Soluble multimer fusion protein, and treatment method using fusion protein

Abstract

To provide a multimer fusion protein, and a method for producing a multimer fusion protein.SOLUTION: A method according to the present invention includes: expressing a nucleic acid encoding an amino acid sequence which includes a signal peptide, (optionally) an antigenic peptide, a CH3 domain of a human IgG1, a (G-P-P)10 collagen-like domain, and a TNF ligand superfamily extracellular domain, from the N terminal to the C terminal, where the extracellular domain lacks a coiled coil trimerization motif, in a mammalian cell; and allowing the nucleic acid-derived polypeptide expressed in the mammalian cell to perform at least one of trimerization and hexamerization to be formed into one or more multimer fusion proteins.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to fusion proteins, and in particular, the present invention relates to soluble multimeric fusion proteins and therapeutic methods using the fusion proteins. [Background technology]

[0002] Antigen-presenting cells, such as dendritic cells, macrophages, and B cells, are immune cells that play a key role in initiating and orchestrating adaptive immune responses by processing antigens and presenting them to T cells. Antigen-presenting cells take up antigens, break them down into smaller peptides, process the peptides, bind the peptides to the surface of major histocompatibility complex (MHC) molecules, and travel to lymph nodes where they encounter T cells. When T cells recognize the antigen-MHC complex, they become activated and initiate an immune response.

[0003] Dendritic cells (DNCs) are the most potent antigen-presenting cells and play an important role in both innate and adaptive immunity. Dendritic cells secrete a variety of proteins, including cytokines, chemokines, and other molecules, and orchestrate immune responses, including T cell activation and inflammation regulation. One major challenge in developing DNC-targeted therapies is effectively delivering antigens to DNCs to promote the activation of antigen-specific CD8+ cytotoxic T cells.

[0004] For example, dendritic cells require two non-self antigens, namely, MHC class I antigens and MHC class II antigens, making them difficult to activate. MHC I is involved in presenting endogenous antigens to CD8+ T cells and is constitutively expressed, while MHC II is involved in presenting foreign antigens to CD4+ T cells and is regulated and upregulated during dendritic cell maturation. Peptide-MHC I complexes on dendritic cells are recognized by CD8+ T cells, leading to their activation and elimination of infected or cancerous cells. MHC II molecules present peptides derived from proteins ingested by cells from foreign antigens. Peptide-MHC II complexes on dendritic cells are recognized by CD4+ T cells, leading to their activation and the development of helper T cell responses.

[0005] CD40 ligand (CD40L, also known as CD154) is a member of the tumor necrosis factor (TNF) superfamily and is primarily expressed on activated CD4+ T cells. CD40 ligand plays a central role in the regulation and activation of antigen-presenting cells. It has been discovered that clustering (or oligomerization) of CD40L is required for effective activation of the cognate CD40 receptor on these immune cells. The use of anti-CD40 agonist antibodies has shown promise in cancer immunotherapy and vaccine adjuvants. However, dose-limiting toxicity associated with Fc receptor cross-linking of anti-CD40 agonist antibodies is a major challenge currently being addressed in clinical trials.

[0006] Studies have shown that CD40 ligation on the surface of dendritic cells bearing CD40L-expressing CD4+ helper T cells is important for priming CD8+ cytotoxic T cells with MHC class I-antigen peptide complexes. Priming CD8+ cytotoxic T cells by binding MHC class II-antigen peptide complexes to dendritic cells in the absence of CD4+ helper T cells can lead to impaired proliferation and cytotoxicity, a phenomenon described as "T cell exhaustion." Ribas et al. reported that CD40 cross-linking can bypass the absolute requirement for CD4+ helper T cells during immunization with melanoma antigen-modified dendritic cells (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0007] Non-Patent Document 1: Ribas A, et al., “CD40 cross-linking bypasses the absolute requirement for CD4 T cells during immunization with melanoma antigen gene-modified dendritic cells”, Cancer Res. 2001 Dec 15;61(24):8787-93. PMID:11751400. Summary of the Invention [Problem to be solved by the invention]

[0008] Based on the foregoing, there remains a need for non-Fc receptor binding anti-CD40 agonist antibodies or CD40 ligands that can cluster CD40 receptors on antigen-presenting cells and reach downstream cell signaling thresholds of immune stimulatory events. [Means for solving the problem]

[0009] The disclosed embodiments solve these and other problems by providing multimeric CD40 ligands configured to cluster CD40 receptors on antigen-presenting cells, thereby reaching downstream cell signaling thresholds of immune stimulatory events. The disclosed embodiments relate to a novel dendritic cell-based cancer vaccine therapy that overcomes the problem of CD8+ T cell exhaustion caused by the lack of CD4+ helper T cells during the dendritic cell activation, or licensing, process by using CD40L fusion proteins bearing soluble multimeric antigenic peptides that can simultaneously activate dendritic cells and prime CD8+ T cells with MHC class I-antigen peptide complexes.

[0010] According to a first embodiment, there is provided a method for producing a multimeric protein in a mammalian cell, the multimeric protein comprising, in N-terminal to C-terminal direction, a signal peptide, optionally an antigenic peptide, the CH3 domain of human IgG1, (GPP) 10 expressing a nucleic acid encoding an amino acid sequence comprising a collagen-like domain and a TNF ligand superfamily extracellular domain, wherein the extracellular domain lacks a coiled-coil trimerization motif; and causing a polypeptide derived from the nucleic acid expressed in the mammalian cell to undergo at least one of trimerization and hexamerization into one or more multimeric fusion proteins.

[0011] The mammalian cell can be an antigen-presenting cell.

[0012] The antigenic peptide may include one selected from the group consisting of epidermal growth factor receptor variant III peptide (PEP3), chicken ovalbumin (257-264) antigenic peptide (OVA), and idiotypic antibody peptide (A20ID) derived from the BALB / c B-cell lymphoma line A20.

[0013] The TNF ligand superfamily extracellular domain can include CD40L.

[0014] Antigenic peptides can form peptide-major histocompatibility complex (MHC) protein complexes on the surface of antigen-presenting cells.

[0015] The antigenic peptide-MHC protein complex can be configured to bind to a T cell receptor on an antigenic peptide-specific T cell and stimulate an immune response.

[0016] The TNF ligand superfamily extracellular domain can include CD137L.

[0017] The antigen-presenting cells can include dendritic cells.

[0018] The nucleotide sequence of the nucleic acid can be SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, or a sequence having at least 90% sequence identity to the nucleotide sequence.

[0019] According to another embodiment, there is provided a method for producing a multimeric protein, the method comprising expressing in an antigen-presenting cell a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7.

[0020] According to another embodiment, from N-terminus to C-terminus, optionally an antigenic peptide, the CH3 domain of human IgG1, (GP-X1) 10 Soluble multimeric fusion proteins are provided that include a collagen-like domain (wherein X1 includes P or O) and a TNF ligand superfamily extracellular domain, wherein the soluble multimeric fusion proteins have a hexameric structure.

[0021] According to another embodiment, an isolated nucleic acid encoding a soluble multimeric protein is provided.

[0022] According to another embodiment, an isolated expression vector comprising the nucleic acid is provided.

[0023] According to another embodiment, an isolated antigen-presenting cell comprising the expression vector is provided.

[0024] According to another embodiment, there is provided an isolated nucleic acid consisting of a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7.

[0025] According to another embodiment, a pharmaceutical composition is provided comprising a nucleic acid encoding the soluble multimeric protein described above.

[0026] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, excipient, or diluent.

[0027] The pharmaceutical composition can be used in the treatment of cancer.

[0028] According to another embodiment, there is provided a method for treating cancer, the method comprising administering to a subject in need thereof the pharmaceutical composition described above.

[0029] The administering step can include delivering the pharmaceutical composition to dendritic cells of the subject ex vivo by transfection.

[0030] The administering step can include delivering the pharmaceutical composition to dendritic cells of the subject in vivo via lipid nanoparticles.

[0031] The cancer can be at least one selected from the group consisting of lymphoma, breast cancer, lung cancer, colon cancer, rectal cancer, prostate cancer, melanoma, brain cancer, spinal cancer, ovarian cancer, pancreatic cancer, uterine cancer, and kidney cancer.

[0032] According to another embodiment, there is provided a pharmaceutical composition for use in the treatment of cancer, the pharmaceutical composition for use in the treatment of cancer comprising a nucleic acid encoding the soluble multimeric protein described above, and administered to a subject in need thereof.

[0033] The administering step can include delivering the pharmaceutical composition for use in treating cancer to dendritic cells of the subject ex vivo by transfection.

[0034] The administering step can include delivering the pharmaceutical composition for use in treating cancer to dendritic cells of the subject in vivo via lipid nanoparticles.

[0035] The cancer can be at least one selected from the group consisting of lymphoma, breast cancer, lung cancer, colon cancer, rectal cancer, prostate cancer, melanoma, brain cancer, spinal cancer, ovarian cancer, pancreatic cancer, uterine cancer, and kidney cancer.

[0036] According to another embodiment, there is provided the use of a nucleic acid encoding the above-described soluble multimeric protein in the manufacture of a pharmaceutical composition, which is administered to a subject in need thereof.

[0037] The administering step can include delivering the pharmaceutical composition to dendritic cells of the subject ex vivo by transfection.

[0038] The administering step can include delivering the pharmaceutical composition to dendritic cells of the subject in vivo via lipid nanoparticles.

[0039] The cancer can be at least one selected from the group consisting of lymphoma, breast cancer, lung cancer, colon cancer, rectal cancer, prostate cancer, melanoma, brain cancer, spinal cancer, ovarian cancer, pancreatic cancer, uterine cancer, and kidney cancer.

[0040] According to another embodiment, there is provided a method of controlling an immune response, the method comprising delivering the pharmaceutical composition described above to dendritic cells of the lymphatic system.

[0041] The pharmaceutical composition can activate CD40 on dendritic cells and induce CD8+ T cells into memory cells through MHC class I antigenic peptide presentation.

[0042] According to another embodiment, there is provided a pharmaceutical composition for use in regulating an immune response, the pharmaceutical composition for use in regulating an immune response comprising a nucleic acid encoding the soluble multimeric protein described above, and delivered to dendritic cells of the lymphatic system.

[0043] The pharmaceutical composition for use in controlling immune responses is capable of activating CD40 on dendritic cells and inducing CD8+ T cells into memory cells through MHC class I antigenic peptide presentation.

[0044] According to another embodiment, there is provided the use of a nucleic acid encoding the above-described soluble multimeric protein in the manufacture of a pharmaceutical composition for the regulation of immune responses, the pharmaceutical composition being delivered to dendritic cells of the lymphatic system.

