Use of carthamus extract in inducing humoral immune responses

Abstract

The present disclosure provides a method for inducing a humoral immune response in a subject in need thereof. The method comprises administering to the subject an effective amount of a safflower plant or an extract thereof, and optionally a pharmaceutically acceptable carrier or excipient. The present disclosure also provides an adjuvant comprising an effective amount of a safflower plant or an extract thereof. The present disclosure also provides an immunogenic composition and method for enhancing a humoral immune response.

Description

[Technical Field]

[0001] <Reference to Related Applications> This application claims priority to and the benefit of U.S. Provisional Patent Application Nos. 63 / 485,175 and 63 / 485,192, filed February 15, 2023, the entireties of which are incorporated herein by reference.

[0002] The present disclosure relates to the use of Crassocephalum rabens, and more particularly to the use of Crassocephalum rabens in clinical applications. [Background technology]

[0003] Humoral immune responses are typically required to protect against viral and bacterial invasion. These responses are characterized by high levels of antibody production by B cells. A growing body of research has demonstrated a strong positive correlation between B cells and cancer-specific survival and response to therapy. Tumor-infiltrating B cells (TIL-B) secreting antitumor autoantibodies (AA) have been reported to improve clinical outcomes [Garaud, S., et al., JCI Insight, 2019. 5(18): e129641; Wang, SS, et al., Cell Mol Immunol, 2019. 16(1): pp. 6-18]. Higher proportions of follicular B cells and tumor-infiltrating plasma cells in tertiary lymphoid tissues correlate with improved long-term survival in cancer patients [Wang, SS, et al., Cell Mol Immunol, 2019. 16(1): pp. 6-18]. The formation of AA plays a protective role in antitumor immunity in cancer patients undergoing treatment.

[0004] Vaccination triggers appropriate immunity against pathogen infection and provides long-term protection. Regardless of the type of vaccine, the addition of an adjuvant is necessary to generate a stronger immune response against poorly immunogenic antigens (e.g., synthetic peptides, subunit antigens, and DNA). Although various adjuvants have been developed, only a few have been approved for clinical use due to their toxicity and side effects.

[0005] Considering their efficiency and safety, numerous research reports have shown that plant-derived components could be developed as adjuvants and used in vaccine formulations. To date, the most promising plant-derived molecules for adjuvant development are saponin (Qiao N, Liu Q, Meng H, Zhao D., International Immunopharmacology. 2014;18(2):333-339), shikonin (Chen HM, Wang PH, Aravindaram K, et al., Journal of Biomedical Science. 2012;19(1):42), polysaccharides (Sun B, Yu S, Zhao D, Guo S, Wang X, Zhao K., Vaccine. 2018;36(35):5226-5234), and lectins (Coffman RL, Sher A, Seder RA. Vaccine adjuvants: putting innate immunity to work. Immunity. 2010;33(4):492-503). Some herbs also enhance humoral immunity by directly stimulating B cells. For example, fractions or components from Echinacea angustifolia (Razin MAF, Osman A, Ali MA, Bahgat MM, Maghraby AS., Acta Microbiologica et Immunologica Hungarica. 2017;64(3):313-330) and Cuscuta cephalanthi seeds (Wang Z, Fang JN, Ge DL, Li XY., Acta Pharmacologica Sinica. 2000;21(12):1136-1140) promoted B cell proliferation. These herbal extracts or components induced higher titers of antibodies against pathogen antigens. Some herbs also have indirect stimulatory effects on B cells.

[0006] Therefore, there is a need to develop therapies or adjuvants to generate strong humoral responses to a variety of antigens. Summary of the Invention

[0007] The present disclosure provides uses of the safflower plant or an extract thereof in inducing a humoral immune response. The present disclosure also provides a safflower plant or an extract thereof for use in a method for treating or preventing a humoral immune response-mediated disease.

[0008] The present disclosure provides a method for inducing a humoral immune response in a subject in need thereof, the method comprising administering to the subject an effective amount of the carthamus plant or an extract thereof, and optionally a pharmaceutically acceptable carrier or excipient.

[0009] In one embodiment, the method is for preventing or treating a humoral immune response-mediated disease. In some further embodiments, the disease is cancer, an infectious disease, or a neurodegenerative disease. Examples of neurodegenerative diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS). Examples of cancer include, but are not limited to, melanoma, adenocarcinoma, thymoma, lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's lymphoma), sarcoma, lung cancer, liver cancer, colon cancer, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, and brain cancer. Examples of infectious diseases include, but are not limited to, bacterial infections (e.g., periodontitis, pneumonia, gastritis, and other bacterial infections), immunodeficiencies (e.g., HIV), papilloma (e.g., HPV), herpes (e.g., HSV), encephalitis, influenza (e.g., human influenza virus A), SARS-CoV-2 virus, and viral infections such as the common cold.

[0010] In some embodiments of the present disclosure, the method is for attracting B cells.

[0011] In some embodiments of the present disclosure, the method is for enhancing antibody production and / or enhancing antibody class switching. In some further embodiments of the present disclosure, the antibody class switching comprises switching from IgM to IgG1, IgG2, or IgG3.

[0012] In some embodiments of the present disclosure, the method is for inducing antibody-dependent cellular cytotoxicity or antibody-dependent cellular phagocytosis.

[0013] In some embodiments of the present disclosure, the method is for increasing serum IFN-γ and IL-21.

[0014] In some embodiments of the present disclosure, the method is for inducing B cell proliferation or B cell differentiation. In some further embodiments of the present disclosure, the method is for inducing B cell proliferation or B cell differentiation via the IL-21R / STAT3 / Blimp-1 pathway.

[0015] In some embodiments of the present disclosure, the humoral immune response is induced against a surface-bound cancer antigen or an antigen associated with a neurodegenerative disease, hi some further embodiments of the present disclosure, the cancer is drug-resistant colorectal cancer.

[0016] In some embodiments of the present disclosure, the safflower ragwort is S. safflower (Benth.) S. Moore.

[0017] In some embodiments of the present disclosure, the safflower plant is a dried or fresh safflower fragment.

[0018] In some embodiments of the present disclosure, the carthamus extract is an alcohol extract of carthamus.

[0019] In some embodiments of the present disclosure, the safflower safflower extract is produced by extracting safflower safflower with an alcohol solution to obtain an alcoholic extract, and fractionating the alcoholic extract to obtain a fraction rich in phytogalactolipids.

[0020] In some embodiments of the present disclosure, the fractionation step is performed by reversed-phase medium-pressure liquid chromatography (RP-MPLC). In some further embodiments of the present disclosure, RP-MPLC is further followed by reversed-phase high-performance liquid chromatography (RP-HPLC).

[0021] The present disclosure also provides a method for inducing a humoral immune response in a subject in need thereof, comprising administering to the subject an effective amount of a galactolipid compound from Carthamus tinctorius or a pharmaceutically acceptable derivative thereof, and optionally a pharmaceutically acceptable carrier or excipient.

[0022] In one embodiment of the present disclosure, the galactolipid compound is 1,2-di-O-α-linolenoyl-3-O-β-galactopyranosyl-sn-glycerol (dLGG) or 1,2-di-(α-linolenoyl)-3-[α-D-galactosyl-(1-6)-β-D-galactosyl]-sn-glycerol (CRDG).

[0023] The present disclosure provides an adjuvant composition comprising an effective amount of the carthamus plant or an extract thereof.

[0024] The present disclosure also provides an immunogenic composition comprising an antigen component and an adjuvant composition described herein.

[0025] In some embodiments of the present disclosure, the antigen component is a peptide or protein.

[0026] The present disclosure also provides a method of enhancing an immune response in a subject in need thereof, comprising administering to said subject an adjuvant composition or an immunogenic composition described herein.

[0027] In some embodiments of the present disclosure, the adjuvant composition or immunogenic composition is administered by the oral route.

[0028] In some embodiments of the present disclosure, the method is for enhancing antibody production.

[0029] In some embodiments of the present disclosure, the method is for enhancing antibody class switching. In some further embodiments of the present disclosure, the method is for increasing IgG2a, IgG2b, or IgG3 in total IgG antibodies.

[0030] In some embodiments of the present disclosure, the method is for triggering the release of IL-21 and / or B cell activating factor (BAFF).

[0031] In some embodiments of the present disclosure, the method is for triggering B cell differentiation.

[0032] In some embodiments of the present disclosure, the method is for enhancing cell proliferation, uptake capacity and / or antigen presentation of phagocytes.

[0033] In some embodiments of the present disclosure, the method is for increasing Th1 and Th2 associated immune responses.

[0034] The present disclosure is described in detail in the following sections. Other features, objects, and advantages of the present disclosure are set forth in the detailed description and claims. [Brief explanation of the drawings]

