay enhancement reagents.

[0028] In the treatment of cancer or infectious diseases, the compound can be administered alone by the AICC therapy or in combination with experimental or regulatory authority-approved anti-cancer / anti-viral antibodies that utilize the mechanisms of action of ADCC or ADCP. Certain anti-cancer and anti-viral antibodies that may benefit from the activation of CD16a and / or the enhancement of the binding of the active compound include, but are not limited to, the following anti-cancer agents. That is, rituximab, trastuzumab, trastuzumab emtansine, cetuximab, YP218, ocrelizumab, daratumumab, elotuzumab, alemtuzumab, necitumumab, pertuzumab, obinutuzumab, nivolumab, ipilimumab, pembrolizumab, ofatumumab, panitumumab, tremelimumab, mosunetuzumab, pemprimumab, amivantamab, margetuximab, naxitamab, tafasitamab, inebilizumab, isatuximab, and durvalumab. Furthermore, the compound can be administered together with anti-infective antibodies including, but not limited to, nirsevimab, tixagevimab, sotrovimab, regdanvimab, and the combination antibody therapy of casirivimab and imdevimab. Antibodies with improved humoral immune function (ADCC or ADCP) can be used in combination therapy with an optimal amount of the compound.

[0029] For therapeutic use, the compound can be administered orally, sublingually, buccally, topically, or parenterally by methods such as intramuscular, intravenous, or subcutaneous.

[0030] The compounds can be formulated to support these various routes of administration. They can also be delivered via liposomal formulations and other formulations. Conventional liposomes are composed of a lipid bilayer consisting of cationic, anionic, or neutral (phosphorylated) lipids and cholesterol, with an aqueous volume enclosed therein. Suitable liposomal formulations include, but are not limited to, those combining guanidinium-cholesterol cationic lipid bis(guanidinium)-tren-cholesterol (BGTC) and colipid dioleoylphosphatidylethanolamine (DOPE). Another example of a suitable liposomal formulation is imidazole-based helper lipid MM27 and related aminoglycoside lipid dioleyl succinyl paromomycin (DOSP). Liposomes can be sterically stabilized, for example, by coating the liposomes with polyethylene glycol. Liposomes can also be targeted to ligands if desired. Suitable ligands include antibodies, peptides, carbohydrates, and proteins.

[0031] Other formulations for parenteral and non-parenteral administration include, but are not limited to, α-tocopherol, cetyl alcohol, cetearyl alcohol, corn oil monoglyceride, Cremophor EL, enteric acrylic resin, ethanol, ethyl oleate, glycerol, isopropyl palmitate, lauryl lactate, liposomes, mono / diglycerides of caprylic / capric acid, oleic acid, PEG, phosphate buffered saline, polysorbate 80, propylene glycol, sorbitan monocrearate, sulfonamides, trehalose, etc.

[0032] In other embodiments of the invention, methods and compositions are provided for developing compounds that can bind and activate any allotype of CD16a-FcR and / or enhance IgG antibody-CD16a affinity binding and downstream activation.

[0033] In some embodiments, the compounds are tested for their ability to enhance CD16a FcR binding and activation of CD16a FcR-expressing cells. Activation of CD16a FcR can be monitored by measuring downstream pathway signaling and downstream markers such as IL-1β, IFN-γ, TNF-α, GM-CSF, and granzyme B. These can be tested in vitro or in vivo.

[0034] In yet other embodiments, analogs of compounds 1a and 1b, 2a and 2b, 3, and compounds 4a and 4b are tested for improvement of CD16a FcR-IgG antibody binding and / or CD16a FcR-mediated cell activation of pathway-expressing cells while minimizing cytotoxicity, for example, in in vitro or in vivo screening. These are performed using various methods known to those skilled in the art.

[0035] In yet another embodiment, the compounds have a modified region to enhance the humoral immune response of test antibodies by improving optimal binding of the compounds to IgG antibodies of CD16a FcR and / or by using any method used by those skilled in the art to measure ADCC and / or ADCP.

[0036] In yet another embodiment, the compounds contain the skeletons of compounds 1a and 1b, 2a and 2b, or 3 and compounds 4a and 4b, while changing the chemical groups of R5 and R6 while retaining the chemical groups of R1, R2, R3, and R4, and can be experimentally tested to enhance the binding of CD16a FcR-IgG antibodies and / or the activation or suppression of immune effector cells.

[0037] In one method of measuring the ability of a compound to enhance FcR-antibody binding or activate CD16a-expressing cells, the compound is tested for direct binding of IgG antibody to CD16a FcR by ELISA, immunohistochemistry, or other methods known to those skilled in the art. Compounds with a binding rate of 25% or more are suitable for further testing.

[0038] In some embodiments, the functional methods use the production of downstream cytokines such as ADCC, ADCP, AICC or IL-1β, IFNγ or TNFα to measure the activity of compounds 1a and 1b, 2a and 2b, or 3, and compounds 4a and 4b, or derived analogs, in the presence or absence of an antibody (where the cells referred to here can be cell lines or primary NK cells, myeloid cells, PBMC cells, or SPL cells). The term "activity" generally refers to a change of 10% or more in the killing of ADCC, ADCP or AICC target cells or cytokine production when the compound is incubated with cells with or without an antibody, compared to cells without the compound. Also, depending on the cell type, antibody, and compound used, it may refer to a change of at least 5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%.

[0039] Compounds can be screened to improve pharmacokinetics (PK), pharmacodynamics (PD), or pharmacological (PL) activity. In some embodiments, the compound is added to cells in vitro and the activation of effector cells is tested for the killing of target cells in the presence or absence of an antibody. Compounds that enhance the killing effect with or without an antibody are now suitable for therapeutic testing using in vivo models. In another embodiment, the compound can be incubated within microsomes of various species to determine modifications of the compound that may result in high metabolic stability. The effect can be a change of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, or more, depending on the compound used.

[0040] In other embodiments, compounds 1a, 1b, 2a, 2b, 3, 4a and / or 4b are used to bind and block the activation and signaling of CD16a, resulting in a reduction of downstream inflammatory signaling via the persistent cytokine production associated with inflammatory diseases.

[0041] Throughout the specification and claims of this document, various terms and technical terms (the "terms") related to the embodiments of the attached description are used. Such terms shall have their ordinary meanings in the relevant technical field, unless otherwise specified. Other specifically defined terms shall be interpreted in a manner consistent with the provided definitions.

[0042] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include the plural forms as well, unless the context clearly dictates otherwise. For example, the expression "a cell" may include combinations of two or more cells. References to "probes" may include poly-histidine tagged human or murine CD16a-FcR, CD32a FcR, or independent probes for monitoring FcR-antibody binding, whereby 2 to 10 histidine codons are incorporated into the mature N-terminal domain or extracellular C-terminal domain of the FcR. These probes are called CD16a-HIS or CD34a-HIS. References to "cell activation" may include the effect of a compound on CD16a FcR-expressing cells by any assay known to those skilled in the art.

[0043] The term "about", when used in reference to an amount, period, and / or similar quantitative values, is meant to encompass variations of up to ±9% from the specified value, as such variations are appropriate for carrying out the disclosed methods. Unless otherwise stated, all values representing amounts of reagents such as molecular weights, molar concentrations, reaction conditions, percentages, etc. used in this specification and the claims are to be understood as being quantified by the term "about" in all instances. Accordingly, unless otherwise specified, the numerical values recited in the following specification and the appended claims are approximate values that may vary depending on the desired characteristics of the compositions, agents, and / or methods to be obtained by the present invention. At the very least, there is no intention to limit the scope of this application, and each numerical value should be evaluated at least by the number of significant digits reported and by the ordinary rounding methods known to those skilled in the art.

[0044] As used herein, the term "antibody" is used in a broad sense and includes immunoglobulins (also referred to as "Igs"), polyclonal antibodies (also referred to as "pAbs"), monoclonal antibodies (also referred to as "mAbs") including mouse, rat, primate, human, humanized and chimeric mAbs, and antibody molecules including full-length bispecific or trispecific antibodies. Generally, an antibody is a protein or polypeptide chain that binds to a specific antigen. An antigen is a structure specifically recognized by a particular antibody. A canonical antibody is composed of a heterotetrameric glycosylated protein in which two light chains and two heavy chains are arranged via a complex of disulfide bonds and hydrogen bonds. The term "its disulfide bridges" refers to the disulfide bridges contained within the heavy chain hinge region, which are generally known to those skilled in the art. Each heavy chain has a variable domain (variable region) (VH), followed by several constant domains (referred to as Fc domains). Each light chain has a variable domain (VL) and a constant domain, and the constant domain of the light chain aligns with the first constant domain of the heavy chain, and the VL of the light chain aligns with the variable domain of the heavy chain. Any type of antibody light chain is classified into one of two different types, namely kappa (κ) and lambda (λ), based on the amino acid sequence within its constant domain.

[0045] Immunoglobulins are classified into classes or isotypes based on the type of Fc domain, namely IgA, IgD, IgE, IgG, IgM, which depend on the sequences contained within their heavy chain constant (Fc) domains. The IgG isotype is further composed of subclasses such as isotype IgG1, IgG2, IgG3, IgG4.

[0046] "Specific binding" or "specifically binds" refers to the ability of an antibody to bind to an antigen (including sequences contained within the antibody itself) with a higher affinity than to other antigens. Typically, a specific antibody or antigen-binding fragment has an equilibrium dissociation constant K -8 of about 5x10 DIt binds to the target antigen. In other examples, it refers to the binding of the antibody Fc domain to the CD16a, CD32a, or CD64 Fc receptor. The equilibrium dissociation constant K D varies depending on the Fc receptor and its allotype.

