FUSION PROTEINS FOR IMMUNOTHERAPY AGAINST CANCER AND INFECTIOUS DISEASES

Abstract

Fusion proteins for immunotherapy against cancer and infectious diseases are disclosed. A fusion protein according to the invention comprises a CD40-binding domain; an antigen; and a translocation domain located between the CD40-binding domain and the antigen, in which a furin and/or cathepsin L cleavage site is present in the fusion protein between the CD40-binding domain and the translocation domain. The antigen is an antigen of a pathogen or a tumor antigen. The furin and/or cathepsin L cleavage site permits removal of the CD40-binding domain away from the fusion protein via furin and/or cathepsin L cleavage. Also disclosed are pharmaceutical compositions, expression vectors and use of the fusion proteins of the invention for eliciting an antigen-specific cell-mediated immune response, treating a tumor and/or a disease caused by a pathogen in a subject in need thereof.

Description

REFERENCE TO RELATED APPLICATION

[0001] The present application claims the priority to U.S. Provisional Application Ser. No. 63/020,545, filed May 6, 2020, which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates generally to fusion proteins, and more specifically to fusion proteins for eliciting T cell-mediated immune responses against tumors and infectious diseases.

BACKGROUND OF THE INVENTION

[0003] The adaptive immune system includes both humoral immunity components and cell-mediated immunity components and destroys invading pathogens. The cells that carry out the adaptive immune response are white blood cells known as lymphocytes. B cells and T cells, two different types of lymphocytes, carry out the main activities: antibody responses, and cell-mediated immune response. The adaptive immunity is activated by exposure to pathogens and leads to an enhanced immune response to future encounters with that pathogen. Vaccines induce antigen-specific memory in adaptive immune cells that enables protection against the target pathogen. There is still a need for novel therapeutic vaccines to treat diseases including cancer and infectious diseases caused by pathogens.

SUMMARY OF THE INVENTION

[0004] In one aspect, the 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.

[0005] In another aspect, the invention relates to a DNA fragment encoding a fusion protein according to the invention. The invention also relates to an expressing vector comprising a DNA fragment encoding a fusion protein of the invention.

[0006] Further in another aspect, the invention relates to a pharmaceutical composition comprising a fusion protein of the invention and a pharmaceutical acceptable carrier and/or an adjuvant.

[0007] Yet in another aspect, the invention relates to a method for eliciting an antigen-specific cell-mediated immune response, comprising administering a therapeutically effective amount of the fusion protein of the invention to a subject in need thereof, and thereby eliciting an antigen-specific cell-mediated immune response in the subject in need thereof.

[0008] The invention also relates to a method for treating a tumor in a subject in need thereof, comprising administering to the subject in need thereof a therapeutically effective amount of the fusion protein of the invention, wherein the antigen of the fusion protein is a tumor antigen, and thereby treating the subject in need thereof.

[0009] The invention also relates to a method for treating a disease caused by a pathogen in a subject in need thereof, comprising administering to the subject in need thereof a therapeutically effective amount of the fusion protein of the invention, wherein the antigen of the fusion protein is an antigen of the pathogen, and thereby treating the disease caused by the pathogen.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 is a vector map.

[0011] FIG. 2 is a vector map. MCS, multiple cloning sites.

[0012] FIG. 3 is a vector map.

[0013] FIG. 4 is a vector map.

[0014] FIGS. 5A-E are schematic drawings illustrating various embodiments of the invention.

[0015] FIG. 6 is a graph showing relative cytokine inductions in each animal group.

[0016] FIG. 7 is a graph showing IFN-.gamma..sup.+ immunospots in the splenocytes from each animal group.

[0017] FIG. 8 is a graph showing serum HPV.sub.16 E7-specific antibody level in each animal group.

[0018] FIG. 9 is a graph showing serum HPV.sub.18 E7-specific antibody level in each animal group.

[0019] FIG. 10 shows an immunization schedule and animal groups treated and untreated with the indicated fusion proteins, respectively.

[0020] FIG. 11 is a graph showing tumor size in each animal group treated or untreated with the fusion protein indicated.

[0021] FIG. 12 is a graph showing survival rate in each animal group treated or untreated with the fusion protein indicated.

[0022] FIG. 13 is a graph showing tumor free rate in each animal group treated or untreated with the fusion protein indicated.

[0023] FIG. 14 is a graph showing tumor size in each animal group treated or untreated with the fusion protein 18sCD40L-T.sup.PE-E7 at various doses indicated.

[0024] FIG. 15 is a graph showing tumor size in each animal group treated or untreated with the fusion protein E7-T.sup.Stx-18sCD4L at various doses indicated.

[0025] FIG. 16 shows an immunization scheme (upper panel), animal groups and respective dosing schedules (lower panel) of the fusion protein HBx-preS1-T.sup.Stx-18sCD40L.

[0026] FIG. 17 is a graph showing IFN-.gamma..sup.+ immunospots in the splenocytes from each animal group in FIG. 16.

[0027] FIG. 18 is a graph showing serum HBx-specific antibody level in each animal group in FIG. 16.

DETAILED DESCRIPTION OF THE INVENTION

[0028] 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 the various terms used to describe the invention.

DEFINITION

[0029] An antigen-presenting cell (APC) or accessory cell is a cell that displays antigen complexed with major histocompatibility complexes (MHCs) on their surfaces; this process is known as antigen presentation. T cells may recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

[0030] Antigen-presenting cells fall into two categories: professional and non-professional. Those that express MHC class II molecules along with co-stimulatory 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. The non-professional APCs express MHC class I molecules, which include all nucleated cell types in the body.

[0031] Professional APCs specialize in presenting antigens to T cells. They are very efficient at internalizing antigens, either by phagocytosis, or by receptor-mediated endocytosis, processing the antigen into peptide fragments and then displaying those peptides (bound to a class II MHC molecule) on their membrane. The T cell recognizes and interacts with the antigen-class II MHC molecule complex on the membrane of the antigen-presenting cell. An additional co-stimulatory signal is then produced by the antigen-presenting cell, leading to activation of the T cell. All professional APCs also express MHC class I molecules as well.

[0032] Professional APCs and non-professional APCs use an MHC class I molecule to display endogenous peptides on the cell membrane. These peptides originate within the cell itself, in contrast to the exogenous antigen displayed by professional APCs using MHC class II molecules. Cytotoxic T cells are able to interact with antigens presented by the MHC class I molecule.

[0033] CD40 is a costimulatory protein expressed on antigen-presenting cells (e.g., dendritic cells, macrophages and B cells). The binding of CD40L to CD40 activates antigen-presenting cells and induces a variety of downstream effects. CD40 is a drug target for cancer immunotherapy.

[0034] The term "a CD40-binding domain" refers to a protein that can recognize and binds to CD40. A CD40-binding domain may be selected from one of the following: "CD40 ligand (CD40L) or a functional fragment thereof", "an anti-CD40 antibody or a functional fragment thereof."

[0035] The terms "CD40L", "CD40 ligand" and "CD154" are interchangeable. CD40L binds to CD40 (protein) on antigen-presenting cells (APC), which leads to many effects depending on the target cell type. CD40L plays a central role in co-stimulation and regulation of the immune response via T cell priming and activation of CD40-expressing immune cells. U.S. Pat. No.5,962,406 discloses the nucleotide and amino acid sequence of CD40L.

[0036] The terms "anti-CD40 antibody" and "CD40-specific antibody" are interchangeable.

[0037] When the term "consist substantially of" or "consisting substantially of" is used in describing an amino acid sequence of a polypeptide, it means that the polypeptide may or may not have a starting amino acid "M" (translated from a start codon AUG) at N-terminal as a part of the polypeptide, depending on protein translation requirements. For example, when the antigen HPV.sub.18 E7 protein (SEQ ID NO: 39) fused to another polypeptide (e.g., another antigen), the starting amino acid "M" could be omitted or kept.

[0038] As used herein, "a translocation domain" is a polypeptide having biological activity in translocating an antigen within a fusion protein across an endosomal membrane into the cytosol of the CD40-expressing cell. The translocation domain guides or facilitates the antigen toward class I major histocompatibility complex (MHC-1) pathway (i.e., a cytotoxic T cell pathway) for antigen presentation.

[0039] The term "a Pseudomonas Exotoxin A (PE) translocation peptide (T.sup.PE)" refers to a PE domain II peptide or a functional fragment thereof that has the biological activity in translocation.

[0040] The term "a Shiga toxin (Stx) translocation peptide (T.sup.Stx)" refers to a Stx translocating domain or a functional fragment thereof that has the biological activity in translocation.

[0041] 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 may be a peptide linker comprising said cleavage site that is introduced into the fusion protein, or an intrinsic protease cleavage site present in the translocation domain of the fusion protein.

[0042] The terms "antigen" and "immunogen" are interchangeable. An antigen refers to an antigenic protein, which may be a tumor antigen (an antigen from a cancer or an antigen associated with a cancer), or an antigen of a pathogen (an antigen from a pathogen).

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

[0044] The terms "an antigen of a cancer cell" and "a tumor antigen" are interchangeable.

[0045] The term "a tumor antigen" refers to a tumor-specific antigen and/or a tumor-associated antigen. A tumor-associated antigen may be a protein or polypeptide expressed on the surface of a tumor cell.

[0046] Cluster of Differentiation 28 (CD28) is a T-cell-specific surface glycoprotein. A CD28 receptor is stimulated during the contact of T cells with antigen-presenting cells. Its function is involved in T-cell activation, the induction of cell proliferation and cytokine production and promotion of T-cell survival.

[0047] The term "an effective amount" refers to the amount of an active fusion protein that is required to confer a therapeutic effect on the treated subject. Effective doses will vary, as recognized by those skilled in the art, depending on rout of administration, excipient usage, and the possibility of co-usage with other therapeutic treatment.

[0048] The term "treating", or "treatment" refers to administration of an effective amount of the fusion protein to a subject in need thereof, who has cancer or infection, or a symptom or predisposition toward such a disease, with the purpose of cure, alleviate, relieve, remedy, ameliorate, or prevent the disease, the symptoms of it, or the predisposition towards it. Such a subject can be identified by a health care professional based on results from any suitable diagnostic method.

[0049] By "0 to 12 repeats" or "2 to 6 repeats", it means that all integer unit amounts within the range "0 to 12" or "2 to 6" are specifically disclosed as part of the invention. Thus, 0, 1, 2, 3, 4, . . . 10, 11 and 12" or "2, 3, 4, 5 and 6" units amounts are included as embodiments of this invention.

[0050] 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 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.

[0051] The fusion proteins of the invention can elicit an antigen-specific T cell immune response via MHC class I antigen presentation pathway. They share a common mechanism of action. Using the fusion protein 18sCD40L-T.sup.PE-E7 as an example, the mechanism of action is illustrated below:

[0052] (1) the fusion protein binds to a CD40-expressing cell (e.g., dendritic cell or macrophage) and is internalized via a CD40-mediated endocytosis;

[0053] (2) the fusion protein is cleaved by furin protease and/or cathepsin L protease within the endosome so as to remove the 18sCD40L fragment away from the T.sup.PE-E7 fragment;

[0054] (3) the T.sup.PE-E7 fragment is translocated across the endosomal membrane of the endosome into the cytosol;

[0055] (4) the T.sup.PE-E7 fragment is digested by cytosol proteasome to generate small E7 antigens with epitopes;

[0056] (5) the E7 antigens are delivered to MHC class I pathway for antigen presentation; and (6) a CD8+ T cell specific immune response is induced or enhanced by T-cell recognizing these presented antigens.

[0057] The above mechanism of action is applicable to the fusion protein E7-T.sup.Stx-18sCD40L, in which case the furin and/or cathepsin L protease cleavage removes the E7-T.sup.Stx fragment away from the 18sCD40L fragment. Thus, the E7-T.sup.Stx fragment is translocated across the endosomal membrane of the endosome into the cytosol, digested by cytosol proteasome to generate small E7 antigens with epitopes; the E7 antigens delivered to MHC class I pathway for antigen presentation; and a CD8+ T cell specific immune response is induced or enhanced by T-cell recognizing these presented antigens.

[0058] According to the invention, no furin and/or cathepsin L cleavage site is present in the fusion protein between the antigen and the translocation domain.

[0059] The presence of the furin and/or cathepsin L cleavage site and its location in the fusion protein permits removal of the CD40-binding domain from the fusion protein after furin and/or cathepsin L cleavage.

[0060] In one embodiment, the fusion protein of the invention further comprises a peptide linker, said linker comprising the furin and/or cathepsin L cleavage site present in the fusion protein between the CD40-binding domain and the translocation domain.