[0035] [Figures 1A-1E]These figures show the tumor growth inhibition of CRA in a CRC tumor allograft mouse model. [Figure 1A] The inhibitory effect of CRA on tumor growth. [Figure 1B] CRA dose-dependently suppressed tumor volume on day 33. CT26.CL25 tumor-bearing mice were orally administered 25, 50, or 100 mg / kg of CRA daily. Tumor volume was recorded weekly (n = 5-10). *: P < 0.05 (compared to the control group). After CRA treatment, (Figure 1C) the percentage of splenic CD19+ B cells and (Figure 1D) different B cell subsets within spleen cells were measured (n = 5). *: P < 0.05 (compared to the control group). [Figure 1E] The expression of TIL-B cells (B220, red) within the tumor by IHC staining. [Figures 2A-2C] Figure 2A shows the titers and recognition of CRA-induced antitumor antibodies. [Figure 2A] Titers of specific anti-CT26.CL25 cell antibodies against control or CRA antisera on days 0 and 33. [Figure 2B] Effect of CRA on class switching of specific anti-CT26 cell antibodies. *: P<0.05 (compared to the control group). The titers of these antisera (1,000x dilution) were measured for IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA. [Figure 2C] Tumor specificity of CRA antisera. Normal serum, control antisera, or CRA antisera bound to CT26.CL25 (black bars) or MG-CAP-A1 cells (white bars) were indirectly detected with FITC-conjugated goat anti-mouse IgG antibody. [Figures 3A-3D]The cytotoxicity and antitumor activity of CRA-induced antisera collected from CT26.CL25 tumor-bearing mice are shown. [Figure 3A] Cytotoxicity of CRA antisera. CT26.CL25 cells were treated with 2 μL of antisera and incubated for 48 hours. Cell viability was measured by MTT assay. To generate sCRA antisera, anti-CT26.CL25 antibodies from the CRA antisera were pre-cleaved by CT26.CL25 cell binding. Data are reported as proliferation index. N is normal serum, Ctrl is control antisera, and sCRA antisera is CRA antisera from which anti-CT26.CL25 antibodies have been removed. [Figure 3B] CRA antisera-mediated ADCC. CSFE-labeled CT26.CL25 cells, which served as target cells, were incubated with mouse splenic NK cells (effector cells) at an E:T ratio of 4:1 with 2 μL of normal serum or antisera for 4 hours. [Figure 3C] CRA antiserum-mediated ADCP. RAW264.7 macrophages were treated with non-opsonized or opsonized CT26.CL25 cells (incubated with 2 μL of antiserum) for 4 hours. The percentage of phagocytosis for normal serum and antiserum is shown. [Figure 3D] CRA antiserum induced CDC activity. Horse complement was used to measure the CDC activity of CRA antiserum. Cell lysis was determined 4 hours after addition of antiserum. *: P<0.05, **: P<0.01, ***: P<0.001 (compared to the control antiserum group). [Figure 4] This figure shows the effect of CRA on ex vivo release of IFN-γ and IL-21. Following different treatments, splenocytes harvested from CT26.CL25 tumor-bearing mice were treated with CT26 cell lysates. The release of IFN-γ and IL-21 in the supernatants was then analyzed. All values ​​are expressed as mean ± SEM (n = 6). *: P < 0.05, **: P < 0.01 (compared to the control group). [Figures 5A-5H]The effect of CRA on B cell differentiation in vitro in splenocytes is shown. [Figure 5A] Cell proliferation of CD19+ B cells. LPS was used as a positive control for B cell proliferation. [Figure 5B] The plasmablast (CD19+CD138+) and plasma cell (CD19- / lowCD138+) cell populations in CRA-treated mouse splenocytes were analyzed by flow cytometry. [Figure 5C] The percentage of plasmablast (CD19+CD138+) and plasma cell (CD19- / lowCD138+) cells. Mouse splenocytes were treated with serial dilutions of CRA for 72 hours. [Figure 5D] CRA increased IL-21 and IFN-γ expression in splenocytes. *: P<0.05, ***: P<0.001 (compared to the control group (n=7)). [Figure 5E] Cell proliferation of CD19+ B cells stimulated with anti-CD40 antibody (1 μg / mL) and mouse IL-4 (100 U / mL). [Figure 5F] Flow cytometry analysis of plasmablast (CD19+CD138+) and plasma cell (CD19- / lowCD138+) cell populations in mouse splenocytes treated with CRA and / or anti-CD40 / IL-4. [Figure 5G] Percentages of plasmablast (CD19+CD138+) and plasma cell (CD19- / lowCD138+) in splenocytes stimulated with anti-CD40 / IL-4. *: P<0.05, **: P<0.01, ***: P<0.001 (compared to the control group). [Figure 5H] CRA increased IL-21 and IFN-γ expression in splenocytes stimulated with anti-CD40 / IL-4. [Figures 6A-6B] These results demonstrate that CRA induced B cell differentiation through upregulation of STAT3 / Prdm1 signaling. CRA increased the expression of STAT3 and Prdm1 in (Figure 6A) unstimulated splenocytes and (Figure 6B) anti-CD40 / IL4-stimulated splenocytes. Unstimulated splenocytes or anti-CD40 / IL4-stimulated splenocytes were treated with CRA for 72 hours, and the expression of STAT3 and Prdm1 was measured by RT-PCR and Western blot. *: P<0.05, **: P<0.01, ***: P<0.001 (compared to the control group). [Figure 7A-7C]The effect of CRA on B cell differentiation in vitro in splenic B cells is shown. [Figure 7A] CRA did not affect IL-21 and IFN-γ expression in splenic B cells. ns: No significant difference (compared to the control group (n=6)). M: Cell culture medium. [Figure 7B] The plasmablast (CD19+CD138+) and plasma cell (CD19- / lowCD138+) cell populations in splenic B cells from CRA-treated mice were analyzed by flow cytometry. [Figure 7C] The percentages of plasmablast (CD19+CD138+) and plasma cell (CD19- / lowCD138+) in splenic B cells from mice after 72 hours of treatment with serial dilutions of CRA. [Figures 8A-8C] Figure 8 shows the effect of CRA on B cell differentiation in anti-CD40 / IL-4-treated splenic B cells in vitro. [Figure 8A] CRA did not affect IL-21 and IFN-γ expression in splenic B cells stimulated with anti-CD40 / IL-4. NC: Splenic B cells without anti-CD40 / IL-4 and CRA treatment. ns: No significant difference (compared to the control group (n=6)). [Figure 8B] Flow cytometry was used to analyze plasmablast (CD19+CD138+) and plasma cell (CD19- / lowCD138+) populations in splenic B cells treated with CRA and / or anti-CD40 / IL-4. Splenic B cells were pre-stimulated with 1 μg / mL anti-CD40 antibody and 100 U / mL mouse IL-4. [Figure 8C] Percentage of plasmablasts (CD19+CD138+) and plasma cells (CD19- / lowCD138+) in splenic B cells stimulated with anti-CD40 / IL-4. *: P<0.05, **: P<0.01 (compared to the control group). [Figure 9] The effect of neutralizing anti-IL21 antibody on CRA-induced B cell differentiation is shown. Spleen cells were treated with CRA and 50 μg of anti-IL21 antibody for 30 hours. The percentage of plasma cells was analyzed by flow cytometry. *: P<0.05 (compared to the control group). ns: no significant difference. [Figures 10A-10E]These results show that CRA promoted IL-21 release from T cells by upregulating the STAT3 / BCL6 / cMaf pathway. [Figure 10A] CRA induced IL-21 release from T cells. After treatment with CRA, the concentration of IL-21 in the cell culture medium was measured by ELISA. The effect of CRA on the expression of (Figure 10B) STAT3, (Figure 10C) BCL6, and (Figure 10D) cMaf in splenic T cells is shown. Splenic T cells were treated with different doses of CRA for 72 hours. The expression of STAT3, BCL6, and cMaf was measured by qPCR. [Figure 10E] Schematic diagram of the proposed mechanism of action of CRA. [Figures 11A-11F] These results demonstrate that CRA-API effectively induced B cell differentiation. [Figure 11A] Plasma cell populations in mouse splenocytes treated with CRA, CRDG, or CRG. [Figure 11B] Plasma cell populations in mouse splenocytes treated with CRA, CRDG, or CRG following anti-CD40 antibody / IL-4 stimulation. [Figure 11C] Effect of CRDG or CRG on IL-21 release from splenic T cells. The effects of CRDG or CRG on the expression of (Figure 11D) STAT3, (Figure 11E) cMaf, and (Figure 11F) BCL6 in splenic T cells are shown. Splenic T cells were treated with different doses of CRDG or CRG for 72 hours. The expression of STAT3, BCL6, and cMaf was measured by qPCR. *: P<0.05, **: P<0.01, ***: P<0.001 (compared to the control group). [Figures 12A-12D]These results show that CRA enhanced antibody production in antigen-pulsed BALB / c mice. [Figure 12A] Total titers of anti-AaHSP60 antibodies were measured weekly after immunization and booster immunization. Female BALB / c mice were immunized and boosted intraperitoneally (ip) with 100 μg of rAaHSP60 mixed with 10 mg / kg CRA or vehicle. Anti-AaHSP60 antibody titers were collected at a 1:25,600 dilution. [Figure 12B] EC50 values ​​of anti-AaHSP60 antibodies on day 35 were calculated. The x-axis shows the serial dilution (log10) of specific antibodies binding to rAaHsp60. The 50% binding rate (EC50) of CRA- or vehicle-induced anti-AaHsp60 antibodies was obtained. [Figure 12C] Antibody isotypes (IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA) were determined on day 35. [Figure 12D] B cell lineages were determined 35 days after immunization. All values ​​are expressed as mean ± SD (n = 5). *: P < 0.05 (compared to the control group). [Figure 13] This figure shows the effect of CRA on the expression of IL-21 and IFN-γ in the serum of immunized mice. Female BLAB / c mice were immunized and boosted with 100 μg of rAaHSP60 mixed with 10 mg / kg of CRA or vehicle by intraperitoneal (ip) injection. The concentrations of IL-21, IFN-γ, and TGF-β in the serum were measured by multiplex cytokine assay. All values ​​are expressed as mean ± SEM (n = 6). *: P < 0.05 (compared to the control group). [Figures 14A-14F]The effects of CRA on cell proliferation and cytokine release in vitro are shown. [Figure 14A] CRA induced cell proliferation in mouse splenocytes. Mouse splenocytes were treated with different concentrations of CRA (1.5, 3, and 6 μg / ml) for 48 hours. Splenocyte proliferation was analyzed by CSFE. The effects of CRA on (Figure 14B) IL-21 and (Figure 14C) IFN-γ release in mouse splenocytes are shown. All values ​​are expressed as mean ± SEM (n = 6). *: P < 0.05 (compared to the control group). [Figure 14D] CRA induced proliferation in AaHSP60-pulsed splenocytes. Mouse splenocytes were treated with different concentrations of CRA (1.5, 3, and 6 μg / ml) and 1 μg / ml of rAaHSP60 for 48 hours. The effect of CRA on the release of (FIG. 14E) IL-21 and (FIG. 14F) IFN-γ from AaHSP60-pulsed mouse splenocytes is shown. #: P<0.05 (compared to the AaHSP60 group). [Figures 15A-15B] Figure 15 shows the effect of CRA on B cell activation in mouse splenocytes. [Figure 15A] Effect of CRA on B cell proliferation. CSFE-labeled splenocytes were treated with different concentrations of CRA (1.5, 3, and 6 μg / ml) for 72 hours in the presence or absence of 1 μg / ml rAaHSP60. The proliferation of CSFE-labeled CD19+ B cells was monitored by flow cytometry. [Figure 15B] CD86 and MHCII expression in CRA-treated splenocytes in the presence or absence of AaHSP60. LPS was used as a positive control. The mean fluorescence fold was evaluated using the following formula: mean fluorescence intensity of the sample ÷ mean fluorescence intensity of the control group. All values ​​are expressed as mean ± SEM (n = 6). *; P < 0.05 (compared to the control group). [Figure 16] This shows the effect of CRA on B cell differentiation in mouse splenocytes. Mouse splenocytes were treated with CRA for 72 hours in the presence or absence of rAaHSP60. The percentage of plasma cells (CD19+CD138- / low) was analyzed by flow cytometry. *: P<0.05 (compared to the control group). #: P<0.05 (compared to the AaHSP60 group). [Figures 17A-17D]The effects of CRA on activation and antigen presentation in macrophages are shown. [Figure 17A] Effect of CRA on macrophage proliferation. *: P<0.05, **: P<0.01 (compared to the control group). [Figure 17B] Antigen uptake ability of macrophages after CRA treatment. Expression of MHC class II (Figure 17D) and CD86 (Figure 17C) in macrophages after CRA treatment is shown. The mean fluorescence fold was evaluated using the following formula: mean fluorescence intensity of the sample ÷ mean fluorescence intensity of the control group. All values ​​are expressed as mean ± SEM (n=6). *: P<0.05 (compared to the control group). #: P<0.05 (compared to the AaHSP60 group). [Figures 18A-18B] Figure 18 shows CRA-induced BAFF released from mouse splenocytes. [Figure 18A] BAFF mRNA expression in CRA-treated mouse splenocytes was detected by RT-PCR. [Figure 18B] BAFF released from CRA-treated mouse splenocytes was detected by ELISA. Mouse splenocytes were treated with different concentrations of CRA (3, 6, 12, and 24 μg / ml) for 24 hours. All values ​​are expressed as mean ± SEM (n = 6). *; P < 0.05 (compared to the control group). [Figure 19] These results demonstrate that oral administration of phytogalactolipid CRA induced specific antibody production in BALB / c mice. Female BALB / c mice were immunized and boosted weekly with 100 μg of rAaHSP60 mixed with 10 mg / kg of CRA or vehicle control. Salivary IgA anti-AaHSP60 antibody titers were measured at a 1:3,200 dilution. DETAILED DESCRIPTION OF THE INVENTION

[0036] The present disclosure may be more readily understood by reference to the following detailed description of various embodiments, examples, and chemical diagrams and tables accompanying the relevant descriptions of the present disclosure. It should be understood that unless otherwise specified in the claims, the present disclosure is not limited to a particular method of preparation, carrier, or formulation, or to a particular mode of formulating the extracts of the present disclosure into products or compositions intended for topical, oral, or parenteral administration, since such may, of course, vary, as is well understood by those skilled in the relevant art. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0037] As used in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0038] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Accordingly, singular terms shall include plurals and plural terms shall include the singular, unless the context clearly dictates otherwise.

[0039] As used herein, the term "or" means "and / or" unless expressly stated to refer to alternatives only or that the alternatives are mutually exclusive.

[0040] As used herein, the terms "subject" and "patient" are used interchangeably herein and are understood to refer to warm-blooded animals, particularly mammals. Non-limiting examples of animals within the scope and meaning of this term include guinea pigs, dogs, cats, rats, mice, horses, goats, cows, sheep, zoo animals, non-human primates, and humans.

