Fusion Proteins for Immunotherapy against Cancer and Infectious Diseases

Abstract

Fusion proteins for immunotherapy against cancer and infectious diseases are disclosed. The fusion proteins of the present invention comprise a CD40-binding domain, an antigen, and a translocation domain located between the CD40-binding domain and the antigen. A furin and / or cathepsin L cleavage site is located between the CD40-binding domain and the translocation domain in the fusion protein. The antigen is a pathogen antigen or a tumor antigen. The furin and / or cathepsin L cleavage site allows for removal of the CD40-binding domain from the fusion protein via furin and / or cathepsin L cleavage. Pharmaceutical compositions, expression vectors, and uses of the fusion proteins of the present invention for inducing an antigen-specific cellular immune response to treat tumors and / or pathogen-induced diseases in a subject are also disclosed.

Description

[Technical field]

[0001] The present invention relates to fusion proteins, in particular to fusion proteins for inducing T cell-mediated immune responses against tumors and infectious diseases. [Background technology]

[0002] The adaptive immune system includes both humoral and cellular immune components and destroys invading pathogens. The cells that execute the adaptive immune response are white blood cells known as lymphocytes. B cells and T cells are two different types of lymphocytes that execute antibody and cellular immune responses. Adaptive immunity is activated by exposure to a pathogen, leading to an enhanced immune response to future encounters with that pathogen. Vaccines induce antigen-specific memory in adaptive immune cells, enabling defense against targeted pathogens. There remains a need for new therapeutic vaccines to treat diseases such as cancer and infectious diseases caused by pathogens. Summary of the Invention

[0003] In one aspect, the present invention relates to a fusion protein comprising (a) a CD40 binding domain, (b) an antigen, and (c) a translocation domain located between the CD40 binding domain and the antigen, wherein a furin and / or cathepsin L cleavage site is present in the fusion protein between the CD40 binding domain and the translocation domain.

[0004] In another aspect, the present invention relates to a DNA fragment encoding the fusion protein according to the invention.The present invention also relates to an expression vector comprising a DNA fragment encoding the fusion protein according to the invention.

[0005] In another aspect, the present invention relates to a pharmaceutical composition comprising a fusion protein according to the invention, a pharma- ceutically acceptable carrier and / or adjuvant.

[0006] In another aspect, the present invention relates to the use of the fusion protein or pharmaceutical composition of the present invention in the manufacture of a medicament for treating a tumor and / or a disease caused by a pathogen in a subject by inducing an antigen-specific cellular immune response. The present invention also relates to the fusion protein or pharmaceutical composition of the present invention used for treating a tumor and / or a disease caused by a pathogen in a subject by inducing an antigen-specific cellular immune response.

[0007] Alternatively, the present invention relates to a method for inducing an antigen-specific cellular immune response to treat a tumor and / or a disease caused by a pathogen in a subject, the method comprising administering to said subject an effective amount of a fusion protein or a pharmaceutical composition according to the present invention. [Brief description of the drawings]

[0008] [Figure 1] This is a vector map.

[0009] [Diagram 2] Vector map. MCS: multiple cloning site.

[0010] [Diagram 3] This is a vector map.

[0011] [Figure 4] This is a vector map.

[0012] [Diagram 5] 5A-5E are schematic diagrams illustrating various embodiments of the present invention.

[0013] [Figure 6] FIG. 1 shows the relative cytokine induction in each animal group.

[0014] [Figure 7] FIG. 1 shows IFN-γ+ immunospots in splenocytes from each group of animals.

[0015] [Figure 8] FIG. 1 shows serum HPV16 E7-specific antibody levels in each animal group.

[0016] [Figure 9] FIG. 1 shows serum HPV18 E7-specific antibody levels in each animal group.

[0017] [Figure 10] The immunization schedule and animal groups treated or not with the indicated fusion proteins are shown.

[0018] [Figure 11] FIG. 1 shows tumor size in each group of animals treated or not with the indicated fusion proteins.

[0019] [Figure 12] FIG. 1 shows the survival rate of each group of animals treated or not with the indicated fusion proteins.

[0020] [Figure 13] FIG. 1 shows the tumor-free rates for each group of animals treated or not with the indicated fusion proteins.

[0021] [Figure 14] FIG. 1 shows the tumor size in each group of animals treated or not with the fusion protein 18sCD40L-TPE-E7 at various doses as indicated.

[0022] [Figure 15] FIG. 1 shows the tumor size in each group of animals treated or not with the fusion protein E7-TStx-18sCD40L at various doses as indicated.

[0023] [Figure 16] The immunization scheme for the fusion protein HBx-preS1-TStx-18sCD40L (upper panel), animal groups and their respective administration schedules (lower panel) are shown.

[0024] [Figure 17] FIG. 17 shows IFN-γ+ immunospots in splenocytes from each group of animals shown in FIG.

[0025] [Figure 18] FIG. 17 shows serum HBx-specific antibody levels in each animal group shown in FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Furthermore, the following definitions are set forth to illustrate and define the meaning and scope of various terms used to describe the present invention.

[0027] definition

[0028] Antigen-presenting cells (APCs) or accessory cells are cells that display antigens on their surface complexed with major histocompatibility complexes (MHC). This process is known as antigen presentation. T cells can recognize these complexes using the T cell receptor (TCR). APCs process and present antigens to T cells.

[0029] Antigen-presenting cells are divided into two categories: professional and non-professional. Cells that express MHC class II molecules along with costimulatory molecules and pattern recognition receptors are called professional antigen-presenting cells. The main types of professional antigen-presenting cells are dendritic cells (DCs), macrophages, and B cells. Non-professional APCs express MHC class I molecules, which includes all nucleated cell types in the body.

[0030] Professional APCs are specialized in presenting antigens to T cells. They are highly efficient at internalizing antigens by either phagocytosis or receptor-mediated endocytosis, processing antigens into peptide fragments, and displaying those peptides (bound to class II MHC molecules) on their membrane. T cells recognize and interact with the antigen-class II MHC molecule complex on the membrane of the antigen-presenting cell. Additional costimulatory signals are then generated by the antigen-presenting cell to activate the T cell. All professional APCs also express MHC class I molecules.

[0031] Professional and non-professional APCs display endogenous peptides on the cell membrane using MHC class I molecules. These peptides are derived from the cell itself, as opposed to exogenous antigens displayed by professional APCs using MHC class II molecules. Cytotoxic T cells can interact with antigens presented by MHC class I molecules.

[0032] CD40 is a costimulatory protein expressed on antigen-presenting cells (e.g., dendritic cells, macrophages, and B cells). Binding of CD40L to CD40 activates the antigen-presenting cell and induces a variety of downstream effects. CD40 is a drug target for cancer immunotherapy.

[0033] The term "CD40 binding domain" refers to a protein capable of recognizing and binding to CD40. The CD40 binding domain may be selected from one of "CD40 ligand (CD40L) or a functional fragment thereof" and "anti-CD40 antibody or a functional fragment thereof".

[0034] The terms "CD40L", "CD40 ligand" and "CD154" are interchangeable. CD40L binds to CD40 (a protein) on antigen-presenting cells (APCs) and has many effects depending on the type of target cell. CD40L plays a central role in costimulation and regulation of immune responses through priming of T cells and activation of CD40-expressing immune cells. US 5,962,406 discloses the nucleic acid and amino acid sequences of CD40L.

[0035] The terms "anti-CD40 antibody" and "CD40-specific antibody" are interchangeable.

[0036] The terms "consisting essentially of" or "consisting essentially of," when used to describe the amino acid sequence of a polypeptide, mean that the polypeptide may or may not have the initiating amino acid "M" (translated from the initiation codon AUG) at the N-terminus as part of the polypeptide, depending on the translation requirements of the protein. For example, the antigen HPV 18 When the E7 protein (SEQ ID NO:39) is fused to another polypeptide (eg, another antigen), the initial amino acid "M" may be omitted or retained.

[0037] As used herein, a "translocation domain" is a polypeptide having the biological activity of translocating an antigen within a fusion protein across the endosomal membrane to the cytosol of a CD40-expressing cell. The translocation domain guides or promotes the antigen toward the class I major histocompatibility complex (MHC-1) pathway for antigen presentation (i.e., the cytotoxic T cell pathway).

[0038] Pseudomonas exotoxin A (PE) translocation peptide (T PE The term "PE domain II peptide" refers to a PE domain II peptide or a functional fragment thereof having translocation biological activity.

