WO2020029968A1 - 活化cd4+t细胞的方法 - Google Patents

活化cd4+t细胞的方法 Download PDF

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WO2020029968A1
WO2020029968A1 PCT/CN2019/099489 CN2019099489W WO2020029968A1 WO 2020029968 A1 WO2020029968 A1 WO 2020029968A1 CN 2019099489 W CN2019099489 W CN 2019099489W WO 2020029968 A1 WO2020029968 A1 WO 2020029968A1
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cells
antigen
multimer
target antigen
subunits
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PCT/CN2019/099489
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English (en)
French (fr)
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侯百东
洪胜
华兆琳
唐宏
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中国科学院生物物理研究所
中国科学院上海巴斯德研究所
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Priority to CN201980052317.0A priority Critical patent/CN112639077B/zh
Priority to US17/266,449 priority patent/US20220111040A1/en
Priority to EP19848673.0A priority patent/EP3835414A4/en
Publication of WO2020029968A1 publication Critical patent/WO2020029968A1/zh

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    • C12N2795/18011—Details ssRNA Bacteriophages positive-sense
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    • C12N2795/00011—Details
    • C12N2795/18011—Details ssRNA Bacteriophages positive-sense
    • C12N2795/18111—Leviviridae
    • C12N2795/18134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure relates to the field of immunotherapy. Specifically, the present disclosure relates to a method for activating CD4 + T cells using a multimer-based antigen complex. The present disclosure also relates to methods of promoting the differentiation of CD4 + T cells into Tfh cells and Th1 cells, and methods of treating diseases by activating CD4 + T cells and / or promoting the differentiation of CD4 + T cells.
  • CD4 + T cells are an important class of T lymphocytes that play an important role in a variety of physiological and pathological processes, including infection-related immune responses, tumor-related immune responses, allergic disease-related immune responses, and autoimmune diseases Class of immune response, etc.
  • the main function of CD4 + T cells is through regulating other immune cells, including regulating B lymphocytes, CD8 + T lymphocytes, monocyte macrophages, and NK cells. Function in the immune response.
  • CD4 + T cells can be divided into several different subgroups according to their functional status, and each subgroup plays a specific role in different immune responses.
  • Follicular helper cells Fficular helper cells (Fficular) helper cells (Tfh) are an important subgroup of CD4 + T cells, which are mainly involved in the germinal center response process. Tfh is essential for the formation and maintenance of germinal centers. Tfh can induce and maintain germinal center B cells to promote antibody production, antibody type conversion, antibody affinity maturation, neutralizing antibody production, and increase the broadness of the antibody map. Therefore, how to effectively generate Tfh cells is a key link for the development of various vaccines.
  • Type 1 helper T cells are another important subgroup of CD4 + T cells, which are antiviral and antibacterial, especially against intracellular bacterial infections, such as tuberculosis Play a key role.
  • CD4 + T cells also play an important role in anti-tumor immunity.
  • CD4 + T cells For CD4 + T cells to perform the above functions, they must first be transformed from a resting state to an activated cell state.
  • the current method for activating CD4 + T cells is to use dendritic immune cells (DC) to initially activate CD4 + T cells in vivo or in vitro, thereby achieving the purpose of generating activated CD4 + T cells.
  • DC dendritic immune cells
  • the activation pathway of CD4 + T cells and its related mechanisms are not clear, and DCs do not have specificity for antigen processing and presentation. There remains a need in the art for new methods for effectively activating CD4 + T cells.
  • the present disclosure is based at least in part on the discovery that by constructing a multimer-based antigen complex, which can be recognized and processed by B cells, activates CD4 + T cells and promotes the differentiation of CD4 + T cells into Tfh and Th1 cells. Because B cells specifically recognize antigens through B cell surface receptors encoded by immunoglobulin receptor genes, they can achieve stronger effects than DCs when B cells are used as antigen-presenting cells to activate CD4 + T cells. .
  • the present disclosure relates to the following aspects.
  • the disclosure relates to a method of activating CD4 + T cells, the method comprising the following steps:
  • the antigen complex comprises:
  • the plurality of subunits comprise or consist of a target antigen, and wherein the immunostimulant is packaged in the polymer, or is attached to the polymer through physical adsorption or chemical connection;
  • antigen complex comprises:
  • the target antigen is attached to the surface of the multimer through physical adsorption or chemical connection, or is fused to at least a part of the plurality of subunits through gene fusion, and the fusion does not affect the assembly of the multimer
  • the target antigen is displayed on the surface of the multimer after the multimer is assembled; and wherein the immunostimulant is packaged in the multimer, or is attached to the multimer by physical adsorption or chemical connection Mentioned multimers,
  • the B cell population is capable of identifying at least one of the subunits
  • the present disclosure relates to a method of promoting the differentiation of CD4 + T cells into follicular helper T cells (Tfh) and / or helper T cells 1 (Th1), the method comprising the following steps:
  • the antigen complex comprises:
  • the plurality of subunits comprise or consist of a target antigen, and wherein the immunostimulant is packaged in the polymer, or is attached to the polymer through physical adsorption or chemical connection;
  • antigen complex comprises:
  • the target antigen is attached to the surface of the multimer through physical adsorption or chemical action, or is fused to at least a part of the plurality of subunits through gene fusion, and the fusion does not affect the assembly of the multimer
  • the target antigen is displayed on the surface of the multimer after the multimer is assembled; and wherein the immunostimulant is packaged in the multimer, or is attached to the multimer by physical adsorption or chemical action Mentioned multimers
  • the B cell population is capable of identifying at least one of the subunits
  • the B cell population may be a B cell population isolated from the peripheral blood or lymphoid organs of the donor .
  • the method may further include performing B cells that recognize the subunit after step b) Screening, enrichment and / or amplification steps.
  • the method may further include performing B cells that recognize the subunit after step b) A step of screening and introducing its gene sequence encoding an immunoglobulin receptor into the B cell population to increase the number of B cells in the population that recognize the subunit.
  • the multimers have a diameter of about 10 nm to about 1000 nm.
  • the multimer may comprise at least 4 subunits.
  • the immune stimulator may be selected from ssRNA derived from bacteria, artificially synthesized ssRNA, or a derivative thereof 3. Synthetic CpG-containing oligonucleotides, interferons, cytokines, and combinations thereof.
  • the ssRNA derived from bacteria is an ssRNA derived from E. coli.
  • the interferon is selected from interferon type I, interferon type II, interferon type III, and combinations thereof.
  • the cytokine is selected from the group consisting of IL-6, IL-12, IL21, and combinations thereof.
  • the multimer is a virus-like particle.
  • the virus-like particle comprises or consists of Q? Protein, HBcAg or AP205.
  • the virus-like particle comprises or consists of a target antigen.
  • the target antigen is selected from Q ⁇ protein, HBcAg and AP205.
  • the antigen complex comprises a loaded target antigen, and the loaded target antigen is a bacterium or a virus Source of antigen.
  • the target antigen is an antigen of M. tuberculosis origin, for example, selected from the group consisting of lens protein and Rv3133c.
  • the target antigen is an antigen of superbacterial origin, such as selected from the group consisting of Klebsiella pneumoniae carbapenemase and penicillin-binding protein.
  • the target antigen is a lentivirus-derived antigen, for example, selected from HBV pre-S1 antigen and EBV LMP1 antigen.
  • the antigen complex comprises a loaded target antigen, and the loaded target antigen is tumor-associated antigen.
  • the tumor-associated antigen is selected from Her2, p53 and neo-antigen.
  • the method is an in vitro method. In other embodiments, step c) of the method occurs in vivo.
  • the disclosure relates to a method of preventing and / or treating a disease in a subject in need thereof, the method comprising:
  • the antigen complex comprises:
  • the plurality of subunits comprise or consist of a target antigen, and wherein the immunostimulant is packaged in the polymer, or is attached to the polymer through physical adsorption or chemical connection;
  • antigen complex comprises:
  • the target antigen is attached to the surface of the multimer through physical adsorption or chemical connection, or is fused to at least a part of the plurality of subunits through gene fusion, and the fusion does not affect the assembly of the multimer
  • the target antigen is displayed on the surface of the multimer after the multimer is assembled; and wherein the immunostimulant is packaged in the multimer, or is attached to the multimer by physical adsorption or chemical connection Mentioned multimer
  • the B cell population is capable of identifying at least one of the subunits
  • the B cell population is a B cell population isolated from a subject's peripheral blood or lymphoid organs.
  • the method further comprises the step of, after step c), screening, enriching, and / or expanding B cells that recognize the subunit.
  • the method further comprises, after step c), screening B cells that recognize the subunit and encoding a gene encoding an immunoglobulin receptor The step of introducing a sequence into the B cell population to increase the number of B cells in the population that recognize the subunit.
  • the multimer has a diameter of 10 nm to 1000 nm.
  • the multimer comprises at least 4 subunits.
  • the immunostimulant comprises a ssRNA derived from a bacterium, a synthetic ssRNA or a derivative thereof, a synthetic CpG-containing oligonucleotide, an interferon, Cytokines, or a combination thereof.
  • the ssRNA derived from bacteria is an ssRNA derived from E. coli.
  • the interferon is selected from interferon type I, interferon type II, interferon type III, and combinations thereof.
  • the cytokine is selected from the group consisting of IL-6, IL-12, IL21, and combinations thereof.
  • the multimer is a virus-like particle.
  • the virus-like particle comprises or consists of a Q ⁇ protein, HBcAg or AP205.
  • the virus-like particle comprises or consists of a target antigen.
  • the target antigen is selected from Q ⁇ protein, HBcAg and AP205.
  • the disease is an infectious disease
  • the antigen complex comprises a loaded target antigen, which is a bacterial or virus-derived antigen.
  • the target antigen is an antigen of M. tuberculosis origin, for example, selected from the group consisting of lens protein and Rv3133c.
  • the target antigen is an antigen of superbacterial origin, such as selected from the group consisting of Klebsiella pneumoniae carbapenemase and penicillin-binding protein.
  • the target antigen is a lentivirus-derived antigen, for example, selected from HBV pre-S1 antigen and EBV LMP1 antigen.
  • the disease is cancer
  • the antigen complex comprises a loaded target antigen
  • the loaded target antigen is a tumor-associated antigen.
  • the tumor-associated antigen is selected from Her2, p53, and tumor nascent antigen.
  • Figure 1 shows a schematic diagram of an example of a multimer-based antigen complex of the invention, showing several different forms of antigen complex. Left: a multimer consisting of the target antigen directly; middle: the target antigen is loaded on the natural multimer backbone; right: the target antigen is loaded on the artificial multimer backbone.
  • the red dots in the figure are immunostimulants in the antigen complex.
  • Figures 2A-2C show strong activation of CD4 + T cells after multimer Q ⁇ -VLP immunization.
  • CFSE-labeled naive OT-II CD4 + T cells were transferred into the mice.
  • days 3 (d3), 5 (d5), and 7 (d7) after immunization spleens were harvested from immunized mice, and spleens (d0) were harvested from non-immunized mice as controls.
  • a representative flow cytometry graph and overall statistics are shown.
  • Figure 2A shows gating of total CD4 + T cells to Thy1.1 + OT-II cells.
  • FIG. 2B shows a histogram of the fluorescence density of CFSE in Thy1.1 + and quantified as a proliferation index.
  • the grey portion in Figure 2B shows data from unimmunized mice.
  • a graph of the up-regulation of the T cell activation marker CD44 and the down-regulation of CD62L (CD44hi and CD62lo) is shown in Figure 2C.
  • the mean ⁇ SD is shown.
  • Figures 3A and 3B show the promotion of CD4 + T cells differentiation into Tfh cells and Th1 cells after multimer Q ⁇ -VLP immunization.
  • CFSE-labeled naive OT-II CD4 + T cells were transferred into the mice.
  • Spleens were harvested from immunized mice on days 3 (d3), 5 (d5), and 7 (d7) after immunization, and spleens (d0) were harvested from unimmunized mice as controls.
  • a representative flow cytometry graph and overall statistics are shown.
  • Thy1.1 + cells were gated for the indicated differentiation markers based on the expression levels of differentiation markers in CFSE undiluted cells from unimmunized mice.
  • FIG. 3A shows that the proportion of CD4 + T cells positive for Tfh cell-specific markers PD1, CXCR5, and Bcl-6 increased significantly, indicating that a large number of CD4 + T cells differentiated to Tfh.
  • Figure 3B shows that the proportion of Th1 cell-specific markers T-bet and CXCR3 positive CD4 + T cells increased significantly, indicating that a large number of CD4 + T cells differentiated into Th1 cells.
  • Figures 4A and 4B show that MyD88 in B cells is necessary for the activation and differentiation of CD4 + T cells after multimer Q ⁇ -VLP immunization.
  • CFSE-labeled naive OT-II CD4 + T cells were transferred into the mice. Spleens were harvested from mice on day 3 (d3) after immunization. A representative flow cytometry graph and overall statistics are shown.
  • Figure 4A Thy1.1 + OT-II cells are gated on total CD4 + T cells.
  • Figure 4B Gating differentiation markers in Thy1.1 + cells from Figure 4A based on expression levels of differentiation markers in naive CD4 + T cells derived from recipient mice (not shown). The mean ⁇ SD is shown. Data analysis was performed using an unpaired Student's test. ns: not significant; ** p ⁇ 0.01; *** p ⁇ 0.001.
  • Figures 5A and 5B show that MyD88 in B cells is not necessary for the activation and differentiation of CD4 + T cells after immunization with the soluble antigen Ova + CpG.
  • CFSE-labeled naive OT-II CD4 + T cells were transferred into the mice. Spleens were harvested from mice on day 3 (d3) after immunization. A representative flow cytometry graph and overall statistics are shown.
  • Figure 5B Gating differentiation markers in Thy1.1 + cells from Figure 5A based on the expression levels of differentiation markers in naive CD4 + T cells derived from recipient mice. The mean ⁇ SD is shown. Data analysis was performed using an unpaired Student's test. ns: Not significant.
  • Figures 6A and 6B show that MyD88 in DC is not necessary for the activation and differentiation of CD4 + T cells after multimer Q ⁇ -VLP immunization.
  • CFSE-labeled naive OT-II CD4 + T cells were transferred into the mice. Spleens were harvested from mice on day 3 (d3) after immunization.
  • Figure 6A Thy1.1 + OT-II cells are gated on total CD4 + T cells.
  • Figure 6B Gating differentiation markers in Thy1.1 + OT-II CD4 + T cells. The mean ⁇ SD is shown. Data analysis was performed using an unpaired Student's test. ns: Not significant.
  • Figures 7A-7C show that mice lacking Q ⁇ -specific B cells were unable to induce a CD4 + T cell response after multimer Q ⁇ -VLP immunization.
  • CFSE-labeled naive OT-II CD4 + T cells were transferred to the mice.
  • a representative flow cytometry graph and overall statistics are shown.
  • Figure 7A Thy1.1 + OT-II cells are gated on total CD4 + T cells.
  • Figures 7B and 7C Gating differentiation markers in Thy1.1 + cells from Figure 7A. The mean ⁇ SD is shown. Data analysis was performed using an unpaired Student's test. ns: not significant; ** p ⁇ 0.01; *** p ⁇ 0.001.
  • Figure 8 shows that DC is not necessary for multimeric Q ⁇ -VLP-induced CD4 + T cell activation.
  • Mice were lethally irradiated to remove immune cells and reconstituted using BM cells from CD11c-DTR / GFP mice. Mice were treated with PBS or DT during OT-II CD4 + T cell transfer. After immunizing mice with Q ⁇ -Qva or Ova mixed with CpG ODN, CFSE-labeled naive OT-II CD4 + T cells were transferred into the mice. Spleens were harvested 24 hours after immunization. Representative flow cytometry plots and total statistics of individual mice are shown.
  • Thy1.1 + CD4 + OT-II T cells were gated for the indicated differentiation markers based on the expression level of differentiation markers in CFSE undiluted cells from unimmunized mice. The mean ⁇ SD is shown. Data analysis was performed using an unpaired Student's test. ns: not significant; *** p ⁇ 0.001.
  • FIGS 9A-9C show that Q ⁇ -VLP is effectively captured by antigen-specific B cells in vivo.
  • Figures 9A and 9B Wild-type or MD4 mice were injected intravenously with Q ⁇ -AF647 or PBS and examined 3 hours later.
  • Figure 9A CD11c + MHCII + DC was first gated from total spleen cells, and then Q ⁇ -AF647 + was further gated.
  • Figure 9B Q ⁇ -AF647 + MHCII + cells are first gated from total spleen cells, and then they are further gated with B220 + B cells and CD11c + DC. The binding of DC and B cells to Q ⁇ -AF647 is shown.
  • Figure 9C Wild-type mice were injected intravenously with Q ⁇ -AF64 and examined at 0.5 and 3 hours after injection. Uninjected mice were also checked as the most control. Total spleen cells were enriched using Q ⁇ -FITC and anti-FITC magnetic beads. Q ⁇ -FITC + B220 + B cells were gated from the enriched fractions, which further showed Q ⁇ -AF647 and CD83. The data represent at least three independent experiments.
  • FIG. 10A Intraperitoneal injection of wild-type mice using unlabeled Q ⁇ -VLP. After 24 hours, the spleen was harvested and the spleen was harvested from unimmunized mice as a control. Total spleen cells were incubated with Q ⁇ -AF647 and Q ⁇ -GFP, and then enriched using anti-AF647 magnetic beads.
  • FIG. 10A gates Q ⁇ -AF647 + B cells from an enriched cell fraction, which is further gated based on Q ⁇ -GFP as AF647 + and Q ⁇ + B cells.
  • CD86 and CCR7 are shown.
  • Figure 10 Mean fluorescence intensity (MFI) of CD86 and CCR7 in BAF647 + and Q ⁇ + B cells. Bars represent the mean. The dots represent data from individual mice. Data analysis was performed using an unpaired Student's test. ns: not significant; ** p ⁇ 0.01.
  • FIG. 11 shows that B cell antigen presentation is necessary for Q ⁇ -VLP-induced CD4 + T cell activation.
  • B-MHCII-/-and control mice were generated by transplanting mixed BM cells from ⁇ MT and MHCII-/-(B-MHCII-/-) or WT (control) mice into lethal irradiated mice. After immunizing the mice with Q ⁇ -Qva, CFSE-labeled naive OT-II CD4 + T cells were transferred into the mice. Spleens were harvested from mice on day 3 (d3) after immunization. A representative flow cytometry graph and overall statistics are shown. Thy1.1 + OT-II cells were gated on total CD4 + T cells.
  • Figures 12A to 12C show that antigen-specific B cells participate in the influenza virus-induced CD4 + T cell response.
  • CFSE-labeled naive OT-II CD4 + T cells were transferred to WT and MD4 mice ( Figures 12A and 12B), or to CD11c-DTR / GFP chimeric mice treated with PBS (control) or DT Medium (C) and then immunized with PR8-Ova. Livers were harvested on day 3 ( Figures 12A and 12B) or 24 hours (Figure 12C) after immunization. Representative flow cytometry plots and overall statistics of individual mice are shown.
  • Figure 12A Thy1.1 + OT-II cells are gated on total CD4 + T cells.
  • Figures 12B and 12C Markers shown for Thy1.1 + CD4 + T cell gating. The mean ⁇ SD is shown. Data analysis was performed using an unpaired Student's test. ns: not significant; * p ⁇ 0.05; ** p ⁇ 0.01; *** p ⁇ 0.001.
  • the disclosure relates to multimer-based antigen complexes.
  • the multimer-based antigen complex comprises:
  • the plurality of subunits comprise or consist of a target antigen, and wherein the immunostimulant is packaged in the multimer, or is attached to the multimer by physical adsorption or chemical linkage.
  • the multimer-based antigen complex comprises:
  • the target antigen is attached to the surface of the multimer through physical adsorption or chemical connection, or is fused to at least a part of the plurality of subunits through gene fusion, and the fusion does not affect the assembly of the multimer
  • the target antigen is displayed on the surface of the multimer after the multimer is assembled, and wherein the immunostimulant is packaged in the multimer, or is attached to the multimer by physical adsorption or chemical connection Mentioned multimers.
  • Figure 1 shows a schematic diagram of an example of a multimer-based antigen complex of the present disclosure, showing several different forms of antigen complexes.
  • multimer-based antigen complex When referring to “multimer-based antigen complexes”, the above term also covers the case of a mixture of multiple multimer-based antigen complexes.
  • the term “multimer-based antigen complex” may mean a mixture of two or more multimer-based antigen complexes.
  • the two or more multimer-based antigen complexes may each have components i) and ii), or components i), ii), and iii) described above.
  • multimer-based antigen complex means two or more multimer-based antigen complexes, some of them may have the above-mentioned components i) and ii ), And other antigen complexes may have the components i), ii) and iii) described above.
  • the multimer-based antigen complex of the present disclosure can be used to activate B cells, or to activate CD4 + T cells through recognition and presentation of B cells, and to promote the differentiation of CD4 + T cells into Tfh and Th1.
  • DCs are the main types of antigen-presenting cells.