[0041] The term "effective amount" of an active ingredient provided herein refers to an amount of the ingredient sufficient to provide the desired modulation of a desired function. As noted below, the exact amount required will vary from subject to subject, depending on the disease state, physical condition, age, sex, species, and weight of the subject, as well as the specific identity and formulation of the composition. The dosage regimen can be adjusted to induce the optimal therapeutic response. For example, the dosage may be administered in divided doses several times daily, or the dosage may be proportionally reduced depending on the exigencies of the therapeutic situation. Therefore, it is impossible to specify an exact "effective amount." However, an appropriate effective amount can be determined by one skilled in the art using only routine experimentation.

[0042] As used herein, the term "humoral immune response" refers to antibody production and associated ancillary processes, such as activation of T helper 1 and 2 (Th1 and Th2) cells and cytokine production, isotype switching, affinity maturation, and activation of memory cells. It also refers to antibody effector functions, such as toxin neutralization, classical complement activation, and promotion of phagocytosis and pathogen clearance. The humoral immune response is mediated by CD4 + Th1 and CD4 + As driven by Th2 cells, the activation or generation of these cell types is also indicative of the humoral immune response referred to herein.

[0043] The phrase "adjuvant composition" refers to a composition that can induce an immune response in a subject when administered to the subject. When administered in combination with an antigen, the "adjuvant composition" can induce an antigen-specific immune response. Adjuvants are generally used to achieve two goals: to delay the release of the antigen from the injection site and to stimulate the immune system.

[0044] An "immune response" to an antigen or composition is the development in a subject of a humoral and / or cellular immune response to molecules present in the antigen or composition of interest. A "humoral immune response" refers to an immune response mediated primarily by antibody molecules, while a "cellular immune response" refers to an immune response mediated primarily by T lymphocytes and / or other white blood cells.

[0045] The term "antigen" refers to any substance that can be recognized by the immune system under appropriate conditions (e.g., bound by an antibody or processed to elicit a cellular immune response, e.g., by a T cell). An antigen contains one or more epitopes. A B cell epitope contains at least about 3-5 amino acids, e.g., 4 or more amino acids. A hapten or polysaccharide can also function as a B cell epitope. A T cell epitope, such as a cytotoxic T cell (CTL) epitope, contains at least about 7-9 amino acids, e.g., 8 or more amino acids. A helper T cell epitope can contain at least about 12-20 amino acids. The term "antigen" refers to subunit antigens (i.e., antigens separate from the whole organism with which the antigen is naturally associated), as well as killed, attenuated, or inactivated bacteria, viruses, fungi, parasites, or other microorganisms, prions, allergens, or other disease-causing agents. Antigens may be modified proteins containing modifications such as deletions, additions, and substitutions (generally conservative in nature) to the native protein sequence. The term antigen also refers to nucleic acids (DNA or RNA) that encode protein or peptide antigens.

[0046] The terms "polypeptide" and "protein" refer to polymers of amino acid residues and are not limited to a minimum length of the product. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included in the definition. Both full-length proteins and fragments thereof are encompassed by this definition. The terms also include post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, "polypeptide" refers to proteins containing modifications such as deletions, additions, and substitutions (generally conservative in nature) to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as mutations of hosts producing the protein or errors due to PCR amplification.

[0047] As used herein, "carrier" or "excipient" refers to any substance, not itself a therapeutic agent, used as a carrier and / or diluent and / or adjuvant, or vehicle for delivery of a therapeutic agent to a subject, or added to a formulation to improve handling or storage, or to enable or facilitate the formation of dosage units of the composition into discrete articles, such as capsules or tablets, suitable for oral administration. Suitable carriers or excipients are well known to those skilled in the art of manufacturing pharmaceutical formulations or food products. Carriers or excipients can include, by way of example and not limitation, buffers, diluents, disintegrants, binders, adhesives, wetting agents, polymers, lubricants, glidants, substances added to mask or neutralize unpleasant tastes or odors, flavors, dyes, fragrances, and substances added to improve the appearance of the composition. Acceptable carriers or excipients include citrate buffer, phosphate buffer, acetate buffer, bicarbonate buffer, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphate and sulfate, magnesium carbonate, talc, gelatin, gum acacia, sodium alginate, pectin, dextrin, mannitol, sorbitol, lactose, sucrose, starch, gelatin, cellulosic materials (such as cellulose esters of alkanoic acids and cellulose alkyl esters), the low melting wax cocoa butter, amino acids, urea, alcohol, ascorbic acid, phospholipids, proteins (e.g., serum albumin), ethylenediaminetetraacetic acid (EDTA), dimethyl sulfoxide (DMSO), sodium chloride or other salts, liposomes, mannitol, sorbitol, glycerol or powder, polymers (such as polyvinylpyrrolidone, polyvinyl alcohol, and polyethylene glycol), and other pharmaceutically acceptable materials. The carrier should not destroy the pharmacological activity of the therapeutic agent and should be non-toxic when administered in amounts sufficient to deliver a therapeutic amount of the agent.

[0048] As used herein, "pharmaceutically acceptable derivative" or "pharmaceutically acceptable derivative" refers to a compound that has been modified from a compound of the present disclosure, but that has the same or superior properties and efficacy as the compound of the present disclosure. Preferably, a pharmaceutically acceptable derivative is a pharmaceutically acceptable salt, solvate, hydrate, or prodrug of a compound of the present disclosure.

[0049] The compounds of the present disclosure can exist as solvates and hydrates.Thus, these compounds can be crystallized with, for example, one, several, or any proportion of water of hydration or molecules of the mother liquor solvent.The solvates and hydrates of such compounds are included in the scope of the present disclosure.

[0050] The present disclosure provides a method for inducing a humoral immune response in a subject in need thereof, the method comprising administering to the subject an effective amount of the carthamus plant or an extract thereof, and optionally a pharmaceutically acceptable carrier or excipient.

[0051] The presently disclosed safflower plant or extract thereof may be able to induce a humoral immune response, thereby protecting a subject from a disease, disorder, or illness associated with an antigen capable of inducing a humoral immune response. This includes, but is not limited to, cancers involving membrane-surface-bound cancer antigens recognized by antibodies, diseases in which it is desirable to sequester antigens in circulation, such as amyloid proteins (e.g., Alzheimer's disease), neutralization of toxins by antibodies, neutralization of viruses or bacteria by antibodies, or neutralization of allergens (e.g., pollen) for the treatment of allergies. In another embodiment, the antigen may be or include a B-cell epitope capable of inducing a humoral immune response. For example, the antigen may be or include a B-cell epitope.

[0052] Humoral immune responses can also be useful in fighting cancer. B cell-mediated responses may target cancer cells through other mechanisms, which may, in some cases, cooperate with cytotoxic CD8 T cells for maximum benefit. Examples of mechanisms of B cell-mediated (e.g., humoral immune response-mediated) anti-tumor responses include, but are not limited to, antibodies: 1) antibodies produced by B cells that bind to surface antigens found on tumor cells or other cells that influence tumorigenesis. Such antibodies may induce target cell killing, e.g., via antibody-dependent cell-mediated cytotoxicity (ADCC) or complement fixation, and trigger the release of additional antigens that are recognized by the immune system. 2) antibodies that bind to receptors on tumor cells and block stimulation, effectively neutralizing their effects. 3) antibodies that bind to factors released by tumor or tumor-associated cells or tumor-associated factors, modulating signaling or cellular pathways that support cancer. 4) antibodies that bind to intracellular targets and mediate anti-tumor activity through currently unknown mechanisms.

[0053] In another embodiment, the antigen may be an antigen associated with a disease in which sequestering the antigen in the circulation is desirable (e.g., Alzheimer's disease), such as, for example, an amyloid protein. Thus, the compositions of the present invention are suitable for use in treating and / or preventing a neurodegenerative disease in a subject in need thereof, where the neurodegenerative disease is associated with expression of the antigen. The subject may be suffering from or at risk of developing a neurodegenerative disease. Neurodegenerative diseases that may be treated and / or prevented by use or administration of the compositions of the present invention include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS).

[0054] As used herein, the terms "cancer," "cancer cell," "tumor," and "tumor cell" (used interchangeably) refer to cells exhibiting abnormal proliferation characterized by a significant loss of control of cell proliferation or immortalization of the cells. The term "cancer" or "tumor" includes metastatic and non-metastatic cancers or tumors. Cancer can be diagnosed using criteria generally accepted in the art, including the presence of malignant tumors.

[0055] In some embodiments of the present disclosure, a phytogalactolipid-rich fraction (CRA) from Benth. S. Moore has been shown to induce humoral immune responses, for example, against cancer. In some embodiments of the present disclosure, CRA treatment attracted large numbers of B cells to target sites, such as tumors. CRA enhanced anti-tumor antibodies in both serum and tumors, and caused class switching of anti-tumor antibodies from IgM to IgG1, IgG2, and IgG3. CRA-induced antisera specifically recognized surface antigens on the plasma membrane of cancer cells. CRA antisera not only exhibit cytotoxicity but also induce antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). In some embodiments, serum IFN-γ and IL-21 significantly increased after CRA treatment. CRAs directly regulate STAT3 and cMaf, induce T cells to secrete IL-21, and induce B cell proliferation and differentiation via the IL-21R / STAT3 / Blimp-1 pathway. Overall, CRAs exhibit potent biological efficacy in regulating T cells and stimulating B cell activation, allowing treated subjects to trigger anti-tumor antibodies and block cancer progression.

[0056] Crassocephalum crepidioides (Benth.) S. Moore, also known as Crassocephalum crepidioides (Benth.) S. Moore, is a common vegetable and a popular folk medicine, and its ethanol extract has been shown to be a safe supplement [Hsu, PK, et al., Toxicol Rep, 2022. 9: pp. 58-63]. C. rabens has attracted attention due to its excellent anti-inflammatory and anti-cancer activities [Hou, CC, et al., Cancer Res, 2007. 67(14): pp. 6907-15; Apaya, MK, et al., Cancers (Basel), 2020. 12(1): pp. 199; Yang, CC, et al., Int J Cancer, 2018. 143(12): pp. 3248-3261]. The glyceroglycolipid 1,2-di-O-α-linolenoyl-3-O-β-galactopyranosyl-sn-glycerol (dLGG), a major component of C. rabens, has chemopreventive activity against cancer and sepsis by inhibiting inflammatory mediators such as TNF-α, IL-6, and bioactive lipid mediators, oxylipins [Hou, CC, et al., Cancer Res, 2007. 67(14): pp. 6907-15]. dLGG suppressed the metastatic potential of melanoma cells by deregulating epithelial-mesenchymal transition (EMT), attenuating tight junction permeability in the pulmonary vasculature, and increasing circulating oxylipins [Yang, CC, et al., Int J Cancer, 2018. 143(12): pp. 3248-3261]. dLGG, used alone or in combination with doxorubicin, effectively attenuates triple-negative breast cancer (TNBC) recurrence and lung metastasis by inhibiting the fatty acid-binding protein (FABP) / epoxyeicosatrienoic acid (EET)-mediated signaling axis [Apaya, MK, et al., Cancers (Basel), 2020. 12(1): p. 199].

[0057] As used herein, the term "safflower ragwort plant" may refer to the whole plant or one or more parts thereof, including, but not limited to, seeds, flowers, leaves, stems, and roots. In one embodiment of the present disclosure, the safflower ragwort plant is the whole plant. In another embodiment of the present disclosure, the safflower ragwort plant is the seeds, flowers, leaves, or any combination thereof.

[0058] The safflower plant may be collected at various stages.

[0059] In some embodiments of the present disclosure, the safflower ragwort plant is a mixture obtained by removing some materials from safflower ragwort. In a preferred embodiment of the present disclosure, the safflower ragwort plant is prepared by drying and grinding safflower ragwort, and is a piece of dried or fresh safflower ragwort.

[0060] In a preferred embodiment of the present disclosure, the safflower safflower extract is prepared by removing the solid content of safflower safflower, and the safflower safflower extract is the liquid of safflower safflower.