[0039] "Shiga toxin (Stx) translocation peptide (T Stx The term "Stx translocation domain" refers to a Stx translocation domain or a functional fragment thereof that has biological activity in translocation.

[0040] The terms "Furin and / or Cathepsin L" or "Furin / Cathepsin L" are interchangeable. A Furin and / or Cathepsin L cleavage site refers to a protease (Furin and / or Cathepsin L) sensitive site. It is a short peptide sequence that can be cleaved by Furin or Cathepsin L, or by both Furin and Cathepsin L. It can be a peptide linker containing said cleavage site introduced into the fusion protein, or a unique protease cleavage site present in the translocation domain of the fusion protein.

[0041] The terms "antigen" and "immunogen" are interchangeable. An antigen refers to an antigenic protein that can be a tumor antigen (an antigen derived from or associated with a cancer) or a pathogen antigen (an antigen derived from a pathogen).

[0042] The terms "tumor" and "cancer" are interchangeable.

[0043] The terms "cancer cell antigen" and "tumor antigen" are interchangeable.

[0044] The term "tumor antigen" refers to a tumor-specific antigen and / or a tumor-associated antigen. A tumor-associated antigen can be a protein or polypeptide expressed on the surface of a tumor cell.

[0045] Cluster of differentiation 28 (CD28) is a T cell-specific surface glycoprotein. The CD28 receptor is stimulated during contact between T cells and antigen-presenting cells. Its functions include T cell activation, induction of cell proliferation, cytokine production, and promotion of T cell survival.

[0046] The term "effective amount" refers to the amount of active fusion protein required to confer a therapeutic effect on a treated subject. Effective doses will vary depending on route of administration, excipient usage, and the possibility of co-use with other therapeutic treatments, as will be recognized by those of skill in the art.

[0047] The term "treat" or "treatment" refers to administering an effective amount of the fusion protein to a subject in need thereof, said subject having cancer or an infectious disease, or a symptom or predisposition to such a disease, for the purpose of curing, alleviating, mitigating, treating, ameliorating or preventing the disease, its symptoms or its predisposition. Such subjects may be identified by a health care professional based on the results from any suitable diagnostic method.

[0048] "0 to 12 repeats" or "2 to 6 repeats" means that all integer unit amounts within the ranges "0 to 12" or "2 to 6" are specifically disclosed as part of the present invention. Thus, amounts of 0, 1, 2, 3, 4, ... 10, 11 and 12" or "2, 3, 4, 5 and 6" units are included as embodiments of the present invention.

[0049] In one aspect, the invention relates to a fusion protein comprising (i) a CD40 binding domain, (ii) an antigen, and (iii) a translocation domain located between said CD40 binding domain and said antigen, wherein a Furin and / or Cathepsin L cleavage site is present between said CD40 binding domain and said translocation domain.

[0050] The fusion proteins of the present invention can induce antigen-specific T cell immune responses via the MHC class I antigen presentation pathway. They share a common mechanism of action. Hereinafter, the fusion protein 18sCD40L-T PE The mechanism of action will be explained using -E7 as an example.

[0051] (1) The fusion protein binds to CD40-expressing cells (e.g., dendritic cells or macrophages) and is internalized by CD40-mediated endocytosis. (2) The fusion protein is cleaved in the endosome by the Furin protease and / or cathepsin L protease, whereby the 18sCD40L fragment is converted to T PE -Removed from E7 fragment. (3) T PEThe -E7 fragment crosses the endosomal membrane of the endosome and enters the cytosol. (4) T PE The -E7 fragment is digested by the cytosolic proteasome to generate a small E7 antigen bearing the epitope. (5) The E7 antigen is delivered to the MHC class I pathway for antigen presentation. (6) CD8+ T cell-specific immune responses are induced or enhanced by T cells recognizing these presented antigens.

[0052] The above mechanism of action is Stx -18sCD40L, in which cleavage by furin and / or cathepsin L proteases results in the formation of E7-T Stx The 18sCD40L fragment is then removed from the E7-T Stx The fragments pass through the endosomal membrane of the endosome into the cytosol, where they are digested by the cytosolic proteasome to generate small epitope-bearing E7 antigens, which are delivered to the MHC class I pathway for antigen presentation. CD8+ T cell-specific immune responses are induced or enhanced by T cell recognition of these presented antigens.

[0053] According to the present invention, in the fusion protein, there are no furin and / or cathepsin L cleavage sites between the antigen and the translocation domain.

[0054] The presence of a Furin and / or Cathepsin L cleavage site and its position in the fusion protein allows removal of the CD40 binding domain from the fusion protein following Furin and / or Cathepsin L cleavage.

[0055] In one embodiment, the fusion protein of the invention further comprises a peptide linker comprising a Furin and / or Cathepsin L cleavage site located between the CD40 binding domain and the translocation domain of the fusion protein.

[0056] The Translocation Domain and antigen are positioned within the fusion protein in an orientation and / or relationship such that the Translocation Domain can translocate the antigen across the endosomal membrane and into the cytosol, and then promote the antigen into the MHC Class I pathway for antigen presentation in CD40-expressing cells.

[0057] In one embodiment, the Translocation Domain is derived from Pseudomonas Exotoxin A (PE). In another embodiment, the Translocation Domain is derived from Shiga Toxin (Stx).

[0058] In one embodiment, the translocation domain is a Pseudomonas exotoxin A (PE) translocation peptide (TPE) when the CD40 binding domain is located at the N-terminus of the fusion protein. PE ).

[0059] In another embodiment, the Translocation Domain is a Shiga Toxin (Stx) Translocation Peptide (T Stx ).

[0060] In another embodiment, the fusion protein of the invention comprises (i) a CD40 binding domain located at the N-terminus of the fusion protein; (ii) a PE translocation peptide (T PE ), and (iii) an antigen located at the C-terminus of the fusion protein. A Furin and / or Cathepsin L cleavage site is present in the fusion protein between the CD40 binding domain and the Translocation Domain.

[0061] In another embodiment, the Translocation Domain is PE wherein said furin and / or cathepsin L cleavage site is a unique furin cleavage site from PE.

[0062] In another embodiment, the fusion protein of the invention comprises (i) a CD40 binding domain located at the N-terminus of the fusion protein, (ii) a peptide linker comprising a furin and / or cathepsin L cleavage site, (iii) a PE translocation peptide (T PE ), and (iv) a pathogen antigen or a tumor antigen.

[0063] In another embodiment, the fusion protein of the present invention comprises (i) an antigen located at the N-terminus of the fusion protein; (ii) an Stx translocation peptide (T Stx ), and (iii) a CD40 binding domain. A Furin and / or cathepsin L cleavage site is present in the fusion protein between the CD40 binding domain and the translocation domain.

[0064] In one embodiment, the Translocation Domain is Stx wherein said furin and / or cathepsin L cleavage site is a unique furin cleavage site from Stx.

[0065] In another embodiment, the fusion protein of the present invention comprises (i) an antigen located at the N-terminus of the fusion protein; (ii) an Stx translocation peptide (T Stx ), (iii) a cleavable linker comprising a furin and / or cathepsin L cleavage site, and (iv) a CD40 binding domain.

[0066] In one embodiment, the furin and / or cathepsin L cleavage site comprises, is or consists of the amino acid sequence of SEQ ID NO:1 or 2.

[0067] In another embodiment, the PE translocation peptide (T PE) is domain II (amino acid residues 253-364; SEQ ID NO:9) of Pseudomonas exotoxin A protein (full length PE; SEQ ID NO:4).

[0068] In another embodiment, the PE translocation peptide (T PE ) contains the minimal functional fragment GWEQLEQCGYPVQRLVALYLAARLSW (SEQ ID NO:5).

[0069] In another embodiment, the PE translocation peptide (T PE ) is composed of 26-112 amino acid residues, and the PE translocation peptide includes the minimal functional fragment GWEQLEQCGYPVQRLVALYLAARLSW (SEQ ID NO:5).

[0070] In another embodiment, the PE translocation peptide (T PE ) comprises an amino acid sequence having at least 90%, 95% or 99% identity to SEQ ID NO:5, 6, 7, 8 or 9.

[0071] In another embodiment, the PE translocation peptide (T PE ) is P.E. 280-305 (SEQ ID NO:5), PE 280-313 (SEQ ID NO: 6), PE 268-313 (SEQ ID NO: 7), PE 253-313 (SEQ ID NO: 8), and PE 253-364 (SEQ ID NO: 9; full length PE domain II).

