FLT3l–flagellin hybrid adjuvant with enhanced antigen cross-presentation efficacy and vaccine composition comprising same

Abstract

The present invention relates to a FLT3L–flagellin hybrid adjuvant with enhanced antigen cross-presentation efficacy and a vaccine composition comprising same. The hybrid adjuvant of the present invention demonstrated significant therapeutic efficacy as an adjuvant for a therapeutic cancer vaccine in a preclinical mouse model of cervical cancer, resulting in complete tumor regression and sustained protection. In addition, the hybrid adjuvant of the present invention induced tumor-specific antigen CD8+ T cell responses and progenitor-exhausted CD8+ T cells (Tpex) due to an increase in cross-presentation by conventional type 1 dendritic cells (cDC1) in tumor-draining lymph nodes and the tumor microenvironment, and the combination of TCV having the hybrid adjuvant applied thereto and anti-PD-1 therapy significantly improves survival outcomes in anti-PD-1–resistant tumors, and thus can be advantageously applied in anticancer immunotherapy using therapeutic cancer vaccines.

Description

FLT3L-flagellin hybrid adjuvant with enhanced antigen cross-presentation efficacy and vaccine composition containing the same

[0001] The present invention was carried out under project number 2020R1A5A2031185 with the support of the Ministry of Science and ICT, the research management agency for the said project is the National Research Foundation of Korea, the research project name is "Basic Research Project (Leading Research Center Support Project)", the research project name is "Combined Cancer Immunotherapy Research Center", the lead institution is Chonnam National University, and the research period is June 1, 2018 – February 28, 2025.

[0002] In addition, the present invention was carried out under project number 2020M3A9G3080282 with the support of the Ministry of Science and ICT, and the research management agency for the said project is the National Research Foundation of Korea, the research project name is "Original Technology Development Project (Establishment of Foundation for Bio R&D Achievement Creation)", the research project name is "Immunotherapy Innovation Research Group", the lead institution is Chonnam National University, and the research period is June 1, 2020 – December 31, 2024.

[0003] The present invention was carried out under project number HV22C0079000023 with the support of the Ministry of Health and Welfare, the research management agency for the said project is the Korea Health Industry Development Institute, the research project name is "Development of Vaccine-based Technology," the research project name is "Development of Universal Platform for Cell Immunity-Inducing Mucosal Immunostimulant," the lead institution is Chonnam National University, and the research period is April 1, 2022 ~ December 31, 2024.

[0004] The present invention was carried out under project number 2019R1A5A2027521 with the support of the Ministry of Science and ICT, the research management agency for the said project is the National Research Foundation of Korea, the research project name is "Basic Research Project (Leading Research Center Support Project)", the research project name is "Hard Tissue Biointerface Research Center", the lead institution is Chonnam National University, and the research period is June 1, 2019 – February 28, 2026.

[0005] The present invention claims priority to Korean Patent Application No. 10-2024-0162190 filed with the Korean Intellectual Property Office on November 14, 2024, and priority to Korean Patent Application No. 10-2025-0086268 filed with the Korean Intellectual Property Office on June 27, 2025, respectively, the contents of which are incorporated herein by reference in their entirety.

[0006] The present invention relates to a Flt3L-flagellin hybrid adjuvant with enhanced antigen cross-presentation efficacy and a vaccine composition containing the same.

[0007] In the field of cancer immunotherapy, therapeutic cancer vaccines (TCVs) are attracting attention as a promising approach that elicits a sustained anti-tumor immune response by inducing and enhancing T-cell responses to tumor antigens. These vaccines operate on the principle of inhibiting tumor growth by stimulating the patient's adaptive immune system through a combination of selected tumor antigens and adjuvants. Currently, various tumor antigens, including personalized neoantigens, are being studied, but the development of effective new adjuvants deserves special attention.

[0008] Adjuvants play a key role in the efficacy of cancer vaccines. Adjuvants enhance anti-tumor immune responses through various mechanisms, such as improving antigen presentation, promoting the presentation of tumor-associated antigens, and increasing the infiltration of immune cells. In particular, bacterial flagellin is a unique pattern recognition receptor (PRR) agonist capable of activating both the extracellular TLR5 and intracellular NLRC4 inflammasome pathways, and has been reported as an excellent adjuvant for various diseases, including cancer (Lee et al., 2016; Nguyen et al., 2013; Gonzalez et al., 2023; Lim et al., 2024). Furthermore, flagellin can generate integrative adjuvants by covalently binding to dendritic cell (DC) target peptides and tumor antigens, or it can be secreted by genetically modified Salmonella to reduce tumor progression.

[0009] Dendritic cells are specialized antigen-presenting cells capable of sampling, storing, and processing soluble and cell-associated antigens, acting as watchdogs of the immune system. Dendritic cells present tumor antigens to naive T cells and provide functional signals necessary for the initiation and maintenance of anti-tumor immune responses. The interaction between the peptide-major histocompatibility complex (MHC) and the T cell receptor (TCR) on dendritic cells constitutes key signals for T cell activation and differentiation. This process of antigen processing and presentation is a critical bottleneck in the targeted destruction of cancer cells by T cells.

[0010] Among the major dendritic cell subpopulations, type I classical dendritic cells (cDC1s) play a pivotal role in initiating anti-tumor immune responses due to their unique ability to activate CD8+ T cells by cross-presenting exogenous antigens to MHC class I molecules. cDC1s can migrate from tumor-draining lymph nodes to tumors via lymphatic vessels, providing survival and activation signals to incoming T cells and delivering antigens to other cDCs. Preclinical data suggest that administration of FMS-tyrosine kinase 3 ligand (Flt3L), a key growth factor regulating DC development in the bone marrow, can promote the expansion of cDC1s in both tumor-draining lymph nodes and the tumor microenvironment. However, single administration of 30 μg of Flt3L intratumorally or intraperitoneally for 9 consecutive days failed to inhibit tumor growth in mouse breast and pancreatic adenocarcinoma models.

[0011] The inventors hypothesized that a fusion protein formed by combining flagellin B (FlaB), a Toll-like receptor 5 (TLR5) ligand, and an FMS-tyrosine kinase 3 ligand (Flt3L) could expand the number of antigen-presenting cells such as cDC1 and maximize their function. As a result of diligent research and effort regarding this, they developed a hybrid adjuvant through a single molecule fusion protein (Flt3L-FlaB: FB) that performs the functions of both FlaB and Flt3L without damaging the stable structure.

[0012] The hybrid adjuvant FB of the present invention demonstrated significant therapeutic efficacy as an adjuvant for therapeutic cancer vaccines in a preclinical mouse model of cervical cancer, leading to complete tumor regression and sustained protection. Furthermore, the hybrid adjuvant FB of the present invention strongly induced tumor-specific antigen CD8+ T cell responses and progenitor-depleted CD8+ T cells (Tpex) due to increased cross-presentation by type 1 classical dendritic cells (cDC1) in tumor-draining lymph nodes and the tumor microenvironment, and the combination of TCV and anti-PD-1 therapy with the hybrid adjuvant FB significantly improved survival outcomes in anti-PD-1 resistant tumors.

[0013] Accordingly, the objective of the present invention is to provide a hybrid adjuvant comprising Flt3L (Fms-related tyrosine kinase 3 ligand) and flagellin, and a vaccine composition comprising the same as an active ingredient.

[0014] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below.

[0015] To achieve the above technical problem, the present invention provides a hybrid immunoadjuvant comprising Flt3L (Fms-related tyrosine kinase 3 ligand) and flagellin.

[0016] In addition, the present invention provides a vaccine composition comprising a hybrid adjuvant comprising Flt3L (Fms-related tyrosine kinase 3 ligand) and flagellin as an active ingredient.

[0017] The hybrid adjuvant of the present invention demonstrated significant therapeutic efficacy as an adjuvant for therapeutic cancer vaccines in a preclinical mouse model of cervical cancer, thereby confirming complete tumor regression and sustained protection. Furthermore, the hybrid adjuvant of the present invention induced tumor-specific antigen CD8+ T cell responses and progenitor-depleted CD8+ T cells (Tpex) due to increased cross-presentation by type 1 classical dendritic cells (cDC1) in tumor-draining lymph nodes and the tumor microenvironment. Since the combination of TCV and anti-PD-1 therapy with the hybrid adjuvant significantly improved survival outcomes in anti-PD-1 resistant tumors, it can be usefully utilized in anticancer therapy using therapeutic cancer vaccines.

[0018] Figure 1 is a vector map of the structure of a recombinant protein prepared using the pSectag2B plasmid.

[0019] Figure 2 shows the 3D structure of the Flt3L-FlaB(FB) fusion protein produced by AlphaFold 3.

[0020] Figure 3 shows the characteristics of deglycosidized recombinant proteins digested by peptide N-glycosidase F (PNGaseF) by SDS-PAGE and WB analysis.

[0021] Figure 4 shows the results of Western blot analysis of recombinant proteins using mouse anti-FlaB serum or rabbit anti-Flt3L antibody generated by SDS-PAGE and intraperitoneal immunization of FlaB containing Freund's adjuvant.

[0022] Fig. 5 is HEK-Blue TM This is a figure analyzing the TLR5-dependent NF-κB stimulating activity of FlaB (B), Flt3L-FlaB (FB), and FlaB-Flt3L (BF) at various protein concentrations using hTLR5 cells.

[0023] Fig. 6 is HEK-Blue TMThis is a figure analyzing the long-term stability of the recombinant protein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and NF-κB stimulation activity using hTLR5 cells (Fig. 6a: 0 day, Fig. 6b: 56 days, Fig. 6c: 98 days).

[0024] Figure 7 is a diagram illustrating a method for generating BMDCs using FlaB (B), Flt3L (F), and Flt3L-FlaB (FB).

