Humoral immune activators for treatment of humoral immunosuppressive pathologies, cancer and infectious diseases and humoral immune inhibitors for treatment of inflammatory diseases

Abstract

Compositions, methods, and kits of small molecular weight agents are capable of binding and stimulating Fc-gamma-activated receptors to enhance humoral immune responses and immune effector cell responses by improving immune effector cell and / or antibody-based efficacy on pathogenic microorganisms as well as viral infected and dysregulated cells. These can be used to treat infectious diseases, cancer and other humoral immunosuppressive diseases. In addition, non-stimulating analogs capable of blocking Fc-gamma activated receptors can also be used to inhibit immune effector cell activation and inflammatory downstream mediators.

Description

Technical Field

[0001] The present invention belongs to the field of humoral immunity and immune effector cell regulation. Specifically, the present invention relates to compositions, methods and kits of small molecular weight agents, which bind and stimulate Fc-γ-activating receptors, thereby enhancing humoral immune responses. Stimulation of Fc-γ-activating receptors improves immune effector cell-mediated and antibody-mediated responses to pathogenic microorganisms, virus-infected cells and dysregulated cells. Small molecular weight agents can be used to treat infectious diseases, cancers and other humoral immunosuppressive diseases. In addition, non-stimulatory analogs that can bind and block CD16a activation can also be used to treat inflammatory diseases mediated by cells expressing CD16a. Background Art

[0002] Humoral immunity is the primary mechanism of vertebrate host organism surveillance and defense against infectious pathogens and dysregulated host cells. In cancer therapy, monoclonal antibodies (mAbs) that can target cancer-specific cell surface antigens have been successfully developed and commercially approved. It has been reported that several of these mAbs, in addition to complement-dependent cytotoxicity (CDC), also exert their tumor-killing effects through humoral-mediated antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP) via engagement with Fc-γ-activating receptors such as CD16a (FCGRIIIA), CD32a (FCGRIIA) and CD64 (FCGRIA) on immune effector cells (NK cells, macrophages, neutrophils, monocytes, dendritic cells, etc.) (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; Van Der Meid KR, et al. al.Cancer Immunol Res 6:1150-1160,2018). Recently, several researchers have reported that tumors produce factors that can inhibit humoral immune pathways and thus inhibit the tumor killing effect of mAbs through ADCC, ADCP and / or CDC mechanisms (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). ADCC, ADCP and CDC humoral immune responses are regulated by the coordination of antibodies with cell surface antigens, which together position the antibodies on the antigen epitopes at a certain distance from the cell surface.In cases where this localization is optimal, cell surface-bound antibodies can engage with Fc-γ-activating receptors on natural killer (NK) or dendritic / myeloid / monocyte cells (any cell that participates in ADCC or ADCP is referred to herein as an "immune effector cell") to initiate ADCC or ADCP and with the C1q complement activating protein to cause death of the antibody-bound cell through the classical complement pathway (Reuschenbach M, et al. Cancer Immunol Immunother 58: 1535-1544, 2009). These effects have been observed for several therapeutic antibodies, such as, but not limited to, rituximab, trastuzumab, cetuximab, pertuzumab, alemtuzumab, and daratumumab (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 are also observed in the patient's own immune response to dysregulated cells and in the response to vaccines. Antibodies with antiproliferative activity and immune-mediated killing activity have been observed in patients with indolent disease (Staff C, et al. J Clin Immunol 32: 855-865; Branden S, et al. Cancer Res 63: 7995-8005, 2003). However, although it has been found that many patients with cancer and viral diseases produce autoantibodies against tumor or viral antigens, their presence is often insufficient to eradicate diseased cells, which may be due to overall low autoimmune induced antibody levels, suboptimal epitope binding, immune effector cell exhaustion and / or humoral immune suppression mechanisms of pathogens, virally infected cells or dysregulated cells (Bi J and Tian Z. Frontiers Immunol 8: 1-10, 2017).

[0003] In cancer (also referred to herein as dysregulated cells), it has been found that the tumor-produced protein MUC16 / CA125 inhibits humoral immune responses by directly binding to a subset of IgG1, IgG3, and IgM type antibodies, which in turn interferes with the Fc region, making them less effective for binding to the Fc-γ-activating receptors FCGR2A (also known as FCGRIIA and CD32a) and / or FCGR3A (also known as FCGRIIIA and CD16a) on immune effector cells or for engaging the C1q protein of CDC (Pantankar MS, et al. Gyncol Oncol 99:704-713, 2005; Kline JB, et al. OncoTarget 8:52045-52060, 2017; Kline JB, et al. J. Clin. Oncol. 5:15, 2018; Wang W, et al. Cytogenet Genome Res152:169-179,2017;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). High affinity FCGR1 (also known as CD64) Fc-γ-receptor (K D =0.5 nM), suggesting that this effect is partly due to low affinity binding to 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] Most of the above findings on humoral immune suppression in cancer point out the importance of NK and myeloid cell activation and antibody binding for effective killing. Several reports have shown that the removal of NK cells reduces the therapeutic efficacy of antibody-mediated anticancer cell responses (Waldhauer I, Steinle A. Oncogene 27: 5932-5943, 2008). Based on these observations, several methods of using NK cells for cancer immunotherapy are currently being explored in experimental models and clinical trial settings (Vivier E, et al. Nat Rev Immunol 12: 239-252, 2012; Shimasaki N, et.al. Nat Rev Drug Dis 19: 200-218, 2020). These methods aim to utilize NK cells to kill host target cells that lack major histocompatibility complex class 1 (MHC1) proteins due to viral infection or dysregulation through ADCC and antibody-independent cellular cytotoxicity (AICC) (Bachanova V and Miller JS. Crit Rev Oncol 19: 133-141, 2014; Paul S and Lal G. Front Immunol 8: 1124, 2017). In addition, NK cell therapy is also being explored for the treatment of virally infected patients to eradicate virally infected host cells, which are downstream sources of viral reactivation (Traylen CM, et al. Future Virol 6: 451-463, 2011, Larkin J, et al. J Interferon Cytokine Res 26: 12, 2006). The main disadvantage of using NK cells directly or through antibody-mediated therapeutic responses is that after administration to patients, a sufficient number of activated cells capable of attacking virally infected cells or dysregulated cells cannot be retained. Several attempts to improve NK cell activation by pre-treatment or co-treatment with cytokine agonists such as IL-15 or stimulators that block NK cell negative regulatory pathways such as NKG2A have brought 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 BCand Antonia SJ. Nature Rev Clin Oncol 16:277-278, 2019). These suboptimal activities may be due to off-target effects that lead to host toxicity or limited in vivo stimulation (Guo, J et al. Cell Res 31:1190-1198, 2021).In view of these shortcomings, there is a need to identify agents that can preferentially or specifically activate and maintain the killing activity of NK and other immune effector cells against virus-infected or dysregulated cells.

[0005] It is also found that cells expressing CD16a are associated with inflammatory diseases and directly involved in inflammatory diseases (Cooper DL, et al. PLOS 7: e28918, 2012). Several studies testing the association of cell infiltration in rheumatoid arthritis found a correlation between cells expressing CD16a and disease severity (Robinson JI, et al. Ann Rheum Dis 69: 1054-1057, 2010). In addition, experimental animal models of inflammatory diseases also show a direct effect of CD16a activity and inflammatory disease progression (Ji H, et al. Immunity 16: 157-168, 2002; Diaz DS, et al. Eur J Immunol 32: 2915-2922, 2002). Based on these findings, the development of agents capable of blocking CD16a activation can be used as a therapeutic approach for CD16a-related inflammatory diseases. Summary of the invention

[0006] The present invention provides naturally occurring and synthetic small molecular weight agents that enhance the binding of IgG antibodies to CD16a-158F Fc receptors (FcR) (SEQ ID NO: 1) and to CD16a-158V Fc receptors (SEQ ID NO: 2), and stimulate CD16a FcR on immune effector cells to achieve cell activation and downstream target cell killing. The small molecular weight agents of the present invention relate to lanostane-8,24-diene-3β-ol (compound 1a) and 9β-19-cyclo-24-lanostane-3β-ol (compound 2a) of the tetracyclic triterpenoid class. They are able to enhance the binding of CD16a-158F FcR and its high affinity allotype CD16a-158V FcR to IgG1 antibodies and are able to activate the CD16a pathway in NK and other immune effector cells. Such therapeutic agents can be used to treat infectious diseases, cancer and other humoral immunosuppression diseases through CD16a activation of immune effector cells (see also Nes WD and Heftmann E. Nat Prod 44: 377-400, 1981). Optionally, the use of these scaffolds can be changed on certain carbons in order to bind CD16a and block its undesirable activation in inflammatory diseases.

[0007] Cholestane-based lanostane-8,24-diene-3β-ol (compounds 1a and 1b); 9β-19-cyclo-24-lanostene-3β-ol (compounds 2a and 2b); compounds 3, 4a and 4b based on biredo; and analogs containing their three cyclohexyl (A, B, C) and cyclopentyl (D) ring skeleton structures as scaffold structures can be used as described to modify CD16a to enhance affinity binding with IgG-type antibodies and / or enhance CD16a receptor activation in the presence or absence of IgG, while other analogs can be used to block CD16a binding to IgG and block its activation. Small molecular weight agents based on the following structures have been analyzed and tested:

[0008]

[0009] Wherein R1 is hydroxyl (OH) or formyl (CH=O);

[0010] wherein R2 and R3 are methyl (CH3);

[0011] Wherein R4 is methyl or 14α-difluoro-methyl (CHF2);

[0012] wherein 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 (CH 3COCH(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); PEGylated amines containing 2 to 12 PEG (polyethylene glycol) units; PEG5 amines; and biotinylated amines, wherein the biotin portion is linked to the amine portion via PEG 2-12.

[0013] Examples of biotinylated PEG2 amines that can be used as R6 are:

[0014]

[0015] Examples of PEG5 amines useful as R6 are:

[0016]

[0017] Another aspect of the invention uses the above compounds and certain other compounds as potent binders of CD16a FcR and stimulators of immune effector cells expressing such receptors to enhance the therapeutic effect on pathogens and virally infected and dysregulated cells, including malignant cells, in patients.

[0018] The present invention also includes therapeutic pharmaceutical compositions for activating immune effector cells in vitro and in vivo in mammals (including humans).The pharmaceutical composition comprises an effective amount of compound 1a, 1b, 2a, 2b, 3, 4a or 4b.

[0019] Several of the above compounds are preferred for reasons such as increased ease of synthesis and / or greater efficacy. Preferred compounds include lanosterol 14α-difluoro-methyl, 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-ol, lanosterol 24-PEG(2-12), lanosterol 24-biotin, lanosterol 24-PEG5-biotin, lanosterol 24-PEG5-lanosterol, lanosterol 24-PEG5-cycloartenol, lanosterol 24-isopropylmethyl ketone, lanosterol 24-methyl-isobutyl ketone, lanosterol 24-methyl, dihydrolanosterol, and 24,25 dihydroxylanosterol.

[0020] Other preferred small molecular weight agents that can be used are: 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-ol, 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.

[0021] Other embodiments include the preparation of the above-mentioned small molecular weight agents and analogs using parenteral and non-parenteral pharmaceutical preparations. Such pharmaceutical preparations may include, but are not limited to, alpha-tocopherol, cetyl alcohol, cetearyl alcohol, corn oil mono-diglyceride, emulsion EL, enteric acrylic resin, ethanol, ethyl oleate, glycerol, isopropyl palmitate, lauryl lactate, liposomes, mono- / diglyceride of caprylic / capric acid, oleic acid, PEG, phosphate buffered saline, polysorbate 80, propylene glycol, sorbitan monooleate, sulfonamides and trehalose.

[0022] Another embodiment of the present invention uses CD16a in a molecular binding assay in the presence of compound 1a, 1b, 2a, 2b, 3, 4a or 4b by any ELISA-based or affinity kinetics-based method (such as surface plasmon resonance (SPR), fluidics-based binding such as Octet TM The binding affinity of CD16a to full-length IgG antibodies or IgG Fc fragments can be measured using a kinetic binding assay or other kinetic binding analysis platform.