[0061] In some embodiments of the present disclosure, the carthamus extract is an alcohol extract of carthamus. In some embodiments, the alcohol is a C1-C4 alcohol. As used herein, the term "C1-C4 alcohol" refers to a straight-chain or branched-chain, substituted or unsubstituted, monofunctional or polyfunctional, saturated or unsaturated alcohol, preferably an unsubstituted, monofunctional, and saturated alcohol. In one embodiment of the present disclosure, the C1-C4 alcohol is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol, and tert-butanol. In some embodiments, the C1-C4 alcohol is methanol or ethanol. The C1-C4 alcohols can be used alone or in combination.

[0062] In some embodiments of the present disclosure, the safflower safflower extract is produced by extracting safflower safflower with an alcohol solution to obtain an alcoholic extract, and fractionating the alcoholic extract to obtain a fraction rich in phytogalactolipids.

[0063] In some embodiments of the present disclosure, the fractionation step is performed by reversed-phase medium-pressure liquid chromatography (RP-MPLC). In some further embodiments of the present disclosure, RP-MPLC is further followed by reversed-phase high-performance liquid chromatography (RP-HPLC).

[0064] The present disclosure also provides a method for inducing a humoral immune response in a subject in need thereof, comprising administering to the subject an effective amount of a galactolipid compound from Carthamus tinctorius or a pharmaceutically acceptable derivative thereof, and optionally a pharmaceutically acceptable carrier or excipient.

[0065] In some embodiments, the active ingredient comprises 1,2-di-O-α-linolenoyl-3-O-(6-O-α-galactopyranosyl-β-galactopyranosyl)-sn-glycerol (referred to as CRDG) and dLGG (referred to as CRG) in CRA.

[0066] The galactolipid compounds of the present disclosure can be further converted into pharmaceutically acceptable derivatives, such as pharmaceutically acceptable salts, solvates, or prodrugs, by any known method.

[0067] An extract of carthamus tinctorius is preferably included in the extract composition.

[0068] The extract composition according to the present disclosure is preferably a pharmaceutical composition or a food composition.

[0069] The pharmaceutical compositions according to the present disclosure are preferably administered locally or systemically by any method known in the art, including, but not limited to, intramuscular, intradermal, intravenous, subcutaneous, intraperitoneal, intranasal, oral, mucosal, or topical routes. The appropriate administration route, formulation, and administration schedule can be determined by those skilled in the art. In the present disclosure, the pharmaceutical compositions can be formulated in various ways, such as liquid solutions, suspensions, emulsions, syrups, tablets, pills, capsules, sustained-release preparations, powders, granules, ampoules, injections, drops, kits, ointments, lotions, liniments, creams, or combinations thereof, depending on the corresponding administration route. If necessary, they can be sterilized or mixed with pharmaceutically acceptable carriers or excipients, many of which are known to those skilled in the art.

[0070] The extract composition can be added to conventional food compositions (i.e., edible foods or their precursors) during the manufacturing process of the food composition. The extract composition of the present disclosure can be added to almost any food composition. Food compositions to which the extract composition of the present disclosure can be added include, but are not limited to, candy, baked goods, ice cream, dairy products, sweet and savory snacks, snack bars, meal replacements, fast food, soup, pasta, noodles, canned foods, frozen foods, dried foods, refrigerated foods, oils and fats, baby foods, or soft foods spread on bread, or mixtures thereof.

[0071] The present disclosure provides the use of the carthamus plant or an extract thereof as an adjuvant for enhancing humoral immune responses.

[0072] The present disclosure also provides an adjuvant composition comprising an effective amount of a galactolipid compound from Carthamus tinctorius.

[0073] In some embodiments of the present disclosure, a galactolipid-rich extract of safflower (referred to as CRA) functions as an adjuvant against pathogen infection. CRA dramatically increased the titer of specific antibodies against antigens, increasing the proportion of IgG2a, IgG2b, and IgG3 in total Ig. In some embodiments, CRA triggers the release of IL-21 and B cell activating factor (BAFF), leading to B cell differentiation. CRA also enhanced cell proliferation, uptake capacity, and antigen-presenting phagocytes. Taken together, CRA possesses immunomodulatory activity and adjuvant effects that enhance mouse immunity to pathogen antigens by specifically increasing both Th1- and Th2-related immune responses. Mechanistic studies demonstrated that CRA induced B cell differentiation through promoting IL-21 and BAFF, resulting in higher titers and affinities of specific antibodies.

[0074] Certain embodiments provided herein include vaccine compositions and immunoadjuvant compositions (including pharmaceutical compositions) that contain, in addition to the safflower plant or an extract thereof, at least one co-adjuvant, which refers to a component of such a composition other than the safflower plant or an extract thereof, that has adjuvant activity. Such co-adjuvants with adjuvant activity include compositions that can alter (i.e., increase or decrease, and in certain preferred embodiments, enhance or increase) the potency and / or longevity of an immune response when administered to a subject, such as a human (e.g., a human patient), a non-human primate, a mammal, or other higher eukaryote with a recognized immune system. In certain embodiments provided herein, the safflower plant or extract thereof and a desired antigen, and optional one or more co-adjuvants, can alter, e.g., induce or enhance, an immune response to a desired antigen, which may be administered simultaneously with the safflower plant or extract thereof or separated in administration time and / or space (e.g., to different anatomical sites). However, certain inventive embodiments are not intended to be so limited and thus also contemplate administering the safflower plant or extract thereof in a composition that does not include a specific antigen, but may include one or more of a TLR agonist, a co-adjuvant, an imidazoquinline immune response modifier, and a double stem-loop immune modifier (dSLIM).

[0075] The present disclosure also provides an immunogenic composition comprising an antigen component and an adjuvant composition described herein.

[0076] In some embodiments of the present disclosure, the antigen component is a peptide or protein.

[0077] The adjuvant compositions of the present disclosure, when combined with an antigen, such as a vaccine or immunogenic composition, provide improved presentation of the antigen portion of the vaccine to the immune system of a vaccine recipient compared to a conventional vaccine or immunogenic composition comprising an adjuvant not provided in the present disclosure. Such improved presentation is compared to the same antigen when combined with an adjuvant composition that is not part of the present disclosure. Preferably, the improved presentation allows for the use of less or a lower amount of antigen to achieve the same level of immune system response. The level of immune system response can be measured by the strength of the response as measured by a marker of immune response, or by the duration of immunization, or by a combination of these two indicators of immune system response. More preferably, the improved antigen presentation is achieved by increasing the antigen content by 95%, more preferably 90%, and even more preferably 85%, 80%, 75%, 60%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5% of the antigen amount in combination with a different adjuvant to achieve the same level of immune system response as the same antigen when administered to an animal of the same species. , 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009, 0.008%, 0.007%, 0.006%, 0.005%, and the range formed by any two members of this group.

[0078] The present disclosure also provides a method of enhancing an immune response in a subject in need thereof, comprising administering to said subject an adjuvant composition or an immunogenic composition described herein.

[0079] The vaccination method of the present disclosure preferably comprises administering a composition comprising an adjuvant of the present disclosure and an antigen, wherein the administration is by needleless or injection. For purposes of the embodiments of the present disclosure, the administration method is preferably oral, intramuscular, subcutaneous, or transdermal administration, although other administration methods can also be employed. In one embodiment, the administration method is selected from the group consisting of topical, intramuscular, intranasal, oral, transdermal, mucosal, needleless administration, and subcutaneous. Needleless administration methods include, but are not limited to, vaccine guns, transdermal patches, aerosols, mucosal administration, skin adhesion, dry particle projectiles, wet projectiles, gold / inert particle guns, and pneumatic guns.

[0080] The following examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0081] Materials and Methods

[0082] Preparation of bioactive fraction CRA from C. rabens plants and purification of two major chemical components in CRA, namely CRDG and CRG.

[0083] Fresh or dried whole plant material of C. rabens was extracted with alcohol, e.g., 50–99.5% ethanol or methanol, to obtain a crude total extract of C. rabens. This was then further fractionated by reversed-phase medium-pressure liquid chromatography (RP-MPLC) to obtain a phytogalactolipid-rich fraction, called CRA. The MPLC mobile phase consisted of primary alcohol (e.g., MeOH, EtOH) and distilled water, and the CRA fraction was eluted with increasing concentrations of alcohol (e.g., 70–100%). The CRA fraction was then subjected to preparative reversed-phase high-performance liquid chromatography (RP-HPLC) to collect pure CRDG and CRG. The HPLC mobile phase consisted of methanol or acetonitrile and distilled water, with a ratio of >90% organic solvent to <10% distilled water, to obtain CRDG and CRG. The purity and structure of CRDG and CRG were investigated using analytical HPLC, high-resolution mass spectrometry, and NMR spectroscopy [Hou, CC, et al., Cancer Res, 2007. 67(14): p. 6907-15].

[0084] <cell>

[0085] Mouse colorectal carcinoma CT26.CL25 cells were purchased from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). CT26.CL25 cells were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 4.5 g / L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate, 0.1 mM non-essential amino acids, 10% fetal bovine serum, and 1% penicillin-streptomycin (Invitrogen). Mouse metastatic castration-resistant prostate cancer MG-Cap A1 cells were provided by Dr. Pei-Wen Hsiao of the Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan, and maintained in DMEM supplemented with 10% fetal bovine serum, 10 μg / mL puromycin, 5 μg / mL blasticidin, and 1% penicillin-streptomycin.

[0086] <Animal>

[0087] Five-week-old female BALB / c mice were used. All animals were health-monitored and maintained in a specific pathogen-free animal facility with controlled temperature (22 ± 2°C) and humidity (55 ± 10%) under a 12:12-h light / dark cycle. Animals were acclimated to a standard rodent diet (LabDiet 5010, St. Louis, MO) and water ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee of Academia Sinica (approved protocol number 20-12-1592) and followed the guidelines for the use of laboratory animals (National Academy Press, Washington, DC).

[0088] <Therapeutic effect of in vivo CRA treatment>

[0089] BALB / c mice were inoculated with 1 × 10 6 CT26.CL25 cells were inoculated subcutaneously. The average tumor volume was 100 mm 3 When tumor-bearing mice reached 100 mg / kg, they were randomly divided into four groups. The animals were treated with 25, 50, and 100 mg / kg CRA by oral administration daily for 33 days. Tumor volumes were measured every 2 days using calipers, and the volume (mm 3 The volume was calculated using the formula: ) = length x width x height. Before sacrificing the mice, serum from the mice was collected for experimental use.

[0090] Phenotypic analysis of splenic and tumor-infiltrating B cell subsets

[0091] Spleens and tumor-infiltrating lymphocytes were collected from CRA-treated or untreated mice. + CD23 + ), activation (CD19 + CD38 + ), Memory B (CD19 + CD27 + ) and plasma cells (CD19- / low CD138 + B cell subsets, such as IgG, IgG, and IgG4, were probed with specific monoclonal antibodies (BioLegend, San Diego, CA) and analyzed by flow cytometry (BD Biosciences). The index of the proportion of different types of B cells was calculated as follows: (number of spleen cells × percentage of each B cell population in treated spleen cells) / (number of spleen cells × percentage of B cell population in untreated spleen cells).

[0092] <Immunohistochemical staining>

[0093] Tumors isolated from CRA-treated mice were fixed overnight with 4% paraformaldehyde at 4°C. After washing with PBS, tumor samples were immersed in 30% sucrose overnight at 4°C. Next, tumor samples were embedded in OCT, sectioned at 10 μm, and processed for immunohistochemical staining. Sections were probed with Alexa Fluor 594-conjugated rat monoclonal anti-B220 (1:50 dilution, Biolegend) and PE-conjugated rat monoclonal anti-CD3 (1:50 dilution, Biolegend) antibodies. Sections were also probed overnight at 4°C with goat polyclonal anti-IL-21 (1:20 dilution, Thermo Fisher Scientific), and the detection antibody was recognized using Alexa Fluor 488-conjugated anti-goat IgG antibody (1:100 dilution, Thermo Fisher Scientific). Immune complexes in the sections were visualized and photographed at 200x magnification using a Zeiss LSM 510 META confocal microscope (Carl Zeiss, Jena, Germany).