  • APC antigen-presenting cells
  • DC Dendritic cells
  • B cells B cells
  • macrophages are the main types of antigen-presenting cells.
  • the initial state of CD4T cells is also called naive CD4 + T cells. Whether they can be effectively activated determines the strength of the subsequent immune response, so this step is an important target for various immune response enhancement measures. Due to the critical role of DCs in this step, DCs are currently targeted as a key target for the development of various vaccines.
  • the present disclosure has surprisingly found that although most antigens activate CD4 + T cells through DCs, they have a special form of antigen, that is, a multimer-based antigen complex, which can pass B cells. Initial activation of naive CD4 + T cells. That is, it is capable of inducing activation and differentiation of CD4 + T cells through antigen presentation in the absence of DC.
  • B cells This ability of B cells is closely related to their expressed immunoglobulin receptor receptors (B cell receptors, BCR) and natural immune signaling pathways.
  • BCR is actually a transmembrane form of immunoglobulin, and its production is the result of the gene rearrangement process that occurs at the DNA level at specific BCR gene-related sites during B cell development, that is, the result of V (D) J rearrangement.
  • V (D) J rearrangement This lymphocyte-specific V (D) J rearrangement process allows different B cells to express different BCRs, and the types of BCRs they can produce can reach as many as 10 12 -10 15 . Due to the richness of BCR, B cells use the B cell population to combat the original higher affinity BCR expression in the recognition of antigens.
  • the diameter of the multimer can be about 10 nm to about 1000 nm, such as about 10 nm to about 500 nm, about 10 nm to about 300 nm, about 10 nm to about 200 nm, about 10 nm to about 100 nm, about 10 nm to about 50nm, about 20nm to about 1000nm, about 20nm to about 500nm, about 20nm to about 300nm, about 20nm to about 200nm, about 20nm to about 100nm, about 20nm to about 50nm, about 50nm to about 1000nm, about 50nm to about 500nm About 50 nm to about 300 nm, about 50 nm to about 200 nm, or about 50 nm to about 100 nm.
  • the multimer can include at least 4 subunits, such as at least 10 subunits, at least 20 subunits, at least 50 subunits, at least 100 subunits, or at least 200 subunits unit.
  • the multimer may have 10 to 1,000 subunits, such as 20 to 500 subunits, 50 to 300 subunits, or 100 to 200 subunits.
  • the immune stimulator may include ssRNA of bacterial origin, artificial ssRNA or a derivative thereof, artificial CpG-containing oligonucleotide, and interferon , Cytokines, or any combination thereof.
  • the ssRNA derived from bacteria may be an ssRNA derived from E. coli.
  • the interferon may be selected from interferon type I, interferon type II, interferon type III, and combinations thereof.
  • the cytokine may be selected from the group consisting of IL-6, IL-12, IL21, and combinations thereof.
  • lymphocytes such as B cells and T cells
  • two important elements are usually required: one is the antigen receptor, which is the stimulation and signaling of BCR and TCR; the other is the stimulation of immune signals or cytokines.
  • antigen receptor which is the stimulation and signaling of BCR and TCR
  • immune stimulants refer to substances capable of exercising secondary stimuli, mainly including ligand stimuli of natural immune receptors and proinflammatory cytokines.
  • the multimer-based antigen complexes of the present disclosure may comprise one immunostimulant, or two or more different immunostimulants.
  • the combination of the immunostimulant and the multimer is not particularly limited.
  • the immunostimulant and the multimer can be packaged in the multimer or attached to the surface of the multimer through physical adsorption or chemical connection to exert its immunostimulatory function.
  • the immunostimulant can be introduced during assembly of the multimer such that the subunits of the multimer package the immunostimulant inside the multimer during assembly.
  • the multimer may be a virus-like particle, other natural multimer, or artificial multimer.
  • Virus-like particles are biological agents that are similar in structure to viruses but do not contain viral genetic material. Virus-like particles usually consist of multiple copies of one or several proteins, and their diameter can vary from tens of nanometers to thousands of nanometers. The surface of virus-like particles presents repeatedly arranged epitopes, which greatly enhances their ability to activate B cells.
  • the virus-like particles can contain natural or artificial nucleic acid materials, and other types of compounds can also be artificially added as immunostimulants.
  • the immune stimulant is important for immune responses induced by virus-like particles, especially B-cell responses.
  • some naturally occurring polysaccharides also belong to natural multimers, and can further form particle-like structures based on them.
  • the multimer can also be used in the antigen complexes of the present disclosure, to obtain B cell activation, and to activate CD4 + T cells through recognition and presentation of B cells, and to promote the differentiation of CD4 + T cells into Tfh and Th1.
  • some artificially involved and engineered proteins can also form multimers.
  • Non-proteinaceous materials can also form multivalent particle formulations, such as synthetic nanoparticles. After the surface of these artificially synthesized multimers is modified with specific chemical groups, the target antigen can be loaded on the multimers through physical adsorption or chemical linkage.
  • a multimer can be assembled from multiple copies of one subunit, or can be assembled from multiple copies of two or more subunits. There are no specific restrictions on the selection of multimers, and those skilled in the art can select various multimeric structures known in the art for constructing the antigen complexes of the present disclosure.
  • the virus-like particle may comprise or consist of a Q ⁇ protein, HBcAg or AP205.
  • Phage Q ⁇ is an icosahedral RNA virus with a diameter of 30 nm. Its host is E. coli. Q ⁇ enters its host cells by binding to F pilus on the surface of bacteria.
  • the first 133 amino acids of the phage Q ⁇ capsid protein can be expressed in other cells such as E. coli or yeast by plasmid transformation, and self-assembled into particles with a diameter of 30 nm.
  • the self-assembly process of phage Q ⁇ capsid protein does not require its own genetic material or other protein assistance.
  • the assembled particles do not have infectivity to any cells (including prokaryotic and eukaryotic cells).
  • HBcAg core antigen
  • core antigen is a hepatitis B virus protein that is an antigen present on the surface of the nucleocapsid core (the innermost layer of hepatitis B virus).
  • packaging of HBcAg and viral nucleic acids is critical.
  • HBcAg can be expressed in other cells such as E. coli or yeast by plasmid transformation, and self-assembles into particles with a diameter of about 30 nm.
  • the self-assembly process of HBcAg does not require its own genetic material or the assistance of other proteins.
  • the assembled particles do not have infectivity to any cells (including prokaryotic and eukaryotic cells).
  • the phage AP205 is an icosahedral RNA virus with a diameter of 30 nm. Its host is Acinetobacter.
  • AP205 capsid protein can be expressed in other cells such as E. coli or yeast by plasmid transformation, and self-assembled into particles with a diameter of 30nm. The self-assembly process of AP205 capsid protein does not require its own genetic material or the assistance of other proteins. The assembled particles do not have infectivity to any cells (including prokaryotic and eukaryotic cells).
  • the Q ⁇ protein may have the amino acid sequence shown below: MAKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQAYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLIDNOY:
  • the HBcAg may have the amino acid sequence shown below: MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGNNLEDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYQRSRPRQPRRSPRQRTRPRTRQRDRQRTQPRTRPRQTRDRQTRDRQR
  • AP205 can have the amino acid sequence shown below: MANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKRPAPKPEGCADACVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAIVSSDTTA (SEQ ID ID: NO)
  • the multimer such as a virus-like particle, comprises or consists of a target antigen. That is, at least one subunit of the multimer itself serves as a target antigen.
  • the subunit as the target antigen may be a Q ⁇ protein, HBcAg or AP205, or another virus-derived protein.
  • Multimers, such as virus-like particles may contain multiple copies of only one subunit, or two or more subunits. In the case where a multimer such as a virus-like particle contains multiple copies of a subunit, the subunit itself may serve as the target antigen. In the case where the multimer contains two or more subunits, at least one of the subunits may serve as a target antigen.
  • the antigen complex comprises a loaded target antigen.
  • the target antigen to be loaded is not particularly limited, and it can be any protein, polypeptide, nucleic acid, or small molecule that is immunogenic and can be specifically recognized by components of the immune system.
  • Various target antigens for immunization are known in the art, and those skilled in the art can select specific target antigens as needed to construct the antigen complex of the present disclosure.
  • the manner of loading the target antigen is not particularly limited, and methods such as physical adsorption, chemical ligation, and gene fusion can be adopted.
  • the timing of loading is not particularly limited.
  • a subunit of a multimer can be contacted with a target antigen (eg, by physical adsorption or chemical interaction), and then assembled into a multimer such that the target antigen is loaded on the surface of the multimer.
  • the binding of the multimer subunit to the target antigen does not affect its assembly into a multimer.
  • the target antigen can be introduced after being assembled into a subunit and assembled into a multimer, such that the target antigen is attached to the surface of the multimer through physical adsorption or chemical linkage.
  • the target antigen When the target antigen is added by gene fusion, that is, the nucleotide sequence encoding the target antigen is fused with the nucleotide sequence encoding the subunit of the multimer by genetic recombination technology and expressed as a fusion protein, the target The antigen may be fused to only a portion of the subunits that make up the multimer. That is, in the case where the multimer contains only multiple copies of one subunit, the target antigen can be fused to all or only a part of the multiple copies.
  • the target antigen may be fused with at least one of the two or more subunits, and further, the target antigen may be fused with the at least one subunit All of them are merged in one step.
  • Methods of constructing fusion proteins by genetic recombination techniques are well known in the art. And those skilled in the art can select a suitable fusion method according to various conditions such as the type, size, immunogenicity of the target antigen used, and the number of copies of the multimer subunit, so that after fusion, the target antigen does not affect Polymer assembly.
  • the loaded target antigen may be an antigen of bacterial or viral origin.
  • the specific kind of bacterial or virus-derived antigen is not particularly limited, and exemplary antigens such as those listed in Table 3 can be used.
  • Tuberculosis DosR Zh Ag85 Zh ESAT6 Zh Crystalline Zh CFP10 Zh Rv2031c Epstein--Barr virus EBNA-1 Zh EBNA-2 Zh EBNA-3A Zh EBNA-3B Zh EBNA-LP Zh LMP-1 Zh LMP-2A Zh LMP-2B Zh EBER Zh Gp350 Hepatitis B virus HBsAg Zh Pre-S1 Plasmodium CSP Zh MSP1 Zh MSP3
  • the bacterial or virus-derived antigen may be an antigen derived from M. tuberculosis, such as selected from the group consisting of lens protein and Rv3133c.
  • the bacterial or virus-derived antigen may be an antigen of superbacterial origin, such as selected from the group consisting of Klebsiella pneumoniae carbapenemase and penicillin-binding protein.
  • the bacterial or virus-derived antigen may be a lentivirus-derived antigen, for example, selected from HBV pre-S1 antigen and EBV LMP1 antigen.
  • the antigen complex comprises a loaded target antigen
  • the loaded target antigen may be a tumor-associated antigen
  • the tumor-associated antigen used is not particularly limited, and may be any antigen that is related to tumor development or aggressiveness.
  • tumor-associated antigen refers to an antigen that is differentially expressed by cancer cells and therefore can be utilized to target cancer cells.
  • Tumor-associated antigens are antigens that can potentially stimulate significant tumor-specific immune responses. Some of these antigens are encoded by normal cells, but not necessarily expressed by normal cells. These antigens can be characterized as those antigens that are usually silent (i.e. not expressed) in normal cells, those antigens that are expressed only at certain stages of differentiation, and those antigens that are expressed in time, such as embryonic and fetal antigens.
  • tumor-associated antigens are encoded by mutant cell genes such as oncogenes (eg, activated ras oncogenes), suppressor genes (eg, mutant p53), and fusion proteins resulting from internal deletions or chromosomal translocations.
  • tumor-associated antigens can be encoded by viral genes, such as genes carried on RNA and DNA tumor viruses.
  • tumors or tumor-associated antigens in the multimer-based antigen complexes herein include, but are not limited to, Her2, prostate stem cell antigen (PSCA), PSMA (Prostate Specific Membrane Antigen), ⁇ -catenin -m, B-cell maturation antigen (BCMA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1 , Epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, Mammaglobin-A, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117, chromogranin, cytokeratin, Desmin, gli
  • the tumor antigen may be selected from Her2, p53, or tumor nascent antigen.
  • the present disclosure relates to a method of activating CD4 + T cells, the method comprising the step of contacting B cells with CD4 + T cells, wherein the B cells have used a multimer-based antigen complex or equivalent The method was activated, the multimer-based antigen complex comprising:
  • the plurality of subunits comprise or consist of a target antigen, and wherein the immunostimulant is packaged in the polymer, or is attached to the polymer through physical adsorption or chemical connection;
  • antigen complex comprises:
  • the target antigen is attached to the surface of the multimer through physical adsorption or chemical connection, or is fused to at least a part of the plurality of subunits through gene fusion, and the fusion does not affect the assembly of the multimer
  • the target antigen is displayed on the surface of the multimer after the multimer is assembled, and wherein the immunostimulant is packaged in the multimer, or is attached to the multimer by physical adsorption or chemical connection Mentioned multimers.
  • the disclosure relates to a method of promoting the differentiation of CD4 + T cells into Tfh and / or Th1 cells, the method comprising the step of contacting B cells with CD4 + T cells, wherein the B cells have been The multimeric antigen complex or equivalent method is activated, said multimer-based antigen complex comprising:
  • the plurality of subunits comprise or consist of a target antigen, and wherein the immunostimulant is packaged in the polymer, or is attached to the polymer through physical adsorption or chemical connection;
  • antigen complex comprises:
  • the target antigen is attached to the surface of the multimer through physical adsorption or chemical connection, or is fused to at least a part of the plurality of subunits through gene fusion, and the fusion does not affect the assembly of the multimer
  • the target antigen is displayed on the surface of the multimer after the multimer is assembled, and wherein the immunostimulant is packaged in the multimer, or is attached to the multimer by physical adsorption or chemical connection Mentioned multimers.
  • the present disclosure relates to a method of activating CD4 + T cells, the method comprising the following steps:
  • the antigen complex comprises:
  • the plurality of subunits comprise or consist of a target antigen, and wherein the immunostimulant is packaged in the polymer, or is attached to the polymer through physical adsorption or chemical connection;
  • antigen complex comprises:
  • the target antigen is attached to the surface of the multimer through physical adsorption or chemical connection, or is fused to at least a part of the plurality of subunits through gene fusion, and the fusion does not affect the assembly of the multimer
  • the target antigen is displayed on the surface of the multimer after the multimer is assembled, and wherein the immunostimulant is packaged in the multimer, or is attached to the multimer by physical adsorption or chemical connection Mentioned multimers,
  • the B cell population is capable of identifying at least one of the subunits
  • the present disclosure relates to a method of promoting the differentiation of CD4 + T cells into Tfh and / or Th1 cells, the method comprising the following steps:
  • the antigen complex comprises:
  • the plurality of subunits comprise or consist of a target antigen, and wherein the immunostimulant is packaged in the polymer, or is attached to the polymer through physical adsorption or chemical connection;
  • antigen complex comprises:
  • the target antigen is attached to the surface of the multimer through physical adsorption or chemical connection, or is fused to at least a part of the plurality of subunits through gene fusion, and the fusion does not affect the assembly of the multimer
  • the target antigen is displayed on the surface of the multimer after the multimer is assembled, and wherein the immunostimulant is packaged in the multimer, or is attached to the multimer by physical adsorption or chemical connection Mentioned multimers,
  • the B cell population is capable of identifying at least one of the subunits
  • T cells or T lymphocytes are the type of lymphocytes that play a central role in cell-mediated immunity. By the presence of cell surface T cell receptors (TCR), they can be distinguished from other lymphocytes such as B cells and natural killer cells (NK cells).
  • TCR cell surface T cell receptors
  • CD4 + T cells are also called helper T helper cells (Th cells), which assist other white blood cells in the immunological process, including the maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages.
  • Th cells express CD4 on their surface. When they present peptide antigens via MHC class II molecules on the surface of antigen presenting cells (APC), Th cells are activated. These cells can differentiate into one of several subtypes, including Th1, Th2, Th3, Th17, Th9, or Tfh, which secrete different cytokines to promote different types of immune responses.
  • Tfh (Follicular Helper T Cells, Tfh) is an important subgroup of CD4 + T cells, which is mainly involved in the germinal center response process. Tfh is essential for the formation and maintenance of germinal centers. By inducing and maintaining the role of germinal center B cells, Tfh can promote a variety of effects such as antibody production, antibody type conversion, antibody affinity maturation, neutralizing antibody production, and increasing the breadth of the antibody map. Therefore, how to effectively generate Tfh cells is a key link for the development of various vaccines.
  • Th1 Type 1 helper T cells (Th1) is another important CD4 + T cell subgroup, which is antiviral and antibacterial, especially against intracellular bacterial infections, such as tuberculosis Play a key role in bacterial infection.
  • CD4 + T cells also play an important role in anti-tumor immunity.
  • the B cell population is a B cell population present in a subject.
  • the B-cell population may be a naturally-occurring B-cell population or a B-cell population that is transferred to the subject.
  • the method of activating CD4 + T cells and the method of promoting CD4 + T cell differentiation of the present disclosure may occur in a subject. That is, by directly administering the multimer-based antigen complex of the present disclosure to a subject, it can be recognized by the B cell population in the subject, and in turn activate CD4 + T cells and promote CD4 + T cells to Tfh and And / or Th1 differentiation.
  • the B cell population may be a B cell population isolated from a donor's peripheral blood or lymphoid organs such as the thymus, spleen, and tonsils.
  • the method may further include a step of screening, enriching and / or expanding B cells that recognize the subunit after step b).
  • the method may further include, after step b), screening B cells that recognize the subunit, and introducing the gene sequence encoding the immunoglobulin receptor into the B cell population to A step of increasing the number of B cells in the population that identifies the subunit.
  • B cells immunoglobulin receptors (B cell receptors, BCR) expressed on their surface.
  • BCR immunoglobulin receptors
  • Different B cells can express different BCRs, and the types of BCRs that can be produced in an individual can reach as many as 10 12 -10 15 .
  • the steps of screening, enriching, and / or expanding B cells that recognize the subunit, or introducing a gene sequence encoding a BCR that recognizes the subunit into the B cell population can increase recognition in the B cell population
  • the number of subunit B cells thereby increasing the efficiency of activating CD4 + T cells and / or promoting CD4 + T cell differentiation.
  • the diameter of the multimer can be about 10 nm to about 1000 nm, such as about 10 nm to about 500 nm, about 10 nm to about 300 nm, about 10 nm to about 200 nm, about 10 nm to about 100 nm, About 10nm to about 50nm, about 20nm to about 1000nm, about 20nm to about 500nm, about 20nm to about 300nm, about 20nm to about 200nm, about 20nm to about 100nm, about 20nm to about 50nm, about 50nm to about 1000nm, about 50nm To about 500 nm, about 50 nm to about 300 nm, about 50 nm to about 200 nm, or about 50 nm to about 100 nm.
  • the multimer may comprise at least 4 subunits, for example comprising at least 10 subunits, at least 20 subunits, at least 50 subunits, at least 100 subunits, or at least 200 subunits.
  • the multimer may have 10 to 1,000 subunits, such as 20 to 500 subunits, 50 to 300 subunits, or 100 to 200 subunits.
  • the immune stimulator may comprise ssRNA of bacterial origin, artificially synthesized ssRNA or a derivative thereof, artificially synthesized CpG-containing oligonucleotide, interferon, cytokine, or any combination.
  • the ssRNA derived from bacteria may be an ssRNA derived from E. coli.
  • the interferon may be selected from interferon type I, interferon type II, interferon type III, and combinations thereof.
  • the cytokine may be selected from the group consisting of IL-6, IL-12, IL21, and combinations thereof.
  • one or more selected from the immune stimuli listed in Tables 1 and 2 above, or other natural or artificial immune stimuli known in the art can be used.
  • the multimer can be a virus-like particle, other natural multimer, or an artificial multimer.
  • a multimer can be assembled from multiple copies of one subunit, or can be assembled from multiple copies of two or more subunits. There are no specific restrictions on the choice of multimers.
  • the virus-like particle may comprise or consist of a Q ⁇ protein, HBcAg or AP205.
  • the Q ⁇ protein may have the amino acid sequence shown below: MAKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQAYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLIDNOY:
  • the HBcAg may have the amino acid sequence shown below: MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGNNLEDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYQRSRPRQPRRSPRQRTRPRTRQRDRQRTQPRTRPRQTRDRQTRDRQR
  • AP205 may have the amino acid sequence shown below: MANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKRPAPKPEGCADACVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAIVSSDTTA (SEQ ID: NO):
  • the multimer such as a virus-like particle, comprises or consists of a target antigen. That is, at least one of the subunits of a multimer can itself serve as a target antigen.
  • the target antigen may be a Q ⁇ protein, HBcAg or AP205, or another virus-derived protein.
  • the antigen complex comprises a loaded target antigen
  • the loaded target antigen may be an antigen of bacterial or viral origin.
  • a bacterial or virus-derived antigen selected from those listed in Table 3 above can be used.
  • the bacterial or virus-derived antigen may be an antigen derived from M. tuberculosis, such as selected from the group consisting of lens protein and Rv3133c.