[0023] Another aspect of the invention is the use of biotinylated compounds 1a, 1b, 2a, 2b, 3, 4a or 4b to monitor the binding of the biotinylated compounds to CD16a domains. This can be used to delineate key binding domains, to improve antibody binding affinity, to improve CD16a FcR activation and / or to determine the bioprocessing of activated FcRs in cells expressing CD16a.

[0024] Another aspect of the invention is the use of compound 1a, 1b, 2a, 2b, 3, 4a or 4b to enhance the binding of CD16a to IgG in a molecular assay. This can be used to facilitate the screening of inhibitors of CD16a binding to IgG Fc. The use of such compounds enhances the binding signal, which can facilitate the screening of such inhibitors.

[0025] Another aspect of the invention is a kit for enhancing the binding of CD16a to the IgG Fc domain for in vitro screening. The kit includes compound 1a, 1b, 2a, 2b, 3, 4a or 4b, which can be used for CD16a and IgG Fc binding assays to enhance the binding signal. The enhanced binding signal can be used to test inhibitors of CD16a binding to IgG Fc. Any method known to those skilled in the art for screening protein-protein antagonists can be used.

[0026] Yet another aspect of the invention is the conjugation of Compound 1a, 1b, 2a, 2b, 3, 4a or 4b to an antibody to enhance the binding of the antibody to CD16a and improve the antibody-mediated therapeutic response.

[0027] Another aspect of the present invention is to apply compound 1a, 1b, 2a, 2b, 3, 4a or 4b to patients with infectious diseases or cancer in the presence or absence of standard-of-care therapy. The compound enhances the humoral immune response of the patient (i.e., activates immune effector cells expressing CD16a), thereby improving clinical outcomes. The compound can be administered orally, by skin administration and / or by injection.

[0028] Other embodiments of the present invention are to use analogs of compounds 1a, 1b, 2a, 2b, 3, 4a and / or 4b that bind to CD16a and block its activation in immune effector cells to inhibit inflammation.

[0029] These and other aspects of the invention, which will be apparent to those skilled in the art by reading the specification, provide the art with methods, compositions and kits for enhancing immune effector cells expressing CD16a, including but not limited to NK cells and myeloid cells. These can be used to improve the patient's response to pathogens or viral infections and to 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 therapies). The antibody may optionally be conjugated to one or more of the compounds. These compounds can improve antibody-mediated humoral immune responses in diseases associated with immunosuppression, including cancer and non-tumor diseases, in which antibody therapeutics are used to stimulate humoral immune responses. A second aspect of the present invention is the use of compounds that block CD16a activation to inhibit immune effector cell activation, which in turn supports undesirable inflammation, to treat inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1A-Figure 1B. Compound 1 and compound 2 specifically enhance the binding of human (Figure 1A) and rodent (Figure 1B) CD16aFc receptor to IgG antibody Fc domain. These data determine rodents as relevant species to test the enhancement of immune effector cell activation expressing CD16a by compounds 1 and 2 and analogs to achieve in vivo therapeutic activity. The experiment represents a minimum of three replicate wells. P values ​​are generated using Student's T test.

[0031] Figure 2. Compound 2 enhances the affinity of low affinity CD16a-158F Fc receptor for human IgG1 antibody. Molecular assays showed that compound 2 was able to significantly improve the binding of CD16a-158F Fc receptor to IgG1, 4.7-fold over the control compound. Experiments represent a minimum of two replicate wells. P values ​​were generated using Student's T test.

[0032] Fig. 3A-Fig. 3B. In the presence and absence of IgG1, compounds 1 and 2 enhance the activation of CD16a Fc receptors in Jurkat-CD16a-158F (JKT-CD16a) luciferase reporter cell lines, demonstrating the usefulness in antibody and non-antibody mediated immune effector activity (Fig. 3A). The experiment represents a minimum of three replicate wells. CD16a activation is demonstrated by enhanced secretion of activation cytokine markers IFNγ and TNFα to Jurkat-CD16a cells rather than CD16a knockout (null) parental Jurkat (JKT) cells in the culture medium (Fig. 3B). P values ​​are generated by comparing compound treatment vs vehicle in each group using Student's T test.

[0033] Figure 4A-Figure 4C. Compound 2 activates primary human peripheral blood mononuclear cells (PBMC), human natural killer cells (NK) and mouse spleen cells (SPL) effector cells and enhances antibody-dependent (ADCC) (Figure 4A) and antibody-independent (AICC) cytotoxicity (Figure 4B) against tumor target cells. Treatment of primary NK cells with compound 2 leads to the production of downstream CD16aFc receptor activation markers TNFα, IFNγ, GM-CSF and granzyme B protein (Figure 4C), thereby supporting evidence of immune effector-mediated cell activation and subsequent target cell killing. Similar cytokine production was found in human PBMC and mouse SPL stimulated cultures. The experiment represents a minimum of three replicate wells. P values ​​are generated using Student's T test.

[0034] FIG. 5A-FIG. 5B. Compound 2 was tested in vivo and shown to be well tolerated at ten times the effective dose calculated from in vitro studies (FIG. 5A). Compound 2 significantly activated serum TNF-α steady-state protein levels in vivo (FIG. 5B). Experiments represent a minimum of three replicate wells. P values ​​were generated using Student's T test.

[0035] Figure 6. Compound 2 activates immune effector cells within PBMC cultures, which in turn are able to kill SARS-CoV-2 virus-infected sibling (sib) PBMC cells and reduce viral load. Experiments represent a minimum of three replicate wells.

[0036] Figure 7A-Figure 7B. Structure activity relationship (SAR) screening reveals key active areas on compound 1, 2, 3 and 4 scaffold structures (Figure 7A). Figure 7B shows the specific compounds tested. These data demonstrate the use of compound 1, 2, 3 and 4 scaffold structures or their analogs for the development of additional compounds that can enhance CD16a Fc receptor-IgG antibody binding and activate or block CD16a pathways in immune effector cells to achieve therapeutic applications. The experiment represents a minimum of three replicate wells. P values ​​are generated using Student's T test.

[0037] Fig. 8. shows that it is possible to achieve other examples of modifications at R5 and R6 to improve CD16a Fc receptor-IgG antibody binding and activation or inhibition. They can also be used as biotinylated probes (e.g., compound 34) to analyze the in vitro and in vivo interactions of compound-CD16a. These modifications can improve solubility, molecular activity and therapeutic activity. Synthesis methods and final compound structures are provided below.

[0038] Fig. 9. R5 / R6 modified compound 2 has the effect on CD16a Fc receptor binding to Pertuzumab. These data show that the use of R5 or R6 modifications present in compounds 26, 27 and 33 can be incorporated into compounds 1, 2, 3 or 4 to further enhance CD16a Fc receptor-IgG antibody binding and activate or block the CD16a pathway in immune effector cells to achieve therapeutic applications. Alternatively, R5 or R6 modifications present in compounds 29, 31 and 32 can be incorporated to inhibit the binding of IgG to CD16a Fc receptors and potentially be used to inhibit inflammation to achieve therapeutic applications. The experiment represents a minimum of three replicate wells. P values ​​are generated using Student's T test.

[0039] Figure 10. Biological and pharmacological analysis of compounds 2, 26, 27 and 33, which show enhanced CD16a-pertuzumab binding in Figure 9, was performed to determine the optimal compound. Human PBMCs were used as effector cells to test the ADCC killing of human SKBR3 (HER2-expressing breast cancer cells) by anti-HER2 pertuzumab (PTZ) antibody test compounds and ethanol (EtOH) vehicles. As shown in the figure, compared with parent compound 2, compounds 27 and 33 significantly enhanced ADCC killing (P<0.0000075). Wells containing cells and PTZ without PBMC or compounds (first bar) were used as negative controls. The experiment represents a minimum of three replicate wells. P values ​​were generated using Student's T test.

[0040] Figure 11. Schematic synthesis of compound 27.

[0041] Figure 12. NK cells are activated in vivo by oral administration of compound 27. Compound 27 or vehicle was administered to mice (N=3 per group). 24 hours after the last treatment, serum was collected and analyzed by ELISA for NK cell activation biomarkers IFNγ and TNFα. As shown in the figure, both biomarkers were significantly increased in mice treated with compound 27 (P<0.0056). The experiment represents a minimum of three replicate wells. P values ​​were generated using Student's T test.

[0042] Figure 13. Development of CD16a-IgG binding antagonists for the treatment of inflammatory diseases. Compound analogs that could potentially block CD16a FcR-IgG binding were tested using ELISA. As shown, compound 29, derived from the scaffold structure of compound 2, was able to significantly inhibit the binding of the CD16a FcR probe to the Pertuzumab (PTZ) antibody coated in a 96-well microplate. The experiment represents a minimum of three replicate wells. P values ​​were generated using a Student's T test. DETAILED DESCRIPTION

[0043] The inventors have identified such small molecular weight agents: it can specifically bind to cross-species CD16a Fc-γ-activating receptors, resulting in NK cells and other immune effector cells carrying CD16a activated in vitro and in vivo. Small molecular weight agents enhance the binding of IgG antibodies to low (158F) and high (158V) affinity CD16a Fc-γ-activating receptors (FcR). The way in which small molecular weight agents exert their effects is by changing the dynamic structure of FcR, resulting in increased FcR-IgG binding, enhanced CD16a receptor signaling, and subsequent immune effector cell activation and target cell killing enhancement. Here, we describe the use of small molecular weight agents and their active cores, which stimulate the activity of FcR, thereby resulting in immune effector cell activation and / or enhanced IgG1 affinity binding and subsequent target cell killing (see also Bruhns P et al. 113: 3716-3725, 2009). Small molecular weight agents that stimulate FcR activation and enhance IgG binding can be used to treat infectious diseases, cancer and other humoral immunosuppression diseases. We also teach the use of compounds such as but not limited to compound 37 to inhibit CD16a activation using compositions containing a key backbone scaffold structure for CD16a binding without a stimulatory domain capable of inhibiting CD16a activation in immune effector cells involved in inflammatory diseases.

[0044] The identified small molecular weight agent can be combined and activated by directly binding to CD16a Fc-γ-activating receptors (FcR) to express rodent and human CD16a Fc receptor immune effector cells. In addition, these compounds can also enhance the combination of IgG type antibodies with high-affinity and low-affinity CD16a receptors, thereby increasing its antibody-mediated killing mechanism (Rugo HS, et al. J Clin Oncol 37: 1000, 2019) to target cells expressing antigens. Alternatively, non-stimulatory compounds provide the opportunity to block CD16a activation, thereby suppressing immune effector cell activation and undesirable inflammatory response. Although not wanting to be limited to any specific theory or mechanism of action, the applicant believes that active small molecular weight compounds are connected with CD16a FcR on immune effector cells (including macrophages, NK cells and any other cell subtypes expressing CD16a FcR). They change the dynamic structure of CD16a, causing receptor signaling and immune effector cell activation to change and to enhance the affinity with the IgG Fc domain, and either or both of these two results lead to enhanced humoral immune cell killing responses for target cells. In addition, one or more of these compounds can be applied to patients to enhance their humoral immune responses for infectious disease pathogens and disordered cells (including cancer). Alternatively, these compounds can be modified to bind and block CD16a activation, resulting in suppressed immune effector cell activation and its associated downstream inflammatory cascade. The application is used to suppress the undesirable inflammation associated with inflammatory diseases well known to those skilled in the art.

[0045] Additional analogs of the above compounds, which further enhance their in vivo therapeutic activity through enhanced CD16a FcR binding and activation or blocking and inactivation, as well as their pharmacokinetic (PK) and pharmacodynamic (PD) properties, can be similarly identified. Such enhancements can improve therapeutic efficacy and patient tolerability, all of which can be further determined through preclinical and human trials.