[0047] The term "allotype" refers to the amino acid sequences of the CD16a or CD32a Fc receptors that have been shown to differ within the human population, each having a different binding affinity for IgG Fc. For CD16a, as is generally known to those skilled in the art, the amino acid at position 158, which usually contains valine (V) or phenylalanine (F), is involved.

[0048] The term "affinity" refers to the measurement of the antibody on-rate and off-rate of antigen binding to the variable domain, and the measurement of the antibody on-rate and off-rate of binding of the antibody Fc domain to Fc receptors (such as CD16a, CD32a, CD64, etc.).

[0049] "Antibody derivative" means an antibody modified by covalently linking another molecule via peptide chemistry (i.e., amidation, etc.), gene fusion, and / or post-translational moieties (i.e., glycosyl, acetyl, and / or phosphoryl) that are not normally associated with antibodies. This may include chemically fusing the compounds taught herein with antibodies to enhance FcR binding activity.

[0050] The term "CD16a dynamic structure" refers to structural changes that can affect the humoral functions, activation, suppression, and / or CD16a-IgG antibody binding of CD16a-expressing immune effector cells.

[0051] "Fc domain" refers to the antibody sequence from the C-terminus to the antibody hinge disulfide region (including this region). This region contains the binding domains for the CD16a, CD32a, and CD64 Fc receptors and is known to those skilled in the art.

[0052] "Antigen" refers to an entity to which an antibody or antibody fragment specifically binds. This includes binding to the antibody or protein of interest.

[0053] The term "mature" refers to an extracellular membrane or secreted protein from which the N-terminal secretion signal sequence has been cleaved, a concept well known to those skilled in the art.

[0054] The terms "cancer", "malignant", and "tumor" are well known in the art and refer to the presence of cells with uncontrolled cell growth and morphological characteristics that are different from normal cell types of similar origin, and are also referred to as "dysregulated cells". Malignant refers to cancer cells that have the potential to cause illness or death. As used herein, "cancer and tumor" includes pre-cancerous and malignant conditions.

[0055] The terms "viral", "pathogenic", and "infectious" are well known in the art and refer to the presence of cells or subjects infected with a microorganism or viral particle that causes disease.

[0056] The term "inflammation" or "inflammatory disease" refers to a patient's disease that causes inflammation in one or more organs of the body due to dysregulation of inflammatory cells.

[0057] As used herein, the term "soluble" refers to a protein or non-protein substance that is not attached to the cell membrane of a cell. For example, a soluble substance may be released, secreted, or excreted from normal or cancer cells into biological fluids such as serum, whole blood, plasma, urine, or the microfluid of cells containing tumors. Natural membrane-bound Fc receptors such as CD16a, CD32a, CD64, or receptors containing a polyhistidine tag can be engineered to be soluble and functional by incorporating a stop codon before the transmembrane domain of the receptor.

[0058] The term "detecting agent" or "detectable agent" refers to any agent that binds to an antibody or FcR and can be detected via an apparatus commonly used in the art. These include, but are not limited to, fluorophores, enzymes and enzyme substrates, radionuclides, heavy metals, colorimetric dyes and substrates, and can be detected by methods such as concentration measurement, spectrophotometry, luminescence, microscopy, radiography, scintillation, etc. Additional reagents may be required to generate a detectable signal.

[0059] The term "biomarker" refers to a protein produced by immune effector cells that is enhanced or suppressed by treatment with a compound. In particular, interferon-γ (also referred to as IFN-gamma or IFNγ), tumor necrosis factor-α (also referred to as TNF-alpha or TNFα), and granzyme B are biomarkers known to be produced by CD16a Fc receptor-stimulated immune effector cells, including NK cells.

[0060] The "level" of a particular protein or compound, such as granzyme B, IFNγ or TNFα as used herein, comprising a CD16a FcR-inducing protein from a target cell, refers to the level of the protein and / or compound determined in vitro or in vivo using any method known in the art for measuring the level of the protein and / or compound in response to treatment with the compound. Such methods include gel electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), superdiffusion chromatography, fluid or gel precipitation reactions, absorbance spectroscopy, colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), solution phase assays, immunoelectrophoresis, western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, fluorescence resonance energy transfer (FRET), Förster resonance energy transfer, electrochemiluminescence immunoassay, and the like. In one embodiment, the level of IFNγ and / or TNFα is determined using a probe-based technology as described in more detail. In another embodiment, the level of the compound is determined using chromatography and ELISA-based techniques.

[0061] The term "humoral immunosuppression, immunosuppression, or humoral immunosuppression" refers to an antibody, antibody fragment, bispecific antibody or trispecific antibody, or immune effector cell (NK, myeloid cell, monocyte, dendritic cell, etc.) in which the maximal biological response is attenuated. The lack of FcR receptor allotypes and activation of immune effector cells has been reported to affect the biological activity of these humoral immune components, including ADCC, ADCP, AICC and / or PK, PD, PL profiles.

[0062] The term "antibody-drug conjugate (ADC)" refers to an antibody conjugated or fused to a compound that enhances the activity of the antibody, which includes enhanced binding to FcR that enhances ADCC and ADCP.

[0063] The term "bispecific or trispecific antibody (BSP)" refers to an antibody that can bind to two or more different antigens. BSPs include, but are not limited to, at least two full-length antibodies, full-length antibodies, or single-chain antibodies that each bind to a different antigen or different epitopes on the same antigen.

[0064] The term "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to an in vitro or in vivo process in which an antibody binds to an antigen on the surface of a cell and engages immune effector cells via sequences within the Fc domain of the antibody, resulting in the release of toxins by the immune effector cells that kill the bound cell.

[0065] The term "antibody-independent cell-mediated cytotoxicity (AICC)" refers to an in vitro or in vivo process in which CD16a-expressing cells are stimulated by non-antibody CD16a FcR activation, resulting in the release of toxins by the CD16a-expressing cells that can bind to dysregulated or infected cells via natural cell surface receptors and kill the bound cells.

[0066] The term "antibody-dependent cell phagocytosis (ADCP) and opsonization" refers to a process in which an antibody binds to an antigen on the surface of a dysregulated cell and then interacts with immune cells via sequences within the Fc domain, resulting in the ingestion and consumption of the antibody-bound cell by the immune cells and ultimately its death.

[0067] The term "screening" may refer to testing compounds and measuring the enhancement or attenuation of a biological response by monitoring ADCC, AICC, ADCP, or the activation of other immune effector cells, with the intention of identifying compounds having such activity. Other references refer to the search for compounds that can inhibit or enhance the binding of the CD16a Fc receptor to IgG antibodies. This term may also be used in other contexts to assay a number of test elements to determine which of the test elements have a particular property. Similarly, it may be used to examine whether a patient's sample has a particular property.

[0068] The term "significant" refers to statistical results where the p-value determined by some programs, including Student's t-test, is less than 0.05.

[0069] The term "pharmacokinetics (PK)" refers to the time for a compound to maintain a steady concentration when administered to a subject.

[0070] The term "pharmacodynamics (PD)" refers to the study of the biochemical and physiological effects of a compound and its mechanism of action, including the correlation between the action and effect when administered to a subject and its structure.

[0071] The term "pharmacology (PL)" refers to the known effects of a compound to manage or kill diseased cells in vitro or in vivo.

[0072] The term "maximum tolerated dose (MTD)" refers to the maximum amount of a compound that can be administered to a patient or test animal until a toxicity signal is experienced. In the case of test animals, MTD is usually defined as a decrease of more than 10% of body weight.

[0073] The term "sample" refers to a collection of similar body fluids, cells, or tissues isolated from a subject, and body fluids, cells, or tissues present in the body of the subject. Body fluids include biological fluids that have come into contact with the subject or biological source, such as cell culture media and organoid culture media, urine, saliva, washings, etc.

[0074] The term "control sample" refers to any clinically or non-clinically relevant control sample, such as a sample from a healthy subject not suffering from a specific cancer type, or cells different from the parental cells.

[0075] The term "control level" refers to an acceptable or pre-determined level of a protein or non-protein agent used for comparison with the level of the same substance in a sample taken from a subject, or used in an in vitro assay.

[0076] When the term "difference" is used between the signals of two compounds that may have a common backbone, it generally refers to a difference that can be statistically determined using the statistical methods commonly used by those skilled in the art, and is a difference of at least 10% compared to the control. Depending on the compounds and assays used, it may also refer to a change of at least 5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, or 75%.

[0077] The terms "inhibit" or "inhibition" mean to reduce by a statistically measurable amount or to prevent completely.

[0078] The term "functional" with respect to a compound in the presence or absence of an antibody used according to the described method means that it enhances or inhibits antibody binding and / or activation of immune effector cells, respectively, leading to enhanced killing of target cells and / or production of downstream proteins, or suppression of inflammatory pathway signaling of immune effector cells in vitro or in vivo.

[0079] The term "pharmaceutically acceptable" refers to substances that are acceptable for administration to a patient from a pharmacological and toxicological perspective and are manufactured by methods known to those skilled in the art. These include drugs approved by federal or state regulatory agencies, or drugs described in the United States Pharmacopeia or other generally recognized pharmacopeias for use in animals or humans. The term "pharmaceutically compatible component" refers to a pharmaceutically acceptable diluent, adjuvant, excipient, or matrix medium used during the administration of an anticancer agent. The term "pharmaceutically acceptable carrier" refers to a matrix that does not inhibit the biological activity of the active ingredient and is non-toxic to the host.