[0025] Figure 8 shows the total number of cells and the number of cell clusters formed in BMDCs after 9 days of culture supplemented with FlaB (B), Flat3L (F), and Flt3L-FlaB (FB).

[0026] Figure 9 shows the results of CD103+ DC generation by recombinant protein through flow cytometry analysis.

[0027] Figure 10 shows the results of quantifying the production of CD103+ DCs by recombinant protein (****P< 0.0001; ***P< 0.001; **P< 0.01; *P< 0.05).

[0028] Figure 11 is a timeline for a study on the therapeutic efficacy of Flt3L-FlaB (FB) fusion proteins using a TC-1 tumor model.

[0029] Figure 12 shows the change in tumor size over time after injection of a primary tumor by a recombinant protein in a TC-1 tumor model.

[0030] Figure 13 shows the mouse overall survival curve after injection of the primary tumor with the recombinant protein in the TC-1 tumor model (****P< 0.0001; ***P< 0.001; **P< 0.01; *P< 0.05; ns, not significant).

[0031] Figure 14 shows an experimental schedule for analyzing tumor-specific immune responses in a tumor-bearing model.

[0032] Figure 15 is a diagram showing the analysis of HPV16 H-2Db-RAHYNIVTF CTL epitope-specific tetramer-positive cells in CD8+ peripheral blood cells.

[0033] Figure 16 shows the results of ELISpot analysis of HPV16 H-2Db-RAHYNIVTF CTL epitope-specific IFN-γ producing cells in tumor drainage lymph nodes (tdLN) (****P< 0.0001; ***P< 0.001; **P< 0.01; *P< 0.05; ns, not significant).

[0034] Figure 17 shows the results of ELISpot analysis of HPV16 H-2Db-RAHYNIVTF CTL epitope-specific IFN-γ producing cells in the spleen (****P< 0.0001; ***P< 0.001; **P< 0.01; *P< 0.05; ns, not significant).

[0035] Figure 18 is a figure analyzing the frequency of memory progenitor effector cells (MPECs; KLRG1-CD127+CD62L-CD44+) and short-lived effector cells (SLECs; KLRG1+CD127-CD62L-CD44+) in tumor drainage lymph nodes (tdLN).

[0036] Figure 19 is a figure analyzing the frequency of memory progenitor effector cells (MPECs; KLRG1-CD127+CD62L-CD44+) and short-lived effector cells (SLECs; KLRG1+CD127-CD62L-CD44+) in the spleen.

[0037] Figure 20 shows a frequency analysis of stem-like memory CD8+ T cells (Tscm; CD44-CD62L+ TCF1+) among CD45+ cells in tumor drainage lymph nodes (tdLN) and a representative staining plot showing TCF1 expression in CD45+ CD8+ CD44-CD62+ T cells.

[0038] Figure 21 is a figure showing the vaccination schedule for the second challenge of TC-1 tumor cells.

[0039] Figure 22 shows the tumor growth curve (mm³) and survival rate (%) of re-challenge mice.

[0040] Figure 23 shows an experimental schedule for analyzing cross-presenting dendritic cells in mice vaccinated with a vaccine containing a Flt3L-FlaB(FB) hybrid adjuvant.

[0041] Figure 24 shows the results of the frequency of total dendritic cells (DCs) in tumor drainage lymph nodes (tdLN).

[0042] Figure 25 shows a flow cytometry plot (right) showing XCR1 and CD103 expression in CD45+ F4 / 80- MHCII+ CD11c+ cells and the results of quantifying the frequency of cDC1 (XCR1+ CD103+) among CD45+ cells of tdLN (left).

[0043] Figure 26 shows the absolute number of total dendritic cells (DCs) and cDC1 per gram of tumor tissue.

[0044] Figure 27 shows Batf3 harboring a TC-1 tumor. - / - Figure showing the tumor growth results of and wild-type (WT) mice (****P< 0.0001; ***P< 0.001; **P< 0.01; *P< 0.05; ns, not significant).

[0045] Figure 28 shows Batf3 harboring a TC-1 tumor. - / - This is a figure showing the survival rates of wild-type (WT) mice (****P< 0.0001; ***P< 0.001; **P< 0.01; *P< 0.05; ns, not significant).

[0046] Figure 29 shows the absolute number of fusion protein progenitor-depleted CD8+ T cells (TCF1+PD-1+TIM3-) within the tumor.

[0047] Figure 30 shows a vaccination schedule for the combination of Flt3L-FlaB (FB) hybrid adjuvant and anti-PD-1 therapy.

[0048] Figure 31 shows the tumor growth curves of individual mice in three different groups (DPBS; antigen(E) plus adjuvant(FB) plus isotype; antigen(E) plus adjuvant(FB) plus anti-PD-1)) (****P< 0.0001; *P< 0.05).

[0049] Figure 32 shows the survival curves of individual mice in three different groups (DPBS; antigen(E) plus adjuvant(FB) plus isotype; antigen(E) plus adjuvant(FB) plus anti-PD-1) and the 60% survival days for each individual group (****P< 0.0001).

[0050] Figure 33 shows the results of the analysis of tumor-specific antigen CD8+ T cells using tetramer staining with peripheral blood cells from re-challenge mice (*P< 0.05).

[0051] Figure 34 shows the degree of tumor development following re-challenge in three different groups (DPBS; antigen (E) plus adjuvant (FB) plus isotype; antigen (E) plus adjuvant (FB) plus anti-PD-1).

[0052] Figure 35 shows the survival rates following re-challenge in three different groups (DPBS; antigen (E) plus adjuvant (FB) plus isotype; antigen (E) plus adjuvant (FB) plus anti-PD-1).

[0053] Figure 36 shows the experimental design for anti-PD-1 treatment before TCV inoculation.

[0054] Figure 37 shows the individual mouse tumor volumes after anti-PD-1 treatment in combination with the hybrid adjuvant Flt3L-FlaB (FB) (****P< 0.0001; **P< 0.01; *P< 0.05).

[0055] Figure 38 shows individual mouse survival curves after anti-PD-1 treatment in combination with the hybrid adjuvant Flt3L-FlaB (FB) (****P< 0.0001; **P< 0.01; *P< 0.05).

[0056] A hybrid adjuvant comprising Flt3L (Fms-related tyrosine kinase 3 ligand) and flagellin.

[0057] Each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention is not to be limited by the specific descriptions provided below.

[0058] Furthermore, a person skilled in the art can recognize or identify a number of equivalents to the specific embodiments of the present invention described in this invention using only ordinary experiments. In addition, such equivalents are intended to be included in the present invention.

[0059] Furthermore, throughout the specification of the present invention, when a part is described as "including" or "containing" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include or contain additional components.

[0060] Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid, unless otherwise noted.

[0061] Furthermore, the term “or” in this specification is intended to mean an implied “or” rather than an exclusive “or.” That is, where the combination or use of the configurations is not otherwise specified or is not evident from the context, i.e., where X includes A; where X includes B; or where X includes both A and B, “X includes A or B” may be applied to either of these cases.

[0062] The present invention will be described in detail below.

[0063] One aspect of the present invention is a hybrid immunoadjuvant comprising Flt3L (Fms-related tyrosine kinase 3 ligand) and flagellin.

[0064] The term "Flt3L (Fms-related tyrosine kinase 3 ligand)" in this specification refers to a hematopoietic cytokine that acts as a ligand for the Fms-related tyrosine kinase 3 receptor (Flt3). Flt3L is a major growth factor that regulates the development, expansion, and function of dendritic cells, and in particular promotes the differentiation and proliferation of CD8+ and CD103+ dendritic cell subtypes specialized for antigen cross-presentation. In the present invention, Flt3L may comprise a recombinant protein, a synthetic peptide, or a derivative, variant, or fragment that preserves the biological activity thereof, and may be used alone or in a fused or combined form with other immunoadjuvants. Flt3L may possess immunoadjuvant activity on its own or exhibit a synergistic effect with other immunoadjuvants to enhance antigen cross-presentation efficacy.

[0065] The term "Flagellin" as used herein refers to a protein monomer that constitutes the filament of a bacterial flagellum and acts as a natural ligand for Toll-like receptor 5 (TLR5). Flagellin can be used as an adjuvant due to its immune-activating ability, and the full-length flagellin protein, a portion thereof (particularly the TLR5 binding domain), or a flagellin-derived peptide may be included in the hybrid adjuvant of the present invention. Flagellin, either alone or in a form fused with Flt3L, can induce a potent antigen-specific immune response and an enhanced effect of antigen cross-presentation through TLR5 activation.

[0066] In the present invention, the flagellin may comprise a full-length flagellin or a fragment thereof.

[0067] The above flagellin may be flagellin B (FlaB) of Vibrio vulnificus.

[0068] The terms "flagellin B" or "FlaB" as used herein refer to one of the major proteins constituting the flagella of Vibrio vulnificus. FlaB is a protein of approximately 40-50 kDa in size that exhibits excellent immunostimulatory activity as a TLR5 ligand. FlaB consists of highly conserved N-terminal and C-terminal domains and a variable central domain, and binding to TLR5 occurs primarily through the conserved domain. In the present invention, FlaB may be used as a full-length protein, a functional fragment thereof, or a variant produced through genetic recombination, and can exert a potent immunostimulatory effect either alone or in a form fused with Flt3L. FlaB is particularly effective in promoting dendritic cell maturation, enhancing antigen cross-presentation, and inducing CD8+ T cell activation, and can exhibit an enhanced anti-tumor immune effect when used in combination with anti-PD-1 therapy prior to TCV.

[0069] The flagellin B (FlaB) of the present invention may include the amino acid sequence of SEQ ID NO. 1.