[0061] The translocation domain and the antigen are located within the fusion protein in such an orientation and/or relation that permits the translocating domain to translocate the antigen across the membrane of the endosome and enter the cytosol, and then facilitate the antigen toward MHC class I pathway for antigen presentation in the CD40-expressing cell.

[0062] In one embodiment, the translocation domain is derived from a Pseudomonas Exotoxin A (PE). In another embodiment, the translocation domain is derived from a Shiga toxin (Stx).

[0063] In one embodiment, the translocation domain comprises or is a Pseudomonas Exotoxin A (PE) translocation peptide (T.sup.PE), with the proviso that the CD40-binding domain is located at the N-terminal of the fusion protein.

[0064] In another embodiment, the translocation domain comprises or is a Shiga toxin (Stx) translocation peptide (T.sup.Stx), with the proviso that the antigen is located at the N-terminal of the fusion protein.

[0065] In another embodiment, a fusion protein of the invention sequentially comprises: (i) a CD40-binding domain located at the N-terminal of the fusion protein; (ii) a translocation domain comprising a PE translocation peptide (T.sup.PE); and (iii) an antigen located at the C-terminal of the fusion protein; wherein a furin and/or cathepsin L cleavage site is present in the fusion protein between the CD40-binding domain and the translocation domain.

[0066] In another embodiment, the translocation domain is a functional moiety of T.sup.PE and the furin and/or cathepsin L cleavage site is an intrinsic furin cleavage site from PE.

[0067] In another embodiment, a fusion protein of the invention sequentially comprises: (i) a CD40-binding domain located at the N-terminal of the fusion protein; (ii) a peptide linker comprising a furin and/or cathepsin L cleavage site; (iii) a translocation domain comprising a PE translocation peptide (T.sup.PE); and (iv) an antigen of a pathogen or a tumor antigen.

[0068] In another embodiment, a fusion protein of the invention sequentially comprises: (i) an antigen located at the N-terminal of the fusion protein; (ii) a translocation domain comprising a Stx translocation peptide (T.sup.Stx); and (iii) a CD40-binding domain; wherein a furin and/or cathepsin L cleavage site is present in the fusion protein between the CD40-binding domain and the translocation domain.

[0069] In one embodiment, the translocation domain is a functional moiety of T.sup.Stx, and said furin and/or cathepsin L cleavage site is an intrinsic furin cleavage site from Stx.

[0070] Further in another embodiment, a fusion protein of the invention sequentially comprises: (i) an antigen located at the N-terminal of the fusion protein; (ii) a translocation domain comprising a Stx translocation peptide (T.sup.Stx); (iii) a cleavable linker comprising a furin and/or cathepsin L cleavage site; and (iv) a CD40-binding domain.

[0071] In one embodiment, a furin and/or cathepsin L cleavage site comprises, or is, or consists of, the amino acid sequence of SEQ ID NO: 1 or 2.

[0072] In another embodiment, a PE translocation peptide (TPE) is the domain II (amino acid residues 253-364; SEQ ID NO: 9) of Pseudomonas Exotoxin A protein (full-length PE, SEQ ID NO: 4).

[0073] In another embodiment, a PE translocation peptide (T.sup.PE) comprises the minimal functional fragment GWEQLEQCGYPVQRLVALYLAARLSW (SEQ ID NO: 5).

[0074] In another embodiment, a PE translocation peptide (T.sup.PE) consists of 26-112 amino acid residues in length, said the PE translocation peptide comprises a minimal functional fragment of GWEQLEQCGYPVQRLVALYLAARLSW (SEQ ID NO: 5).

[0075] In another embodiment, a PE translocation peptide (T.sup.PE) comprises an amino acid sequence that is at least 90%, 95% or 99% identical to SEQ ID NO: 5, 6, 7, 8 or 9.

[0076] In another embodiment, a PE translocation peptide (TPF) is selected from the group consisting of PE.sub.280-305 (SEQ ID NO: 5), PE.sub.280-313 (SEQ ID NO: NO: 6), PE.sub.268-313 (SEQ ID NO: NO: 7), PE.sub.253-313 (SEQ ID NO: 8), and PE.sub.253-364 (SEQ ID NO: 9; full-length PE domain II).

[0077] In one embodiment, a Stx translocation peptide (T.sup.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 invention, a Stx translocation peptide has translocation function but no cytotoxic effect of subunit A. Sequence identify between Shiga toxin (Stx) subunit A and Slt-I subunit A is 99% and the two proteins has only one amino acid difference.

[0078] In another embodiment, a Stx translocation peptide (T.sup.Stx) consists of 8-84 amino acid residues in length.

[0079] In another embodiment, a Stx translocation peptide (T.sup.Stx) comprises a minimal functional fragment of LNCHHHAS (SEQ ID NO: 12).

[0080] In another embodiment, a Stx translocation peptide (T.sup.Stx) consists of 8-84 amino acid residues in length, said T.sup.Stx comprising a minimal fragment of LNCHHHAS (SEQ ID NO: 12).

[0081] In another embodiment, a Stx translocation peptide (T.sup.Stx) comprises an amino acid sequence that is at least 90%, 95% or 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: l2, 13, 14, 15 and 16.

[0082] In another embodiment, a Stx translocation peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15 and 16.

[0083] In another embodiment, a Stx translocation peptide (T.sup.Stx) is seleted from the group consisting of Stx.sub.240-247 (SEQ ID NO: 12), Stx.sub.240-251 (SEQ ID NO: 13), Stx.sub.211-247 (SEQ ID NO: 14), Stx.sub.211-251 (SEQ ID NO: 15) and Stx.sub.168-251 (SEQ ID NO: 16) of Stx subunit A.

[0084] A CD40-binding domain is a polypeptide having biological activity in binding to CD40 protein on a CD40-expressing cell. A CD40-binding domain permits a fusion protein of the invention to bind to a CD40 receptor on a CD40-expressing cell (e.g., dendritic cell or macrophage).

[0085] In one embodiment, a 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.

[0086] The CD40L, the anti-CD40 antibody, and the respective functional fragments thereof all have biological activity in binding to CD40 protein on a CD40-expressing cell.

[0087] A functional fragment of CD40L is a truncated CD40L with biological activity, substantially lacking transmembrane and cytoplasmic regions of the full-length CD40L.sub.1-261 protein (SEQ ID NO: 17).

[0088] In another embodiment, a CD40L or a functional fragment thereof consists of 154-261 amino acid residues in length.

[0089] In another embodiment, a truncated CD40L with functional activity is selected from the group consisting of CD40L.sub.47-261 (SEQ ID NO: 18) and CD40L.sub.108-261 (named 18sCD40L; SEQ ID NO: 19).

[0090] In another embodiment, a CD40 ligand (CD40L) or a functional fragment thereof consists of 154-261 amino acid residues in length, said functional fragment thereof comprises CD40L.sub.108-261 (SEQ ID NO: 19).

[0091] In another embodiment, a CD40L comprises or consists of an amino acid sequence that is at least 90%, 95% or 99% identical to SEQ ID NO: 17, 18 or 19, said CD40L having biological activity in binding to CD40 protein on a CD40-expressing cell.

[0092] In another embodiment, a CD40 ligand (CD40L) is selected from the group consisting of CD40L.sub.47-261 (SEQ ID NO: 18), CD40L.sub.108-261 (SEQ ID NO: 19; referred to as 18sCD40L) and CD40L.sub.1-261 (SEQ ID NO: 17).

[0093] In another embodiment, a CD40-binding domain is a CD40-specific antibody (or anti-CD40 antibody). A CD40-specific antibody is an antibody specific against CD40 protein. A CD40-specific antibody can bind to CD40 protein on a CD40-expressing cell.

[0094] In one embodiment, the CD40-specific antibody comprises a heavy chain variable domain (V.sub.H) and a light chain variable domain (V.sub.L), wherein the Vu comprises the amino acid sequence of SEQ ID NO: 22; and the V.sub.L comprises the amino acid sequence of SEQ ID NO: 23.

[0095] In another embodiment, the CD40-specific antibody according to the invention is selected from the group consisting of 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), Fab.sub.2, a minibody and a fully antibody.

[0096] In another embodiment, a CD40-binding domain according to the invention is a CD40-specific scFv (anti-CD40 scFv) comprising a heavy chain variable domain (V.sub.H), a light chain variable domain (V.sub.L) and a flexible linker (L) connecting the V.sub.H and the V.sub.L.

[0097] In one embodiment, a CD40-specific scFv comprises the amino acid sequence of SEQ ID NO: 20 or 21.

[0098] In another embodiment, the CD40-binding domain according to the invention is (i) a CD40-specific antibody or a binding fragment thereof, or (ii) a CD40-specific single chain variable fragment (scFv) or a binding fragment thereof; said CD40-specific antibody or said CD40-specific scFv comprising a V.sub.H and a V.sub.L, wherein: (a) the V.sub.H comprises the amino acid sequence of SEQ ID NO: 22; and (b) the V.sub.L comprises the amino acid sequence of SEQ ID NO: 23.

[0099] In another embodiment, the CD40-specific antibody or CD40-specific scFv comprises a V.sub.H and a V.sub.L, the V.sub.H comprising V.sub.H CDR1, V.sub.H CDR2 and V.sub.H CDR3; and the V.sub.L comprising V.sub.L CDR1, V.sub.L CDR2 and V.sub.L CDR3, wherein: (i) the V.sub.H CDR1, V.sub.H CDR2 and V.sub.H CDR3 comprises the amino acid sequence of SEQ ID NOs: 24, 25 and 26, respectively; and (ii) the V.sub.L CDR1, V.sub.L CDR2 and V.sub.L CDR3 comprises the amino acid sequence of SEQ ID NOs: 27, 28 and 29, respectively.

[0100] In another embodiment, the CD40-binding domain is a CD40-specific scFv comprising a V.sub.H and a V.sub.L, wherein: (a) the V.sub.H comprises the amino acid sequence of SEQ ID NO: 22; and (b) the V.sub.L comprises the amino acid sequence of SEQ ID NO: 23.

[0101] In another embodiment, the fusion protein of the invention further comprises an endoplasmic reticulum (ER) retention sequence located at the C-terminal of the antigen, with the proviso that the translocation domain comprises a PE translocation peptide (T.sup.PE).

[0102] In another embodiment, the ER retention sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 30, 31, 32, 33 and 34.

[0103] In another embodiment, the fusion protein of the invention further comprises a CD28-activating peptide located between the CD40-binding domain and the furin and/or cathepsin L cleavage site.

[0104] In another embodiment, the CD28-activating peptide consisting of 28-53 amino acid residues in length.

[0105] In another embodiment, the CD28-activating peptide has a length of 28-53 amino acid residues, said CD28-activating peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 35, 36 and 37.

[0106] In another embodiment, the CD28-activating peptide has a length of 28-53 amino acid residues, said CD28-activating peptide comprising the amino acid sequence of SEQ ID NO: 35.

[0107] In another embodiment, the CD28-activating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 35, 36 and 37.

[0108] In another embodiment, the CD28-activating peptide comprises an amino acid sequence that is at least 90%, 95% or 99% identical to SEQ ID NO: 35, 36 or 37.

[0109] An antigen in the fusion protein of the invention is an antigen of a pathogen or a tumor antigen.

[0110] The pathogen may be selected from the group consisting of 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-associated coronavirus (SARS-CoV), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), Epstein-Barr virus (EBV), Zika Virus, Rabies Virus, Variola virus, Chikungunya Virus, West Nile virus, Poliovirus, Measles virus, Rubella virus, Hantavirus, Japanese encephalitis virus, Coxsackievirus, Echovirus, Enterovirus, Mumps virus, Varicella-zoster virus (VZV), Cercopithecine herpesvirus-1 (CHV-1), Yellow fever virus (YFV), Rift Valley Fever Virus, Lassa virus, Marburg virus, Ebolavirus, 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 Botulinum, Corynebacterium diphtheriae, Salmonella enterica serovar Typhi, Salmonella enterica serovar Paratyphi A, Shiga toxin-producing E. coli (STEC), Shigella dysenteriae, Shigella flexneri, Shigella boydii, Shigella sonnet, Entamoeba histolytica, Vibrio cholerae, Mycobacterium tuberculosis, Neisseria meningitidis, Bordetella pertusis, Haemophilus influenzae type B (HiB), Clostridium tetani, Listeria monocytogenes and Streptococcus pneumoniae.

[0111] 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 particularly, the pathogen is selected from the group consisting of HPV, HBV, HCV and SARS-CoV-2.

[0112] In another embodiment, the antigen is a pathogenic antigen selected from the group consisting of HPV.sub.16 E7 protein, HPV.sub.18 E7 protein, HBV X protein (HBx), HBV preS1 protein, HCV core protein (HCVcore) and SARS-CoV-2 spike protein (CoV2S).