[0080] The term "formulation" refers to a solvent, excipient, or matrix medium added to support the administration of a compound to a patient.

[0081] The terms "patient" and "subject" are used interchangeably to refer to humans and other animals (including veterinary subjects) that are treated with a therapeutic agent. The term "non-human animals" includes all vertebrates. In one embodiment, the subject is a human.

[0082] The terms "effective amount" and "therapeutically effective" are used interchangeably and are used in the context of administering an amount of a medicament sufficient to provide a favorable clinical outcome to a patient. An effective amount of the agent is administered according to an "effective regimen" as described herein. The term "effective regimen" refers to a combination of the amount and frequency of administration of an agent sufficient to improve the clinical outcome for a particular cancer patient. An improvement in efficacy is an improved clinical outcome when a patient is administered an agent that can restore the morbidity rate more than the parent compound, or an agent that improves the clinical outcome of an effective regimen. The effective amount referred to herein is the amount of the compound required to show efficacy or a difference as compared to the case where the compound is not included.

[0083] A "therapeutic agent" is typically substantially free of undesirable contaminants. This means that the agent typically has a purity of at least about 50% w / w (weight / weight) and is substantially free of interfering proteins and contaminants.

[0084] The term "target cell" refers to a eukaryotic or prokaryotic cell, or cell population, that expresses an antigen for a particular antibody or antibody-containing moiety and is bound by CD16a Fc receptor-expressing cells through direct antibody engagement for ADCC or ADCP activity, or indirect antibody interaction by AICC.

[0085] The term "immune effector cell" refers to any cell line or natural cell including, but not limited to, NK, myeloid cells, monocytes, neutrophils, dendritic cells, which can confer antibody - dependent cell - mediated cytotoxicity (ADCC) or phagocytosis (ADCP or opsonization) by binding to antibody - bound target cells, and can confer antibody - independent cell - mediated cytotoxicity (AICC) by CD16a FcR stimulation. Natural cells may be purified in the form of peripheral blood mononuclear cells (PBMCs) or may exist as a mixture. Cell lines may be naturally CD16a - expressing or may be modified to express CD16a.

[0086] "Dysregulated cells" refer to cells that are considered abnormal relative to the parental cells. This includes transformed cells, malignant cells, virus - infected cells, cells that self - proliferate by self - regulatory functions, or prokaryotic pathogens.

[0087] The term "humoral immune response" refers to ADCC or ADCP of target cells by immune effector cells in the presence of a test antibody. The term "cellular response" refers to AICC of target cells by immune effector cells in the absence of a test antibody.

[0088] The term "compound" refers to a compound described in this document that can bind to CD16a FcR and change its activity, or a compound understood from the formula. In particular, it refers to the use of cholesterol - based lanosta - 8,24 - dien - 3β - ol (Compound 1a), 9β - 19 - cyclo - 24 - lanosten - 3β - ol (Compound 2a) and compounds derived from bisnor alcohol (Compound 3) and analogs (Compound 4a) containing their three cyclohexyl (rings A, B, C) and cyclopentyl (ring D) ring backbone structures as scaffolds. The term "compound" also refers to a compound described in this specification that can bind to CD16a FcR and enhance or inhibit its activity, or a compound understood from the formula, including Compound 1a, 1b, 2a, 2b, and analogs (Compound 3, 4a or 4b) constructed with their three cyclohexyl (rings A, B, C) and cyclopentyl (ring D) ring backbone structures as scaffolds.

[0089] The term "analog" refers to a compound derived from compound 1a, 2a, 3, 4a, 1b, 2b, or 4b, in which one or more modifications have been made to R1, R2, R3, R4, R5, and / or R6.

[0090] The term "food" refers to a substance that is used in the body of an organism to grow, repair, maintain life activities, and supply energy, and is essentially composed of proteins, carbohydrates, and / or fats. Food may also contain auxiliary substances such as minerals, vitamins, and seasonings. See Merriam-Webster's Collegiate Dictionary, 10th Edition, 1993. The term "food" includes beverages consumed by humans and animals. As used herein, "food additive" is based on the definition in 21 C.F.R. 170.3(e)(1) of the FDA and includes direct additives and indirect additives.

[0091] The term "nutritional supplement" refers to a product (excluding tobacco) intended to supplement the diet and containing any one or more of vitamins, minerals, herbs or other plants, amino acids, nutrients used by humans to supplement the diet by increasing the total daily intake, or concentrates, metabolites, constituents, extracts, or combinations of these components.

[0092] Therapeutic compounds, kits, and methods for enhancing humoral immunity-mediated therapy Provided herein are compositions, kits, and methods for identifying small molecule compounds that can effectively enhance or suppress the binding of the CD16a Fc receptor to IgG antibodies and / or activate an immune effector cell response against pathogens, virus-infected cells or dysregulated cancer cells, or suppress dysregulated inflammatory cells. Examples thereof are schematically shown in FIGS. 1-11. The kit is composed of a compound whose chemical composition is taught by the formula of the present invention and a schematic method for synthesizing an appropriate composition described in Example 3. The active compound can be identified using molecular CD16a FcR-IgG antibody binding and / or effector cell activation or suppression assays as described through the following examples.

[0093] In a method for identifying an active compound by cell-based screening, the compound is added to a culture of target cells expressing the target antigen of the test antibody, regardless of the presence or absence of the antibody. Immune effector cells are added to the culture, and the humoral immune responses of the cells with and without the added compound are compared, and the humoral response is monitored using standard ADCC, ADCP, or AICC killing assays. Usually, a change of at least 10% is considered a significant effect on ADCC, ADCP or AICC function. Depending on the assay employed, a significant effect can also be defined as a change of at least 5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, or 75%.

[0094] In other methods of identifying activating compounds, the test antibody is added to the culture medium of target cells in a state where the target cells naturally express the antigen of the test antibody. Modified human Jurkat cells (JKT-CD16a) expressing the human CD16a Fc receptor linked to a luciferase reporter are added, and the luciferase level is monitored 12 to 24 hours later. Usually, a change of at least 10% is considered a significant effect on CD16a activation. Depending on the antibody and assay employed, a significant effect may be defined as a change of at least 5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, or 75%.

[0095] Also provided is a method of treating a cancer subject with an active compound. For example, a patient may be suffering from CD20, HER2, EGFR, or mesothelin-expressing cancers such as lymphoma, breast cancer, mesothelioma, colorectal cancer, lung cancer, ovarian cancer, pancreatic cancer, cholangiocarcinoma, endometrial cancer, etc. Some antibodies targeting these antigens have been reported to be more effective in patients in whom immune effector cells express the high-affinity CD16a-158V FcR allotype (Rugo HS, et al. J Clin Oncol 37:1000, 2019). Unfortunately, since more than 50% of the world's population expresses the low CD16a-158F FcR, a compound that can enhance the binding affinity of a test antibody for CD16a-158F FcR is a pharmaceutically desirable entity (Wang W, et al. Cytogenet Genome Res 152:169-179, 2017). One such antibody is anti-CD20 rituximab, but in clinical trials, it has been found that the therapeutic effect is significantly lower in patients with heterozygous or homozygous CD16a-158F alleles (Treon SP, et al. J Clin Oncol 23:474-481, 2016). Using an effective compound for this antibody is highly desirable pharmaceutically.

[0096] In some embodiments of the methods of treating a subject with an active compound, a patient with an infectious disease or cancer can be treated with the compound alone or in combination with a standard treatment. In some embodiments of the methods of treating a subject described herein, the compound is administered to the subject alone. In yet another embodiment, the present compound is co-administered with a standard therapeutic agent. Standard therapeutic agents include all agents considered standard for the disease indication at the time the subject is being treated.

[0097] In a method for identifying a CD16a pathway blocking compound, a test antibody is added to the culture medium of target cells in a state where the target cells naturally express the antigen of the test antibody. Modified human Jurkat cells (JKT-CD16a) expressing the human CD16a Fc receptor linked to a luciferase reporter are added, and the luciferase level is monitored 12 to 24 hours later. Usually, when compared with a culture not treated with the compound, at least a 10% inhibitory signal is considered a significant effect on CD16a inhibition. Depending on the antibody and assay employed, a significant effect may be defined as at least 5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, or 75% inhibition.

[0098] In some embodiments of the methods of treating a subject with an active compound, a patient with an inflammatory disease can be treated with the compound alone or in combination with a standard treatment. In some embodiments of the methods of treating a subject described herein, the compound is administered to the subject alone. In yet another embodiment, the present compound is co-administered with a standard therapeutic agent. Standard therapeutic agents include all agents considered standard for the disease indication at the time the subject is being treated.

[0099] In some embodiments of the treatment methods described herein, the compound is composed of a chemical skeleton derived from Compound 1, 2, 3, or 4, retains the chemical groups of R1, R2, and R3, while the R4, R5, and / or R6 regions are modified using the formulas taught or exemplified herein.

[0100] The method of the present invention can be combined with alternative treatments such as surgery, radiation therapy, targeted therapy, chemotherapy, immunotherapy, use of growth factor inhibitors, use of anti-angiogenic factors, etc. The compound can be administered simultaneously to a patient undergoing alternative treatment. The patient can also receive alternative treatment before or after administration of standard treatment, for at least 1 hour to several months after administration of the compound, for example, at least 1 hour, 5 hours, 12 hours, 1 day, 1 week, 1 month, or 3 months. Some embodiments of the treatment methods provided herein include, in addition to the compound, administering to the subject a therapeutically effective antibody specific for an antigen expressed by the pathogen, virus, cancer, or inflammatory mediator.