[0070] [Sequence No. 1]

[0071] MAVNVNTNVAAMTAQRYLNNANSAQQTSMERLSSGFKINSAKDDAAGLQISNRLNVQSRGLDVAVRNANDGISIAQTAEGAMAETTNILQRMRDLSLQSAAGSNSKSERVAIQEEVTALNDELNRIAETTSFGGNKLLAGTYGTKAMQIGADNGEAVMLSLKDMRSDNVMMGGVSYQAEEGKDKNWNV AAGDNDLTIALTDSFGNEQEIEINAKAGDDIEELATYINGQTDLVKASVGEGGKLQIFAGNNKVQGEIAFSGSLAGELGLGEGKNVTVDTIDVTTVQGAQESVAIVDAALKYVDSHRAELGAFQNRFNHAISNLDNINENVNASKSRIKDTDFAKETTQLTKTQILSQASSSILAQAKQAPNSALSLLG

[0072]

[0073] The term "hybrid" as used herein refers to a structure in which two or more substances or components of different origins or different biological functions are integrated into a single entity through chemical bonding, fusion, covalent bonding, non-covalent bonding, or physical bonding. In this invention, a hybrid refers to a complex or fused molecule designed at the molecular level, rather than a simple physical mixture. Hybrid structures can exhibit synergistic effects or display novel functional characteristics while maintaining the individual activities of their components.

[0074] The term "adjuvant" as used herein refers to a substance that quantitatively and / or qualitatively enhances, accelerates, prolongs, or modulates the immune response to an antigen. An adjuvant may act through mechanisms such as (i) recruitment, activation, and maturation of antigen-presenting cells (e.g., dendritic cells); (ii) promotion of antigen capture, processing, and presentation; (iii) induction of cytokine and chemokine production; (iv) promotion of T cell and B cell activation; (v) enhancement of immune memory formation; or (vi) induction of immune bias toward specific immune response types (e.g., Th1, Th2, Th17, or cytotoxic T cell responses). Adjuvants include, but are not limited to, inorganic salts (e.g., aluminum salts), lipids (e.g., MF59, AS03), lipopolysaccharide derivatives (e.g., MPL), saponins (e.g., QS-21), nucleic acids (e.g., CpG-ODN), proteins / peptides, virus-like particles, cytokines, chemically synthesized substances, or combinations thereof.

[0075] The term "hybrid adjuvant" as used in this specification refers to an adjuvant in which two or more immunostimulating active substances having different mechanisms of action are chemically or physically combined to form a single molecule or complex.

[0076] The hybrid adjuvant of the present invention demonstrated significant therapeutic efficacy as an adjuvant for a therapeutic cancer vaccine in a preclinical mouse model of cervical cancer, thereby confirming complete tumor regression and sustained protection (Example 3). In addition, the hybrid adjuvant of the present invention induced tumor-specific antigen CD8+ T cell responses and progenitor-depleted CD8+ T cells (Tpex) due to increased cross-presentation by type 1 classical dendritic cells (cDC1) in tumor-draining lymph nodes and the tumor microenvironment (Examples 4 to 7), and the combination of TCV and anti-PD-1 therapy with the hybrid adjuvant significantly improved survival outcomes in anti-PD-1 resistant tumors (Example 8), so it can be usefully utilized in anticancer therapy using a therapeutic cancer vaccine.

[0077] The Flt3L and flagellin of the present invention may exist in the form of a fusion protein.

[0078] The term "fusion protein" as used herein refers to a protein in which two or more different proteins or protein domains are combined at the genetic level to form a single polypeptide chain. A fusion protein can perform multiple biological functions simultaneously as a single molecule while maintaining or improving the functional characteristics of its constituent components. In the present invention, the fusion protein of Flt3L and flagellin can be prepared using recombinant DNA technology. The Flt3L-flagellin fusion protein can be designed in a configuration where Flt3L is located at the N-terminus and flagellin at the C-terminus (Flt3L-flagellin) or in the opposite direction (flagellin-Flt3L), and each configuration may influence specific functional characteristics or efficacy.

[0079] The above fusion protein may be Flt3L-flagelline, in which Flt3L is located at the N-terminus and flagellin is located at the C-terminus, but is not limited thereto. In one embodiment of the present invention, the above fusion protein may be Flt3L-FlaB, in which Flt3L is located at the N-terminus and FlaB is located at the C-terminus.

[0080] The fusion protein may additionally include 3 to 10, 3 to 9, 3 to 8, 3 to 7, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 5 to 10, 5 to 9, 5 to 8, and 5 to 7 histidine residues (6xHis-tag) at the C-terminus, for example, may additionally include 6 histidine residues (6xHis-tag), but is not limited thereto.

[0081] The term "histidine residue (6xHis-tag)" as used herein generally refers to a short amino acid sequence consisting of six consecutive histidine amino acids (HHHHHH). This is genetically fused to the N-terminus or C-terminus of a recombinant protein to facilitate protein purification, detection, and characterization. In the present invention, the 6xHis-tag is located at the C-terminus of the Flt3L and flagellin fusion protein and enables efficient purification via Immobilized Metal Ion Affinity Chromatography (IMAC). The histidine tag is designed to have minimal impact on the function of the target protein and can be removed using a protease if necessary.

[0082] The above hybrid adjuvant may activate Toll-like receptor 5 (TLR5), but is not limited thereto.

[0083] The term “Toll-like receptor 5 (TLR5)” as used herein refers to a transmembrane protein belonging to the pattern recognition receptor (PRR) family that recognizes pathogen-associated molecular patterns (PAMPs). TLR5 primarily recognizes flagellin, a bacterial flagellar protein, and is expressed in various cell types, including dendritic cells, monocytes, macrophages, neutrophils, and intestinal epithelial cells. Activation of TLR5 induces NF-κB and MAP kinase activation through the MyD88-dependent signaling pathway, thereby promoting the expression of pro-inflammatory cytokines, chemokines, and co-stimulatory molecules.

[0084] The above-mentioned hybrid adjuvant may induce the generation of CD103+ dendritic cells, but is not limited thereto.

[0085] The term "CD103+ dendritic cells" as used in this specification refers to a specialized subgroup of dendritic cells that express CD103 (integrin αE, ITGAE) on their cell surface. CD103+ dendritic cells are primarily found in tissues such as the intestines, lungs, skin, and lymph nodes. They possess specialized functions for cross-presentation of antigens and are known to co-express XCR1 (XC Motif chemokine receptor 1) and CLEC9A (c-type lectin domain family 9 member A). CD103+ dendritic cells are particularly capable of capturing external antigens and efficiently presenting them to CD8+ T cells via MHC class I, playing an important role in anti-tumor immune responses and defense against viral infections.

[0086] The CD103+ dendritic cells of the present invention may be derived from bone marrow, bone marrow progenitor, peripheral blood mononuclear cell, cord blood, lymph node, skin, intestine, lung, CD34+ hematopoietic stem cell, monocyte, or in vitro differentiated dendritic cell, but are not limited thereto. In one embodiment of the present invention, the CD103+ dendritic cells may be derived from bone marrow or a bone marrow progenitor.

[0087] The above-mentioned hybrid adjuvant may induce an increased antigen-specific CD8+ T cell response compared to Flt3L alone or flagellin alone, but is not limited thereto.

[0088] The term "increased antigen-specific CD8+ T cell response" as used herein refers to a phenomenon promoted by a hybrid adjuvant, in which the activation, proliferation, functional maturation, and effective cytotoxic activity of CD8+ T cells responding to a specific antigen are statistically significantly enhanced compared to a control group, and can be measured by the following indicators: (i) an increase in the number of antigen-specific CD8+ T cells; (ii) an increase in the production of effector molecules such as IFN-γ, TNF-α, granzyme B, or perforin upon antigen stimulation; (iii) an enhancement in antigen-specific cytotoxic activity (cell lytic ability); (iv) an increase in the expression of activation markers (CD25, CD69, CD44high, CD62Llow, etc.); (v) an increase in the formation of memory T cell subpopulations; and / or (vi) an increase in CD8+ T cell infiltration into the tumor microenvironment. The hybrid adjuvant of the present invention effectively activates the immune response, particularly to tumor antigens, by inducing an increased antigen-specific CD8+ T cell response through the enhancement of cross-presentation of antigens and the induction of CD103+ dendritic cell generation, and can exhibit a synergistic anti-tumor effect when combined with anti-PD-1 therapy.

[0089] The above-mentioned hybrid adjuvant may increase the number of IFN-γ-secreting cells, but is not limited thereto.

[0090] The term "IFN-γ (Interferon-gamma)" as used herein refers to a type II interferon cytokine produced primarily by activated T cells, NK cells, NKT cells, etc. IFN-γ possesses potent immunomodulatory functions and performs various immunological functions, such as (i) macrophage activation, (ii) increased expression of MHC class I and II, (iii) promotion of Th1 cell differentiation, (iv) enhancement of antiviral and antitumor immune responses, (v) induction of enzymes and reactive oxygen species necessary for pathogen elimination, and (vi) enhancement of the antigen presentation process. In antitumor immunity, IFN-γ plays an important role in promoting the recognition and elimination of tumor cells, enhancing antigen presentation, and inhibiting tumor angiogenesis.

[0091] The above hybrid adjuvant may increase the frequency of one or more cell populations selected from the group consisting of memory precursor effector cells (MPECs; KLRG1-CD127+CD62L-CD44+), short-lived effector cells (SLECs; KLRG1+CD127-CD62L-CD44+), and stem-like memory T cells (Tscm; CD44-CD62L+TCF-1+), but is not limited thereto. In one embodiment of the present invention, the hybrid immunoadjuvant may increase the frequency of populations of memory precursor effector cells (MPECs; KLRG1-CD127+CD62L-CD44+), short-lived effector cells (SLECs; KLRG1+CD127-CD62L-CD44+), and stem-like memory T cells (Tscm; CD44-CD62L+TCF-1+).