[0113] In another embodiment, said antigen comprises at least one epitope for inducing a desired immune response, preferably containing 1 to 30 epitopes, more preferably containing 1 to 15 epitopes.

[0114] In another embodiment, the antigen is a pathogenic antigen comprising or consisting substantially of an amino acid sequence that is at least 70%, 80%, 90%, 95% or 99% identical to SEQ ID NO: 38, 39, 40, 41, 42 or 43.

[0115] In another embodiment, the antigen is a pathogenic antigen comprising or consisting substantially of an amino acid sequence that is at least 80% identical to SEQ ID NO: 38, 39, 40, 41, 42 or 43.

[0116] 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.

[0117] In another embodiment, the antigen is a tumor antigen. A tumor antigen is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA).

[0118] In one embodiment, the tumor or cancer is selected from the group consisting of breast cancer, colon cancer, rectal cancer, bladder cancer, endometrial cancer, kidney cancer, gastric cancer, glioblastoma, hepatocellular carcinoma, bile duct cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), melanoma, ovarian cancer, cervical cancer, pancreatic cancer, prostate cancer, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), non-Hodgkin's lymphoma, and thyroid cancer.

[0119] In another embodiment, a tumor-associated antigen is selected from the group consisting of SSX2, MAGE-A3, NY-ESO-1, iLRP, WT12-281, RNF43, CEA-NE3, AFP, ALK, Anterior gradient 2 (AGR2), BAGE proteins, .beta.-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EphA2, Fra-1, FOLR1, GAGE proteins (e.g., GAGE-1, -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, HLA/B-raf, HLA/k-ras, HLA/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-.beta., TMPRSS2, Tn, TRP-1, TRP-2, tyrosinase, and uroplakin-3.

[0120] 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.

[0121] In another embodiment, the antigen is a tumor-associated antigen comprising an amino acid sequence that is at least 70%, 80%, 90%, 95% or 99% identical to SEQ ID NO: 44, 45, 46, 47, 48, 49 or 50.

[0122] 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.

[0123] An antigen may be a single antigen or an antigenic fragment thereof, or a fusion antigen comprising at least two antigens fused together. For example, an antigen may be a single antigen of HPV.sub.16 E7 protein or a fusion antigen comprising HPV.sub.16 E7 and HPV.sub.18 E7 proteins. A fusion antigen may or may not have a linker connecting different antigens.

[0124] In another embodiment, the antigen is a fusion antigen having at least one linker connecting different antigens.

[0125] In another embodiment, the antigen is a fusion antigen having a rigid linker, (EAAAAK).sub.n, connecting different antigens, wherein n is an integer from 0-12, preferably from 2-6, more preferably from 3-4. in other words, the rigid linker comprises 0 to 12 repeats, 2 to 6 repeats or 3-4 repeats of the sequence EAAAAK (SEQ ID NO: 56).

[0126] 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 liner comprising 0 to 12 repeats of the amino acid sequence EAAAAK (SEQ ID NO: 56).

[0127] The rigid linker may be (EAAAAK).sub.n, or (SEQ ID NO: 56).sub.n, wherein n is an integer from 0-12, preferably from 2-6, more preferably from 3-4.

[0128] In another embodiment, the rigid linker comprises 2 to 6 repeats or 3-4 repeats of SEQ ID NO: 56.

[0129] In another embodiment, the fusion protein of the invention comprises, or consists substantially of, an amino acid sequence that is at least 90%, 95% or 99% identical to SEQ ID NO: 51, 52, 53, 54 or 55.

[0130] Further in another embodiment, the fusion protein of the invention comprises, or consists substantially of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 51, 52, 53, 54 and 55.

[0131] In another aspect, the invention relates to a DNA fragment encoding a fusion protein of the invention. The invention also relates to an expressing vector comprising a DNA fragment encoding a fusion protein of the invention. The invention further relates to a pharmaceutical composition comprising a fusion protein of the invention and a pharmaceutically acceptable carrier and/or an adjuvant.

[0132] The pharmaceutical composition may be an enteral or a parenteral dosage form, suitable for transdermal, transmucosal, nasopharyngeal, pulmonary or direct injection, or for systemic (e.g., parenteral) or local (e.g., intratumor or intralesional injection) administration. Parenteral injection may be via intravenous, intraperitoneal, intramuscular, subcutaneous or intradermal routes.

[0133] Suitable adjuvants include, but not limited to, a saponin-based adjuvant or a Toll-like receptor (TLR) agonist adjuvant. A saponin-based adjuvant may be GPI-0100, Quil A or QS-21. A TLR agonist adjuvant may be Poly 1:C, monophosphoryl lipid A (MPL) or CpG oligonucleotide (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 adjuvant is a CpG oligonucleotide.

[0134] The pharmaceutical composition may also be administered orally, e.g., in the form of tablets, coated tablets, drages, hard and soft gelatine capsules.

[0135] The dosage of the fusion protein may vary, depending on the disease to be controlled, the age and the individual condition of the patient and the mode of administration. The dosage may be fitted to individual requirements in each particular case so as to obtain a therapeutically effective amount of the fusion protein of the invention to achieve a desired therapeutic response.

[0136] For adult patients, a single dosage of about 0.1 to 50 mg, especially about 0.1 to 5 mg, comes into consideration. Depending on severity of the disease and the precise pharmacokinetic profile, the fusion protein may be administered with one dosage unit per week, bi-week or month, and totally give 1 to 6 dosage units per cycle to satisfy such treatment.

[0137] In another aspect, the invention relates to use of the fusion protein or the pharmaceutical composition of the invention in the manufacture of a medicament for eliciting an antigen-specific T cell immune response, protecting against and/or treating an infectious disease or a tumor in a subject in need thereof.

[0138] Abbreviations: Rap1, Ras-proximate-1 or Ras-related protein 1; CD40, Cluster of differentiation 40; CDR, Complementarity-determining region.

EXAMPLES

Animal Tumor Model

[0139] An HPV.sub.16 E6- and E7-expressing tumor cell line from lung epithelial cells of C57BL/6 mice was used to establish a mouse HPV.sub.16 tumor model for in vivo efficacy assays in the examples 6-8. The tumor cells were grown in RPMI 1640 medium containing FBS (10%) and penicillin/streptomycin/Amphotericin B (50 units/mL) at 37.degree. C., 5% CO2.

SEQ ID NOs. and Components

[0140] Table 1 shows SEQ ID NOs. and corresponding peptides, polypeptides and fusion proteins.

TABLE-US-00001 TABLE 1 SEQ ID No. Component name or sequence (N.fwdarw.C) Length (aa) 1 Cleavable linker 1 4 RX.sup.1X.sup.2R, wherein X.sup.1 and X.sup.2 are any amino acid residue. 2 Cleavable linker 2 6 RX.sup.1RX.sup.2X.sup.3R, wherein X.sup.1 and X.sup.2 are any amino acid residue, and X.sup.3 is K, F or R. 3 Rigid linker 1 (EAAAAK).sub.3 18 4 Full length PE 613 5 PE translocation peptide (PE.sub.280-305, minimal) 26 6 PE translocation peptide (PE.sub.280-313) 34 7 PE translocation peptide (PE.sub.268-313) 46 8 PE translocation peptide (PE.sub.253-313) 61 9 PE translocation peptide (PE.sub.253-364) 112 10 Full length Shiga toxin (Stx) subunit A 293 11 Full length Shiga-like toxin I (Slt-I) subunit A 293 12 Stx translocation peptide (Stx.sub.240-247, minimal) 8 13 Stx translocationpsptide (Stx.sub.240-251) 12 14 Stx translocation peptide (Stx.sub.211-247) 37 15 Stx translocation peptide (Stx.sub.211-251) 41 16 Stx translocation peptide (Stx.sub.168-251) 84 17 Full length CD40 ligand (CD40L.sub.1-261) 261 18 Truncated CD40 ligand (CD40L.sub.47-261) 215 19 Truncated CD40 ligand (CD40L.sub.108-261, also referred to as 154 18sCD40L) 20 Anti-CD40 scFv (V.sub.H-L-V.sub.L) 246 21 Anti-CD40 scFv (V.sub.L-L-V.sub.H) 246 22 V.sub.H of the anti-CD40 scFv 119 23 V.sub.L of the anti-CD40 scFv 112 24 V.sub.H CDR1 GFTFSTYGMH 10 25 V.sub.H CDR2 GKGLEWLSYISGGSSYIFYADSVRGR 26 26 V.sub.H CDR3 CARILRGGSGMDL 13 27 V.sub.L CDR1 CTGSSSNIGAGYNVY 15 28 V.sub.L CDR2 GNINRPS 7 29 V.sub.L CDR3 CAAWDKSISGLV 12 30 ER retention sequence KDEL 4 31 ER retention sequence KKDLRDELKDEL 12 32 ER retention sequence KKDELRDELKDEL 13 33 ER retention sequence KKDELRVELKDEL 13 34 ER retention sequence KDELKDELKDEL 12 35 CD28 consensus sequence 28 T.sup.1D.sup.2I.sup.3Y.sup.4F.sup.5C.sup.6K.sup.7X.sup.8E.sup.9X.sup.10X.- sup.11Y.sup.12P.sup.13P.sup.14P.sup.15Y.sup.16X.sup.17D.sup.18N.sup.19 E.sup.20K.sup.21S.sup.22N.sup.23G.sup.24T.sup.25I.sup.26I.sup.27H.sup.28, wherein X.sup.8 is I or L, X.sup.10 is V, F or A, X.sup.11 is M or L, X.sup.17 is L or I. 36 CD28-activating peptide (minimal) 28 37 CD28-activating peptide 53 38 Antigen HPV.sub.16 E7 protein 98 39 Antigen HPV.sub.18 E7 protein 104 40 Antigen HBV X protein (HBx; full length) 154 41 Antigen HBV preS1 protein 108 42 Antigen HCV core protein (full length) 190 43 Antigen SARS-CoV-2 spike protein 1273 44 Antigen SSX2 187 45 Antigen MAGE-A3 314 46 Antigen NY-ESO-1 180 47 Antigen iLRP 296 48 Antigen WT12-281 279 49 Antigen RNF43 406 50 Antigen CEA-NE3 284 51 Fusion protein CD40L.sub.47-261-T.sup.PE-E7 528 52 Fusion protein 18sCD40L-T.sup.PE-E7 467 53 Fusion protein E7-T.sup.Stx-CD40L.sub.47-261 535 54 Fusion protein E7-T.sup.Stx-18sCD40L 474 55 Fusion protein HBx-preS1-T.sup.Stx-18sCD40L 541 56 Rigid linker EAAAAK 6

[0141] Flow cytometry. Splenocytes were stimulated with an antigenic stimulator for 2 hours at 37.degree. C., followed by treating with 50 .mu.g/mL of Brefeldin A and Monensin at 37.degree. C. for 2 hours. The cells were harvested, washed with PBS containing 0.5% BSA, and stained with APC/Cy7-conjugated anti-CD3 antibody, PerCP/Cy5.5-conjugated anti-CD4 antibody, FITC-conjugated anti-CD8 antibody, PE-conjugated anti-CD44 antibody and APC-conjugated anti-CD62L antibody simultaneously. After wash, the cells were permeabilized, fixed and intracellularly stained with PE-conjugated anti-IFN-.gamma. antibody, PE/Cy7-conjugated anti-IL-2 antibody and eFluor450-conjugated anti-TNF-.alpha. antibody simultaneously. The intracellular cytokine characterization (IFN-.gamma., IL-2 or TNF-.alpha.) of splenocytes with CD8+ or CD4+ memory T cell phenotypes (CD3.sup.+CD44.sup.hiCD62L.sup.lo) were further analyzed by Gallios flow cytometer and Kaluza software.

[0142] Enzyme-linked immunospot (ELISpot) assay. Splenocytes were seeded in triplicate in a pretreated murine IFN-.gamma. capturing 96-well plate (CTL IMMUNOSPOT.RTM.) at a cell density of 2.times.10.sup.5 cells/well in the presence or absence of an antigenic stimulator. The cells were discarded after 24 hours of incubation at 37.degree. C. After wash, the captured IFN-.gamma. was detected by biotin-conjugated anti-murine IFN-.gamma. antibody at room temperature for 2 hours and the IFN-.gamma.-immunospots were developed according to the manufacturer's instructions. The scanning and counting of IFN-.gamma.-immunospots was performed by IMMUNOSPOT.RTM. S5 Micro analyzer (CTL).