  • the antigen of bacterial or viral origin may be an antigen of super bacterial origin, such as selected from the group consisting of Klebsiella pneumoniae carbapenemase and penicillin-binding protein.
  • the antigen of bacterial or viral origin may be an antigen of lentiviral origin, for example, selected from HBV pre-S1 antigen and EBV LMP1 antigen.
  • the antigen complex comprises a loaded target antigen
  • the loaded target antigen may be a tumor-associated antigen.
  • the tumor-associated antigen used is not particularly limited, and may be any antigen that is related to tumor development or aggressiveness.
  • a tumor-associated antigen as described above in the multimer-based antigen complex of the present disclosure can be used.
  • the tumor antigen may be selected from Her2, p53, or tumor nascent antigen.
  • the method may be an in vitro method.
  • an isolated B cell population can be contacted with a multimer-based antigen complex in vitro, so that the B cell recognizes and processes the antigen complex, and presents the target antigen to CD4 + T cells, thereby activating CD4 + T Cells and promote the differentiation of CD4 + T cells into Tfh and / or Th1 cells.
  • some steps of the method can occur in vitro, while the rest occur in vivo.
  • an isolated B cell population can be contacted with a multimer-based antigen complex in vitro, so that the B cell recognizes and processes the antigen complex, and presents a complex of the target antigen and MHC II on the cell surface.
  • the B cell population can be administered to a subject such that the B cell population activates CD4 + T cells and promotes the differentiation of CD4 + T cells into Tfh and / or Th1 cells in the subject.
  • the method occurs in a subject.
  • a subject by directly administering the multimer-based antigen complex of the present disclosure to a subject, it can be recognized by the B cell population in the subject, and thereby activate CD4 + T cells and promote CD4 + T cells to Tfh and And / or Th1 differentiation.
  • the B-cell population may be a naturally-occurring B-cell population or a B-cell population that is transferred to the subject.
  • the present disclosure provides a method of preventing and / or treating a disease in a subject in need thereof, the method comprising the step of administering to the subject an effective amount of a multimer-based antigen complex
  • the multimeric antigen complex comprises:
  • the plurality of subunits comprise or consist of a target antigen, and wherein the immunostimulant is packaged in the polymer, or is attached to the polymer through physical adsorption or chemical connection;
  • antigen complex comprises:
  • the target antigen is attached to the surface of the multimer through physical adsorption or chemical connection, or is fused to at least a part of the plurality of subunits through gene fusion, and the fusion does not affect the assembly of the multimer
  • the target antigen is displayed on the surface of the multimer after the multimer is assembled, and wherein the immunostimulant is packaged in the multimer, or is attached to the multimer by physical adsorption or chemical connection Mentioned multimers.
  • the present disclosure provides a method of preventing and / or treating a disease in a subject in need thereof, the method comprising:
  • the plurality of subunits comprise or consist of a target antigen, and wherein the immunostimulant is packaged in the polymer, or is attached to the polymer through physical adsorption or chemical connection;
  • antigen complex comprises:
  • the target antigen is attached to the surface of the multimer through physical adsorption or chemical connection, or is fused to at least a part of the plurality of subunits through gene fusion, and the fusion does not affect the assembly of the multimer
  • the target antigen is displayed on the surface of the multimer after the multimer is assembled, and wherein the immunostimulant is packaged in the multimer, or is attached to the multimer by physical adsorption or chemical connection Mentioned multimer
  • the B cell population is capable of identifying at least one of the subunits
  • the multimer-based antigen complex of the present disclosure it is possible to activate CD4 + T cells and promote differentiation of CD4 + T cells into Tfh and / or Th1 cells by being recognized and presented by B cells. Due to the function of CD4 + T cells, especially Tfh and Th1 in immune responses, especially adaptive immune responses, the method of activating CD4 + T cells and promoting their differentiation into Tfh and / or Th1 can be used for prevention and And / or treat a disease.
  • the B cell population is a B cell population present in a subject.
  • the B-cell population may be a naturally-occurring B-cell population or a B-cell population that is transferred to the subject.
  • the method of activating CD4 + T cells and the method of promoting CD4 + T cell differentiation of the present disclosure may occur in a subject.
  • the B cell population may be a B cell population isolated from a donor's peripheral blood or lymphoid organs such as the thymus, spleen, and tonsils.
  • the method may further include, after step c), screening, enriching, and identifying B cells that recognize the subunit. Collection and / or amplification steps.
  • the method may further include, after step c), screening B cells that recognize the subunit, and introducing the gene sequence encoding the immunoglobulin receptor into the B cell population to A step of increasing the number of B cells in the population that recognize the target antigen.
  • the diameter of the multimer can be about 10 nm to about 1000 nm, such as about 10 nm to about 500 nm, about 10 nm to About 300nm, about 10nm to about 200nm, about 10nm to about 100nm, about 10nm to about 50nm, about 20nm to about 1000nm, about 20nm to about 500nm, about 20nm to about 300nm, about 20nm to about 200nm, about 20nm to about 100nm About 20 nm to about 50 nm, about 50 nm to about 1000 nm, about 50 nm to about 500 nm, about 50 nm to about 300 nm, about 50 nm to about 200 nm, or about 50 nm to about 100 nm.
  • the multimer may comprise at least 4 subunits, such as at least 10 subunits, at least 20 subunits Unit, at least 50 subunits, at least 100 subunits, or at least 200 subunits.
  • the multimer may have 10 to 1,000 subunits, such as 20 to 500 subunits, 50 to 300 subunits, or 100 to 200 subunits.
  • the immune stimulator may comprise a ssRNA derived from a bacterium, a synthetic ssRNA or a derivative thereof, a synthetic containing CpG oligonucleotides, interferons, cytokines, or any combination thereof.
  • the ssRNA derived from bacteria may be an ssRNA derived from E. coli.
  • the interferon may be selected from interferon type I, interferon type II, interferon type III, and combinations thereof.
  • the cytokine may be selected from the group consisting of IL-6, IL-12, IL21, and combinations thereof.
  • one or more selected from the immune stimuli listed in Tables 1 and 2 above, or other natural or artificial immune stimuli known in the art can be used.
  • the multimer can be a virus-like particle, other natural multimer, or an artificial multimer.
  • a multimer can be assembled from multiple copies of one subunit, or can be assembled from multiple copies of two or more subunits. There are no specific restrictions on the choice of multimers.
  • the virus-like particle may comprise or consist of a Q ⁇ protein, HBcAg or AP205.
  • the Q ⁇ protein may have the amino acid sequence shown below: MAKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQAYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLIDNOY:
  • the HBcAg may have the amino acid sequence shown below: MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGNNLEDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYQRSRPRQPRRSPRQRTRPRTRQRDRQRTQPRTRPRQTRDRQTRDRQR
  • AP205 may have the amino acid sequence shown below: MANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKRPAPKPEGCADACVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAIVSSDTTA (SEQ ID: NO: 3)
  • the multimer such as a virus-like particle, comprises or consists of a target antigen.
  • the target antigen may be a Q ⁇ protein, HBcAg or AP205, or another virus-derived protein.
  • the disease is an infectious disease
  • the antigen complex comprises a loaded target antigen
  • the loaded target antigen is an antigen of bacterial or viral origin.
  • a bacterial or virus-derived antigen selected from those listed in Table 3 above can be used.
  • the antigen of bacterial or viral origin is an antigen of Mycobacterium tuberculosis, for example selected from the group consisting of lens protein and Rv3133c.
  • the antigen of bacterial or viral origin is an antigen of super bacterial origin, such as selected from the group consisting of Klebsiella pneumoniae carbapenemase and penicillin-binding protein.
  • the bacterial or virus-derived antigen is a lentivirus-derived antigen, such as selected from the group consisting of HBV pre-S1 antigen and EBV LMP1 antigen.
  • the disease is cancer.
  • cancer include, but are not limited to: basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; peritoneal cancer; cervical cancer Cholangiocarcinoma; Choriocarcinoma; Colon and rectal cancer; Connective tissue cancer; Digestive cancer; Endometrial cancer; Esophagus cancer; Eye cancer; Head and neck cancer; Gastric cancer (including gastrointestinal cancer); Glioblastoma; Liver cancer Liver cancer; neoepithelial neoplasms; kidney cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (for example, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); lymphoma, including Hodgkin lymphoma And non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma;
  • the loaded target antigen is a tumor-associated antigen
  • the tumor-associated antigen used is not particularly limited, and may be any antigen related to tumor development or aggressiveness.
  • a tumor-associated antigen as described above in the multimer-based antigen complex of the present disclosure can be used.
  • the tumor-associated antigen may be selected from Her2, p53, and tumor nascent antigen.
  • MAGE 1, 2 and 3 defined by immunity
  • MART-1 / Melan-A gp100
  • CEA Carcinoembryonic Antigen
  • HER-2 HER-2
  • Mucin i.e. MUC-1
  • PSA Prostate specific antigen
  • PAP prostate acid phosphatase
  • viral proteins such as hepatitis B (HBV), Eba (EBV), and human papilloma (HPV) have been shown to play important roles in the development of hepatocellular carcinoma, lymphoma, and cervical cancer, respectively.
  • HBV hepatitis B
  • Eba Eba
  • HPV human papilloma
  • cancer antigens commonly associated with spermatocytes or spermatogonia of the testes, placenta and ovary include the cancer-testis (CT) antigens BAGE, GAGE, MAGE-1 and MAGE-3, NY-ESO-1, SSX. These antigens are found in melanoma, lymphoma, lung cancer, bladder cancer, colon cancer, and breast cancer.
  • CT cancer-testis
  • Tumor-associated antigens commonly found in melanocytes, epithelial tissue, prostate and colon also include differentiation antigens Gp100, Melan-A / Mart-1, tyrosinase, PSA, CEA, and Mammaglobin-A. These antigens are found in melanoma, prostate and colon and breast cancers. Some tumor-associated antigens are shared antigens that are commonly expressed at low levels but overexpressed in cancer. Examples of overexpressed tumor-associated antigens include p53, HER-2 / neu, livin, and survivin found in esophagus, liver, pancreas, colon, breast, ovary, bladder, and prostate cancer.
  • tumor-associated antigens are unique, such as ⁇ -catenin-m, ⁇ -actin / 4 / m, myosin /, related to one or more of melanoma, non-small cell lung cancer, and kidney cancer m, HSP70-2 / m and HLA-A2-R170J.
  • Other tumor-associated antigens are tumor-associated carbohydrate antigens commonly found in epithelial tissues such as kidney, intestine, and colorectal tissue.
  • tumor-associated antigens include GM2, GD2, GD3, MUC-1, sTn, abd globo-H, which can be found in melanoma, neuroblastoma, colorectal cancer, lung cancer, breast cancer, ovarian cancer, and prostate cancer.
  • treatment refers to a therapeutic treatment in which the purpose is to reverse, reduce, ameliorate, suppress, slow or stop the progression or severity of a condition associated with a disease or disorder.
  • treatment includes reducing or alleviating at least one side effect or symptom of a disease or condition.
  • Treatment is usually “effective” if one or more symptoms or clinical markers are reduced.
  • treatment is “effective” if the progression of the disease is reduced or stopped, that is, “treatment” includes not only an improvement in symptoms or markers, but also the expected progression or worsening of symptoms in the absence of treatment Stop, at least slow down.
  • Beneficial or desired clinical results include, but are not limited to, alleviating one or more symptoms, reducing the extent of the disease, stabilizing (i.e., not exacerbating) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and remission (whether partial or partial All), whether detectable or undetectable.
  • treatment also includes alleviating the symptoms or side effects of the disease (including palliative care).
  • the terms “subject” and “individual” are used interchangeably herein and refer to animals and include mammals such as rats, mice, rabbits, sheep, cats, dogs, cattle, pigs, and Non-human primates.
  • the term “subject” also includes any vertebrate, including but not limited to mammals, reptiles, amphibians and fish.
  • the subject is a mammal such as a human or other mammal, such as a domestic mammal, such as a dog, cat, horse, and the like. Production mammals such as cattle, sheep, pigs, etc. are also included in the term subject.
  • the method may further include administering to the subject another therapy, such as an anti-cancer therapy, chemotherapeutic agent, or immunomodulatory agent.
  • the immunomodulatory agent comprises an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor binds one or more of the following: PD1, PDL1, PDL2, CTLA4, LAG3, TIM3, TIGIT, and / or CD103.
  • the immune checkpoint inhibitor is a PD1, PDL1, and / or PDL2 inhibitor.
  • anticancer therapy refers to a therapy that can be used to treat cancer.
  • anticancer therapeutic agents include, but are not limited to, for example, surgery, chemotherapeutic agents, growth inhibitors, cytotoxic agents, radiation therapy and agents for radiation therapy, antiangiogenic agents, apoptotic agents, antitubulin agents, and Other cancer treatment agents, such as anti-HER-2 antibodies, anti-CD20 antibodies, epidermal growth factor receptor antagonists, HER1 / EGFR inhibitors, platelet-derived growth factor inhibitors, COX-2 inhibitors, interferons, cytokines, Antagonists that bind to one or more of the following targets PD1, PDL1, PDL2; CTLA4; LAG3; CD103; TIM-3 and / or other TIM family members; CEACAM-1 and / or other CEACAM family members, ErbB2, ErbB3 , ErbB4, PDGFR- ⁇ , BlyS, APRIL, BCMA or VEGF receptor, TRAIL / Ap
  • anti-cancer therapies include immunotherapy such as adoptive cell transfer.
  • adoptive cell transfer refers to immunotherapy that involves genetically engineering a subject or patient's own T cells to produce a special receptor called a chimeric antigen receptor (CAR) on its surface.
  • CAR is a protein that allows T cells to recognize specific proteins (antigens) on tumor cells. These engineered CAR T cells are then cultured in the laboratory until they are counted in the billions. An expanded population of CAR T cells is then infused into the patient. After infusion, T cells multiply in the subject's body and, under the guidance of their engineered receptors, recognize and kill cancer cells that carry antigens on their surface.
  • chemotherapeutic agent refers to any chemical agent that has therapeutic utility in treating a disease characterized by abnormal cell growth. Such diseases include tumors, new organisms and cancers, as well as diseases characterized by proliferative growth.
  • chemotherapeutic agents include chemical agents and biological agents. These agents function to inhibit the cellular activity that cancer cells rely on to achieve sustained survival.
  • the class of chemotherapeutic agents includes alkylating / alkaloids, antimetabolites, hormones or hormone analogs, and a wide variety of anti-neoplastic drugs. Most, if not all of these agents are directly toxic to cancer cells and do not require immune stimulation.
  • the chemotherapeutic agent is an agent for treating a new organism such as a solid tumor.
  • the chemotherapeutic agent is a radioactive molecule.
  • the chemotherapeutic agent is a radioactive molecule.
  • Random therapy refers to the use of directional gamma or beta rays to induce sufficient damage to cells to limit their ability to function properly or completely destroy cells. It should be understood that there will be many known methods in the art to determine the dosage and duration of treatment.
  • the present disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a multimer-based antigen complex of the present disclosure, and optionally one or more other therapeutic agents and / or a pharmaceutically acceptable carrier .
  • the pharmaceutical composition containing the peptide of the present disclosure may be formulated by a conventional formulation method as necessary.
  • the pharmaceutical composition of the present disclosure may include carriers, excipients, and the like generally used in medicine without particular limitation.
  • carriers that can be used in the pharmaceutical composition of the present disclosure include sterilized water (eg, water for injection), physiological saline, phosphate buffered saline, phosphate buffered saline, Tris buffered saline, 0.3% glycine, culture fluid, and the like.
  • the pharmaceutical composition of the present disclosure may contain a stabilizer, a suspending agent, a preservative, a surfactant, a solubilizer, a pH adjuster, an aggregation inhibitor, and the like as necessary.
  • the pharmaceutical composition of the present disclosure can induce specific immunity against cancer cells expressing URLC10, and thus can be used for the purpose of cancer treatment or prevention (prevention).
  • pharmaceutically acceptable carrier refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, medium, encapsulating material, manufacturing aid (for example, lubricants, talc magnesium, calcium stearate or zinc or stearic acid) or solvent encapsulating materials, which are involved in maintaining the stability, solubility or activity of the LAP binder.
  • a pharmaceutically acceptable material such as a liquid or solid filler, diluent, excipient, solvent, medium, encapsulating material, manufacturing aid ( For example, lubricants, talc magnesium, calcium stearate or zinc or stearic acid) or solvent encapsulating materials, which are involved in maintaining the stability, solubility or activity of the LAP binder.
  • manufacturing aid for example, lubricants, talc magnesium, calcium stearate or zinc or stearic acid
  • solvent encapsulating materials which are involved in maintaining the stability, solubility or activity of
  • materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as Sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) shaping Agents such as cocoa butter and suppository waxes; (8) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (9) glycols such as propylene glycol; (10) polyols, Such as glycerol, sorbitol, mannitol and polyethylene glycol (PEG); (11) esters, such as ethyl oleate and ethyl laurate; (12) agar; (13) buffering agents, such as magnesium hydroxide and hydrogen
  • compositions of the present disclosure may also include adjuvants known to effectively establish cellular immunity.
  • An adjuvant refers to a compound that enhances an immune response against an immunologically active antigen when administered together (or continuously) with the antigen.
  • Known adjuvants described in the literature can be used, for example Clin Microbiol Rev 1994, 7: 277-89.
  • Suitable adjuvants include aluminum salts (aluminum phosphate, aluminum hydroxide, aluminum hydroxide, etc.), alum, cholera toxin, salmonella toxin, IFA (incomplete Freund's adjuvant), CFA (complete Freund's adjuvant), ISCOMatrix GM-CSF and other immune-stimulating cytokines, oligodeoxynucleotides (CpG7909, etc.) containing CpG motifs, oil-in-water emulsions, saponins or derivatives thereof (QS21, etc.), lipopolysaccharides such as lipid A or derivatives thereof (MPL, RC529, GLA, E6020, etc.), lipopeptide, lactoferrin, flagellin, double-stranded RNA or its derivative (poli IC, etc.), bacterial DNA, imidazoquinoline (Imiquimod, R848, etc.), C Type lectin ligand (trehalose-6
  • the adjuvant may be contained in another container separate from the pharmaceutical composition containing the peptide of the present disclosure.
  • the adjuvant and the pharmaceutical composition may be administered to the subject continuously or mixed together immediately before administration to the subject.
  • the disclosure also provides such kits comprising a peptide pharmaceutical composition and an adjuvant comprising the disclosure.
  • the kit may further include a redissolution solution.
  • the present disclosure provides a kit including a container containing a pharmaceutical composition of the present disclosure and a container storing an adjuvant.
  • the kit may further include a container storing a re-dissolution solution as necessary.
  • compositions according to the present disclosure may be by any common route, as long as the target tissue is available through that route.
  • suitable methods for administering the peptides or pharmaceutical compositions of the present disclosure include oral, intradermal, subcutaneous, intramuscular, intraosseous, intraperitoneal, and intravenous injections, as well as systemic or topical application near the target site, but Not limited to this.
  • a pharmaceutical composition of the present disclosure is packaged with or stored in a device for administration.
  • Devices for injectable preparations include, but are not limited to, injection ports, autoinjectors, syringe pumps, and pens.
  • Devices for atomizing or powdering formulations include, but are not limited to, inhalers, insufflators, aspirators, and the like. Accordingly, the present disclosure includes an application device comprising a pharmaceutical composition of the present disclosure for treating or preventing one or more conditions described herein.
  • the present disclosure relates to the use of the multimer-based antigen complex or pharmaceutical composition of the present disclosure to activate CD4 + T cells and / or promote CD4 + T cells to differentiate into Tfh and / or Th1 cells.
  • the present disclosure relates to the use of the multimer-based antigen complex or pharmaceutical composition of the present disclosure for the prevention and / or treatment of a disease.
  • the disease is selected from infectious diseases and cancer.
  • infectious diseases and cancers described above in the methods for preventing and / or treating diseases are examples of infectious diseases and cancers described above in the methods for preventing and / or treating diseases.
  • the present disclosure relates to the use of the multimer-based antigen complex of the present disclosure in the preparation of a pharmaceutical composition for activating CD4 + T cells and / or promoting the differentiation of CD4 + T cells into Tfh and / or Th1 cells .
  • the present disclosure relates to the use of the multimer-based antigen complex of the present disclosure in the preparation of a pharmaceutical composition for treating a disease.
  • the disease is selected from infectious diseases and cancer.
  • infectious diseases and cancers described above in the methods for preventing and / or treating diseases are infectious diseases and cancers described above in the methods for preventing and / or treating diseases.
  • Example 1 Multimer-based antigen complex capable of potently activating CD4 + T cells
  • Q ⁇ -VLP Bacteriophage Q ⁇ -derived VLP
  • the immunostimulant can be ssRNA derived from host bacteria or synthetic CpG-containing oligodeoxynucleotide (CpG ODN), which acts as a ligand for TLR7 or TLR9 to enhance the immune response (Jegerlehner, A. et al., (2007). TLR9 signalling in cells, classifiers, switches, and recombination to IgG2a. J Immunol 178, 2415-2420).