[0046] Some of these methods, compositions and kits can be divided into three categories. In one category, the compound is developed for use as a therapeutic agent. It is well known in the art that stronger antibody binding affinity to CD16a Fc receptors, especially to low-affinity human CD16a-158F FcR allotypes, leads to improved therapeutic responses of target cells for specific antigens expressing antibodies (Stavenhagen, JB et al. Cancer Res 67: 8882-8890, 2007). In addition, activated NK cells are also known in the art to be effective in killing virus-infected cell types 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 both activate NK cells and enhance binding to CD16a Fc receptor antibodies. In yet another category, it is known to those skilled in the art that dysregulated inflammatory cells expressing CD16a may cause undesirable inflammation, thereby causing various inflammatory diseases (Cooper DL, et al. PLOS 7: e28918, 2012). The use of analogs of the compositions and methods described herein enables the development of compounds that can bind to and block immune effector cell activation and its downstream inflammatory cascade. Finally, another category is the use of active compounds as molecular biological reagents to enhance CD16a-IgG antibody binding in ELISA and other binding assay formats to promote the screening of CD16a FcR-IgG antibody binding inhibitors or activators and subsequent pathway inhibition / activation. These compounds can be used in kits or as independent assay enhancement reagents.

[0047] For cancer and infectious disease treatment uses, the compound can be administered alone to achieve AICC therapy, and can also be administered in combination with experimental or regulatory approved anticancer / antiviral antibodies that utilize ADCC or ADCP mechanisms of action. Certain anticancer and antiviral antibodies that may benefit from enhanced CD16a activation and / or binding of active compounds include, but are not limited to, anticancer agents: rituximab, trastuzumab, trastuzumab emtansine, cetuximab, YP218, ocrelizumab, daratumumab, elotuzumab, alemtuzumab, necitumumab, pertuzumab, obinutuzumab, nivolumab, ipilimumab ), pembrolizumab, ofatumumab, panitumumab, tremelimumab, mosunetuzumab, penpulimab, amivantamab, margetuximab, naxitamab, tafasitamab, inebilizumab, isatuximab, and durvalumab. In addition, the compound can also be administered with anti-infective disease antibodies, including but not limited to: nirsevimab, tixagevimab, sotrovimab, regdanvimab and combination antibody therapy, i.e., casirivimab + imdevimab. Antibodies that improve humoral immune function (ADCC or ADCP) can be used to achieve combination therapy with the optimal amount of the compound.

[0048] For therapeutic use, the compounds can be administered orally, sublingually, buccally, topically or parenterally, via intramuscular, intravenous or subcutaneous methods.

[0049] Compounds can be formulated to support these different routes of administration. They can be delivered via liposome preparations or other preparations. Conventional liposomes are composed of a lipid bilayer composed of cations, anions or neutral (phosphoric acid) lipids and cholesterol, and the lipid bilayer surrounds the water volume. Suitable compositions of liposomes include but are not limited to guanidine-cholesterol cationic lipids bis (guanidine)-tren-cholesterol (BGTC) combined with lipid dioleoylphosphatidylethanolamine (DOPE). Another example of suitable liposome preparations is aminoglycoside lipid dioleyl succinyl paromomycin (DOSP) associated with imidazole-based auxiliary lipid MM27. Liposomes can be spatially stable, for example, using polyethylene glycol to coat liposomes. If desired, liposomes can be ligand-targeted. Suitable ligands include antibodies, peptides, carbohydrates and proteins.

[0050] Still other formulations for parenteral and non-parenteral administration include, but are not limited to, alpha-tocopherol, cetyl alcohol, cetearyl alcohol, corn oil mono-diglycerides, emulsion EL, enteric acrylic resins, ethanol, ethyl oleate, glycerol, isopropyl palmitate, lauryl lactate, liposomes, caprylic / capric mono / diglycerides, oleic acid, PEG, phosphate-buffered saline, polysorbate 80, propylene glycol, sorbitan monooleate, sulfonamides, and trehalose.

[0051] In other embodiments of the invention, we provide methods and compositions for developing compounds that are capable of binding and activating either allotype of CD16a-FcR and / or enhancing IgG antibody-CD16a affinity binding and downstream activation.

[0052] In some embodiments, the ability of the test compound to enhance CD16a FcR binding and activation of cells expressing CD16a FcR. CD16a FcR activation can be monitored by measurement of downstream pathway signaling and downstream markers such as, but not limited to, IL-1β, IFN-γ, TNF-α, GM-CSF, and granzyme B. These can be tested in vitro or in vivo.

[0053] In yet other embodiments, analogs of compounds 1a and 1b, 2a and 2b, 3 and compounds 4a and 4b are tested for improved CD16a FcR-IgG antibody binding and / or cellular activation of CD16a FcR-mediated pathway-expressing cells, while minimizing cytotoxicity, such as in in vitro or in vivo screening. These are accomplished using various methods known to those skilled in the art.

[0054] In yet another embodiment, the compound has modified regions to improve optimal binding of the CD16a FcR to an IgG antibody and / or enhance the humoral immune response of the test antibody using any method used by one of skill in the art to measure ADCC and / or ADCP.

[0055] In yet another embodiment, the compound comprises the backbone of compounds 1a and 1b, 2a and 2b, or 3 and compounds 4a and 4b, while retaining the chemical groups at R1, R2, R3 and R4, and modified chemical groups at R5 and R6 - these modified chemical groups can be empirically tested for their enhancement of CD16a FcR-IgG antibody binding and / or immune effector cell activation or inhibition.

[0056] In a method for measuring the ability of a compound to effectively enhance FcR antibody binding or activation of cells expressing CD16a; compounds are tested for direct binding of IgG antibodies to CD16a FcR by ELISA, immunohistochemistry, or other methods known to those skilled in the art. Compounds with 25% or greater binding are suitable for further testing.

[0057] In some embodiments, the activity of compounds 1a and 1b, 2a and 2b, or 3 and compounds 4a and 4b or derived analogs is measured using a functional method using ADCC, ADCP, AICC or downstream cytokines such as IL-1β, IFNγ or TNFα in the presence or absence of antibodies, wherein the cells can be cell lines or primary NK, myeloid, PBMC or SPL cells. The term "activity" generally refers to a 10% or greater change in ADCC, ADCP or AICC target cell killing or cytokine production compared to cells without the compound when the compound is incubated with the cell with or without the antibody. Depending on the cell type, antibody and compound used, this term may also refer to a change of at least 5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70% or 75%.

[0058] Compounds with improved pharmacokinetic (PK), pharmacodynamic (PD) or pharmacological (PL) activity can be screened. In some embodiments, compounds are added to cells in vitro and tested for killing of target cells by effector cell activation in the presence or absence of antibodies. Compounds with enhanced killing with or without antibodies are now suitable for therapeutic testing using in vivo models. In another embodiment, compounds can be incubated in microsomes from various species to determine compound modifications that may affect such compounds (one or more) with higher metabolic stability. Depending on the compound used, this effect can be at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75% or greater changes.

[0059] Other embodiments employ Compounds 1a, 1b, 2a, 2b, 3, 4a and / or 4b to bind and block CD16a activation and signaling, which in turn leads to a reduction in downstream inflammatory signaling via sustained cytokine production associated with inflammatory diseases.

[0060] Various terms and expressions ("terms") related to various aspects of the attached description are used throughout the specification and claims of this document. Unless otherwise specifically indicated, these terms are to be given their ordinary meanings in the art. Other specifically defined terms should be understood in a manner consistent with the definitions provided.

[0061] As used in this specification and the appended claims, the singular forms "a", "one / kind", and "the / said" also include plural references unless the content clearly dictates otherwise. As an example, reference to "a cell" may include a combination of two or more cells, and so on. Reference to "a probe" may include polyhistidine-tagged human or rodent CD16a-FcR, CD32a FcR, or an independent probe for monitoring FcR-antibody binding, whereby 2-10 histidine codons are engineered at the mature N-terminal or extracellular C-terminal domain of the FcR. These probes are referred to as CD16a-HIS or CD34a-HIS. Reference to "cell activation" may include the effect of a compound on cells expressing CD16a FcR by any analytical method known to those skilled in the art.

[0062] The term "about" when used in reference to quantitative values ​​such as amounts, time periods, and / or the like is intended to encompass variations of up to ±9% from the specified value, as such variations are appropriate for practicing the disclosed methods. Unless otherwise indicated, all values ​​used in the specification and claims indicating amounts of reagents (such as molecular weights, molar concentrations, reaction conditions, percentages, etc.) are to be understood as being quantified in all cases by the term "about". Therefore, unless otherwise indicated, the numerical values ​​listed in the following specification and the enumerated claims are approximate values ​​that may vary depending on the desired composition properties and / or the methods sought to be obtained by the present invention. At the very least, and without attempting to limit the scope of this application, each numerical value should at least be rounded to the reported significant figures and by ordinary rounding methods known to those skilled in the art.

[0063] The term "antibody" as used is broad and includes immunoglobulins (also referred to as "Ig") or antibody molecules, including polyclonal antibodies (also referred to as pAbs), monoclonal antibodies (also referred to as mAbs), including mouse, rat, primate, human, humanized and chimeric mAbs, and full-length bispecific or trispecific antibodies. In general, an antibody is a protein or polypeptide chain that binds to a specific antigen. The antigen is the structure that is specifically recognized by a given antibody. A typical antibody comprises a heterotetrameric glycosylated protein, consisting of two light chains and two heavy chains arranged by a complex of disulfide bonds and hydrogen bonds. The term "its disulfide bridges" refers to the disulfide bridges contained in the hinge region of the heavy chain, which is well known to those skilled in the art. Each heavy chain has a variable domain (variable region) (VH) followed by multiple constant domains (referred to as Fc domains). Each light chain has a variable domain (VL) and a constant domain; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain VL is aligned with the variable domain of the heavy chain. The light chains of antibodies from any species are assigned to one of two different types, kappa (κ) and lambda (λ), based on their amino acid sequences within their constant domains.

[0064] Immunoglobulins are classified into classes or isotypes, depending on the type of Fc domain, namely IgA, IgD, IgE, IgG and IgM, depending on the sequence contained in their heavy chain constant (Fc) domain. The IgG isotype is further composed of the following subclasses, such as isotypes IgG1, IgG2, IgG3 and IgG4.

[0065] "Specific binding" or "specifically binds" refers to the binding of an antibody or antigen (including sequences contained in the antibody itself) with greater affinity than for other antigens. In general, a specific antibody or antigen-binding fragment has a specific affinity of about 5×10 -8 The equilibrium dissociation constant K is M or smaller DBinding to the target antigen. In other cases, it refers to the binding of the antibody Fc domain to the CD16a, CD32a or CD64 Fc receptor. The equilibrium dissociation constant K D The range can depend on the Fc receptor and its allotype.

[0066] The term "allotype" refers to the amino acid sequence of the CD16a or CD32a Fc receptors, which have been shown to differ in the human population, each with a different binding affinity for IgG Fc. For CD16a, it generally refers to the amino acid at position 158, containing either valine (V) or phenylalanine (F), as is well known to those skilled in the art.

[0067] The term "affinity" refers to the measurement of the on vs off ratio of binding of an antibody to its variable domain for antigen and the measurement of the on vs off ratio of binding of an antibody to its Fc domain for Fc receptors including CD16a, CD32a and CD64.

[0068] An "antibody derivative" refers to an antibody as defined above that has been modified by covalent attachment of another molecule, such as by peptide chemistry (i.e., amidation, etc.), genetic fusion, and / or by post-translational moieties not normally associated with antibodies (i.e., carbohydrate, acetyl, and / or phosphoryl groups), etc. This may include a compound taught herein that is a chemical fusion to an antibody that enhances the FcR binding activity of the antibody.

[0069] The term "CD16a dynamic structure" refers to any structural changes that can affect the humoral function, activation, inhibition and / or CD16a-IgG antibody binding of immune effector cells expressing CD16a.

[0070] "Fc domain" refers to any antibody sequence at the C-terminus of and including the antibody hinge disulfide region. This region includes the binding domains of CD16a, CD32a and CD64 Fc receptors and is known to those skilled in the art.