[0143] Indirect enzyme-linked immunosorbent assay (ELISA). Collected whole blood samples were left undisturbed at 4.degree. C. for 30-60 minutes followed by centrifugation at 5,000 g for 10 minutes to pellet the clot. The serum samples were stored at -20.degree. C. The purified coating protein for antigen-specific antibody binding was diluted in guanidine coating buffer (2 M guanidine hydrochloride, 500 mM Na.sub.2HPO.sub.4, 25 mM citrate, pH 4.0-4.4) and distributed into 96-well plate at 1 .mu.g/well. After overnight incubation at 4.degree. C., the 96-well plate was blocked with 1% BSA in PBS at 37.degree. C. for 1 hour. The serum samples were thawed, and subsequently 10-fold serial diluted in PBS with 1% BSA. The coated protein was incubated with 100 .mu.l of 1000-fold diluted serum sample at 37.degree. C. for 2 hours. After 4 times washing with phosphate buffered saline TWEEN.RTM.-20 (PBST), the antigen-specific antibodies were detected by horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (at a dilution of 1:10,000, Cat #31430, Thermo Fisher Science) at 37.degree. C. for 30 minutes. Following 4 times of washing with PBST, the HRP-mediated color development was catalyzed in the presence of 100 .mu.L of TMB substrate and quenched by 100 .mu.L of 1 N HCl. The relative titers of antigen-specific antibody in the serum samples were determined by the absorbance at 450 nm.

[0144] Statistical analysis. The significance of all comparisons was calculated by using t-test, and results considered significant when p<0.05.

Example 1

Constructions of Expression Vectors for CD40L.sub.47-261-T.sup.PE-E7 and 18sCD40L-T.sup.PE-E7

[0145] FIGS. 5A-E illustrates various embodiments of the fusion protein according to the invention.

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

[0147] An expression vector for CD40L.sub.47-261-T.sup.PE-E7 (FIG. 1) is constructed as follows: A DNA fragment encoding .sup.HindIIICD40L-Linker-PE.sup.Ncol, XhoI, SalI, comprising the CD40L.sub.47-261, the cleavable linker and the PE translocation peptide (PE.sub.280-305), was PCR synthesized, digested by HindIII/SalI and then ligated into the plasmid pTAC-MAT-Tag-2 having HindIII/XhoI cutting sites to obtain the plasmid P07-His-pNC (FIG. 2). Then, a DNA fragment encoding a fusion antigen HPV.sub.16/18 E7 carrying a His tag was inserted into the plasmid P07-His-pNC (FIG. 2) via restriction enzymes NcoI/XhoI to generate the expression vector for the fusion protein CD40L.sub.47-261-T.sup.PE-E7 (FIG. 1).

[0148] A cleavable linker allows furin and/or cathepsin L protease to cut the fusion protein for releasing the T.sup.PE-E7 fragment from the fusion protein.

[0149] Applying a similar method as described above, any other antigen(s) of interest may be used to replace E7 and be inserted into the plasmid of FIG. 2 to generate an expression vector similar to the plasmid of FIG. 1 for a fusion protein comprising the antigen of interest according to the invention.

[0150] An expression vector for the fusion protein 18sCD40L-T.sup.PE-E7 (SEQ ID NO: 52; FIG. 5B) was constructed using a similar method described above, in which the truncated CD40 ligand: CD40L.sub.47-261 (SEQ ID NO: 18) was replaced by 18sCD40L, another truncated CD40 ligand: CD40L.sub.108-261 (SEQ ID NO: 19).

Example 2

Construction of Expression Vectors for E7-T.sup.Stxx-CD40L.sub.47-261 and E7-T.sup.Stx-18sCD40L

[0151] The fusion protein E7-T.sup.Stx-CD40L.sub.47-261 (SEQ ID NO: 53; FIG. 5C) comprises (a) a fusion antigen HPV.sub.16/18 E7 (comprising HPV.sub.16 E7 protein (SEQ ID NO: 38) and HPV.sub.18 E7 protein (SEQ ID NO: 39)), (b) a Stx translocation peptide of SEQ ID NO: 14 (Stx.sub.211-247), (c) a cleavable linker comprising RX.sup.1X.sup.2R of SEQ ID NO: 1 (wherein X.sup.1 is V, X.sup.2 is A) and (EAAAAK).sub.3 of SEQ ID NO: 3, and (d) a truncated CD40 ligand of SEQ ID NO: 18 (CD40L.sub.47-261).

[0152] An expression vector for E7-T.sup.Stx-CD40L.sub.47-261 (FIG. 3) is constructed as follows:

[0153] A DNA fragment encoding .sup.HindIII, XhoIStx-Linker-CD40L.sup.SalI, comprising the Stx translocation peptide (Stx.sub.211-247), the cleavable linker and the CD40L.sub.47-261, was PCR synthesized, digested by HindIII/SalI, then ligated into plasmid pTAC-MAT-Tag-2 backbone having HindIII/XhoI cutting sites to obtain the plasmid P08(RP)-His-pNC (FIG. 4). Then, another DNA fragment encoding a fusion antigen HPV.sub.16/18 E7 carrying a His tag was inserted into the plasmid P08(RP)-His-pNC (FIG. 4) via restriction enzymes HindIII/XhoI to generate the expression vector E7-T.sup.stx-CD40L.sub.47-261 (FIG. 3).

[0154] The cleavable linker is vital for the fusion protein of the invention because it allows the fusion protein to be cut by furin and/or cathepsin L protease so as to release the E7-T.sup.Stx fragment from the fusion protein. For example, see FIG. 5C.

[0155] Applying a similar method as described above, any other antigen(s) of interest from various pathogens or cancer may replace E7 and be inserted into the plasmid of FIG. 4 to generate an expression vector similar to the plasmid of FIG. 3 for a fusion protein comprising the antigen of interest according to the invention.

[0156] Using a similar method described above, an expression vector for the fusion protein E7-T.sup.Stx-18sCD40L (SEQ ID NO: 54; FIG. 5D) was constructed, in which the truncated CD40 ligand: CD40.sub.47-261 (SEQ ID NO: 18) was replaced by 18sCD40L, another truncated CD40 ligand: CD40L.sub.108-261 (SEQ ID NO: 19).

[0157] For a comparison purpose, we have constructed the fusion protein RAP1-CD28.sub.convPE.sub.t-E7-K3 (referred to as "RAP1-E7" in the present application), which was almost identical to the prior construct disclosed in U.S. Pat. No. 9,481,714 B2, Example 1. The RAP1-CD28.sub.convPE.sub.t-E7-K3 (referred as "RAP1-E7" in the application) comprises a RAP1 domain III, a CD28 sequence, a linker, a PE translocation domain II (PE.sub.268-313), an antigen E7 protein and an endoplasmic reticulum retention sequence. The antigen E7 protein used here is a fusion antigen HPV.sub.16/18 E7, which comprises a HPV.sub.16 E7 protein (SEQ ID NO: 38) and HPV.sub.18 E7 protein (SEQ ID NO: 39), while the antigen E7 protein used in the prior art is HPV.sub.16 E7 protein.

Example 3

Construction of Expression Vectors for HBx-preS1-T.sup.Stx-18sCD40L

[0158] The fusion protein HBx-preS1-T.sup.Stx-18sCD40L (SEQ ID NO: 55; FIG. 5E) comprises (a) a fusion antigen HBx-preS1 comprising a HBx protein of SEQ ID NO: 40 and a HBV preS1 protein of SEQ ID No.41, (b) a Stx translocation peptide of SEQ ID NO: 14 (Stx.sub.211-247), (c) a cleavable linker comprising RX.sup.1X.sup.2R of SEQ ID NO: 1 (wherein X.sup.1 is V, X.sup.2 is A) and (EAAAAK).sub.3 of SEQ ID NO: 3, and (d) a truncated CD40 ligand of SEQ ID NO: 19 (18sCD40L).

[0159] Using a similar method described in Example 2, an expression vector for the fusion protein HBx-preS1-T.sup.Stx-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.

Example 4

Protein Expression

[0160] E. coli BL21 cells harboring the protein expression vector CD40L.sub.47-261-T.sup.PE-E7 were inoculated in ZY media (10 g/L tryptone and 5 g/L yeast extract) containing a selected antibiotic at an appropriate concentration at 37.degree. C. When the culture reached an early log phase, (OD.sub.600=2 to 5), the expression of fusion protein was induced by isopropyl-1-thio-.beta.-D-galactopyranoside (IPTG) (0.5 to 2 mM). Cells were harvested after 4 hours of IPTG induction and disrupted by sonication. The inclusion bodies were isolated and solubilized in solubilisation buffer (6 M guanidine hydrochloride, 20 mM potassium phosphate, 500 mM NaCl, 20 mM imidazole, 1 mM DTT, pH 7.4) for the recovery of overexpressed fusion protein. After purification, the refolding of the fusion protein was performed by dialysis against 20- to 50-fold volume of dialysis buffer (10 mM PBS) at 4.degree. C. overnight. The refolded fusion proteins were subject to SOS-PAGE analyses under reduced (with dithiothreitol; +DTT) and non-reduced (without dithiothreitol; -DTT) conditions to evaluate whether they were properly refolded.

[0161] The following fusion proteins were also expressed and refolded by using a similar method as described above: (1) 18sCD40L-T.sup.PE-E7; (2) E7-T.sup.Stx-CD40L.sub.47-261; (3) E7-T.sup.Stx-18sCD40L; (4) RAP1-E7; (5) CD40L.sub.47-261-T.sup.PE-HBx-preS1; (6) 18sCD40L-T.sup.PE-HBx-preS1; (7) HBx-preS1-T.sup.Stx-CD40L.sub.47-261; (8) HBx-preS1-T.sup.Stx-18sCD40L; (9) CD40L.sub.47-261-T.sup.PE-HCVcore; (10) 18sCD40L-T.sup.PE-HCVcore; (11) HCVcore-T.sup.Stx-CD40L.sub.47-261; (12) HCVcore-T.sup.Stx-18sCD40L; (13) CD40L.sub.47-261-T.sup.PE-CoV2S; (14) 18sCD40L-T.sup.PE-CoV2S; (15) CoV2S-T.sup.Stx-CD40L.sub.47-261; (16) CoV2S-T.sup.Stx-18sCD40L; (17) CD40L.sub.47-261-T.sup.PE-SSX2; (18) 18sCD40L-T.sup.PE-SSX2; (19) SSX2-T.sup.Stx-CD40L.sub.47-261; (20) SSX2-T.sup.Stx-18sCD40L. The fusion proteins CD40L.sub.47-261-T.sup.PE-E7, 18sCD40L-T.sup.PE-E7, E7-T.sup.stx-18sCD40L, RAP1-E7 and HBx-preS1-T.sup.Stx-18sCD40L were further subjected to an immunogenicity analysis or an efficacy analysis in the following experiments.

Example 5

Immunogenicity Analysis of Fusion Proteins

[0162] Female C57BL/6NerlBltw mice (5 to 6-week-old) were randomly divided into 5 groups (n=5): (A) placebo (i.e., PBS); (B) fusion protein CD40L.sub.47-261-T.sup.PE-E7 (100 .mu.g); (C) fusion protein 18sCD40L-T.sup.PE-E7 (100 .mu.g); (D) fusion protein E7-T.sup.Stx-18sCD40L (100 .mu.g); and (E) fusion protein RAP1-E7 (100 .mu.g). The fusion proteins were dialyzed into PBS. CpG1826 (50 .mu.g) was used as an adjuvant to animal groups B to E. Each group received three immunizations subcutaneously (s.c.) at 7 days interval from day 0. Blood samples were collected on day 0, 7 and 14. On day 21, the blood samples were harvested and the splenocytes were resuspended in RPMI 1640 medium containing FBS (10%) and PSA.

[0163] The splenocytes were used to analyze intracellular cytokine induction (IFN-.gamma., IL-2 and TNF-.alpha.) in the CD8.sup.+ and CD4.sup.+ memory T cells in the presence or absence of antigen stimulation. Briefly, splenocytes from each animal group were treated with or without antigen E7 protein (2 .mu.g/mL of HPV.sub.16 E7 peptide pool) and then analyzed by flow cytometry. The degree or the level of the intracellular cytokine induction in each mouse group was presented as relative cytokine induction, which was obtained by normalizing the frequency of cytokine.sup.+/CD8.sup.+ and cytokine.sup.+/CD4.sup.+ splenocytes in the presence of the stimulating antigen E7 to that of the unstimulated (untreated) control.

[0164] The splenocytes were also used to analyze the frequency of IFN-.gamma.-secreting splenocytes in the presence or absence of antigen stimulation (2 .mu.g/mL of HPV.sub.16 E7 peptide pool) by using Enzyme-linked immunospot (ELISpot) assay. The results were presented as IFN-.gamma..sup.+ immunospots per million splenocytes.

[0165] The blood samples were used to analyze the level of serum HPV.sub.16 E7-specific and HPV.sub.18 E7-specific antibody by using ELISA, in which the purified HPV.sub.16 E7 and HPV.sub.18 E7 recombinant proteins were used as coating proteins, respectively.