[0101] In some embodiments of the treatment methods described herein, the subject may have received alternative therapy and antibody-based standard treatment prior to administration of the compound.

[0102] Administration of the therapeutic agent and the compound according to the treatment methods described herein can be carried out by any means known in the art.

[0103] The amount of a therapeutic agent or compound effective for the treatment or prevention of cancer or non-cancer (virus, pathogen, inflammatory cell)-mediated diseases can be determined by standard clinical techniques. Further, in vitro assays can be employed as needed to identify the optimal dosage range required for the compound using the standard or unique screening methods taught herein. The effective amount can be estimated from the dose-response curve of the compound obtained from in vitro or animal model test systems.

[0104] For example, the toxicity and therapeutic effects of a drug can be determined in cell culture or experimental animals by standard pharmaceutical procedures for determining the LD 50 (lethal dose for 50% of the population) value and the ED 50 (therapeutically effective dose for 50% of the population) value. The dose ratio between the toxic effect and the therapeutic effect is the therapeutic index, and LD 50 / ED 50It can be expressed as a ratio. Agents showing a therapeutic index exceeding 2 are suitable. When the agent shows toxic side effects, by using a delivery system that targets the agent to the site of the diseased tissue, potential damage to the cells of healthy tissues can be minimized, thereby reducing side effects.

[0105] Dosage and dosing schedule may vary depending on the active agent concentration and may vary depending on the needs of the subject.

[0106] Kit for optimizing CD16a FCR binding activity to IgG-type antibodies for screening Furthermore, kits are provided for enhancing the binding of active compounds to IgG antibodies of human or murine CD16a FcR in order to rapidly screen for inhibitors or activators that may affect the pathway. It is taught here that the active compositions provided by the present invention can significantly improve CD16a FcR-IgG antibody binding in ELISA and other binding assay formats. This improvement enhances the ability to screen for agents that have a positive or negative impact on the CD16a FcR biological pathway in immune effector cells in order to develop therapeutic agents useful for the treatment of diseases where modulation of the CD16a FcR pathway may provide clinical benefit. One teaching involves adding active compounds (such as, but not limited to, Compound 1, 2, 4, 14, 26, 27, 30, 33, or 36) added to high-throughput screening assays using CD16a FcR and antibodies to find low-molecular-weight or high-molecular-weight inhibitors (Mandelboim O, et al. Proc Natl Acad Sci USA 96:5640-5644, 1999). Other screening may involve screening Fc domain-modified antibodies or Fc fusion proteins for enhancement of CD16a FcR binding and may be used to improve pharmacological activity (Saunders KO. Front Immunol 10:1-20, 2019). Examples of this function are shown in FIGS. 1 and 2.

[0107] The kit may contain both the compound and the reagents commonly used in ELISA, or may contain only the compound accompanied by instructions for use for enhancing the binding of the CD16a FcR-IgG antibody in a molecular binding assay or a cell binding assay.

[0108] The above disclosure outlines the present invention. All references disclosed herein are hereby incorporated by explicit reference. A more complete understanding can be obtained by referring to the following specific examples, which are provided herein for illustrative purposes only and are not intended to limit the scope of the present invention.

Example

[0109] Example 1 - Identification of Compounds 1 and 2 that Enhance Immunoglobulin Binding to Rodent and Human CD16a-Fc Receptors and Immune Effector Cell Activation In Vitro and In Vivo As part of an effort to identify compounds that may enhance the binding of low-affinity human CD16a-158F FcR to IgG-type immunoglobulins, a semi-high-throughput screening assay was developed to screen a commercially available library of natural small-molecule compounds. These libraries contain over 1,100 low-molecular-weight chemical substances derived from various natural sources and are composed of a vast variety of chemical substances with diverse structures and biological properties. In the primary assay, a 96-well ELISA-based format was employed, and the plates were coated with 2.5 μg / mL of human IgG1 or human serum albumin (HSA) as a negative control. CD16a-158F-HIS (SEQ ID NO:1), CD16a-158V-HIS (SEQ ID NO:2), or CD32a-131H-HIS (SEQ ID NO:3) FcR binding was tested using 2.5 μg / mL of each probe. The cDNA of each probe lacking the transmembrane domain and the C-terminal polyhistidine tag for purification was incorporated into a mammalian expression vector containing a blasticidin selection marker. The vector was stably introduced into HEK293 cells, and a high-titer production of the FcR protein in blasticidin-resistant clones was screened using an ELISA-based screening method and an anti-polyhistidine (HIS)-horseradish peroxidase (HRP)-conjugated antibody as a detection probe. The secreted mature CD16a or CD32a protein (leader sequence cleaved during secretion) was in serum-free BalanCD HEK293 medium (Irvine Scientific) at 1.5×10 6Isolated from a 10-day culture grown to 10⁶ cells / mL and isolated on a HisPur™ Ni-NTA resin column according to the supplier's (Thermo Scientific) recommendations. After primary probing of the compound library, the wells were washed and IgG binding was monitored with a secondary anti-HIS-HRP detection antibody (Sino Biologicals). Well signals corresponding to individual clones were quantified at 450 nm using a multi-well plate reader (Varioskan, Thermo Fisher). Screening of over 1,100 compounds resulted in only one compound that enhanced the binding of huCD16a-158F's FcR to the IgG1 antibody with better reproducibility than the control group. Compound analysis identified this as cholestane-based lanosta-8,24-dien-3β-ol (Compound 1). Structure analysis led to the discovery of a highly related 9β-19-cyclo-24-lanosten-3β-ol compound (Compound 2), and this compound was obtained and tested to compare its molecular and cellular activities against CD16a and CD32a FcRs with Compound 1. As shown in Figure 1A, both Compounds 1 and 2 significantly enhanced the binding of huCD16a-158F and huCD16a-158V FcRs to the IgG1 antibody, in contrast to huCD32a-131H FcR or HSA, demonstrating the target specificity of both compounds. Compound 2 was further tested for its ability to enhance the binding of human IgG to other CD16a species. As shown in Figure 1B, Compound 2 enhanced the binding of human IgG1 to mouse CD16a homolog 1 (FCGR3) (SEQ ID NO:4) (muFCGR3-HIS), but not to mouse homolog 2 (FCGR4) (SEQ ID NO:5) (muFCGR4-HIS). These analyses used the same ELISA method as above, with HIS-tagged mouse FCGR3 and FCGR4 as probes (Sino Biologics), and anti-HIS-HRP for detection. These data identified rodents as a suitable species for testing Compounds 1, 2, 3, 4, and derived analogs that enhance in vivo immune effector activation for treatment confirmation and proof of concept.Furthermore, the data shown in Figure 1 support the use of Compound 1 or 2, and their related active analogs shown in Figures 7 and 8. Any compound that is a potential enhancer of CD16a FcR-IgG binding can be used as an assay reagent in molecular biology research, facilitating the screening of compounds that may have a positive or negative impact on the biological pathway of the CD16a FcR-IgG antibody, and is useful for therapeutic indications related to this pathway. When these compounds are applied as molecular biology assay reagents, a reliable ELISA signal can be read in 3 - 5 minutes, while it takes 15 - 30 minutes in the basic CD16a FcR-IgG antibody binding format, thus facilitating the screening platform. Plates with 3 or more wells were used in the experiment. The p-value was determined using Student's T-test.

[0110] To further evaluate the enhancing effect of CD16a FcR-IgG antibody binding, a titration curve was generated to measure the amount of binding enhancement that compound 2 could confer on the low-affinity huCD16a-158F FcR. Briefly, ELISA plates were coated with 2.5 μg / mL of human IgG1 and probed with 2.5 μg / mL of huCD16a-158F-HIS FcR in the presence of various concentrations of compound 2 or an inert analog (compound 5, see Figure 7). After probing, the plates were washed and secondarily probed with anti-HIS-HRP for detection. The wells were quantified at 450 nm using a multi-well plate reader (Varioskan). As shown in Figure 2, compound 2 was able to improve CD16a-158F-IgG antibody binding 4.7-fold compared to the control compound 5, suggesting a significant improvement in affinity binding compared to CD16a-158F alone. This improvement increased the affinity of huCD16a-158F for IgG1 binding from approximately 1 μM to 200 nM, comparable to the affinity of the high-affinity huCD16a-158V FcR allotype, which has a published affinity of approximately 415 nM (Nordstrom JL, et al. Breast Cancer Res 13:1-14, 2011). This is an unexpected finding and a highly desirable feature in the development of CD16a-based therapies (Bowles JA, et al. Blood 108:2648-2654, 2006). Plates with three or more wells were used for the experiment. The p-value was determined using Student's T-test.