[0045] The pharmaceutical composition can activate CD40 on dendritic cells and induce CD8+ T cells into memory cells through MHC class I antigenic peptide presentation. [Brief explanation of the drawings]

[0046] [Figure 1] FIG. 1 is a schematic diagram of direct priming and activation of T cells by mRNA-transfected dendritic cells, according to one embodiment. [Figure 2]Schematic diagrams of various structural formats of CD40L fusion proteins are shown. Format A: PepCSCD40L; Format B: PepCS6CD40L; Format C: CD40LCS6Pep; Format D: CS6-TNFL. Pep represents the antigenic peptide, ECD represents the extracellular domain, CS is a collagen-like scaffold peptide (GPP)10, and CS6 contains the CH3 domain of human IgG1, a short linker, and a collagen-like peptide (GPP)10. TNFL represents the TNF ligand superfamily. [Figure 3] Domain structure and purification of the recombinant CD40L fusion protein, PEP3CSCD40L. (A) Schematic diagram of the PEP3CSCD40L protein domain. The signal peptide (SP), epidermal growth factor receptor variant III (EGFRvIII) peptide (PEP3), collagen-like scaffold peptide (CS), extracellular domain of CD40L, and C-terminal affinity tag are shown. The amino acid sequence of PEP3 is shown. (B) SDS-PAGE analysis of PEP3CSCD40L protein purified from culture medium using a Strep-Tactin XT Superflow column. Lane 1: benchmark; Lane 2: PEP3CSCD40L (non-reduced); Lane 3: PEP3CSCD40L (reduced). The protein monomer and trimer are depicted and labeled with arrows. [Figure 4]Domain organization and purification of the recombinant CD40L fusion protein, PEP3CS6CD40L. (A) Schematic diagram of the PEP3CS6CD40L protein domain. Shown are the signal peptide (SP), epidermal growth factor receptor variant III (EGFRvIII) peptide (PEP3), the CS6 domain consisting of the CH3 domain of human IgG1 (CH3) and the (GPP)10 collagen-like scaffold peptide, the extracellular domain of CD40L, and the C-terminal affinity tag. The amino acid sequence of PEP3 is shown. (B) SDS-PAGE analysis of PEP3CS6CD40L and PEP3CSCD40L proteins purified from culture medium using a Strep-Tactin XT Superflow column. Lane 1: benchmark; lane 2: PEP3CS6CD40L (non-reduced); lane 3: PEP3CS6CD40L (reduced); lane 4: PEP3CSCD40L (non-reduced); lane 5: PEP3CSCD40L (reduced). The monomer, trimer, and hexamer of the protein are depicted and labeled with arrows. [Figure 5] Size-exclusion (SEC)-HPLC analysis of purified PEP3CS6CD40L and PEP3CSCD40L proteins is shown. 20 micrograms each of purified PEP3CS6CD40L and PEP3CSCD40L proteins was injected onto a SEC-HPLC column (Zenix-C SEC-300) and monitored by absorbance at 280 nm. Exemplary hexameric and trimeric CD40L fusion proteins, PEP3CS6CD40L and PEP3CSCD40L, respectively, are depicted. After the Strep-Tactin XT column, the elution peak contained biotin in the sample buffer. [Figure 6]The domain structure and purification of the hexameric CD40L fusion protein are shown. (A) Schematic diagram of the PepCS6CD40L protein domain, where Pep represents the antigenic peptide. The signal peptide (SP), the idiotypic antibody peptide (A20ID) derived from the BALB / c B-cell lymphoma line A20 or the chicken ovalbumin (257-264) antigenic peptide (OVA), the CS6 domain consisting of the human CH3 domain of IgG1 (CH3) and the (GPP)10 collagen-like scaffold peptide, the extracellular domain of CD40L, and the C-terminal affinity tag are shown. The amino acid sequences of the A20ID and OVA peptides are shown. (B) SDS-PAGE analysis of A20IDCS6CD40L and OVACS6CD40L proteins purified from culture medium using a Strep-Tactin XT Superflow column. Lane 1: benchmark; lane 2: A20IDCS6CD40L (non-reduced); lane 3: A20IDCS6CD40L (reduced); lane 4: OVACS6CD40L (non-reduced); lane 5: OVACS6CD40L (reduced). The protein monomer and hexamer are depicted and labeled by arrows. [Figure 7] Domain organization and purification of hexameric TNF ligand family fusion proteins of mouse CD40L (CS6CD40L) and human CD137L (CS6hCD137L). (A) Schematic diagram of CS6CD40L and CS6hCD137L, consisting of the human CH3 domain of IgG1 (CH3) and the (GPP)10 collagen-like scaffold peptide, and the extracellular domains of mouse CD40L and human CD137L, respectively. C-terminal affinity tags are shown for purification. (B) SDS-PAGE analysis of CS6CD40L and CS6hCD137L proteins purified from culture medium using a Strep-Tactin XT Superflow column. Lane 1: benchmark; Lane 2: CS6CD40L (non-reduced); Lane 3: CS6CD40L (reduced); Lane 4: CS6hCD137L (non-reduced); Lane 5: CS6hCD137L (reduced). The protein monomer and hexamer are depicted and labeled with arrows. [Figure 8]CD40 activation assays using various CD40 agonist proteins, including PEP3CSCD40L (△), PEP3CS6CD40L (▲), and an anti-CD40 monoclonal antibody (clone G28.5) (□), are shown. The assays were performed using a CD40 cell-based reporter system to assess CD40 cell activation, which was quantified by measuring firefly luciferase activity. [Figure 9] CD137 activation assays with various CD137 agonist proteins, including CS6hCD137L (●) and anti-CD137 monoclonal antibody (urelumab analog) (○), are shown. The assays were performed using a CD137 cell-based reporter system to assess CD137 cell activation, which was quantified by measuring firefly luciferase activity. [Figure 10] Surface marker expression is shown. (A) Expression levels of surface markers: MHC-II, CD40, and CD86 in MutuDC1940 cells after treatment with 1 μg / mL of recombinant mouse CD40L trimer, PEP3CSCD40L trimer, and PEP3CS6CD40L hexamer for 24 hours. (B) Quantification of relative expression levels, measured by mean fluorescence intensity, of the three CD40L fusions measured by flow cytometry. [Figure 11] This shows the presentation of foreign antigens by dendritic cells using the OVA257-264 antigen peptide and OVACS6CD40L hexamer protein together with the anti-OVA-H2Kb monoclonal antibody clone 25D1.16, which specifically reacts with the OVA257-264 peptide that binds to H-2Kb of MHC class I. [Figure 12]Structural and functional characterization of secreted CD40L fusion proteins after mRNA delivery into mammalian cells is shown. (A) HEK293T cells were individually transfected with 1 μg of PEP3CSCD40L (lanes 2-4) and PEP3CS6CD40L (lanes 5-7) mRNA using MesengerMax transfection reagent. Culture supernatants were collected at the indicated days posttransfection and analyzed by immunoblotting with an EGFRvIII-specific monoclonal antibody. Mock represents cells treated with transfection reagent alone. (B) Three days after transfection, culture supernatants from mock-, PEP3CSCD40L-, and PEP3CS6CD40 mRNA-transfected HEK293T cells were analyzed by a cell-based CD40 activation assay. [Figure 13] Figure 1 shows the mean fluorescence intensity of flow cytometry for various embodiments. MutuDC1940 cells were transfected with the indicated amount of mRNA encoding the PEP3CS6CD40L hexamer for 24 hours. Quantification of the relative expression levels of dendritic cell activation surface markers MHC-II, CD40, and CD86 was measured by flow cytometry mean fluorescence intensity. Mock represents cells treated with transfection reagent only. Data from three independent experiments are presented as mean ± SD. Statistical significance was determined using a single two-tailed t-test (*p<0.05; ***p<0.0001; ns: not significant). [Figure 14] Figure 1 shows antigen presentation of various embodiments. Mock represents cells transfected with transfection reagent only (without RNA). [Figure 15]Figure 1 shows T cell priming and activation according to various embodiments. (A) A schematic diagram of direct priming and activation of T cells by dendritic cells transfected with OVACS6CD40L mRNA. mRNA encoding OVACS6CD40L is formulated with lipid nanoparticles and delivered to dendritic cells. (B) MutuDC1940 cells were transfected with OVACS6CD40L and OVACS6CD40L mRNA for 24 hours and subsequently co-cultured with CD8+ T cell hybridoma RF33.70 cells for an additional 48 hours. IL-2 in the culture supernatant was quantified using an IL-2 reporter cell assay kit. Mock represents cells treated with transfection reagent alone. (C) MutuDC1940 cells were treated with 2 μg / mL of OVA257-264 peptide or 10 μg / mL of purified OVACS6CD40L protein for 24 hours, followed by co-culture with CD8+ T cell hybridoma RF33.70 cells for an additional 48 hours. IL-2 in the culture supernatant was quantified using an IL-2 reporter cell assay kit. [Figure 16] Figures A-C represent tumor cell studies according to embodiments. (A) Schematic of the animal study design. mRNA encoding PEP3CS6CD40L was formulated with lipid nanoparticles (in vivo-jetRNA®+) and immunized intravenously into mice. Mice were immunized subcutaneously with PEP3-KLH protein mixed with complete Freund's adjuvant. (B) Exemplary sections of brain tumor size stained with H&E. (C) Total tumor volume was calculated by multiplying the sum of tumor areas in each section by the thickness and spacing per section. Column heights represent the mean ± SEM, and each point represents an individual mouse. Statistical significance was determined using a single two-tailed t-test (*p<0.05, ***p<0.001). Tumor incidence and tumor growth inhibition (TGI) rates for each study group are shown in the right panel. [Figure 17-1]Figures A-C show E.G7-OVA tumor growth in mice according to embodiments. (A) Schematic of the animal study design. C57BL / 6 mice were immunized with mRNA encoding OVACS6CD40L formulated with lipid nanoparticles (in vivo-jetRNA®+) by iv injection on days -14, -7, and -1, or with OVA peptide mixed with poly(I:C) adjuvant by ip injection on days -14 and -1, or with PBS (control) by iv injection on days -14, -17, and -1. (B) The mean tumor volume in each group is shown. Tumor volumes for mice immunized with PBS (●), peptide / poly(I:C) (▲), and mRNA-LNP (■) are shown. (C) The left panel shows the mean tumor volume in each group. 180 days after initial tumor inoculation, eight mRNA-LNP-immunized, tumor-free mice (■) in (B) were re-challenged with 3 × 105 E.G7-OVA cells. Six naive control mice (●) were implanted with the same amount of tumor cells. The right panel shows individual tumor growth curves for each mouse after tumor challenge. Results are shown as mean ± SEM. Statistical significance was determined using a single two-tailed t-test (***p<0.001). CR indicates complete rejection. [Figure 17-2] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0047] In the following detailed description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. However, it will be apparent that one or more embodiments may be practiced without these specific details.

[0048] Disclosed embodiments relate to multimeric fusion proteins, methods for producing multimeric fusion proteins, nucleic acids, expression vectors, antigen-presenting cells, pharmaceutical compositions, methods for treating cancer, and methods for regulating immune responses.

[0049] The disclosed embodiments are applicable to any suitable antigen-presenting cells, including, but not limited to, dendritic cells, macrophages, and B cells. For purposes of this disclosure, embodiments will be described with respect to dendritic cells. However, it will be understood that the disclosure is not intended to be limited to dendritic cells, and that the embodiments are applicable to other antigen-presenting cells.

[0050] Furthermore, the disclosed embodiments are applicable to any suitable target of the immune system. For purposes of this disclosure, embodiments are described that relate specifically to the lymphatic system, including lymphoid organs or lymph nodes and lymphoid cells. Lymphoid structures have been shown to be well suited to stimulating robust immune responses in response to vaccines or other therapeutic treatments.

[0051] FIG. 1 illustrates the development of a dendritic cell-based cancer vaccine using mRNA encoding a multimeric fusion protein, such as a neoantigenic peptide fusion protein with soluble multimeric CD40L, according to an embodiment.