[0072] In one embodiment, the Stx translocation peptide (T Stx ) is a functional fragment of Shiga toxin (Stx) subunit A (SEQ ID NO: 10) or Shiga-like toxin I (Slt-I) subunit A (SEQ ID NO: 11). According to the present invention, the Stx translocation peptide has the translocation function but does not have the cytotoxic effect of subunit A. The sequence identity between Shiga toxin (Stx) subunit A and Slt-I subunit A is 99%, with only one amino acid difference between the two.

[0073] In another embodiment, the Stx translocation peptide (T Stx ) are comprised of 8-84 amino acid residues in length.

[0074] In another embodiment, the Stx translocation peptide (T Stx ) contains the minimal functional fragment of LNCHHHAS (SEQ ID NO: 12).

[0075] In another embodiment, the Stx translocation peptide (T Stx ) is composed of 8-84 amino acid residues in length, Stx contains the minimal fragment of LNCHHHAS (SEQ ID NO: 12).

[0076] In another embodiment, the Stx translocation peptide (T Stx ) comprises an amino acid sequence having at least 90%, 95% or 99% identity to an amino acid sequence selected from SEQ ID NOs: 12, 13, 14, 15 and 16.

[0077] In another embodiment, the Stx translocation peptide comprises an amino acid sequence selected from SEQ ID NOs: 12, 13, 14, 15 and 16.

[0078] In another embodiment, the translocation peptide (T Stx ) is Stx of Stx subunit A 240-247 (SEQ ID NO: 12), Stx 240-251 (SEQ ID NO: 13), Stx 211-247 (SEQ ID NO: 14), Stx 211-251 (SEQ ID NO: 15) and Stx 168-251 (SEQ ID NO: 16).

[0079] A CD40 binding domain is a polypeptide having biological activity that binds to the CD40 protein on a CD40-expressing cell. The CD40 binding domain enables the fusion protein of the invention to bind to the CD40 receptor on a CD40-expressing cell (e.g., a dendritic cell or macrophage).

[0080] In one embodiment, the CD40 binding domain is selected from the group consisting of: (i) a CD40 ligand (CD40L) or a functional fragment thereof, and (ii) an anti-CD40 antibody or a functional fragment thereof.

[0081] CD40L, anti-CD40 antibodies and their respective functional fragments all have the biological activity of binding to CD40 protein on CD40-expressing cells.

[0082] A functional fragment of CD40L is a biologically active truncated CD40L that substantially lacks the transmembrane and cytoplasmic domains of the full-length CD40L1-261 protein (SEQ ID NO: 17).

[0083] In another embodiment, CD40L, or a functional fragment thereof, consists of 154-261 amino acid residues in length.

[0084] In another embodiment, the functionally active truncated CD40L is 47-261 (SEQ ID NO: 18) and CD40L 108-261 (18sCD40L; SEQ ID NO: 19).

[0085] In another embodiment, the CD40 ligand (CD40L) or functional fragment thereof consists of 154-261 amino acid residues in length, said functional fragment being CD40L 108-261 (SEQ ID NO: 19).

[0086] In another embodiment, the CD40L comprises or consists of an amino acid sequence having at least 90%, 95% or 99% identity to SEQ ID NO: 17, 18 or 19. Said CD40L has the biological activity of binding to CD40 protein on CD40-expressing cells.

[0087] In another embodiment, the CD40 ligand (CD40L) is 47-261 (SEQ ID NO: 18), CD40L 108-261 (SEQ ID NO: 19; referred to as 18sCD40L) and CD40L 1-261 (SEQ ID NO: 17).

[0088] In another embodiment, the CD40 binding domain is a CD40-specific antibody (or an anti-CD40 antibody). A CD40-specific antibody is an antibody specific for the CD40 protein. A CD40-specific antibody is capable of binding to the CD40 protein on a CD40-expressing cell.

[0089] In one embodiment, the CD40-specific antibody comprises a heavy chain variable domain (V H ) and the light chain variable domain (V L ) is included. V H comprises the amino acid sequence of SEQ ID NO: 22, L comprises the amino acid sequence of SEQ ID NO:23.

[0090] In another embodiment, the CD40-specific antibody of the invention is a single chain variable fragment (scFv), a diabody (dscFv), a triabody, a tetrabody, a bispecific scFv, a scFv-Fc, a scFc-CH3, a single chain antigen-binding fragment (scFab), an antigen-binding fragment (Fab), a Fab 2 , a minibody, and a complete antibody.

[0091] In another embodiment, the CD40 binding domain of the invention comprises a heavy chain variable domain (V H ), light chain variable domain (V L ), and the V H and VL and a CD40-specific scFv (anti-CD40 scFv) comprising a flexible linker (L) linking

[0092] In one embodiment, the CD40-specific scFv comprises the amino acid sequence of SEQ ID NO: 20 or 21.

[0093] In another embodiment, the CD40 binding domain of the invention is (i) a CD40-specific antibody or binding fragment thereof, or (ii) a CD40-specific single chain variable fragment (scFv) or binding fragment thereof. The CD40-specific antibody or CD40-specific scFv is H and V L (a) The above V H (b) the V L comprises the amino acid sequence of SEQ ID NO:23.

[0094] In another embodiment, the CD40-specific antibody or CD40-specific scFv is H and V L The above V H V H CDR1, V H CDR2 and V H CDR3, L V L CDR1, V L CDR2 and V L (i) the V H CDR1, V H CDR2 and V H (ii) the V L CDR1, V L CDR2 and V L CDR3 comprises the amino acid sequences of SEQ ID NOs: 27, 28 and 29, respectively.

[0095] In another embodiment, the CD40 binding domain is H and V L (a) a CD40-specific scFv comprising said V H(b) the V L comprises the amino acid sequence of SEQ ID NO:23.

[0096] In another embodiment, the fusion protein of the invention comprises a fusion domain that is a PE translocation peptide (T PE ), it further comprises an endoplasmic reticulum (ER) retention sequence located at the C-terminus of the antigen.

[0097] In another embodiment, the ER retention sequence comprises an amino acid sequence selected from SEQ ID NOs: 30, 31, 32, 33 and 34.

[0098] In another embodiment, a fusion protein of the invention further comprises a CD28 activation peptide positioned between the CD40 binding domain and the Furin and / or cathepsin L cleavage site.

[0099] In another embodiment, the CD28 activating peptide is 28-53 amino acid residues in length.

[0100] In another embodiment, the CD28 activating peptide is 28-53 amino acid residues in length, and the CD28 activating peptide comprises an amino acid sequence selected from SEQ ID NOs: 35, 36, and 37.

[0101] In another embodiment, the CD28 activating peptide is 28-53 amino acid residues in length, and the CD28 activating peptide comprises the amino acid sequence of SEQ ID NO:35.

[0102] In another embodiment, the CD28 activating peptide comprises an amino acid sequence selected from SEQ ID NOs: 35, 36 and 37.

[0103] In another embodiment, the CD28 activating peptide comprises an amino acid sequence having at least 90%, 95% or 99% identity to SEQ ID NO:35, 36 or 37.

[0104] The antigen of the fusion protein of the present invention is a pathogen antigen or a tumor antigen.

[0105] The pathogens include human papillomavirus (HPV), human immunodeficiency virus-1 (HIV-1), influenza virus, dengue virus, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), severe acute respiratory syndrome-related coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MSRS-CoV-3), and the like. MERS-CoV, Epstein-Barr virus (EBV), Zika virus, rabies virus, smallpox virus, chikungunya virus, West Nile virus, poliovirus, measles virus, rubella virus, hantavirus, Japanese encephalitis virus, coxsackievirus, echovirus, enterovirus, mumps virus, varicella zoster virus (VZV), cercopithecin herpesvirus-1 (CHV-1), yellow fever virus (YFV), V), Rift Valley fever virus, Lassa virus, Marburg virus, Ebola virus, Norovirus, Rotavirus, Adenovirus, Sapovirus, Astrovirus, Rickettsia prowazekii, Rickettsia typhi, Orientia tsutsugamushi, Borrelia burgdorferi, Yersinia pestis, Plasmodium vivax, Plasmodium malariae, Plasmodium falciparum, Plasmodium ovale, Bacillus anthracis, Clostridium difficile, Clostridium The bacterial strain may be selected from the group consisting of M. botulinum, Corynebacterium diphtheriae, Salmonella enterica serovar Typhi, Salmonella enterica serovar Paratyphi A, Shiga toxin-producing Escherichia coli (STEC), Shigella disenteriae, Shigella dysenteriae, Clostridium flexneri, Clostridium shiga sonnei, Entamoeba histolytica, Vibrio cholerae, Mycobacterium tuberculosis, Neisseria meningitidis, Bordetella pertussis, Haemophilus influenzae type B (HiB), Clostridium tetani, Listeria monocytogenes, and Streptococcus pneumoniae.