[0111] To confirm that enhanced CD16a FcR-IgG antibody binding has biological relevance, a bioassay was established using the Jurkat-CD16a-158F-luciferase (JKT-CD16a) reporter cell line (Promega), and the ability of compounds 1 and 2 to enhance and activate the low-affinity CD16a-158F FcR was tested in the presence or absence of IgG1, with target cells, target cells only, or JKT-CD16a cells only. The target cells used were OV-CD20, a human ovarian cancer cell line (OVCAR3) that was stably transfected to express the human CD20 antigen. The test antibody used was a chimeric (mouse variable region fused to the human IgG1 Fc domain) anti-CD20 rituximab. Briefly, 0 to 5,000 target cells were seeded into black 96-well microplates in RPMI (R7.5) containing 7.5% fetal bovine serum (FBS) and 1% L-glutamine growth medium and cultured overnight at 37°C and 5% CO2. The next day, the wells were washed and cultured with 50 μL of assay medium (clear RPMI, 1% low IgG FBS, L-glutamine (R1)) containing various concentrations of compound 1 or 2, with or without 2.5 μg / mL of the test rituximab antibody, which has previously been shown to specifically bind to OV-CD20 cells (Grasso L, et al. Oncol Letters 23:1-10, 2022). Finally, 9,400 JKT-CD16a cells in R1 medium were added to each well, and the plates were incubated at 37°C and 5% CO2 for 12 to 18 hours. For each well, CD16a FcR activation was examined using the BIO-GLO (Promega) luciferase substrate according to the manufacturer's recommendations, and relative luciferase production (RLU) was analyzed using a multi-well plate reader (Varioskan). As shown in Figure 3A, both compounds 1 and 2 significantly enhanced JKT-CD16a activation in the presence of IgG, in contrast to the control wells without medium or compound (central bar graph group). CD16a-158F FcR activation was also tested for JKT-CD16a cells and target cells without antibody in the same manner as described above.As shown in the bar graph group on the right side of Panel A, both Compounds 1 and 2 stimulated JKT-CD16a-158F FcR activation in the absence of IgG antibody. This indicates the ability of these compounds to directly stimulate CD16a FcR activation, although the level was lower than in the presence of IgG antibody. In wells without the addition of JKT-CD16a cells (left bar), no enhancement of CD16a activation was observed under any treatment conditions. To confirm the CD16a FcR specificity of Compounds 1 and 2, ELISA was used to test for the production of the downstream CD16a FcR activation markers IL-1β and TNFα in the medium using parental Jurkat cells that do not express CD16a FcR and JKT-CD16a cells. 50,000 cells / well were seeded in an opaque 96-well microplate in R1 medium, and 10 μM of Compound 2 or its inactive analog, Compound 5, was added to each well and incubated at 37 °C, 5% CO2 for 18 hours. The plates were centrifuged to pellet the cells, and the supernatants were collected and tested for the production of INFγ (Sino Biologicals) and TNFα (R&D Systems) by ELISA according to the manufacturer's recommendations. As shown in Figure 3B, both cytokines were produced in JKT-CD16a-158F cells grown in the presence of Compound 2, but little cytokine was produced in any of the cells treated with parental Jurkat or Compound 5. This indicated that CD16a FcR is required for the induction of cytokine production by Compound 2. These data show that by using compounds and similar analogs, 1) it is possible to enhance the binding of CD16a-158F FcR to IgG1 antibody, and 2) it is possible to stimulate the activation of CD16a FcR in the absence of antibody binding. Both features are useful for the pharmaceutical development of CD16a FcR-based therapies. Plates with 3 or more wells were used for the experiments. The p-values were generated by comparing the compound treatment group with the vehicle group for each group using Student's T-test.

[0112] To demonstrate the ability of active compounds such as Compound 2 to stimulate the immune effector activity of primary immune cells, human peripheral blood mononuclear cells (PBMC), human NK, and mouse spleen cells (SPL) effector cells were tested for activation by Compound 2 by measuring antibody-dependent (ADCC) or antibody-independent (AICC) cytotoxicity against tumor target cells. Briefly, 5,000 human breast cancer cells (SKBR3 or OE19) or OV-CD20 target cells were seeded into black 96-well microplates in R7.5 medium for 48 hours. The wells were washed and 50,000 PBMC, NK, or SPL effector cells were added into R1 assay medium with or without the target test antibody and 25 or 50 μM of Compound 2. The vehicle is the solvent in which Compound 2 is dissolved. In assays using the SKBR3 human breast cancer cell line or the OE19 esophageal adenocarcinoma cell line, 2.5 μg / mL of the HER2-target trastuzumab test antibody was used for the ADCC assay. In the OV-CD20 assay, 2.5 μg / mL of the CD20-target rituximab test antibody was used for the ADCC assay. Cultures were performed in triplicate for 24 hours at 37 °C and 5% CO2. After culture, the culture supernatant was collected for cytokine analysis, the wells were washed with phosphate-buffered saline, and the viability of the adherent target cells was quantified with Cell Titer-GLO (Promega) using a Varioskan plate reader. As shown in Figure 4A, in wells treated with Compound 2 in combination with the target antibody, it was demonstrated that Compound 2 could enhance the effector cells of humans (PBMC and NK cells) and mice (SPL cells) that kill target cells via ADCC compared to treatment with the antibody alone (vehicle). The ADCC data were graphed as the killing rate of the antibody treated with Compound 2 or vehicle and the culture not treated with the antibody. Using the same method as above, Figure 4B shows that in the absence of the antibody, Compound 2 activates human primary NK cells, resulting in the ability of NK cells to kill OV-CD20 tumor cells via the AICC mechanism. The AICC results were graphed as a comparison of the killing rate by Compound 2 and the killing rate by the culture treated with vehicle only.To show that activation of the CD16a FcR pathway in NK cells is involved in the AICC effect, the supernatants of the compound 2 and vehicle-treated wells from the experiment in Figure 2B were analyzed for cytokines and granzyme related to effector cell activation via CD16a FcR by ELISA using the combination of anti-cytokine antibodies recommended by each manufacturer. As shown in the figure, in the wells treated with compound 2, it was found that TNFα, IFNγ, and GM-CSF (Abcam) were all produced and secreted by NK cells in the absence of antibodies, and the production of the cytotoxic granzyme B protein (Sino Biologics) was also confirmed. This supports the evidence of cell activation via the CD16a FcR effector by compound 2 and subsequent target cell killing (Wang R, et al. J Leukoc Biol 91:299-309, 2012; Louis C, et al. J Exp Med 217:e20191421; Caligiuri MA. Blood 112:461-469, 2008). Kinetic analysis revealed that activation and cytokine production occurred within 30 minutes after exposure. Similar cytokine production was also observed in human PBMC and mouse SPL cultures treated with compound 2. These data indicate that compound 2 and active analogs can enhance antibody-mediated antibody-based target cell killing and non-antibody-based stimulation of human and mouse immune effector cells, and have the potential as therapeutic agents for diseases targeting CD16a FcR. Plates with 3 or more wells were used in the experiment. The p-value was determined using Student's T-test.

[0113] To demonstrate the ability of an active compound such as Compound 2 to activate immune effector cells in vivo, athymic nude mice were used to measure tolerance and systemic immune effector cell activation. In previous in vivo tests, Compound 2 was found to tolerate i.v. administration at at least 7 mg / kg per day in athymic nude mice. To examine the ability of Compound 2 to activate immune effector cells in vivo, 7 mg / kg of Compound 2 or an equal amount of vehicle (ethanol) was administered daily for 5 days (from day 1 to day 5), and whole serum was separated on day 11 to measure immune effector cell activation markers. As shown in Figure 5A, no weight loss was observed, indicating that the 5-day administration showed good tolerance in all mice of each cohort. The TNFα (R&D Systems) immune effector cell activation marker was monitored in the sera of treated mice by ELISA in the same manner as above. It was found that in Compound 2-administered mice (298 pg / mL), it was significantly upregulated compared to the vehicle control cohort (11 pg / mL) (P = 0.00000026). These data suggest that Compound 2 and its active analogs can be used to evaluate their potential therapeutic uses in mouse models of human diseases, and as potential therapies for treating human diseases involving CD16a FcR such as cancer and infectious diseases, regardless of the presence or absence of therapeutic antibody-based therapies, they show the ability to activate immune effector cells in vivo. Plates with 3 or more wells were used in the experiment. The p-value was determined using Student's T-test.

[0114] Example 2 - Identification of Compounds that Suppress the Killing of Human Peripheral Blood Mononuclear Cells by Viruses It has previously been reported that NK cells have the ability to kill virus-infected cells (Bjorkstrom NK, et al. Nat Reviews Immunol 22:112-123, 2022). In particular, previous studies have shown that the SARS-CoV-2 virus infects human PBMCs and that this infection has a fatal impact on the survival rate of PBMCs as a whole (Manunta MDI, et al. Scientific Reports 11:4904-4916, 2021). The low-molecular-weight agent that activates CD16a FcR described here was tested against human PBMCs exposed to the SARS-CoV-2 virus to examine its ability to activate effector cells within PBMCs, kill PBMC sibling cells infected with the virus, and reduce the virus load. Briefly, IL2-activated PBMCs grown in RPMI medium (R10) supplemented with 10% heat-inactivated FBS were seeded at 100,000 cells / well in a 96-well U-bottom microplate. Next, the appropriate wells with or without 25 μM of compound 2 were added with a virus titer (previously determined in plaque-forming units, PFU). The cultures were carried out at 37 °C and 5% CO2 for 72 hours. The plates were centrifuged and the supernatants were collected for future cytokine and virus titer analysis. The cells were resuspended in RPMI plus 10% FBS, transferred to a 96-well black microplate, and the viability was tested using Cell Titer-Glo (Promega), and the viability was read using a 96-well microplate reader. As shown in Figure 6, the viability of PBMC cultures treated with virus and vehicle was 77%, whereas the cultures treated with 25 μM of compound 2 maintained 100% viability. These data support the usefulness of compound 2 and related analogs for the treatment of virus-infected cells and infected patients. More than 3 wells of plates were used in the experiment. 5 The virus titer (previously determined in plaque-forming units, PFU) was added. The cultures were carried out at 37 °C and 5% CO2 for 72 hours. The plates were centrifuged and the supernatants were collected for future cytokine and virus titer analysis. The cells were resuspended in RPMI plus 10% FBS, transferred to a 96-well black microplate, and the viability was tested using Cell Titer-Glo (Promega), and the viability was read using a 96-well microplate reader. As shown in Figure 6, the viability of PBMC cultures treated with virus and vehicle was 77%, whereas the cultures treated with 25 μM of compound 2 maintained 100% viability. These data support the usefulness of compound 2 and related analogs for the treatment of virus-infected cells and infected patients. More than 3 wells of plates were used in the experiment.