[0166] FIG. 6 shows cytokine induction results after antigen stimulation of the splenocytes with HPV.sub.16 E7 peptide pool. The relative cytokine induction of IFN-.gamma. and TNF-.alpha., but not IL2, in CD8.sup.+ memory T cells from the animals immunized with the fusion protein CD40L.sub.47-261-T.sup.PE-E7, 18sCD40L-T.sup.PE-E7 or E7-T.sup.Stx-18sCD40L (Groups B-D) all significantly increased as compared to that from the RAP1-E7-treated group (Group E) or the placebo group (Group A). The relative cytokine induction of IL-2 in CD8.sup.+ memory T cells, and the cytokines IFN-.gamma., IL-2 or TNF-.alpha. in CD4.sup.+ memory T cells in the animal groups B-E slightly increased, however, showed no significant difference as compared to placebo group (Group A).

[0167] Nonetheless, it can be concluded that the fusion protein of the invention is superior to the prior art fusion protein in inducing the expression of IFN-.gamma. and TNF-.alpha. in CD8.sup.+ memory T cells in response to the stimulation of the antigen HPV.sub.16 E7.

[0168] FIG. 7 shows IFN-.gamma..sup.+ immunospots in the splenocytes stimulated with the HPV.sub.16 E7 peptide pool in vitro. The frequency of IFN-.gamma.-secreting splenocytes from the animal groups immunized with CD40L.sub.47-261-T.sup.PE-E7, 18sCD40L-T.sup.PE-E7, E7-T.sup.Stx-18sCD40L and RAP1-E7 (Groups B-E), respectively, significantly increased as compared to the placebo group. Particularly, E7-T.sup.Stx-18sCD40L induced significantly higher frequency of IFN-.gamma.-secreting cells than CD40L.sub.47-261-T.sup.PE-E7 (p=0.035).

[0169] The results indicate that the fusion protein of the invention can significantly increase IFN-.gamma.-secreting T cell population upon or after stimulation with the antigenic HPV.sub.16 E7 peptide pool.

[0170] FIG. 8 shows the serum HPV.sub.16 E7-specific antibody levels in the animals immunized with various fusion proteins on day 0, 7 and 14. The HPV.sub.16 E7-specific antibody level started to increase after the second vaccination on day 7, and further rose after the third vaccination on day 14 in animals vaccinated with CD40L.sub.47-261-T.sup.PE-E7, 18sCD40L-T.sup.PE-E7 or E7-T.sup.Stx18sCD40L (Groups B-D respectively). On day 21, the serum HPV.sub.16 E7-specific antibody levels in Groups B-D animals were higher than the placebo and the animal group vaccinated with RAP1-E7 (i.e., RAP1-CD28convPEt-E7-K3).

[0171] The fusion protein RAP1-E7 (RAP1-CD28convPEt-E7-K3) failed to elicit HPV.sub.16 E7-specific antibody level after two vaccinations (on day 0 and 7). It started to induce HPV.sub.16 E7-specific antibody after the third vaccination on day 14, and the serum antibody level was only modest on day 21 as compared to Groups B-D. In contrast, the fusion protein of the invention elicited serum HPV.sub.16 E7-specific antibody level after two shots of the vaccine on day 0 and 7.

[0172] A similar effect in inducing HBx-specific antibody was also observed when animals were vaccinated with RAP1-CD28convPEt-HBx-K3 (referred to as "RAP1-HBx"), using the same regimen and immunization schedule described above. The fusion protein RAP1-HBx was generated by using HBx antigen to replace the E7 antigen in the RAP1-E7 (RAPI-CD28convPEt-E7-K3). The fusion protein RAP1-HBx induced serum HBx-specific antibody level after the third vaccination on day 14, and the serum antibody level on day 21 was only modest as compared to animals vaccinated with the fusion protein of the invention (data not shown).

[0173] FIG. 9 shows the serum HPV.sub.18 E7-specific antibody level in the animals immunized with various fusion proteins on day 0, 7 and 14. The fusion proteins CD40L.sub.47-261-T.sup.PE-E7, 18sCD40L-T.sup.PE-E7 and E7-T.sup.Stx-18sCD40L (Groups B-D, respectively) significantly increased the serum HPV.sub.18 E7-specific antibody level as compared to the placebo group.

[0174] Thus, the fusion protein of the invention is effective in inducing antigen-specific antibodies and the antibody induction occurs after twice vaccinations.

[0175] In summary, the fusion protein of the invention can induce antigen-specific T cell response, increase the expression of proinflammatory cytokines, e.g., IFN-.gamma. and TNF-.alpha., and generate antigen-specific antibody response.

Example 6

In Vivo Efficacy Assay of fusion proteins

[0176] Female C57BL/6NCrlBltw mice (5 to 6-week-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.sub.47-261-T.sup.PE-E7 (25 .mu.g; n=5); Group C, 18sCD40L-T.sup.PE-E7 (25 .mu.g; n=4); Group D, E7-T.sup.Stx-18sCD40L (25 .mu.g; n=5); and Group E, RAP1-E7 (25 .mu.g; n=5). The fusion proteins were dissolved in PBS and CpG1826 (50 .mu.g) was used as an adjuvant in vaccinating animals in Groups B to E. FIG. 10 shows an immunization schedule, fusion proteins and the dosages.

[0177] To challenge mice, tumor cells (1.times.10.sup.5 in 0.1 mL) were injected s.c. into the left flank of each mouse on day 0. Three immunizations were s.c. given on day 7, 14 and 21. The tumor size was determined twice a week by multiplication of caliper measurements based on the modified ellipsoidal formula: Termor volume=1/2 (length.times.width.sup.2). The survival rate and tumor free rate were calculated. Mice with tumor length over 2 cm were considered dead and mice without measurable or palpable tumor masses were considered tumor-free.

[0178] The inoculated tumor developed rapidly in the placebo group, in which two animals died on day 25 and thus the data for the placebo group were shown only until day 21 (FIG. 11). The tumor masses in the Groups C and D animals (immunized with 18sCD40L-T.sup.PE-E7 and E7-T.sup.Stx-18.sub.sCD40L, respectively) were almost completely suppressed at least during the entire experimental period (last day is Day 39). The tumors in Group B and E animals (immunized with CD40L.sub.47-261-T.sup.PE-E7 and RAP1-E7, respectively) were initially well controlled, however, gradually grew after ceasing immunization.

[0179] The results indicate that the fusion protein of the invention, particularly the fusion proteins 18sCD40L-T.sup.PE-E7 and E7-T.sup.Stx-18sCD40L, can effectively suppress tumor growth.

[0180] The survival rate in the animal groups B-E (immunized with CD40L.sub.47-261-T.sup.PE-E7, 18sCD40L-T.sup.PE-E7, E7-T.sup.Stx-18sCD40L and RAP1-E7, respectively) remained 100% on day 35 as compared to the placebo group, which declined to 0% on day 35 (FIG. 12).

[0181] The results indicate that the fusion protein of the invention can effectively maintain the survival rate in the animal tumor model.

[0182] No tumor-free animals could be found in Groups A, B and E animals during the entire experimental period (day 39) (FIG. 13). One animal (25%) in group C and three animals (60%) in group D (immunized with 18sCD40L-T.sup.PE-E7 and E7-T.sup.Stx-18.sub.sCD40L, respectively) were found surviving without measurable or palpable tumors. Notably, in those tumor-free mice the tumor masses were all eliminated soon after completion of three times immunizations with 18sCD40L-T.sup.PE-E7 or E7-T.sup.Stx-18sCD40L.

[0183] The results indicated that the fusion proteins of the invention are more potent than the prior art fusion protein RAP1-E7 in increasing tumor free rate in animals having tumors.

Example 7

In Vivo Efficacy Analysis on Different Doses of 18sCD40L-T.sup.PE-E7

[0184] Female C57BL/6NCrlBl tw mice (4 to 6-week-old) were randomly divided into 5 groups (n=5 per group): (A) placebo (PBS); (B) 18sCD40L-T.sup.PE (100 .mu.g; without the fusion antigen E7); (C) 18sCD40L-T.sup.PE-E7 (100 .mu.g); (D) 18sCD40L-T.sup.PE-E7 (50 .sub.lig); (E) 18sCD40L-T''-E7 (25 .mu.g). The fusion proteins were dissolved in PBS and CpG1826 (50 .mu.g) used as an adjuvant in Groups B to E. Tumor cells (1.times.10.sup.6in 0.1 mL) were injected s.c. into the left flank of each mouse on day 0. Two weeks after the challenge, tumor mice were vaccinated three times s.c. on day 14, 21 and 28.

[0185] The tumor volume was determined. AU the dosages (25 .mu.g, 50 .mu.g or 100 .mu.g) of the fusion protein 18sCD40L-T.sup.PE-E7 showed potent effects in suppressing tumor growth. The inhibition of the tumor size by the fusion protein was seen after the first shot on day 14, sustained through the entire experimental period until the last day of observation on day 34. The placebo and 18sCD40L-T.sup.PE, both lacking the antigen E7, had no effect in suppressing tumor growth (FIG. 14).

Example 8

In Vivo Efficacy Analysis on Different Doses of E7-T.sup.Stx-18sCD40L

[0186] Mice were grouped, challenged with tumor cells, and dosed on day 14, 21, 28 with the fusion protein, and the tumor size measured using a method similar to Example 7, except that Groups B-E mice were vaccinated with (B) T.sup.Stx-18sCD40L (100 .mu.g; without the fusion antigen E7); (C) E7-T.sup.Stx-18sCD40L (100 .mu.g); (D) E7-T.sup.Stx-18sCD40L (50 .mu.g); and (E) E7-T.sup.Stx-18sCD40L (25 .mu.g), respectively. All the dosages (25 .mu.g, 50 .mu.g or 100 .mu.g) of the fusion protein E7-T.sup.Stx-18sCD40L showed potent effects in suppressing tumor growth (FIG. 15). The inhibition of the tumor size by the fusion protein was seen after the first shot, sustained through the entire experimental period until the last day of observation on day 34. The placebo and T.sup.Stx-18sCD40L, both lacking the antigen E7, had no effect in suppressing tumor growth (FIG. 15).

[0187] Thus, the fusion protein of the invention has potent effects in suppressing tumor growth with outstanding therapeutic efficacy.

Example 9

Number of Vaccine Doses: Immunogenicity Analysis of HBx-preS1-T.sup.Stx-18sCD40L

[0188] FIG. 16 shows each animal group's dosing schedule. C57BL/6JNarl female mice (5 weeks old) were randomly divided into four groups (n=5 per group): (1) placebo group; (2) D0-D7-D14 group (three doses, vaccinated on days 0, 7, 14); (3) D7-D14 (two doses, vaccinated on days 7, 14); and (4) D14 group (one dose, vaccinated on day 14). The placebo group received PBS via s.c. on Day 0, 7 and 14. Mice in other groups received HBx-preS1-T.sup.Stx-18sCD40L (100 .mu.g) adjuvanted with CpG1826 ODN (50 .mu.g) via s.c. according to the dosing schedule in FIG. 16. Blood samples were collected on day 0, 7, 14 and 21. On day 21, the animals were sacrificed, splenocytes harvested and cultured. The frequency of IFN-.gamma.-secreting splenocytes in the presence and absence of an antigenic stimulator (a HBx-specific peptide pool, i.e., HBV 32aa overlap 9 peptide) was analyzed by ELISpot assay, respectively. The levels of serum HBx-specific antibodies were assayed by ELISA, in which purified HBx recombinant proteins were used as coating proteins.

[0189] FIG. 17 shows the IFN-.gamma..sup.+ immunospots in the splenocytes stimulated with the HBV 32aa overlap 9 peptide pool in vitro in each animal group. The results indicate that the splenocytes from animal groups immunized with three doses, two doses and one dose (groups D0-D7-D14, D7-D14 and D14, respectively) all show a significant increase in the frequency of IFN-.gamma.-secreting splenocytes as compared to the placebo. The frequency of IFN-.gamma.-secreting splenocytes was positively correlated with the number of immunizations. The group D0-D7-D14 (vaccinated three times) showed the best induction of IFN-.gamma.-secreting splenocytes.

[0190] In contrast, a single priming dose (D14 group) of HBx-preS1-T.sup.Stx-18sCD40L did not apparently induce HBx-specific antibody response. However, the second immunization boosted the antibody level moderately (D7-D14 group) and the third dose further boosted the antibody level even higher as shown in the animal group D0-D7-D14 (FIG. 18). The dosing-number-dependent effect in inducing humoral response is consistent with that in inducing cell-mediated immune responses. The fusion protein is effective in inducing IFN-.gamma. production in a dosing number dependent manner (FIG. 17).