[0106] In another embodiment, the pathogen is selected from the group consisting of HPV, HIV-1, influenza virus, dengue virus, HAV, HBV, HCV, SARS-CoV, SARS-CoV-2. More specifically, the pathogen is selected from the group consisting of HPV, HBV, HCV, and SARS-CoV-2.

[0107] In another embodiment, the antigen is HPV 16 E7 protein, HPV 18 The pathogenicity antigen is selected from the group consisting of E7 protein, HBV X protein (HBx), HBV preS1 protein, HCV core protein (HCVcore) and SARS-CoV-2 spike protein (CoV2S).

[0108] In another embodiment, the antigen comprises at least one epitope, preferably 1 to 30 epitopes, more preferably 1 to 15 epitopes to induce a desired immune response.

[0109] In another embodiment, the antigen is a pathogenic antigen comprising or consisting essentially of an amino acid sequence having at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO: 38, 39, 40, 41, 42 or 43.

[0110] In another embodiment, the antigen is a pathogenic antigen comprising or consisting essentially of an amino acid sequence having at least 80% identity to SEQ ID NO:38, 39, 40, 41, 42 or 43.

[0111] In another embodiment, the antigen comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40, 41, 42 and 43.

[0112] In another embodiment, the antigen is a tumor antigen. The tumor antigen is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA).

[0113] In one embodiment, the tumor or cancer is selected from the group consisting of breast cancer, colon cancer, rectal cancer, bladder cancer, endometrial cancer, renal cancer, gastric cancer, glioblastoma, hepatocellular carcinoma, cholangiocarcinoma, small cell lung cancer, non-small cell lung cancer (NSCLC), melanoma, ovarian cancer, cervical cancer, pancreatic cancer, prostate cancer, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), non-Hodgkin's lymphoma, and thyroid cancer.

[0114] In another embodiment, the tumor associated antigen is SSX2, MAGE-A3, NY-ESO-1, iLRP, WT12-281, RNF43, CEA-NE3, AFP, ALK, anterior gradient 2 (AGR2), BAGE protein, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CD40, CDK4, CEA, CTLA4, cyclin B1, CYP1B1, EGFR, EGFRvIll, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EphA2, Fra-1, FOLR1, GAGE ​​protein (e.g., GAGE-1, -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, HLA / B-raf, HLA / k-ras, HL A / MAGE-A3, hTERT, LMP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6 and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ES01, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, Steap-1, Steap-2, survivin, TAG-72, TGF-β, TMPRSS2, Tn, TRP-1, TRP-2, tyrosinase and uroplakin-3.

[0115] In another embodiment, the antigen is a tumor-associated antigen selected from the group consisting of SSX2, MAGE-A3, NY-ESO-1, iLRP, WT12-281, RNF43 and CEA-NE3.

[0116] In another embodiment, the antigen is a tumor associated antigen comprising an amino acid sequence having at least 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO: 44, 45, 46, 47, 48, 49 or 50.

[0117] In another embodiment, the antigen is a tumor associated antigen comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 44, 45, 46, 47, 48, 49 and 50.

[0118] The antigen may be a single antigen or an antigenic fragment thereof, or a fusion antigen in which at least two antigens are fused together. For example, the antigen may be an HPV 16 Single antigen of E7 protein or HPV 16 E7 and HPV 18 It may be a fusion antigen comprising the E7 protein. The fusion antigen may or may not have a linker connecting the different antigens.

[0119] In another embodiment, the antigen is a fusion antigen having at least one linker that connects the different antigens.

[0120] In another embodiment, the antigen is a rigid linker (EAAAAK) that links different antigens. n where n is an integer between 0 and 12, preferably between 2 and 6, more preferably between 3 and 4. In other words, the rigid linker comprises 0-12, 2-6 or 3-4 repeats of the sequence EAAAAK (SEQ ID NO: 56).

[0121] In another embodiment, the fusion protein of the invention further comprises a rigid linker between the CD40 binding domain and the Furin and / or cathepsin L cleavage site. The rigid linker may be a peptide linker comprising 0-12 repeats of the amino acid sequence EAAAAK (SEQ ID NO:56).

[0122] The rigid linker is (EAAAAK) nOr it may be (SEQ ID NO: 56), where n is an integer from 0 to 12, preferably from 2 to 6, more preferably from 3 to 4.

[0123] In another embodiment, the rigid linker comprises 2-6 or 3-4 repeats of SEQ ID NO:56.

[0124] In another embodiment, a fusion protein of the invention comprises or consists essentially of an amino acid sequence having at least 90%, 95% or 99% identity to SEQ ID NO:51, 52, 53, 54 or 55.

[0125] In another embodiment, a fusion protein of the invention comprises or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs:51, 52, 53, 54 and 55.

[0126] In another aspect, the present invention relates to a DNA fragment encoding the fusion protein of the present invention.The present invention relates to an expression vector comprising the DNA fragment encoding the fusion protein of the present invention.The present invention further relates to a pharmaceutical composition comprising the fusion protein of the present invention and a pharma- ceutically acceptable carrier and / or adjuvant.

[0127] The pharmaceutical compositions may be in enteral or parenteral dosage forms adapted for transdermal, transmucosal, nasopharyngeal, pulmonary or direct injection, or for systemic (e.g., parenteral) or local (e.g., intratumoral or intralesional injection) administration. Parenteral injection may be via intravenous, intraperitoneal, intramuscular, subcutaneous or intradermal routes.

[0128] Suitable adjuvants include, but are not limited to, saponin-based adjuvants or toll-like receptor (TLR) agonist adjuvants. Saponin-based adjuvants may be GPI-0100, Quil A or QS-21. TLR agonist adjuvants may be PolyI:C, monophosphoryl lipid A (MPL) or CpG oligonucleotides (e.g., class A CpG: CpG1585, CpG2216 or CpG2336; class B CpG: CpG1668, CpG1826, CpG2006, CpG2007, CpG BW006 or CpG D-SL01; class C CpG: CpG2395, CpG M362 or CpG D-SL03). In one embodiment, the adjuvants are CpG oligonucleotides.

[0129] The pharmaceutical compositions may also be administered orally, for example, in the form of tablets, coated tablets, dragees, hard and soft gelatin capsules.

[0130] The dosage of the fusion protein may vary depending on the disease to be controlled, the age and individual condition of the patient, and the mode of administration. The dosage can be tailored to the individual requirements in each particular case in order to obtain a therapeutically effective amount of the fusion protein of the invention to achieve the desired therapeutic response.

[0131] For adult patients, a single dosage of about 0.1 to 50 mg, in particular about 0.1 to 5 mg, is considered. Depending on the severity of the disease and the exact pharmacokinetic profile, the fusion protein can be administered in weekly, biweekly or monthly dosage units, giving a total of 1 to 6 dosage units per cycle to fulfill such a treatment.

[0132] In another aspect, the present invention relates to the use of the fusion protein or pharmaceutical composition of the present invention in the manufacture of a medicament for inducing an antigen-specific T cell immune response to protect against and / or treat an infection or tumor in a subject.

[0133] Abbreviation Rap1: Ras-proximate-1 or Ras-associated protein 1 CD40: differentiation antigen group 40 CDR: Complementarity determining region

[0134] Working Example

[0135] Animal tumor models

[0136] For the in vivo efficacy assays of Examples 6-8, HPV was isolated from lung epithelial cells of C57BL / 6 mice. 16 Using mouse HPV E6 and E7 expressing tumor cell lines 16 Tumor models were established. Tumor cells were cultured in RPMI 1640 medium containing FBS (10%) and penicillin / streptomycin / amphotericin B (50 units / mL) at 37°C and 5% CO 2 and grown in .