[0115] Example 3 - Screening of Active Analogs and Definition of Key Regions for Enhancing CD16a FC Receptor-Antibody Binding and Maintaining Immune Effector Cell Activation Using a molecular-based recombinant CD16a-HIS FcR and IgG1 antibody ELISA assay, screening is facilitated to identify optimal regions within compounds 1 and 2, as well as related 3-cyclohexyl,1-cyclopentyl related bisnor alcohols (compounds 3 and 4), in addition to new analogs that may further enhance or suppress the specific activation of CD16a FcR and downstream immune effector cell activity. Lead compounds identified by the molecular screening described herein are tested in in vitro bioassays for confirmation and then in vivo to identify the most optimal, pharmacologically and therapeutically tolerable compounds. Here, first, several compounds within the public domain having a structure common with compound 1 or 2 were tested. As shown in Figure 7A, these studies demonstrate the importance of a hydroxyl group or a formyl group as R1. Here, cycloartenol ferulate (compound 12, Figure 7B), a very similar analog of compound 2, lacks a hydroxyl group at R1 and is completely inactive, while the 3-formylcycloartenol 24-ketone analog (compound 20) containing a formyl group at R1 enhances CD16a FcR-IgG antibody binding, similar to the 3-β-hydroxy-4,4-dimethyl-5-α-bisnor alcohol analog (compound 4), and in contrast to 3-α-hydroxy-4,4-dimethyl-5-α-bisnor alcohol (compound 17), also demonstrating the importance of stereospecificity in the R1 region.

[0116] Furthermore, the importance of the methyl groups at R2 and R3 in the enhanced binding and activation of the CD16a FcR-IgG antibody is shown by the loss of activity of 5α-cholesta-8,24-dien-3β-ol (compound 6) compared to compound 1, while as shown by compound 13, the stereospecificity at C13 and C14 has some adverse effect on the activity. Exchanging the methyl group with fluorine at R4 of compound 1 also seems to have little effect on the activity.

[0117] The synthesis of analogs using the cholestane or compound 3 skeleton based on the above findings further demonstrated the flexibility to create diverse analogs in the R5 and R6 regions, whereby PEG dimerization of two cholestane-derived 9β-19-cyclo-24-lanosten-3β-ol in the R6 region (compound 14), or substitution of the ketone at C24 of cholestane-derived compound 20, demonstrated that the enhanced CD16a binding activity was hardly or not at all lost. This is similar to the retained binding activity of compound 4 derived from compound 3, leading to intensive efforts to further expand the structure-activity relationships of various chemical classes for further optimization in the R5 / R6 region. These and additional structure-activity relationships found in compounds 1-20, compound 30, and compounds 36-38 provide additional teaching of the active regions for generating additional active analogs for the treatment of disease indications involving CD16a FcR such as cancer and infectious diseases. Alternatively, non-enhanced compounds such as compound 37 or 38 can be used as CD16a activation inhibitors for treating CD16a-mediated inflammatory diseases.

[0118] Based on the discovery that analogs derived from the active compounds 1, 2, 3, or 4 exhibit extreme variability in the R5 and R6 regions, additional modifications can be realized to improve the binding and / or activation of CD16a FcR-IgG antibodies. Additionally, they can be used as biotinylated probes (compound 34, Figure 8) to analyze compound-CD16a FcR interactions in vitro and in vivo (Figure 8). These modifications may improve solubility, molecular activity, PK / PD properties, and therapeutic activity. Synthetic methods for other similar types of compound structures are also provided.

[0119] A general scheme was implemented to produce further analogs of compound 2 having variations at R5 / R6 and which met the requirements necessary to enhance the binding of CD16a-FcR to IgG1 antibody in therapeutic applications and the FcR activation of effector cells expressing CD16a. To prepare these compounds, compound 12 was generated and used as a starting material to generate compound 2 and its analogs. In Scheme 1, compound 21 was generated by base hydrolysis, protection of the 3-hydroxyl group, cleavage of the isopropylidine, followed by removal of the 3-hydroxyl protecting group, and this was used as an active analog and common intermediate for reductive amination at C24 (Scheme 2), generation of carboxylic acid analogs by oxidation (Scheme 3), and acetylation and sulfation of the intermediate (Scheme 4) to generate additional R5 / R6 analogs based on compound 2.

[0120] In the following Scheme 1, compound 2 is generated by hydrolyzing compound 12 in the presence of an aqueous potassium hydroxide (KOH) solution in ethanol (EtOH). The resulting secondary alcohol is protected by acetylation with acetic anhydride. The double bond is then oxidized using O3, Zn, AcOH, or potassium osmate and sodium periodate to generate aldehyde 1-2, and after hydrolysis with an 80% NaOH / EtOH aqueous solution, compound 21 is generated. Since compound 12 is of natural origin, from previous experience, it is known that the C24 ketone isomer consistently exists, and in the same way, but purified by supercritical fluid chromatography (SFC) and confirmed by nuclear magnetic resonance (NMR), compound 30 (Figure 8) can be generated.

[0121] Scheme 1 TIFF2025522432000006.tif52160

[0122] In the following Scheme 2, compound 25 is prepared from compound 21 and commercially available dimethylamine hydrochloride using reductive amination in MeOH and / or DCM.

[0123] Scheme 2 TIFF2025522432000007.tif40156

[0124] Another analog generated by Scheme 2 is Compound 26, which is prepared from Compound 21 and commercially available isopropylamine using reductive amination in MeOH and DCM. TIFF2025522432000008.tif40155

[0125] Compound 27 was prepared according to Scheme 2 from Compound 21 and commercially available piperidine using a reductive amination reaction in MeOH and DCM. TIFF2025522432000009.tif41155

[0126] Analog 28 of Compound 2 generated by the method of Scheme 2 was prepared using reductive amination of Compound 21 and commercially available morpholine in MeOH and DCM. TIFF2025522432000010.tif41155

[0127] In another Scheme 2 synthesis, Compound 29 is produced by oxidizing Compound 21 in the presence of NaClO4, NaH2PO4, and 2-methyl-2-butene in t-BuOH and THF. TIFF2025522432000011.tif41155

[0128] As the last example of analog synthesis of Scheme 2, the PEGylated R6 analog of Compound 2 (with a polyethylene glycol (PEG) unit length of 2 - 12) is synthesized from Compound 21 and commercially available NH2-PEG2-OH (Compound 32), NH2-PEG12-OH (33), and intermediate PEG lengths using reductive amination in MeOH and DCM. TIFF2025522432000012.tif41155TIFF2025522432000013.tif41155

[0129] In the following Scheme 3, compound 22 was prepared from compound 21 and commercially available BnNH2 using reductive amination in MeOH and DCM, followed by hydrogenation in the presence of Pd(OH)2 / C, H2 atmosphere, and MeOH. Next, compound 22 was sulfonylated with methylsulfonyl chloride under basic conditions to obtain compound 23, while compound 24 was prepared from compound 22 by condensation with acetic acid in the presence of EDCI and DMAP in DCM.

[0130] Schemes 3 and 4 TIFF2025522432000014.tif51158

[0131] In another method, compound 21 was reduced in the presence of NaBH4 in EtOH to produce compound 31, which was then condensed with D-(+)-biotin in the presence of EDCI and DMAP in DCM to produce biotinylated compound 34. TIFF2025522432000015.tif39161

[0132] Using a similar method, analogs of compound 1 with similar R5 and R6 modifications were generated.

[0133] Example 4 - Identification of Compounds with Optimal Pharmacological Properties for in vivo Use Analysis of analogs derived from compound 2 described in Example 3 was performed by testing the enhancement of CD16a FcR binding to IgG by ELISA. Briefly, 96-well plates were coated with PTZ as described above, probed with CD16a-158F-HIS in the presence of 10 μM of the parent compound 2 and R5 / R6 compound analogs 22 - 34, and assayed for those mediating the enhancement of CD16a-IgG binding. As shown in Figure 9, compounds 26, 27, and 33 significantly enhanced the binding of CD16a-158F to IgG (P < 0.005). To further evaluate the effect of the enhanced binding, these compounds were tested for the enhancement of human PBMC ADCC as described above. Human breast cancer SKBR3 cells were seeded at 5,000 cells / well in black 96-well microplates in R7.5 and cultured overnight at 37 °C and 5% CO2. The next day, 50,000 PBMCs in R1 medium were added per well (3 wells) in the presence of each compound or ethanol (EtOH) vehicle control 1 or 5 μM. Wells treated with EtOH and PTZ without PBMC were used as negative controls. Cultures were performed in triplicate at 37 °C and 5% CO2 for 24 hours. After incubation, the viability of target cells was quantified with Cell Titer-GLO (Promega) using a Varioskan plate reader. The units are expressed as relative light units (RLU). As shown in Figure 10, compounds 27 and 33 most significantly enhanced ADCC (P < 0.0000075).