[0052] Multimeric fusion proteins Multimeric fusion proteins according to embodiments can include an antigenic peptide domain, such as a neoantigenic peptide fusion protein domain, human IgG, a collagen-like scaffold peptide, and a TNF ligand protein. The multimeric fusion protein can have a trimeric or hexameric structure. In a preferred embodiment, the multimeric fusion protein has a hexameric structure. The hexameric structure can be formed by dimerization of a trimeric structure via a dimerization domain and a trimerization domain. In a preferred embodiment, the dimerization domain is human IgG, and the trimerization domain is a collagen-like scaffold peptide.

[0053] In embodiments, stable trimeric or hexameric structures can be formed even in the presence of molecules that tend to form dimers. Trimeric dimerization requires careful design of the two driving forces from the dimerization and trimerization domains, as described in U.S. Pat. No. 8,669,350, the entire contents of which are incorporated herein by reference. For example, stabilizing the trimeric structure of a multimeric fusion protein can include, but is not limited to, increasing the number of GPP triplet repeats and / or incorporating a trimerization motif. This results in a thermally stable triple-helical structure that promotes the formation of the trimeric fusion protein despite the presence of a strong dimerization domain. To obtain a pure trimeric Fc-fusion protein, a method can involve destabilizing the dimerization force of the dimerization domain without interfering with the trimeric assembly of the fusion partner.

[0054] The antigenic peptide according to the embodiment can be any suitable antigenic peptide. Antigenic peptides are short segments of amino acids that can be recognized by the immune system, particularly by T cell receptors. These peptides are derived from larger proteins or synthetic peptides. Antigenic peptides are usually presented on the surface of antigen-presenting cells (APCs), particularly dendritic cells, by major histocompatibility complex (MHC) molecules. This presentation is important for T cell activation. In embodiments, antigenic peptides can include, but are not limited to, epidermal growth factor receptor variant III peptide (PEP3), idiotypic antibody peptide (A20ID) derived from the BALB / c B cell lymphoma line A20, or chicken ovalbumin (257-264) antigenic peptide (OVA).

[0055] In embodiments, the human IgG can be IgG1, IgG2, IgG3, and / or IgG4. In preferred embodiments, the human IgG comprises the CH3 domain of human IgG1.

[0056] The CH3 domain of the human IgG1 heavy chain constant region can form homodimers as described by Ying et al. (2013). "Engineered Soluble Monomeric IgG1 CH3 Domain." J Biol Chem. 288(35): 25154-25164, which is incorporated herein by reference in its entirety.

[0057] Collagen-like scaffold peptides according to embodiments can be, for example, multivalent Fab fragments having collagen-like peptides, as described in U.S. Patent No. 10,329,350, the entire contents of which are incorporated herein by reference. Collagen-like scaffold peptides according to embodiments can include at least one extension consisting of at least five, or at least ten, consecutive repeats of a Gly-Pro-Pro or Gly-Pro-Hyp triplet. Collagen-like scaffold peptides can include Gly-Pro-Pro or Gly-Pro-Hyp motifs and / or other Gly-Xaa-Yaa motifs, where Xaa and Yaa are any amino acid residues. Collagen-like scaffold peptides can also include perfectly repeating Gly-Xaa-Yaa triplet sequences interrupted by short defects in which a Gly residue at the first position or a Yaa residue at the third position is missing, as found in many naturally occurring collagens and proteins containing collagen-like domains. This scaffold peptide allows for self-trimerization. The dimer of the trimeric fusion protein, i.e., the hexameric fusion protein, allows for superior clustering effects as discussed herein, without which sufficient dendritic cell activation would not occur.

[0058] In embodiments, the assembled trimer comprises three monomers: a first, second, and third multivalent antibody fragment. In embodiments, the assembled hexamer comprises a dimer of trimers: a first, second, third, fourth, fifth, and sixth multivalent antibody fragment. In one embodiment, the first, second, third, fourth, fifth, and sixth multivalent antibody fragments are substantially identical and have at least 75% sequence identity with each other (e.g., any number between 75% and 100%, inclusive, e.g., 75%, 76%, ... 95%, 96%, 97%, 98%, or 99%). A complex formed by three or six identical multivalent antibody fragments is a homotrimer or homohexamer, respectively. The three or six multivalent antibody fragments described herein can be functionally equivalent. "Functional equivalent" refers to a polypeptide derivative of a generic polypeptide, e.g., a protein having one or more point mutations, insertions, deletions, truncations, fusion proteins, or combinations thereof, that substantially retains the activity of the heterologous domain, such as the ability to form a triple helix coil and binding to a ligand. In one embodiment, there are three copies of the first monomeric multivalent antibody fragment structure and three copies of the second multivalent antibody fragment structure. In one embodiment, there can be two copies of the first multivalent antibody fragment structure, two copies of the second multivalent antibody fragment structure, and two copies of the third polypeptide structure.

[0059] In a hexameric structure according to an embodiment, each of the six monomeric polypeptide sequences can be substantially identical. In one embodiment, there are three copies of the first monomeric fusion polypeptide sequence and three copies of the second fusion polypeptide sequence. In one embodiment, there can be two copies of the first fusion polypeptide sequence, two copies of the second fusion polypeptide sequence, and two copies of the third polypeptide sequence.

[0060] In embodiments, each monomer polypeptide independently comprises: (a) an extracellular domain of a TNF receptor family or a single domain antibody; (b) a collagen-like domain comprising at least eight GP-X1 blocks (wherein X1 can be P or O) and a trimerization motif; (c) optionally, a hinge region of IgG or a glycine linker; and (d) an Fc domain comprising the CH2 and CH3 regions of human IgG.

[0061] For example, percent identity can be determined by comparing sequence information using the GAP computer program (version 6.0) described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math 2:482, 1981). Default parameters for the GAP program include: (1) a one-component comparison matrix (containing a value of 1 for identity and a value of 0 for non-identity) and the weighted comparison matrix for nucleotides of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

[0062] TNF superfamily proteins can include any one or more of 19 structurally unique ligands capable of binding to 29 TNF receptor superfamily members within the TNF superfamily, each encoded by a unique gene. TNF proteins exhibit a characteristic protein fold, shared TNF domains, and trimeric structure and are expressed as type II transmembrane proteins. Soluble cytokines are obtained by proteolytic cleavage of some membrane-bound family members, exemplified by the classical family member TNF (formerly TNF-α), originally characterized for its ability to impair endothelial integrity and its role in tumor necrosis. TNF release is also mediated by neutrophils, CD4 + Following are T cells and innate lymphoid cells. Membrane-expressed TNF superfamily members have key roles in ensuring adaptive immune responses, including CD40 ligand (CD40L), which activates antigen-presenting cells; OX40L, which provides T cell costimulation; FasL, CD27L, and CD30L, which regulate B or T lymphocyte homeostasis and apoptosis, respectively; and BLyS, which affects B cell proliferation and differentiation and has been successfully targeted for SLE therapy.

[0063] For purposes of this disclosure, embodiments will be described with respect to CD40L, however, it will be understood that the disclosure is not intended to be limited to CD40L and that the embodiments are applicable to other TNF ligand proteins.

[0064] As discussed herein, CD40L is a T helper cell membrane-bound trimer that activates CD40 on dendritic cells and induces cytotoxic CD8+ T cells in memory cells. Furthermore, without intending to be bound by theory, CD40L is believed to induce long-term B cell memory by clustering multimeric antigen-binding agents to B cell receptors. The clustering effect, facilitated by the hexameric structure of the disclosed multimeric fusion proteins, is a key feature of the disclosed embodiments. Clustering of multivalent soluble CD40L and CD40 on the surface of dendritic cells triggers surprisingly potent downstream cell signaling events, resulting in dendritic cell maturation, induction of the Th1 pathway via IL-12 secretion, and promotion of CD8+ T cell activation via MHC class I-antigenic peptide presentation.

[0065] Ligation of CD137 by its natural ligands, CD137L or 4-1BBL, activates CD8+ T cells and natural killer cells, leading to increased cell proliferation and survival, proinflammatory cytokine secretion, cytolytic function, and antibody-dependent cellular cytotoxicity.

[0066] The ligand can bind by intermolecular forces with a certain functional affinity. In one embodiment, the multimeric protein has 10 -6 In one embodiment, the multimeric protein has a functional affinity for its ligand that is greater than 10 -8 In one embodiment, the multimeric protein has a functional affinity for its ligand that is greater than 10 -10 In certain embodiments, the soluble trimeric or hexameric fusion protein has a functional affinity for its ligand of greater than 10 -7 M~10 -12 M, 10 -8 M~10 -11 M, 10 -7 M~10 -10 M, 10 -8 M~10 -10 M, and 10-9 M~10 -10 It has a functional affinity (or affinity) of M.

[0067] In embodiments, CD40L can be soluble. A soluble protein is one that is soluble under physiological conditions. In one embodiment, the soluble trimeric or hexameric construct of the multimeric fusion protein is a secreted protein. A secreted fusion protein is one that is secreted by cells. Protein secretion can be targeted by including a signal sequence or signal peptide on the polypeptide comprising the multimeric protein domain.

[0068] Soluble multimeric CD40L can significantly reduce the cytotoxicity caused by excessive expression of transmembrane CD40L on the cell surface compared with the expression of transmembrane forms or anti-CD40 antibodies. Multimeric CD40L fusions can differentiate activated CD8+ T cells into memory CD8+ T cells. Multimeric CD40L-CD40 clustering induces potent downstream cell signaling, bypassing the absolute requirement for CD4+ helper T cells for dendritic cell maturation.

[0069] The multimeric proteins may further comprise a TSMH affinity tag (LVPRGS) containing a signal peptide, a furin cleavage site, a thrombin cleavage site, a Strep-tag II (WSHPQFEK), a c-myc epitope tag (EQKLISEEDL), and a polyhistidine tag (HHHHHH). In further embodiments, the multimeric proteins may comprise other conjugates, such as labeling agents (i.e., "marking agents"), e.g., fluorescent or radioactive agents. Marker proteins include, but are not limited to, luciferase, green fluorescent protein, and enhanced green fluorescent protein. The multimeric fusion proteins of the present embodiments comprising marker proteins can be used in diagnostic and molecular imaging. In embodiments, multimeric fusion proteins comprising marker proteins or radioactive ions or other fusion moieties can be packaged into kits containing the multimeric proteins and other reagents necessary for imaging specific molecules. These reagents may include, but are not limited to, reagents for preparing biological samples and reagents for visualizing the marker proteins.

[0070] In further embodiments, the multimeric proteins can be conjugated to polymers, including, but not limited to, polyethylene glycol, polypropylene glycol, and polyoxyethylated polyols.

[0071] Multimeric proteins according to embodiments can further comprise additional conjugates or amino acid sequences or residues that link the above-described domains. Selective addition or removal of such conjugates or amino acid sequences or residues is encompassed by the present disclosure. It will be appreciated that such selection can be used to optimize the functionality of the multimeric protein under desired conditions.

[0072] Embodiments also include isolated nucleic acids containing sequences encoding multimeric proteins, or the complement of the sequences. Nucleic acid refers to DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), or DNA or RNA analogs. DNA or RNA analogs can be synthesized from nucleotide analogs. Nucleic acid molecules can be single-stranded or double-stranded, but in one embodiment are double-stranded DNA. An "isolated nucleic acid" is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of naturally occurring genomic nucleic acid. Thus, the term encompasses, for example, (a) DNA that has the sequence of a portion of a naturally occurring genomic DNA molecule, but that sequence is not flanked by both of the coding sequences that flank that portion of the molecule in the genome of the organism in which it occurs in nature; (b) nucleic acid that has been incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in such a way that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) separate molecules, such as cDNA, genomic fragments, fragments generated by polymerase chain reaction (PCR), or restriction fragments; and (d) recombinant nucleotide sequences that are part of a hybrid gene, i.e., a gene that encodes a fusion protein.