[0137] Sequence number and composition

[0138] Table 1 shows the SEQ ID NOs, corresponding peptides, polypeptides and fusion proteins. [Table 1] JPEG0007643741000002.jpg66132

[0139] Flow cytometry Spleen cells were stimulated with antigenic stimuli for 2 h at 37°C, and then treated with 50 μg / mL brefeldin A and monensin for 2 h at 37°C. Cells were harvested, washed with PBS containing 0.5% BSA, and stained simultaneously with APC / Cy7-conjugated anti-CD3, erCP / Cy5.5-conjugated anti-CD4, FITC-conjugated anti-CD8, PE-conjugated anti-CD44, and APC-conjugated anti-CD62L. After washing, cells were permeabilized, fixed, and stained intracellularly with PE-conjugated anti-IFN-γ, PE / Cy7-conjugated anti-IL-2, and eFluor450-conjugated anti-TNF-α. CD8+ or CD4+ memory T cell phenotype (CD3 + CD44 hi CD62L loThe intracellular cytokine signature (IFN-γ, IL-2, or TNF-α) of spleen cells with IFN-γ, IL-2, or TNF-α was further analyzed by a Gallios flow cytometer and Kaluza software.

[0140] Enzyme-linked immunospot (ELISpot) assay Spleen cells were plated at 2x10 in pretreated mouse IFN-γ capture 96-well plates (CTL IMMUNOSPOT®) in the presence or absence of antigenic stimuli. 5 Cells were seeded in triplicate at a cell density of 1000 cells / well. After 24 h incubation at 37°C, the cells were discarded. After washing, the captured IFN-γ was detected by biotin-conjugated anti-mouse IFN-γ antibody at room temperature for 2 h, and IFN-γ immunospots were generated according to the manufacturer's instructions. Scanning and counting of IFN-γ-immunospots were performed by an IMMUNOSPOT® S5 microanalyzer (CTL).

[0141] Indirect enzyme-linked immunosorbent assay (ELISA) Collected whole blood samples were left at 4 °C for 30–60 min and then centrifuged at 5,000 g for 10 min to pellet the clots. Samples were stored at -20 °C. Purified coating proteins for antigen-specific antibody binding were coated with guanidine coating buffer (2 M guanidine hydrochloride, 500 mM NaCl). 2 HPO 4, 25 mM citric acid, pH 4.0-4.4) and distributed at 1 μg / well into a 96-well plate. After overnight incubation at 4 °C, the 96-well plate was blocked with 1% BSA in PBS for 1 h at 37 °C. After thawing the serum samples, they were serially diluted 10-fold in PBS containing 1% BSA. The coated proteins were incubated with 100 μl of serum samples diluted 1000-fold at 37 °C for 2 h. After washing four times with phosphate-buffered saline TWEEN®-20 (PBST), antigen-specific antibodies were detected by horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:10,000 dilution, catalog no. 31430, Thermo Fisher Science) for 30 min at 37 °C. After washing four times with PBST, HRP-mediated color development was catalyzed in the presence of 100 μL of TMB substrate and quenched with 100 μL of 1 N HCl. The relative titers of antigen-specific antibodies in serum samples were determined by absorbance at 450 nm.

[0142] statistical analysis The significance of all comparisons was calculated by t test, and results were considered significant when p<0.05.

[0143] Example 1

[0144] CD40L 47-261 -T PE -E7 and 18sCD40L-T PE Construction of expression vector for -E7

[0145] 5A-E show various embodiments of the fusion proteins of the present invention.

[0146] Fusion protein CD40L 47-261 -T PE -E7 (SEQ ID NO:51; FIG. 5A) is a truncated CD40 ligand, CD40L 47-261( SEQ ID NO: 18), (b) (EAAAAK) 3 (SEQ ID NO: 3) and RX 1 RX 2 X 3 R (SEQ ID NO:2) (wherein X 1A, X 2 is Y, X 3 is K), (c) a cleavable linker comprising a PE translocation peptide of SEQ ID NO:5 (PE 280-305 ), and (d) HPV of SEQ ID NO: 38 16 E7 protein and HPV of SEQ ID NO:39 18 Fusion antigen HPV containing E7 protein 16 / 18 Includes E7.

[0147] CD40L as below 47-261 -T PE Construct an expression vector for -E7 (Figure 1). CD40L 47-261 , a cleavable linker and a PE translocation peptide (PE 280-305 ) HindIII CD40L-Linker-PE NcoI,XhoI,SalI The DNA fragment encoding the His-tagged fusion antigen HPV was synthesized by PCR, digested with HindIII / SalI, and ligated into the plasmid pTAC-MAT-Tag-2 with HindIII / XhoI cleavage sites to obtain the plasmid P07-His-pNC (Figure 2). 16 / 18 The DNA fragment encoding E7 was inserted into the plasmid P07-His-pNC (Figure 2) via the restriction enzymes NcoI / XhoI to produce the fusion protein CD40 L 47-261 -T PE An expression vector for -E7 was generated (Figure 1).

[0148] The cleavable linker allows the Furin and / or Cathepsin L proteases to cleave the fusion protein to produce the T PE The -E7 fragment can be released from the fusion protein.

[0149] In a similar manner to that described above, other antigens of interest can be used to replace E7 and inserted into the plasmid of FIG. 2 to generate an expression vector similar to the plasmid of FIG. 1 for a fusion protein containing the antigen of interest of the present invention.

[0150] The fusion protein 18sCD40L-T was synthesized by the same method as above. PEAn expression vector for CD40-E7 (SEQ ID NO: 52; FIG. 5B) was constructed, in which the truncated CD40 ligand CD40L 47-261 (SEQ ID NO: 18), other truncated CD40 ligands, CD40L 108-261 The 18sCD40L shown in FIG.

[0151] Example 2

[0152] E7-T Stx -CD40L 47-261 and E7-T Stx Construction of expression vector for -18sCD40L

[0153] Fusion protein E7-T Stx -CD40L 47-261 (SEQ ID NO: 53; FIG. 5C) is (a) a fusion antigen HPV 16 / 18 E7(HPV 16 E7 protein (SEQ ID NO: 38) and HPV 18 E7 protein (SEQ ID NO: 39)), (b) the Stx translocation peptide of SEQ ID NO: 14 (Stx 211-247 ), (c) RX of SEQ ID NO: 1 1 X 2 R(X 1 V, X 2 is A) and (EAAAAK) of SEQ ID NO:3 3 and (d) a truncated CD40 ligand (CD40L) of SEQ ID NO: 18. 47-261 ).

[0154] As shown below, E7-T Stx -CD40L 47-261 An expression vector (Figure 3) is constructed.

[0155] Stx Translocation Peptide (Stx 211-247 ), a cleavable linker and CD40L 47-261 Includes HindIII, XhoI Stx-Linker-CD40L SalIThe DNA fragment encoding the His-tagged fusion antigen HPV was PCR synthesized, digested with HindIII / SalI, and ligated into the plasmid pTAC-MAT-Tag-2 backbone with HindIII / XhoI cleavage sites to obtain the plasmid P08(RP)-His-pNC (Figure 4). 16 / 18 Another DNA fragment encoding E7 was inserted into the plasmid P08(RP)-His-pNC (Figure 4) via the restriction enzymes HindIII / XhoI to produce the expression vector E7-T Stx -CD40L 47-261 was produced (Figure 3).

[0156] The cleavable linker is important for the fusion protein of the present invention. The cleavable linker allows the fusion protein to bind to the E7-T Stx The fragment can be cleaved by Furin and / or Cathepsin L proteases to be released from the fusion protein. See, e.g., Figure 5C.

[0157] In a similar manner to that described above, other antigens of interest from various pathogens or cancers can be used to replace E7 and inserted into the plasmid of FIG. 4 to generate expression vectors similar to the plasmid of FIG. 3 for fusion proteins containing the antigen of interest of the present invention.

[0158] The fusion protein E7-T was isolated by the same method as above. Stx An expression vector for -18sCD40L (SEQ ID NO: 54; FIG. 5D) was constructed, in which the truncated CD40 ligand CD40L 47-261 (SEQ ID NO: 18), other truncated CD40 ligands, CD40L 108-261 The 18sCD40L shown in FIG.