[0191] Thus, the fusion protein HBx-preS1-T.sup.Stx-18scD40L could effectively elicit HBx-specific T cell-mediated immune response and HBx-specific humoral immune response after twice immunizations, which could be further boosted by multiple vaccinations.

[0192] The foregoing description of the exemplary embodiments has been presented only for the purposes of illustration and description and is not intended to be exhaustive to limit the invention to the precise forms disclosed. All references cited and discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference.

Sequence CWU 1

1

5614PRTArtificial SequenceCleavable linker 1MISC_FEATURE(2)..(3)Xaa can be any naturally occurring amino acid 1Arg Xaa Xaa Arg126PRTArtificial SequenceCleavable linker 2MISC_FEATURE(2)..(2)Xaa can be any naturally occurring amino acidMISC_FEATURE(4)..(4)Xaa can be any naturally occurring amino acidMISC_FEATURE(5)..(5)Xaa is K, F or R. 2Arg Xaa Arg Xaa Xaa Arg1 5318PRTArtificial SequenceRigid linker 1 3Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala1 5 10 15Ala Lys4613PRTPseudomonas aeruginosa 4Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val1 5 10 15Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro 20 25 30Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met Val 35 40 45Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala Leu 50 55 60Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu65 70 75 80Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser 85 90 95Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn 100 105 110Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His 115 120 125Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala Lys 130 135 140Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn Glu145 150 155 160Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly Val Ser Val Val Met 165 170 175Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser 180 185 190Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Tyr 195 200 205Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr Trp Glu Gly Lys Ile 210 215 220Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His Asp Leu Asp Ile Lys225 230 235 240Pro Thr Val Ile Ser His Arg Leu His Phe Pro Glu Gly Gly Ser Leu 245 250 255Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro Leu Glu Thr Phe 260 265 270Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly 275 280 285Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser 290 295 300Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly305 310 315 320Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala 325 330 335Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg 340 345 350Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val 355 360 365Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp 370 375 380Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe385 390 395 400Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn 405 410 415Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg 420 425 430Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln 435 440 445Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala 450 455 460Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly465 470 475 480Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly 485 490 495Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr 500 505 510Arg Thr Gly Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu 515 520 525Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly 530 535 540Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu545 550 555 560Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg 565 570 575Asn Val Gly Gly Asp Leu Ala Pro Ser Ser Ile Pro Asp Gln Glu Gln 580 585 590Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Ser 595 600 605Arg Glu Asp Leu Lys 610526PRTArtificial SequencePE translocation peptide (PE280-305, minimal) 5Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val1 5 10 15Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp 20 25634PRTArtificial SequencePE translocation peptide (PE280-313) 6Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val1 5 10 15Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val 20 25 30Ile Arg746PRTArtificial SequencePE translocation peptide (PE268-313) 7Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln1 5 10 15Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu 20 25 30Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg 35 40 45861PRTArtificial SequencePE translocation peptide (PE253-313) 8Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro1 5 10 15Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu 20 25 30Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala 35 40 45Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg 50 55 609112PRTArtificial SequencePE translocation peptide (PE253-364) 9Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro1 5 10 15Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu 20 25 30Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala 35 40 45Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu 50 55 60Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln65 70 75 80Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu 85 90 95Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn 100 105 11010293PRTShigella dysenteriae 10Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp Ser1 5 10 15Leu Asn Val Ile Arg Ser Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser 20 25 30Ser Gly Gly Thr Ser Leu Leu Met Ile Asp Ser Gly Thr Gly Asp Asn 35 40 45Leu Phe Ala Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg Phe 50 55 60Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val Thr Gly65 70 75 80Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe Ser 85 90 95His Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Gly Asp Ser 100 105 110Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly Met 115 120 125Gln Ile Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu Met Ser 130 135 140His Ser Gly Thr Ser Leu Thr Gln Ser Val Ala Arg Ala Met Leu Arg145 150 155 160Phe Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg 165 170 175Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met 180 185 190Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser 195 200 205Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg Ile 210 215 220Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu225 230 235 240Asn Cys His His His Ala Ser Arg Val Ala Arg Met Ala Ser Asp Glu 245 250 255Phe Pro Ser Met Cys Pro Ala Asp Gly Arg Val Arg Gly Ile Thr His 260 265 270Asn Lys Ile Leu Trp Asp Ser Ser Thr Leu Gly Ala Ile Leu Met Arg 275 280 285Arg Thr Ile Ser Ser 29011293PRTEscherichia coli 11Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp Ser1 5 10 15Leu Asn Val Ile Arg Ser Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser 20 25 30Ser Gly Gly Thr Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp Asn 35 40 45Leu Phe Ala Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg Phe 50 55 60Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val Thr Gly65 70 75 80Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe Ser 85 90 95His Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Gly Asp Ser 100 105 110Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly Met 115 120 125Gln Ile Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu Met Ser 130 135 140His Ser Gly Thr Ser Leu Thr Gln Ser Val Ala Arg Ala Met Leu Arg145 150 155 160Phe Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg 165 170 175Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met 180 185 190Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser 195 200 205Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg Ile 210 215 220Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu225 230 235 240Asn Cys His His His Ala Ser Arg Val Ala Arg Met Ala Ser Asp Glu 245 250 255Phe Pro Ser Met Cys Pro Ala Asp Gly Arg Val Arg Gly Ile Thr His 260 265 270Asn Lys Ile Leu Trp Asp Ser Ser Thr Leu Gly Ala Ile Leu Met Arg 275 280 285Arg Thr Ile Ser Ser 290128PRTArtificial SequenceStx translocation peptide (Stx240-247, minimal) 12Leu Asn Cys His His His Ala Ser1 51312PRTArtificial SequenceStx translocation peptide (Stx240-251) 13Leu Asn Cys His His His Ala Ser Arg Val Ala Arg1 5 101437PRTArtificial SequenceStx translocation peptide (Stx211-247) 14Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg Ile Ser Phe1 5 10 15Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu Asn Cys 20 25 30His His His Ala Ser 351541PRTArtificial SequenceStx translocation peptide (Stx211-251) 15Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg Ile Ser Phe1 5 10 15Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu Asn Cys 20 25 30His His His Ala Ser Arg Val Ala Arg 35 401684PRTArtificial SequenceStx translocation peptide (Stx168-251) 16Ala Leu Arg Phe Arg Gln Ile Gln Arg Gly Phe Arg Thr Thr Leu Asp1 5 10 15Asp Leu Ser Gly Arg Ser Tyr Val Met Thr Ala Glu Asp Val Asp Leu 20 25 30Thr Leu Asn Trp Gly Arg Leu Ser Ser Val Leu Pro Asp Tyr His Gly 35 40 45Gln Asp Ser Val Arg Val Gly Arg Ile Ser Phe Gly Ser Ile Asn Ala 50 55 60Ile Leu Gly Ser Val Ala Leu Ile Leu Asn Cys His His His Ala Ser65 70 75 80Arg Val Ala Arg17261PRTHomo sapiens 17Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly1 5 10 15Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu 20 25 30Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg 35 40 45Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val 50 55 60Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser65 70 75 80Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys 85 90 95Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu 100 105 110Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser 115 120 125Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly 130 135 140Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln145 150 155 160Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr 165 170 175Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser 180 185 190Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala 195 200 205Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His 210 215 220Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn225 230 235 240Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe 245 250 255Gly Leu Leu Lys Leu 26018215PRTArtificial SequenceTruncated CD40 ligand (CD40L47-261) 18His Arg Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp1 5 10 15Phe Val Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser 20 25 30Leu Ser Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe 35 40 45Val Lys Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser 50 55 60Phe Glu Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val65 70 75 80Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu 85 90 95Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly 100 105 110Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln 115 120 125Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile 130 135 140Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu145 150 155 160Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser 165 170 175Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe 180 185 190Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr 195 200 205Ser Phe Gly Leu Leu Lys Leu 210 21519154PRTArtificial SequenceTruncated CD40 ligand (CD40L108-261, also referred to as 18sCD40L) 19Glu Asn Ser Phe Glu Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala1 5 10 15Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln 20 25 30Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu 35 40 45Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile 50 55 60Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala65 70 75 80Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg 85 90 95Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly 100 105 110Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala 115 120 125Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr

130 135 140Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu145 15020246PRTArtificial SequenceAnti-CD40 scFv (VH-L-VL) 20Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30Gly Met His Val Trp Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Ser Tyr Ile Ser Gly Gly Ser Ser Tyr Ile Phe Tyr Ala Asp Ser Val 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu Asn Ala Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ile Leu Arg Gly Gly Ser Gly Met Asp Leu Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala 130 135 140Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser145 150 155 160Ser Asn Ile Gly Ala Gly Tyr Asn Val Tyr Trp Tyr Gln Gln Leu Pro 165 170 175Gly Thr Ala Pro Lys Leu Leu Ile Tyr Gly Asn Ile Asn Arg Pro Ser 180 185 190Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser 195 200 205Leu Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys 210 215 220Ala Ala Trp Asp Lys Ser Ile Ser Gly Leu Val Phe Gly Gly Gly Thr225 230 235 240Lys Leu Thr Val Leu Gly 24521246PRTArtificial SequenceAnti-CD40 scFv (VL-L-VH) 21Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30Tyr Asn Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45Leu Ile Tyr Gly Asn Ile Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu65 70 75 80Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Lys Ser 85 90 95Ile Ser Gly Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 115 120 125Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 130 135 140Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr Gly145 150 155 160Met His Val Trp Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Ser 165 170 175Tyr Ile Ser Gly Gly Ser Ser Tyr Ile Phe Tyr Ala Asp Ser Val Arg 180 185 190Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu Asn Ala Leu Tyr Leu 195 200 205Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 210 215 220Arg Ile Leu Arg Gly Gly Ser Gly Met Asp Leu Trp Gly Gln Gly Thr225 230 235 240Leu Val Thr Val Ser Ser 24522119PRTArtificial SequenceVH of the anti-CD40 scFv 22Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30Gly Met His Val Trp Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Ser Tyr Ile Ser Gly Gly Ser Ser Tyr Ile Phe Tyr Ala Asp Ser Val 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu Asn Ala Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ile Leu Arg Gly Gly Ser Gly Met Asp Leu Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11523112PRTArtificial SequenceVL of the anti-CD40 scFv 23Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30Tyr Asn Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45Leu Ile Tyr Gly Asn Ile Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu65 70 75 80Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Lys Ser 85 90 95Ile Ser Gly Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 1102410PRTArtificial SequenceVH CDR1 24Gly Phe Thr Phe Ser Thr Tyr Gly Met His1 5 102526PRTArtificial SequenceVH CDR2 25Gly Lys Gly Leu Glu Trp Leu Ser Tyr Ile Ser Gly Gly Ser Ser Tyr1 5 10 15Ile Phe Tyr Ala Asp Ser Val Arg Gly Arg 20 252613PRTArtificial SequenceVH CDR3 26Cys Ala Arg Ile Leu Arg Gly Gly Ser Gly Met Asp Leu1 5 102715PRTArtificial SequenceVL CDR1 27Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asn Val Tyr1 5 10 15287PRTArtificial SequenceVL CDR2 28Gly Asn Ile Asn Arg Pro Ser1 52912PRTArtificial SequenceVL CDR3 29Cys Ala Ala Trp Asp Lys Ser Ile Ser Gly Leu Val1 5 10304PRTArtificial SequenceER retention sequence 30Lys Asp Glu Leu13112PRTArtificial SequenceER retention sequence 31Lys Lys Asp Leu Arg Asp Glu Leu Lys Asp Glu Leu1 5 103213PRTArtificial SequenceER retention sequence 32Lys Lys Asp Glu Leu Arg Asp Glu Leu Lys Asp Glu Leu1 5 103313PRTArtificial SequenceER retention sequence 33Lys Lys Asp Glu Leu Arg Val Glu Leu Lys Asp Glu Leu1 5 103412PRTArtificial SequenceER retention sequence 34Lys Asp Glu Leu Lys Asp Glu Leu Lys Asp Glu Leu1 5 103528PRTArtificial SequenceCD28 consensus sequenceMISC_FEATURE(8)..(8)Xaa is I or LMISC_FEATURE(10)..(10)Xaa is V, F or AMISC_FEATURE(11)..(11)Xaa is M or LMISC_FEATURE(17)..(17)Xaa is L or I 35Thr Asp Ile Tyr Phe Cys Lys Xaa Glu Xaa Xaa Tyr Pro Pro Pro Tyr1 5 10 15Xaa Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His 20 253628PRTArtificial SequenceCD28-activating peptide (minimal) 36Thr Asp Ile Tyr Phe Cys Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr1 5 10 15Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His 20 253753PRTArtificial SequenceCD28-activating peptide 37Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr Phe Tyr Leu Gln1 5 10 15Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys Lys Ile Glu Val 20 25 30Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile 35 40 45Ile His Val Lys Gly 503898PRTHuman papillomavirus type 16 38Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln1 5 10 15Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser 20 25 30Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp 35 40 45Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr 50 55 60Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu65 70 75 80Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser Gln 85 90 95Lys Pro39104PRTHuman papillomavirus type 18 39His Gly Pro Lys Ala Thr Leu Gln Asp Ile Val Leu His Leu Glu Pro1 5 10 15Gln Asn Glu Ile Pro Val Asp Leu Leu Cys His Glu Gln Leu Ser Asp 20 25 30Ser Glu Glu Glu Asn Asp Glu Ile Asp Gly Val Asn His Gln His Leu 35 40 45Pro Ala Arg Arg Ala Glu Pro Gln Arg His Thr Met Leu Cys Met Cys 50 55 60Cys Lys Cys Glu Ala Arg Ile Lys Leu Val Val Glu Ser Ser Ala Asp65 70 75 80Asp Leu Arg Ala Phe Gln Gln Leu Phe Leu Asn Thr Leu Ser Phe Val 85 90 95Cys Pro Trp Cys Ala Ser Gln Gln 10040154PRTHepatitis B virus 40Met Ala Ala Arg Met Cys Cys Gln Leu Asp Pro Ala Arg Asp Val Leu1 5 10 15Cys Leu Arg Pro Val Gly Ala Glu Ser Arg Gly Arg Pro Leu Pro Gly 20 25 30Pro Leu Gly Ala Leu Pro Pro Ser Ser Ala Ser Ala Val Pro Ala Asp 35 40 45His Gly Ser His Leu Ser Leu Arg Gly Leu Pro Val Cys Ser Phe Ser 50 55 60Ser Ala Gly Pro Cys Ala Leu Arg Phe Thr Ser Ala Arg Arg Met Glu65 70 75 80Thr Thr Val Asn Ala Pro Trp Ser Leu Pro Thr Val Leu His Lys Arg 85 90 95Thr Ile Gly Leu Ser Gly Arg Ser Met Thr Trp Ile Glu Glu Tyr Ile 100 105 110Lys Asp Cys Val Phe Lys Asp Trp Glu Glu Leu Gly Glu Glu Ile Arg 115 120 125Leu Lys Val Phe Val Leu Gly Gly Cys Arg His Lys Leu Val Cys Ser 130 135 140Pro Ala Pro Cys Asn Phe Phe Thr Ser Ala145 15041108PRTHepatitis B virus 41Met Gly Gly Trp Ser Ser Lys Pro Arg Gln Gly Met Gly Thr Asn Leu1 5 10 15Ser Val Pro Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro 20 25 30Ala Phe Gly Ala Asn Ser Asn Asn Pro Asp Trp Asp Phe Asn Pro Asn 35 40 45Lys Asp His Trp Pro Glu Ala Asn Gln Val Gly Ala Gly Ala Phe Gly 50 55 60Pro Gly Phe Thr Pro Pro His Gly Gly Leu Leu Gly Trp Ser Pro Gln65 70 75 80Ala Gln Gly Ile Leu Thr Thr Val Pro Ala Ala Pro Pro Pro Ala Ser 85 90 95Thr Asn Arg Gln Ser Gly Arg Gln Pro Thr Pro Ile 100 10542190PRTHepatitis C virus 42Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn1 5 10 15Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 20 25 30Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala 35 40 45Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro 50 55 60Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly65 70 75 80Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Met Gly Trp Ala Gly Trp 85 90 95Leu Leu Ser Pro Arg Gly Ser Arg Pro Asn Trp Gly Pro Thr Asp Pro 100 105 110Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys 115 120 125Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu 130 135 140Gly Gly Val Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp145 150 155 160Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile 165 170 175Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Thr Pro Ala Ser 180 185 190431273PRTArtificial SequenceAntigen SARS-CoV-2 spike protein 43Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val1 5 10 15Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp65 70 75 80Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr145 150 155 160Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr225 230 235 240Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val305 310 315 320Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe385 390 395 400Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys465 470 475 480Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu545 550 555 560Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser625 630 635 640Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665

670Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala 675 680 685Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser 690 695 700Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile705 710 715 720Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val 725 730 735Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu 740 745 750Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr 755 760 765Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln 770 775 780Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe785 790 795 800Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser 805 810 815Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly 820 825 830Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp 835 840 845Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu 850 855 860Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly865 870 875 880Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile 885 890 895Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr 900 905 910Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn 915 920 925Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala 930 935 940Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn945 950 955 960Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val 965 970 975Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln 980 985 990Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val 995 1000 1005Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn 1010 1015 1020Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys 1025 1030 1035Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 1040 1045 1050Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val 1055 1060 1065Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His 1070 1075 1080Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn 1085 1090 1095Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln 1100 1105 1110Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val 1115 1120 1125Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro 1130 1135 1140Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn 1145 1150 1155His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn 1160 1165 1170Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu 1175 1180 1185Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu 1190 1195 1200Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu 1205 1210 1215Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met 1220 1225 1230Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys 1235 1240 1245Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro 1250 1255 1260Val Leu Lys Gly Val Lys Leu His Tyr Thr 1265 127044187PRTHomo sapiens 44Met Asn Gly Asp Asp Ala Phe Ala Arg Arg Pro Thr Val Gly Ala Gln1 5 10 15Ile Pro Glu Lys Ile Gln Lys Ala Phe Asp Asp Ile Ala Lys Tyr Phe 20 25 30Ser Lys Glu Glu Trp Glu Lys Met Lys Ala Ser Glu Lys Ile Phe Tyr 35 40 45Val Tyr Met Lys Arg Lys Tyr Glu Ala Met Thr Lys Leu Gly Phe Lys 50 55 60Ala Thr Leu Pro Pro Phe Met Cys Asn Lys Arg Ala Glu Asp Phe Gln65 70 75 80Gly Asn Asp Leu Asp Asn Asp Pro Asn Arg Gly Asn Gln Val Glu Arg 85 90 95Pro Gln Met Thr Phe Gly Arg Leu Gln Gly Ile Ser Pro Lys Ile Met 100 105 110Pro Lys Lys Pro Ala Glu Glu Gly Asn Asp Ser Glu Glu Val Pro Glu 115 120 125Ala Ser Gly Pro Gln Asn Asp Gly Lys Glu Leu Cys Pro Pro Gly Lys 130 135 140Pro Thr Thr Ser Glu Lys Ile His Glu Arg Ser Gly Pro Lys Arg Gly145 150 155 160Glu His Ala Trp Thr His Arg Leu Arg Glu Arg Lys Gln Leu Val Ile 165 170 175Tyr Glu Glu Ile Ser Asp Pro Glu Glu Asp Asp 180 18545314PRTHomo sapiens 45Met Pro Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu1 5 10 15Glu Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala 20 25 30Thr Glu Glu Gln Glu Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val 35 40 45Thr Leu Gly Glu Val Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser 50 55 60Pro Gln Gly Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp65 70 75 80Ser Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser 85 90 95Thr Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys 100 105 110Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu 115 120 125Pro Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln 130 135 140Tyr Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu145 150 155 160Val Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr 165 170 175Ile Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp 180 185 190Asn Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile 195 200 205Ile Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu 210 215 220Leu Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly225 230 235 240Asp Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu 245 250 255Glu Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu 260 265 270Trp Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His 275 280 285His Met Val Lys Ile Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu 290 295 300His Glu Trp Val Leu Arg Glu Gly Glu Glu305 31046180PRTHomo sapiens 46Met Gln Ala Glu Gly Arg Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp1 5 10 15Gly Pro Gly Gly Pro Gly Ile Pro Asp Gly Pro Gly Gly Asn Ala Gly 20 25 30Gly Pro Gly Glu Ala Gly Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala 35 40 45Gly Ala Ala Arg Ala Ser Gly Pro Gly Gly Gly Ala Pro Arg Gly Pro 50 55 60His Gly Gly Ala Ala Ser Gly Leu Asn Gly Cys Cys Arg Cys Gly Ala65 70 75 80Arg Gly Pro Glu Ser Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe 85 90 95Ala Thr Pro Met Glu Ala Glu Leu Ala Arg Arg Ser Leu Ala Gln Asp 100 105 110Ala Pro Pro Leu Pro Val Pro Gly Val Leu Leu Lys Glu Phe Thr Val 115 120 125Ser Gly Asn Ile Leu Thr Ile Arg Leu Thr Ala Ala Asp His Arg Gln 130 135 140Leu Gln Leu Ser Ile Ser Ser Cys Leu Gln Gln Leu Ser Leu Leu Met145 150 155 160Trp Ile Thr Gln Cys Phe Leu Pro Val Phe Leu Ala Gln Pro Pro Ser 165 170 175Gly Gln Arg Arg 18047296PRTHomo sapiens 47Phe Ser Gly Ala Leu Asp Val Leu Gln Met Lys Glu Glu Asp Val Leu1 5 10 15Lys Phe Leu Ala Ala Gly Thr His Leu Gly Gly Thr Asn Leu Asp Phe 20 25 30Gln Met Glu Gln Tyr Ile Tyr Lys Arg Lys Ser Asp Gly Ile Tyr Ile 35 40 45Ile Asn Leu Lys Arg Thr Trp Glu Lys Leu Leu Leu Ala Ala Arg Ala 50 55 60Ile Val Ala Ile Glu Asn Pro Ala Asp Val Ser Val Ile Ser Ser Arg65 70 75 80Asn Thr Gly Gln Arg Ala Val Leu Lys Phe Ala Ala Ala Thr Gly Ala 85 90 95Thr Pro Ile Ala Gly Arg Phe Thr Pro Gly Thr Phe Thr Asn Gln Ile 100 105 110Gln Ala Ala Phe Arg Glu Pro Arg Leu Leu Val Val Thr Asp Pro Arg 115 120 125Ala Asp His Gln Pro Leu Thr Glu Ala Ser Tyr Val Asn Leu Pro Thr 130 135 140Ile Ala Leu Cys Asn Thr Asp Ser Pro Leu Arg Tyr Val Asp Ile Ala145 150 155 160Ile Pro Cys Asn Asn Lys Gly Ala Ala His Ser Val Gly Leu Met Trp 165 170 175Trp Met Leu Ala Arg Glu Val Leu Arg Met Arg Gly Thr Ile Ser Arg 180 185 190Glu His Pro Trp Glu Val Met Pro Asp Leu Tyr Phe Tyr Arg Asp Pro 195 200 205Glu Glu Ile Glu Lys Glu Glu Gln Ala Ala Ala Glu Lys Ala Val Thr 210 215 220Lys Glu Glu Phe Gln Gly Glu Trp Thr Ala Pro Ala Pro Glu Phe Thr225 230 235 240Ala Thr Gln Pro Glu Val Ala Asp Trp Ser Glu Gly Val Gln Val Pro 245 250 255Ser Val Pro Ile Gln Gln Phe Pro Thr Glu Asp Trp Ser Ala Gln Pro 260 265 270Ala Thr Glu Asp Trp Ser Ala Ala Pro Thr Ala Gln Ala Thr Glu Trp 275 280 285Val Gly Ala Thr Thr Asp Trp Ser 290 29548279PRTHomo sapiens 48Gly Ser Asp Val Arg Asp Leu Asn Ala Leu Leu Pro Ala Val Pro Ser1 5 10 15Leu Gly Gly Gly Gly Gly Cys Ala Leu Pro Val Ser Gly Ala Ala Gln 20 25 30Trp Ala Pro Val Leu Asp Phe Ala Pro Pro Gly Ala Ser Ala Tyr Gly 35 40 45Ser Leu Gly Gly Pro Ala Pro Pro Pro Ala Pro Pro Pro Pro Pro Pro 50 55 60Pro Pro Pro His Ser Phe Ile Lys Gln Glu Pro Ser Trp Gly Gly Ala65 70 75 80Glu Pro His Glu Glu Gln Cys Leu Ser Ala Phe Thr Val His Phe Ser 85 90 95Gly Gln Phe Thr Gly Thr Ala Gly Ala Cys Arg Tyr Gly Pro Phe Gly 100 105 110Pro Pro Pro Pro Ser Gln Ala Ser Ser Gly Gln Ala Arg Met Phe Pro 115 120 125Asn Ala Pro Tyr Leu Pro Ser Cys Leu Glu Ser Gln Pro Ala Ile Arg 130 135 140Asn Gln Gly Tyr Ser Thr Val Thr Phe Asp Gly Thr Pro Ser Tyr Gly145 150 155 160His Thr Pro Ser His His Ala Ala Gln Phe Pro Asn His Ser Phe Lys 165 170 175His Glu Asp Pro Met Gly Gln Gln Gly Ser Leu Gly Glu Gln Gln Tyr 180 185 190Ser Val Pro Pro Pro Val Tyr Gly Cys His Thr Pro Thr Asp Ser Cys 195 200 205Thr Gly Ser Gln Ala Leu Leu Leu Arg Thr Pro Tyr Ser Ser Asp Asn 210 215 220Leu Tyr Gln Met Thr Ser Gln Leu Glu Cys Met Thr Trp Asn Gln Met225 230 235 240Asn Leu Gly Ala Thr Leu Lys Gly Val Ala Ala Gly Ser Ser Ser Ser 245 250 255Val Lys Trp Thr Glu Gly Gln Ser Asn His Ser Thr Gly Tyr Glu Ser 260 265 270Asp Asn His Thr Thr Pro Ile 27549406PRTHomo sapiens 49Ser Gly Gly His Gln Leu Gln Leu Ala Ala Leu Trp Pro Trp Leu Leu1 5 10 15Met Ala Thr Leu Gln Ala Gly Phe Gly Arg Thr Gly Leu Val Leu Ala 20 25 30Ala Ala Val Glu Ser Glu Arg Ser Ala Glu Gln Lys Ala Ile Ile Arg 35 40 45Val Ile Pro Leu Lys Met Asp Pro Thr Gly Lys Leu Asn Leu Thr Leu 50 55 60Glu Gly Val Phe Ala Gly Val Ala Glu Ile Thr Pro Ala Glu Gly Lys65 70 75 80Leu Met Gln Ser His Pro Leu Tyr Leu Cys Asn Ala Ser Asp Asp Asp 85 90 95Asn Leu Glu Pro Gly Phe Ile Ser Ile Val Lys Leu Glu Ser Pro Arg 100 105 110Arg Ala Pro Ala His Pro Leu Ile Cys Gly Pro Pro Gly Leu Asp Lys 115 120 125Arg Leu Leu Pro Glu Thr Pro Gly Pro Cys Tyr Ser Asn Ser Gln Pro 130 135 140Val Trp Leu Cys Leu Thr Pro Arg Gln Pro Leu Glu Pro His Pro Pro145 150 155 160Gly Glu Gly Pro Ser Glu Trp Ser Ser Asp Thr Ala Glu Gly Arg Pro 165 170 175Cys Pro Tyr Pro His Cys Gln Val Leu Ser Ala Gln Pro Gly Ser Glu 180 185 190Glu Glu Leu Glu Glu Leu Cys Glu Gln Ala Val Ser Gly Gly His Gln 195 200 205Leu Gln Leu Ala Ala Leu Trp Pro Trp Leu Leu Met Ala Thr Leu Gln 210 215 220Ala Gly Phe Gly Arg Thr Gly Leu Val Leu Ala Ala Ala Val Glu Ser225 230 235 240Glu Arg Ser Ala Glu Gln Lys Ala Ile Ile Arg Val Ile Pro Leu Lys 245 250 255Met Asp Pro Thr Gly Lys Leu Asn Leu Thr Leu Glu Gly Val Phe Ala 260 265 270Gly Val Ala Glu Ile Thr Pro Ala Glu Gly Lys Leu Met Gln Ser His 275 280 285Pro Leu Tyr Leu Cys Asn Ala Ser Asp Asp Asp Asn Leu Glu Pro Gly 290 295 300Phe Ile Ser Ile Val Lys Leu Glu Ser Pro Arg Arg Ala Pro Ala His305 310 315 320Pro Leu Ile Cys Gly Pro Pro Gly Leu Asp Lys Arg Leu Leu Pro Glu 325 330 335Thr Pro Gly Pro Cys Tyr Ser Asn Ser Gln Pro Val Trp Leu Cys Leu 340 345 350Thr Pro Arg Gln Pro Leu Glu Pro His Pro Pro Gly Glu Gly Pro Ser 355 360 365Glu Trp Ser Ser Asp Thr Ala Glu Gly Arg Pro Cys Pro Tyr Pro His 370 375 380Cys Gln Val Leu Ser Ala Gln Pro Gly Ser Glu Glu Glu Leu Glu Glu385 390 395 400Leu Cys Glu Gln Ala Val 40550284PRTHomo sapiens 50Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly Lys Glu1 5 10 15Val Leu Leu Leu Val His Asn Leu Pro Gln His Leu Phe Gly Tyr Ser 20 25 30Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln Ile Ile Gly Tyr 35 40 45Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser Gly Arg 50 55 60Glu Ile Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Ile Ile Gln65 70 75 80Asn Asp Thr Gly Phe Tyr Thr Leu His Val Ile Lys Ser Asp Leu Val 85 90 95Asn Glu Glu Ala Thr Gly Gln Phe Arg Val Tyr Pro Glu Leu Pro Lys 100 105 110Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro Val Glu Asp Lys Asp Ala 115 120 125Val Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Ala Thr Tyr Leu Trp 130 135 140Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln Leu Ser145 150 155 160Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn Val Thr Arg Asn Asp Thr 165 170 175Ala Ser Tyr Lys Cys Glu Thr Gln Asn Pro Val Ser Ala Arg Arg Ser 180 185