[0073] The nucleic acids described above can be used to express the polypeptides of the embodiments. For this purpose, the nucleic acids can be operably linked to suitable regulatory sequences to generate expression vectors. A vector refers to a nucleic acid molecule that can transport another nucleic acid to which it is linked. A vector can be autonomously replicating or can integrate into host DNA. Examples of vectors include plasmids, cosmids, or viral vectors. A vector according to the embodiments can include a nucleic acid in a form suitable for expression of a nucleic acid in a host cell. A vector includes one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed.

[0074] "Regulatory sequences" include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include sequences that direct constitutive expression of nucleotide sequences, as well as tissue-specific control sequences and / or derivative sequences. The design of the expression vector may depend on factors such as the choice of host cell to be transformed and the level of expression of the desired protein. The expression vector can be introduced into dendritic cells to produce the polypeptides of the embodiments. The dendritic cells can contain the nucleic acids described above.

[0075] The sequences encoding the disclosed constructs can also contain nucleotide or protein sequences that allow for identification and purification. Such sequences can include restriction enzyme cleavage sites, tags, spacers, and other methods for purifying or identifying the nucleotide or protein sequence. Often, such sequences are contained within nucleotides and encode short amino acid sequences of 4-6 amino acids in length. These often represent intermediate domains of multivalent antibody fragments as artifacts, but do not materially affect the basic and novel features of the present invention.

[0076] To produce the multimeric protein, dendritic cells can be cultured in culture medium under conditions that allow expression of the polypeptide encoded by the nucleic acid, and the polypeptide can be purified from the cultured cells or from the cell culture medium. Peptides, including collagen-like peptides, can be difficult to purify in the absence of an affinity tag.

[0077] Loading of dendritic cells with mRNA nucleic acids can be achieved by any suitable means known in the art. In this regard, both in vivo and ex vivo applications of dendritic cell-based cancer vaccines are contemplated by the present disclosure. Induction of Th1 responses may occur through endogenous mRNA expression pathways. As shown in Figure 1, loading of mRNA can be achieved by ex vivo transfection according to methods known in the art. Loading of mRNA can also be achieved by in vivo application with lipid nanoparticles according to methods known in the art, such as cationic lipid-mediated delivery, electroporation, and polymer-based nanoparticles.

[0078] In embodiments, mRNA nucleic acids can be loaded via the endogenous protein expression pathway by delivering DNA or mRNA encoding an MHC class I-specific peptide fused to multivalent CD40L to dendritic cells; the secreted multivalent CD40L fusion protein can effectively activate dendritic cells, and the MHC class I-specific peptide processed by the dendritic cells can then prime and activate their cognate CD8+ T cells. CD40L-based dendritic cell adjuvants can be delivered to lymphoid organs using an mRNA-nanoparticle approach, thereby avoiding the systemic liver toxicity commonly associated with CD40-activating antibodies.

[0079] The disclosed embodiments also provide methods for treating, preventing, or alleviating symptoms of cancer and T cell-mediated immune diseases, such as autoimmune diseases, by using the multimeric proteins. The disclosed embodiments also provide methods for regulating immune responses.

[0080] In particular, the disclosed methods provide for administering mRNA encoding a multimeric fusion protein secreted outside of dendritic cells while at least some of the multimeric fusion protein is degraded intracellularly. Intracellular degradation of the fusion protein is handled by the proteasome. After trimming by cytosolic proteases, the peptide is transported to the endoplasmic reticulum and loaded onto the shallow groove of the MHC I complex, which then transports the peptide-loaded MHC I complex to the surface of the dendritic cell. The secreted, intact multimeric fusion protein is particularly well suited for achieving CD40 clustering within dendritic cells, followed by dendritic cell activation and priming of peptide-specific CD8+ T cells via ligation of the peptide-loaded MHC I complex, as discussed herein.

[0081] As used herein, cancer may include, but is not limited to, lymphoma, breast cancer, lung cancer, colon cancer, rectal cancer, prostate cancer, melanoma, brain cancer, spinal cancer, ovarian cancer, pancreatic cancer, uterine cancer, and / or kidney cancer.

[0082] As used herein, an autoimmune disease may refer to a disease in which the immune system mistakenly attacks and destroys healthy body tissues, resulting in tissue damage. Autoimmune diseases include, but are not limited to, rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, type 1 diabetes, inflammatory bowel disease, systemic lupus erythematosus, mixed connective tissue disease, progressive systemic sclerosis, antiphospholipid syndrome, psoriasis, scleroderma, glomerulonephritis, dermatomyositis, Hashimoto's thyroiditis, and Graves' disease.

[0083] Effector functions can be demonstrated by apoptosis of tumor cells or phagocytosis and destruction of antibody-coated particles by complement-dependent cytotoxicity (CDC); lysis of antibody-coated target cells by crosslinking of antibody Fc fragments with Fcγ receptors on activated effector cells, such as natural killer cells, by antibody-dependent cellular cytotoxicity (ADCC); destruction of cell membranes; release of inflammatory mediators, including IL-1α, IL-1β, IL-6, and TNFα; and control of immunoglobulin production.

[0084] T cell activation can be demonstrated by measuring T cell proliferation upon stimulation of the T cell receptor (TCR) of a T cell by antigen or agonist antibody. TCR activation can lead to the initiation of signaling pathways, including the induction of specific protein tyrosine kinases (PTKs), the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), the activation of protein kinase C (PKC), and an increase in intracellular calcium ion concentration. These initial events are translocated to the nucleus and result in T cell clonal expansion; upregulation of activation markers on the cell surface; differentiation into effector cells; induction of cytotoxic or cytokine secretion, such as IL-2; and induction of apoptosis.

[0085] Cytokine release syndrome is manifested, for example, by headache, nausea, vomiting, fever, muscle pain, joint pain, and tremors, and can be caused by increased serum levels of, for example, IL-1α, IL-1β, IL-2, IL-6, IL-10, TNFα, and IFNγ.

[0086] Disclosed are methods of treating a disorder, such as, for example, by administering an effective amount of a multimeric protein to a subject in need of treatment to treat the disorder. The subject to be treated can be identified as having the disorder, having a condition characterized by the disorder, or at risk for the disorder or a condition characterized by the disorder. The method can be performed alone or in combination with other drugs or therapies.

[0087] In one embodiment, the treatment is used to treat disorders caused or exacerbated by T cell receptor / alloantigen interactions, thus controlling T cell-mediated toxicity associated with autoimmune diseases. In another embodiment, the treatment is used to modulate the biological activity of CD3, modulate the level of CD3 signaling, or modulate T cell receptor / alloantigen interactions in a subject in need thereof. In one embodiment, the treatment reduces the level of unbound CD3 or CD3 signaling.

[0088] Cytotoxic T cell activation can occur when the CD3 antigen as an effector antigen binds to the surface of the cytotoxic T cell via a multimeric fusion protein. Other lymphoid cell-associated effector antigens include human CD16, NKG2D, NKp46, CD2, CD28, CD25, CD64, and CD89 antigens. Binding to these effector antigens results in the activation of effector cells such as monocytes, neutrophil granulocytes, and dendritic cells. These activated cells then exert cytotoxic or apoptotic effects on target cells.

[0089] The term "treating" is defined as administering a composition to a subject to cure, alleviate, ameliorate, treat, prevent, or reduce a disorder, a symptom of a disorder, a secondary condition of a disorder, a disorder exacerbated by a ligand of a multimeric protein, or a predisposition to a disorder. An "effective amount" is an amount of a composition capable of producing a medically desirable result, e.g., as described above, in a treated subject.

[0090] The disclosed embodiments also provide therapeutic compositions (e.g., compositions containing multimeric fusion proteins) for administration to a subject. Generally, the complexes are suspended in a pharmaceutically acceptable carrier (e.g., saline) and administered orally or intravenously, or by subcutaneous, intramuscular, intrathecal, intraperitoneal, rectal, vaginal, intranasal, intragastric, intratracheal, or intrapulmonary injection or implantation. As discussed herein, the pharmaceutical compositions activate CD40 on dendritic cells and induce CD8+ T cells into memory cells through MHC class I antigenic peptide presentation.

[0091] The required dosage will depend on the route of administration; the nature of the formulation; the nature of the subject's illness; the subject's size, weight, surface area, age, and sex; other medications being administered; and the judgment of the attending physician. A suitable dosage is in the range of 0.01 to 100.0 mg / kg. Suitable dosages are 0.01 to 100.0 mg / kg, or, more specifically, 0.1 to 100, 0.1 to 75, 0.1 to 50, 0.1 to 25, 0.1 to 10, 0.5 to 100, 0.5 to 75, 0.5 to 50, 0.5 to 25, 0.5 to 10, 1 to 100, 1 to 75, 1 to 50, or 1 to 25 mg / kg. Doses can include 1 to 10, 10 to 100, 10 to 75, 10 to 50, 10 to 25, 25 to 50, 50 to 75, 25 to 100, 25 to 50, 50 to 100, or 75 to 100 mg / kg, or can be in the range of 1 to 2, 3 to 4, 5 to 6, 7 to 8, or 9 to 10 mg / kg.

[0092] Therapeutic compositions of the disclosed embodiments can be administered daily, once, twice, or three or more times per week for about 1 to 10 weeks, e.g., 2 to 8 weeks, or about 3 to 7 weeks, and even about 4, 5, or 6 weeks. Variations in dosage requirements can be expected in light of the types of compositions available and differences in efficacy among various routes of administration. For example, oral administration is expected to require higher dosages than administration by intravenous injection. As is well understood in the art, variations in these dosage levels can be adjusted using empirical routines for standard optimization. Encapsulating the composition in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) can increase the efficiency of delivery, particularly oral delivery.

[0093] Pharmaceutically acceptable carriers include solvents, dispersion media, coating agents, antibacterial and antifungal agents, and isotonic and absorption retarding agents.Specifically, these agents include saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and tonicity adjusters such as sodium chloride or dextrose.The pH of the pharmaceutical composition can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide.

[0094] The disclosed embodiments also provide pharmaceutical compositions containing a pharmaceutically acceptable carrier and an effective amount of the multimeric fusion protein of the embodiments. The pharmaceutical compositions can be used to treat the disorders listed above. Pharmaceutically acceptable carriers include solvents, dispersion media, coating agents, antibacterial and antifungal agents, and isotonic and absorption delaying agents. Conventional methods can be used to formulate pharmaceutical compositions into dosage forms for various administration routes.

[0095] The efficacy of the compositions of the disclosed embodiments can be evaluated both in vitro and in vivo. For in vivo studies, the compositions can be injected into animals (e.g., mouse models), and then their therapeutic effects are investigated. Based on the results, appropriate dosage ranges and administration routes can be determined.

[0096] As used herein, the terms "directed against" and "specifically binds to" refer to a fusion protein of the present application that includes an antibody domain and that the antibody, or fragment of the antibody, binds specifically to its ligand with at least 10 -6 This means that it has a functional affinity of M.