[0159] For comparison, the fusion protein RAP1-CD28, which is nearly identical to the conventional construct disclosed in U.S. Pat. No. 9,481,714 B2 (Example 1), conv PE t -E7-K3 (also referred to as "RAP1-E7" in the present invention) was constructed. conv PEt -E7-K3 (also referred to as "RAP1-E7" in the present invention) contains RAP1 domain III, CD28 sequence, linker, PE translocation domain II (PE 268-313 ), the antigen E7 protein, and the endoplasmic reticulum retention sequence. As used herein, the antigen E7 protein includes the fusion antigen HPV 16 / 18 E7, HPV 16 E7 protein (SEQ ID NO: 38) and HPV 18 E7 protein (SEQ ID NO: 39), whereas the antigen E7 protein used in the prior art is HPV 16 The E7 protein.

[0160] Example 3

[0161] HBx-preS1-T Stx Construction of expression vector for -18sCD40L

[0162] Fusion protein HBx-preS1-T Stx -18sCD40L (SEQ ID NO: 55; FIG. 5E) binds to (a) the fusion antigen HBx-preS1, which comprises the HBX protein of SEQ ID NO: 40 and the HBV preS1 protein of SEQ ID NO: 41; (b) the Stx translocation peptide (Stx 211-247 ), (c) RX of SEQ ID NO: 1 1 X 2 R(X 1 V, X 2 is A) and (EAAAAK) of SEQ ID NO:3 3 and (d) a truncated CD40 ligand (18sCD40L) of SEQ ID NO: 19.

[0163] The fusion protein HBx-preS1-T was synthesized by the same method as in Example 2. Stx An expression vector for -18sCD40L was constructed, in which the truncated CD40 ligand used was 18sCD40L and the antigen used was the fusion antigen HBx-preS1, as described above.

[0164] Example 4

[0165] Protein expression

[0166] Protein expression vector CD40L 47-261 -T PE E. coli BL21 cells containing -E7 were inoculated at 37°C in ZY medium (10 g / L tryptone and 5 g / L yeast extract) containing the appropriate concentration of the selected antibiotic. When the culture reached early logarithmic phase (OD600 = 2-5), expression of the fusion protein was induced with isopropyl-1-thio-β-D-galactopyranoside (IPTG) (0.5-2 mM). After 4 h of IPTG induction, cells were harvested and disrupted by sonication. To recover the overexpressed fusion protein, inclusion bodies were isolated and solubilized in solubilization buffer (6 M guanidine hydrochloride, 20 mM potassium phosphate, 500 mM NaCl, 20 mM imidazole, 1 mM DTT, pH 7.4). After purification, the fusion protein was refolded by dialysis overnight at 4°C against 20-50 volumes of dialysis buffer (10 mM PBS). The refolded fusion proteins were subjected to SDS-PAGE analysis under reducing (with dithiothreitol; +DTT) and non-reducing (without dithiothreitol; -DTT) conditions to assess whether they were properly refolded.

[0167] The following fusion proteins were also expressed and refolded in the same manner as above: (1) 18sCD40L-T PE -E7, (2)E7-T Stx -CD40L 47-261 , (3) E7-T Stx -18sCD40L, (4)RAP1-E7, (5)CD40L 47-261 -T PE -HBx-preS1, (6)18sCD40L-T PE -HBx-preS1, (7)HBx-preS1-T Stx -CD40L 47-261 , (8)HBx-preS1-T Stx -18sCD40L, (9)CD40L 47-261 -T PE -HCV core, (10)18sCD40L-TPE -HCV Core, (11)HCV Core-T Stx -CD40L 47-261 , (12)HCV core-T Stx -18sCD40L, (13)CD40L 47-261 -T PE -CoV2S, (14)18sCD40L-T PE -CoV2S, (15)CoV2S-T Stx -CD40L 47-261 , (16)CoV2S-T Stx -18sCD40L, (17)CD40L 47-261 -T PE -SSX2, (18)18sCD40L-T PE -SSX2, (19)SSX2-T Stx -CD40L 47-261 , (20)SSX2-T Stx -18sCD40L. Fusion protein CD40L 47-261 -T PE -E7, 18sCD40L-T PE -E7, E7-T Stx -18sCD40L, RAP1-E7 and HBx-preS1-T Stx -18sCD40L was further subjected to immunogenicity and efficacy analysis in the following experiments.

[0168] Example 5

[0169] Immunogenicity analysis of fusion proteins

[0170] Female C57BL / 6NCrlBltw mice (5-6 weeks old) were randomly divided into 5 groups (n=5): (A) placebo (i.e., PBS), (B) fusion protein CD40L 47-261 -T PE -E7 (100μg), (C) Fusion protein 18sCD40L-T PE -E7 (100 μg), (D) fusion protein E7-T Stx-18sCD40L (100 μg), and (E) the fusion protein RAP1-E7 (100 μg). The fusion protein was dialyzed into PBS. In animal groups B to E, CpG1826 (50 μg) was used as adjuvant. Each group received three immunizations subcutaneously (sc) at 7-day intervals starting from day 0. Blood samples were collected on days 0, 7 and 14. On day 21, blood samples were taken and spleen cells were resuspended in RPMI1640 medium containing FBS (10%) and PSA.

[0171] Spleen cells were used to generate CD8 + and CD4 + Intracellular cytokine induction (IFN-γ, IL-2, and TNF-α) in memory T cells was analyzed. Briefly, spleen cells from each group of animals were transfected with antigen E7 protein (2 μg / mL of HPV 16 The extent or level of intracellular cytokine induction in each mouse group was expressed as relative cytokine induction, which is the increase in cytokines in the presence of the stimulating antigen E7. + / CD8 + and cytokines + / CD4 + It was obtained by normalizing the splenocyte frequency to that of the unstimulated (untreated) control.

[0172] Spleen cells were cultured using an enzyme-linked immunospot (ELISpot) assay after antigen stimulation (2 μg / mL of HPV 16 The results were also used to analyze the frequency of IFN-γ-secreting splenocytes in the presence or absence of a 100-fold increase in the number of IFN-γ-secreting cells per million splenocytes. + Expressed as immunospots.

[0173] Serum HPV detected by ELISA using blood samples 16 E7-specific and HPV 18 The levels of E7-specific antibodies were analyzed. 16 E7 and HPV 18The E7 recombinant protein was used as the coating protein, respectively.

[0174] Figure 6 shows HPV 16 Cytokine induction results after antigen stimulation of splenocytes with E7 peptide pool. Fusion protein CD40L 47-261 -T PE -E7, 18sCD40L-T PE -E7 or E7-T Stx CD8 from animals immunized with -18sCD40L (group BD) + The relative cytokine induction of IFN-γ and TNF-α, but not IL2, in memory T cells was all significantly increased compared to the RAP1-E7 treated group (Group E) or the placebo group (Group A). + IL-2 and CD4 in memory T cells + The relative cytokine induction of the cytokines IFN-γ, IL-2 or TNF-α in memory T cells was slightly increased but did not show significant differences compared to the placebo group (Group A).

[0175] Nevertheless, the fusion protein of the present invention is capable of detecting the antigen HPV 16 CD8 in response to E7 stimulation + It can be concluded that it is superior to the prior art fusion proteins in inducing expression of IFN-γ and TNF-α in memory T cells.

[0176] Figure 7. In vitro HPV 16 IFN-γ+ immunospots in splenocytes stimulated with E7 peptide pools. 47-261 -T PE -E7, 18sCD40L-T PE -E7, E7-T Stx The frequency of IFN-γ-secreting splenocytes from animals stimulated with -18sCD40L and RAP1-E7 (group BE) was significantly increased compared to the placebo group. Stx -18sCD40L is CD40L 47-261 -T PE-E7 induced a significantly higher frequency of IFN-γ secreting cells compared to E7 (p=0.035).

[0177] This result shows that the fusion protein of the present invention is capable of inhibiting antigenic HPV 16 It is shown that the IFN-γ-secreting T cell population can be significantly increased upon or after stimulation with the E7 peptide pool.

[0178] FIG. 8 shows serum HPV in animals challenged with various fusion proteins on days 0, 7, and 14. 16 The levels of E7-specific antibodies are shown. 47-261 -T PE -E7, 18sCD40L-T PE -E7 or E7-T Stx In animals vaccinated with -18sCD40L (groups B-D, respectively), HPV 16 The levels of E7-specific antibodies began to increase after the second vaccination on day 7 and further increased after the third vaccination on day 14. On day 21, in group BD animals, serum HPV 16 The levels of E7-specific antibodies are higher than in the groups of animals vaccinated with placebo and RAP1-E7 (ie, RAP1-CD28convPEt- E7-K3).