[0071] An "antigen" is an entity to which an antibody or antibody fragment specifically binds. This includes binding to an antibody or protein of interest.

[0072] The term "mature" refers to an extracellular membrane or secretory protein in which the N-terminal secretion signal sequence is cleaved, which is a concept well known to those skilled in the art.

[0073] The terms "cancer," "malignant," and "tumor" are well known in the art and refer to the presence of cells having unregulated cell growth and morphological characteristics that are different from normal cell types of similar origin, also referred to as "dysregulated cells." Malignant refers to those cancer cells that are capable of causing morbidity and / or mortality. As used, "cancer and tumor" include both premalignant and malignant types.

[0074] The terms "viral," "pathogenic," and "infectious" are art-recognized and refer to the presence of a cell or object that has been infected with a disease-causing microorganism or viral particle.

[0075] The terms "inflammation" or "inflammatory disease" refer to a disease in a patient whereby dysregulation of inflammatory cells results in inflammation of one or more organs of the body.

[0076] As used, the term "soluble" refers to a protein or non-protein agent that is not attached to the cell membrane of a cell. For example, a soluble agent can be shed, secreted or exported from a normal or cancerous cell into a biological fluid, including serum, whole blood, plasma, urine or a microfluidic of cells (including tumors). Native membrane-bound Fc receptors such as CD16a, CD32a or 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.

[0077] The term "detection agent" or "detectable agent" refers to any agent that can be linked to an antibody or FcR and detected by equipment commonly used in the art. These include, but are not limited to, fluorophores, enzymes and enzyme substrates, radionuclides, heavy metals and colorimetric dyes, and substrates that can be detected by methods such as, but not limited to, densitometry, spectrophotometry, luminescence, microscopy, radiography, and scintillation. Additional reagents may be required to produce a detectable signal.

[0078] The term "biomarker" refers to a protein produced by immune effector cells that is enhanced or suppressed by treatment with a compound. Specifically, interferon-γ (also known as IFN-gamma or IFNγ), tumor necrosis factor-α (also known as TNF-alpha or TNFα), and granzyme B are known biomarkers produced by immune effector cells (including NK cells) stimulated by the CD16a Fc receptor.

[0079] The "level" of a specific protein or compound (such as, but not limited to, granzyme B, IFNγ or TNFα) used, including a protein induced by CD16a FcR from a target cell, refers to the level or levels of a protein or compound determined by any method known in the art for measuring protein and / or compound levels in vitro or in vivo in response to treatment with a compound. Such methods include gel electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), superdiffusion chromatography, fluid or gel precipitation reaction, absorption spectroscopy, colorimetric assay, spectrophotometric assay, flow cytometry, immunodiffusion (single or double), solution phase assay, immunoelectrophoresis, Western blot, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, fluorescence resonance energy transfer (FRET), Forster resonance energy transfer, electrochemiluminescence immunoassay, etc. In one embodiment, the level of IFNγ and / or TNFα is determined using a probe-based technique, as described in more detail. In another embodiment, the level of the compound is determined using chromatography-based and ELISA-based techniques.

[0080] The term "humoral immuno-suppression", immunosuppression or humoral immune suppression" refers to any antibody, antibody fragment, bispecific or trispecific antibody or immune effector cell (i.e., NK cells, myeloid cells, monocytes, dendritic cells, etc.) that silences the maximum biological response. It is reported that FcR receptor allotypes and lack of immune effector cell activation can affect the biological activity of these humoral immune components, including ADCC, ADCP, AICC and / or PK, PD and PL profiles.

[0081] The term "antibody drug conjugate (ADC)" refers to any antibody that is conjugated or fused to a compound that results in enhanced activity of the antibody, including enhanced binding to FcRs, thereby resulting in enhanced ADCC and ADCP.

[0082] The term "bispecific or trispecific antibody (BSP)" refers to any antibody capable of binding to two or more different antigens. A BSP can include at least, but not limited to, two full-length antibodies, one full-length antibody, or one single-chain antibody, each of which binds to a different antigen or a different epitope on the same antigen.

[0083] The term "antibody-dependent cellular cytotoxicity (ADCC)" refers to an in vitro or in vivo process in which an antibody is able to bind to an antigen on the surface of a cell and then engage with an immune effector cell through sequences within the Fc domain of the antibody, resulting in the release of toxins by the immune effector cell that are able to kill the bound cell.

[0084] The term "antibody-independent cellular cytotoxicity (AICC)" refers to an in vitro or in vivo process in which cells expressing CD16a are stimulated by non-antibody CD16a FcR activation, which in turn results in the binding of CD16a-expressing cells to deregulated or infected cells via the native cell surface receptor, leading to the release of toxins that are capable of killing the bound cells.

[0085] The terms "antibody-dependent cellular phagocytosis (ADCP) and opsonization" refer to a process in which antibodies are able to bind to antigens on the surface of dysregulated cells and then engage immune cells through sequences within their Fc domains, resulting in the immune cells engulfing, consuming, and ultimately killing the antibody-bound cell.

[0086] The term "screening" may refer to testing compounds and measuring enhanced or diminished biological responses by monitoring ADCC, AICC, ADCP or other immune effector cell activation, with the goal of identifying compounds with such activity. Other references relate to finding compounds that can block or enhance the binding of CD16a Fc receptors to IgG antibodies. The term may be used in other contexts where a large number of test elements are assayed to determine which elements in the test element have a certain property. Similarly, it can be used to refer to assaying patient samples with specific properties.

[0087] The term "significantly" refers to a statistical result having a P value less than 0.05 as determined by various procedures including Student's T-test.

[0088] The term "pharmacokinetics (PK)" refers to the time over which a compound maintains its steady-state concentration when administered to a subject.

[0089] The term "pharmacokinetics (PD)" refers to the study of the biochemical and physiological effects of compounds and their mechanism(s) of action, including the correlation of their actions and effects with their biochemical structure when administered to a subject.

[0090] The term "pharmacology (PL)" refers to the known effects of a compound on the regulation or killing of diseased cells in vitro or in vivo.

[0091] The term "maximum tolerated dose (MTD)" refers to the maximum dose of a compound that can be administered to a patient or test animal before the patient or test animal experiences signs of toxicity. In test animals, the MTD is generally defined as a body weight loss of more than 10%.

[0092] The term "sample" refers to a general term for similar fluids, cells or tissues isolated from a subject, as well as fluids, cells or tissues present in a subject. Fluids may include biological fluids, which include liquid solutions in contact with a subject or biological source, including cell and organoid culture media, urine, saliva, lavage fluids, etc.

[0093] As used, the term "control sample" refers to any clinically or non-clinically relevant control sample, including, for example, samples from healthy subjects not suffering from a particular cancer type or cells other than their parental cells.

[0094] The term "control level" refers to an accepted or predetermined level of a protein or non-protein agent that is used for comparison with the level of the same agent in a sample derived from a subject or used in an in vitro assay.

[0095] As used, a "difference" between the signals of two compounds that may have a common backbone is generally any difference that can be determined statistically using statistical methods commonly used by those skilled in the art, and is at least 10% or greater compared to a control. Depending on the compound and assay used, a change of at least 5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70% or 75% may also be referred to.

[0096] The term "inhibit" or "inhibition thereof" means to reduce by a statistically measurable amount, or to prevent completely.

[0097] The term "functional" in the context of a compound, in the presence or absence of an antibody used according to the method, indicates that the compound is able to enhance or block, respectively, the binding of the antibody and / or the activation of immune effector cells, resulting in enhanced killing of target cells in vitro or in vivo and / or the production of downstream proteins or inhibition of inflammatory pathway signaling of immune effector cells.

[0098] The term "pharmaceutically acceptable" refers to a substance that is pharmacologically and toxicologically acceptable and is administered to a patient and is manufactured using methods known to those skilled in the art. These include agents for animals and humans that are approved by regulatory agencies of the federal or state governments or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias. The term "pharmaceutically compatible ingredients" refers to a pharmaceutically acceptable diluent, adjuvant, excipient, or matrix vehicle that is administered with an anticancer agent. A "pharmaceutically acceptable carrier" refers to a matrix that does not interfere with the effectiveness of the biological activity of the active ingredient (one or more) and is nontoxic to the host.

[0099] The term "formulation" refers to solvents, excipients, or base vehicles to which a compound is added to support its administration to a patient.

[0100] The terms "patient" and "subject" are used interchangeably to refer to humans and other non-human animals, including veterinary subjects, who are treated with a therapeutic agent. The term "non-human animal" includes all vertebrates. In one embodiment, the subject is a human.

[0101] The terms "effective amount" and "therapeutically effective" are used interchangeably and are used in the context of administering an agent in an amount sufficient to produce an enhanced clinical outcome in a patient. An effective amount of an agent is administered according to the methods described herein in an "effective regimen". The term "effective regimen" refers to a combination of an amount of an agent and a dosage frequency sufficient to achieve an enhanced clinical outcome for a patient with a specific cancer. When an agent that can overcome morbidity better than the parent compound or an agent that can enhance the clinical outcome of an effective regimen is administered to a patient, the enhanced efficacy is an improved clinical outcome. For example, in the context of this article, an effective amount refers to the amount of the compound required to demonstrate efficacy or difference when compared to no compound.

[0102] A "therapeutic agent" is generally substantially free of undesirable contaminants. This means that the agent is generally at least about 50% w / w (weight / weight) pure and is substantially free of interfering proteins and contaminants.

[0103] The term "target cell" refers to a eukaryotic or prokaryotic cell or cell population that expresses a specific antibody or antigen comprising an antibody portion and is bound by a cell expressing the CD16a Fc receptor through direct antibody engagement to achieve ADCC or ADCP activity or through indirect antibody interaction via AICC.

[0104] The term "immune effector cell" refers to any cell line or natural cell, including but not limited to NK cells, myeloid cells, monocytes, neutrophils, dendritic cells, which can confer antibody-dependent cellular cytotoxicity (ADCC) or phagocytosis (ADCP or opsonization) when bound to antibody-bound target cells and antibody-independent cellular cytotoxicity (AICC) when stimulated by CD16a FcR. Natural cells can be purified or mixed in the form of peripheral blood mononuclear cells (PBMC). Cell lines can naturally express CD16a or be engineered to express CD16a.

[0105] The term "dysregulated cell" refers to any cell that is considered abnormal to the parent cell. These include transformed cells, malignant cells, virally infected cells, cells that grow autonomously through autoregulation, or prokaryotic pathogens.

[0106] The term "humoral 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.

[0107] The term "compound" refers to any compound or structural formula compound described in this document that can bind to and change the activity of CD16a FcR. Specifically, it refers to cholestane-based lanostane-8,24-diene-3β-ol (compound 1a); 9β-19-cyclo-24-lanostene-3β-ol (compound 2a) and bi-decyl alcohol-derived compounds (compound 3) and analogs (compound 4a) composed of their three cyclohexyl (ring A, ring B, ring C) and cyclopentyl (ring D) ring skeleton structures as scaffold structures. The term "compound" also refers to any compound or structural formula compound described in this document that can bind to and enhance or block the activity of CD16a FcR, including compounds 1a, 1b, 2a, 2b and analogs (compounds 3, 4a or 4b) composed of their three cyclohexyl (ring A, B, C) and cyclopentyl (ring D) ring skeleton structures as scaffold structures.

[0108] The term "analog" refers to any compound derived from compound 1a, 2a, 3, 4a, 1b, 2b or 4b, wherein single or multiple modifications are made at R1, R2, R3, R4, R5 and / or R6.

[0109] The term "food" refers to a substance consisting mainly of protein, carbohydrate and / or fat, which is used in an organism to maintain growth, repair and life processes and provide energy. Food may also contain supplementary substances such as minerals, vitamins and condiments. See Merriam-Webster's Collegiate Dictionary, 10th Edition, 1993. The term food includes beverages suitable for human or animal consumption. As used herein, "food additives" are defined by FDA in 21 C.FR170.3 (e) (1), including direct and indirect additives.