[0097] Structure of multimeric fusion proteins Figure 2 is a schematic diagram of various structural formats of multimeric fusion proteins containing TNF family ligands. In both structural formats A and B of Figure 2, the ECD of CD40 ligand is placed at the C-terminus, and the peptide is placed at the N-terminus of the CS and CS6 domains, respectively. In structural format C, the ECD of CD40 ligand is placed at the N-terminus of the CS6 domain, followed by the peptide. In structural format D, the N-terminal peptide motif is removed, and the ECD of either mouse CD40L or human CD137L is placed at the C-terminus of CS6, respectively. In the disclosed embodiments, placing CD40L at the C-terminus is important to achieve proper secretion of the protein from dendritic cells.

[0098] The sequence of a multimeric protein according to an embodiment can include the polypeptide sequence of PEP3CS6CD40L (SEQ ID NO: 3) and a cDNA sequence encoding the polypeptide sequence (SEQ ID NO: 4). The coding region of PEP3CS6CD40L contains, from the N-terminus to the C-terminus, a signal peptide (underlined), a furin cleavage site (double underlined), epidermal growth factor receptor variant III peptide (PEP3)-LEEKKGNYVVTDH (underlined and bold), the CH3 domain of human IgG1 (bold italics), and (GPP) 10It contained the collagen-like domain (bold), the extracellular domain (ECD) of mouse CD40L (amino acid residues Gly115 to Leu260, UniProt accession number P27548) (italicized), lacking the coiled-coil trimerization motif, followed by a TSMH affinity tag containing a thrombin cleavage site (LVPRGS), a Strep-tag II (WSHPQFEK), a c-myc epitope tag (EQKLISEEDL), and a polyhistidine tag (HHHHHH). [Table 1] [Table 2]

[0099] The sequence of a multimeric protein according to an embodiment can include the polypeptide sequence of A20IDCS6CD40L (SEQ ID NO: 5) and a cDNA sequence encoding the polypeptide sequence (SEQ ID NO: 6). The coding region of A20IDCS6CD40L contains, from the N-terminus to the C-terminus, a signal peptide (underlined), a furin cleavage site (double underlined), an idiotypic antibody peptide (A20ID)-DYWGQGTEL (underlined and bold) derived from the BALB / c B-cell lymphoma line A20, the CH3 domain of human IgG1 (bold italics), and (GPP). 10 It contained the collagen-like domain (bold), the extracellular domain (ECD) of mouse CD40L (amino acid residues Gly115 to Leu260, UniProt accession number P27548) (italicized), lacking the coiled-coil trimerization motif, followed by a TSMH affinity tag containing a thrombin cleavage site (LVPRGS), a Strep-tag II (WSHPQFEK), a c-myc epitope tag (EQKLISEEDL), and a polyhistidine tag (HHHHHH). [Table 3] [Table 4]

[0100] The sequence of a multimeric protein according to an embodiment can include the polypeptide sequence of OVACS6CD40LL (SEQ ID NO: 7) and a cDNA sequence encoding the polypeptide sequence (SEQ ID NO: 8). The coding region of OVACS6CD40L contains, from the N-terminus to the C-terminus, a signal peptide (underlined), a furin cleavage site (double underlined), chicken ovalbumin antigen peptide-SIINFEKL (underlined and bold), the CH3 domain of human IgG1 (bold italics), (GPP) 10 It contained the collagen-like domain (bold), the extracellular domain (ECD) of mouse CD40L (amino acid residues Gly115 to Leu260, UniProt accession number P27548) (italicized), lacking the coiled-coil trimerization motif, followed by a TSMH affinity tag containing a thrombin cleavage site (LVPRGS), a Strep-tag II (WSHPQFEK), a c-myc epitope tag (EQKLISEEDL), and a polyhistidine tag (HHHHHH). [Table 5] [Table 6]

[0101] The amino acid and nucleotide sequences according to the embodiments can further include additional amino acid sequences / residues and nucleotide sequences / residues that link or are linked between the above-described multiple domains, so long as the peptides in formats A and B of Figure 2, i.e., the ECD of CD40 ligand located at the C-terminus and the peptides located at the N-terminus of the CS and CS6 domains, respectively, are preserved. Selective addition or removal of such sequences or residues is encompassed by the present disclosure. It will be understood that such selection can be used to optimize the functionality of the multimeric protein under desired conditions.

[0102] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 3, 5, and 7.

[0103] In some embodiments, the nucleotide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 4, 6, and 8.

[0104] The signal peptide sequence is (SEQ ID NO: 15) [Table 7] may include:

[0105] The signal peptide can be cleaved during the expression, assembly, and / or secretion process.

[0106] The furin cleavage site sequence is (SEQ ID NO: 16) [Table 8] may include:

[0107] The PEP3 sequence is (SEQ ID NO: 17) [Table 9] may include:

[0108] The A20ID sequence is (SEQ ID NO: 18) [Table 10] may include:

[0109] The OVA sequence is (SEQ ID NO: 19) [Table 11] may include:

[0110] The CH3 domain of the human IgG1 sequence is (SEQ ID NO: 20) [Table 12] may include:

[0111] The collagen-like polypeptide sequence is (SEQ ID NO: 21) [Table 13] may include:

[0112] The CD40L sequence is (SEQ ID NO: 22) [Table 14] may include:

[0113] The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. [Example]

[0114] Example 1 Construction of plasmids expressing soluble multimeric CD40 ligand fusion proteins—PEP3CSCD40L, PEP3CS6CD40L, A20IDCS6CD40L, OVACS6CD40L, and CD40LCS6OVA The polypeptide sequence of PEP3CSCD40L (SEQ ID NO: 1) and the cDNA sequence encoding said polypeptide sequence (SEQ ID NO: 2) are listed below. The coding region of PEP3CSCD40L contains, from the N-terminus to the C-terminus, a signal peptide (underlined), a furin cleavage site (double underlined), epidermal growth factor receptor variant III peptide (PEP3)-LEEKKGNYVVTDH (underlined and bold), (GPP) 10 The extracellular domain (ECD) of mouse CD40L (amino acid residues Gly115 to Leu260, UniProt accession number P27548) lacking the collagen-like domain (bold) and coiled-coil trimerization motif (italic) was included, followed by a TSMH affinity tag containing a thrombin cleavage site (LVPRGS), a Strep-tag II (WSHPQFEK), a c-myc epitope tag (EQKLISEEDL), and a polyhistidine tag (HHHHHH). The cDNA synthesis sequence of the above polypeptide chain (SEQ ID NO: 2) was codon-optimized by overlap PCR. The PCR product was subcloned into a modified expression vector, pSecTag2 / Hygro (catalog number: V90020, Thermo Fisher Scientific Inc.). In the sequence, the original C-terminal affinity tag sequence was replaced with a TSMH affinity tag for detection and purification. [Table 15] [Table 16]

[0115] PEP3CS6CD40L was constructed according to SEQ ID NO: 3. The cDNA synthesis sequence of the polypeptide chain (SEQ ID NO: 4) was codon-optimized by overlap PCR. The PCR product was subcloned into a modified expression vector, pSecTag2 / Hygro (Cat. No.: V90020, Thermo Fisher Scientific Inc.). In the sequence, the original C-terminal affinity tag sequence was replaced with a TSMH affinity tag for detection and purification.

[0116] A20IDCS6CD40L was constructed according to SEQ ID NO: 5. The cDNA synthesis sequence of the polypeptide chain (SEQ ID NO: 6) was codon-optimized by overlap PCR. The PCR product was subcloned into a modified expression vector, pSecTag2 / Hygro (Cat. No.: V90020, Thermo Fisher Scientific Inc.). In the sequence, the original C-terminal affinity tag sequence was replaced with a TSMH affinity tag for detection and purification.

[0117] OVACS6CD40L was constructed according to SEQ ID NO: 7. The cDNA synthesis sequence of the polypeptide chain (SEQ ID NO: 8) was codon-optimized by overlap PCR. The PCR product was subcloned into a modified expression vector, pSecTag2 / Hygro (Cat. No.: V90020, Thermo Fisher Scientific Inc.). In the sequence, the original C-terminal affinity tag sequence was replaced with a TSMH affinity tag for detection and purification.

[0118] The polypeptide sequence of CD40LCS6OVA (SEQ ID NO: 9) and the cDNA sequence encoding said polypeptide sequence (SEQ ID NO: 10) are listed below. The coding region of CD40LCS6OVA contains, from the N-terminus to the C-terminus, a signal peptide (underlined), the extracellular domain (ECD) of mouse CD40L (amino acid residues Gly115 to Leu260, UniProt accession number P27548) (italics), lacking the coiled-coil trimerization motif, the CH3 domain of human IgG1 (bold italics), and the cDNA sequence encoding the cDNA (GPP). 10The construct contained a collagen-like domain (bold), a chicken ovalbumin antigen peptide-SIINFEKL (underlined and bold), followed by a TSMH affinity tag (LVPRGS) containing a thrombin cleavage site, a Strep-tag II (WSHPQFEK), a c-myc epitope tag (EQKLISEEDL), and a polyhistidine tag (HHHHHH). The cDNA synthesis sequence of the above polypeptide chain (SEQ ID NO: 10) was codon-optimized by overlap PCR. The PCR product was subcloned into a modified expression vector, pSecTag2 / Hygro (catalog number: V90020, Thermo Fisher Scientific Inc.). The original C-terminal affinity tag sequence was replaced with a TSMH affinity tag for detection and purification. [Table 17] [Table 18]

[0119] The polypeptide sequence of CS6CD40L (SEQ ID NO: 11) and the cDNA sequence encoding said polypeptide sequence (SEQ ID NO: 12) are listed below. The coding region of CS6CD40L contains, from the N-terminus to the C-terminus, a signal peptide (underlined), a furin cleavage site (double underlined), the CH3 domain of human IgG1 (bold italics), and (GPP) 10The extracellular domain (ECD) of mouse CD40L (amino acid residues Gly115 to Leu260, UniProt accession number P27548) (italicized) lacking the collagen-like domain (bold), coiled-coil trimerization motif, was included, followed by a TSMH affinity tag (LVPRGS) containing a thrombin cleavage site, a Strep-tag II (WSHPQFEK), a c-myc epitope tag (EQKLISEEDL), and a polyhistidine tag (HHHHHH). The cDNA synthesis sequence of the above polypeptide chain (SEQ ID NO: 12) was codon-optimized by overlap PCR. The PCR product was subcloned into a modified expression vector, pSecTag2 / Hygro (catalog number: V90020, Thermo Fisher Scientific Inc.). In the sequence, the original C-terminal affinity tag sequence was replaced with a TSMH affinity tag for detection and purification. [Table 19] [Table 20]

[0120] Example 2 Construction of a plasmid expressing the soluble multimeric CD137 ligand fusion protein-CS6hCD137L The polypeptide sequence of CS6hCD137L (SEQ ID NO: 13) and the cDNA sequence encoding said polypeptide sequence (SEQ ID NO: 14) are listed below. The coding region of CS6hCD137L contains, from the N-terminus to the C-terminus, a signal peptide (underlined), a furin cleavage site (double underlined), the CH3 domain of human IgG1 (bold italics), and (GPP) 10The extracellular domain (ECD) of human CD137L (amino acid residues Ala93 to Glu254, UniProt accession number P41273) (italicized) lacking the collagen-like domain (bold), coiled-coil trimerization motif, was included, followed by a TSMH affinity tag (LVPRGS) containing a thrombin cleavage site, a Strep-tag II (WSHPQFEK), a c-myc epitope tag (EQKLISEEDL), and a polyhistidine tag (HHHHHH). The cDNA synthesis sequence of the above polypeptide chain (SEQ ID NO: 14) was codon-optimized by overlap PCR. The PCR product was subcloned into a modified expression vector, pSecTag2 / Hygro (catalog number: V90020, Thermo Fisher Scientific Inc.). In the sequence, the original C-terminal affinity tag sequence was replaced with a TSMH affinity tag for detection and purification. [Table 21] [Table 22]