[0134] Next, the metabolic stabilities of Compounds 2, 26, 27, and 33 were tested using microsomes from humans and mice. For each test compound, the sample was diluted to a final concentration of 2 μg / mL with 25 mM potassium phosphate buffer, and various liver microsomes were added at a final concentration of 0.5 mg / mL. The enzyme reaction was initiated by adding NADPH reagent at a final concentration of 1 mM. The NADPH reagent was not added to the negative control samples. The samples were incubated at 37 °C on an orbital shaker at 50 RPM, and aliquots were taken at predetermined time points (0 min, 5 min, 10 min, 15 min, 30 min, 60 min). Thereafter, the samples were precipitated with 3 volumes of acetonitrile containing propranolol as an internal standard, centrifuged at 2000 g for 10 minutes, and then analyzed by LC / MS / MS. Note that the compounds undergo only Phase I metabolism in liver microsomes in the presence of the NADPH cofactor. The interpretation of the resulting data is based on the residual ratio of the parent compound relative to the sample incubated for 0 minutes, from which the elimination half-life is calculated based on a natural logarithm plot of compound residual ratio versus time. To estimate the in vitro metabolic stability of the compounds, the following parameters were calculated. Cmp = microsomal protein concentration (mg / mL), t 1 / 2 = half-life (minutes), where t 1 / 2 is equal to 0.693 / slope, CLint = intrinsic hepatic clearance (μL / min / mg), where CLint is equal to 0.693 / (t 1 / 2 ×Cmp). Based on these analyses, as shown in Table 1, Compounds 26 and 27 had significantly improved metabolic stability in both mouse and human microsomes, in contrast to Compounds 2 and 33.

[0135] (Table 1) Metabolic profiles of Compounds 2, 26, 27, and 33 in liver microsomes TIFF2025522432000016.tif31128

[0136] Based on the excellent metabolic stability of compound 27 (Table 1) and the enhanced ADCC by PBMC (Figure 10), the in vivo activity of the compound was further analyzed. First, as described below and shown in Figure 11, the synthesis of compound 27 was scaled up. 25 grams of gamma-oryzanol was hydrolyzed under basic conditions with an aqueous solution of 0.415 M KOH in EtOH to obtain secondary alcohol N. Next, 37.8 mM of Ac2O was used to protect secondary alcohol N with an acetyl group, followed by ozonolysis to obtain aldehyde intermediate 2. Subsequently, 27.1 mM of piperidine was dissolved in AcOH, and NaBH(OAc)3 was added for reductive amination to obtain intermediate 3. The free base of compound 27 was obtained by hydrolyzing intermediate 3 under the conditions of an aqueous solution of NaOH in MeOH and THF. The free base of compound 27 was converted to the HCl salt using a 4 M HCl dioxane solution, and the precipitated product was collected by filtration. The final product was analyzed by mass spectrometry and H-NMR. Mass spectrometry analysis revealed the major compound with the predicted molecular weight of 470.6 for compound 27 (see below), along with the exact H-NMR spectral profile.

[0137] Mass spectrometry analysis of compound 27 revealed the major species with a molecular weight of 470.6, as predicted for compound 27.

[0138] To further investigate the pharmacological properties of Compound 27, NK cell activation markers IFNγ and TNFα were measured to test oral bioavailability and the ability to systemically activate NK cells in mice. For oral bioavailability, mice were fed a standard laboratory rodent diet and housed individually in cages under a 12-hour light / dark cycle at 22 ± 30°C and 50 ± 20% relative humidity. Animals were fasted overnight before dosing, refed 6 hours later, and blood samples were collected at that time. During the test period, water was available ad libitum. Compound 27 was prepared by grinding the precipitated powder into fine particles and suspending it in an aqueous solution of 0.5% hydroxypropylmethylcellulose (HPMC). Three animals were dosed orally with 100 mg / kg (10 mL / kg) via a nasogastric needle for oral administration. Blood samples (25 - 30 μL per sample) were collected from the jugular vein at 5, 15, 30 minutes, 1, 2, 4, 6, 8, and 24 hours after dosing. Additional blood samples (150 - 200 μL per sample) were collected 24 hours later for cytokine analysis. Blood samples were collected into Greiner MiniCollect K2EDTA tubes, placed on ice, centrifuged at 15,000 g for 5 minutes within 30 minutes to obtain plasma samples, and 50 - 100 μL of each plasma sample was collected 24 hours later. All plasma samples were stored at -70°C until analysis.

[0139] The bioanalysis of plasma samples was performed as follows. Plasma samples were prepared using 3 volumes of internal standard-containing acetonitrile per 1 volume of plasma to precipitate proteins. The samples were centrifuged at 3000 g for 10 minutes, the supernatant was removed, and analyzed by LC-MS / MS. Calibration standards and quality control solutions were prepared by preparing a stock solution at 1 mg / mL, followed by a series of working solutions of methanol:water (1:1, v / v), which were added to blank plasma to obtain a series of calibration standard samples in the range of 1 ng / mL to 10 μg / mL and quality control samples at three concentration levels (low, medium, high). All collected plasma samples were treated in the same manner as calibration standards and quality control samples. LC-MS / MS analysis was performed using multiple reaction monitoring to detect characteristic ions of each drug candidate, additional relevant analytes, and internal standard substances. Plasma concentrations were measured as described above, and concentration-time profiles were determined. The area under the plasma concentration-time curve (AUC) was calculated using the linear trapezoidal method known to those skilled in the art. Fitting of the data to obtain pharmacokinetic (PK) parameters was performed using non-compartmental analysis.

[0140] The main PK parameters reported after intravenous administration were the terminal half-life t 1 / 2 , the initial plasma concentration C0, the area under the plasma concentration-time curve AUC, the volume of distribution at steady state Vss, the total plasma clearance CLp, and the mean residence time MRT.

[0141] The main PK parameters reported after extravascular administration were the terminal half-life t 1 / 2 , the maximum plasma concentration Cmax, the time to reach the maximum plasma concentration tmax, the area under the plasma concentration-time curve AUC, the mean residence time MRT, and the bioavailability F. All parameters were presented for individual animals along with the mean, standard deviation, and coefficient of variation, and are generally known to those skilled in the art.

[0142] After oral administration of a 100 mg / kg suspension, the test compound gradually reached a maximum plasma concentration (Cmax) of 511 ng / mL within 6 hours (Tmax), after which the plasma concentration decreased in a polyphasic manner, and the final measurable concentration was 242 ng / mL after 24 hours, and the terminal phase half-life (t 1 / 2 ) was 18.3 hours. The systemic exposure (AUClast) was 7.75 h*μg / mL, and the oral bioavailability (F) was 11.7%. These data indicate the oral bioavailability of compound 27.

[0143] To examine the tolerance and systemic activation ability of NK cells in mice, a test was conducted in which compound 27 was orally administered to mice. Six mice were randomly divided into a vehicle control (water) and a treatment group of 100 mg / kg of compound 27 (both were suspended in 0.5% hydroxypropyl methylcellulose). The mice were orally administered on days 1 to 5, 7, 9, 11, 13, and 15, and sacrificed on day 16, and plasma was collected at that time. Tolerance was based on the body weight of each mouse on day 1. The maximum tolerated dose was determined when the weight loss exceeded 20% or more than 10% of the animals in the group died. After 10 courses of administration, it was confirmed that all the mice in both groups were healthy with the same body weight, and multiple oral administrations of 100 mg / kg of compound 27 were confirmed to have high tolerance.

[0144] To confirm that oral administration of compound 27 activated NK cells, the NK activation markers IFNγ and TNFα were measured by ELISA from the plasma collected on day 16 using the method described in Example 1. As shown in Figure 12, in the mice administered compound 27, in contrast to the mice administered only the vehicle, both NK cell activation markers were increased, and oral ingestion of compound 27 and activation of target NK cells were confirmed. These scaffolds embody the main inventive steps taught within this application.

[0145] Example 5 - Development of analogs using the scaffolds of compounds 1, 2, 3, and / or 4 for developing CD16a pathway inhibitors that suppress inflammation While many examples of analogs synthesized from the scaffolds of Compounds 1, 2, 3, and 4 show enhanced activity in CD16a FcR binding and activation of immune effector cells, other analogs have been found to inhibit IgG-C16a FcR binding. This class of analogs may be useful in suppressing inflammatory diseases. It has been revealed in several reports that activated CD16a signaling in inflammatory diseases may be a fundamental cause of a subset of inflammatory diseases (Steel AW, et al. Aliment Pharmacol Ther 33:115-126, 2011). The teachings herein show that chemical groups enhancing CD16a-stimulating activity can be added using the scaffolds of Compounds 1, 2, 3, and 4. Herein, methods for developing CD16a antagonists that suppress CD16a-IgG interactions and downstream activities (including production of inflammatory cytokines such as IFNγ) using these scaffolds are exemplified. Figures 10 and 13 show the ELISA binding assays described in Example 1. Briefly, 96-well clear microplates were coated with 2.5 μg / mL pertuzumab (PTZ) IgG or HSA as a negative control (without PTZ). Next, wells were probed with human CD16a-158F-HIS with or without the compound added to monitor CD16a-PTZ binding. As shown in Figures 10 and 13, multiple analogs (Compounds 29, 31, 32) derived from the scaffold of Compound 2 were able to suppress the CD16a-PTZ interaction by approximately 49% (P≤0.005), resulting in suppression of signaling in the CD16a pathway. By expanding the use of the scaffolds taught herein, it is possible to develop more potent CD16a inhibitors, similar to the approach for developing CD16a activators taught herein. These scaffolds embody the key inventive steps taught within this application.

[0146] The methods described in the examples provided herein teach the technology of novel compositions useful in the treatment and therapy of CD16a FcR-mediated diseases.