  • CpG ODN synthetic CpG-containing oligodeoxynucleotide
  • VLP assembled from both Q ⁇ protein and a fusion protein of Q ⁇ protein and ovalbumin-derived peptide, which can be replaced by CD4 + TCR transgenic T cells (OT-II) recognition.
  • OT-II CD4 + TCR transgenic T cells
  • T cell activation In addition to cell proliferation, we evaluated markers of T cell activation, including upregulation of CD44 and downregulation of CD62L (CD44hi and CD62lo). As shown by the results in Figure 2C, up-regulation of CD44 and down-regulation of CD62L were observed in most CFSE-diluted (i.e., cells undergoing proliferation) cells, indicating the activation of these T cells.
  • TLR signals in B cells but not DC are required for Q ⁇ -VLP-induced activation and differentiation of CD4 + T cells
  • Example 3 Mice lacking Q [beta] -specific B cells were unable to initiate Q [beta] -VLP immune-induced CD4 + T cell activation.
  • B cells bind to antigens through a specific B-cell antigen receptor (BCR). Therefore, we tested whether mice lacking BCR capable of specifically binding to Q ⁇ -VLP were defective in Q ⁇ -Ova-induced activation and differentiation of CD4 + T cells.
  • BCR transgenic mice MD4 which express BCR that recognizes egg lysozyme (Goodnow, CCetal., (1988).
  • antigen-specific B cells are necessary for multimer-based antigen complexes such as Q ⁇ -Ova-induced activation and differentiation of CD4 + T cells.
  • CD11c-DTR / GFP mice express the fusion protein of diphtheria toxin receptor and GFP under the control of the CD11c promoter (Jung, S. et al., (2002). Invivo depletion of CD11c + dendritic cells abrogates priming of CD8 + Tcells by exogenous cell-associated antigens. Immunity 17,211-220). In this chimeric mouse, diphtheria toxin receptor is expressed on the surface of DC cells.
  • Example 5 Q ⁇ -VLP efficiently captured by antigen-specific B cells in vivo.
  • CD83 is a molecule that is up-regulated after B cell activation and participates in post-translational regulation of MHC II (Tze, LEE et al., (2011). CD83 increases MHC II and CD86 on dendritic cells by opposing IL-10-driven MARCH1-mediated ubiquitination and degradation. J Exp Med 208, 149-165). The above results indicate that Q ⁇ -VLP can effectively activate antigen-specific B cells. In addition, Q ⁇ + B cells enriched from mice immunized for 24 hours showed significant up-regulation of the co-stimulatory molecule CD86 and the chemokine receptor CCR7 ( Figure 10), indicating that they stimulate T cells and migrate to the T cell region Enhanced capabilities.

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Abstract

本公开提供了使用基于多聚体的抗原复合物活化CD4+ T细胞的方法，所述方法包括将基于多聚体的抗原复合物与B细胞接触以使B细胞对抗原复合物进行处理和呈递，然后使所述B细胞与CD4+ T细胞接触以活化CD4+ T细胞。本公开还提供了使用所述抗原复合物促进CD4+ T细胞分化为Tfh细胞和Th1细胞的方法，以及通过活化CD4+ T细胞和/或促进CD4+ T细胞的分化来治疗疾病的方法。

Description

活化CD4+T细胞的方法
本申请要求于2018年08月07日递交的申请号为CN 201810892647.8的中国专利申请的优先权，在此全文引用上述中国专利申请公开的内容以作为本申请的一部分。
发明领域
本公开涉及免疫治疗领域。具体而言，本公开涉及使用基于多聚体的抗原复合物活化CD4+T细胞的方法。本公开还涉及促进CD4+T细胞分化为Tfh细胞和Th1细胞的方法，以及通过活化CD4+T细胞和/或促进CD4+T细胞的分化来治疗疾病的方法。
发明背景
CD4+T细胞是一类重要的T淋巴细胞，其在多种生理和病理过程中发挥重要作用，包括感染相关的免疫反应，肿瘤相关的免疫反应，过敏性疾病相关的免疫反应，自身免疫病类的免疫反应等。CD4+T细胞的主要功能是通过调控其它免疫细胞的来发挥作用，包括调控B淋巴细胞，CD8+T淋巴细胞，单核巨噬细胞，和NK细胞等多种免疫细胞在适应性免疫和天然免疫反应中的功能。
CD4+T细胞根据其功能状态可分为数个不同亚群，各亚群在不同免疫反应中发挥特异性的作用。滤泡辅助性T细胞(Follicular helper T cells,Tfh)是CD4+T细胞的一个重要亚群，主要参与生发中心反应过程。Tfh对于生发中心的形成和维持都必不可少。Tfh通过诱导和维持生发中心B细胞的作用可以促进抗体产生，抗体类型转换，抗体亲和力成熟，中和性抗体的产生，增加抗体图谱的广泛性等多种效应。因此如何有效地产生Tfh细胞对于多种疫苗研发都属于关键环节。1型辅助性T细胞(Type 1 helper T cells,Th1)是另一类重要的CD4+T细胞亚群，其在抗病毒反应和抗细菌反应，特别是对抗胞内细菌感染，如结核菌感染中发挥关键作用。此外，CD4+T细胞对于抗肿瘤免疫 也起到重要作用。
CD4+T细胞要发挥上述功能，必须先由静息状态转化为活化的细胞状态。目前用于活化CD4+T细胞的方法主要是利用树突状免疫细胞(DC)在体内或体外对CD4+T细胞进行初始活化，进而达到产生活化CD4+T细胞的目的。然而，对于CD4+T细胞的活化途径及其相关机制尚不明确，且DC对抗原的处理和呈递不具备特异性。本领域仍然需要用于有效活化CD4+T细胞的新的方法。
发明概述
本公开至少部分基于以下发现，即通过构建基于多聚体的抗原复合物，其能够被B细胞识别并处理，进而活化CD4+T细胞，并促进CD4+T细胞分化为Tfh和Th1细胞。由于B细胞通过由免疫球蛋白受体基因编码的B细胞表面受体对抗原进行特异性识别，因此在利用B细胞作为抗原呈递细胞对CD4+T细胞进行活化时能够获得比DC更强的效果。
相应的，本公开涉及以下方面。
在一方面，本公开涉及一种活化CD4+T细胞的方法，所述方法包括以下步骤：
a)使基于多聚体的抗原复合物与B细胞群体接触，
所述抗原复合物包含：
i)由多个亚单位组装的多聚体；和
ii)免疫刺激物，
其中所述多个亚单位包含目标抗原或由目标抗原组成，且其中所述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学连接附接于所述多聚体；
或者所述抗原复合物包含：
i)由多个亚单位组装的多聚体；
ii)加载的目标抗原；和
iii)免疫刺激物，
其中所述目标抗原通过物理吸附或化学连接附接于所述多聚体 的表面，或通过基因融合与所述多个亚单位的至少一部分融合，所述融合不影响所述多聚体的组装，且在所述多聚体组装后所述目标抗原展示在所述多聚体的表面；并且其中所述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学连接附接于所述多聚体，
其中所述B细胞群体的至少一部分能够识别所述亚单位的至少一种；
b)将所述抗原复合物与所述B细胞群体温育，以允许B细胞识别和处理所述抗原复合物，并将所述目标抗原呈递于细胞表面；
c)使所述B细胞群体与CD4+T细胞接触，以活化所述CD4+T细胞。
在另一方面，本公开涉及一种促进CD4+T细胞分化为滤泡辅助性T细胞(Tfh)和/或辅助性T细胞1(Th1)的方法，所述方法包括以下步骤：
a)使基于多聚体的抗原复合物与B细胞群体接触，
所述抗原复合物包含：
i)由多个亚单位组装的多聚体；和
ii)免疫刺激物，
其中所述多个亚单位包含目标抗原或由目标抗原组成，且其中所述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学连接附接于所述多聚体；
或者所述抗原复合物包含：
i)由多个亚单位组装的多聚体；
ii)加载的目标抗原；和
iii)免疫刺激物，
其中所述目标抗原通过物理吸附或化学作用附接于所述多聚体的表面，或通过基因融合与所述多个亚单位的至少一部分融合，所述融合不影响所述多聚体的组装，且在所述多聚体组装后所述目标抗原展示在所述多聚体的表面；并且其中所述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学作用附接于所述多聚体，
其中所述B细胞群体的至少一部分能够识别所述亚单位的至少一 种；
b)将所述抗原复合物与所述B细胞群体温育，以允许B细胞识别和处理所述抗原复合物，并将所述目标抗原呈递于细胞表面；
c)使所述B细胞与CD4+T细胞接触，以促进所述CD4+T细胞分化为Tfh和/或Th1。
在上述活化T细胞的方法和促进CD4+T细胞分化为Tfh和/或Th1细胞的方法的一些实施方案中，所述B细胞群体可以是从供体的外周血或淋巴器官分离的B细胞群体。
在上述活化T细胞的方法和促进CD4+T细胞分化为Tfh和/或Th1细胞的方法的一些实施方案中，所述方法还可以包括在步骤b)之后对识别所述亚单位的B细胞进行筛选、富集和/或扩增的步骤。
在上述活化T细胞的方法和促进CD4+T细胞分化为Tfh和/或Th1细胞的方法的一些实施方案中，所述方法还可以包括在步骤b)之后对识别所述亚单位的B细胞进行筛选，并将其编码免疫球蛋白受体的基因序列导入所述B细胞群体，以增加所述群体中识别所述亚单位的B细胞的数量的步骤。
在上述活化T细胞的方法和促进CD4+T细胞分化为Tfh和/或Th1细胞的方法的任意实施方案中，所述多聚体的直径为约10nm至约1000nm。
在上述活化T细胞的方法和促进CD4+T细胞分化为Tfh和/或Th1细胞的方法的任意实施方案中，所述多聚体可以包含至少4个亚单位。
在上述活化T细胞的方法和促进CD4+T细胞分化为Tfh和/或Th1细胞的方法的任意实施方案中，所述免疫刺激物可以选自细菌来源的ssRNA、人工合成的ssRNA或其衍生物、人工合成的含CpG的寡核苷酸，干扰素，细胞因子，及其组合。在一些实施方案中，所述细菌来源的ssRNA是大肠杆菌来源的ssRNA。在一些实施方案中，所述干扰素选自I型干扰素、II型干扰素、III型干扰素，及其组合。在一些实施方案中，所述细胞因子选自IL-6、IL-12、IL21，及其组合。
在上述活化T细胞的方法和促进CD4+T细胞分化为Tfh和/或Th1细胞的方法的一些实施方案中，所述多聚体是病毒样颗粒。在一些实 施方案中，所述病毒样颗粒包含Qβ蛋白，HBcAg或AP205，或由Qβ蛋白，HBcAg或AP205组成。
在一些实施方案中，所述病毒样颗粒包含目标抗原，或由目标抗原组成。例如，所述目标抗原选自Qβ蛋白，HBcAg和AP205。
在上述活化T细胞的方法和促进CD4+T细胞分化为Tfh和/或Th1细胞的方法的一些实施方案中，所述抗原复合物包含加载的目标抗原，所述加载的目标抗原是细菌或病毒来源的抗原。在一些实施方案中，所述目标抗原是结核分枝杆菌来源的抗原，例如选自晶状体蛋白和Rv3133c。在另一些实施方案中，所述目标抗原是超级细菌来源的抗原，例如选自肺炎克雷伯菌碳青霉烯酶和青霉素结合蛋白。在再一些实施方案中，所述目标抗原是慢病毒来源的抗原，例如选自HBV pre-S1抗原和EBV LMP1抗原。
在上述活化T细胞的方法和促进CD4+T细胞分化为Tfh和/或Th1细胞的方法的另一些实施方案中，所述抗原复合物包含加载的目标抗原，所述加载的目标抗原是肿瘤相关抗原。在一些实施方案中，所述肿瘤相关抗原选自Her2、p53和肿瘤新生抗原(neo-antigen)。
在上述活化T细胞的方法和促进CD4+T细胞分化为Tfh和/或Th1细胞的方法的一些实施方案中，所述方法是体外方法。在另一些实施方案中，所述方法的步骤c)发生在体内。
在另一个方面，本公开涉及一种预防和/或治疗有此需要的受试者中的疾病的方法，所述方法包括：
a)从所述受试者分离B细胞群体；
b)使基于多聚体的抗原复合物与所述B细胞群体接触；
所述抗原复合物包含：
i)由多个亚单位组装的多聚体；和
ii)免疫刺激物，
其中所述多个亚单位包含目标抗原或由目标抗原组成，且其中所述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学连接附接于所述多聚体；
或者所述抗原复合物包含：
i)由多个亚单位组装的多聚体；
ii)加载的目标抗原；和
iii)免疫刺激物，
其中所述目标抗原通过物理吸附或化学连接附接于所述多聚体的表面，或通过基因融合与所述多个亚单位的至少一部分融合，所述融合不影响所述多聚体的组装，且在所述多聚体组装后所述目标抗原展示在所述多聚体的表面；并且其中所述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学连接附接于所述多聚体；
其中所述B细胞群体的至少一部分能够识别所述亚单位的至少一种；
c)将所述抗原复合物与所述B细胞群体温育，以允许B细胞识别和处理所述抗原复合物，并将所述目标抗原呈递于细胞表面；
d)将所述B细胞群体施用至所述受试者。
在上述预防和/或治疗疾病的方法的一些实施方案中，所述B细胞群体是从受试者的外周血或淋巴器官分离的B细胞群体。
在上述预防和/或治疗疾病的方法的一些实施方案中，所述方法还包括在步骤c)之后对识别所述亚单位的B细胞进行筛选、富集和/或扩增的步骤。
在上述预防和/或治疗疾病的方法的另一些实施方案中，所述方法还包括在步骤c)之后对识别所述亚单位的B细胞进行筛选，并将其编码免疫球蛋白受体的基因序列导入所述B细胞群体，以增加所述群体中识别所述亚单位的B细胞的数量的步骤。
在上述预防和/或治疗疾病的方法的任意实施方案中，所述多聚体的直径为10nm至1000nm。
在上述预防和/或治疗疾病的方法的任意实施方案中，所述多聚体包含至少4个亚单位。
在上述预防和/或治疗疾病的方法的任意实施方案中，所述免疫刺激物包含细菌来源的ssRNA、人工合成的ssRNA或其衍生物、人工合成的含CpG的寡核苷酸，干扰素，细胞因子，或其组合。在一些实施方案中，所述细菌来源的ssRNA是大肠杆菌来源的ssRNA。在一些 实施方案中，所述干扰素选自I型干扰素、II型干扰素、III型干扰素，及其组合。在一些实施方案中，所述细胞因子选自IL-6、IL-12、IL21，及其组合。
在上述预防和/或治疗疾病的方法的一些实施方案中，所述多聚体是病毒样颗粒。在一些实施方案中，所述病毒样颗粒包含Qβ蛋白，HBcAg或AP205，或由Qβ蛋白，HBcAg或AP205组成。
在一些实施方案中，所述病毒样颗粒包含目标抗原，或由目标抗原组成。例如，所述目标抗原选自Qβ蛋白，HBcAg和AP205。
在上述预防和/或治疗疾病的方法的一些实施方案中，所述疾病是感染性疾病，并且所述抗原复合物包含加载的目标抗原，所述加载的目标抗原是细菌或病毒来源的抗原。在一些实施方案中，所述目标抗原是结核分枝杆菌来源的抗原，例如选自晶状体蛋白和Rv3133c。在另一些实施方案中，所述目标抗原是超级细菌来源的抗原，例如选自肺炎克雷伯菌碳青霉烯酶和青霉素结合蛋白。在再一些实施方案中，所述目标抗原是慢病毒来源的抗原，例如选自HBV pre-S1抗原和EBV LMP1抗原。
在上述预防和/或治疗疾病的方法的另一些实施方案中，所述疾病是癌症，并且所述抗原复合物包含加载的目标抗原，所述加载的目标抗原是肿瘤相关抗原。在一些实施方案中，所述肿瘤相关抗原选自Her2、p53和肿瘤新生抗原。
附图简述
图1显示了本发明的基于多聚体的抗原复合物的实例的示意图，其中显示了几种不同的抗原复合物形式。左图：由目标抗原直接组成的多聚体；中间图：目标抗原加载在天然多聚体骨架上；右图：目标抗原加载在人工构建的多聚体骨架上。图中的红点为抗原复合物中的免疫刺激物。
图2A-图2C显示了在多聚体Qβ-VLP免疫后，CD4+T细胞的强烈活化。使用Qβ-Qva对野生型小鼠进行免疫后，将CFSE标记的幼稚OT-II CD4+T细胞转移到小鼠体内。在免疫后第3天(d3)、第5天(d5) 和第7天(d7)对免疫的小鼠收获脾脏，并对未免疫的小鼠收获脾脏(d0)作为对照。显示了代表性的流式细胞术图和总的统计数据。图2A显示了将总的CD4+T细胞对Thy1.1+OT-II细胞进行门控。图2B显示了将Thy1.1+中的CFSE的荧光密度的直方图，并作为增殖指数进行定量。图2B中的灰色部分显示了来自未免疫小鼠的数据。图2C中显示了T细胞活化标志物CD44的上调和CD62L的下调(CD44hi和CD62lo)的图。显示了平均值±SD。
图3A和图3B显示了在多聚体Qβ-VLP免疫后，促进CD4+T细胞向Tfh细胞和Th1细胞分化。使用Qβ-Qva对野生型小鼠进行免疫后，将CFSE标记的幼稚OT-II CD4+T细胞转移到小鼠体内。在免疫后第3天(d3)、第5天(d5)和第7天(d7)从免疫的小鼠收获脾脏，并从未免疫的小鼠收获脾脏(d0)作为对照。显示了代表性的流式细胞术图和总的统计数据。基于来自未免疫小鼠的CFSE未稀释的细胞中分化标志物的表达水平，将Thy1.1+细胞对于所示的分化标志物进行门控。d3图中的灰色部分显示了来自未免疫小鼠的CFSE未稀释的细胞中分化标志物的表达水平。图3A显示了Tfh细胞特异性标志物PD1、CXCR5和Bcl-6阳性的CD4+T细胞比例显著上升，表明大量的CD4+T细胞向Tfh分化。图3B显示了Th1细胞特异性标志物T-bet和CXCR3阳性的CD4+T细胞比例显著上升，表明大量的CD4+T细胞向Th1细胞分化。