[0110] The term "dietary supplement" means a product (other than tobacco) intended to supplement the diet that carries or contains one or more of the following dietary ingredients: vitamins, minerals, herbs or other plants, amino acids, dietary substances intended for human use to supplement the diet by increasing the total daily intake, or concentrates, metabolites, components, extracts, or combinations of these ingredients.

[0111] Therapeutic compounds, kits and methods for enhancing humoral immunity-mediated therapy

[0112] Provided herein are compositions, kits and methods for identifying small molecular weight compounds that are effective in enhancing or inhibiting CD16a Fc receptor binding to IgG antibodies and / or activating immune effector cell responses to pathogens, virus-infected cells or dysregulated cancer cells or inhibiting dysregulated inflammatory cells. Figures 1-11 schematically show examples. The kits consist of compounds whose chemical compositions are taught by the structural formulas in the present invention and the schematic methods for synthesizing appropriate compositions described in Example 3. Active compounds can be identified using molecular CD16a FcR-IgG antibody binding and / or effector cell activation or inhibition assays, as described in the following examples.

[0113] In the method for identifying an activating compound by cell-based screening, the compound is added to a target cell culture expressing the target antigen of the test antibody with or without an antibody. Immune effector cells are added to the culture and the humoral immune response when there is a compound and no compound is compared, and the humoral response is monitored using standard ADCC, ADCP or AICC killing assays. It is generally believed that at least 10% of the change is a significant impact on ADCC, ADCP or AICC function. Depending on the assay used, a significant impact can also be defined as a change of at least 5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70% or 75%.

[0114] In other methods for identifying activated compounds, the test antibody is added to the culture of target cells, wherein the target cells naturally express the antigen of the test antibody. Add the human Jurkat cells of the human CD16a Fc receptor (JKT-CD16a) of engineered expression connected to the luciferase reporter gene, and monitor the luciferase level after 12-24 hours. It is generally believed that at least 10% of the change is a significant impact on CD16a activation. According to the antibody used and the determination, the significant impact can also be defined as at least 5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70% or 75% change.

[0115] Also provided herein is a method for treating a cancer subject with an active compound. For example, a patient may suffer from cancers such as lymphoma, breast cancer, mesothelioma, colorectal cancer, lung cancer, ovarian cancer, pancreatic cancer, bile duct cancer, or endometrial cancer that express CD20, HER2, EGFR, or mesothelin. It is reported that several antibodies targeting these antigens are more effective in patients whose immune effector cells express high-affinity CD16a-158V FcR allotypes (Rugo HS, et al. J Clin Oncol 37: 1000, 2019). Unfortunately, more than 50% of the world's population expresses low CD16a-158F FcR, so that compounds capable of enhancing the binding affinity of test antibodies to CD16a-158F FcR become a pharmaceutically desirable entity (Wang W, et al. Cytogenet Genome Res 152: 169-179, 2017). One such antibody is anti-CD20 rituximab, and clinical studies have found that the therapeutic effect of this antibody is significantly reduced in patients with heterozygous or homozygous CD16a-158F alleles (Treon SP, et al. J Clin Oncol 23:474-481, 2016). It is also highly desirable to use effective compounds against this antibody in medicine.

[0116] In some embodiments of the methods of treating a subject with an active compound, a patient suffering from an infectious disease or cancer can be treated with the compound alone or in combination with a standard of care therapy. In some embodiments of the methods of treating a subject described herein, the compound is administered alone to the subject. In yet another embodiment, the compound is administered in combination with a standard of care therapy, which includes all agents that are considered standard of care for the disease indication when treating the subject.

[0117] In the method for identifying a compound that blocks CD16a pathways, a test antibody is added to a culture of a target cell, wherein the target cell naturally expresses the antigen of the test antibody. Human Jurkat cells expressing an engineered human CD16a Fc receptor (JKT-CD16a) connected to a luciferase reporter gene are added, and luciferase levels are monitored after 12-24 hours. It is generally believed that a signal inhibition of at least 10% when compared to cultures not treated with a compound is a significant effect on CD16a inhibition. Depending on the antibody used and assay, a significant effect may also be defined as an inhibition of at least 5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70% or 75%.

[0118] In some embodiments of the methods of treating a subject with an active compound, a patient suffering from an inflammatory disease can be treated with the compound alone or in combination with a standard of care therapy. In some embodiments of the methods of treating a subject described herein, the compound is administered alone to the subject. In yet another embodiment, the compound is administered in combination with a standard of care therapy, which includes all agents that are considered standard of care for the disease indication when treating the subject.

[0119] In some embodiments of the methods of treatment described herein, the compound is composed of a chemical skeleton derived from Compound 1, 2, 3 or 4 and retains the chemical groups at R1, R2 and R3, while modifying the R4, R5 and / or R6 regions using the structural formulas taught or exemplified herein.

[0120] The present method can be combined with alternative treatments such as surgery, radiation, targeted therapy, chemotherapy, immunotherapy, growth factor inhibitors or the use of anti-angiogenic factors. The compound can be administered to patients who are receiving alternative treatments at the same time. Before or after the standard of care therapy is administered, the compound is administered for at least one hour to several months, for example, at least one hour, five hours, 12 hours, one day, one week, one month or three months, and the patient can also receive alternative treatment. Some embodiments of the methods of treatment provided herein relate to administering to the subject a therapeutically effective antibody specific for an antigen expressed by the pathogen, virus, cancer or inflammatory mediator in addition to the compound.

[0121] In some embodiments of the treatment methods described herein, the subject may have received both alternative therapies and antibody-based standard of care treatments prior to administration of the compound.

[0122] Administration of therapeutic agents and compounds according to the methods of treatment described herein can be carried out by any means known in the art.

[0123] The amount of therapeutic agents or compounds effective in the treatment or prevention of cancer or non-tumor (virus, pathogen or inflammatory cell) mediated diseases can be determined by standard clinical techniques. In addition, in vitro assays can be optionally used to help determine the optimal dosage range required for the compound using standard or proprietary screening methods taught herein. The effective dose can be extrapolated from the dose-response curve of the compound derived from an in vitro or animal model test system.

[0124] For example, the toxicity and therapeutic efficacy of an agent can be determined in cell cultures or experimental animals by determining the LD 50 (a dose lethal to 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as LD 50 / ED 50A ratio of 1:1 to 2:1. An agent that exhibits a therapeutic index greater than 2-fold is suitable. When an agent exhibits toxic side effects, a delivery system that targets the agent to the site of the affected tissue can be used to minimize potential damage to cells in healthy tissue, thereby reducing side effects.

[0125] Dosing and dosage regimens may vary depending on the active drug concentration, which in turn may depend on the needs of the subject.

[0126] A panel for optimizing the binding activity of CD16a FcR and IgG antibodies for screening

[0127] Also provided herein is a kit for rapidly screening inhibitors or activators that can affect pathways using active compounds to enhance human or mouse CD16a FcR binding to IgG antibodies. It is taught herein that the active composition provided in the present invention can significantly improve CD16a FcR-IgG antibody binding in ELISA and other binding assay formats. This improvement promotes the screening of agents that can positively or negatively affect CD16a FcR biological pathways in immune effector cells to develop therapeutic agents for the treatment of diseases, thereby regulating the CD16a FcR pathway to have clinical benefits. A teaching includes the addition of active compounds (such as, but not limited to, compounds 1, 2, 4, 14, 26, 27, 30, 33 or 36) to be added to high-throughput screening assays-using CD16a FcR and antibodies to find small or large molecular weight inhibitors (Mandelboim O, et al. Proc Natl Acad Sci USA 96: 5640-5644, 1999). Other screening can be used to screen for antibodies or Fc fusion proteins with modified Fc domains to achieve enhanced CD16a FcR binding, thereby improving pharmacological activity (Saunders KO. Front Immunol 10: 1-20, 2019). An example of this feature is provided in Figures 1 and 2.

[0128] The kits may include the compound with reagents commonly used in ELISA, or may simply include the compound alone with instructions for use to enhance CD16a FcR-IgG antibody binding in a molecular or cellular binding assay.

[0129] The above disclosure generally describes the present invention. All references disclosed herein are incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples, which are provided herein only for illustrative purposes and are not intended to limit the scope of the present invention.

[0130] Example 1 - Identification of Compounds 1 and 2 for Enhancing Immunoglobulin Binding to Rodent and Human CD16a-Fc Receptor and Immune Effector Cell Activation in Vitro and in Vivo

[0131] To identify compounds that could potentially bind and enhance low affinity human CD16a-158F FcR binding to IgG type immunoglobulins, semi-high throughput screening assays were performed to screen natural small molecule chemical libraries obtained from commercial sources. These libraries contain more than 1,100 small molecular weight chemicals from various natural sources and consist of a large number of chemicals with different structural and biological properties. The primary assay was based on a 96-well ELISA format, whereby plates were coated with 2.5 μg / mL of human IgG1 or human serum albumin (HSA) - used as a negative control - and 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 detected using 2.5 μg / mL of each probe. The cDNA of each probe lacking a transmembrane domain and purified with a C-terminal polyhistidine tag were engineered into a mammalian expression vector containing a blasticidin selection marker. The vector was stably transduced into HEK293 cells, and blasticidin-resistant clones with high titer production of FcR proteins were screened using an ELISA-based screening method and an anti-polyhistidine (HIS)-horseradish peroxidase (HRP) conjugated antibody as a detection probe. 6 Secreted mature CD16a or CD32a protein (leader sequence cleaved during secretion) was isolated from 10-day cultures of 10 cells / mL and purified by HisPur TMNi-NTA resin column was used for separation. After preliminary exploration of the compound library, the wells were washed and IgG binding was monitored by a second anti-HIS-HRP detection antibody (Sino Biologicals). The well signals corresponding to individual clones were quantified at 450nm using a multi-well plate reader (Varioskan, ThermoFisher). After screening more than 1,100 compounds, only one compound was found to reproducibly enhance the binding of huCD16a-158F FcR to IgG1 antibodies compared to the control group. Compound analysis identified this compound as cholestane-based lanostane-8,24-diene-3β-ol (compound 1). Structural analysis discovered a highly related 9β-19-cyclo-24-lanostane-3β-ol compound (compound 2), which was subsequently obtained and tested to compare the molecular and cellular activities of compound 1 against CD16a FcR and CD32a FcR. As shown in Figure 1A, both compounds 1 and 2 were able to significantly enhance the binding of huCD16a-158F FcR and huCD16a-158V FcR to IgG1 antibodies compared 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 murine CD16a homolog 1 (FCGR3) (SEQ ID NO: 4) (muFCGR3-HIS) to human IgG1, but did not enhance the binding of murine homolog 2 (FCGR4) (SEQ ID NO: 5) (muFCGR4-HIS) to human IgG1. These analyses were performed using an ELISA method similar to that above and using HIS-labeled murine FCGR3 and FCGR4 as probes (Sino Biologics), followed by detection using anti-HIS-HRP. These data identify rodents as relevant species to test compounds 1, 2, 3 and 4 and derivative analogs for the enhancement of in vivo immune effector activation to achieve therapeutic confirmation and proof of concept. In addition, the data shown in Figure 1 support the use of compound 1 or 2 and its related active analogs shown in Figures 7 and 8. Any and all compounds as potential CD16a FcR-IgG binding enhancers can be used as assay reagents in molecular biology studies to facilitate screening of compounds that can positively or negatively affect the CD16a FcR-IgG antibody biological pathway for therapeutic indications involving this pathway. Applying these compounds as molecular biology assay reagents will produce a robust ELISA signal readout within 3 to 5 minutes, compared to 15 to 30 minutes in the basic CD16a FcR-IgG antibody binding format, thereby promoting the development of screening platforms. The experiment represents a minimum of three replicate wells. P values ​​are generated using Student's T test.