[0121] Example 3 Expression, purification, and characterization of soluble multimeric CD40 ligand fusion proteins—PEP3CSCD40L, PEP3CS6CD40L, A20IDCS6CD40L, OVACS6CD40L, CD40LCS6OVA, CS6CD40L, and CS6hCD137L The above expression plasmid constructs for PEP3CSCD40L, PEP3CS6CD40L, A20IDCS6CD40L, OVACS6CD40L, CD40LCS6OVA, CS6CD40L, and CS6hCD137L were individually expressed in HEK293 cells using the Expi293 Expression System Kit (Cat. No.: A14635, Thermo Fisher Scientific Inc.) according to the manufacturer's instructions. Expi293 cells were cultured at 2.9 × 10 6The cells were cultured in Expi293 expression medium until they reached a density of 100 cells / mL. These cultures were maintained at 37°C on an orbital shaker (125 rpm) in a humidified atmosphere incubator containing 5% CO2. For transfection, 30 μg of pure plasmid DNA and ExpiFectamine 293 reagent were added separately to 1.5 mL aliquots of Opti-MEM reduced serum medium at 37°C. After mixing, the solution was incubated for 20 minutes at 25°C and then added to the Expi293 cell culture. The cultures were then incubated for 48–72 hours at 37°C on an orbital shaker (125 rpm) in a humidified atmosphere incubator containing 5% CO2. Cells were then harvested by centrifugation at 4000 x g for 20 minutes at 4°C. For purification, approximately 30 mL of each filtered medium was applied to a Strep-Tactin XT Superflow column (catalog number: 2-1208-010, IBA Lifesciences GmbH) pre-equilibrated with 1x Buffer W (catalog number: 2-1003-100, IBA Lifesciences GmbH) at a flow rate of 60 mL / h. After washing with the same buffer, the protein was eluted using Buffer 1x BXT (catalog number: 2-1042-025, IBA Lifesciences GmbH). SDS-PAGE was performed using a 4-12% NuPAGE Bis-Tris polyacrylamide gel (catalog number: NP0002, Thermo Fisher Scientific Inc.) containing MES as the running buffer. Proteins were stained with InstantBlue (catalog number: 516938, Expedeon, United States Biological). Bench Mark (catalog number: 10747012, Thermo Fisher Scientific Inc.) was used as a molecular size standard.Size-exclusion (SEC)-HPLC analysis of the purified proteins was performed by injecting 20 μg of each of the purified PEP3CS6CD40L and PEP3CSCD40L proteins onto a Zenix® SEC-300 gel filtration column, 3 μm (Cat. No. Z777092, Merck KGaA) and monitoring the absorbance at 280 nm.

[0122] Table 1 below summarizes the structural characteristics of several trimeric fusion proteins after transient expression in Expi293 cells. [Table 23]

[0123] Both structural formats A and B, in which the ECD of CD40 ligand (lacking the coiled-coil trimerization motif) is located C-terminal to the CS and CS6 domains, respectively, can be expressed as soluble secreted proteins in Expi293 cells (see Figure 2). SDS-PAGE and SEC-HPLC analysis of format A of PEP3CSCD40L under native and physiological conditions, respectively, reveals the presence of a collagen-like peptide (GPP). 10 It was confirmed that the trimerization motif of CS containing the β-CD40L motif can form stable CD40L trimers, as shown in lane 2 of Figure 3B, lane 4 of Figure 4B, and the dotted line in Figure 5. Under reducing conditions, the PEP3CSCD40L trimer dissociated into monomers (lane 3 of Figure 3B and lane 5 of Figure 4B). SDS-PAGE and SEC-HPLC analyses of PEP3CS6CD40L format B under native and physiological conditions, respectively, revealed the presence of the human IgG1 CH3 dimerization domain and the trimerized collagen-like peptide (GPP). 10The CS6 domain containing the PEP3CSCD40L trimer can form stable CD40L hexamers, as shown in lane 2 of Figure 4B and the solid line in Figure 5. Under reducing conditions, the PEP3CSCD40L trimer dissociated into monomers (lane 3 of Figure 4B). Similar results were obtained with format B of A20IDCS6CD40L and OVACS6CD40L, which form stable hexamers when analyzed by SDS-PAGE under non-denaturing conditions (Figure 6). In structural format D (Figure 7A), in which the N-terminal peptide motif is removed and the ECD of either mouse CD40L or human CD137L (which lacks the coiled-coil trimerization motif) is placed at the C-terminus of the CS6 domain, both purified fusion proteins can form stable hexamers when analyzed by SDS-PAGE under non-denaturing conditions (Figure 7B, lanes 2 and 4).

[0124] Unexpectedly, the ECD of CD40 ligand is located at the N-terminus of the CS6 domain, which binds to OVA. 257-264 The structural format C of CD40LCS6OVA in Table 1, followed by the peptide, was found to be non-secreted in Expi293 cells. The TNF family of ligands are type II transmembrane proteins in which the ECD is located in the C-terminal region of the assembled protein trimer. Therefore, the correct domain structural orientation is required for proper assembly of the CS6-CD40L fusion protein into a hexamer.

[0125] Example 4 CD40 reporter gene potency assay of soluble multimeric CD40L fusion proteins The efficacy of various CD40 agonist proteins, including PEP3CSCD40L, PEP3CS6CD40L, and an anti-CD40 monoclonal antibody (clone G28.5), in activating cell signaling was performed by a bioluminescent cell-based assay using a CD40 Bioassay kit (Promega, #JA2151) according to the manufacturer's protocol. Briefly, frozen CD40 effector cells expressing CD40 and a downstream NF-κB promoter-driven firefly luciferase gene were thawed and cultured at 2.5 × 10 cells / mL in 100 μL of reconstitution medium (90% RPMI 1640 / 10% FBS). 4 Cells were seeded at a density of 1000 cells / well. The lid was placed on the assay plate and incubated overnight (18-22 hours) in a 37°C, 5% CO2 incubator. On the day of the assay, all media was carefully removed from the cells in the wells. Serial dilutions of CD40 agonist protein (70 μL volume) were added to each well. The cells and CD40 agonist were incubated at 37°C for 6 hours. 70 μL of One-Glo reagent was then added, and the plate was incubated at room temperature for 15 minutes. The reaction mixture was then transferred to a white plate for luminescence measurement.

[0126] As shown in Figure 8, upon binding of reporter cells to CD40, the soluble PEP3CS6CD40L hexamer exhibits the strongest downstream expression of firefly luciferase compared to the soluble PEP3CSCD40L trimer and a bivalent CD40 agonist monoclonal antibody (G28.5). The results also confirm that the functional folding of the hexameric PEP3CS6CD40L and trimeric PEP3CSCD40L fusion protein structures allows for the induction of potent downstream cell signaling effects in response to CD40 cell activation.

[0127] Example 5 CD137 reporter gene potency assay of soluble hexameric CD137L fusion protein Urelumab is an anti-human CD137 agonist monoclonal antibody intended to stimulate T cells to promote antitumor immunity. The efficacy of the soluble hexameric CD137L fusion protein, CS6hCD137L, and the CD137 agonist monoclonal antibody (urelumab analog) in activating cell signaling was determined by a bioluminescent cell-based assay using the CD137 Bioassay Kit (Promega, #JA2351) according to the manufacturer's protocol. Briefly, frozen CD137 effector cells expressing CD137 and a downstream NF-κB promoter-driven firefly luciferase gene were thawed and cultured at 2.5 × 10 cells / mL in 100 μL of reconstitution medium (95% RPMI 1640 / 5% FBS). 4 Cells were seeded at a density of 1000 cells / well. The lid was placed on the assay plate and incubated overnight (18-22 hours) in a 37°C, 5% CO2 incubator. On the day of the assay, all media was carefully removed from the cells in the wells. Serial dilutions of CD40 agonist protein (70 μL volume) were added to each well. The cells and CD40 agonist were incubated at 37°C for 6 hours. 70 μL of One-Glo reagent was then added, and the plate was incubated at room temperature for 15 minutes. The reaction mixture was then transferred to a white plate for luminescence measurement.

[0128] As shown in Figure 9, upon binding of reporter cells to CD137, soluble CS6hCD137L hexamers exhibit stronger downstream expression of firefly luciferase than a bivalent CD137 agonist monoclonal antibody (a urelumab analog). The results also confirm that the functional folding of the hexameric CS6hCD137L structure allows CD137-CD137L clustering to elicit potent downstream cell signaling effects on the CD137 response to cell activation.

[0129] Example 6 Activation of dendritic cells by soluble multimeric CD40L fusion proteins CD40 +We compared the effects of various CD40L fusion proteins on dendritic cell activation. MutuDC1940 dendritic cells (Cat. No. T0528, Applied Biological Materials Inc.) 13 5 x 10 cells in a 12-well plate 5 Cells were plated at 100 cells / well and incubated in the presence of 1 μg / mL recombinant mouse CD40L (active trimer, catalog number: CDL-M52D5, Acro Biosystems), PEP3CSCD40L, and PEP3CS6CD40L for 24 hours at 37°C in RPMI-1640 medium supplemented with L-glutamine, 50 μM β-mercaptoethanol, 10 μg / mL gentamicin, and 10% heat-inactivated fetal bovine serum. Cells were washed, resuspended in staining buffer, and stained with PE-conjugated anti-mouse MHC class II mAb, PerCP / Cy5.5-conjugated anti-mouse CD40, and APC-conjugated anti-mouse CD86 prior to flow cytometry analysis.

[0130] As shown in Figure 10A, only the soluble PEP3CS6CD40L hexamer, but not the recombinant mouse CD40L (active trimer) or the PEP3CSCD40L trimer, was able to significantly upregulate the expression of dendritic cell activation surface markers, MHC class II, CD40, and CD86, and flow cytometry data analysis showed that the induction level for PEP3CS6CD40L was approximately 1.5-fold greater than that for the recombinant mouse CD40L reference control (Figure 10B).

[0131] The results indicate that multivalent cross-linking of CD40L to the CD40 receptor on dendritic cells is essential for dendritic cell activation and maturation. The results further demonstrate that a hexameric CD40L fusion protein (PEP3CS6CD40L) has more potent stimulatory activity for MutuDC1940 dendritic cells than either native soluble trimeric CD40L or recombinant PEP3CSCD40L trimer.

[0132] Example 7 Presentation of exogenous antigens by dendritic cells treated with OVA257-264 antigen peptide and OVACS6CD40L hexamer protein Various concentrations of OVA 257-264 5 x 10 MutuDC1940 dendritic cells were cultured in 12-well plates in the presence of peptide and OVACS6CD40L hexameric protein for 24 hours. 5 Cells were washed, resuspended in staining buffer, and incubated with OVA peptide. 257-264 , and H-2K of the MHC class I complex. b The cells were stained with APC-conjugated 25D1.16 monoclonal antibody (catalog number: 141605, Biolegend), which specifically binds to , and then subjected to flow cytometry analysis.