图4A和图4B显示了B细胞中的MyD88对于多聚体Qβ-VLP免疫后CD4+T细胞的活化和分化是必要的。使用Qβ-Qva对野生型小鼠或B-MyD88-/-小鼠进行免疫后，将CFSE标记的幼稚OT-II CD4+T细胞转移到小鼠体内。在免疫后第3天(d3)从小鼠收获脾脏。显示了代表性的流式细胞术图和总的统计数据。图4A：对总的CD4+T细胞门控Thy1.1+OT-II细胞。图4B：基于来源于受体小鼠的幼稚CD4+T细胞中分化标志物的表达水平(未显示)对来自图4A的Thy1.1+细胞中的分化标志物进行门控。显示了平均值±SD。使用未配对的Student’s t检验进行数据分析。ns:不显著；**p<0.01；***p<0.001。
图5A和图5B显示了B细胞中的MyD88对于可溶性抗原Ova+CpG免疫后CD4+T细胞的活化和分化不是必要的。使用Ova+CpG对野生 型小鼠或B-MyD88-/-小鼠进行免疫后，将CFSE标记的幼稚OT-II CD4+T细胞转移到小鼠体内。在免疫后第3天(d3)从小鼠收获脾脏。显示了代表性的流式细胞术图和总的统计数据。图5A：对总的CD4+T细胞门控Thy1.1+OT-II细胞。图5B：基于来源于受体小鼠的幼稚CD4+T细胞中分化标志物的表达水平，对来自图5A的Thy1.1+细胞中的分化标志物进行门控。显示了平均值±SD。使用未配对的Student’s t检验进行数据分析。ns:不显著。
图6A和6B显示了DC中的MyD88对于多聚体Qβ-VLP免疫后CD4+T细胞的活化和分化不是必要的。使用Qβ-Qva对野生型小鼠或DC-MyD88-/-小鼠进行免疫后，将CFSE标记的幼稚OT-II CD4+T细胞转移到小鼠体内。在免疫后第3天(d3)从小鼠收获脾脏。图6A：对总的CD4+T细胞门控Thy1.1+OT-II细胞。图6B：对Thy1.1+OT-II CD4+T细胞中的分化标志物进行门控。显示了平均值±SD。使用未配对的Student’s t检验进行数据分析。ns:不显著。
图7A-图7C显示了缺乏Qβ特异性B细胞的小鼠在多聚体Qβ-VLP免疫后不能诱导CD4+T细胞应答。使用Qβ-Qva或与CpG ODN混合的Ova对野生型小鼠和MD4小鼠进行免疫后，将CFSE标记的幼稚OT-II CD4+T细胞转移到小鼠体内。显示了代表性的流式细胞术图和总的统计数据。图7A：对总的CD4+T细胞门控Thy1.1+OT-II细胞。图7B和图7C：对来自图7A的Thy1.1+细胞中的分化标志物进行门控。显示了平均值±SD。使用未配对的Student’s t检验进行数据分析。ns:不显著；**p<0.01；***p<0.001。
图8显示了DC对于多聚体Qβ-VLP诱导的CD4+T细胞活化不是必要的。对小鼠进行致死照射以去除免疫细胞，并使用来自CD11c-DTR/GFP小鼠的BM细胞进行重构。在进行OT-II CD4+T细胞转移时使用PBS或DT对小鼠进行处理。使用Qβ-Qva或与CpG ODN混合的Ova对小鼠进行免疫后，将CFSE标记的幼稚OT-II CD4+T细胞转移到小鼠体内。在免疫后24小时收获脾脏。显示了个体小鼠的代表性流式细胞术图和总的统计数据。基于来自未免疫小鼠的CFSE未稀释的细胞中分化标志物的表达水平，将Thy1.1+CD4+OT-II T细胞对于 所示的分化标志物进行门控。显示了平均值±SD。使用未配对的Student’s t检验进行数据分析。ns:不显著；***p<0.001。
图9A-图9C显示了在体内抗原特异性B细胞有效地捕获Qβ-VLP。图9A和图9B：使用Qβ-AF647或PBS静脉内注射野生型或MD4小鼠，并于3小时后进行检查。图9A：首先从总的脾脏细胞中门控CD11c+MHCII+DC，然后对其进一步门控Qβ-AF647+。图9B：首先从总的脾脏细胞中门控Qβ-AF647+MHCII+细胞，然后对其进一步门控B220+B细胞和CD11c+DC。显示了DC和B细胞对Qβ-AF647的结合。图9C：使用Qβ-AF64静脉内注射野生型小鼠，并于注射后0.5小时和3小时进行检查。还检查了未注射的小鼠最为对照。总的脾脏细胞使用Qβ-FITC和抗FITC磁珠进行富集。从富集的级分中门控Qβ-FITC+B220+B细胞，其进一步显示Qβ-AF647和CD83。数据代表了至少三次独立的实验。
图10A和图10B：使用未标记的Qβ-VLP腹膜内注射野生型小鼠。24小时后收获脾脏，并从未免疫的小鼠收获脾脏作为对照。将总的脾脏细胞与Qβ-AF647和Qβ-GFP温育，然后使用抗AF647磁珠进行富集。图10A从富集的细胞级分门控Qβ-AF647+B细胞，其进一步根据Qβ-GFP作为AF647+和Qβ+B细胞进行门控。显示了CD86和CCR7。图10BAF647+和Qβ+B细胞中CD86和CCR7的平均荧光强度(MFI)。柱代表了平均值。点代表了来自个体小鼠的数据。使用未配对的Student’s t检验进行数据分析。ns:不显著；**p<0.01。
图11显示了B细胞的抗原呈递对于Qβ-VLP诱导的CD4+T细胞活化时必要的。通过将来自μMT和MHCII-/-(B-MHCII-/-)或WT(对照)小鼠的混合的BM细胞移植到致死照射的小鼠中生成了B-MHCII-/-和对照小鼠。使用Qβ-Qva对小鼠进行免疫后，将CFSE标记的幼稚OT-II CD4+T细胞转移到小鼠体内。在免疫后第3天(d3)从小鼠收获脾脏。显示了代表性的流式细胞术图和总的统计数据。对总的CD4+T细胞门控Thy1.1+OT-II细胞。对于B-MHCII-/-显示了两个不同的代表性图，其中#1显示出低水平的CFSE稀释。显示了平均值±SD。使用未配对的Student’s t检验进行数据分析。ns:不显著；*p<0.05；**p<0.01； ***p<0.001。
图12A至图12C显示了抗原特异性B细胞参与流感病毒诱导的CD4+T细胞应答。将CFSE标记的幼稚OT-II CD4+T细胞转移到WT和MD4小鼠中(图12A和图12B)，或转移到使用PBS(对照)或DT处理的CD11c-DTR/GFP BM嵌合小鼠中(C)，然后使用PR8-Ova进行免疫。在免疫后第3天(图12A和图12B)或24小时(图12C)收获肝脏。显示了个体小鼠的代表性的流式细胞术图和总的统计数据。图12A：对总的CD4+T细胞门控Thy1.1+OT-II细胞。图12B和图12C：对Thy1.1+CD4+T细胞门控所示的标志物。显示了平均值±SD。使用未配对的Student’s t检验进行数据分析。ns:不显著；*p<0.05；**p<0.01；***p<0.001。
发明详述
基于多聚体的抗原复合物
在一个方面，本公开涉及基于多聚体的抗原复合物。
在一些实施方案中，所述基于多聚体的抗原复合物包含：
i)由多个亚单位组装的多聚体；和
ii)免疫刺激物，
其中所述多个亚单位包含目标抗原或由目标抗原组成，且其中所述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学连接附接于所述多聚体。
在另一些实施方案中，所述基于多聚体的抗原复合物包含：
i)由多个亚单位组装的多聚体；
ii)加载的目标抗原；和
iii)免疫刺激物，
其中所述目标抗原通过物理吸附或化学连接附接于所述多聚体的表面，或通过基因融合与所述多个亚单位的至少一部分融合，所述融合不影响所述多聚体的组装，且在所述多聚体组装后所述目标抗原展示在所述多聚体的表面，并且其中所述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学连接附接于所述多聚体。
图1显示了本公开的基于多聚体的抗原复合物的实例的示意图，其中显示了几种不同的抗原复合物形式。
当提及“基于多聚体的抗原复合物时”，上述术语还涵盖多种基于多聚体的抗原复合物的混合物的情况。例如，术语“基于多聚体的抗原复合物”可以意指两种或更多种基于多聚体的抗原复合物的混合物。在一些实施方案中，所述两种或更多种基于多聚体的抗原复合物可以各自具有上述组分i)和ii)，或组分i)、ii)和iii)。也就是说，在“基于多聚体的抗原复合物时”意指两种或更多种基于多聚体的抗原复合物的情况下，其中一些抗原复合物可以具有上述组分i)和ii)，而其它抗原复合物可以具有上述组分i)、ii)和iii)。
本公开的基于多聚体的抗原复合物可以用于活化B细胞，或通过B细胞的识别和呈递进而活化CD4+T细胞，并促进CD4+T细胞分化为Tfh和Th1。
在生物体内，大多数细胞都不表达MHC II。能够表达MHC II并与CD4+T细胞发生抗原介导的特异性结合的细胞称为抗原呈递细胞(APC)。树突状细胞(DC)、B细胞和巨噬细胞是几类主要的抗原呈递细胞。虽然这几类细胞均表达MHC II，并且在特定情况下能够刺激CD4+T细胞的活化，但目前的普遍观点认为只有DC能够通过抗原呈递(通过MHC II类分子)和产生细胞因子活化初始状态的CD4+T细胞。初始状态的CD4T细胞又称为幼稚CD4+T细胞，其是否可以被有效活化决定了后续免疫反应的强度，因此这一步是各种增强免疫反应措施的重要靶向对象。由于DC在这一步骤中的关键作用，因而目前都将DC作为各类疫苗研发的重点靶向目标。
在本申请中，本公开人令人惊讶的发现，尽管大多数抗原是通过DC对CD4+T细胞进行活化，但是具有特殊形式的抗原，即基于多聚体的抗原复合物，可以通过B细胞对幼稚CD4+T细胞进行初始活化。即，其能够在不存在DC的情况下通过抗原呈递诱导CD4+T细胞的活化和分化。
B细胞的这种能力与其表达的免疫球蛋白受体受体(B细胞受体，BCR)和天然免疫信号通路密切相关。BCR实际上是跨膜形式的免疫 球蛋白，其产生是由B细胞发育过程中在特定BCR基因相关位点发生DNA水平的基因重排过程，即V(D)J重排后的结果。这种淋巴细胞特有的V(D)J重排过程使不同的B细胞可以表达不同的BCRs，其可能产生的BCR种类可以达到10 12-10 15之多。由于BCR的丰富程度，B细胞在识别抗原时是利用B细胞群中对抗原有较高亲和力BCR表达的细胞群体。这些细胞在接触抗原后，可以被活化，并进一步向免疫效应细胞分化。我们的研究发现，当这些细胞被本文中所述的基于多聚体的抗原复合物活化时，它们作为APC的能力大大增强，可以完全取代DC的作用。
在一些实施方案中，所述多聚体的直径可以是约10nm至约1000nm，例如约10nm至约500nm，约10nm至约300nm，约10nm至约200nm，约10nm至约100nm，约10nm至约50nm，约20nm至约1000nm，约20nm至约500nm，约20nm至约300nm，约20nm至约200nm，约20nm至约100nm，约20nm至约50nm，约50nm至约1000nm，约50nm至约500nm，约50nm至约300nm，约50nm至约200nm，或约50nm至约100nm。
在一些实施方案中，所述多聚体可以包含至少4个亚单位，例如包含至少10个亚单位，至少20个亚单位，至少50个亚单位，至少100个亚单位，或至少200个亚单位。所述多聚体可以具有10至1000个亚单位，例如20至500个亚单位，50至300个亚单位，或者100至200个亚单位。
在上述基于多聚体的抗原复合物的任意实施方案中，所述免疫刺激物可以包含细菌来源的ssRNA、人工合成的ssRNA或其衍生物、人工合成的含CpG的寡核苷酸，干扰素，细胞因子，或其任意组合。在一些实施方案中，所述细菌来源的ssRNA可以是大肠杆菌来源的ssRNA。在一些实施方案中，所述干扰素可以选自I型干扰素、II型干扰素、III型干扰素，及其组合。在一些实施方案中，所述细胞因子可以选自IL-6、IL-12、IL21，及其组合。
在淋巴细胞例如B细胞和T细胞活化的过程中，通常需要两重要素：一是抗原受体，也就是BCR和TCR的刺激和信号传导；二是免疫 信号或细胞因子的刺激。这两重刺激共同决定了淋巴细胞是否可以活化，及活化后的功能分化方向。在本文中，免疫刺激物指能够行使第二重刺激的物质，主要包括天然免疫受体的配体刺激物和促炎类的细胞因子两大类。
在以下表1中列出了可以在本申请的抗原组合物中使用的免疫刺激物类型的实例。
表1：免疫刺激物
天然免疫受体刺激物 Toll样受体的配体刺激物 TLR1配体刺激物
    TLR2配体刺激物
    TLR3配体刺激物
    TLR4配体刺激物
    TLR5配体刺激物
    TLR6配体刺激物
    TLR7配体刺激物
    TLR8配体刺激物
    TLR9配体刺激物
    TLR10配体刺激物
    TLR11配体刺激物
    TLR12配体刺激物
    TLR13配体刺激物
  NOD样受体的配体刺激物  
  RIG-I样受体的配体刺激物  
细胞因子 白介素 IL-6
    IL-12
    IL-21
    IL-4
  干扰素 IFN-a
    IFN-b
    IFN-g
  肿瘤坏死因子家族 TNF
    CD70
    TNFSF8
    TNFSF13
    TNFSF13B
在以下表2中列出了Toll样受体的配体刺激物的具体实例。
表2：Toll样受体的配体刺激物
Figure PCTCN2019099489-appb-000001
本公开的基于多聚体的抗原复合物可以包含一种免疫刺激物，或者两种或更多种不同的免疫刺激物。
此外，除上述具体例举的免疫刺激物外，本领域已知各种用于促进免疫应答的天然和人工免疫刺激物，并且可以选择用于构建本公开的抗原复合物。免疫刺激物和多聚体的组合方式没有特定限制，例如可以包装在所述多聚体中，或通过物理吸附或化学连接附接于多聚体的表面，以发挥其免疫刺激功能。在免疫刺激物包装在多聚体内的情况下，可以在多聚体组装期间引入免疫刺激物，使得多聚体的亚单位在组装期间将免疫刺激物包装在多聚体的内部。
在上述基于多聚体的抗原复合物的进一步的实施方案中，所述多聚体可以是病毒样颗粒，其它天然多聚体或人工合成的多聚体。
病毒样颗粒是结构上与病毒类似，但不含有病毒遗传物质的生物制剂。病毒样颗粒通常由一种或几种蛋白的多个拷贝组成而成，其直径大小可以从数十纳米至上千纳米变化。病毒样颗粒的表面呈现重复排列的抗原表位，使得其活化B细胞的能力大大增强。病毒样颗粒内部可以容纳天然或人工的核酸物质，也可以人工添加其它种类的化合物，作为免疫刺激物。该免疫刺激物对于病毒样颗粒诱导的免疫应答，尤其是B细胞应答是重要的。
除了病毒样颗粒外，一些天然存在的多糖类化合物也属于天然多聚体，并能够在其基础上进一步形成颗粒样结构。该多聚体也可以用于本公开的抗原复合物，用于B细胞获得活化，和通过B细胞的识别和呈递进而活化CD4+T细胞，并促进CD4+T细胞分化为Tfh和Th1。此外，一些经人工涉及和改造的蛋白也可以形成多聚体。非蛋白物质也可以形成多价颗粒制剂，例如人工合成的纳米颗粒。将这些人工合成的多聚体表面使用特定的化学基团修饰后，可以将目标抗原通过物理吸附或化学连接加载在所述多聚体上。
多聚体可以由一种亚单位的多个拷贝组装而成，或者可以由两种或更多种亚单位的多个拷贝组装。对多聚体的选择没有具体限制，并且本领域技术人员可以选择本领域已知的各种多聚体结构用于构建本公开的抗原复合物。
在一些实施方案中，所述病毒样颗粒可以包含Qβ蛋白，HBcAg或AP205，或由Qβ蛋白，HBcAg或AP205组成。
噬菌体Qβ是二十面体RNA病毒，直径为30nm。其宿主是大肠杆菌。Qβ通过结合细菌表面的F菌毛后进入其宿主细胞。噬菌体Qβ衣壳蛋白的前133个氨基酸可以通过质粒转化在大肠杆菌或酵母等其它细胞内表达，并自组装为直径为30nm的颗粒。噬菌体Qβ衣壳蛋白的自组装过程不需要其本身的遗传物质，也不需要其它蛋白辅助，组装成的颗粒不具有对任何细胞(包括原核和真核细胞)的感染性。
HBcAg(核心抗原)是一种乙型肝炎病毒蛋白，其是存在于核衣壳核心(乙型肝炎病毒最内层)表面的抗原。在乙型肝炎病毒感染的细胞中，HBcAg与病毒核酸的包装由关。虽然乙型肝炎病毒只感染真核细胞，HBcAg可以通过质粒转化在大肠杆菌或酵母等其它细胞内表达，并自组装为直径约30nm的颗粒。HBcAg的自组装过程不需要其本身的遗传物质，也不需要其它蛋白辅助，组装成的颗粒不具有对任何细胞(包括原核和真核细胞)的感染性。
噬菌体AP205是二十面体RNA病毒，直径为30nm。其宿主是Acinetobacter。AP205衣壳蛋白可以通过质粒转化在大肠杆菌或酵母等其它细胞内表达，并自组装为直径为30nm的颗粒。AP205衣壳蛋白的自组装过程不需要其本身的遗传物质，也不需要其它蛋白辅助，组装成的颗粒不具有对任何细胞(包括原核和真核细胞)的感染性。
在一些实施方案中，Qβ蛋白可以具有以下所示的氨基酸序列：MAKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQAYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY(SEQ ID NO:1)
在一些实施方案中，HBcAg可以具有以下所示的氨基酸序列：MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGNNLEDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC(SEQ ID NO:2)
在一些实施方案中，AP205可以具有以下所示的氨基酸序列： MANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKRPAPKPEGCADACVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAIVSSDTTA(SEQ ID NO:3)
在一些实施方案中，所述多聚体例如病毒样颗粒包含目标抗原，或由目标抗原组成。即，多聚体的至少一种亚单位本身作为目标抗原。例如，在多聚体是病毒样颗粒的情况下，所述作为目标抗原的亚单位可以是Qβ蛋白，HBcAg或AP205，或其它病毒来源的蛋白。多聚体例如病毒样颗粒可以仅包含一种亚单位的多个拷贝，或包含两种或更多种亚单位。在多聚体例如病毒样颗粒包含一种亚单位的多个拷贝的情况下，所述亚单位本身可以作为目标抗原。在多聚体包含两种或更多种亚单位的情况下，所述述亚单位中的至少一种可以作为目标抗原。
在另一些实施方案中，所述抗原复合物包含加载的目标抗原。加载的目标抗原没有具体限制，其可以是任何具有免疫原性并能够被免疫系统的组分特异性识别的蛋白质、多肽、核酸或小分子。本领域已知各种用于免疫的目标抗原，并且本领域技术人员能够根据需要选择具体的目标抗原来构建本公开的抗原复合物。
此外，目标抗原的加载方式没有特别限制，并且可以采用例如物理吸附、化学连接和基因融合的方式。当使用物理吸附或化学连接的方式加载目标抗原时，加载的时机没有特别限制。例如，在一些实施方案中，可以将多聚体的亚单位与目标抗原接触(例如发生物理吸附或化学作用)，然后组装成多聚体，使得目标抗原加载在多聚体的表面。在这种情况下，多聚体亚单位与目标抗原的结合不影响其组装成多聚体。在另一些实施方案中，可以在组装成亚单位组装成多聚体后引入目标抗原，使得目标抗原通过物理吸附或化学连接附接于多聚体的表面。
当采用基因融合的方式加目标抗原时，即通过遗传重组技术将编码目标抗原的核苷酸序列与编码多聚体的亚单位的核苷酸序列融合，并作为融合蛋白表达的情况下，目标抗原可以仅与组成多聚体的亚单位的一部分融合。也就是说，在多聚体仅包含一种亚单位的多个拷贝的情况下，目标抗原可以与所述多个拷贝的全部或仅一部分进行融 合。在多聚体包含两种或更多种亚单位的情况下，目标抗原可以与所述两种或更多种亚单位的至少一种融合，此外，目标抗原可以与所述至少一种亚单位的全部仅一步分进行融合。
通过遗传重组技术构建融合蛋白的方法是本领域公知的。并且本领域技术人员可以根据各种条件例如使用的目标抗原种类、大小、免疫原性，以及多聚体的亚单位的拷贝数等选择适合的融合方式，使得在融合后，目标抗原不影响多聚体的组装。
在一些实施方案中，所述加载的目标抗原可以是细菌或病毒来源的抗原。细菌或病毒来源的抗原的具体种类没有特别限制，并且可以使用如表3中列出的那些示例性抗原。
表3：细菌或病毒来源的抗原
结核菌 DosR
  Ag85
  ESAT6
  Crystalline
  CFP10
  Rv2031c
Epstein–Barr病毒 EBNA-1
  EBNA-2
  EBNA-3A
  EBNA-3B
  EBNA-LP
  LMP-1
  LMP-2A
  LMP-2B
  EBER
  Gp350
乙型肝炎病毒 HBsAg
  Pre-S1
疟原虫 CSP
  MSP1
  MSP3
  DBP
在一些实施方案中，所述细菌或病毒来源的抗原可以是结核分枝杆菌来源的抗原，例如选自晶状体蛋白和Rv3133c。
在另一些实施方案中，所述细菌或病毒来源的抗原可以是超级细菌来源的抗原，例如选自肺炎克雷伯菌碳青霉烯酶和青霉素结合蛋白。