[0132] To further evaluate the effect of CD16a FcR-IgG antibody binding enhancement, a titration curve was generated to determine the amount of low-affinity huCD16a-158F FcR enhanced binding that compound 2 could confer. In brief, in the presence of different concentrations of compound 2 or inactive analogs (compound 5, see Figure 7), ELISA plates were coated with 2.5 μg / mL of human IgG1 and probed with 2.5 μg / mL of huCD16a-158F-HIS FcR. After probing, the plates were washed and secondary probing was performed using 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 increase CD16a-158F-IgG antibody binding by 4.7 times compared to control compound 5, and showed that affinity binding was significantly improved compared to CD16a-158F alone. This improvement was calculated to increase the affinity of huCD16a-158F for IgG1 binding from approximately 1 μM to 200 nM, making its affinity similar to that of the high-affinity huCD16a-158VFcR allotype, which has a published affinity of approximately 415 nM (Nordstrom JL, et al. Breast Cancer Res 13: 1-14, 2011), an unexpected finding and a highly desirable feature in the development of CD16a-based therapies (Bowles JA, et al. Blood 108: 2648-2654, 2006). Experiments represent a minimum of three replicate wells. P values ​​were generated using Student's T test.

[0133] To confirm that the enhanced CD16a FcR-IgG antibody binding is biologically relevant, a bioassay was established using a Jurkat-CD16a-158F-luciferase (JKT-CD16a) reporter cell line (Promega) to test the ability of compounds 1 and 2 to enhance and activate low-affinity CD16a-158F FcR in the presence or absence of IgG1 plus: target cells; target cells alone; or JKT-CD16a cells alone. The target cells used were OV-CD20, a human ovarian cancer cell line (OVCAR3) stably transduced to express the human CD20 antigen. The assay antibody used was a chimeric (mouse variable region fused to a human IgG1 Fc domain) anti-CD20 rituximab. Briefly, 0 to 5,000 target cells were seeded into RPMI (containing 7.5% fetal bovine serum (FBS) and 1% L-glutamine growth medium (R7.5)) in a black 96-well microplate and grown overnight at 37°C in 5% CO2. The next day, the wells were washed and plated with 50 μL of assay medium (clear RPMI, 1% low IgG FBS and L-glutamine (R1)) containing different concentrations of compound 1 or 2 with or without 2.5 μg / mL test rituximab antibodies. The test rituximab antibodies have previously been shown to specifically engage with 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 plate was incubated at 37°C in 5% CO2 for 12-18 hours. According to the manufacturer's advice, CD16a FcR activation in the test wells was tested using BIO-GLO (Promega) luciferase substrate and its relative luciferase production (relative luciferase production, RLU) was analyzed using a multi-well plate reader (Varioskan). As shown in Figure 3A, compared with the vehicle control wells or the no-compound control wells, compounds 1 and 2 showed significant enhancement of JKT-CD16a activation in the presence of IgG (a group of bars in the middle). JKT-CD16a cells plus target cells were also tested for CD16a-158F FcR activation in the absence of antibodies using a method similar to the above. As shown in a group of bars on the right in A, compounds 1 and 2 stimulated JKT-CD16a-158F FcR activation in the absence of IgG antibodies, demonstrating the ability of these compounds to directly stimulate CD16a FcR activation, but the level of stimulation was lower than that in the presence of IgG antibodies. Under any treatment conditions, the wells (left side bars) without the addition of JKT-CD16a cells did not show CD16a activation enhancement.To confirm the CD16a FcR specificity of compounds 1 and 2, parental Jurkat cells that do not express CD16a FcR and JKT-CD16a cells were tested by ELISA for the production of downstream CD16a FcR activation markers IL-1β and TNFα in culture medium. 50,000 cells / well in R1 medium were inoculated into an opaque 96-well microplate and 10 μM compound 2 or its inactive compound 5 analog was added to each well and incubated at 37°C in 5% CO2 for 18 hours. The plate was centrifuged to pellet the cells and the supernatant was collected and tested for the production of INFγ (SinoBiologicals) and TNFα (R&D Systems) by ELISA according to the manufacturer's recommendations. As shown in Figure 3B, when grown in the presence of compound 2, both cytokines were produced by JKT-CD16a-158F cells, while parental Jurkat or any cells treated with compound 5 produced almost no cytokines, thus demonstrating that CD16a FcR is required for compound 2 to induce cytokine production. These data demonstrate that the use of compounds and close analogs can: 1) enhance the binding of CD16a-158F FcR to IgG1 antibodies, and 2) stimulate CD16a FcR activation in the absence of antibody binding, both of which are useful for drug development of CD16a FcR-based therapies. The experiment represents a minimum of three replicate wells. P values ​​are generated using Student's T test to compare compound treatment vs vehicle in each group.

[0134] To demonstrate the ability of active compounds such as Compound 2 to stimulate the immune effector activity of primary immune cells, Compound 2 was tested for activation of human peripheral blood mononuclear cells (PBMC), human NK and mouse spleen cells (SPL) effector cells by measuring antibody-dependent (ADCC) or antibody-independent (AICC) cytotoxicity against tumor target cells. Briefly, 5000 human breast cancer (SKBR3 or OE19) or OV-CD20 target cells were plated in R7.5 medium in black 96-well microplates for 48 hours. The wells were washed and 50,000 PBMC, NK or SPL effector cells were added to R1 assay medium with or without targeted test antibodies and 25 or 50 μM of Compound 2. The vehicle is the solvent in which Compound 2 is dissolved. For assays using SKBR3 human breast cancer or OE19 esophageal adenocarcinoma cell lines, 2.5 μg / mL of trastuzumab test antibodies targeting HER2 were used for ADCC assays. For OV-CD20 determination, 2.5 μg / mL of rituximab test antibody targeting CD20 was used for ADCC analysis. The culture was incubated at 37°C for 24 hours in 5% CO2, three times. After incubation, the culture supernatant was harvested for cytokine analysis, and the wells were washed with phosphate-buffered saline and the viability of the attached target cells was quantified by Cell Titer-GLO (Promega) using a Varioskan plate reader. As shown in Figure 4A, the wells treated with compound 2 plus the targeted antibody showed that compound 2 was able to enhance the killing of target cells by human (PBMC cells and NK cells) and mouse (SPL cells) effector cells by ADCC compared to antibody treatment alone (vehicle). ADCC data are plotted as the percentage of killing of the culture treated with compound 2 or vehicle vs. not treated with antibody. Using a similar method as above, Figure 4B shows that compound 2 activates human primary NK cells in the absence of antibodies, thereby enabling NK cells to kill OV-CD20 tumor cells through the AICC mechanism. AICC results were plotted as percent killing of cultures treated with compound 2 vs vehicle alone. To demonstrate the involvement of CD16a FcR pathway activation in NK cells in the AICC effect, supernatants from compound 2 and vehicle-treated wells from the experiment in Figure 2B were analyzed by ELISA for cytokines and granzymes associated with CD16a FcR-mediated effector cell activation using a combination of anti-cytokine antibodies recommended by each manufacturer.As shown in the figure, in addition to the production of cytotoxic granzyme B protein (SinoBiologics), TNFα, INFγ and GM-CSF (Abcam) were found to be produced and secreted by NK cells in the wells treated with compound 2 in the absence of antibodies, supporting the evidence that compound 2 causes CD16a FcR effector-mediated cell activation and subsequent killing of target cells (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). Time course analysis found that activation and cytokine production occurred within 30 minutes after exposure. Similar cytokine production was found in human PBMC and mouse SPL cultures treated with compound 2. These data demonstrate the ability of compound 2 and active analogs to enhance antibody-based target cell killing and non-antibody-based stimulation of human and mouse immune effector cells by antibodies, and their potential as treatments for diseases targeting CD16a FcR. Experiments represent a minimum of three replicate wells. P values ​​were generated using Student's T test.

[0135] To prove the ability of active compounds such as compound 2 to activate immune effector cells in vivo, we use athymic nude mice to determine tolerance and systemic immune effector cell activation.Previous in vivo assays found that in athymic nude mice, compound 2 was the highest tolerated daily iv administration of at least 7mg / kg.To determine the ability of compound 2 to activate immune effector cells in vivo, we administered 7mg / kg compound 2 or an approximate volume of vehicle (ethanol) daily for 5 days (1-5 days), and separated total serum on the 11th day to measure immune effector cell activation markers.As shown in Figure 5A, all mice in each cohort were well tolerated for 5 days of treatment by not losing weight.Using a method similar to the description above, TNFα (R&D Systems) immune effector cell activation markers were monitored in serum from treated mice by ELISA and found to be significantly upregulated in mice (298pg / mL) treated with compound 2-P=0.00000026 compared to vehicle control cohort (11pg / mL). These data demonstrate the ability of Compound 2 and its active analogs to activate immune effector cells in vivo for evaluation of potential therapeutic applications in mouse models of human disease and as potential therapies for the treatment of human diseases involving CD16a FcR, such as cancer or infectious diseases, with or without therapeutic antibody-based therapies. Experiments represent a minimum of three replicate wells. P values ​​were generated using Student's T test.

[0136] Example 2 - Identification of compounds capable of inhibiting viral killing of human peripheral blood mononuclear cells

[0137] It has been previously reported that NK cells can kill virus-infected cells (Bjorkstrom NK, et al. Nat Reviews Immunol 22: 112-123, 2022). Specifically, previous studies have shown that the SARS-CoV-2 virus can infect human PBMCs, and this infection may be fatal to overall PBMC survival (Manunta MDI, et al. Scientific Reports 11: 4904-4916, 2021). To test the ability of the small molecular weight agent of CD16a FcR activation described herein to activate effector cells in PBMCs, thereby being able to kill virus-infected PBMC sibling cells and reduce viral load, compound 2 was tested for human PBMCs exposed to SARS-CoV-2 virus. In brief, IL2-activated PBMCs grown in RPMI medium containing 10% heat-inactivated FBS (R10) were plated in 96-well U-bottom microplates at 100,000 cells / well. Next, 2.7×10 5 Each virus titer was added to the appropriate wells containing or not containing 25 μM compound 2. The culture was incubated at 37°C for 72 hours in 5% CO2. The plate was centrifuged and the supernatant was collected for future cytokine and virus titer analysis. The cells were resuspended in RPMI+10% FBS, transferred to a 96-well black microplate, and tested for viability using Cell Titer-Glo (Promega), and read out viability on a 96-well microplate reader. As shown in Figure 6, the PBMC culture treated with virus and vehicle had 77% viability compared to the PBMC culture treated with 25 μM compound 2 retaining 100% viability. These data support the role of compound 2 and related analogs in treating virus-infected cells and patients with infectious diseases. The experiment represents a minimum of three replicate wells.

[0138] Example 3 - Screening of active analogs and identification of key regions that maintain enhanced CD16a Fc receptor-antibody binding and immune effector cell activation

[0139] The use of molecular-based recombinant CD16a-HIS FcR and IgG1 antibody ELISA assays enables facilitated screening to determine the optimal region within compounds 1 and 2 and related 3-cyclohexyl, 1-cyclopentyl related bidecyl alcohols (compounds 3 and 4), in addition to novel analogs that may further enhance or inhibit CD16a FcR specific activation and downstream immune effector cell activity. Lead compounds identified by the molecular screening taught herein can then be tested in in vitro bioassays to confirm, and then tested in vivo to identify the best and best tolerated compound (one or more) in terms of pharmacokinetic, pharmacodynamic, and therapeutic. Here, we first tested a large number of compounds with a common structure with compound 1 or 2 in the public domain. As shown in Figure 7A, these studies demonstrated the importance of a hydroxyl or formyl group as R1, where a highly similar analog of compound 2, cycloartenol ferulate (compound 12, Figure 7B), which lacks a hydroxyl group at R1, was completely inactive, while a 3-formylcycloartenol 24-one analog (compound 20) containing a formyl group at R1 retained enhanced CD16aFcR-IgG antibody binding, as did a 3-β-hydroxy-4,4-dimethyl-5-α-bis-orbitol analog (compound 4), in contrast to 3-α-hydroxy-4,4-dimethyl-5-α-bis-orbitol (compound 17), again showing the importance of stereospecificity at the R1 region.