[0094] <Titer and Ig class of anti-tumor autoantibodies>

[0095] Blood was collected from CT26.CL25-bearing mice treated with PBS or 25, 50, or 100 mg / kg of CRA, and the titer of CT26.CL25-specific antibodies in the serum was measured as follows: CT26.CL25 (10 6Cells were seeded at 100 μL / well. The next day, cells were fixed with 4% paraformaldehyde, washed, and blocked with 300 μL of 2% skim milk in PBST (PBS buffer containing 0.05% Tween-20) for 1 hour. 100 μL of serum diluted 1:1000 in PBS containing 0.5% skim milk was added to each well and incubated for 2 hours at room temperature. After three washes, 100 μL of HRP-conjugated anti-mouse Ig antibody (1:5000 dilution, Sigma-Aldrich, St. Louis, MO) was added to each well and incubated for 1 hour. After three washes, 100 μL of NeA-Blue (Clinical Science Products, Mansfield, MA) was added to each well and incubated for 20 minutes. The reaction was stopped with 100 μL of 1N HCl. The optical density was measured at 450 nm using an ELISA reader (Tecan, Mannedorf, Switzerland). The isotype of specific anti-CT26.CL25 cell antibodies in the antisera (1:1600 dilution) was determined by using HRP-conjugated specific anti-mouse IgM, IgG1, IgG2a, IgG2b, IgG3, or IgA antibodies (Acris, Herford, Germany).

[0096] Immunofluorescence analysis of antisera binding to tumor antigens on cell surfaces

[0097] CT26.CL25 or MG-Cap-A1 cells were incubated with 2 μL of normal serum, PBS antiserum (control antiserum), or CRA antiserum for 1 h and detected with an FITC-conjugated secondary antibody (Molecular Probes, Eugene, USA). Antiserum binding to tumor antigens on the surface of CT26.CL25 and MG-Cap-A1 cells was analyzed by flow cytometry (BD Biosciences).

[0098] In vitro ADCC, ADPC, and CDC assays

[0099] In vitro ADCC assays yielded a total of 5 × 10 3CSFE-labeled CT26.CL25 target cells were preincubated with 2 μL of antiserum. Murine NK cells purified from mouse splenocytes were added at an effector to target cell (E:T) ratio of 4:1. The assay plate was incubated at 37°C for 4 hours. Cytolysis was measured using an ELISA reader (BioTek, Winooski, VT, USA).

[0100] In the ADCP assay, a total of 1 × 10 5 CSFE-labeled CT26.CL25 cells were incubated with 2 μL of antiserum for 30 min, and then 3 × 10 5 The cells were added to the macrophages. After 4 hours, the macrophages were probed with Alexa Fluor 647-conjugated rat monoclonal F4 / 80 antibody (1:100 dilution, Biolegend). Phagocytosis was determined by counting double-labeled cells by flow cytometry (BD Biosciences).

[0101] For the CDC assay, 1 × 10 cells were cultured in 100 μL of serum-free medium. 4 CT26.CL25 cell suspensions containing 100 cells were mixed with 2 μL of antiserum and incubated on ice for 30 minutes. 200 μL of non-heat-treated Gibco horse serum (Thermo Fisher) was added and incubated at 37°C for 2 hours. After washing with PBS, cell viability was measured by MTT assay.

[0102] <Cytokine assay>

[0103] Mouse spleen cells isolated from CRA-treated mice were placed in each well of a 96-well microplate at 3 × 10 5 The cells were seeded at 1 cell / 300 μL and then incubated for 72 hours. Cell culture supernatants were collected and analyzed by cytokine multiplex assay (Inflammation Core Facility, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan).

[0104] For analysis of IL-21 and IFN-γ concentrations, spleen cells or splenic T cells were plated at 3 x 10 in a 96-well culture plate. 5 Cells were seeded at 1000 x g for 72 h and then treated with various doses of CRA, CRG, or CRDG for 24 h. IL-21 levels in the culture medium were determined using an ELISA kit (R&D System, Minneapolis, MN) according to the manufacturer's instructions.

[0105] <In vitro B cell proliferation and differentiation>

[0106] Spleen cells (1 × 10 5 1 × 10 cells / well) were labeled with CSFE for 15 minutes at 37°C. Then, the cells were washed with PBS and suspended in fresh medium. CSFE-labeled cells were treated with different concentrations of CRA for 72 hours. B cell proliferation was analyzed by anti-CD19 antibody staining in CSFE-labeled cells. LPS (1 μg / mL) was used as a positive control. In a separate in vitro proliferation experiment, spleen cells (1 × 10 5 Cells (100 cells / well) were stimulated with 1 μg / mL anti-CD40 antibody and 100 U / mL IL-4 and treated with CRA for 72 hours. B cell proliferation was analyzed as described above. For B cell differentiation, plasmablasts (CD19 + CD138 + ) and plasma cells (CD19 - / low CD138 + ) were probed with specific monoclonal antibodies (BioLegend) and analyzed by flow cytometry (BD Biosciences).

[0107] <Quantitative polymerase chain reaction (qPCR)>

[0108] Spleen cells or splenic T cells were treated with the indicated concentrations of CRA, CRG, or CRDG, and then mRNA expression of Stat3, IL-21, Bcl6, and cMaf was measured by qPCR. Briefly, total cellular RNA was extracted with Trizol reagent (Invitrogen) and reverse transcribed into cDNA using the Superscript RT kit (Invitrogen). Primers for Stat3, Prdm-1, Bcl6, cMaf, and β-actin were designed using Primer-BLAST on the NCBI website. The Stat3 primers were the forward primer 5'-AGGAGTCTAACAACGGCAGCCT-3' (SEQ ID NO: 1) and the reverse primer 5'-GTGGTACACCTCAGTCTCGAAG-3' (SEQ ID NO: 2). The primers for IL-21 were forward primer 5'-TAGACGCTCACGAATGCAGG-3' (SEQ ID NO: 3) and reverse primer 5'-GTCTGTGCAGGGAACCACAA-3' (SEQ ID NO: 4). The primers for Bcl6 were forward primer 5'-CAGAGATGTGCCTCCATACTGC-3' (SEQ ID NO: 5) and reverse primer 5'-CTCCTCAGAGAAACGGCAGTCA-3' (SEQ ID NO: 6). The primers for cMaf were forward primer 5'-AGCAGTTGGTGACCATGTCG-3' (SEQ ID NO: 7) and reverse primer 5'-TGGAGATCTCCTGCTTGAGG-3' (SEQ ID NO: 8). All PCR reagents used for cDNA amplification were purchased from Promega (Madison, WI, USA). β-actin was used as an endogenous control. qPCR was performed using Fast SYBR Green Master Mix (Applied Biosystems, Waltham, MA, USA) and detected with an ABI 7500 Fast Real-Time PCR System (Applied Biosystems). Relative expression levels were calculated using the 2 − ΔΔCT method using the following formula: ΔCT (sample) = CT target gene − CT control gene.

[0109] <Statistical analysis>

[0110] Results were expressed as mean ± SD and analyzed using the SAS statistical software package (SAS Institute, Cary, NC). ANOVA tests were used for comparisons of multiple samples. Differences with a P value of less than 0.05 were considered statistically significant.

[0111] <Animal immunity>

[0112] Female BLAB / c mice were immunized intraperitoneally (ip) with 100 μg of recombinant AaHSP60 mixed with 10 mg / kg CRA. Subsequent immunizations of mice continued with weekly ip injections of the AaHSP60 and CRA mixture for 35 days. Blood was collected from the mice before and after immunization. Antiserum titers were measured by ELISA.

[0113] <Titer and Ig class of anti-AaHSP60 antibodies>

[0114] Mice immunized with AaHSP60 / CRA were bled weekly. The titer of anti-AaHSP60 antibody in the serum was measured as follows. Each well of a 96-well plate was coated with 100 ng of AaHSP60 overnight at room temperature. Further, the wells were blocked with 300 μl of 2% skim milk PBST (PBS buffer containing 0.05% tween-20) for 1 hour. 100 μl of serum diluted with PBS containing 0.5% skim milk was placed in each well and incubated at room temperature for 2 hours. After washing three times, 100 μl of HRP-conjugated anti-mouse Ig antibody (1:10,000 dilution, Sigma-Aldrich, St. Louis, MO, USA) was added to each well and incubated for 1 hour. After washing three times, 100 μL of NeA-Blue (Clinical Science Products, Inc., Mansfield, MA, USA) was added to each well, incubated for 20 minutes, and the reaction was stopped using 100 μL of 1N HCl. The optical density was measured at 450 nm using an ELISA reader (Tecan, Mannedorf, Switzerland). The isotype of the specific anti-AaHSP60 antibody in the serum (1:2000 dilution) was determined by using HRP-conjugated specific anti-mouse IgM, IgG1, IgG2a, IgG2b, IgG3 or IgA antibodies (Acris, Herford, Germany).

[0115] <Changes in the B cell lineage in AaHSP60 / CRA-immunized mice>

[0116] After AaHSP60 immunization, mouse spleen cells were obtained according to the method described above. Spleen cells were probed with fluorescent dye-conjugated anti-CD19, anti-CD-23, anti-CD-38, anti-CD27 and anti-CD138 antibodies and measured using flow cytometry (BD Biosciences). B cell types were defined as follows: naive B cells (CD19 + [[ID=1[]0]]CD23[[ID=1[]1]] + [[ID=1[]2]])、activated B cells (CD19[[ID=1[]3]] + [[ID=1[]4]]CD38[[ID=1[]5]] + [[ID=1[]6]])、memory B cells (CD19[[ID=1[]7]] + [[ID=1[]8]]CD27[[ID=1[]9]] + [[ID=2[]0]]) and plasma cells (CD19low / - CD138 + )。

[0117] <Cytokine profile in sera from AaHSP60 / CRA-immunized mice>

[0118] Sera isolated from the blood of AaHSP60 / CRA-immunized mice were analyzed by cytokine multiplex assay according to Bio-Plex Pro Mouse Cytokine Standard Group I 23-Plex (Bio-Rad, Hercules, CA, USA) and Milliplex mouse IL-21, IL-33, and TGFβ (Inflammation Core Facility, Academia Sinica, Taipei, Taiwan). IL-21, IFN-γ, and TGFβ in mouse sera or the culture medium of CRA-treated cells were analyzed by ELISA assay kits (R&D System, Minneapolis, MN) according to the manufacturer's protocol.

[0119] <Cells and cell culture>

[0120] Spleen cells were isolated from BALB / c mice according to the following procedure. Briefly, BALB / c mice were sacrificed and their spleens were collected. The spleens were placed in a cell strainer and homogenized into a Petri dish through the cell strainer. Red blood cells in the homogenized spleen were washed with PBS and then lysed with ACK lysis buffer at room temperature for 5 minutes. After spinning down the cells, they were resuspended and incubated in RPMI1640 growth medium (Gibco / Invitrogen) supplemented with heat-inactivated 10% fetal bovine serum (Gibco / Invitrogen) and 1% penicillin / streptomycin (Gibco / Invitrogen).

[0121] RAW264.7 mouse macrophages were purchased from BCRC (Hsinchu, Taiwan, ROC). RAW264.7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco / Invitrogen) supplemented with heat-inactivated 10% fetal bovine serum (Gibco / Invitrogen) and 1% penicillin / streptomycin (Gibco / Invitrogen) at 37°C in 5% CO.

[0122] <Cell proliferation assay>

[0123] Spleen cells (1 × 10 5 100 cells / well) were labeled with CSFE for 15 minutes at 37°C, then washed with PBS and suspended in fresh medium. CSFE-labeled cells were treated with 1 μg / ml AaHSP60 and different concentrations of CRA for 72 hours. Splenocyte proliferation was measured by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA). B cell proliferation was analyzed by anti-CD19 antibody staining in CSFE-labeled cells.