在再一些实施方案中，所述细菌或病毒来源的抗原可以是慢病毒来源的抗原，例如选自HBV pre-S1抗原和EBV LMP1抗原。
在上述基于多聚体的抗原复合物的另一些实施方案中，所述抗原复合物包含加载的目标抗原，所述加载的目标抗原可以是肿瘤相关抗原。
使用的肿瘤相关抗原没有特别限制，并且可以是对于肿瘤发展或侵袭性相关的任何抗原。术语“肿瘤相关抗原”是指由癌细胞差异表达，因此可以利用以靶向癌细胞的抗原。肿瘤相关抗原是可以潜在地刺激明显的肿瘤特异性免疫应答的抗原。这些抗原中的一些由正常细胞编码，但不一定由正常细胞表达。这些抗原可以表征为在正常细胞中通常是沉默(即不表达)的那些抗原、仅在某些分化阶段表达的那些抗原和那些在时间上表达的那些抗原，如胚胎和胎儿抗原。其它肿瘤相关抗原由突变细胞基因如癌基因(例如激活的ras癌基因)、抑制基因(例如突变型p53)和由内部缺失或染色体易位产生的融合蛋白编码。其它肿瘤相关抗原可以由病毒基因编码，如RNA和DNA肿瘤病毒上携带的基因。
在一些实施方案中，使用完整的癌抗原，而在其它实施方案中，使用癌抗原的肽表位(通过蛋白水解消化或重组制备)。因此，本文的基于多聚体的抗原复合物中的肿瘤或肿瘤相关抗原的非限制性实例包括但不限于Her2、前列腺干细胞抗原(PSCA)、PSMA(前列腺特异性膜抗原)、β-连环蛋白-m、B细胞成熟抗原(BCMA)、甲胎蛋白(AFP)、癌胚抗原(CEA)、癌抗原-125(CA-125)、CA19-9、钙网膜蛋白(calretinin)、MUC-1，上皮膜蛋白(EMA)、上皮肿瘤抗原(ETA)、酪氨酸酶、Mammaglobin-A、黑素瘤相关抗原(MAGE)、CD34、CD45、 CD99、CD117、嗜铬粒蛋白、细胞角蛋白、结蛋白、胶质纤维酸性蛋白(GFAP)、巨囊性病的液状蛋白(gross cystic disease fluid protein)(GCDFP-15)、EBV、gp100、HMB-45抗原、蛋白melan-A(被T淋巴细胞识别的黑素瘤抗原；MART-1)、livin、存活蛋白、myo-D1、肌肉特异性肌动蛋白(MSA)、神经丝、神经元特异性烯醇化酶(NSE)、胎盘碱性磷酸酶、突触囊泡蛋白、甲状腺球蛋白、甲状腺转录因子-1、丙酮酸激酶同工酶M2型的二聚体形式(肿瘤M2-PK)，CD19，CD22，
CD27，CD30，CD70，GD2(神经节苷脂G2)EphA2,CSPG4,CD138,FAP(成纤维细胞活化蛋白),CD171,kappa,lambda,5T4,α vβ 6整联蛋白,B7-H3,B7-H6,CAIX,CD19,CD20,CD22,CD30,CD33,CD44,CD44v6,CD44v7/8,CD70,CD123,EGFR,EGP2,EGP40,EpCAM,fetal AchR,FRα,GAGE,GD3,HLA-A1+MAGE1,MAGE-3,HLA-A1+NY-ESO-1,IL-11Rα,IL-13Rα2,Lewis-Y,Muc16,NCAM,NKG2D Ligands,NY-ESO-1,PRAME,ROR1,SSX,存活蛋白,TAG72,TEMs,VEGFR2,EGFRvIII(表皮生长因子变体III)，精子蛋白17(Sp17)，间皮蛋白(mesothelin)，PAP(前列腺酸性磷酸酶)，prostein，TARP(T细胞受体γ交替读码框蛋白)，Trp-p8，STEAP1(前列腺六跨膜上皮抗原1(six-transmembrane epithelial antigen of the prostate 1))，HSP70-2/m和HLA-A2-R170J，酪氨酸酶，异常ras蛋白或异常p53蛋白。
在一些实施方案中，所述肿瘤抗原可以选自Her2、p53或肿瘤新生抗原。
活化T细胞和促进T细胞分化的方法
在一方面，本公开涉及一种活化CD4+T细胞的方法，所述方法包括使B细胞与CD4+T细胞接触的步骤，其中所述B细胞已经使用基于多聚体的抗原复合物或等同方法进行了活化，所述基于多聚体的抗原复合物包含：
i)由多个亚单位组装的多聚体；和
ii)免疫刺激物，
其中所述多个亚单位包含目标抗原或由目标抗原组成，且其中所 述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学连接附接于所述多聚体；
或者所述抗原复合物包含：
i)由多个亚单位组装的多聚体；
ii)加载的目标抗原；和
iii)免疫刺激物，
其中所述目标抗原通过物理吸附或化学连接附接于所述多聚体的表面，或通过基因融合与所述多个亚单位的至少一部分融合，所述融合不影响所述多聚体的组装，且在所述多聚体组装后所述目标抗原展示在所述多聚体的表面，并且其中所述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学连接附接于所述多聚体。
在另一个方面，本公开涉及一种促进CD4+T细胞分化为Tfh和/或Th1细胞的方法，所述方法包括使B细胞与CD4+T细胞接触的步骤，其中所述B细胞已经使用基于多聚体的抗原复合物或等同方法进行了活化，所述基于多聚体的抗原复合物包含：
i)由多个亚单位组装的多聚体；和
ii)免疫刺激物，
其中所述多个亚单位包含目标抗原或由目标抗原组成，且其中所述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学连接附接于所述多聚体；
或者所述抗原复合物包含：
i)由多个亚单位组装的多聚体；
ii)加载的目标抗原；和
iii)免疫刺激物，
其中所述目标抗原通过物理吸附或化学连接附接于所述多聚体的表面，或通过基因融合与所述多个亚单位的至少一部分融合，所述融合不影响所述多聚体的组装，且在所述多聚体组装后所述目标抗原展示在所述多聚体的表面，并且其中所述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学连接附接于所述多聚体。
在再一个方面，本公开涉及一种活化CD4+T细胞的方法，所述 方法包括以下步骤：
a)使基于多聚体的抗原复合物与B细胞群体接触，
所述抗原复合物包含：
i)由多个亚单位组装的多聚体；和
ii)免疫刺激物，
其中所述多个亚单位包含目标抗原或由目标抗原组成，且其中所述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学连接附接于所述多聚体；
或者所述抗原复合物包含：
i)由多个亚单位组装的多聚体；
ii)加载的目标抗原；和
iii)免疫刺激物，
其中所述目标抗原通过物理吸附或化学连接附接于所述多聚体的表面，或通过基因融合与所述多个亚单位的至少一部分融合，所述融合不影响所述多聚体的组装，且在所述多聚体组装后所述目标抗原展示在所述多聚体的表面，并且其中所述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学连接附接于所述多聚体，
其中所述B细胞群体的至少一部分能够识别所述亚单位的至少一种；
b)将所述抗原复合物与所述B细胞群体温育，以允许B细胞识别和处理所述抗原复合物，并将所述目标抗原呈递于细胞表面；
c)使所述B细胞群体与CD4+T细胞接触，以活化所述CD4+T细胞。
在另一个方面，本公开涉及一种促进CD4+T细胞分化为Tfh和/或Th1细胞的方法，所述方法包括以下步骤：
a)使基于多聚体的抗原复合物与B细胞群体接触，
所述抗原复合物包含：
i)由多个亚单位组装的多聚体；和
ii)免疫刺激物，
其中所述多个亚单位包含目标抗原或由目标抗原组成，且其中所 述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学连接附接于所述多聚体；
或者所述抗原复合物包含：
i)由多个亚单位组装的多聚体；
ii)加载的目标抗原；和
iii)免疫刺激物，
其中所述目标抗原通过物理吸附或化学连接附接于所述多聚体的表面，或通过基因融合与所述多个亚单位的至少一部分融合，所述融合不影响所述多聚体的组装，且在所述多聚体组装后所述目标抗原展示在所述多聚体的表面，并且其中所述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学连接附接于所述多聚体，
其中所述B细胞群体的至少一部分能够识别所述亚单位的至少一种；
b)将所述抗原复合物与所述B细胞群体温育，以允许B细胞识别和处理所述抗原复合物，并将所述目标抗原呈递于细胞表面；
c)使所述B细胞与CD4+T细胞接触，以使所述CD4+T细胞分化为Tfh和/或Th1。
T细胞或T淋巴细胞是在细胞介导的免疫中发挥中心作用的淋巴细胞类型。通过细胞表面T细胞受体(TCR)的存在，它们可以从其它淋巴细胞例如B细胞和自然杀伤细胞(NK细胞)区分。
CD4+T细胞又称为辅助性T辅助细胞(Th细胞)，其在免疫学过程中辅助其他白细胞，包括B细胞成熟为浆细胞和记忆B细胞，以及活化细胞毒性T细胞和巨噬细胞。Th细胞在其表面表达CD4。当它们通过抗原呈递细胞(APC)表面上的MHC II类分子呈递肽抗原时，Th细胞被活化。这些细胞可以分化成几种亚型之一，包括Th1，Th2，Th3，Th17，Th9或Tfh，其分泌不同的细胞因子以促进不同类型的免疫应答。
Tfh即滤泡辅助性T细胞(Follicular helper T cells,Tfh)是CD4+T细胞的一个重要亚群，主要参与生发中心反应过程。Tfh对于生发中心的形成和维持都必不可少。Tfh通过诱导和维持生发中心B细胞的作 用可以促进抗体产生，抗体类型转换，抗体亲和力成熟，中和性抗体的产生，增加抗体图谱的广泛性等多种效应。因此如何有效地产生Tfh细胞对于多种疫苗研发都属于关键环节。
Th1即1型辅助性T细胞(Type 1 helper T cells,Th1)是另一类重要的CD4+T细胞亚群，其在抗病毒反应和抗细菌反应，特别是对抗胞内细菌感染，如结核菌感染中发挥关键作用。此外，CD4+T细胞对于抗肿瘤免疫也起到重要作用。
在上述方法的一些实施方案中，所述B细胞群体是存在于受试者体内的B细胞群体。例如，所述B细胞群体可以是受试者体内天然存在的B细胞群体，或者是转移到受试者体内的B细胞群体。在这种情况下，本公开的活化CD4+T细胞的方法和促进CD4+T细胞分化的方法可以发生在受试者体内。即，通过直接向受试者施用本公开的基于多聚体的抗原复合物，其可以被受试者体内的B细胞群体识别，并进而活化CD4+T细胞和促进CD4+T细胞向Tfh和/或Th1分化。
在上述方法的另一些实施方案中，所述B细胞群体可以是从供体的外周血或淋巴器官例如胸腺、脾脏和扁桃体等分离的B细胞群体。
在上述方法的进一步的实施方案中，所述方法还可以包括在步骤b)之后对识别所述亚单位的B细胞进行筛选、富集和/或扩增的步骤。在另一些实施方案中，所述方法还可以包括在步骤b)之后对识别所述亚单位的B细胞进行筛选，并将其编码免疫球蛋白受体的基因序列导入所述B细胞群体，以增加所述群体中识别所述亚单位的B细胞的数量的步骤。
B细胞对抗原的特异性识别能力来源于其表面表达的免疫球蛋白受体(B细胞受体，BCR)。不同的B细胞可以表达不同的BCR，并且个体内可能产生的BCR种类可以达到10 12-10 15之多。通过对识别所述亚单位的B细胞进行筛选、富集和/或扩增的步骤，或者将编码识别所述亚单位的BCR的基因序列导入所述B细胞群体，能够增加B细胞群体中识别亚单位的B细胞的数量，从而提高活化CD4+T细胞和/或促进CD4+T细胞分化的效率。
在上述方法的任意实施方案中，所述多聚体的直径可以是约10 nm至约1000nm，例如约10nm至约500nm，约10nm至约300nm，约10nm至约200nm，约10nm至约100nm，约10nm至约50nm，约20nm至约1000nm，约20nm至约500nm，约20nm至约300nm，约20nm至约200nm，约20nm至约100nm，约20nm至约50nm，约50nm至约1000nm，约50nm至约500nm，约50nm至约300nm，约50nm至约200nm，或约50nm至约100nm。
在上述方法的任意实施方案中，所述多聚体可以包含至少4个亚单位，例如包含至少10个亚单位，至少20个亚单位，至少50个亚单位，至少100个亚单位，或至少200个亚单位。所述多聚体可以具有10至1000个亚单位，例如20至500个亚单位，50至300个亚单位，或者100至200个亚单位。
在上述方法的任意实施方案中，所述免疫刺激物可以包含细菌来源的ssRNA、人工合成的ssRNA或其衍生物、人工合成的含CpG的寡核苷酸，干扰素，细胞因子，或其任意组合。在一些实施方案中，所述细菌来源的ssRNA可以是大肠杆菌来源的ssRNA。在一些实施方案中，所述干扰素可以选自I型干扰素、II型干扰素、III型干扰素，及其组合。在一些实施方案中，所述细胞因子可以选自IL-6、IL-12、IL21，及其组合。
在一些实施方案中，可以使用选自以上表1和表2中列出的免疫刺激物的一种或多种，或者本领域已知的其它天然或人工免疫刺激物。
在上述方法的进一步的实施方案中，所述多聚体可以是病毒样颗粒、其它天然多聚体或人工合成的多聚体。多聚体可以由一种亚单位的多个拷贝组装而成，或者可以由两种或更多种亚单位的多个拷贝组装。对多聚体的选择没有具体限制。在一些实施方案中，所述病毒样颗粒可以包含Qβ蛋白，HBcAg或AP205，或者由Qβ蛋白，HBcAg或AP205组成。
在一些实施方案中，Qβ蛋白可以具有以下所示的氨基酸序列：MAKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQAYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY(SEQ ID NO:1)
在一些实施方案中，HBcAg可以具有以下所示的氨基酸序列：MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGNNLEDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC(SEQ ID NO:2)
在一些实施方案中，AP205可以具有以下所示的氨基酸序列：MANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKRPAPKPEGCADACVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAIVSSDTTA(SEQ ID NO:3)
在上述方法的一些实施方案中，所述多聚体例如病毒样颗粒包含目标抗原，或由目标抗原组成。也就是说，多聚体的至少一种亚单位本身可以作为目标抗原。例如，所述目标抗原可以是Qβ蛋白，HBcAg或AP205，或者其它病毒来源的蛋白。
在上述方法的另一些实施方案中，所述抗原复合物包含加载的目标抗原，所述加载的目标抗原可以是细菌或病毒来源的抗原。例如，可以使用选自以上表3中列出的细菌或病毒来源的抗原。
在一些实施方案中，所述细菌或病毒来源的抗原可以是结核分枝杆菌来源的抗原，例如选自晶状体蛋白和Rv3133c。
在一些实施方案中，所述细菌或病毒来源的抗原可以是超级细菌来源的抗原，例如选自肺炎克雷伯菌碳青霉烯酶和青霉素结合蛋白。
在一些实施方案中，所述细菌或病毒来源的抗原可以是慢病毒来源的抗原，例如选自HBV pre-S1抗原和EBV LMP1抗原。
在上述方法的另一些实施方案中，所述抗原复合物包含加载的目标抗原，所述加载的目标抗原可以是肿瘤相关抗原。使用的肿瘤相关抗原没有特别限制，并且可以是对于肿瘤发展或侵袭性相关的任何抗原。例如，可以使用如上文中关于本公开的基于多聚体的抗原复合物中描述的肿瘤相关抗原。
在一些实施方案中，所述肿瘤抗原可以选自Her2、p53或肿瘤新生抗原。
在上述方法的任意实施方案中，所述方法可以是体外方法。例如， 可以在体外使分离的B细胞群体与基于多聚体的抗原复合物接触，使得B细胞识别并处理所述抗原复合物，并将目标抗原呈递至CD4+T细胞，进而活化CD4+T细胞并促进CD4+T细胞分化为Tfh和/或Th1细胞。
在另一些实施方案中，所述方法的一些步骤可以发生在体外，而其余部分发生于体内。例如，可以在体外使分离的B细胞群体与基于多聚体的抗原复合物接触，使得B细胞识别并处理所述抗原复合物，并将目标抗原与MHC II的复合物呈递于细胞表面。随后，可以将B细胞群体施用于受试者，使得B细胞群体在受试者体内活化CD4+T细胞并促进CD4+T细胞分化为Tfh和/或Th1细胞。
在再一些实施方案中，所述方法发生在受试者体内。例如，通过直接向受试者施用本公开的基于多聚体的抗原复合物，其可以被受试者体内的B细胞群体识别，并进而活化CD4+T细胞和促进CD4+T细胞向Tfh和/或Th1分化。例如，所述B细胞群体可以是受试者体内天然存在的B细胞群体，或者是转移到受试者体内的B细胞群体。
预防和/或治疗疾病的方法
在一个方面，本公开提供了预防和/或治疗有此需要的受试者中的疾病的方法，所述方法包括向所述受试者施用有效量的基于多聚体的抗原复合物的步骤，所述多聚体抗原复合物包含：
i)由多个亚单位组装的多聚体；和
ii)免疫刺激物，
其中所述多个亚单位包含目标抗原或由目标抗原组成，且其中所述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学连接附接于所述多聚体；
或者所述抗原复合物包含：
i)由多个亚单位组装的多聚体；
ii)加载的目标抗原；和
iii)免疫刺激物，
其中所述目标抗原通过物理吸附或化学连接附接于所述多聚体 的表面，或通过基因融合与所述多个亚单位的至少一部分融合，所述融合不影响所述多聚体的组装，且在所述多聚体组装后所述目标抗原展示在所述多聚体的表面，并且其中所述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学连接附接于所述多聚体。
在另一个方面，本公开提供了预防和/或治疗有此需要的受试者中的疾病的方法，所述方法包括：
a)从所述受试者分离B细胞群体；
b)使基于多聚体的抗原复合物与所述B细胞群体接触；
i)由多个亚单位组装的多聚体；和
ii)免疫刺激物，
其中所述多个亚单位包含目标抗原或由目标抗原组成，且其中所述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学连接附接于所述多聚体；
或者所述抗原复合物包含：
i)由多个亚单位组装的多聚体；
ii)加载的目标抗原；和
iii)免疫刺激物，
其中所述目标抗原通过物理吸附或化学连接附接于所述多聚体的表面，或通过基因融合与所述多个亚单位的至少一部分融合，所述融合不影响所述多聚体的组装，且在所述多聚体组装后所述目标抗原展示在所述多聚体的表面，并且其中所述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学连接附接于所述多聚体；
其中所述B细胞群体的至少一部分能够识别所述亚单位的至少一种；
c)将所述抗原复合物与所述B细胞群体温育，以允许B细胞识别和处理所述抗原复合物，并将所述目标抗原呈递于细胞表面；
d)将所述B细胞群体施用至所述受试者。
如上文所述，使用本公开的基于多聚体的抗原复合物，能够通过被B细胞识别并呈递，进而活化CD4+T细胞，并促进CD4+T细胞分化为Tfh和/或Th1细胞。由于CD4+T细胞，特别是Tfh和Th1在免疫应 答，尤其是适应性免疫应答中的功能，本公开的活化CD4+T细胞并促进其分化为Tfh和/或Th1的方法可以用于预防和/或治疗疾病。本领域技术人员还将理解，对于预防和/或治疗的疾病的种类没有限制，只要其涉及机体内的免疫应答，包括天然免疫应答和适应性免疫应答即可，并且疾病的实例可以特别是包括各种感染性疾病和癌症。
在上述方法的一些实施方案中，所述B细胞群体是存在于受试者体内的B细胞群体。例如，所述B细胞群体可以是受试者体内天然存在的B细胞群体，或者是转移到受试者体内的B细胞群体。在这种情况下，本公开的活化CD4+T细胞的方法和促进CD4+T细胞分化的方法可以发生在受试者体内。
在上述方法的另一些实施方案中，所述B细胞群体可以是从供体的外周血或淋巴器官例如胸腺、脾脏和扁桃体等分离的B细胞群体。
在上述预防和/或治疗有此需要的受试者中的疾病的方法的进一步的实施方案中，所述方法还可以包括在步骤c)之后对识别所述亚单位的B细胞进行筛选、富集和/或扩增的步骤。在另一些实施方案中，所述方法还可以包括在步骤c)之后对识别所述亚单位的B细胞进行筛选，并将其编码免疫球蛋白受体的基因序列导入所述B细胞群体，以增加所述群体中识别所述目标抗原的B细胞的数量的步骤。