[0092] The term "memory precursor effector cell (MPECs)" in this specification refers to a subpopulation of CD8+ T cells having the KLRG1-CD127+CD62L-CD44+ phenotype. These cells perform initial effector functions after antigen stimulation and possess the potential to survive for a long time and differentiate into memory T cells. MPECs are characterized by high expression of the IL-7 receptor (CD127) and low expression of KLRG1 (Killer Cell Lectin Like Receptor G1), a terminal differentiation marker. Since these cells survive even after antigen removal and provide sustained immune protection and rapid reactivation capabilities, they play an important role in effective vaccine responses and the formation of long-term immune memory. The hybrid adjuvant of the present invention can induce an enhanced anti-tumor immune response and sustained immune memory by significantly increasing the frequency of MPECs in the spleen and tumor-draining lymph nodes (tdLN).

[0093] The term "short-lived effector cells (SLECs)" in this specification refers to a subpopulation of CD8+ T cells having a KLRG1+ CD127- CD62L- CD44+ phenotype. These cells rapidly proliferate after antigen stimulation and perform potent effector functions (cytotoxicity, cytokine production, etc.), but most of them disappear through apoptosis after antigen removal. They also have the characteristic of highly expressing KLRG1 and low expressing the survival factor receptor CD127 (IL-7Rα). These cells are known to play an important role in immediate immune responses and the efficient removal of pathogens or tumor cells. The hybrid adjuvant of the present invention can promote an immediate and potent anti-tumor effector response by significantly increasing the frequency of SLECs in the spleen and tumor-draining lymph nodes (tdLN).

[0094] The term "stem-like memory T cells (Tscm)" as used herein refers to a subpopulation of CD8+ T cells having a CD44-CD62L+ TCF-1+ phenotype. Tscm cells are the most primitive subpopulation of memory T cells and possess self-renewal capabilities as well as the ability to differentiate into various effectors and memory T cell subtypes. These cells express the transcription factor TCF-1 (T-cell factor 1), possess the ability to maintain a long-lasting immune response and rapidly expand upon antigen re-exposure, and play an important role in vaccine responses and sustained anti-tumor immunity. The hybrid adjuvant of the present invention can induce a long-term and sustained anti-tumor immune response by significantly increasing the frequency of Tscm cells in tumor-draining lymph nodes (tdLN).

[0095] The term "tumor-draining lymph nodes (tdLN)" as used herein refers to the first lymph node or group of lymph nodes that receive lymph fluid from tumor tissue. TdLNs are the primary sites where tumor antigen presentation and tumor-specific T cell activation occur, and they regulate the initiation and development of anti-tumor immune responses. The immune environment within the tdLN has a significant influence on determining the efficacy and persistence of the anti-tumor immune response. The hybrid adjuvant of the present invention can effectively enhance tumor-specific immune responses by significantly increasing the frequency of various CD8+ T cell subpopulations (MPECs, SLECs, Tscm) in the tdLNs.

[0096] The term "increase in frequency of cell populations" as used herein refers to a phenomenon in which the proportion or absolute number of immune cell subpopulations with a specific phenotype increases statistically significantly compared to the control group after treatment with a hybrid adjuvant. This can be quantified through flow cytometry, immunohistochemistry, or other cell counting methods based on the expression patterns of specific cell surface markers. The hybrid adjuvant of the present invention can induce enhanced anti-tumor immunity by promoting both immediate effector responses and long-term memory responses through an increase in the frequency of functionally important CD8+ T cell subpopulations, such as MPECs, SLECs, and Tscms. In particular, these results observed in the spleen and tumor-draining lymph nodes suggest that the hybrid adjuvant induced a greater expansion of both the functional and memory characteristics of CD8+ T cell populations. This balanced increase in various CD8+ T cell subpopulations reflects the characteristics of an ideal adjuvant capable of simultaneously providing an effective initial immune response and long-term immune protection.

[0097] The above-mentioned hybrid adjuvant may increase the cross-presentation of antigens by classical dendritic cell type 1 (cDC1), but is not limited thereto.

[0098] The term "classical dendritic cell type 1 (cDC1)" as used herein refers to a specialized subgroup of dendritic cells having a CD8α+ (mouse) or CD141+ (human) phenotype. cDC1 is a dendritic cell specialized in cross-presentation of antigens, effectively inducing cytotoxic T cell responses by presenting external antigens or tumor antigens to CD8+ T cells via MHC class I molecules. cDC1 is particularly known for its excellent ability to capture and process antigens from apoptotic cells, is essential for anti-tumor immunity and defense against viral infections, and plays a decisive role in CD8+ T cell infiltration within the tumor microenvironment and the efficacy of anti-tumor immune responses.

[0099] The term "cDC1-mediated antigen cross-presentation" as used herein refers to a specific process in which cDC1 captures and processes externally introduced antigens and presents them to CD8+ T cells via MHC class I molecules. This process is distinguished from the traditional pathway in which external antigens are generally presented via MHC class II and is essential for inducing cytotoxic T cell responses. cDC1 is known to be more efficient at antigen cross-presentation than other dendritic cell subgroups through specialized intracellular antigen processing mechanisms and signaling pathways.

[0100] The above-mentioned hybrid adjuvant is intratumoral progenitor-exhausted T cells (Tpex; TCF-1 + PD-1 + TIM3 - It may increase the frequency of ), but is not limited to this.

[0101] The term "progenitor-exhausted T cells (Tpex)" in this specification refers to TCF-1+ PD-1 + TIM3 - It refers to a CD8+ T cell subpopulation with a specific phenotype. Tpex cells are a specialized T cell population found in environments of chronic antigen stimulation (such as tumors or chronic infections). Unlike terminal exhausted T cells, they partially preserve self-renewal capabilities and anti-tumor effector functions. Tpex cells are characterized by expressing the transcription factor TCF-1 (T-cell factor 1) and the exhaustion marker PD-1, but not the terminal exhaustion marker TIM3. As a key cell population that determines responsiveness to PD-1 blockade immunotherapy, they possess the potential to proliferate upon anti-PD-1 treatment and differentiate into functional effector T cells.

[0102] The term "T cell exhaustion" as used herein refers to a state in which, under conditions of chronic antigen stimulation, T cells progressively lose effector functions (cytotoxicity, cytokine production, etc.), the expression of inhibitory receptors such as PD-1, TIM3, LAG3, and CTLA-4 increases, and proliferative capacity decreases. T cell exhaustion is commonly observed in chronic viral infections or tumor environments and serves as a limiting factor in effective immune responses. The exhaustion process proceeds gradually, forming subpopulations of exhausted T cells at various differentiation stages, among which Tpex represents a relatively early stage of exhaustion.

[0103] The term "TCF-1 (T-cell factor 1)" in this specification refers to a protein encoded by the TCF7 gene. TCF-1 plays an important role in the regulation of T cell development, differentiation, and function, and is involved in the self-renewal ability of T cells and the formation of memory T cells. CD8+ T cells expressing TCF-1 in a tumor environment have characteristics similar to stem cells, are highly responsive to anti-PD-1 therapy, and are important for maintaining a T cell pool for a sustained anti-tumor immune response. Furthermore, the expression of TCF-1 in Tpex cells is known to be an important indicator that these cells are not completely depleted and maintain their potential for functional recovery.

[0104] The term "increase in tumor precursor-depleted T cell frequency" in this specification refers to TCF-1 in tumor tissue after treatment with a hybrid adjuvant. + PD-1 + This refers to a phenomenon in which the proportion or absolute number of CD8+ T cells (Tpex) with the TIM3- phenotype increases statistically significantly compared to the control group. An increase in Tpex cells within a tumor is closely associated with improved responsiveness to immune checkpoint inhibitors (particularly anti-PD-1 antibodies) and provides a continuous supply of T cells for an effective anti-tumor immune response. The hybrid adjuvant of the present invention significantly increases the frequency of Tpex cells in the tumor microenvironment by increasing the cDC1 population, thereby inducing an enhanced anti-tumor effect when combined with anti-PD-1 therapy.

[0105] Another aspect of the present invention is a vaccine composition comprising the hybrid immunoadjuvant of the present invention as an active ingredient.

[0106] The term "vaccine" as used in this specification refers to the prevention of infection or reinfection by a said pathogen, the reduction of the severity of symptoms caused by the said pathogen, or the elimination of symptoms, or the substantial or complete elimination of the said pathogen or the disease caused by said pathogen, by inducing an immune response against said pathogen in animals, including humans, that serve as hosts. Accordingly, the "vaccine composition" of the present invention may be administered to animals, including humans, prophylactically before infection by said pathogen or therapeutically after infection by said pathogen.

[0107] The vaccine composition of the present invention may be prepared in any suitable, pharmaceutically acceptable formulation. For example, it may be prepared in the form of an immediate-administration solution or suspension, a concentrated stock solution suitable for dilution prior to administration, or a reconstituteable form such as a freeze-dried, lyophilized, or frozen formulation.

[0108] The vaccine composition of the present invention may be formulated by including a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier typically includes a diluent, an excipient, a stabilizer, a preservative, etc. For example, diluents that may be included in the vaccine composition of the present invention may include non-aqueous solvents such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil and peanut oil, or aqueous solvents such as brine and water containing a buffer medium; excipients may include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol, propylene glycol, water, ethanol, etc.; and stabilizers may include carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran, glutamate, glucose, etc., or proteins such as milk powder, serum albumin, casein, animal, plant, or microbial proteins. In addition, preservatives include thimerosal, merthioleate, gentamicin, neomycin, nystatin, amphotericin B, tetracycline, penicillin, streptomycin, polymyxin B, etc.