[0147] Table 1. Array identification (all arrays are from N-terminus to C-terminus) underline indicates the poly-histidine tag Bold indicates the polymorphic changes of human CD16a 158F and 158V SEQ ID NO:1 Mature human CD16a-158F-HIS (accession number P08637) TIFF2025522432000017.tif18153 SEQ ID NO:2 Mature human CD16a-158V-HIS (accession number P08637) TIFF2025522432000018.tif19153 SEQ ID NO:3 Mature human CD32a-131H-HIS (accession number P12318) TIFF2025522432000019.tif22154 SEQ ID NO:4 Mature mouse CD16a-1 (FCRG3)-HIS (accession number P08508) TIFF2025522432000020.tif18152 SEQ ID NO:5 Mature mouse CD16a-2 (FCRG4)-HIS (accession number 653142) TIFF2025522432000021.tif18153

Claims

1. A method of treating a subject having a viral infection, cancer, parasitic infection, bacterial infection, yeast infection, or inflammatory disease, comprising: administering to the subject an effective amount of compound 1a, 1b, 2a, 2b, 4a, or 4b, whereby the immune response of the subject against viral infection, cancer, parasitic infection, bacterial infection, yeast infection is increased, or whereby inflammation is reduced; comprising, wherein the compound is represented by: where, R1 is hydroxyl (OH) or formyl (CH=O); R2 and R3 are methyl (CH 3 ) and R4 is methyl or 14a-difluoromethyl (CHF 2 ), and R5 and R6 are independently aldehyde (CH 3 CHO), amine (NH 2 ), dimethylamine ((CH 3 ) 2 NH), piperidine ((CH 2 ) 5 NH), isopropylamine ((CH 3 ) 2 CHNH 2 ), morpholine (O(CH 2 CH 2 ) 2 NH), carboxylic acid (C(=O)OH), sulfonamide (CH 3 SO 2 NH 2 ), acetamide (CH 3 CONH 2 ), or (C 2 H 5 NO), alcohol (OH), ketone (CH 3 C(O)CH 3 ), isopropyl methyl ketone (CH 3 COCH(CH 3 ) 2 ), or methyl isobutyl ketone ((CH 3 ) 2 CHCH 2 COCH 3 ), methyl (CH 3 ), 2-methylheptane (CH 3 CH(CH 2 ) 3 CH(CH 3 ) 2 ), 2-methylheptane 2,3-diol (CH 3 CH(CH 2 )CH(OH)C(OH)(CH 3 ) 2 ), a pegylated amine containing 2 to 12 PEG (polyethylene glycol) units, a PEG5 amine that binds to compounds 1a, 1b, 2a, 2b, 4a or 4b to form a dimer, and a biotinylated amine in which a biotin moiety is linked to an amine moiety by PEG2 - 12, a method.

2. The method according to claim 1, further comprising administering a therapeutic antibody to the subject.

3. A method of assaying an antibody for binding to Fc receptor CD16a, comprising: contacting Fc receptor CD16a with compound 1a, 1b, 2a, 2b, 4a, or 4b; contacting the antibody with Fc receptor CD16a; and determining the amount of binding of the antibody to Fc receptor CD16a. a method.

4. The method according to claim 3, wherein Fc receptor CD16a is on the cell surface.

5. The method according to claim 3, wherein Fc receptor CD16a is in a cell-free preparation.

6. A method of screening a compound for modification of the binding of an antibody to Fc receptor CD16a, comprising: contacting Fc receptor CD16a with the compound; contacting the antibody with Fc receptor CD16a that has been contacted with the compound; and determining a first amount of binding of the antibody to Fc receptor CD16a that has been contacted with the compound; and comparing the first amount of binding with a second amount of binding of the antibody to Fc receptor CD16a that has not been contacted with the compound, wherein a compound that increases the amount of binding is a candidate for a therapeutic immunomodulator and a compound that decreases the amount of binding is a candidate for a therapeutic agent for inflammatory disease. a method.

7. The method according to claim 6, wherein Fc receptor CD16a is on the cell surface.

8. The method according to claim 6, wherein Fc receptor CD16a is in a cell-free preparation.

9. A method of screening a compound for activation or inhibition of CD16a-expressing cells, comprising: culturing a sample of CD16a-expressing cells in the presence of the compound; culturing a sample of CD16a-expressing cells in the absence of the compound; A step of measuring the amount of cell activation or suppression of a sample cultured in the presence of the compound and a sample cultured in the absence of the compound, wherein a compound that increases cell activation is a candidate for a therapeutic immunomodulator, and a compound that decreases cell activation is a candidate for a therapeutic agent for inflammatory diseases, the step of measuring A method comprising the above.

10. The method according to claim 9, wherein the CD16a-expressing cells are selected from the group consisting of a recombinant cell line expressing the CD16a receptor, innate immune cells, natural killer (NK) cells, myeloid cells, dendritic cells, and monocyte cells.

11. A therapeutic antibody chemically linked to Compound 1a, 1b, 2a, 2b, 4a, or 4b.

12. Compound 1a, 1b, 2a, 2b, 4a, or 4b, and A therapeutic antibody A kit comprising the above.

13. A composition for regulating the immune response in a human or rodent subject, comprising the following:[[]] Compound 1a, 1b, 2a, 2b, 4a, or 4b, which is not lanosta-8,24-dien-3β-ol or 9β-19-cyclo-24-lanosten-3β-ol, and A pharmaceutically acceptable carrier A composition comprising the above.

14. A dietary supplement comprising Compound 1a, 1b, 2a, 2b, 4a, or 4b, wherein the concentration of the compound exceeds 700 mg per 100 g of the supplement.

15. The dietary supplement according to claim 14, wherein the compound is not lanosta-8,24-dien-3β-ol or 9β-19-cyclo-24-lanosten-3β-ol.

16. The dietary supplement according to claim 14, wherein the concentration of the compound exceeds 1 g per 100 g of the supplement.

17. The dietary supplement according to claim 14, which is a liquid beverage.

18. The dietary supplement according to claim 14, which is a solid snack bar.

19. A method for manufacturing a fortified food for enhancing the immune response, comprising the following:[[]] A step of mixing a dietary supplement containing Compound 1a, 1b, 2a, 2b, 4a, or 4b with a food substance to form a fortified food suitable for enhancing the immune response, wherein the compound is not lanosta-8,24-dien-3β-ol or 9β-19-cyclo-24-lanosten-3β-ol, and the concentration of the compound in the dietary supplement exceeds 700 mg per 100 g of the supplement, the step of mixing A method comprising the above.

20. The method according to claim 19, wherein the food substance is a dietary fiber supplement.

21. A method for treating inflammation in a subject, comprising: administering to the subject an effective amount of a compound, whereby the inflammatory response of the subject is reduced; comprising wherein the compound is selected from the group consisting of bisnor alcohol, 5α-cholesta-8,24-dien-3β-ol, cholesta-5,24-dien-3β-ol, 5α-cholesta-7-en-3β-ol, 3β-chloro-cholesta-5-ene, cycloartenyl ferulate, 3-hydroxy-4,4-dimethyl-5-ene-bisnor alcohol, 3-α-hydroxy-4,4-dimethyl-5-ene-bisnor alcohol, 2-methyl-3-β-hydroxy-4,4-dimethyl-5-ene-bisnor alcohol, 3β-lanosta-8,24,25-trihydroxy-ene-3-ol, lanosta-8,24-dien-3β-ol-25-morpholino, and 9β-19-cyclo-24-lanosten-3β-ol-25-morpholino.

22. The method according to claim 21, wherein the subject has an inflammatory disease or inflammatory syndrome.

23. The method according to claim 21, wherein the inflammatory disease is selected from the group consisting of ankylosing spondylitis (AS), antiphospholipid antibody syndrome (APS), gout, inflammatory arthritis, myositis, multiple sclerosis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus (SLE, lupus), and vasculitis.

24. A method for assaying a compound for inhibition or enhanced activation of Fc receptor CD16a on the cell surface, comprising: contacting Fc receptor CD16a on the cell surface with compound 1a, 1b, 2a, 2b, 4a, or 4b; and determining activation or inhibition of Fc receptor CD16a. comprising

25. The method according to claim 24, wherein activation is determined by antibody-dependent cell cytotoxicity (AICC).

26. The method according to claim 24, wherein activation or inhibition is determined by measuring luciferase production from a CD16a-luciferase reporter cell line in the presence or absence of IgG antibody.

27. The method according to claim 24, wherein activation is determined by measuring TNFα, IFNγ, granzyme B, GM-CSF, or IL1β production in the cells.

28. A composition comprising compound 27.

29. A method for producing compound 27, the method comprising a step of reductively aminating compound 21 in methanol, dichloromethane and piperidine.

30. A composition comprising compound 26.

31. A method for producing compound 26, the method comprising a step of reductively aminating compound 21 and isopropylamine in methanol and dichloromethane.

32. A composition comprising compound 29.

33. A method for producing compound 29, the method comprising a step of oxidizing compound 21 in t-butyl alcohol in tetrahydrofuran in the presence of sodium perchlorate, sodium dihydrogen phosphate and 2-methyl-2-butene.

34. A reagent for enhancing an in vitro antibody-based assay that utilizes the binding of Fc receptor CD16a and an antibody, comprising a compound selected from compound 1a, 1b, 2a, 2b, 4a, and 4b, or a combination thereof, wherein the compound is not lanosta-8,24-dien-3β-ol or 9β-19-cyclo-24-lanosten-3β-ol.

35. A kit comprising a reagent for enhancing an in vitro antibody-based assay that utilizes the binding of Fc receptor CD16a and an antibody, the kit comprising: a compound selected from compound 1a, 1b, 2a, 2b, 4a, and 4b, or a combination thereof, and a cell-free preparation of Fc receptor CD16a

36. The kit according to claim 35, comprising 3,3',5,5'-tetramethylbenzidine (TMB). ​

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