[0179] The fusion protein RAP1-E7 (RAP1-CD28convPEt-E7-K3) inhibited HPV after two vaccinations (days 0 and 7). 16 The HPV vaccine was not able to induce E7-specific antibody levels after the third vaccination on day 14. 16 The fusion protein of the present invention started to induce E7-specific antibodies, and serum antibody levels were slight on day 21 compared to groups B to D. In contrast, the fusion protein of the present invention did not significantly increase serum HPV after two vaccinations on days 0 and 7. 16 E7-specific antibody levels were induced.

[0180] A similar effect in the induction of HBx-specific antibodies was observed when animals were vaccinated with RAP1-CD28convPEt-HBx-K3 (referred to as "RAP1-HBx") using the same regimen and immunization schedule as described above. The fusion protein RAP1-HBx was generated using HBx antigen instead of E7 antigen in RAP1-E7 (RAP1-CD28convPEt-E7-K3). The fusion protein RAP1-HBx induced serum HBx-specific antibody levels after the third vaccination on day 14, and serum antibody levels on day 21 were modest compared to animals vaccinated with the fusion protein of the invention (data not shown).

[0181] FIG. 9 shows serum HPV levels in animals immunized with various fusion proteins on days 0, 7, and 14. 18 The levels of E7-specific antibodies are shown. Fusion protein CD40L 47-261 -T PE -E7, 18sCD40L-T PE -E7 and E7-T Stx -18sCD40L (groups BD, BD, and BD, respectively) were significantly associated with increased serum HPV infection compared with the placebo group. 18 The levels of E7-specific antibodies were significantly increased.

[0182] Thus, the fusion proteins of the present invention are effective in inducing antigen-specific antibodies, with the induction of antibodies occurring after two vaccinations.

[0183] As described above, the fusion protein of the present invention can induce antigen-specific T cell responses, increase the expression of proinflammatory cytokines such as IFN-γ and TNF-α, and induce antigen-specific antibody responses.

[0184] Example 6

[0185] In vivo potency assay of fusion proteins

[0186] Female C57BL / 6NCrlBltw mice (5 to 6 weeks old) were randomly divided into 5 groups and treated with PBS (group A, placebo, n=4) or one of the following fusion proteins: group B, CD40L 47-261 -T PE -E7 (25 μg; n=5); group C, 18sCD40L-T PE -E7 (25 μg; n = 4); group D, E7-T Stx -18sCD40L (25 μg; n=5); and group E, RAP1-E7 (25 μg; n=5). Fusion proteins were dissolved in PBS and CpG1826 (50 μg) was used as adjuvant for vaccination of animals in groups B to E. Figure 10 shows the immunization schedule, fusion proteins and doses.

[0187] To challenge the mice, tumor cells (1 × 10 5 Each mouse was subcutaneously injected with 0.1 mL / mL of IgG1 in the left flank on day 0. Three immunizations were performed subcutaneously on days 7, 14, and 21. Tumor size was determined using a modified ellipsoid formula (tumor size = 1 / 2 (length × width)). 2 Tumor masses were determined twice weekly by multiplying caliper measurements based on the mean tumor mass index (mM) and mean tumor mass index (TMA). Survival and tumor-free rates were calculated. Mice with tumor lengths >2 cm were considered dead, and mice without measurable or palpable tumor masses were considered tumor-free.

[0188] The inoculated tumors developed rapidly in the placebo group, and two animals died on day 25, so data for the placebo group are shown only up to day 21 (Figure 11). Animals in groups C and D (18sCD40L-T, respectively) PE -E7 and E7-T Stx The tumor masses in the animals of groups B and E (immunized with CD40L-18sCD40L, respectively) were almost completely suppressed, at least for the entire experimental period (the final day was day 39). 47-261 -T PE Tumors immunized with RAP1-E7 and RAP1-E7 were initially well controlled but grew gradually when immunization was discontinued.

[0189] The above results indicate that the fusion protein of the present invention, in particular the fusion protein 18sCD40L-TPE -E7 and E7-T Stx These results indicate that -18sCD40L can effectively suppress tumor growth.

[0190] Animal groups B and C (CD40L 47-261 -T PE -E7, 18sCD40L-T PE -E7, E7-T Stx The survival rate of mice immunized with -18sCD40L and RAP1-E7 remained at 100% on day 35 compared to the placebo group, which dropped to 0% on day 35 (Figure 12).

[0191] The above results demonstrate that the fusion protein of the present invention can effectively maintain survival in animal tumor models.

[0192] Throughout the entire experimental period (39 days), no tumor-free animals were found in groups A, B, and E (Figure 13). One (25%) in group C and three (60%) in group D (18sCD40L-T PE -E7 and E7-T Stx We found that mice immunized with 18sCD40L-T (immunized with 18sCD40L-T) survived without measurable or palpable tumors. Notably, in these tumor-free mice, PE -E7 or E7-T Stx Immediately after completion of three immunizations with -18sCD40L, all tumor masses were eliminated.

[0193] The above results show that the fusion protein of the present invention is more effective in improving the tumor-free rate of tumor-bearing animals than the prior art fusion protein RAP1-E7.

[0194] Example 7

[0195] 18sCD40L-T PE -In vivo efficacy analysis at different doses of E7

[0196] Female C57BL / 6NCrlBltw mice (4 to 6 weeks old) were randomly divided into 5 groups (n=5 / group): (A) placebo (PBS); (B) 18sCD40L-T PE (100μg; no fusion antigen E7); (C)18sCD40L-T PE -E7(100μg);(D)18sCD40L-T PE -E7(50μg);(E)18sCD40L-T PE -E7 (25 μg). In groups B to E, the fusion protein was dissolved in PBS, and CpG1826 (50 μg) was used as an adjuvant. Tumor cells (1 × 10 6 / 0.1 mL) was injected subcutaneously into the left flank of each mouse on day 0. Two weeks after challenge, tumor-bearing mice were vaccinated three times on days 14, 21, and 28.

[0197] Tumor volumes were determined. Fusion protein 18sCD40L-T PE All doses of 18sCD40L-T7 (25 μg, 50 μg or 100 μg) showed a strong tumor growth inhibitory effect. The inhibition of tumor size by the fusion protein was seen after the first injection on day 14 and continued throughout the entire experimental period until the last observation day on day 34. PE , both of which lacked the antigen E7, were ineffective in suppressing tumor growth (Figure 14).

[0198] Example 8

[0199] E7-T Stx In vivo efficacy analysis of different doses of -18sCD40L

[0200] Mice were divided into groups, challenged with tumor cells, and administered the fusion protein on days 14, 21, and 28, and tumor size was measured using the same method as in Example 7. Note that mice in groups B to E were vaccinated as follows: (B) T Stx -18sCD40L (100μg; no fusion antigen E7); (C)E7-T Stx -18sCD40L(100μg);(D)E7-T Stx-18sCD40L (50 μg); and (E) E7-T Stx -18sCD40L (25μg). Fusion protein E7-T Stx All doses of -18sCD40L (25 μg, 50 μg or 100 μg) showed a strong effect on suppressing tumor growth (Figure 15). The suppression of tumor size by the fusion protein was seen after the first inoculation and continued throughout the entire experimental period until the last observation day, day 34. Stx -18sCD40L, both of which lack the antigen E7, was ineffective in suppressing tumor growth (Figure 15).

[0201] Therefore, the fusion protein of the present invention has a strong effect of suppressing tumor growth with excellent therapeutic effect.

[0202] Example 9

[0203] Number of vaccinations: HBx-preS1-T Stx -Immunogenicity analysis of 18sCD40L

[0204] Figure 16 shows the administration schedule for each animal group. C57BL / 6JNarl female mice (5 weeks old) were randomly divided into 4 groups (n=5 / group): (1) placebo group; (2) D0-D7-D14 group (3 doses, vaccinated on days 0, 7, 14); (3) D7-D14 group (2 doses, vaccinated on days 7, 14); and (4) D14 group (1 dose, vaccinated on day 14). For the placebo group, PBS was administered subcutaneously on days 0, 7, and 14. For the other groups of mice, HBx-preS1-T adjuvanted with CpG1826ODN (50 μg) was administered according to the administration schedule shown in Figure 16. Stx-18sCD40L (100 μg) was administered subcutaneously. Blood samples were taken on days 0, 7, 14 and 21. On day 21, animals were euthanized and spleen cells were collected and cultured. The frequency of IFN-γ-secreting spleen cells in the presence and absence of antigenic stimuli (HBx-specific peptide pool, i.e., HBV32aa overlapping 9 peptides) was analyzed by ELISpot assay, respectively. Serum HBx-specific antibody levels were analyzed by ELISA using purified HBx recombinant protein as coating protein.