[0109] The vaccine composition of the present invention may be administered parenterally or orally depending on the intended method, and the dosage varies depending on the patient's body weight, age, gender, health status, diet, time of administration, method of administration, excretion rate, and severity of the disease. In addition, the amount of the composition that is prophylactically or therapeutically effective may vary depending on the method of administration, target site, and the patient's condition, and when used in the human body, the dosage should be determined as an appropriate amount by considering both safety and efficacy.

[0110] The above vaccine composition may further include an anti-PD-1 antibody, but is not limited thereto.

[0111] The above anti-PD-1 antibody may be BioXCell’s BP0273, BE0273, BP0146, BE0146, BP0033-2, BE0033-2, or a combination thereof, for example, BP0146, but is not limited thereto.

[0112] The above vaccine adjuvant can be manufactured by conventional methods well known in the art and may optionally include various additives available in the art for vaccine manufacturing.

[0113] The present invention will be explained in more detail below through the following examples. However, these examples are intended to explain the invention more specifically, and the scope of the invention is not limited to these examples.

[0114] <Example>

[0115] Example 1. Experimental Materials and Methods

[0116] 1.1 Mice, Cell Lines, and Media

[0117] BALB / c and C57BL / 6 female mice were purchased from ORIENT (Seongnam-si, Gyeonggi-do, South Korea). C57BL6 female TLR5 - / - and Batf3 - / -Mice were bred in animal facilities, and all animals were matched in age (6–8 weeks) at the start of the experiment and were raised in the animal facilities of the National Center for Immunotherapy Innovations under conditions free of specific pathogens.

[0118] TC-1 cell lines were cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY, USA) containing 10% fetal bovine serum (HyClone, Logan, UT) and 1% penicillin / streptomycin (Life Technologies, Grand Island, NY, USA). Expi293 TM (Thermo Fisher Scientific) Expi293 at 37°C, 8% CO2, and a stirring speed of 125 rpm TM It was retained in Expression Medium (Thermofisher, A1435101). HEK-Blue TM hTLR5 cells (InvivoGen, hκb-help-5) were maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin / streptomycin, and 100 µg / ml Normocin.

[0119] 1.2 Plasmid Construction and Recombinant Protein Purification

[0120] All proteins were generated using the pSectag2B vector (Invitrogen, V900-20) via restriction enzyme digestion and ligation. Inserted DNA fragments were generated using the wild-type mouse Flt3L extracellular domain (UniProtKB, P49772) or the synthesized DNA sequence of codon-optimized FlaB (Bioneer Inc., South Korea) (Table 1).

[0121] Cell / plasmidDescriptionSource of referenceExpi293F TMTransient eukaryotic expression cell system based on highdensity suspension culture of Expi293F™ Cells inExpi293™ expression medium.Thermo Fisher ScientificInc.HEK-Blue ™ hTLR5 CellsHEK-Blue ™-hTLR5 cells were obtained by co-transfection of the human TLR5 gene and an inducible SEAP (secretedembryonic alkaline phosphatase) reporter gene into HEK293cells araD139 Δ(ara-leu)7697 galU galK rpsL (StrR) endA1nupGInvitrogenE. coli BL 21 (DE3)hsdS gal (λcIts857 ind1 Sam7 nin5 lacUV5-T7 gene1)Laboratory collectionpET30a+:4xE7ΔNLSN-terminal fusion expression vector in which the N terminus of a target protein is a fused His-tag; KmrDr Sao PaperBiomaterials 2022 Jul; 286:121542.pSectag2BEukaryotic expression vector for the secretion of C terminally 6xHis-tagged proteins; AmprInvitrogen pSectag2B::FlaBpSectag2B vector carrying an 1134 pb HindIII-NotI fragment of the codon-optimized site-directed mutant FlaB (N83A,N101A, and N139A)Dr Chhoy PaperNPJ Vaccines. 2023 Sep 26;8(1):139.pSectag2B::Flt3lpSectag2B vector carrying a 489 pb HindIII-NotI fragment of a wildtype mouse Flt3l extracellular domainThis studypSectag2B::Flt3l-FlaBpSectag2B vector carrying a 489 pb fused 1134 Kb (HindIIIBamHI-NotI) fragment containing Flt3l-FlaBThis studypSectag2B::FlaB-Flt3lpSectag2B vector carrying a 1134 pb fused 489 Kb (HindIIIBamHI-NotI) fragment containing FlaB-Flt3lThis study.

[0122] Primers with overhangs recognized by restriction enzymes HindIII, BamHI, or NotI (Table 2) were designed to amplify the insertion fragment via PCR.

[0123] PrimerNucleotide sequence (5' to 3') No. (SEQ ID NO) FlaB-HindIII-FORCCCAAGCTTATGGCCGTCAACGTGAACAC2FlaB-NotI-REVAAGGAAAAAAGCGGCCGCGCCCAGCAGGGAGAGGG3FlaB-BamHI-FORCGCGGATCCATGGCCGTCAACGTGAACAC4FlaB-BamHI-REVCGCGGATCCGCCCAGCAGGGAGAGGG5Flt3l -HindIII-FORCCCAAGCTTGGGACACCTGACTGTTACTTCAGCCACAG6Flt3l-NotI-REVATAAGAATGCGGCCGCCTGCCTGGGCCGAGGCTC7Flt3l-BamHI-FORCGCGGATCCGGGACACCTGACTGTTACTTCAGCCACAG8Flt3l-BamHI-REVCGCGGATCCCTGCCTGGGCCGAGGCTCT9

[0124] The recognition sites of the restriction enzyme are indicated byunderlined sequence.

[0125]

[0126] The amplified fragments were treated with restriction enzymes at 37°C for 3 hours. Subsequently, the ligation process between the pSectag2B vector and the insert fragment for a single protein or two insert fragments for the production of a fusion protein was performed overnight at 16°C using T4 DNA ligase (Enzynomics, M001S). The ligated plasmids were transformed into E. coli TOP10 responsive cells and screened on LB agar plates containing ampicillin. The DNA sequences of the resulting expression vectors were verified using the Macrogen online sequencing system (http: / dna.macrogen.com / kor / ). Three-dimensional predictions of all constructs were obtained from the AlphaFold 3 online website (https: / golgi.sandbox.google.com / about).

[0127] For the protein purification process, the verified plasmid is prepared according to the manufacturer's protocol using ExpiFectamine TM Expi293FTM cells were transformed using the 293 Transfection Kit (Thermofisher, A14524). Specifically, transformation was performed using the 2nd to 4th passages of Expi293FTM cells. On the day of transformation, 25 µg of purified plasmid was added to 25 ml of Expi293FTM cell culture suspension (3 × 10⁶). 6Cells ( / ml) were transformed. After 18 hours of transformation, enhancers I and II were added to the transformed cell suspension. Proteins were buffered with PBS using a dialysis membrane (Fisher Scientific, 08-667E) overnight at 4°C, and the purity of the recombinant protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent Western blot analysis using anti-FlaB antibody or anti-Flt3L antibody (Abcam, ab231192) generated in mice using full Freund adjuvant (Sigma, CAS9007-81-2).

[0128] To produce an antigen for a therapeutic cancer vaccine model, E7ΔNLS(E) was prepared and purified by a known method (Puth, S., et al., An all-in-one adjuvanted therapeutic cancer vaccine targeting dendritic cell cytosol induces long-lived tumor suppression through NLRC4 inflammasome activation. Biomaterials, 2022. 286: p. 121542.).

[0129] 1.3 Determination of TLR5 Stimulation Activity

[0130] To evaluate the TLR5 stimulating activity of the recombinant fusion protein, HEK-Blue according to the manufacturer's instructions TM hTLR5 cells (InvivoGen, hκb-help-5) and HEK-Blue TM The TLR5-dependent NF-κB stimulating activity of recombinant proteins was evaluated using the Detection (InvivoGen, hb-det2) assay system. Specifically, HEK-Blue TM Collect hTLR5 cells at 2.5 × 10⁶ per well of a 96-well flat-bottom plate. 4 HEK-Blue in cells TMIt was resuspended in the detection solution. An appropriate amount of recombinant protein was added to the cells to generate the total dose-response curve. After 16 hours, the level of secreted embryonic alkaline phosphatase (SEAP) was measured at 620 nm using an Epoch Microplate Spectrophotometer (Biotek) according to the manufacturer's protocol.

[0131] 1.4 Generation of Bone Marrow-Derived Dendritic Cells (FL-DCs)

[0132] Bone marrow (BM) cells were obtained by washing the femur and tibia of C57BL / 6 mice, and red blood cells (RBCs) were removed using ACK lysis (Gibco, A10492-01). After centrifugation at 1500 rpm for 3 minutes to remove ACK, the cells were passed through a 40 μm cell filter (Falcon, 352340) and then resuspended in complete RPMI medium (RPMI 1640 containing 10% heat-inactivated FBS, 100 units / ml penicillin, and streptomycin).

[0133] Afterwards, count the cells and place 5 × 10⁶ cells in 10 ml of dendritic cell medium (DC-medium) (complete RPMI supplemented with 3 ng / ml mGM-CSF (R&D system, 415-ML-010) and an appropriate amount of recombinant protein). 6 The cells were diluted. Then, the cell suspension was dispensed into a 100-mm dish (ID) and cultured at 37°C and 5% CO2 for 5 days. On the 5th day, 5 ml of freshly prepared DC medium was added to the culture medium and cultured for an additional 4 days, and on the 9th day, the suspension cells were collected and used for the experiment.

[0134] To evaluate the production of CD103+ DCs by flow cytometry, cells were washed and stained with IR885-live / dead, APC-CD11c, FITC-MHCII, and PE-CD103 antibodies, and CD103+ DCs were gated from viable cells with CD11c+MHCII. The total cell count on day 9 was measured by collecting suspended cells by light pipetting and using a cell counter (BECKMAN COULTER, Vi-Cell Blue, C19196). Plates cultured for 9 days were EVOS TM Cell cluster formation was observed using the M5000 Imaging System (Invitrogen, AMS5000).