[0133] In Figure 11, the 25D1.16 monoclonal antibody inhibited OVA binding to H-2Kb of MHC class I. 257-264 This indicates that the OVA peptide reacted specifically with the 257-264 The peptide inhibited the H-2K binding of MHC class I to MutuDC1940 cells at a saturating peptide concentration of approximately 2 μg / mL. b This indicates that the OVA in OVACS6CD40L was successfully loaded onto the cells. No binding was observed in MutuDC1940 cells treated with OVACS6CD40L. Although the soluble hexameric CD40L fusion protein of OVACS6CD40L was able to activate MutuDC1940 cells, the OVA in OVACS6CD40L was not. 257-264 Peptides cannot be exogenously processed by dendritic cells and are bound to H-2K receptors on the MHC class I receptors on the dendritic cell surface. b The results confirm that class I MHC molecules utilize the cytosolic pathway for processing antigens.

[0134] Example 8 In vitro synthesis of 5'-capped pseudouridine-modified mRNA encoding a soluble multimeric CD40L fusion protein Intact secreted hexameric OVACS6CD40L protein and OVA 257-264To achieve the dual function of OVACS6CD40L in simultaneously activating the same dendritic cells by processing antigenic peptides and presenting them on class I MHC molecules, we attempted to endogenously express the protein by either introducing DNA encoding OVACS6CD40L or by directly delivering mRNA containing the OVACS6CD40L open reading frame into dendritic cells. cDNA open reading frame sequences encoding soluble multimeric CD40L fusion proteins, including PEP3CSCD40L, PEP3CS6CD40L, OVACS6CD40L, and CS6CD40L (lacking the N-terminal antigenic peptide sequence), were subcloned in frame into the multiple cloning site (MCS) of the pUC-T7ag mRNA expression vector. The pUC-T7ag vector contains the dinucleotide "AG" immediately downstream of the T7 promoter for CleanCap® AG 5'-end cap formation, followed by the 5'UTR, MCS, TGA translation termination codon, 3'UTR, and poly(A) tail. In vitro transcription of various soluble multimeric CD40L mRNAs was performed using the RiboMAX™ Large-Scale RNA Production System (Cat. No. P1300, Promega, Corp.). Each transcription reaction contained 10 μg of linearized pUC-T7ag DNA template, 1× reaction buffer, 4 mM CleanCap® AG (Cat. No. N-7113, TriLink BioTechnologies), 5 mM each of ATP, GTP, CTP, and N1-methylpseudo-UTP (Cat. No. NU-890L, Jena Biosciences), 100 units of RNasin® Plus Ribonuclease Inhibitor (Cat. No. N2615, Promega Corp.), and 100 units of T7 RNA polymerase in a final volume of 100 μL. The reaction mixture was incubated at 37° C. for 3 hours, followed by digestion with 10 units of DNase I (Cat. No. M6101, Promega Corp.) for 15 minutes at 37° C. to remove the DNA template.The mRNA was purified using the Embark™ mRNA Purification Kit (Cat. No. EMBARK50, Messenger Bio) according to the manufacturer's instructions. The amount of RNA was measured by UV absorbance at 260 nm.

[0135] Example 9 mRNA transfection and Western blot analysis Because the efficacy of mRNA therapeutics depends on the level and duration of protein translation after delivery of mRNA to target cells, we evaluated the mRNA translation level and duration of soluble multimeric CD40L fusion proteins, including PEP3CSCD40L and PEP3CS6CD40L. HEK293T cells were transfected with 1 μg of in vitro-transcribed PEP3CSCD40L and PEP3CS6CD40L mRNA, respectively, in 24-well plates using Lipofectamine MessengerMAX (catalog number: LMRNA015, Thermo Fisher Scientific Inc.) according to the manufacturer's protocol. Cells were cultured at 37°C in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS). Thirty microliters of culture supernatant was collected on days 1, 2, and 3 posttransfection and run on a 4-12% NuPAGE Bis-Tris SDS-polyacrylamide gel (catalog no. NP0002, Thermo Fisher Scientific Inc.) under non-denaturing conditions using MES as the running buffer. Proteins were then transferred to a nitrocellulose membrane (GE Healthcare) according to the manufacturer's protocol. After blocking with 5% nonfat milk overnight at 4°C, the membrane was incubated overnight at 4°C with an EGFRvIII-specific rabbit monoclonal antibody, clone RM419 (catalog no. 31-1305-00, RevMAb Biosciences). After washing, the membrane was incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (catalog no. 7074S, Cell Signaling) for 1 hour at room temperature. The membrane was treated with ECL Western blotting detection reagent (Invitrogen) and imaged using a Fujifilm LAS-4000 detection system.

[0136] Figure 12A shows the results of immunoblot imaging. As expected from the recombinant expression of both PEP3CSCD40L and PEP3CS6CD40L by transient transfection of DNA plasmid constructs into Expi293 cells, direct delivery of mRNA encoding the two proteins into HEK293T cells successfully secreted intact trimeric and hexameric PEP3 antigenic peptide-containing CD40L fusions, respectively, after probing with an EGFRvIII-specific antibody. Notably, a trimeric form, half the molecular size of the PEP3CS6CD40L hexamer, was detected by the antibody. This trimeric species was likely caused by SDS-induced dissociation of non-interchain disulfide-bonded CH3 dimers under electrophoresis. Comparison of protein expression levels over time in HEK293T cells transfected with 1 μg of mRNA demonstrated that the mRNA-transfected cells were capable of constitutively expressing both proteins for up to 3 days.

[0137] Example 10 CD40 reporter gene potency assay of mRNA-transfected cell culture supernatants To further evaluate the CD40 activation potency of the above-described cell-secreted multimeric CD40L fusion proteins synthesized with cognate mRNAs, including PEP3CSCD40L and PEP3CS6CD40L, a cell-based luciferase reporter gene assay for activating cell signaling was performed using a CD40 Bioassay Kit (Promega, #JA2151) as described in Example 4. Three days after transfection, 40 microliters of PEP3CSCD40L and PEP3CS6CD40L mRNA-transfected cell culture supernatant was added to each well. The cells were cultured at 37°C for 6 hours. Then, 70 μL of One-Glo reagent was added, and the plate was incubated at room temperature for 15 minutes. The reaction solution was then transferred to a white plate for luminescence measurement.

[0138] Figure 12B shows that culture supernatants from HEK293T cells transfected with PEP3CSCD40L and PEP3CS6CD40L mRNA can activate CD40-bearing reporter cells. Similar to the results in Example 4, using purified proteins, mRNA encoding hexameric PEP3CS6CD40L exerts a CD40 activation potency threefold higher than that of trimeric PEP3CSCD40L.

[0139] Example 11 Activation of dendritic cells by cell-transfected mRNA encoding a soluble multimeric CD40L fusion protein. CD40 + MutuDC1940 dendritic cells (Cat. No.: T0528, Applied Biological Materials Inc.) were cultured in a 12-well plate at 5 × 10 5 Cells were plated at 1000 cells / well and incubated at 37°C in RPMI-1640 medium supplemented with L-glutamine, 50 μM β-mercaptoethanol, 10 μg / mL gentamicin, and 10% heat-inactivated fetal bovine serum. Cells were transfected with either 1 or 2 μg of in vitro-transcribed PEP3CS6CD40L mRNA in 24-well plates using Lipofectamine MessengerMAX (catalog number: LMRNA015, Thermo Fisher Scientific Inc.) according to the manufacturer's protocol. 24 hours after transfection, cells were washed, resuspended in staining buffer, and stained with PE-conjugated anti-mouse MHC class II mAb, PerCP / Cy5.5-conjugated anti-mouse CD40, and APC-conjugated anti-mouse CD86 prior to flow cytometry analysis.

[0140] As shown in Figure 13, MutuDC1940 dendritic cells could be activated after transfection with mRNA encoding a hexameric CD40L fusion protein (PEP3CS6CD40L). Flow cytometry data analysis showed that MutuDC1940 dendritic cells transfected with either 1 or 2 μg of mRNA encoding the PEP3CS6CD40L hexamer could significantly upregulate the expression of dendritic cell activation surface markers, MHC class II, CD40, and CD86, and the induction levels of these dendritic cell activation surface markers for PEP3CS6CD40L were approximately 1.8-fold, 2.0-fold, and 1.7-fold greater than those of the untransfected cell control (Ctrl), respectively. The mRNA encoding the PEP3CS6CD40L hexamer could be successfully secreted as a soluble protein, and CD40 + The results show that it is able to bind to and activate dendritic cells.

[0141] Example 12 Presentation of endogenous antigens by dendritic cells transfected with mRNA encoding OVACS6CD40L MutuDC1940 dendritic cells were seeded into 12-well plates at 5 x 10 5 Cells were plated at 100 cells / well and incubated at 37°C in RPMI-1640 medium supplemented with L-glutamine, 50 μM β-mercaptoethanol, 10 μg / mL gentamicin, and 10% heat-inactivated fetal bovine serum. Cells were transfected with 1 μg of either PEP3CS6CD40L mRNA or OVACS6CD40L mRNA for 24 hours using Lipofectamine MessengerMAX. Cells were washed, resuspended in staining buffer, and transfected with OVA peptide. 257-264 , and H-2K of the MHC class I complex. b The cells were stained with APC-conjugated 25D1.16 monoclonal antibody (catalog number: 141605, Biolegend), which specifically binds to , and then subjected to flow cytometry analysis.

[0142] In Figure 14, the 25D1.16 monoclonal antibody specifically reacts with MutuDC1940 dendritic cells transfected with OVACS6CD40L mRNA. 257-264 The peptide was successfully loaded and the H-2K complex of the MHC class I-peptide complex was b The results show that delivery of OVACS6CD40L mRNA to MutuDC1940 dendritic cells enabled the OVA antigenic peptide to be processed by the exogenous antigen presentation pathway and transferred to the MHC class I complex on the cell surface. No binding was observed in MutuDC1940 cells treated with mock (transfection reagent only) or PEP3CS6CD40L mRNA, confirming the antibody specificity of 25D1.16.

[0143] Treatment of MutuDC1940 cells with intact OVACS6CD40L protein resulted in the uptake of OVA peptide. 257-264 The previous results in Example 7 indicate that antigenic peptides cannot be processed and loaded onto class I MHC molecules on the cell surface. However, by directly delivering mRNA containing the open reading frame of OVACS6CD40L into dendritic cells, OVA can be expressed via the endogenous protein expression pathway (i.e., intracellularly produced protein). 257-264 The peptides can be successfully processed and loaded onto class I MHC molecules at the cell surface.

[0144] Example 13 Antigen presentation and CD8+ T cell activation by dendritic cells In Example 12, OVA was administered to MutuDC1940 cells. 257-264 The amount of peptide / MHC class I complexes is 257-264 Peptide-specific CD8 + To further demonstrate that OVACS6CD40L mRNA can prime and activate T cells, MutuDC1940 cells transfected with OVACS6CD40L mRNA were transfected with CD8 +MutuDC1940 dendritic cells were co-cultured with T cells to confirm whether T cells could be activated by IL-2 secretion. The day before transfection, 5 × 10 5 MutuDC1940 cells were plated at 100 cells / well and incubated in RPMI-1640 medium supplemented with L-glutamine, 50 μM β-mercaptoethanol, 10 μg / mL gentamicin, and 10% heat-inactivated fetal bovine serum at 37°C. MutuDC1940 cells were transfected with 1 μg of CS6CD40L mRNA or OVACS6CD40L mRNA using Lipofectamine MessengerMAX, or with 2 μg / mL OVA. 257-264 MutuDC1940 cells were stimulated with peptide or 10 μg / mL of OVACS6CD40L. After 24 hours of incubation, OVA 257-264 Peptide-specific CD8 + T cell hybridoma RF33.70 cells (kindly provided by Kenneth Rock, University of Massachusetts) were cultured at 1 × 10 6 The cells were added to 12-well plates at a density of 1 cells / well and co-cultured with MutuDC1940 cells for an additional 48 hours. The supernatants from these cultures were analyzed for the presence of IL-2 using HEK-Blue™ IL-2 cells (InvivoGen) according to the manufacturer's protocol.