[0205] FIG. 17 shows the IFN-γ expression in spleen cells stimulated in vitro with HBV 32aa overlapping 9 peptide pools in each animal group. + Immunospots are shown. As a result, spleen cells from animal groups immunized with 3 doses, 2 doses and 1 dose (groups D0-D7-D14, D7-D14 and D14, respectively) all showed a significant increase in the frequency of IFN-γ secreting spleen cells compared to the placebo group. The frequency of IFN-γ secreting spleen cells was positively correlated with the number of immunizations. Group D0-D7-D14 (3 vaccinations) showed the best induction of IFN-γ secreting spleen cells.

[0206] In contrast, HBx-preS1-T Stx A single initial dose of -18sCD40L (group D14) did not clearly induce an HBx-specific antibody response. However, a second immunization moderately increased the antibody levels (group D7-D14), and a third dose further increased the antibody levels, as shown in the animal group D0-D7-D14 (Figure 18). The dose-time-dependent effect in inducing humoral responses is consistent with the effect in inducing cellular immune responses. The fusion protein is effective in inducing IFN-γ production in a dose-time-dependent manner (Figure 17).

[0207] Therefore, the fusion protein HBx-preS1-T Stx -18sCD40L can effectively induce HBx-specific T cell-mediated and HBx-specific humoral immune responses after two immunizations, which can be further enhanced by multiple vaccinations.

[0208] The foregoing description of exemplary embodiments has been presented for purposes of illustration and description only and is not intended to be exhaustive or to limit the invention to the precise form disclosed. All references cited and discussed herein are incorporated by reference in their entirety and to the same extent as if each reference was individually incorporated by reference.

Claims

1. A fusion protein comprising: (a) a CD40 binding domain; (b) an antigen; and (c) a translocation domain positioned between the CD40 binding domain and the antigen; Including, The fusion protein, wherein a furin and / or cathepsin L cleavage site is present between the CD40 binding domain and the translocation domain.

2. The fusion protein of claim 1 , wherein the translocation domain is a Pseudomonas exotoxin A (PE) translocation peptide and the CD40 binding domain is located at the N-terminus of the fusion protein.

3. The fusion protein of claim 1, wherein the translocation domain is a PE translocation peptide consisting of 26-112 amino acid residues in length, and the PE translocation peptide comprises the amino acid sequence of SEQ ID NO:

5.

4. The fusion protein of claim 1 , wherein the translocation domain is a Shiga toxin (Stx) translocation peptide and the antigen is located at the N-terminus of the fusion protein.

5. The fusion protein of claim 1, wherein the translocation domain is a Stx translocation peptide consisting of 8-84 amino acid residues in length, and the Stx translocation peptide comprises the amino acid sequence of SEQ ID NO:

12.

6. The fusion protein of claim 1 , wherein the furin and / or cathepsin L cleavage site allows for removal of the CD40 binding domain from the fusion protein via furin and / or cathepsin L cleavage.

7. The fusion protein of claim 1 , wherein the furin and / or cathepsin L cleavage site comprises the amino acid sequence of SEQ ID NO: 1 or 2.

8. The fusion protein of claim 1, further comprising a peptide linker comprising a furin and / or cathepsin L cleavage site located between the CD40 binding domain and the translocation domain.

9. The fusion protein of claim 1 , wherein the CD40 binding domain is CD40 ligand (CD40L) or a functional fragment thereof.

10. The fusion protein of claim 1, wherein the CD40 binding domain is a CD40 ligand (CD40L) comprising the amino acid sequence of SEQ ID NO: 19, or a functional fragment thereof, and the CD40L or functional fragment thereof has 154-261 amino acid residues in length.

11. the CD40 binding domain is a CD40-specific antibody or binding fragment thereof, or a single chain variable fragment (scFv); The CD40-specific antibody or scFv is Ｈ and V Ｌ Including, (a) The V Ｈ comprises the amino acid sequence of SEQ ID NO: 22, (b) the V Ｌ The fusion protein of claim 1, comprising the amino acid sequence of SEQ ID NO:

23.

12. The CD40 binding domain is a CD40-specific antibody or a binding fragment thereof, the CD40-specific antibody being Ｈ and V Ｌ and V Ｈ V Ｈ CDR1, V Ｈ CDR2 and V Ｈ CDR3, Ｌ V Ｌ CDR1, V Ｌ CDR2 and V Ｌ comprising CDR3, (i) The V Ｈ CDR1, V Ｈ CDR2 and V Ｈ CDR3 comprises the amino acid sequences of SEQ ID NOs: 24, 25 and 26, respectively; (ii) the V Ｌ CDR1, V Ｌ CDR2 and V Ｌ The fusion protein of claim 1, wherein CDR3 comprises the amino acid sequences of SEQ ID NOs: 27, 28 and 29, respectively.

13. 2. The fusion protein of claim 1, wherein the antigen is a tumor antigen and the tumor is selected from the group consisting of breast cancer, colon cancer, rectal cancer, bladder cancer, endometrial cancer, renal cancer, gastric cancer, glioblastoma, hepatocellular carcinoma, cholangiocarcinoma, small cell lung cancer, non-small cell lung cancer (NSCLC), melanoma, ovarian cancer, cervical cancer, pancreatic cancer, prostate cancer, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), non-Hodgkin's lymphoma, and thyroid cancer.

14. The antigens include human papillomavirus (HPV), human immunodeficiency virus-1 (HIV-1), influenza virus, dengue virus, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), severe acute respiratory syndrome-related coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MRSA), and pulmonary circulation virus (PVC). ERS-CoV), Epstein-Barr virus (EBV), Zika virus, rabies virus, smallpox virus, chikungunya virus, West Nile virus, poliovirus, measles virus, rubella virus, hantavirus, Japanese encephalitis virus, coxsackievirus, echovirus, enterovirus, mumps virus, varicella zoster virus (VZV), cercopithecin herpesvirus-1 (CHV-1), yellow fever virus (YFV), Rift Valley fever virus Viruses that may be of concern include: Russa virus, Marburg virus, Ebola virus, Norovirus, Rotavirus, Adenovirus, Sapovirus, Astrovirus, Rickettsia prowazekii, Rickettsia typhi, Orientia tsutsugamushi, Borrelia burgdorferi, Yersinia pestis, Plasmodium vivax, Plasmodium falciparum, Plasmodium ovale, Bacillus anthracis, Clostridium difficile, Clostridium botulinum, Corynebacterium erythroides, The fusion protein of claim 1, which is an antigen of a pathogen selected from the group consisting of Salmonella diphtheriae, Salmonella enterica serovar Typhi, Salmonella enterica serovar Paratyphi A, Shiga toxin-producing Escherichia coli (STEC), Shigella dysenteriae, Shigella dysenteriae, Clostridium flexneri, Clostridium shiga sonnei, Entamoeba histolytica, Vibrio cholerae, Mycobacterium tuberculosis, Neisseria meningitidis, Bordetella pertussis, Haemophilus influenzae type B (HiB), Clostridium tetani, Listeria monocytogenes and Streptococcus pneumoniae.

15. 15. Use of a fusion protein according to any one of claims 1 to 14 in the manufacture of a medicament for treating a tumor and / or a disease caused by a pathogen in a subject by inducing an antigen-specific cellular immune response.

16. The fusion protein of claim 2, further comprising an endoplasmic reticulum (ER) retention sequence located at the C-terminus of the antigen.

17. 5. The fusion protein of claim 2 or 4, further comprising a CD28 activation peptide located between the CD40 binding domain and the Furin and / or cathepsin L cleavage site, wherein the CD28 activation peptide is 28-53 amino acid residues in length and comprises an amino acid sequence selected from SEQ ID NOs: 35, 36 and 37.

18. An expression vector comprising a DNA fragment encoding the fusion protein of claim 1.

19. The fusion protein of claim 2, wherein the PE translocation peptide comprises an amino acid sequence selected from SEQ ID NOs: 5, 6, 7, 8 and 9.

20. The fusion protein of claim 4, wherein the Stx translocation peptide comprises an amino acid sequence selected from SEQ ID NOs: 12, 13, 14, 15 and 16.

Images