[0124] 1 x 10 macrophage RAW264.7 cells in a 96-well microculture plate 4 Cells were seeded at 100 cells / well overnight. After 48 hours of treatment with serial concentrations of CRA in DMSO (final DMSO concentration was 0.1%), macrophage proliferation was measured by the MTT assay. Cell viability (%) was calculated using the following formula: % viability = absorbance of test sample / absorbance of control × 100%.

[0125] <Surface marker expression>

[0126] After 48 hours of treatment with AaHSP60 and CRA, the treated cells were probed with FITC-conjugated anti-mouse CD86 and anti-mouse MHC II monoclonal antibodies, and cell surface fluorescence was measured using flow cytometry (Becton Dickinson). Data were expressed as mean fold fluorescence.

[0127] <Phagocytosis assay>

[0128] RAW 264.7 cells (3×10 5 cells / well) were treated with 1 μg / ml of AaHSP60 and a predetermined concentration of CRA for 48 hours. The phagocytosis activity of RAW264.7 cells was measured using a phagocytosis assay kit (Cayman, Ann Arbor, MI) according to the product manual. Briefly, the cells were incubated with latex beads-rabbit IgG-FITC (1:400) at 37°C for 1 hour. Next, 50 μL of trypan blue quenching solution (diluted 1:50 in Cell-Based Assay Buffer) was added to quench the surface fluorescence. Phagocytosis was analyzed by flow cytometry (Becton Dickinson).

[0129] <Analysis of mRNA expression of mBAFF by RT-PCR>

[0130] Spleen cells (1×10 6 cells / well) were treated with CRA for 16 hours. Next, total cellular RNA was extracted using TRIzol (Invitrogen Life Technologies, Carlsbad, CA), and Superscript TM-III kit (Invitrogen) was used for reverse transcription into cDNA. For detecting gene expression, PCR analysis was performed on aliquots of the cDNA preparations. Next, the cDNA of mBAFF was amplified by PCR. The primers for mouse BAFF were forward primer 5'-TGGTGAGGCAAACAGGCTAT-3' (SEQ ID NO: 9) and reverse primer 5'-AGAAGGTGTCGTCTCCGT TG-3' (SEQ ID NO: 10). All PCR reagents used for amplifying cDNA were purchased from Promega (Madison, WI, USA). The GAPDH cDNA in the samples was used to normalize the loading amount of each reaction. Finally, the PCR products were separated by electrophoresis on a 2% agarose gel, stained with ethidium bromide, and photographed using the Uni-photo band tool (EZ lab, Taipei, Taiwan).

[0131] <Analysis of protein expression of BAFF by ELISA>

[0132] Spleen cells (1×10 6 cells / well) were treated with CRA for 24 hours. Using an ELISA kit (R&D System, Minneapolis, MN), the levels of BAFF in the culture medium were measured according to the manufacturer's instructions.

[0133] <Statistical analysis>

[0134] The results were presented as mean ± SEM. Student's t-test was performed to test the differences between two groups, or one-way ANOVA was performed to test the differences between groups, followed by Newman-Keuls post hoc analysis to evaluate statistical significance. P < 0.05 was determined to be significant.

[0135] <Example 1> Tumor growth inhibition by CRA and activation of systemic and tumor-infiltrating B cells

[0136] To investigate whether CRA triggered immunomodulatory activity to suppress tumor growth, we orally administered 25, 50, or 100 mg / kg of CRA to CT26.CL25 tumor-bearing BALB / c mice and monitored its effects. After 21 days of treatment, 50 and 100 mg / kg of CRA significantly suppressed the growth of CT26.CL25 tumors in BALB / c mice (Figure 1A). At the end of treatment (day 33), significantly smaller tumor masses were observed in the CRA_50 (40.9%) and CRA_100 (44.3%) groups compared with the control (100%) group (Figure 1B). Control and treated mice maintained similar body weights throughout the experiment (Figure 1C).

[0137] To confirm whether B cell activation is involved in the antitumor activity of CRA, we analyzed the percentage of splenic CD19-positive B cells and their functional activity. No significant differences were observed in the levels of splenic CD19-positive B cells between control and CRA-treated mice (Figure 1D). We further investigated different B cell subsets. CRA reduced the percentage of splenic naive B cells but increased the percentages of splenic activated cells, memory cells, and plasma cells (Figure 1E). These results revealed that CRA did not alter the total B cell level, but altered the percentages of B cell subsets. CRA enhanced the functional activity and promoted the differentiation of systemic B cells. Using immunohistochemistry (IHC) assays, we demonstrated that CRA treatment resulted in the infiltration of B cells into the tumor area (Figure 1F). These results suggest that CRA-induced B cell activation and differentiation may play an adjuvant role in CRA-induced tumor suppression.

[0138] Example 2: Enhancement of anti-tumor autoantibody production and activity by CRA

[0139] Characterization of the antisera revealed that the titer of specific anti-CT26CL25 antibodies in the CRA antisera was higher than that in the control antisera. Even a low dose (25 mg / kg) of CRA elevated the anti-CT26CL25 antibody titer (Figure 2A). The main classes of antibodies elevated in the CRA antisera were IgG1, IgG2a, IgG2b, and IgG3 (Figure 2B). Similar to normal mouse serum, the control antisera weakly recognized CT26CL25 cells, whereas the CRA antisera showed stronger binding affinity for CT26CL25 cells (Figure 2C). The CRA antisera did not recognize MG-Cap A1 cells (Figure 2C). This suggests that the CRA-induced antibodies in the antisera have tumor specificity.

[0140] Example 3: Tumor growth suppression by humoral immune response activated by CRA

[0141] To confirm whether CRA-triggered humoral immunity is involved in antitumor activity, we performed cytotoxicity assays using CRA antisera or control antisera. CRA antisera significantly inhibited cell proliferation in CT26.CL25 cells, whereas sCRA antisera (previously removed anti-CT26.CL25 cell antibodies) had no effect (Figure 3A). CRA antisera triggered major ADCC and ADCP responses and minor CDC responses to kill tumor cells (Figures 3B, 3C, and 3D). Previous removal of anti-CT26.CL25 cell antibodies reduced these three antibody-mediated cell death responses. These results indicate that CRA-activated humoral immunity contributes to the therapeutic effect of CRA treatment in cancer therapy.

[0142] Example 4: Role of cytokine expression in CRA-induced B cell activation

[0143] Cytokine expression in spleen cells isolated from CRA-treated mice was examined, and it was shown that the levels of IFN-γ and IL-21 were significantly increased (Fig. 4). These results suggest that IL-21 may be an important cytokine in regulating B cell activation and differentiation.

[0144] Example 5: CRA promoted B cell differentiation in vitro In vitro promotion of B cell differentiation by CRA

[0145] To examine the effect of CRA on B cell activation, we measured cell proliferation and differentiation in mouse splenocytes after CRA treatment. CRA did not induce B cell proliferation in mouse splenocytes (Figure 5A), but it did increase the proportion of plasma (CD19low / -CD138+) cells (Figures 5B and 5C). This indicates that CRA primarily promotes B cell differentiation rather than proliferation. CRA increased the release of IL-21 and IFN-γ in splenocytes (Figure 5D). In another set of in vitro studies, splenocytes were stimulated with anti-CD40 antibody and IL-4 and then treated with CRA. The results also showed that CRA did not affect splenic B cell proliferation (Figure 5E), but accelerated B cell differentiation in anti-CD40 antibody and IL-4-stimulated splenocytes (Figures 5F and 5G). In the culture medium, CRA treatment also increased the levels of IL-21 and IFN-γ in splenocytes stimulated with anti-CD40 antibody and IL-4 (Figure 5H). Furthermore, CRA treatment increased the gene and protein expression of STAT3 and Blimp, which are downstream of IL-21 / IL-21 receptor signaling in B cells (Figures 6A and 6B).

[0146] To understand whether CRA can directly induce B cell differentiation or whether IL-21 mediation is required, splenic B cells were purified and then treated with CRA. In the absence of IL-21, CRA did not trigger a differentiation response in splenic B cells, regardless of the presence or absence of anti-CD40 antibody and IL-4 stimulation (Figures 7 and 8). Furthermore, when IL-21 released from CRA-treated spleen cells was neutralized with anti-IL-21 antibody, CRA-induced differentiation was reduced (Figure 9). These results suggest that CRA-induced B cell activation requires the presence of IL-21, which can trigger IL-21 / IL-21R signaling, leading to B cell differentiation.

[0147] Example 6: Role of T cells in CRA-induced B cell activation Role of T cells in CRA-induced B cell activation

[0148] Immune cells producing both IL-21 and IFN-γ should be important reactive cells for CRA-induced B cell activation. It has been reported that T cells produce IL-21 via STAT3 / cMaf / BCL6 signaling. To examine whether CRA activates T cells to release IL-21, we measured the expression of STAT3, cMaf, and BCL6 in splenic T cells. CRA not only increased IL-21 release (Figure 10A) but also increased the expression of STAT3 and cMaf in a dose-dependent manner, whereas CRA increased BCL6 expression only at the highest dose (Figures 10B, 10C, and 10D). Thus, CRA induced T cells to produce IL-21 via the STAT3 / cMaf / BCL6 pathway. IL-21 released from CRA-activated T cells enabled B cell differentiation and produced high titers and affinity of antitumor autoantibodies, suppressing tumor growth (Figure 10E).

[0149] Example 7: Effect of the active ingredient of CRA on B cell differentiation

[0150] CRA consists of two major compounds, CRDG and CRG. To investigate which compound is the active component of CRA on B cell differentiation, we measured the changes in the percentage of plasma cells in splenocytes treated with CRDG or CRG. In this study, the effect of CRA on B cell differentiation was elicited from the two compounds, CRDG and CRG (Figures 11A and 11B). Both CRDG and CRG induced splenic T cells to release IL-21 via the STAT3 / cMaf / BCL6 pathway (Figures 11C, 11D, 11E, and 11F). This suggests that CRDG and CRG are the active compounds in CRA-promoted B cell differentiation.

[0151] <Consideration>

[0152] This study revealed that CRA, a phytogalactolipid-rich fraction isolated from C. rabens (Benth.) S. Moorecan, provides antitumor activity by activating humoral responses (Figure 1) and producing antitumor autoantibodies (AAs) with high titer, affinity, and cytotoxic activity (Figures 2 and 3). These antibodies were able to eliminate CRC tumor growth in BLAB / c mice (Figure 1). The proposed mechanism of CRA is shown in Figure 8E. CRA increased STAT3, cMaf, and BCL6 activity in T cells, inducing IL-21 release (Figures 8A–D), which then bound to the IL-21 receptor on B cells (Figures 6A–D), activating STAT3 / BLIMP-1 signaling, and promoting B cell maturation and differentiation. Blockade of CRA-induced IL-21 with a neutralizing monoclonal anti-IL-21 antibody significantly inhibited B cell differentiation (Figure 6E). These results indicate that CRA-triggered IL-21 is a key cytokine for mediating B cell differentiation and antibody production. IL-21 is essential for B cell differentiation into plasma cells and promotes functional germinal centers and immunoglobulin production [Spolski, R. and WJ Leonard, Nat Rev Drug Discov, 2014. 13(5): pp. 379-95]. The primary mechanism of IL-21-induced B cell differentiation is the STAT3-mediated induction of BLIMP-1, a transcriptional repressor for plasma cell generation and the establishment of long-lived antibody responses [Avery, DT, et al., J Exp Med, 2010. 207(1): pp. 155-71]. It has been reported that IL-21 can have both positive and negative effects on B cells in vitro. IL-21 increased the proliferation of splenic B cells in mice stimulated with anti-IgM and anti-CD40 [Konforte, D., N. Simard, and CJ Paige, J Immunol, 2009. 182(4): p. 1781-7].In contrast, B cell proliferation induced by anti-IgM and IL-4 was inhibited by IL-21 [Konforte, D., N. Simard, and CJ Paige, J Immunol, 2009. 182(4): pp. 1781-7]. In this study, CRA treatment did not affect the proliferation of unstimulated splenocytes (Figure 5A). CRA treatment tended to inhibit B cell proliferation in splenocytes stimulated with anti-CD40 / IL4 (Figure 5E). These results are similar to those observed when IL-21 was added to splenic B cells stimulated in vitro with anti-IgM and IL-4 [Konforte, D., N. Simard, and CJ Paige, J Immunol, 2009. 182(4): pp. 1781-7]. However, B cell differentiation is promoted in both unstimulated and anti-CD40 / IL4-stimulated cells (Figures 5B, 5C, 5F, and 5G). IL-21 signaling primes CD40-stimulated human naive B cells and promotes their differentiation into plasmablasts via STAT3 [Berglund, LJ, et al., Blood, 2013. 122(24): pp. 3940-50]. Therefore, the role of CRA-induced IL-21 contributes to the promotion of B cell differentiation.