[0135] 1.5 Determination of Tumor Transplantation and Antitumor Activity

[0136] For primary tumor transplantation and treatment, 5 × 10⁶ per mouse were implanted in the right mid-flank of female C57BL / 6 mice. 4 TC-1 cancer cells were injected subcutaneously (sc). After 7 days (when the tumor size reached a diameter of approximately 2-4 mm), tumor-bearing mice were randomly assigned to different treatment groups and subcutaneously vaccinated in the peritumoral region with 100 μl of DPBS (DPBS, n=15), 8 μg of E7ΔNLS (E; n=6), 8 μg of E7ΔNLS with an additional 2 μg of Flt3L (E + F; n=10), 8 μg of E7ΔNLS with an additional 4 μg of FlaB (E + B; n=10), and 8 μg of E7ΔNLS with an additional 6 μg of Flt3L-FlaB (E + FB; n=15). Vaccination was performed three times at 5-day intervals, and tumor growth was monitored every 5 days. Tumor volume was calculated using the formula V = (tumor length) × (tumor width) × (tumor height) / 2, and survival of tumor-carrying mice was observed up to 110 days, with a tumor burden area of ​​2000 mm 3Mice were euthanized when [the threshold] was reached. For re-experimentation, mice that survived on the specified date after the first tumor transplant were given a second subcutaneous injection of TC-1 cells into the contralateral flank, and age-matched purebred mice transplanted with TC-1 cells were used as a control. The tumor transplantation and vaccination schedule was Batf3 - / - The same was maintained in mice. For the anti-PD-1 combination experiment, anti-PD-1 (BioxCell, BE0146) was administered intraperitoneally (ip) at a dose of 200 ng per mouse three times during the experiment as indicated. In the anti-PD-1 combination experiment, tumor-free mice were given a 10-fold higher dose (5 x 10 per mouse). 5 The experiment was repeated using TC-1 cells of the cell.

[0137] 1.6 CD8+ T cell tetramer staining

[0138] To analyze TC-1 tumor-antigen-specific CD8 T cells, tetramer staining specific to HPV16 H-2Db restriction epitope E7 (RAHYNIVTF) was performed. Peripheral blood was obtained from vaccinated mice via retroorbital hemorrhage, and PE-conjugated HPV16 H-2Db-RAHYNIVTF tetramer reagent (TB-5008-1, MBL) was stained with surface-stained CD3 and CD8 antibodies on ice for 45 minutes. Live / dead cell differentiation was performed. TM Fixable Near-IR Dead Cell Stain Kit (Invitrogen TMThe procedure was performed using ACK lysis buffer (Gibco, A10492-01). Subsequently, red blood cells were lysed using ACK lysis buffer (Gibco, A10492-01), and the cells were washed with FACS buffer (DPBS containing 3% FBS) before fixation with 4% paraformaldehyde (T&I, BPP-9004). Data were analyzed by flow cytometry using Cytoflex LX (Beckman Coulter) and analyzed with FlowJo software (Tree Star, Ashland, OR).

[0139] 1.7 IFN-γ ELISpot Analysis

[0140] To measure the frequency of T cells producing IFN-γ after antigen-specific stimulation ex vivo, spleen and tdLN cells were collected 7 days after the last immunization and used for ELISpot-forming cell analysis. Spleen and tdLN were passed through a 70 μm cell filter (FALCON, 352350) and pulverized to release single cells using the flat end of the plunger of a sterile 3 cc syringe, after which the cells were collected in a 15 ml tube containing 7 ml of complete RPMI medium (RPMI 1640 containing 10% heat-inactivated FBS, 100 units / ml penicillin, and streptomycin).

[0141] Red blood cells from the spleen were lysed using ACK lysis buffer (Gibco, A10492-01). The cells were filtered again using a 40 μm cell filter before counting. Total 5 × 10 6 Splenocytes or tdLN cells were seeded into 96-well filtered ELISpot plates (Merck, HAMAS4510) coated with purified anti-IFN-γ antibody (clone R4-6A2; BD Biosciences) and stimulated with 1 μg / ml E7 CTL short peptide (amino acids 49-57: RAHYNIVTF) for 3-5 days for in vitro stimulation.

[0142] Cells stimulated with 10 ng / ml concanavalin were used as a positive control, and cells not stimulated with E7 CTL short peptides were used as a negative control. IFN-γ producing cells were detected using the Mouse IFN-γ ELISpot Set (BD Bioscience, 551083) according to the manufacturer's instructions, and spots were developed using the AEC substrate set (BD, 551951). IFN-γ producing cell analysis was performed using the CTL-ImmunoSpot Analyzer and ImmunoSpot Professional Software version 5.0 (Cellular Technology, Shaker Heights, OH, USA).

[0143] 1.8 Flow Cytometry

[0144] Mice were euthanized at designated times, and subsequently, spleen, tdLN, and tumor tissues were collected for flow cytometry analysis. Spleen and tdLN were digested into single cells by passing them through a 70 μm cell filter, and erythrocytes from the spleen were lysed using ACK lysis buffer. Tumors were collected, weighed, and processed using a razor blade. Tumor tissues were digested with collagenase D (Roche, 11088858001) and DNase I (Roche, 11284932001) supplemented in enzyme medium (RPMI 1640 containing 15% FBS, 100 units / ml penicillin and streptomycin, HEPS, sodium pyruvate, and MEM NEAA) with continuous shaking for 40 minutes at 37°C, and the tumor cell suspension was filtered through a 40 μm cell filter before washing with FACS buffer.

[0145] Subsequently, tumor-infiltrating lymphocytes (TILs) were enriched using CD45(TIL) MicroBeads, mouse (Miltenyi Biotec, 130-110-618) according to the manufacturer's instructions. Single-cell suspensions derived from the spleen, tdLN, and tumors were counted, followed by LIVE / DEAD TM The cells were stained on ice for 40 minutes using the Fixable Near-IR Dead Cell Stain Kit. Afterward, the cells were washed with FACS buffer and stained with extracellular antibodies (Table 3) at 4°C for 30 minutes.

[0146] AntibodyCloneSouceIdentifierCD8 alpha Monoclonal Antibody (KT15), FITCKT15Invitrogen# MA5-16759PE anti-mouse CD8a Antibody53-6.7Biolegend100707APC anti-mouse CD8a Antibody53-6.7Biolegend100711FITC anti-mouse CD8a Antibody53-6.7Biolegend100706LIVE / DEAD™ Fixable Near-IR Dead Cell Stain Kit, for 633 or 635 nm excitationInvitrogenL34975Brilliant Violet 510™ anti-mouse CD45 Antibody30-F11Biolegend103137FITC anti-mouse I-Ab AntibodyAF6-120.1Biolegend116405APC anti-mouse CD103 Antibody2E7Biolegend121413PE anti-mouse CD103 Antibody2E7Biolegend121405Brilliant Violet 605™ anti-mouse CD4 AntibodyGK1.5Biolegend100451PE / Cyanine5 anti-mouse / human CD44 AntibodyIM7Biolegend103009PE / Cyanine7 anti-mouse CD62L AntibodyMEL-14Biolegend104417BD Horizon™ BUV395 Rat Anti-Mouse CD62L (L-Selectin)MEL-14BD Biosciences569400TCF1 / TCF7 (C63D9) Rabbit mAb (Alexa Fluor® 647 Conjugate) #6709C63D9Cell SignalingTechnology6709SBD Pharmingen™ PE Mouse Anti-TCF-7 / TCF-1S33-96BD Biosciences564217Brilliant Violet 510™ anti-mouse CD279 (PD-1) Antibody29F.1A12Biolegend135241APC anti-mouse CD279 (PD-1) Antibody29F.1A12Biolegend135210Brilliant Violet 785™ anti-mouse CD366 (Tim-3) AntibodyRMT3-23Biolegend119725Brilliant Violet 421™ anti-mouse CD3ε Antibody145-2C11Biolegend100341Brilliant Violet 650™ anti-mouse F4 / 80 AntibodyBM8Invitrogen123149Brilliant Violet 785™ anti-mouse / human KLRG1 (MAFA) Antibody2F1 / KLRG1Biolegend138429BD Pharmingen™ PE-Cy™7 Hamster Anti-Mouse CD11cHL3BD Biosciences558079APC anti-mouse CD127 (IL-7Rα) AntibodySB / 199Biolegend121122Brilliant Violet 421™ anti-mouse / rat XCR1 AntibodyZETBiolegend148216iTAg Tetramer / PE - H-2 Db HPV 16 E7 (RAHYNIVTF)MBLTB-5008-1CD45 (TIL) MicroBeads, mouseMiltenyi Biotec130-110-618CD8a+ T Cell Isolation Kit, mouseMiltenyi Biotec130-104-075CellTrace™ CFSE Cell Proliferation Kit, for flow cytometryInvitrogenC34554.

[0147] Cells were fixed and permeated for intracellular staining using the FoxP3 / Transcription Factor Staining Buffer Set (eBioscience, 00-5523-00) according to the manufacturer's instructions. All flow cytometry data were collected using Cytoflex LX (Beckman Coulter) and analyzed with FlowJo software (Tree Star, Ashland, OR).

[0148] Immune cell phenotypes were defined by pre-gating singlets and viable cells and determined as follows:

[0149] CD8+ T cell population: memory precursor effector cell (MPEC; KLRG1- CD127+ CD62L- CD44+), short-lived effector cell (SLEC; KLRG1+ CD127- CD62L- CD44+), progenitor-exhausted cell (Tpex; TCF1+PD-1+TIM3-), stem-like memory cell (Tscm, CD44- CD62L+ TCF1+).