[0140] Furthermore, the importance of the methyl groups at R2 and R3 for enhanced binding and activation of the CD16a FcR-IgG antibody was demonstrated by the loss of activity of 5α-cholesta-8,24-dien-3β-ol (compound 6) compared to compound 1, while stereospecificity at C13 and C14 had a somewhat negative impact on activity, as shown in compound 13. The exchange of the methyl group for fluorine at R4 in compound 1 appeared to have little effect on activity.

[0141] Based on the above findings, the use of cholestane or compound 3 backbones to synthesize analogs further demonstrates the flexibility of preparing a variety of analogs at the R5 and R6 regions, whereby PEG dimerization of two cholestane-derived 9β-19-cyclo-24-lanostene-3β-ol (compound 14) at the R6 region or substitution of ketone at C24 on cholestane-derived compound 20 shows that the enhanced CD16a binding activity is almost not lost, similar to the retained binding activity of compound 4 derived from compound 3, leading to concentrated efforts to further expand the structure-activity relationship of different chemical classes to achieve further optimization of the R5 / R6 region. These and additional structure-activity relationships found in compounds 1 to 20, compound 30, and compounds 36 to 38 provide additional guidance on the active region, thereby generating additional active analogs for treating disease indications involving CD16a FcR (including cancer and infectious diseases). Alternatively, non-enhanced compounds such as compound 37 or 38 can be used as inhibitors of CD16a activation to treat CD16a-mediated inflammatory diseases.

[0142] Based on the discovery that analogs derived from active compounds 1, 2, 3 or 4 have extreme variability at the R5 and R6 regions, additional modification examples can be implemented to improve CD16a FcR-IgG antibody binding and / or activation and to be used as biotinylated probes (compound 34, Figure 8) to analyze compound-CD16a FcR interactions in vitro and in vivo (Figure 8). These modifications are used to improve solubility, molecular activity, PK / PD quality and therapeutic activity. Methods for synthesizing other similar types of compound structures are provided.

[0143] The overall scheme was implemented to generate additional analogs of compound 2 with changes at R5 / R6 and to accommodate the necessary requirements for enhanced CD16a-FCR binding to IgG1 antibodies and FcR activation of effector cells expressing CD16a to achieve therapeutic applications. To prepare these compounds, compound 12 was generated and used as a source for generating compound 2 and its analogs. In Scheme 1, base hydrolysis, protection of the 3-hydroxyl group, cleavage of isopropylidine, followed by removal of the 3-hydroxyl protecting group, generated compound 21, which was then used as an active analog and common intermediate to generate additional R5 / R6 analogs based on compound 2 by: reductive amination at C24 (Scheme 2); oxidation to generate a carboxylic acid analog (Scheme 3); and acetylation and sulfation of the intermediate (Scheme 4).

[0144] In the following Scheme 1, compound 2 is generated by hydrolysis of compound 12 in the presence of potassium hydroxide (KOH) in ethanol (EtOH) in water. The resulting secondary alcohol is protected by acetylation using acetic anhydride. The double bond is then oxidized using O3, Zn and AcOH or potassium osmate and sodium periodate to generate aldehyde 1-2, which is then hydrolyzed in 80% NaOH / EtOH aqueous solution to produce compound 21. Since compound 12 is from a natural source, previous experience has found that the C24 keto isomer persists, which enables the use of the same method to generate compound 30 (Figure 8), but it must be purified by supercritical fluid chromatography (SFC) and confirmed by nuclear magnetic resonance (NMR).

[0145] Solution 1

[0146]

[0147] In Scheme 2 below, compound 25 was prepared from compound 21 and commercial dimethylamine hydrochloride in MeOH and / or DCM using reductive amination.

[0148] Solution 2

[0149]

[0150] Another analog generated by Scheme 2 is compound 26, which was prepared from compound 21 and commercial isopropylamine using reductive amination in MeOH and DCM.

[0151]

[0152] Compound 27 was prepared by Scheme 2 from compound 21 and commercial piperidine in MeOH and DCM using reductive amination.

[0153]

[0154] Compound 2 analog 28 generated by the method of Scheme 2 was prepared by reductive amination of compound 21 in MeOH and DCM with commercial morpholine.

[0155]

[0156] In another Scheme 2 synthesis, compound 29 is prepared by oxidation of compound 21 in t-BuOH and THF in the presence of NaClO4, NaH2PO4 and 2-methyl-2-butene to produce compound 29.

[0157]

[0158] As a final example of the synthesis of analogs from Scheme 2, PEGylated R6 analogs of compound 2 with different lengths ranging from 2 to 12 polyethylene glycol (PEG) units were synthesized from compound 21 and commercial NH2-PEG2-OH (compound 32), NH2-PEG12-OH (33), and PEG lengths in between by reductive amination in MeOH and DCM.

[0159]

[0160] In the following Scheme 3, compound 22 was prepared from compound 21 and commercial BnNH2 in MeOH and DCM using reductive amination followed by hydrogenation in the presence of Pd(OH)2 / C, H2 atmosphere and MeOH. Compound 22 was then sulfonylated with methanesulfonyl chloride under basic conditions to give compound 23, while compound 24 was prepared from compound 22 by condensation with acetic acid in the presence of EDCI and DMAP in DCM.

[0161] Schemes 3 and 4

[0162]

[0163] In another method, compound 21 was reduced in the presence of NaBH4 in EtOH to give compound 31, which was then condensed with D-(+)-biotin in the presence of EDCI and DMAP in DCM to give biotinylated compound 34.

[0164]

[0165] Analogs of Compound 1 with similar R5 and R6 modifications were generated using similar methods.

[0166] Example 4 - Identification of Compounds with Optimal Pharmacological Properties for In Vivo Use

[0167] Analysis of the compound 2 derivative analogs described in Example 3 was performed, and the enhanced CD16aFcR binding to IgG was tested by ELISA. In brief, 96-well plates were coated with PTZ as described above, and probed with CD16a-158F-HIS in the presence of 10 μM parent compound 2 and R5 / R6 compound analogs 22-34 to determine those compounds that mediate enhanced 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 impact of enhanced binding, these compounds were tested for enhancement of human PBMC ADCC as described above. Human breast cancer SKBR3 cells were plated in R7.5 in a black 96-well microplate at 5,000 cells / well and cultured overnight at 37°C in 5% CO2. The next day, 50,000 PBMCs in R1 medium were added to each well in triplicate in the presence of 1 or 5 μM of each compound or ethanol (EtOH) vehicle control. Wells treated with EtOH and PTZ without PBMCs were used as negative controls. The cultures were incubated at 37°C for 24 hours in 5% CO2 in triplicate. After incubation, target cell viability was quantified by Cell Titer-GLO (Promega) using a Varioskan plate reader. Units are displayed in relative luminescence units (RLU). As shown in Figure 10, compounds 27 and 33 enhanced ADCC most significantly (P<0.0000075).

[0168] Next, we use human and mouse microsomes to test the metabolic stability of compounds 2, 26, 27 and 33. For each test compound, the sample was diluted to a final concentration of 2 μg / mL in 25mM potassium phosphate buffer, while the final concentration of the liver microsomes of each species was 0.5mg / mL. The enzyme reaction was initiated by adding the NADPH reagent with a final concentration of 1mM. For the negative control sample, the NADPH reagent was not added. The sample was incubated at 37°C on a 50RPM orbital shaker, and aliquots were taken out at predetermined time points of 0, 5, 10, 15, 30 and 60 minutes. The sample was then precipitated with three volumes of acetonitrile containing propranolol as an internal standard, and centrifuged at 2000g for 10min, and then analyzed by LC / MS / MS. Note that in the presence of NADPH cofactors, compounds undergo only Phase I metabolism in liver microsomes. Data interpretation of the results was based on the percentage of parent compound remaining relative to the 0 minute incubation sample, from which the elimination half-life was calculated based on the natural logarithm of the % compound remaining vs. time plot. The following parameters were calculated to assess the in vitro metabolic stability of the compounds: Cmp = concentration of microsomal protein (mg / mL); t 1 / 2= half-life (min), where t 1 / 2 =0.693 / slope; CLint = intrinsic hepatic clearance (μL / min / mg), where CLint is equal to 0.693 / (t 1 / 2 Based on these analyses, as shown in Table 1, compounds 26 and 27 have significantly improved metabolic stability in both mouse and human microsomes compared to compounds 2 and 33.

[0169]

[0170] Based on the excellent metabolic stability of compound 27 in PBMC (Table 1) and ADCC enhancement (Figure 10), we further analyzed the in vivo activity of the compound. We first scaled up the synthesis of compound 27, as described below and depicted in Figure 11. A batch of 25 grams of γ-oryzanol was hydrolyzed under alkaline conditions of 0.415M KOH in EtOH aqueous solution to obtain secondary alcohol N. Secondary alcohol N was then protected with acetyl group using 37.8mM Ac2O, followed by ozonolysis to obtain aldehyde intermediate 2. Subsequently, reductive amination was performed using 27.1mM piperidine in AcOH solution, followed by addition of NaBH(OAc)3 to obtain intermediate 3. The free base of compound 27 was obtained by hydrolyzing intermediate 3 under the conditions of NaOH in MeOH and THF aqueous solution. 4M HCl was used to dihydrate the intermediate 3. The free base of compound 27 was converted to its HCl salt by 2% ethanol and the precipitated product was collected by filtration. The final product was analyzed by mass spectrometry and H-NMR. Mass spectrometry showed a major compound, the expected compound 27 molecular weight of 470.6 (see below) and with accurate H-NMR spectral characteristics.

[0171] Mass spectrometry analysis of compound 27 showed that the molecular weight of the main species was 470.6, as predicted for compound 27.

[0172] To further characterize the pharmacological properties of compound 27, we tested its oral bioavailability and ability to systemically activate NK cells in mice, as determined by measuring the NK cell activation markers IFNγ and TNFα. For oral bioavailability, mice were fed a standard laboratory rodent diet and housed at 22 + 30℃ and relative humidity 50 +20%, 12 hours of light and 12 hours of dark cycle in a separate cage. Before administration, the animals were fasted overnight, and food supply was restored after 6 hours, when blood samples were collected. Water was provided ad libitum throughout the study. Compound 27 was prepared by grinding the precipitated powder of compound 27 into fine particles and suspending it in an aqueous solution of 0.5% hydroxypropyl methylcellulose (HPMC). Three animals were administered 100 mg / kg (10 mL / kg) via a gavage needle for oral administration. Blood samples (25-30 μL per sample) were collected via the saphenous vein 5, 15, 30 minutes and 1, 2, 4, 6, 8, 24 hours after administration. At 24 hours, blood samples (150-200 μL per sample) were additionally collected for cytokine analysis. Blood samples were collected in Greiner MiniCollect K2 EDTA tubes, placed on ice for no longer than 30 minutes, centrifuged at 15,000 g for 5 minutes, and plasma samples were obtained. At 24 hours, 50-100 μL of each plasma sample was collected. All plasma samples were stored at -70°C before analysis.

[0173] The bioanalysis of plasma samples was performed as follows. Plasma samples were prepared using three volumes of acetonitrile containing internal standards and one volume of plasma to precipitate proteins. The samples were centrifuged at 3000 g for 10 minutes and the supernatant was removed for LC-MS / MS analysis. Calibration standards and quality control products were prepared by preparing a 1 mg / mL stock solution and a subsequent series of working solutions in methanol: water (1: 1, v / v). These working solutions were incorporated into blank plasma to obtain a series of calibration standard samples ranging from 1 ng / mL to 10 μg / mL and quality control samples at three concentration levels (low, medium and high). The treatment methods of all produced plasma samples were the same as those of calibration standards and quality control samples. LC-MS / MS analysis was performed using multiple reaction monitoring to detect the characteristic ions of each candidate drug, other related analytes and internal standards. Plasma concentrations were measured as described above to determine concentration vs. time spectra. The area under the plasma concentration vs. time curve (AUC) was calculated using the linear trapezoidal method known to those skilled in the art. Non-compartmental analysis was used to fit the data to obtain pharmacokinetic (PK) parameters.