[0153] CRA enhanced the titer of antitumor autoantibodies (Figure 2A), and the increased antibody isotypes were IgG1, IgG2a, IgG2b, and IgG3 (Figure 2B). Cytokines secreted by activated helper T cells determine Ig class switching. CRA treatment induced the release of IL-21 and IFN-γ (Figures 4A-E). IL-21 regulates B cells to produce IgG1 and IgG3 antibodies, while IFN-γ promotes the production of IgG2a antibodies [Pene, J., et al., J Immunol, 2004. 172(9): pp. 5154-7; Bossie, A. and ES Vitetta, Cell Immunol, 1991. 135(1): pp. 95-104]. Somatic hypermutation (SHM) and class switch recombination (CSR) generate antibody diversity. Stimulation with anti-CD40 mAb and recombinant IL-21 induces the expression of activation-induced cytidine deaminase (AID), leading to CSR production of IgG1 and IgG3 antibodies through the induction of γ1 and γ3 germline transcripts and Sγ / Sμ switch circular DNA [Konforte, D., N. Simard, and CJ Paige, J Immunol, 2009. 182(4): pp. 1781-7; Pene, J., et al., J Immunol, 2004. 172(9): pp. 5154-7]. IFN-γ produced by Th1 cells promotes the secretion of IgG2a. Our results suggest that CRA-induced IL-21 and IFN-γ not only induce antibody class switching but also activate both Th1 and Th2 responses.

[0154] IL-21 is produced by T cell populations, with production highest in T follicular helper (Tfh) cells and Th17 cells, and at lower levels in natural killer T (NKT) cells and CD8+ T cells [Spolski, R. and WJ Leonard, Nat Rev Drug Discov, 2014. 13(5): p. 379-95]. In this study, CRA primarily activated STAT3 / c-Maf signaling and slightly activated Bcl6 signaling to produce IL-21. STAT3 expression in T cells was required for IL-21 production by multiple T helper subsets. In Tfh and Th17 cells, STAT3 activated by IL-6 and / or IL-21 is required for IL-21 production upon T cell receptor stimulation [Wei, L., et al., J Biol Chem, 2007. 282(48): pp. 34605-10; Dienz, O., et al., J Exp Med, 2009. 206(1): pp. 69-78]. STAT3 can directly bind to the IL-21 promoter for IL-21 production. Furthermore, c-Maf is required for IL-21 production via transactivation of the IL-21 promoter in Tfh and Th17 cells. c-Maf- / - mice exhibit insufficient IL-21 expression, suggesting that IL-21 and c-Maf expression are interdependent [Bauquet, AT, et al., Nat Immunol, 2009. 10(2): pp. 167-75]. Bcl6 increases IL-21 production by inhibiting Krüppel-like transcription factor 2 (Klf2), which suppresses IL-21 expression [Choi, J. and S. Crotty, Trends Immunol, 2021. 42(4): pp. 336-349]. This Bcl6 repressor-of-repressors mechanism functions to regulate IL-21 production in Tfh [Choi, J. and S. Crotty, Trends Immunol, 2021. 42(4): pp. 336-349].These findings suggest that CRA is a STAT3 activator that increases c-Maf expression or directly activates c-Maf to induce IL-21 production. Furthermore, IL-21 contributes to the proliferation and survival of T lymphocytes through both autocrine and paracrine IL-21 pathways [Liu, S. M. and C. King, J. Immunol., 2013. 191(7): pp. 3501-6]. Generally, IL-21 also affects the expression of transcription factors such as STAT3, c-Maf, and Bcl-6, which play central roles in Tfh development [Liu, S. M. and C. King, J. Immunol., 2013. 191(7): pp. 3501-6]. Therefore, the data herein demonstrate that CRA triggers IL-21 release by activating STAT3, c-Maf, and Bcl-6 signaling, thereby promoting the development of Th subsets.

[0155] Cancer patients have been shown to produce autoantibodies in their serum that recognize their own tumor antigens. These autoantibodies offer no therapeutic benefit for tumor elimination. In some cases, they accelerate tumor growth. In the present invention, CRA improves the quality of autoantibodies by inducing an Ig class switch from IgM to IgG, thereby improving their binding affinity (Figure 2B). IgG has a longer circulating half-life in the human body. IgG efficiently increases killing by NK cells and phagocytosis by macrophages. Therefore, CRA-induced class switching may be the reason why CRA antisera inhibit tumor growth more efficiently than control antisera. CRA may enhance the somatic hypermutation process in B cells, which allows autoantibodies in CRA antisera to have higher antigen-binding affinity than those in control antisera. As a result, antitumor autoantibodies can stably bind to surface antigens to provide cytotoxicity (Figure 3A) and trigger more efficient ADCC and ADCP (Figures 3B and 3C).

[0156] <Conclusion>

[0157] The present invention discovered a novel pharmacological function of CRA on the dominant humoral response, which involves T cell-dependent IL-21 induction via the IL-21R / STAT3 / Blimp-1 pathway. CRA triggers systemic and tumor responses to produce higher titers and better quality of antitumor autoantibodies compared to untreated mice, resulting in tumor elimination in the animals.

[0158] Example 8: Adjuvant effect of CRA on antibody production in vivo

[0159] To evaluate the efficacy of CRA as an effective adjuvant in vivo, mice were immunized with AaHsp60 as the antigen. After the immunization protocol, anti-AaHsp60 antibody titers in the serum were measured. Figure 12A shows that CRA significantly induced specific anti-AaHsp60 antibody titers after 21 days of treatment. While the dilution range of vehicle-induced anti-AaHsp60 antibodies was 1:12595 to 1:64028, the EC50 range of CRA-induced anti-AaHsp60 antibodies was 1:37301 to 1:64028 (Figure 12B). The isotypes of the increased specific anti-AaHsp60 antibodies were IgM, IgG2a, IgG2b, and IgG3 (Figure 12C). Furthermore, CRA increased the proportion of plasma cells in splenocytes isolated from mice immunized with AaHsp60 (Figure 12D). These results indicate that CRAs enhance the production and binding affinity of specific antibodies. CRA-induced antibody class switching is the reason for the improved antibody binding affinity, and CRA-induced B cell differentiation leads to greater antibody production.

[0160] Antibody class switching and B cell differentiation depend on the type of cytokines produced after antigen stimulation. To determine which immune pathway CRA induces, we examined the different cytokines induced by antigen stimulation after immunization. Table 1 and Figure 13 show that CRA significantly increased serum levels of IL-21, IFN-γ, and TGF-β in mice immunized with AaHSP60, whereas CRA did not affect Th2 cytokines (IL-4, IL-5, and IL-10) associated with B cell differentiation and antibody production. The cytokine results indicated that CRA induces B cell proliferation by upregulating IL-21 expression. Antigen immunization with CRA also increased the number of IFN-γ-expressing T cells that produced IgG2a and IgG3 antibodies, and TGF-β-expressing T cells that induced IgG2b antibodies.

[0161] JPEG2026507504000002.jpg127170*P<0.05 (compared to the control group)

[0162] Example 9: Effect of CRA on cell proliferation and cytokine production in splenocytes

[0163] To evaluate the adjuvant effect of CRA, we analyzed cell proliferation and cytokine release in splenocytes. CRA efficiently induced cell proliferation in mouse splenocytes, regardless of AaHsp60 stimulation (Figures 14A and 14D). IL-21 and IFN-γ release from CRA-treated splenocytes was significantly increased (Figures 14B, 14C, 14E, and 14F). Taken together, we demonstrated that CRA has potent immunomodulatory activity and adjuvant effects.

[0164] Example 10: Effect of CRA on B cell activation and differentiation in vitro

[0165] To assess the effect of CRA on B cell activation and differentiation, we analyzed cell proliferation and surface marker expression in CRA-treated B cells. Unexpectedly, CRA had no effect on B cell proliferation in mouse splenocytes, regardless of the presence or absence of AaHsp60 (Figure 15A). CD86 and MHCII expression are associated with B cell activation and the ability to present antigen (act as APCs). CRA also had no effect on CD86 and MHCII expression in CD19+ B cells (Figure 15B). After CRA treatment, the phenotype (CD19 + CD138 - / low The proportion of IgG4-associated (IgG4-associated) cells significantly increased (Figure 16). These results indicate that CRA efficiently induces B cell differentiation, despite not affecting B cell proliferation or their ability to act as APCs. Therefore, the adjuvant effect of CRA on antibody production arises from CRA-induced B cell differentiation.

[0166] Example 11: Effect of CRA on antigen presentation by macrophages

[0167] To evaluate the adjuvant effect of CRA on the efficiency of antigen presentation, we analyzed cell proliferation, phagocytic activity, and surface marker expression in macrophages, a type of antigen-presenting cell. CRA induced 20-30% cell proliferation in macrophages (Figure 17A). CRA did not affect the antigen uptake capacity of macrophages without antigen stimulation. However, CRA / AaHSP60 promoted antigen uptake in macrophages by phagocytosis (Figure 17B). 12 μg / ml CRA enhanced the uptake capacity in AaHSP60-treated macrophages compared with vehicle control (Figure 17B). Increased CD86 and MHC II expression are involved in APC maturation, which leads to efficient antigen presentation. We analyzed the effect of CRA on CD86 and MHC II expression, which are involved in antigen presentation. Although CRA did not affect CD86 expression (Figure 17C), it effectively increased cell surface MHC II expression in macrophages, regardless of whether they were stimulated with AaHSP60 (Figure 17D). Interestingly, CRA effectively induced BAFF mRNA expression and protein production in macrophages (Figure 18). These results demonstrated that CRA not only activated macrophages to become APCs but also produced BAFF to support B cell differentiation.

[0168] Example 12: Effect of CRA on antigen production via oral administration

[0169] Female BLAB / c mice were immunized by oral administration of vehicle / rAaHSP60 (100 μg of rAaHSP60) or CRA / rAaHSP60 (10 mg / kg of CRA mixed with 100 μg of rAaHSP60), followed by weekly administration of CRA / rAaHSP60 for 5 weeks to boost immunity. Saliva was analyzed at a dilution of 1:3,200 for IgA-type anti-AaHSP60 antibodies. As shown in Figure 19, CRA fully achieved the effect of inducing antigen production via oral administration.