[0150] Dendritic cell population: total dendritic cell (DC; CD45+, F4 / 80-, MHCII+, CD11c+), type 1 conventional dendritic cell (cDC1; CD45+, F4 / 80-, MHCII+, CD11c+, XCR1+, CD103+).

[0151] 1.9 Statistical Analysis

[0152] Results were expressed as mean ± standard error (SEM), and Student's t-test or one-way ANOVA was used to directly compare single variables. The statistical significance of tumor suppression was calculated using two-way ANOVA, and the statistical significance of survival was determined by the log-rank (Mantel-Cox) test. Statistical analysis was performed using Prism 10.00 software for Windows (GraphPad Software, San Diego, CA). A P-value <0.05 was considered statistically significant.

[0153]

[0154] Example 2. Preparation of the protein-based adjuvant Flt3L-FlaB (FB)

[0155] The inventors demonstrated that the Vibrio vulnificus flagellin (FlaB) adjuvant significantly enhances the E7 tumor-specific CD8+ T cell immune response in the TC-1 model via CD11c+ DCs (Lee et al., 2016; Nguyen et al., 2013; Puth et al., 2022). Batf3-dependent type 1 classical dendritic cells are important for the activation and expansion of antigen-specific CD8+ T cells in the tumor microenvironment (TME) (Broz et al., 2014; Spranger et al., 2000). cDC1s can be expanded and recruited by systemic or local administration of the Fms-like tyrosine kinase 3 ligand (Flt3L) (Hegde et al., 2020; Oba et al., 2021; Oba et al., 2020). Therefore, the efficiency of therapeutic cancer vaccines (TCVs) was further enhanced by targeting DCs, specifically cDC1, using a novel hybrid adjuvant consisting of a combination of FlaB and Flt3L. To this end, the hybrid adjuvant was designed using the extracellular domain of mouse Flt3L and FlaB. Two candidates were distinguished by the FlaB position at the C-terminus (Flt3L-FlaB: FB) or N-terminus (FlaB-Flt3L: BF). Only FlaB (B) or Flt3L (F) was generated as a control (Figs. 1, 2, and Table 2). Recombinant protein validation was performed using SDS-PAGE and Western blot with mouse anti-FlaB serum and rabbit anti-Flt3L antibodies (Fig. 4). All recombinant proteins showed increased molecular weight compared to calculations based on amino acid content due to post-translational glycosylation of the recombinant proteins provided in the eukaryotic system (Fig. 3).

[0156] A fusion protein was prepared by combining two structures and different polypeptide functions; to demonstrate the appropriate structure and functionality of the fusion protein, TLR5 stimulating activity was rigorously evaluated. Both FB and BF showed dose-dependent TLR5 stimulating activity using HekBlue hTLR5 cells (Fig. 5), and their function was maintained stably even after storage at -80°C for up to 98 days (Figs. 6a, 6b, and 6c).

[0157] The lower EC50 value of FB compared to BF indicates that the fusion protein with FlaB located at the C-terminus exhibits higher TLR5 stimulating activity and potentially exposes more TLR5 binding motifs. Therefore, FB was selected as a fusion protein candidate. Mouse bone marrow-derived dendritic cells (BMDCs) cultured under Flt3L supplementation produced a large number of DCs in vitro (Brasel, et al., 2000).

[0158] To verify whether Flt3L function is maintained in the fusion protein construct, BMDCs were examined using recombinant F or FB. Specifically, bone marrow cells from C57BL / 6 mice were collected and cultured under supplements B, F, or FB using 8.14 nM recombinant protein for 9 days, after which the suspended cells were harvested by gently pipetting (Fig. 7). As can be seen in Fig. 8, BMDCs cultured with F and FB showed significantly higher cell numbers than the B or untreated groups. Additionally, a significant increase in cell cluster formation was observed in the FB group, whereas only a few cell clusters were detected in the F group (Fig. 8). In terms of total cell number and cluster formation, FB demonstrated a significant improvement compared to F.

[0159] In addition, we investigated whether FB could induce bone marrow-derived CD103+ DCs, as demonstrated by Flt3L in previous studies (Oba et al., 2021; Mayer et al., 2014). Cells cultured for 9 days were washed and stained to detect CD103+ DCs by flow cytometry. As can be seen in Figures 9 and 10, FB-treated bone marrow cells stimulated CD103+ DC production comparable to that of F-treated cells in a dose-dependent manner not observed at any concentration of B. These findings strongly suggest that the functionality of FlaB and Flt3L is effectively preserved within the FB (Flt3L-FlaB) single molecule.

[0160]

[0161] Example 3. Induction of anti-tumor immune response of a therapeutic cancer vaccine using a hybrid protein Flt3L-FlaB adjuvant

[0162] To determine the adjuvant activity of FB for therapeutic cancer vaccines, a previously well-established TC-1 tumor cell transplant mouse model was used (Lee et al., 2016; Puth et al., 2022).

[0163] First, after subcutaneously (sc) injecting TC-1 cells into mice, tumor-bearing mice were vaccinated three times each in the peritumoral region with antigen alone (E), E plus FlaB (E + B), E plus Flt3L (E + F), or E plus Flt3L-FlaB (E + FB) (Fig. 11). As can be seen in Fig. 12, mice vaccinated with E + F and E + B showed equivalent tumor volume reduction (p = 0.9998), and both showed significantly superior results compared to the E group (p = 0.0002 for E vs. E + F; p = 0.0001 for E vs. E + B). On the other hand, mice immunized with antigen and FB significantly delayed tumor growth compared to the antigen plus F (p = 0.0373) and antigen plus B (p = 0.0477) groups. When observing the survival of vaccinated tumor-bearing mice (Fig. 13), there was no significant difference in survival time between the E and E + F groups, but E + B vaccinated mice showed significantly longer survival than the E group (p = 0.0255). These results imply that FlaB can extend survival, which is consistent with recent studies (Giles et al, 2023). In particular, only the E + FB treatment group completely eliminated tumor lesions and provided a long-term survival benefit to 6 out of 15 vaccinated mice by the end of the experiment (Figs. 12 and 13).

[0164]

[0165] Example 4. Induction of tumor antigen-specific CD8+ T cell responses by cancer vaccine using Flt3L-FlaB adjuvant

[0166] To determine the underlying mechanism of FB-induced tumor suppression and long-term survival in tumor-carrying vaccinated mice, we evaluated whether FB could promote the expansion of tumor antigen-specific CD8+ T cells in blood, spleen, and tumor drainage lymph nodes (tdLN) (Fig. 14).

[0167] First, tumor antigen E7-specific CD8+ T cells were evaluated in peripheral blood using tetramers for 4 days following the last vaccination. As can be seen in Figure 15, the frequency of E7 tetramer+ CD8+ T cells in the FB vaccination group was found to be significantly higher than in the DPBS-, E+F-, or E+B vaccination groups. Subsequently, CTL antigen-specific IFN-γ producing cells were determined in the spleen and tdLN using enzyme-linked immunospot (ELISpot) for 7 days following the last vaccination. In the tdLN, mice were immunized with antigen plus adjuvant F or antigen plus adjuvant B. Both adjuvants significantly increased the number of immune cell clusters (spots), and this response was significantly higher than that observed in the control group treated with DPBS. In particular, mice in the group immunized with E + FB induced about 4 times higher numbers of IFN-γ+ secreting cells compared to the E + F or E + B groups (Fig. 16).

[0168] Next, the systemic effects of vaccination were confirmed by analyzing the spleen cells of immunized mice. As determined by IFN-γ ELISpot analysis, this analysis resulted in the generation of the strongest detectable T cell responses in the group immunized with antigen plus FB (Fig. 17). In conclusion, the FB vaccination group strongly induced local (tdLN) and systemic (blood and spleen) antigen-specific CTL cell responses.

[0169]

[0170] Example 5. Induction of CD8+ T cell activation by Flt3L-FlaB adjuvant

[0171] To further confirm the effect of FB on the CD8+ T cell population, the phenotype was evaluated in the tumor-draining lymph nodes and spleens of vaccinated tumor-bearing mice for 7 days after the last vaccination.

[0172] Evaluation results confirmed that the memory precursor effector cell (MPECs; KLRG1-CD127+CD62L-CD44+) phenotype was significantly induced by FB treatment in both the tdLN (Fig. 18) and spleen (Fig. 19) compared to F or B treatments. On the other hand, E+F and E+B immunized mice induced a higher MPEC population in the tdLN (Fig. 18) compared to the DPBS group but failed in the spleen (Fig. 19). It was confirmed that the short-lived effector cell (SLECs; KLRG1+CD127-CD62L-CD44+) population was present in significantly higher percentages in both the tdLN and spleen only in the FB treatment group (Figs. 18 and 19). In addition, FB immunization in tumor drainage lymph nodes (tdLN) significantly induced the frequency of stem-like memory T cells (Tscm; CD44-CD62L+ TCF-1+) (Fig. 20). These results suggest that FB induced a greater expansion of both functional and memory characteristics of the CD8+ T cell population.

[0173] To further verify the above results, we evaluated whether FB could induce long-term protective immunity to prevent tumor recurrence. Tumor-free mice (6) immunized with FB were transplanted with a second set of TC-1 cells 110 days after the initial tumor cell transplant, and pure mice of the same age were used as a control (Fig. 21). Upon administration of the FB vaccine, 100% of the mice did not develop tumors after the second TC-1 challenge with a double dose of cancer cells. In stark contrast, the control group showed the onset of tumor growth just 10 days after the TC-1 transplant (left graph in Fig. 22). Furthermore, all re-challenged mice in the FB group survived until the end of the observation period (164 days), whereas the control group of the same age died within 51 days after the second transplant (right graph in Fig. 22). These results indicate that the effective activation and memory properties of CD8+ T cells were induced by the FB adjuvant.