在上述预防和/或治疗有此需要的受试者中的疾病的方法的任意实施方案中，所述多聚体的直径可以是约10nm至约1000nm，例如约10nm至约500nm，约10nm至约300nm，约10nm至约200nm，约10nm至约100nm，约10nm至约50nm，约20nm至约1000nm，约20nm至约500nm，约20nm至约300nm，约20nm至约200nm，约20nm至约100nm，约20nm至约50nm，约50nm至约1000nm，约50nm至约500nm，约50nm至约300nm，约50nm至约200nm，或约50nm至约100nm。
在上述预防和/或治疗有此需要的受试者中的疾病的方法的任意实施方案中，所述多聚体可以包含至少4个亚单位，例如包含至少10个亚单位，至少20个亚单位，至少50个亚单位，至少100个亚单位，或至少200个亚单位。所述多聚体可以具有10至1000个亚单位，例如 20至500个亚单位，50至300个亚单位，或者100至200个亚单位。
在上述预防和/或治疗有此需要的受试者中的疾病的方法的任意实施方案中，所述免疫刺激物可以包含细菌来源的ssRNA、人工合成的ssRNA或其衍生物、人工合成的含CpG的寡核苷酸，干扰素，细胞因子，或其任意组合。在一些实施方案中，所述细菌来源的ssRNA可以是大肠杆菌来源的ssRNA。在一些实施方案中，所述干扰素可以选自I型干扰素、II型干扰素、III型干扰素，及其组合。在一些实施方案中，所述细胞因子可以选自IL-6、IL-12、IL21，及其组合。
在一些实施方案中，可以使用选自以上表1和表2中列出的免疫刺激物的一种或多种，或者本领域已知的其它天然或人工免疫刺激物。
在上述方法的进一步的实施方案中，所述多聚体可以是病毒样颗粒、其它天然多聚体或人工合成的多聚体。多聚体可以由一种亚单位的多个拷贝组装而成，或者可以由两种或更多种亚单位的多个拷贝组装。对多聚体的选择没有具体限制。在一些实施方案中，所述病毒样颗粒可以包含Qβ蛋白，HBcAg或AP205，或者由Qβ蛋白，HBcAg或AP205组成。
在一些实施方案中，Qβ蛋白可以具有以下所示的氨基酸序列：MAKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQAYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY(SEQ ID NO:1)
在一些实施方案中，HBcAg可以具有以下所示的氨基酸序列：MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGNNLEDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC(SEQ ID NO:2)
在一些实施方案中，AP205可以具有以下所示的氨基酸序列：MANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKRPAPKPEGCADACVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAIVSSDTTA(SEQ ID NO:3)
在上述预防和/或治疗有此需要的受试者中的疾病的方法的一些 实施方案中，所述多聚体例如病毒样颗粒包含目标抗原，或由目标抗原组成。例如，所述目标抗原可以是Qβ蛋白，HBcAg或AP205，或者其它病毒来源的蛋白。
在上述预防和/或治疗有此需要的受试者中的疾病的方法的另一些实施方案中，所述疾病是感染性疾病，并且所述抗原复合物包含加载的目标抗原，所述加载的目标抗原是细菌或病毒来源的抗原。例如，可以使用选自以上表3中列出的细菌或病毒来源的抗原。
在一些实施方案中，所述细菌或病毒来源的抗原是结核分枝杆菌来源的抗原，例如选自晶状体蛋白和Rv3133c。
在一些实施方案中，所述细菌或病毒来源的抗原是超级细菌来源的抗原，例如选自肺炎克雷伯菌碳青霉烯酶和青霉素结合蛋白。
在一些实施方案中，所述细菌或病毒来源的抗原是慢病毒来源的抗原，例如选自HBV pre-S1抗原和EBV LMP1抗原。
在上述方法的另一些实施方案中，所述疾病是癌症，癌症的实例包括但不限于：基底细胞癌、胆道癌；膀胱癌；骨癌；脑和CNS癌；乳腺癌；腹膜癌；宫颈癌；胆管癌；绒毛膜癌；结肠和直肠癌；结缔组织癌；消化系统癌症；子宫内膜癌；食道癌；眼癌；头颈癌；胃癌(包括胃肠癌)；胶质母细胞瘤；肝癌；肝癌；上皮内新生物；肾癌；喉癌；白血病；肝癌；肺癌(例如，小细胞肺癌、非小细胞肺癌、肺腺癌和肺鳞状细胞癌)；淋巴瘤，包括霍奇金淋巴瘤和非霍奇金淋巴瘤；黑素瘤；骨髓瘤；神经母细胞瘤；口腔癌(例如唇、舌、口、和咽)；卵巢癌；胰腺癌；前列腺癌；视网膜母细胞瘤；横纹肌肉瘤；直肠癌；呼吸系统癌；唾液腺癌；肉瘤；皮肤癌；鳞状细胞癌；胃癌；畸胎癌；睾丸癌；甲状腺癌；子宫或子宫内膜癌；泌尿系统癌症；外阴癌；以及其它癌和肉瘤；以及B细胞淋巴瘤(包括低级别/滤泡型非霍奇金淋巴瘤(NHL)；小淋巴细胞性(SL)NHL；中级/滤泡型NHL；中等级弥漫性NHL；高级免疫母细胞性NHL；高级免疫母细胞性NHL；高等级小非裂解细胞NHL；大体积疾病NHL(bulky disease NHL)；套细胞淋巴瘤；AIDS相关淋巴瘤；和沃尔登斯特伦(Waldenstrom)巨球蛋白血症)；慢性淋巴细胞性白血病(CLL)；急性成淋巴细胞性白血病 (ALL)；毛细胞白血病；慢性成髓细胞性白血病；和移植后淋巴增生性疾病(PTLD)，以及与斑痣性错构瘤病相关的异常血管增生，水肿(如与脑肿瘤相关的水肿)、原始起源肿瘤和梅格斯(Meigs)氏综合征等。
在治疗的疾病是癌症的情况下，所述加载的目标抗原是肿瘤相关抗原，使用的肿瘤相关抗原没有特别限制，并且可以是对于肿瘤发展或侵袭性相关的任何抗原。例如，可以使用如上文中关于本公开的基于多聚体的抗原复合物中描述的肿瘤相关抗原。在一些实施方案中，所述肿瘤相关抗原可以选自Her2、p53和肿瘤新生抗原。
许多肿瘤抗原已经根据多种实体瘤定义：MAGE 1、2和3，由免疫定义；MART-1/Melan-A、gp100、癌胚抗原(CEA)、HER-2、粘蛋白(即MUC-1)、前列腺特异性抗原(PSA)和前列腺酸性磷酸酶(PAP)。此外，已经显示了乙肝(HBV)、埃巴(EBV)和人乳头瘤(HPV)等病毒蛋白分别在肝细胞癌、淋巴瘤和子宫颈癌的发展中起重要作用。然而，肿瘤使用或受益于一系列不同的免疫逃避机制，使得癌症患者的免疫系统通常不能对肿瘤抗原响应。通常与睾丸、胎盘和卵巢的精母细胞或精原细胞相关的癌抗原的一些实例包括癌症-睾丸(CT)抗原BAGE、GAGE、MAGE-1和MAGE-3、NY-ESO-1、SSX。这些抗原存在于黑素瘤、淋巴瘤、肺癌、膀胱癌、结肠癌和乳腺癌中。通常在黑素细胞、上皮组织、前列腺和结肠中发现的肿瘤相关抗原还包括分化抗原Gp100、Melan-A/Mart-1、酪氨酸酶、PSA、CEA和Mammaglobin-A。这些抗原存在于黑素瘤、前列腺癌和结肠癌和乳腺癌中。一些肿瘤相关抗原是以低水平普遍表达但在癌症中过表达的共享抗原。过表达的肿瘤相关抗原的实例包括在食道、肝脏、胰腺、结肠、乳腺、卵巢、膀胱和前列腺癌中发现的p53、HER-2/neu、livin和存活蛋白。其它肿瘤相关抗原是独特的，如与黑素瘤、非小细胞肺癌和肾癌的一种或多种相关的β-连环蛋白-m、β-肌动蛋白/4/m、肌球蛋白/m、HSP70-2/m和HLA-A2-R170J。其它肿瘤相关抗原是通常在上皮组织如肾、肠和结肠直肠组织中发现的肿瘤相关的碳水化合物抗原。这些肿瘤相关抗原包括可以在黑素瘤、成神经细胞瘤、结肠直肠癌、肺癌、乳腺癌、卵巢癌和前列腺癌中发现的GM2、GD2、GD3、MUC-1、sTn、abd  globo-H。
如本文所用，术语“治疗”是指治疗性处理，其中目的是反转、减轻、改善、抑制、减缓或停止与疾病或病症相关的状况的进展或严重性。术语“治疗”包括减少或减轻疾病或病症的至少一种副作用或症状。如果减少一种或多种症状或临床标志物，则治疗通常是“有效的”。或者，如果疾病的进展减少或停止，则治疗是“有效的”，也就是说，“治疗”不仅包括症状或标志物的改善，而且还包括在缺乏治疗的情况下预期的症状的进展或恶化的停止，至少减慢。有益或期望的临床结果包括但不限于减轻一种或多种症状、减少疾病程度，稳定(即不恶化)疾病状态、延迟或减缓疾病进展、改善或缓解疾病状态、和缓解(不管是部分还是全部)，无论是可检测的还是检测不到的。疾病的术语“治疗”还包括缓解疾病的症状或副作用(包括姑息治疗)。
如本文所用，术语“受试者”和“个体”在本文中可互换使用，并且是指动物，并且包括哺乳动物如大鼠、小鼠、兔、绵羊、猫、狗、牛、猪和非人灵长类动物。术语“受试者”还包括任何脊椎动物，包括但不限于哺乳动物、爬行动物、两栖动物和鱼。然而，有利地，受试者是哺乳动物如人或其它哺乳动物，例如驯养的哺乳动物，例如狗、猫、马等。生产哺乳动物，如牛、绵羊、猪等也包括在术语受试者中。
在上述预防和/或治疗疾病的方法的任意实施方案中，所述方法可以进一步包括对所述受试者施用其它疗法，例如抗癌疗法、化疗剂或免疫调节剂。在一个实施方案中，所述免疫调节剂包含免疫检查点抑制剂。在一个实施方案中，免疫检查点抑制剂结合下列一项或多项：PD1、PDL1、PDL2、CTLA4、LAG3、TIM3、TIGIT和/或CD103。在一个实施方案中，免疫检查点抑制剂是PD1、PDL1、和/或PDL2抑制剂。
术语“抗癌疗法”是指可用于治疗癌症的疗法。抗癌治疗剂的实例包括但不限于例如手术、化疗剂、生长抑制剂、细胞毒性剂、放射疗法和用于放射疗法的试剂、抗血管生成剂、凋亡剂、抗微管蛋 白剂、和其它治疗癌症的试剂，如抗HER-2抗体、抗CD20抗体、表皮生长因子受体拮抗剂、HER1/EGFR抑制剂、血小板衍生生长因子抑制剂、COX-2抑制剂、干扰素、细胞因子、拮抗剂，其结合一种或多种以下靶物PD1、PDL1、PDL2；CTLA4；LAG3；CD103；TIM-3和/或其它TIM系列成员；CEACAM-1和/或其它CEACAM家族成员、ErbB2、ErbB3、ErbB4、PDGFR-β、BlyS、APRIL、BCMA或VEGF受体、TRAIL/Apo2和其它生物活性和有机化学剂等。本文所述的方法也特别考虑其组合。
在一些实施方案中，抗癌疗法包括诸如过继性细胞转移的免疫治疗。本文所用，“过继性细胞转移”是指涉及遗传工程化受试者或患者自身的T细胞以在其表面上产生称为嵌合抗原受体(CAR)的特殊受体的免疫疗法。CAR是允许T细胞识别肿瘤细胞上的特定蛋白质(抗原)的蛋白质。然后，将这些工程化的CAR T细胞在实验室中培养直至它们以数十亿计数。然后，将CAR T细胞的扩大群体输注入患者中。输注后，T细胞在受试者的身体中繁殖，并在其工程受体的指导下识别并杀死其表面上携带抗原的癌细胞。
如本文所用，术语“化学疗法”或“化疗剂”是指具有在治疗以异常细胞生长为特征的疾病中的治疗有用性的任何化学试剂。此类疾病包括肿瘤、新生物和癌症以及以增生性生长为特征的疾病。如本文所用的化学治疗剂包括化学剂和生物剂。这些试剂发挥功能以抑制癌细胞实现持续存活所依赖的细胞活性。化疗剂的类别包括烷化/生物碱剂、抗代谢物、激素或激素类似物，以及各种各样的抗新生物药物。大多数(如果不是全部)这些试剂对癌细胞是直接毒性的，并且不需要免疫刺激。在一个实施方案中，化疗剂是用于治疗新生物如实体瘤的试剂。在一个实施方案中，化疗剂是放射性分子。本领域技术人员可以容易地鉴定使用的化学治疗剂。
“放射疗法”是指使用定向性γ射线或β射线来诱导对细胞的足够损伤，以限制其正常发挥功能或完全破坏细胞的能力。应当理解，本领域中将存在许多已知的方法来确定治疗的剂量和持续时间。
药物组合物和用途
在一个方面，本公开涉及药物组合物，所述药物组合物包含本公开的基于多聚体的抗原复合物，以及任选的一种或多种其它治疗剂和/或药学上可接受的载体。
包含本公开的肽的药物组合物可以根据需要通过常规制剂方法配制。除了本公开的肽之外，本公开的药物组合物可以包含通常用于药物中的载体，赋形剂等，而没有特别限制。可用于本公开的药物组合物中的载体的例子包括灭菌水(例如注射用水)，生理盐水，磷酸盐缓冲液，磷酸盐缓冲盐水，Tris缓冲盐水，0.3％甘氨酸，培养液，等等。此外，本公开的药物组合物可根据需要含有稳定剂、悬浮剂、防腐剂、表面活性剂、增溶剂、pH调节剂、聚集抑制剂，等等。本公开的药物组合物可以诱导针对表达URLC10的癌细胞的特异性免疫，因此可以用于癌症治疗或预防(防范)的目的。
短语“药学上可接受的载体”是指药学上可接受的材料、组合物或媒介物，如液体或固体填充剂、稀释剂、赋形剂、溶剂、介质、包封材料、制造助剂(例如润滑剂、滑石镁、硬脂酸钙或锌或硬脂酸)或溶剂包封材料，其涉及维持LAP结合剂的稳定性、溶解度或活性。在与配制剂的其它成分相容并且不对患者有害的意义上，每种载体必须是“可接受的”。可充当药学上可接受的载体的材料的一些实例包括：(1)糖，如乳糖、葡萄糖和蔗糖；(2)淀粉，如玉米淀粉和马铃薯淀粉；(3)纤维素及其衍生物，如羧甲基纤维素钠、甲基纤维素、乙基纤维素、微晶纤维素和乙酸纤维素；(4)粉状黄蓍胶；(5)麦芽；(6)明胶；(7)赋形剂，如可可脂和栓剂蜡；(8)油，如花生油、棉籽油、红花油、芝麻油、橄榄油、玉米油和大豆油；(9)二醇，如丙二醇；(10)多元醇，如甘油、山梨糖醇、甘露醇和聚乙二醇(PEG)；(11)酯，如油酸乙酯和月桂酸乙酯；(12)琼脂；(13)缓冲剂，如氢氧化镁和氢氧化铝；(14)海藻酸；(15)无热原水；(16)等张盐水；(17)林格氏溶液；(19)pH缓冲溶液；(20)聚酯、聚碳酸酯和/或聚酐；(21)填充剂，如多肽和氨基酸(22)血清组分，如血清白蛋白、HDL和LDL；(23)C2-C12醇，如乙醇；和(24)药物配制剂中使用的其它无毒相容物质。释放剂、包衣剂、防腐 剂和抗氧化剂也可以存在于药物配制剂中。诸如“赋形剂”、“载体”、“药学上可接受的载体”等的术语在本文中可互换使用。
本公开的药物组合物还可以包含已知用于有效建立细胞免疫的佐剂。佐剂是指当与抗原一起(或连续)施用时增强针对具有免疫学活性的抗原的免疫应答的化合物。可以使用描述于文献中的已知佐剂，例如Clin Microbiol Rev 1994,7:277-89。合适佐剂的例子包括铝盐(磷酸铝，氢氧化铝，羟基氧化铝等),明矾,霍乱毒素,沙门氏菌毒素,IFA(不完全弗氏佐剂),CFA(完全弗氏佐剂),ISCOMatrix,GM-CSF和其他免疫刺激细胞因子，含有CpG基序的寡脱氧核苷酸(CpG7909等)，水包油乳液，皂苷或其衍生物(QS21等)，脂多糖例如脂质A或其衍生物(MPL,RC529,GLA,E6020等)，脂肽，乳铁蛋白，鞭毛蛋白，双链RNA或其衍生物(poli IC等)，细菌DNA，咪唑并喹啉(Imiquimod,R848等)，C型凝集素配体(海藻糖-6,6’-二山嵛酸/酯(TDB)等)，CD1d配体(alpha-半乳糖神经酰胺等)，角鲨烯乳液(MF59,AS03,AF03等),PLGA等，但不限于此。在包含本公开的药物组合物的试剂盒中，佐剂可以包含在与包含本公开的肽的药物组合物分开的另一容器中。在这种情况下，佐剂和药物组合物可以连续地施用于受试者，或者在施用给受试者之前立即混合在一起。本公开还提供了包含含有本公开的肽药物组合物和佐剂的此类试剂盒。当本公开的药物组合物是冷冻干燥制剂时，试剂盒还可以包含再溶解溶液。此外，本公开提供试剂盒，其包括容纳本公开的药物组合物的容器和存储佐剂的容器。试剂盒可以根据需要进一步包含储存再溶解溶液的容器。
根据本公开的这些组合物的施用可以通过任何常见途径，只要靶组织可通过该途径可用。用于施用本公开的肽或药物组合物的合适方法的实例包括口服、皮内、皮下、肌肉内、骨内、腹膜内和静脉内注射，以及全身施用或局部施用至靶位点附近，但不限于此。
在本公开的某些实施方案中，本公开的药物组合物与用于施用的装置包装在一起或储存在用于施用的装置中。用于可注射制剂的装置包括但不限于注射口，自动注射器，注射泵和注射笔。用于雾化或粉 末制剂的装置包括但不限于吸入器，吹入器，抽吸器等。因此，本公开包括包含本公开的药物组合物的施用装置，用于治疗或预防本文所述的一种或多种病症。
在另一个方面，本公开涉及本公开的基于多聚体的抗原复合物或药物组合物在活化CD4+T细胞和/或促进CD4+T细胞分化为Tfh和/或Th1细胞中的用途。
在再一个方面，本公开涉及本公开的基于多聚体的抗原复合物或药物组合物在预防和/或治疗疾病中的用途。在一些实施方案中，所述疾病选自感染性疾病和癌症。例如，在上文关于预防和/或治疗疾病的方法中所述的感染性疾病和癌症。
在一个方面，本公开涉及本公开的基于多聚体的抗原复合物在制备用于活化CD4+T细胞和/或促进CD4+T细胞分化为Tfh和/或Th1细胞的药物组合物中的用途。
在另一个方面，本公开涉及本公开的基于多聚体的抗原复合物在制备用于治疗疾病的药物组合物中的用途。在一些实施方案中，所述疾病选自感染性疾病和癌症。例如，在上文关于预防和/或治疗疾病的方法中所述的感染性疾病和癌症。
实施例
通过参考以下实施例进一步说明本公开的实施方案。然而，应该注意的是，这些实施例与上述实施方案一样是说明性的且不应被解释为以任何方式限制本公开的范围。
实施例1：基于多聚体的抗原复合物能够强力活化CD4+T细胞
细菌噬菌体Qβ衍生的VLP(Qβ-VLP)由单一类型的单体组装而成，并在内部含有核酸。先前的研究表明Qβ-VLP能够在不使用任何常规佐剂的情况下诱导强力的抗体应答，包括GC应答(Gatto,D.et al.,(2004).Rapid response of marginal zone B cells to viral particles.J Immunol 173,4308-4316；Liao,W.et al.,(2017).Characterization of T-Dependent and T-Independent B Cell Responses to a Virus-like Particle.J Immunol 198,3846-3856)。已知Qβ-VLP包封的核酸作为免 疫刺激物，对于其强免疫原性是重要的，该免疫刺激物可以是来源于宿主细菌的ssRNA或人工合成的含有CpG的寡脱氧核苷酸(CpG ODN)，其作为TLR7或TLR9的配体增强免疫应答(Jegerlehner,A.et al.,(2007).TLR9 signaling in B cells determines class switch recombination to IgG2a.J Immunol 178,2415-2420)。
为了探索CD4+T细胞如何响应于Qβ-VLP而活化，我们生成了由Qβ蛋白以及Qβ蛋白和卵清蛋白衍生肽的融合蛋白两者组装的VLP，所述卵清蛋白衍生肽可以被CD4+TCR转基因T细胞(OT-II)识别。组装的VLP多聚体的180个单体中约10-15％被融合蛋白替换，其对应于每个颗粒中约20-30个拷贝的OT-II CD4+T细胞表位。生成的这种VLP命名为Qβ-Ova。
为了探索Qβ-VLP如何活化CD4+T细胞，我们将5x10 5个CFSE标记的幼稚OT-II CD4+T细胞(CD4+CD44lo CD62Lhi)过继转移到野生型(WT)小鼠中，然后在一天后用Qβ-Ova进行腹膜内免疫。随后，处死小鼠并分离其脾脏细胞进行流式细胞术检测。将来自OT-II供体的CD4+T细胞作为Thy 1.1+从脾脏的总CD4+T细胞中进行门控。结果表明，在免疫后的不同时间点，OT-II CD4+T细胞显示出强烈的扩增(图2A)，其与这些细胞中广泛的CFSE稀释一致(图2B)。在该结果中，将CFSE稀释度定量为增殖指数，其反应了免疫后细胞分裂的平均数量。
除了细胞增殖外，我们评估了T细胞活化的标志物，包括CD44的上调和CD62L的下调(CD44 hi和CD62 lo)。如图2C中的结果所示，在大部分CFSE稀释(即经历增殖)的细胞中观察到CD44的上调和CD62L的下调，表明这些T细胞的活化。
为了进一步Qβ-VLP免疫是否引起CD4+T细胞的分化，我们检查了OT-II CD4+T细胞中与不同Th谱系的分化相关的分子标志物。结果表明，在使用Qβ-VLP后，OT-II CD4+T细胞中的CXCR5和PD-1均显著上调，并且在一部分OT-II CD4+T细胞中，Bcl-6也显著上调(图3A)，表明它们向Tfh谱系的分化。此外，在大量OT-II CD4+T细胞中，向Th1分化的转录因子T-bet，及其靶基因CXCR3也被显著诱导，表明 Qβ-Ova也促进了CD4+T细胞向Th1细胞分化(图3B)。
实施例2.B细胞而非DC中的TLR信号是Qβ-VLP诱导的CD4+T细胞的活化和分化所需的