[0170] <Consideration>

[0171] We demonstrated that CRA, a plant galactolipid-based extract, has an adjuvant effect that enhances the immunogenicity of antigens. CRA not only enhanced specific antibody production but also induced class switching (Figures 12A and 12C). CRA-induced IL-21 and BAFF release enabled B cell differentiation (Figures 12D, 13, and 18). CRA induced Th1 responses, including elevated levels of IgG2a (Figure 12) and IFN-γ (Figure 13), as well as Th2 responses, including elevated levels of IgG2b (Figure 12C) and IL-21 (Figure 13). Unexpectedly, the levels of typical Th2 cytokines, such as IL-4 and IL-10, in mouse serum were unchanged by CRA treatment. Therefore, IL-21 plays an important role in CRA-induced B cell differentiation. IL-21 is produced by follicular Th (Tfh) cells in primary and subsequent responses to T cell-dependent antigens, initiating and maintaining long-term humoral immunity (Bryant VL, Ma CS, Avery DT, et al., Journal of Immunology. 2007;179(12):8180-8190). IL-21 stimulates CD4+ to differentiate B cells into Ab-secreting plasmablasts. +IL-21 induces T cell-mediated help (Bryant VL, Ma CS, Avery DT, et al. Journal of Immunology. 2007;179(12):8180-8190). IL-21 supports the generation and differentiation of Th2 cells through autocrine regulation. IL-21 belongs to the Th2 cytokines that theoretically inhibit Th1 differentiation and GM-CSF production (Kastirr I, Maglie S, Paroni M, et al., Journal of Immunology. 2014;193(7):3322-3331). However, IL-21, acting synergistically with IL-12, IL-15, or IL-18, enhances IFN-γ production in NK cells and T cells, subsequently increasing Th1-polarized immune responses (Strengell M, Sareneva T, Foster D, Julkunen I, Matikainen S., Journal of Immunology. 2002;169(7):3600-3605). CRA-treated mice showed elevated IgG2a antibody and IFN-γ levels (Figure 1C). This indicates that Th1 cells can be activated by CRA treatment. Although IL-21 signaling has been proposed to promote Th17 differentiation (Huber M, Brustle A, Reinhard K, et al., Proceedings of the National Academy of Sciences of the United States of America. 2008;105(52):20846-20851; Korn T, Bettelli E, Gao W, et al., Nature. 2007;448(7152):484-487), our results showed that CRA did not affect IL-17 production (Table 1), indicating that Th17 was not altered by CRA treatment in mice.

[0172] CRA induced Ig class switching from IgM to IgG2a, IgG2, and IgG3 (Figure 12C). CRA enhanced the process of somatic hypermutation in B cells, resulting in antibodies with higher antigen-binding affinity. The mechanism of CRA-induced Ig class switching and somatic hypermutation may involve the release of IL-21, IFN-γ, and TGF-β. IL-21 is not only a cytokine for B and T cell activation but also a switch factor for IgG1 and IgG3 antibody production. Stimulation of CD19+ B cells with recombinant IL-21 and anti-CD40 mAb resulted in the production of Sγ / Sμ switched circular DNA by enhancing γ1 and γ3 germline transcripts and cytidine deaminase activation (Pene J, Gauchat JF, Lecart S, et al., Journal of Immunology. 2004;172(9):5154-5157). IFN-γ promotes IgG2a antibody production, whereas TGF-β regulates B cells to produce IgG2b and IgA antibodies (Bossie A, Vitetta ES., Cellular Immunology. 1991;135(1):95-104; Snapper CM, Waegell W, Beernink H, Dasch JR., Journal of Immunology. 1993;151(9):4625-4636). The present disclosure observed that increased BAFF levels are also a regulator of CRA-induced Ig class switching.BAFF upregulates the expression of the transcription factor Pax5 / BSAP, which in turn increases the transcription of activation-induced cytidine deaminase (AID), an RNA-editing enzyme responsible for IgH class switch recombination and somatic hypermutation (Xu Z, Pone EJ, Al-Qahtani A, Park SR, Zan H, Casali P., Critical reviews in immunology. 2007;27(4):367-397; Stavnezer J, Schrader CE., Journal of Immunology. 2014;193(11):5370-5378; Muramatsu M, Kinoshita K, Fagarasan S, Yamada S, Shinkai Y, Honjo T., Cell. 2000;102(5):553-563). Furthermore, in the murine immune system, IgG2a and IgG2b efficiently sensitize NK cells for cell killing (Clemenceau B, Vivien R, Pellat C, Foss M, Thibault G, Vie H., MAbs. 2013;5(4):587-594). Regarding IgG2a and IgG2b, the most potent IgG subclasses can bind to FcγRs (IgG2a binds to FcγR1, III, and IV; IgG2b binds to FcγRIII and IV) and trigger NK cell-mediated ADCC (Nimmerjahn F, Bruhns P, Horiuchi K, Ravetch JV., Immunity. 2005;23(1):41-51). IgG2a and IgG2b, as well as IgG1, readily diffuse to extravascular sites to access antigens and have higher potency for activating the complement system.In some cases, IgG2a and IgG2b have been found to be more effective than IgG1 in triggering ADCC and complement-mediated neutralization (Clemenceau B, Vivien R, Pellat C, Foss M, Thibault G, Vie H., MAbs. 2013;5(4):587-594; Ishizaka ST, Piacente P, Silva J, Mishkin EM., The Journal of Infectious Diseases. 1995;172(4):1108-1111). Mouse IgG3 binds to FcγRI on monocytes / macrophages and triggers antibody-dependent cellular phagocytosis (ADCP). Human IgG3 is the most effective subclass in terms of activating effector functions due to its high binding affinity to C1q and FcγRs (Stapleton NM, Andersen JT, Stemerding AM, et al., Nature Communications. 2011;2:599). Therefore, it is conceivable that CRA-induced class switching may be exploited in the future to inactivate antibodies against pathogens.

[0173] BAFF has also been reported to be a potent cytokine with stimulatory effects on B and T cells (Smulski CR, Eibel H., Frontiers in Immunology. 2018;9:2285). Increased systemic BAFF levels can enhance pathogen-specific immune responses upon immunization with heat-killed Pseudomonas aeruginosa (Tertilt C, Joh J, Krause A, et al., Infection and Immunity. 2009;77(7):3044-3055). BAFF has been used as an immunostimulator because it has trimerization properties that enhance immune responses (Gupta S, Clark ES, Termini JM, et al., Journal of Virology. 2015;89(8):4158-4169). Transient overexpression of BAFF in vivo enhances antigen-specific humoral immunity, suggesting its potential role as a vaccine adjuvant. CRA induced BAFF release and promoted anti-AaHSP60 antibody production, suggesting that the immunostimulatory effects of CRA may be utilized as an adjuvant for pathogen vaccines.

[0174] <Conclusion>

[0175] CRA has been shown to have an adjuvant effect, enhancing mouse immunity against pathogen antigens, particularly increasing both Th1- and Th2-related immune responses. CRA promotes B cell differentiation and produces antibodies with higher titers and higher affinity against pathogen antigens. CRA has been demonstrated to be a promising adjuvant for pathogen vaccines.

[0176] While the present disclosure has been described and illustrated with reference to specific embodiments thereof, these descriptions and illustrations are not intended to be limiting. Those skilled in the art will recognize that various changes may be made and equivalents substituted without departing from the true spirit and scope of the present disclosure, as defined by the appended claims. Illustrations are not necessarily drawn to scale. Differences between the representations in the drawings of this disclosure and the actual devices due to manufacturing processes and tolerances are possible. There may be other embodiments in the present disclosure not specifically illustrated. The specification and drawings are to be considered illustrative, not restrictive. Changes may be made to adapt a particular situation, material, composition of matter, method, or process to the objective, spirit, and scope of the present disclosure. All such modifications are intended to be within the scope of the claims appended hereto. While the methods disclosed herein are described with reference to particular operations performed in a particular order, it will be understood that these operations may be combined, sub-divided, or reordered to form equivalent methods without departing from the teachings of the present disclosure. Accordingly, unless specifically indicated herein, the order and grouping of operations is not intended to limit the present disclosure.

Claims

1. A method for inducing a humoral immune response in a subject in need thereof, comprising administering to the subject an effective amount of the safflower ragwort plant or an extract thereof, or a galactolipid compound from the safflower ragwort or a pharmaceutically acceptable derivative thereof, and optionally a pharmaceutically acceptable carrier or excipient.

2. 2. The method of claim 1, wherein the method is for attracting B cells, enhancing antibody production, enhancing antibody class switching, inducing antibody-dependent cellular cytotoxicity, inducing antibody-dependent cellular phagocytosis, increasing serum IFN-γ and IL-21, inducing B cell proliferation, or inducing B cell differentiation.

3. 3. The method of claim 2, wherein the antibody class switching comprises switching from IgM to IgG1, IgG2, or IgG3.

4. The method of claim 2, wherein the method is for inducing B cell proliferation or inducing B cell differentiation via the IL-21R / STAT3 / Blimp-1 pathway.

5. 2. The method of claim 1, wherein the humoral immune response is induced against a surface-bound cancer antigen or an antigen associated with a neurodegenerative disease.

6. 6. The method of claim 5, wherein the cancer is drug-resistant colorectal cancer.

7. 2. The method of claim 1, wherein the safflower ragwort is Safflower ragwort (Benth.) S. Moore.

8. 2. The method of claim 1, wherein the safflower plant is a piece of dried or fresh safflower.

9. 2. The method of claim 1, wherein the extract of safflower safflower is an alcohol extract of safflower safflower.

10. The method of claim 1, characterized in that the safflower ragwort extract is produced by extracting safflower ragwort with an alcohol solution to obtain an alcoholic extract, and fractionating the alcoholic extract to obtain a fraction rich in phytogalactolipids.

11. 11. The method of claim 10, wherein the fractionation step is performed by reversed-phase medium pressure liquid chromatography (RP-MPLC).

12. 12. The method of claim 11, wherein the RP-MPLC is followed by a further reversed-phase high performance liquid chromatography (RP-HPLC).

13. 2. The method of claim 1, wherein the galactolipid compound is 1,2-di-O-α-linolenoyl-3-O-β-galactopyranosyl-sn-glycerol (dLGG) or 1,2-di-(α-linolenoyl)-3-[α-D-galactosyl-(1-6)-β-D-galactosyl]-sn-glycerol (CRDG).

14. An adjuvant composition comprising an effective amount of the carthamus plant or an extract thereof, or a galactolipid compound from the carthamus plant or a pharmaceutically acceptable derivative thereof.

15. The adjuvant composition according to claim 14, wherein the safflower safflower is Safflower safflower (Benth.) S. Moore.

16. 15. The adjuvant composition of claim 14, wherein the safflower plant is a dried or fresh safflower fragment.

17. The adjuvant composition according to claim 14, wherein the extract of safflower safflower is an alcohol extract of safflower safflower.

18. The adjuvant composition of claim 14, characterized in that the carthamus extract is produced by extracting carthamus with an alcohol solution to obtain an alcohol extract, and fractionating the alcohol extract to obtain a fraction rich in phytogalactolipids.

19. The adjuvant composition of claim 18, wherein the fractionation is carried out by reversed-phase medium pressure liquid chromatography (RP-MPLC).

20. The adjuvant composition of claim 19, wherein RP-MPLC is followed by reversed-phase high performance liquid chromatography (RP-HPLC).

21. The adjuvant composition according to claim 14, wherein the galactolipid compound is 1,2-di-O-α-linolenoyl-3-O-β-galactopyranosyl-sn-glycerol (dLGG) or 1,2-di-(α-linolenoyl)-3-[α-D-galactosyl-(1-6)-β-D-galactosyl]-sn-glycerol (CRDG).

22. An immunogenic composition comprising an antigen component and the adjuvant composition of claim 14.

23. 23. The immunogenic composition of claim 22, wherein the antigen component is a peptide or protein.

24. 24. A method for enhancing an immune response in a subject in need thereof, comprising administering to said subject an adjuvant composition according to any one of claims 14 to 21, or an immunogenic composition according to claim 22 or 23.

25. 25. The method of claim 24, wherein the adjuvant composition or the immunogenic composition is administered by oral route.

26. 25. The method of claim 24, wherein the method is for enhancing antibody production, enhancing antibody class switching, increasing the proportion of IgG2a, IgG2b, or IgG3 in total IgG antibodies, triggering the release of IL-21 and / or B cell activating factor (BAFF), triggering B cell differentiation, enhancing phagocyte proliferation, uptake capacity, and / or antigen presentation, or increasing Th1 and Th2-associated immune responses.

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