[0145] A schematic diagram of direct priming and activation of T cells by OVACS6CD40L mRNA-transfected dendritic cells is shown in Figure 15A. MutuDC1940 dendritic cells transfected with OVACS6CD40L mRNA can prime and activate OVA antigen peptide-specific CD8+ T cells. Dendritic cells transfected with the mRNA produce secreted hexameric OVACS6CD40L protein, which self-activates by binding to their own CD40 receptor. During this process, a portion of the cytosolic OVACS6CD40L protein is degraded by the proteasome system, followed by OVA. 257-264The antigen peptide was presented to class I MHC molecules. 257-264 -MHC class I complexes are expressed by OVA 257-264 It is able to react with (prime) antigenic peptide-specific CD8+ T cells, leading to T cell activation and proliferation by secreting the cytokine IL-2.

[0146] After transfection of MutuDC1940 cells, only OVACS6CD40L mRNA, but not mock or CS6CD40L mRNA carrying a non-OVA antigen peptide, was able to prime and activate RF33.70 cells (Fig. 15B). 257-264 As expected from exogenous peptide treatment, peptide-specific CD8 + T cells were able to bind the TCR to the unprocessable OVACS6CD40L protein but not to the loaded OVA on MutuDC1940 cells. 257-264 It could be activated by binding to peptide / MHC class I complexes (Fig. 15C).

[0147] Example 14 Antitumor efficacy of PEP3CS6CD40L mRNA-LNPs in a syngeneic mouse glioblastoma multiforme (GBM) tumor model For the syngeneic mouse GBM tumor model experiment, 6-8 week-old female C57BL / 6 mice were randomly divided into three groups (n=8 per group) and administered as shown in Figure 16A. In Group 1, each mouse received 100 μL of PBS (control) via iv injection on days -14, -17, and -1. In Group 2, each mouse received 20 μg of PEP3CS6CD40L mRNA formulated by in vivo-jetRNA®+ (Polyplus, catalog number: 101000122) via iv injection on days -14, -17, and -1. In Group 3, each mouse received 50 μg of PEP3-KLH protein mixed with complete Freund's adjuvant on day −14, followed by another 50 μg of PEP3-KLH protein mixed with incomplete Freund's adjuvant on day −1, by sc injection. On day 0 after the last administration, 1×10 mice were injected with 1×10 EGFRvIII stably expressing the EGFRvIII gene (CT-2A-EGFRvIII). 5 Mouse CT-2A glioblastoma multiforme (GBM) cells (EMD Millipore Corporation, catalog number: SCC194) were intracranially implanted into mice. Mice were sacrificed on day 18, and total tumor volume was calculated by multiplying the sum of the tumor areas in each section by the thickness and spacing per section (FIG. 16B, exemplary section).

[0148] The results shown in Figures 16B and 16C demonstrate that after immunization, both the PEP3CS6CD40L mRNA-LNP and PEP3-KLH / adjuvant vaccines effectively prevented the growth of CT-2A-EGFRvIII GBM in C57BL / 6 mice compared with the PBS-untreated group. Notably, the antitumor effect of the PEP3CS6CD40L mRNA-LNP vaccine was far superior to that of the PEP3-LKH / adjuvant vaccine in terms of tumor size and tumor incidence, and four of eight mice in the PEP3CS6CD40L mRNA-LNP-vaccinated group were found to be tumor-free.

[0149] Example 15 Antitumor efficacy of OVACS6CD40L mRNA-LNP in a syngeneic lymphoma tumor model For the syngeneic mouse lymphoma tumor model experiment, 6-8 week-old female C57BL / 6 mice were randomly divided into three groups and administered as shown in Figure 17A. In Group 1 (n = 6), each mouse received 100 μL of PBS (control) via iv injection. In Group 2 (n = 8), each mouse received 20 μg of OVACS6CD40L mRNA formulated with in vivo-jetRNA®+ (Polyplus, catalog number: 101000122) via iv injection. In Group 3 (n = 6), each mouse received 100 μg of OVA mixed with 50 μg of poly(I:C) adjuvant (catalog number: vac-pic, InvivoGen) via ip injection. 257-264 Mice in groups 1 and 2 were repeatedly dosed on days -14, -7, and -1, while mice in group 3 were dosed on days -14 and -1, followed by 3 x 10 5 E.G7-OVA cells (Cat. No.: 60418, Bioresource Collection and Research Center, Taiwan) were subcutaneously implanted. Mice were sacrificed on day 14. To monitor tumor progression, tumor size was continuously measured and measured as 1 / 2×L×W. 2 Tumor volume was expressed as (cubic millimeters), where L is the longest tumor diameter and W is the shortest tumor diameter.

[0150] The tumor rechallenge study was designed as follows: Tumor-free mice in the OVACS6CD40L mRNA-LNP-treated group were monitored for 180 days to ensure there were no signs of tumor growth. Mice (n=8) were then administered 3x10 5 E.G7-OVA cells were implanted subcutaneously, and tumor growth was monitored individually by measuring tumor size once a week for 35 days. As a control group, six naive female C57BL / 6 mice, 6–8 weeks old, were implanted with the same amount of tumor cells.

[0151] The results shown in Figure 17B demonstrate that the antitumor effect of the OVACS6CD40L mRNA-LNP vaccine is far superior to that of the OVA peptide / adjuvant vaccine. All eight mice pretreated with three doses of OVACS6CD40L mRNA-LNP showed complete tumor rejection for up to 180 days after E.G7-OVA cell inoculation. Of the six mice in the OVA peptide / poly(I:C) adjuvant group, two were found to have positive tumor growth on day 14. In the PBS control group, four were found to have positive tumor growth on day 14.

[0152] In Figure 17C, the eight OVACS6CD40L mRNA-LNP-treated mice that had been immunized against E.G7-OVA cells were rechallenged with E.G7-OVA cells. After monitoring tumor growth for 35 days, only one of the eight mice was found to have tumor growth. In contrast, all six naive mice receiving E.G7-OVA cells were found to have exponential tumor growth and tumor volume increase over time.

[0153] These results demonstrate that OVACS6CD40L mRNA-LNPs can not only prevent the growth of syngeneic E.G7-OVA lymphoma cells in C57BL / 6 mice, but also exert long-term cytotoxic T memory effects, which are presumably caused by the activation of CD40 on dendritic cells through the clustering of secreted forms of the hexameric CD40L fusion encoded by OVACS6CD40L mRNA.

[0154] Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety.

Claims

1. 1. A method for producing a multimeric fusion protein, said method comprising: In mammalian cells, from the N-terminus to the C-terminus, a signal peptide, optionally an antigen peptide, human IgG １ CH3 domain of (GPP) １０ expressing a nucleic acid encoding an amino acid sequence comprising a collagen-like domain and a TNF ligand superfamily extracellular domain, wherein the extracellular domain lacks a coiled-coil trimerization motif; causing said polypeptides expressed in said mammalian cells to at least one of trimerize and hexamerize into one or more multimeric fusion proteins; A method comprising:

2. The method of claim 1 , wherein the mammalian cell is an antigen-presenting cell.

3. 2. The method of claim 1, wherein the antigenic peptide comprises one selected from the group consisting of epidermal growth factor receptor variant III peptide (PEP3), chicken ovalbumin (257-264) antigenic peptide (OVA), and idiotypic antibody peptide (A20ID) derived from BALB / c B-cell lymphoma line A20.

4. The method of claim 1, wherein the TNF ligand superfamily extracellular domain comprises CD40L.

5. The method of claim 1, wherein the antigenic peptide forms a peptide-major histocompatibility complex (MHC) protein complex on the surface of an antigen-presenting cell.

6. 6. The method of claim 5, wherein the antigenic peptide-MHC protein complex is configured to bind to a T cell receptor on an antigenic peptide-specific T cell and stimulate an immune response.

7. The method of claim 1, wherein the TNF ligand superfamily extracellular domain comprises CD137L.

8. The method of claim 2 , wherein the antigen-presenting cells comprise dendritic cells.

9. 2. The method of claim 1, wherein the nucleotide sequence of the nucleic acid is SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, or a sequence having at least 90% sequence identity to the nucleotide sequence.

10. A method for producing a multimeric fusion protein, the method comprising expressing in an antigen-presenting cell a nucleic acid encoding an amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:

7.

11. From the N-terminus to the C-terminus, optionally an antigenic peptide, human IgG １ CH3 domain of (GP-X1) １０ A soluble multimeric fusion protein comprising a collagen-like domain (wherein X1 comprises P or O) and a TNF ligand superfamily extracellular domain, wherein the extracellular domain lacks a coiled-coil trimerization motif, and wherein the soluble multimeric fusion protein has a hexameric structure.

12. 12. An isolated nucleic acid encoding the soluble multimeric fusion protein of claim 11.

13. 13. An isolated expression vector comprising the nucleic acid of claim 12.

14. An isolated antigen-presenting cell comprising the expression vector of claim 13.

15. An isolated nucleic acid comprising a nucleic acid encoding an amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:

7.

16. A pharmaceutical composition comprising a nucleic acid encoding the soluble multimeric protein of claim 11.

17. 17. The pharmaceutical composition of claim 16, further comprising a pharmaceutically acceptable carrier, excipient, or diluent.

18. 17. The pharmaceutical composition of claim 16 for use in the treatment of cancer.

19. 12. A pharmaceutical composition for use in the treatment of cancer, said pharmaceutical composition comprising a nucleic acid encoding a soluble multimeric fusion protein of claim 11, wherein said pharmaceutical composition is administered to a subject with cancer in need thereof.

20. 20. The pharmaceutical composition for use in treating cancer of claim 19, wherein said administering comprises delivering said pharmaceutical composition for use in treating cancer to dendritic cells of said subject ex vivo by transfection.

21. 20. The pharmaceutical composition for use in treating cancer of claim 19, wherein said administering comprises delivering said pharmaceutical composition for use in treating cancer to dendritic cells of said subject in vivo via lipid nanoparticles.

22. 20. The pharmaceutical composition for use in treating cancer of claim 19, wherein the cancer is at least one selected from the group consisting of lymphoma, breast cancer, lung cancer, colon cancer, rectal cancer, prostate cancer, melanoma, brain cancer, spinal cancer, ovarian cancer, pancreatic cancer, uterine cancer, and kidney cancer.

23. 12. A pharmaceutical composition for use in controlling an immune response, said pharmaceutical composition for use in controlling an immune response comprising a nucleic acid encoding a soluble multimeric protein of claim 11, and delivered to dendritic cells of the lymphatic system.

24. The pharmaceutical composition for use in controlling an immune response according to claim 23, wherein the pharmaceutical composition for use in controlling an immune response activates CD40 on the dendritic cells and induces CD8+ T cells into memory cells through MHC class I antigenic peptide presentation.

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