我们进一步研究了B细胞在Qβ-VLP免疫后OT-II CD4+T细胞的活化和分化中的功能。具体而言，我们研究了B细胞中TLR信号传导的下游的衔接蛋白MyD88的缺陷是否会影响Qβ-VLP免疫后OT-II CD4+T细胞的活化。为了该目的，我们使用了B细胞中缺乏MyD88的小鼠(MyD88 fl/fl CD79a-Cre，称为B-MyD88-/-)。结果发现，在免疫后第3天与WT小鼠相比，B-MyD88-/-小鼠中OT-II CD4+T细胞扩增明显减少，增殖指数显著降低(图4A)。更重要的是，在B-MyD88-/-小鼠的CFSE稀释细胞中，Qβ-VLP免疫对Tfh和Th1标志物分子的诱导存在显著缺陷(图4B)。上述结果表明，B细胞中的TLR信号传导对于Qβ-Ova诱导的CD4+T细胞活化和分化是必需的。
为了确定B细胞在抗原诱导的B细胞活化中的该作用是否依赖于抗原的形式，在OT-II CD4+T细胞转移后我们使用可溶性Ova与CpG ODN的混合物对小鼠进行免疫，并如上文所述检测OT-II CD4+T细胞中T细胞活化和分化的标志物是否被诱导。发现WT和B-MyD88-/-小鼠之间的OT-II CD4+T细胞增殖或细胞分化没有明显差异(图5A和5B)。该结果表明，在使用可溶性抗原的情况下，B细胞中的TLR信号不是诱导CD4+T细胞活化所必需的。
由于目前已知DC广泛地参与起始CD4+T细胞应答(Iwasaki,A.,and Medzhitov,R.(2010).Regulation of adaptive immunity by the innate immune system.Science 327,291-295)，我们进一步研究了DC中的TLR信号传导是否也能够促进由Qβ-Ova诱导的CD4T细胞活化。令人惊讶地发现，在Qβ-Ova免疫后，DC中缺乏MyD88的小鼠(MyD88 fl/fl CD11c-Cre，称为DC-MyD88-/-)与野生型小鼠相比在OT-II CD4+T细胞增殖和分化方面没有显示出缺陷(图6A和6B)。上述结果表明，DC中的TLR信号不是诱导CD4+T细胞活化所必需的。
实施例3.缺乏Qβ特异性B细胞的小鼠无法起始Qβ-VLP免疫诱导的CD4+T细胞活化。
Qβ-Ova在B细胞而非DC中引发TLR信号来促进CD4+T细胞的活化和分化这一事实使我们考虑B细胞是否直接参与早期T细胞应答期间CD4+T细胞的活化。与DC不同，B细胞通过特异性B细胞抗原受体(BCR)与抗原结合。因此，我们测试了缺乏能够与Qβ-VLP特异性结合的BCR的小鼠在Qβ-Ova诱导的CD4+T细胞活化和分化方面是否存在缺陷。在该实验中，我们使用了先前发现BCR转基因小鼠(MD4)，其表达识别鸡蛋溶菌酶的BCR(Goodnow,C.C.et al.,(1988).Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice.Nature 334,676-682)，并含有极少的Qβ特异性B细胞(Liao,W.et al.,(2017).Characterization of T-Dependent and T-Independent B Cell Responses to a Virus-like Particle.J Immunol 198,3846-3856)。
我们将幼稚OT-II CD4+T细胞转移到MD4小鼠中，然后使用Qβ-Ova进行免疫，并在免疫后第3天评估T细胞的活化和分化。令人惊讶的是，用Qβ-Ova免疫后，MD4小鼠中的OT-II CD4+T细胞应答存在严重缺陷。具体而言，MD4小鼠中的增殖指数和OT-II CD4+T细胞百分比远低于WT小鼠，表明细胞增殖存在严重缺陷(图7A)。此外，MD4小鼠中仅有极少数OT-II CD4+T细胞响应Qβ-Ova免疫而表现出CD4+T细胞分化标志物的上调(图7B和7C)。
为了确定MD4小鼠是否可能存在影响CD4+T细胞活化的任何其它因素，我们在OT-II CD4+T细胞转移后用可溶性Ova与CpG ODN的混合物对MD4小鼠进行免疫。结果表明，相比于野生型小鼠，MD4小鼠中响应可溶性Ova的CD4+T细胞活化没有明显缺陷(图7A-图7C)。该结果表明，在使用可溶性抗原形式进行免疫的情况下，CD4+T细胞的活化不依赖于抗原特异性B细胞的存在。由于在MD4小鼠中，其他APC例如DC是完全功能性的，因此推测其能够起始可溶性抗原诱导的CD4+T细胞活化。
综上所述，对于基于多聚体的抗原复合物例如Qβ-Ova诱导的 CD4+T细胞的活化和分化，抗原特异性B细胞是必需的。
实施例4.DC对于Qβ-VLP诱导的CD4+T细胞活化不是必需的
为了进一步证明Qβ-VLP诱导的CD4+T细胞活化依赖于B细胞而非DC，我们产生了嵌合体小鼠，其具有来自CD11c-DTR/GFP小鼠的骨髓(BM)。CD11c-DTR/GFP小鼠在CD11c启动子的控制下表达白喉毒素受体和GFP的融合蛋白(Jung,S.et al.,(2002).In vivo depletion of CD11c+dendritic cells abrogates priming of CD8+T cells by exogenous cell-associated antigens.Immunity 17,211-220)。在该嵌合体小鼠中，在DC细胞表面表达白喉毒素受体。在OT-II T细胞转移时，使用白喉毒素处理该嵌合体，随后在一天后进行免疫。该处理有效地删除了嵌合体小鼠中的DC。在免疫后24小时，我们检查了嵌合体小鼠中T细胞的活化状况，并发现T细胞活化的标志物，包括CD69、CD62L和CD25发生显著变化(图8)。该结果出乎意料的表明，DC的删除对于CD4+T细胞活化没有明显影响，表明DC对于Qβ-VLP免疫诱导的CD4+T细胞活化不是必需的。为了确认在上述嵌合体小鼠中白喉毒素处理对DC的删除，我们测试了可溶性Ova免疫诱导的CD4+T细胞活化。如所预期的，当DC删除时，可溶性抗原诱导的CD4+T细胞活化被大大抑制。
实施例5.在体内抗原特异性B细胞有效地捕获的Qβ-VLP。
上述结果强烈表明抗原特异性B细胞在基于多聚体的抗原复合物诱导的CD4+T细胞活化中发挥功能。我们进一步研究了在体内，B细胞如何捕获此类基于多聚体的抗原复合物。为了跟踪免疫后Qβ-VLP如何被抗原呈递细胞结合和识别，我们将AF647标记的Qβ-VLP(Qβ-AF647)或PBS静脉内注射到WT小鼠中，并在注射后3小时处死小鼠并进行脾脏检测。
结果表明，脾脏中只有非常低百分比的DC在注射后表现出Qβ-AF647的结合(图9A)。通过检查MHCII+细胞(包括B细胞和DC)内的Qβ-AF647+细胞，我们发现在WT小鼠中绝大多数Qβ-AF647+ MHCII+细胞是B细胞非DC(图9B)。此外，在Qβ-AF647+MHCII+的B细胞中，约5％表现出高水平的Qβ-AF647结合。这种高水平的Qβ-AF647结合是由特异性BCR介导的，因为在MD4小鼠中不存在这些高水平的Qβ-AF647结合的细胞(图9B)。
我们接下来检查了显示高水平Qβ-AF647结合的B细胞是否表现出有利于抗原呈递的特征。我们对这些细胞进行了富集。具体而言，将来自注射了Qβ-AF647的小鼠的脾细胞与FITC标记的Qβ-VLP(Qβ-FITC)温育，然后与抗FITC缀合的磁珠温育，以富集Qβ+B细胞。在富集的细胞中，绝大部分表现出与Qβ-AF647的高水平结合(图9C)。更值得注意的是，来自富集级分的这些Qβ-AF647+B细胞中的大多数是CD83+(图9C)。CD83是一种B细胞活化后上调的分子，并参与MHC II的翻译后调节(Tze,L.E.et al.,(2011).CD83increases MHC II and CD86 on dendritic cells by opposing IL-10-driven MARCH1-mediated ubiquitination and degradation.J Exp Med 208,149-165)。上述结果表明Qβ-VLP能够有效地活化抗原特异性B细胞。此外，从免疫24小时的小鼠中富集的Qβ+B细胞表现出共刺激分子CD86和趋化性受体CCR7的显著上调(图10)，表明其刺激T细胞和迁移至T细胞区的能力增强。
实施例6.Qβ-VLP诱导的CD4+T细胞活化需要B细胞的抗原呈递
上述结果强烈提示活化的Qβ特异性B细胞充当抗原呈递细胞(APC)来活化T细胞的能力。为了进一步评估同源T细胞-B细胞相互作用在Qβ-Ova免疫后CD4+T细胞活化中的重要性，我们通过将来自MHC II-/-(20％)和μMT(80％)小鼠的混合骨髓细胞移植到致死照射的WT小鼠，生成了在B细胞中选择性缺失MHC II的小鼠(B-MHCII-/-)。正如我们预期的，转移至B-MHCII-/-小鼠的OT-II CD4+T细胞响应于Qβ-Ova免疫的细胞扩增的显著降低(图11)。上述结果证明，抗原特异性B细胞和CD4+T细胞之间的同源相互作用在Qβ-VLP诱导的CD4+T细胞活化中占主导地位。
实施例7.抗原特异性B细胞参与由流感病毒诱导的CD4+T细胞活化
上述实施例证明了B细胞对于基于聚合物的抗原复合物诱导的T细胞活化和分化中发挥重要作用。我们推测这种机制对于防御病毒方面是进化上保守的途径，特别是在病毒血症期间。在病毒血症的情况下，病毒抗原可以直接传播到脾脏并通过鼻窦和边缘带到达B细胞滤泡。因此，抗原特异性B细胞，其也具有用于感测病毒核苷酸的TLR，可能对血液中的病原体极其敏感，并且负责启动适应性免疫系统。为了验证该假设，我们使用携带OT-II CD4+T细胞表位的经修饰的甲型流感病毒株(PR8-OVA)(Hua,L.et al.,(2013).Cytokine-dependent induction of CD4+T cells with cytotoxic potential during influenza virus infection.J Virol 87,11884-11893)进行免疫。在OT-II CD4+T细胞转移到WT或MD4小鼠中后，将福尔马林灭活的PR8-OVA腹膜内注射到小鼠中。结果显示，与WT小鼠相比，MD4小鼠在PR8-OVA免疫后表现出CD4+T细胞扩增和向辅助性T分化的显著降低(图12A和12B)。为了测试B细胞是否足以在不存在DC的情况下起始CD4+T细胞活化，我们在OT-II CD4T细胞转移和白喉毒素处理后用灭活的PR8-OVA免疫CD11c-DTR/GFP嵌合小鼠。对照和白喉毒素处理的小鼠之间的CD4+T细胞活化没有差异(图12C)，表明DC对于甲型流感病毒诱导的CD4+T细胞应答不是必需的。
在不脱离本公开的范围和精神的情况下，本公开所述方法和系统的各种修改和变化对于本领域技术人员来说是显而易见的。尽管已经结合特定的优选实施方案描述了本公开，但是应该理解，要求保护的本公开不应过度限于这些特定的实施方案。实际上，对于分子生物学、免疫学或相关领域的技术人员显而易见的用于实施本公开的所述模式的各种修改旨在落入所附权利要求书的范围内。

Claims (47)

  1. 一种活化CD4+T细胞的方法，所述方法包括以下步骤：
    a)使基于多聚体的抗原复合物与B细胞群体接触，
    所述抗原复合物包含：
    i)由多个亚单位组装的多聚体；和
    ii)免疫刺激物，
    其中所述多个亚单位包含目标抗原或由目标抗原组成，且其中所述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学连接附接于所述多聚体；
    或者所述抗原复合物包含：
    i)由多个亚单位组装的多聚体；
    ii)加载的目标抗原；和
    iii)免疫刺激物，
    其中所述目标抗原通过物理吸附或化学连接附接于所述多聚体的表面，或通过基因融合与所述多个亚单位的至少一部分融合，所述融合不影响所述多聚体的组装，且在所述多聚体组装后所述目标抗原展示在所述多聚体的表面；并且其中所述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学连接附接于所述多聚体，
    其中所述B细胞群体的至少一部分能够识别所述亚单位的至少一种；
    b)将所述抗原复合物与所述B细胞群体温育，以允许B细胞识别和处理所述抗原复合物，并将所述目标抗原呈递于细胞表面；
    c)使所述B细胞群体与CD4+T细胞接触，以活化所述CD4+T细胞。
  2. 一种促进CD4+T细胞分化为滤泡辅助性T细胞(Tfh)和/或辅助性T细胞1(Th1)的方法，所述方法包括以下步骤：
    a)使基于多聚体的抗原复合物与B细胞群体接触，
    所述抗原复合物包含：
    i)由多个亚单位组装的多聚体；和
    ii)免疫刺激物，
    其中所述多个亚单位包含目标抗原或由目标抗原组成，且其中所述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学连接附接于所述多聚体；
    或者所述抗原复合物包含：
    i)由多个亚单位组装的多聚体；
    ii)加载的目标抗原；和
    iii)免疫刺激物，
    其中所述目标抗原通过物理吸附或化学作用附接于所述多聚体的表面，或通过基因融合与所述多个亚单位的至少一部分融合，所述融合不影响所述多聚体的组装，且在所述多聚体组装后所述目标抗原展示在所述多聚体的表面；并且其中所述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学作用附接于所述多聚体，
    其中所述B细胞群体的至少一部分能够识别所述亚单位的至少一种；
    b)将所述抗原复合物与所述B细胞群体温育，以允许B细胞识别和处理所述抗原复合物，并将所述目标抗原呈递于细胞表面；
    c)使所述B细胞与CD4+T细胞接触，以促进所述CD4+T细胞分化为Tfh和/或Th1。
  3. 如权利要求1或2所述的方法，其中所述B细胞群体是从供体的外周血或淋巴器官分离的B细胞群体。
  4. 如权利要求1-3中任一项所述的方法，其中所述方法还包括在步骤b)之后对识别所述亚单位的B细胞进行筛选、富集和/或扩增的步骤。
  5. 如权利要求1-3中任一项所述的方法，其中所述方法还包括在步骤b)之后对识别所述亚单位的B细胞进行筛选，并将其编码免疫球蛋白受体的基因序列导入所述B细胞群体，以增加所述群体中识别所述亚单位的B细胞的数量的步骤。
  6. 如权利要求1-5中任一项所述的方法，其中所述多聚体的直径为10nm至1000nm。
  7. 如权利要求1-6中任一项所述的方法，其中所述多聚体包含至少4个亚单位。
  8. 如权利要求1-7中任一项所述的方法，其中所述免疫刺激物选自细菌来源的ssRNA、人工合成的ssRNA或其衍生物、人工合成的含CpG的寡核苷酸，干扰素，细胞因子，及其组合。
  9. 如权利要求8所述的方法，其中所述细菌来源的ssRNA是大肠杆菌来源的ssRNA。
  10. 如权利要求8所述的方法，其中所述干扰素选自I型干扰素、II型干扰素、III型干扰素，及其组合。
  11. 如权利要求8所述的方法，其中所述细胞因子选自IL-6、IL-12、IL21，及其组合。
  12. 如权利要求1-11中任一项所述的方法，其中所述多聚体是病毒样颗粒。
  13. 如权利要求12所述的方法，其中所述病毒样颗粒包含Qβ蛋白，HBcAg或AP205，或由Qβ蛋白，HBcAg或AP205组成。
  14. 如权利要求12或13所述的方法，其中所述病毒样颗粒包含目标抗原，或由目标抗原组成。
  15. 如权利要求1-13中任一项所述的方法，其中所述抗原复合物包含加载的目标抗原，所述加载的目标抗原是细菌或病毒来源的抗原。
  16. 如权利要求15所述的方法，其中所述目标抗原是结核分枝杆菌来源的抗原。
  17. 如权利要求16所述的方法，其中所述目标抗原选自晶状体蛋白和Rv3133c。
  18. 如权利要求15所述的方法，其中所述目标抗原是超级细菌来源的抗原。
  19. 如权利要求18所述的方法，其中所述目标抗原选自肺炎克雷伯菌碳青霉烯酶和青霉素结合蛋白。
  20. 如权利要求15所述的方法，其中所述目标抗原是慢病毒来源的抗原。
  21. 如权利要求20所述的方法，其中所述目标抗原选自HBV pre-S1抗原和EBV LMP1抗原。
  22. 如权利要求1-12中任一项所述的方法，其中所述抗原复合物包含加载的目标抗原，所述加载的目标抗原是肿瘤相关抗原。
  23. 如权利要求22所述的方法，其中所述肿瘤相关抗原选自Her2、p53和肿瘤新生抗原。
  24. 如权利要求1-23中任一项所述的方法，其中所述方法是体外方法。
  25. 如权利要求1-23中任一项所述的方法，其中所述方法的步骤c)发生在体内。
  26. 预防和/或治疗有此需要的受试者中的疾病的方法，所述方法包括：
    a)从所述受试者分离B细胞群体；
    b)使基于多聚体的抗原复合物与所述B细胞群体接触；
    所述抗原复合物包含：
    i)由多个亚单位组装的多聚体；和
    ii)免疫刺激物，
    其中所述多个亚单位包含目标抗原或由目标抗原组成，且其中所述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学连接附接于所述多聚体；
    或者所述抗原复合物包含：
    i)由多个亚单位组装的多聚体；
    ii)加载的目标抗原；和
    iii)免疫刺激物，
    其中所述目标抗原通过物理吸附或化学连接附接于所述多聚体的表面，或通过基因融合与所述多个亚单位的至少一部分融合，所述融合不影响所述多聚体的组装，且在所述多聚体组装后所述目标抗原展示在所述多聚体的表面；并且其中所述免疫刺激物包装在所述多聚体内，或通过物理吸附或化学连接附接于所述多聚体；
    其中所述B细胞群体的至少一部分能够识别所述亚单位的至少一 种；
    c)将所述抗原复合物与所述B细胞群体温育，以允许B细胞识别和处理所述抗原复合物，并将所述目标抗原呈递于细胞表面；
    d)将所述B细胞群体施用至所述受试者。
  27. 如权利要求26所述的方法，其中所述B细胞群体是从受试者的外周血或淋巴器官分离的B细胞群体。
  28. 如权利要求26或27所述的方法，其中所述方法还包括在步骤c)之后对识别所述亚单位的B细胞进行筛选、富集和/或扩增的步骤。
  29. 如权利要求26-28中任一项所述的方法，其中所述方法还包括在步骤c)之后对识别所述亚单位的B细胞进行筛选，并将其编码免疫球蛋白受体的基因序列导入所述B细胞群体，以增加所述群体中识别所述亚单位的B细胞的数量的步骤。
  30. 如权利要求26-29中任一项所述的方法，其中所述多聚体的直径为10nm至1000nm。
  31. 如权利要求26-30中任一项所述的方法，其中所述多聚体包含至少4个亚单位。
  32. 如权利要求26-31中任一项所述的方法，其中所述免疫刺激物包含细菌来源的ssRNA、人工合成的ssRNA或其衍生物、人工合成的含CpG的寡核苷酸，干扰素，细胞因子，或其组合。
  33. 如权利要求32所述的方法，其中所述细菌来源的ssRNA是大肠杆菌来源的ssRNA。
  34. 如权利要求32所述的方法，其中所述干扰素选自I型干扰素、II型干扰素、III型干扰素，及其组合。
  35. 如权利要求32所述的方法，其中所述细胞因子选自IL-6、IL-12、IL21，及其组合。
  36. 如权利要求26-35中任一项所述的方法，其中所述多聚体是病毒样颗粒。
  37. 如权利要求36所述的方法，其中所述病毒样颗粒包含Qβ蛋白，HBcAg或AP205，或由Qβ蛋白，HBcAg或AP205组成。
  38. 如权利要求36或37所述的方法，其中所述病毒样颗粒包含目 标抗原，或由目标抗原组成。
  39. 如权利要求26-37中任一项所述的方法，其中所述疾病是感染性疾病，并且所述抗原复合物包含加载的目标抗原，所述加载的目标抗原是细菌或病毒来源的抗原。
  40. 如权利要求39所述的方法，其中所述目标抗原是结核分枝杆菌来源的抗原。
  41. 如权利要求40所述的方法，其中所述目标抗原选自晶状体蛋白和Rv3133c。
  42. 如权利要求39所述的方法，其中所述目标抗原是超级细菌来源的抗原。
  43. 如权利要求42所述的方法，其中所述目标抗原选自肺炎克雷伯菌碳青霉烯酶和青霉素结合蛋白。
  44. 如权利要求39所述的方法，其中所述目标抗原是慢病毒来源的抗原。
  45. 如权利要求44所述的方法，其中所述目标抗原选自HBV pre-S1抗原和EBV LMP1抗原。
  46. 如权利要求26-36中任一项所述的方法，其中所述疾病是癌症，并且所述抗原复合物包含加载的目标抗原，所述加载的目标抗原是肿瘤相关抗原。
  47. 如权利要求46所述的方法，其中所述肿瘤相关抗原选自Her2、p53和肿瘤新生抗原。
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