[0174] The key PK parameters reported after intravenous administration are as follows: terminal half-life t 1 / 2 , initial plasma concentration C0, area under the plasma concentration vs. time curve AUC, volume of distribution at steady state Vss, total plasma clearance CLp and mean residence time MRT.

[0175] The key PK parameters reported after extravascular administration are as follows: terminal half-life t 1 / 2, maximum plasma concentration Cmax, time to reach maximum plasma concentration tmax, area under the plasma concentration vs. time curve AUC, mean residence time MRT and bioavailability F. All parameters are expressed for individual animals as well as mean values, standard deviations and coefficients of variation and are well known to those skilled in the art.

[0176] After oral administration of 100 mg / kg suspension, the test compound gradually reached a peak plasma concentration (Cmax) of 511 ng / mL (Tmax) within 6 hours, and its plasma concentration decreased in a multiphasic manner, with the last measurable concentration of 242 ng / mL at 24 hours and a long terminal half-life (t 1 / 2 ) was 18.3 hours. The total systemic exposure (AUClast) was 7.75h*μg / mL, and the oral bioavailability (F) was 11.7%. These data indicate the oral bioavailability of compound 27.

[0177] To determine the tolerance and systemic activation of NK cells in mice, we tested compound 27 administered orally to mice. Six mice were randomly divided into treatment groups: vehicle control (water) and 100 mg / kg compound 27, all suspended in 0.5% hydroxypropyl methylcellulose. The mice were orally administered on days 1-5, 7, 9, 11, 13, and 15, and the mice were killed on day 16, when plasma was collected. Tolerance is based on the body weight of each mouse on day 1. The maximum tolerated dose was determined as a group of animals that died with a weight loss of more than 20% or >10%. After 10 dosing courses, all mice in both groups were found to be healthy and of similar weight, confirming that multiple oral administrations of 100 mg / kg of compound 27 were well tolerated.

[0178] To confirm that oral administration of compound 27 activates NK cells, NK activation markers IFNγ and TNFα were measured by ELISA from plasma harvested on day 16 using the method described in Example 1. As shown in Figure 12, both NK cell activation markers were upregulated in mice treated with compound 27 compared to mice treated with the vehicle control, confirming the oral uptake of compound 27 and the activation of target NK cells. These scaffold structures embody the basic creativity taught in this application.

[0179] Example 5 - Development of analogs using Compound 1, 2, 3 and / or 4 scaffolds to develop CD16a pathway inhibitors to inhibit inflammation

[0180] Although multiple examples of analogs synthesized by compound 1, 2, 3 and 4 scaffold structures have shown enhanced CD16aFcR binding and immune effector cell activation activity, it is found that other analogs inhibit IgG-C16a FcR binding. This class of analogs can be used to inhibit inflammatory diseases. Several reports have found that active CD16a signaling in inflammatory diseases may be a potential cause of a subset of inflammatory diseases (Steel AW, et al.Aliment Pharmacol Ther 33: 115-126, 2011). The teachings herein show that compound 1, 2, 3 and 4 scaffold structures can be used to add chemical groups that can enhance their activity to CD16a stimulation. Here, we provide examples of how these scaffold structures can be used to develop CD16a antagonists to inhibit CD16a-IgG interactions and downstream activities (including the production of proinflammatory cytokines, such as IFNγ). Figures 10 and 13 show the ELISA binding assay 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 (no PTZ). The wells were then probed with human CD16a-158F-HIS in the presence or absence of the compound to monitor CD16a-PTZ binding. As shown in Figures 10 and 13, multiple analogs derived from the scaffold structure of compound 2 (compounds 29, 31, 32) were able to inhibit CD16a-PTZ interactions by approximately 49% (P≤0.005), which in turn would inhibit CD16a pathway signaling. The use of the scaffold structure taught herein can be expanded to develop even more potent CD16a inhibitors, similar to the methods for developing CD16a activators taught herein. These scaffold structures embody the basic creativity taught in this application.

[0181] The methods described in the Examples provided herein teach novel compositions and therapeutic techniques for treating CD16a FcR-mediated diseases.

[0182] Table 1. Sequence ID (all sequences from N to C terminus)

[0183] Underline Polyhistidine tag

[0184] Bold indicates the polymorphisms of human CD16a 158F and 158V

[0185] SEQ ID NO: 1 Mature human CD16a-158F-HIS (Accession No. P08637)

[0186]

[0187] SEQ ID NO:2 Mature human CD16a-158V-HIS (Accession No. P08637)

[0188]

[0189] SEQ ID NO:3 Mature human CD32a-131H-HIS (Accession No. P12318)

[0190] QAAAPPKAVLKLEPPWINVLQEDSVTLTCQGARSPESDSIQWFHNGNLIPTHTQPSYRFKANNNSGEYTCQTGQTSLSDPVHLTVLSEWLVLQTPHLEFQEGETIMLRCHSWKDKPLVKVTFFQNGKSKFSHLDPTFSIPQANHSHSGDY HCTGNIGYTLFSSKPVTITVQVPSMGSSSPMGIIVAVVIATAVAAVAAVVALIYCRKKRISANSTDPVKAAQFEPPGRQMIAIRKRQLEETNNDYETADGGYMTLNPRADDDKNIYLTLPPNDHVNSNNGLNDIFEAQKIEWHEHHHHHH

[0191] SEQ ID NO:4 Mature mouse CD16a-1 (FCRG3)-HIS (Accession No. P08508)

[0192] ALPKAVVKLDPPWIQVLKEDMVTLMCEGTHNPGNSSTQWFHNGRSIRSQVQASYTFKATVNDSGEYRCQMEQTRLSDPVDLGVISDWLLLQTPQRVFLEGETITLRCHSWRNKLLNRISFFHNEKSVRYHHYKSNFSIPKANHSHSGDYYCKGSLGSTQHQSKPVTITVQDPATT GLNDIFEAQKIEWHEHHHHHH

[0193] SEQ ID NO:5 Mature mouse CD16a-2 (FCRG4)-HIS (Accession No. 653142)

[0194] GLQKAVVNLDPKWVRVLEEDSVTLRCQGTFSPEDNSIKWFHNESLIPHQDANYVIQSARVKDSGMYRCQTALSTISDPVQLEVHMGWLLLQTTKWLFQEGDPIHLRCHSWQNRPVRKVTYLQNGKGKKYFHENSELLIPKATHNDSGSYFCRGLIGHNNKSSASFRISLGDPGSPSMFPPWHQ GLNDIFEAQKIEWHEHHHHHH

Claims

1. A method of treating a subject suffering from a viral infection, cancer, parasitic infection, bacterial infection, yeast infection, or inflammatory disease, comprising: An effective amount of Compound 1a, 1b, 2a, 2b, 4a or 4b is administered to the subject, thereby increasing the subject's immune response to the viral infection, the cancer, the parasitic infection, the bacterial infection, the yeast infection, or thereby reducing inflammation, wherein the compound is represented by: Wherein R1 is hydroxyl (OH) or formyl (CH=O); wherein R2 and R3 are methyl (CH3); Wherein R4 is methyl or 14a-difluoro-methyl (CHF2); wherein 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 (CH3CH(CH2)3CH(CH3)2); 2-methylheptane 2,3-diol (CH3CH(CH2)CH(OH)C(OH)(CH3)2); PEGylated amines comprising 2 to 12 PEG (polyethylene glycol) units; PEG5 amines linked to compounds 1a, 1b, 2a, 2b, 4a or 4b to form dimers; and biotinylated amines, wherein the biotin portion is linked to the amine portion via PEG 2-12.

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

3. A method for determining an antibody that binds to the Fc receptor CD16a, comprising: contacting the Fc receptor CD16a with compound 1a, 1b, 2a, 2b, 4a or 4b; contacting said antibody with said Fc receptor CD16a; and The amount of binding of the antibody to the Fc receptor CD16a is determined. The method of claim 3 , wherein the Fc receptor CD16a is on the cell surface.

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

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

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

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

9. A method for screening a compound for activating or inhibiting cells expressing CD16a, comprising: culturing a sample of cells expressing CD16a in the presence of a compound; culturing a sample of cells expressing CD16a in the absence of the compound; The amount of cell activation or inhibition is determined for samples cultured in the presence of the compound and samples cultured in the absence of the compound, wherein compounds that increase cell activation are candidate therapeutic immunomodulators and compounds that decrease cell activation are candidate therapeutic agents for inflammatory diseases.

10. The method of claim 9, wherein the cell expressing CD16a is selected from a recombinant cell line expressing the CD16a receptor, an innate immune cell, a natural killer (NK) cell, a myeloid cell, a dendritic cell, and a monocyte.

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

12. A set comprising: Compound 1a, 1b, 2a, 2b, 4a or 4b; and Therapeutic antibodies.

13. A composition for modulating an immune response in a human or rodent subject, comprising: Compound 1a, 1b, 2a, 2b, 4a or 4b, wherein the compound is not lanosta-8,24-dien-3β-ol or 9β-19-cyclo-24-lanosten-3β-ol; and A pharmaceutically acceptable carrier.

14. A nutritional supplement comprising Compound 1a, 1b, 2a, 2b, 4a or 4b, wherein the concentration of the compound is greater than 700 mg / 100 g of the supplement.

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

16. The nutritional supplement of claim 14, wherein the concentration of the compound is greater than 1 g / 100 g of the supplement.

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

18. The nutritional supplement of claim 14 which is a solid snack bar.

19. A method of preparing a fortified food for increasing an immune response, comprising: A nutritional supplement comprising compound 1a, 1b, 2a, 2b, 4a or 4b is mixed with a food substance to form a fortified food suitable for increasing an immune response, wherein the compound is not lanosta-8,24-diene-3β-ol or 9β-19-cyclo-24-lanostene-3β-ol, and wherein the concentration of the compound in the nutritional supplement is greater than 700 mg / 100 g of the supplement.

20. The method of claim 19, wherein the food substance is a fiber supplement.

21. A method of treating inflammation in a subject, comprising: An effective amount of a compound is administered to the subject, thereby reducing the inflammatory response of the subject, wherein the compound is selected from the group consisting of binorol, 5α-cholesta-8,24-dien-3β-ol, cholesta-5,24-dien-3β-ol, 5α-cholesta-7-ene-3β-ol, 3β-chloro-cholesta-5-ene, cycloartenol ferulate, 3-hydroxy-4,4-dimethyl-5-ene-binorol, 3-α-hydroxy-4,4-dimethyl-5-ene-binorol, 2-methyl-3-β-hydroxy-4,4-dimethyl-5-ene-binorol, 3β-lanosta-8,24,25-dihydroxy-ene-3-ol, lanosta-8,24-dien-3β-ol-25-morpholinyl, and 9β-19-cyclo-24-lanosta-3β-ol-25-morpholinyl.

22. The method of claim 21, wherein the subject suffers from an inflammatory disease or inflammatory syndrome.

23. The method of 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, Sjögren's syndrome, systemic lupus erythematosus (SLE, lupus), and vasculitis.

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

25. The method of claim 24, wherein activation is determined by antibody independent cellular cytotoxicity (AICC).

26. The method of 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 antibodies.

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

28. A composition comprising compound 27.

29. A method for preparing compound 27, comprising: Compound 21 was reductively aminized in methanol, dichloromethane and piperidine.

30. A composition comprising Compound 26.

31. A process for preparing compound 26, which comprises reductive amination of compound 21 and isopropylamine in methanol and dichloromethane.

32. A composition comprising Compound 29.

33. A method for preparing compound 29, comprising: Compound 21 was oxidized in tert-butyl alcohol, 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 utilizing binding of the Fc receptor CD16a to an antibody, comprising a compound selected from compounds 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 reagents for enhancing an in vitro antibody-based assay utilizing binding of the Fc receptor CD16a to an antibody, the kit comprising: A compound selected from compounds 1a, 1b, 2a, 2b, 4a and 4b, or a combination thereof; and Cell-free preparation of the Fc receptor CD16a.

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

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