[0174]

[0175] Example 6. Induction of increased cDC1-mediated cross-presentation of antigens by Flt3L-FlaB adjuvant

[0176] Within the dendritic cell (DC) subpopulation, cDC1 possesses the ability to cross-present antigens to major histocompatibility complex class I (MHC-I), facilitating the promotion of CD8+ T cell responses (Roberts et al., 2016). To evaluate the relevance of cDC1 to the antitumor efficacy of FB, tumors and tumor drainage lymph nodes (tdLN) were collected 24 hours after the last vaccination, and the frequency of the cDC1 subpopulation was determined by flow cytometry (Fig. 23). FB-vaccinated mice showed a significantly increased frequency of cDC1 in tumor drainage lymph nodes (tdLN) rather than total DCs (Fig. 24) (Fig. 25). Furthermore, in the tumor microenvironment, a strong induction of the absolute number of both total DCs and cDC1 subpopulations was detected in FB-vaccinated mice (Fig. 26). In contrast, no significant differences were detected between the DPBS, F-, and B treatment groups. To evaluate the pivotal role of cDC1 in the therapeutic effect of FB, Batf3 knockout (Batf3 - / - ) Subcutaneous transplantation of TC-1 cells was performed on mice, and antitumor efficacy was evaluated compared to wild-type C57BL / 6(WT) mice. Batf3 - / - There are 5 x 10 per mouse 4 Cell transplantation and 8 μg E plus 6 μg FB (Batf3) 7 days after tumor transplantation - / - _E + FB) or DPBS(Batf3 - / - The first vaccine was administered with _DPBS. E + FB treated Batf3 compared to WT mice. - / -Tumor development in mice was significantly accelerated (Fig. 27), failing to mediate complete tumor regression and long-term survival (Fig. 28). These results strongly suggest that the loss of Batf3 significantly reduced the therapeutic efficiency of E+ FB treatment compared to WT mice. Taken together, these results indicate that cDC1 is a significant contributor to the response to FB treatment, leading to enhanced antigen-specific T cell stimulation for tumor elimination and long-term survival.

[0177]

[0178] Example 7. Induction of expansion of precursor-depleted T cells (Tpex) by Flt3L-FlaB adjuvant

[0179] The relationship between progenitor-exhausted T cells (Tpex), a key factor in the reasonable prognosis of cDC1 and PD-1 blockage therapy, has been highlighted in many studies (Schenkel et al., 2021; Im et al., 2016). To determine whether the FB adjuvant can increase Tpex cells by increasing the cDC1 population in tumor-draining lymph nodes (tdLN) and tumors, tumors were collected 7 days after the third vaccination and the Tpex cell population (TCF-1) was analyzed by flow cytometry. + PD-1 + TIM3 - ) was detected. In the tumor microenvironment of E + FB-treated mice, significantly higher levels of Tpex cells were found than in the DPBS, E + F, and E + B groups (Fig. 29).

[0180] Although anti-PD-1 therapy has shown promise as a cancer treatment, its effects were observed in only a small number of patients (Shen et al., 2018). To improve clinical outcomes, a therapeutic cancer vaccine containing an FB adjuvant was conjugated with an anti-PD-1 antibody in TC-1 tumors resistant to anti-PD-1 therapy (Verma et al., 2019; Lee et al., 2022). Anti-PD-1 was administered three times via intraperitoneal injection at a dose of 200 μg per mouse, starting 4 days after the first vaccination (Fig. 30). E+FB conjugated with the anti-PD-1 antibody significantly delayed tumor growth compared to vaccine monotherapy (Fig. 31). Notably, the median survival for the E+FB+α-PD-1 group was not reached at follow-up after 120 days, while the DPBS and TCV monotherapy groups were 44.5 and 62 days, respectively (Fig. 32).

[0181] Although therapeutic cancer vaccines alone can induce long-term protection (Figs. 12 and 13), to verify whether co-administration of TCV and anti-PD-1 can enhance primary tumor treatment efficacy and yield higher memory protection, two groups of tumor-free mice were challenged on the contralateral side with a dose of TC-1 cells 10 times higher than that of the primary tumor transplant. Pure mice of the same age were used as a control. Seven days after the second tumor transplant, PBMCs were harvested from the challenged mice, and tumor antigen (E7)-specific immune memory CD8+ T cell responses were determined by tetramer staining.

[0182] As can be seen in Fig. 33, the E + FB immunization group and the E + FB + α-PD-1 combination group showed tumor antigen-tetramer+ CD8+ T cell frequencies approximately 10-fold and 33-fold higher, respectively, than the age-identical tumor transplant control group. When comparing TCVs with and without anti-PD-1, tumor-specific antigen CD8+ T cells were significantly induced approximately 4-fold in favor of E7 + FB combined with α-PD-1. Furthermore, the combination of anti-PD-1 treatment confirmed complete protection against tumor development even when the TC-1 dose was increased tenfold for the second challenge (Figs. 34 and 35). Conversely, the E + FB group without anti-PD-1 treatment exhibited a single instance of tumor development after the re-challenge (Fig. 34). Due to the significant number of TC-1 cell transplants, age-identical control mice showed noticeably accelerated tumor progression, which reduced the median survival to 37 days (Fig. 35). Importantly, all mice in the PD-1 combination group survived until the end of observation (Fig. 35), and these results indicate that combining a therapeutic cancer vaccine utilizing an FB adjuvant and anti-PD-1 therapy significantly enhances the therapeutic effect.

[0183]

[0184] Example 8. Induction of antitumor immune effects of anti-PD-1 therapy prior to TCV

[0185] Anti-PD-1 therapy has transformed the landscape of cancer immunotherapy, and various anti-PD-1 antibodies from several companies have received FDA approval for the treatment of various cancer types (Upadhaya et al., 2022). However, further research is needed regarding the optimal schedule for anti-PD-1 antibody therapy for successful clinical application (Verma et al., 2019). As shown in the results of Example 7 above, the combination of TCV administered with an FB adjuvant and anti-PD-1 therapy demonstrated favorable therapeutic efficacy (Figs. 31 to 35).

[0186] Considering the optimal timing of CD8+ T cell exposure to antigens in the cancer vaccine prior to the administration of the anti-PD-1 antibody, a strategy was developed to administer anti-PD-1 at different time points. For this strategy, vaccinated tumor-bearing mice received the first anti-PD-1 antibody 4 days after the first or second vaccination (Fig. 36). As expected, after the initial administration of the cancer vaccine, vaccinated mice treated with anti-PD-1 showed a more pronounced antitumor effect than the group that received only E+ FB (Fig. 37). In particular, when the initiation of anti-PD-1 administration was delayed until the end of the second vaccination, approximately 83.3% of vaccinated tumor-bearing mice achieved complete tumor eradication (Fig. 37), which resulted in a notable difference in long-term survival compared to other groups (Fig. 38).

[0187] The observed effect may be attributed to adequate exposure of CD8+ T cells to tumor antigens following two administrations of the cancer vaccine. This data highlights the critical role of the timing of PD-1 blockade therapy when combined with TCV. In conclusion, administering a PD-1 inhibitor after the second administration of the cancer vaccine improved tumor clearance and overall survival.

[0188] Although the present invention has been described in detail above through representative embodiments, those skilled in the art will understand that various modifications can be made to the above-described embodiments without departing from the scope of the present invention. Therefore, the scope of the rights of the present invention should not be limited to the described embodiments, but should be determined by the claims set forth below as well as all modifications or variations derived from the claims and equivalent concepts.

[0189] The hybrid adjuvant FB of the present invention demonstrated significant therapeutic efficacy as an adjuvant for therapeutic cancer vaccines in a preclinical mouse model of cervical cancer, leading to complete tumor regression and sustained protection. Furthermore, the hybrid adjuvant FB of the present invention strongly induced tumor-specific antigen CD8+ T cell responses and progenitor-depleted CD8+ T cells (Tpex) due to increased cross-presentation by type 1 classical dendritic cells (cDC1) in tumor-draining lymph nodes and the tumor microenvironment, and the combination of TCV and anti-PD-1 therapy with the hybrid adjuvant FB significantly improved survival outcomes in anti-PD-1 resistant tumors.

[0190] Accordingly, the objective of the present invention is to provide a hybrid adjuvant comprising Flt3L (Fms-related tyrosine kinase 3 ligand) and flagellin, and a vaccine composition comprising the same as an active ingredient.

Claims

1. A hybrid adjuvant comprising Flt3L (Fms-related tyrosine kinase 3 ligand) and flagellin.

2. In Paragraph 1, The above flagellin is a hybrid adjuvant, which is flagellin B (FlaB) of Vibrio vulnificus.

3. In Paragraph 1, A hybrid immunoadjuvant in which the above Flt3L and the above flagellin exist in the form of fusion proteins.

4. In Paragraph 3, The above fusion protein is a hybrid adjuvant, wherein Flt3L-Flagellin is located at the N-terminus and Flagellin is located at the C-terminus.

5. In Paragraph 1, A hybrid adjuvant, wherein the fusion protein further comprises six histidine residues (6xHis-tag) at the C-terminus.

6. In Paragraph 1, The above-mentioned adjuvant is a hybrid adjuvant that activates Toll-like receptor 5 (TLR5).

7. In Paragraph 1, The above-mentioned adjuvant is a hybrid adjuvant that induces the production of CD103+ dendritic cells.

8. In Paragraph 1, The above hybrid adjuvant is a hybrid adjuvant that induces an increased antigen-specific CD8+ T cell response compared to Flt3L alone or flagellin alone.

9. In Paragraph 1, The adjuvant is a hybrid adjuvant that increases the number of IFN-γ secreting cells.

10. A vaccine composition comprising the hybrid adjuvant of claim 1 as an active ingredient.

Images