190Asp Ser Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Thr Pro Ile Ile 195 200 205Ser Pro Pro Asp Ser Ser Tyr Leu Ser Gly Ala Asn Leu Asn Leu Ser 210 215 220Cys His Ser Ala Ser Asn Pro Ser Pro Gln Tyr Ser Trp Phe Val Asn225 230 235 240Gly Thr Phe Gln Gln His Thr Gln Val Leu Leu Ile Ala Lys Ile Gln 245 250 255Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe Val Ser Asn Leu Ala Thr 260 265 270Gly Arg Asn Asn Ser Ile Val Lys Ser Ile Thr Val 275 28051528PRTArtificial SequenceFusion protein CD40L47-261-TPE-E7 51Met His Arg Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu1 5 10 15Asp Phe Val Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg 20 25 30Ser Leu Ser Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly 35 40 45Phe Val Lys Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn 50 55 60Ser Phe Glu Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His65 70 75 80Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala 85 90 95Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn 100 105 110Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala 115 120 125Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe 130 135 140Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu145 150 155 160Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln 165 170 175Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val 180 185 190Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe 195 200 205Thr Ser Phe Gly Leu Leu Lys Leu Glu Phe Gly Ser Glu Leu Glu Ala 210 215 220Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys225 230 235 240Arg Ala Arg Tyr Lys Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr 245 250 255Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp 260 265 270Thr Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val 275 280 285Pro Arg Gly Ser His Met His Gly Asp Thr Pro Thr Leu His Glu Tyr 290 295 300Met Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln305 310 315 320Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly 325 330 335Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys 340 345 350Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His Val Asp 355 360 365Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys 370 375 380Pro Ile Cys Ser Gln Lys Pro Ala Glu Ala Ala Ala Ala Lys Glu Ala385 390 395 400Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys 405 410 415Ala His Gly Pro Lys Ala Thr Leu Gln Asp Ile Val Leu His Leu Glu 420 425 430Pro Gln Asn Glu Ile Pro Val Asp Leu Leu Cys His Glu Gln Leu Ser 435 440 445Asp Ser Glu Glu Glu Asn Asp Glu Ile Asp Gly Val Asn His Gln His 450 455 460Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg His Thr Met Leu Cys Met465 470 475 480Cys Cys Lys Cys Glu Ala Arg Ile Lys Leu Val Val Glu Ser Ser Ala 485 490 495Asp Asp Leu Arg Ala Phe Gln Gln Leu Phe Leu Asn Thr Leu Ser Phe 500 505 510Val Cys Pro Trp Cys Ala Ser Gln Gln Thr Arg Ala Lys Asp Glu Leu 515 520 52552467PRTArtificial SequenceFusion protein 18sCD40L-TPE-E7 52Met Glu Asn Ser Phe Glu Met Gln Lys Gly Asp Gln Asn Pro Gln Ile1 5 10 15Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu 20 25 30Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr 35 40 45Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr 50 55 60Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln65 70 75 80Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu 85 90 95Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys 100 105 110Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly 115 120 125Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly 130 135 140Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Glu Phe Gly Ser Glu145 150 155 160Leu Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala 165 170 175Ala Ala Lys Arg Ala Arg Tyr Lys Arg Gly Trp Glu Gln Leu Glu Gln 180 185 190Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg 195 200 205Leu Ser Trp Thr Met Gly Ser Ser His His His His His His Ser Ser 210 215 220Gly Leu Val Pro Arg Gly Ser His Met His Gly Asp Thr Pro Thr Leu225 230 235 240His Glu Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu Tyr Cys 245 250 255Tyr Glu Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly 260 265 270Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr 275 280 285Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr 290 295 300His Val Asp Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr Leu Gly305 310 315 320Ile Val Cys Pro Ile Cys Ser Gln Lys Pro Ala Glu Ala Ala Ala Ala 325 330 335Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala 340 345 350Ala Ala Lys Ala His Gly Pro Lys Ala Thr Leu Gln Asp Ile Val Leu 355 360 365His Leu Glu Pro Gln Asn Glu Ile Pro Val Asp Leu Leu Cys His Glu 370 375 380Gln Leu Ser Asp Ser Glu Glu Glu Asn Asp Glu Ile Asp Gly Val Asn385 390 395 400His Gln His Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg His Thr Met 405 410 415Leu Cys Met Cys Cys Lys Cys Glu Ala Arg Ile Lys Leu Val Val Glu 420 425 430Ser Ser Ala Asp Asp Leu Arg Ala Phe Gln Gln Leu Phe Leu Asn Thr 435 440 445Leu Ser Phe Val Cys Pro Trp Cys Ala Ser Gln Gln Thr Arg Ala Lys 450 455 460Asp Glu Leu46553535PRTArtificial SequenceFusion protein E7-TStx-CD40L47-261 53Met Lys Leu Thr Met Gly Ser Ser His His His His His His Ser Ser1 5 10 15Gly Leu Val Pro Arg Gly Ser His Met His Gly Asp Thr Pro Thr Leu 20 25 30His Glu Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu Tyr Cys 35 40 45Tyr Glu Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly 50 55 60Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr65 70 75 80Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr 85 90 95His Val Asp Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr Leu Gly 100 105 110Ile Val Cys Pro Ile Cys Ser Gln Lys Pro Ala Glu Ala Ala Ala Ala 115 120 125Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala 130 135 140Ala Ala Lys Ala His Gly Pro Lys Ala Thr Leu Gln Asp Ile Val Leu145 150 155 160His Leu Glu Pro Gln Asn Glu Ile Pro Val Asp Leu Leu Cys His Glu 165 170 175Gln Leu Ser Asp Ser Glu Glu Glu Asn Asp Glu Ile Asp Gly Val Asn 180 185 190His Gln His Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg His Thr Met 195 200 205Leu Cys Met Cys Cys Lys Cys Glu Ala Arg Ile Lys Leu Val Val Glu 210 215 220Ser Ser Ala Asp Asp Leu Arg Ala Phe Gln Gln Leu Phe Leu Asn Thr225 230 235 240Leu Ser Phe Val Cys Pro Trp Cys Ala Ser Gln Gln Thr Arg Ala Pro 245 250 255Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg Ile Ser Phe Gly 260 265 270Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu Asn Cys His 275 280 285His His Ala Ser Arg Val Ala Arg Glu Phe Gly Ser Glu Leu Glu Ala 290 295 300Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys305 310 315 320His Arg Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp 325 330 335Phe Val Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser 340 345 350Leu Ser Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe 355 360 365Val Lys Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser 370 375 380Phe Glu Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val385 390 395 400Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu 405 410 415Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly 420 425 430Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln 435 440 445Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile 450 455 460Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu465 470 475 480Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser 485 490 495Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe 500 505 510Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr 515 520 525Ser Phe Gly Leu Leu Lys Leu 530 53554474PRTArtificial SequenceFusion protein E7-TStx-18sCD40L 54Met Lys Leu Thr Met Gly Ser Ser His His His His His His Ser Ser1 5 10 15Gly Leu Val Pro Arg Gly Ser His Met His Gly Asp Thr Pro Thr Leu 20 25 30His Glu Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu Tyr Cys 35 40 45Tyr Glu Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly 50 55 60Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr65 70 75 80Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr 85 90 95His Val Asp Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr Leu Gly 100 105 110Ile Val Cys Pro Ile Cys Ser Gln Lys Pro Ala Glu Ala Ala Ala Ala 115 120 125Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala 130 135 140Ala Ala Lys Ala His Gly Pro Lys Ala Thr Leu Gln Asp Ile Val Leu145 150 155 160His Leu Glu Pro Gln Asn Glu Ile Pro Val Asp Leu Leu Cys His Glu 165 170 175Gln Leu Ser Asp Ser Glu Glu Glu Asn Asp Glu Ile Asp Gly Val Asn 180 185 190His Gln His Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg His Thr Met 195 200 205Leu Cys Met Cys Cys Lys Cys Glu Ala Arg Ile Lys Leu Val Val Glu 210 215 220Ser Ser Ala Asp Asp Leu Arg Ala Phe Gln Gln Leu Phe Leu Asn Thr225 230 235 240Leu Ser Phe Val Cys Pro Trp Cys Ala Ser Gln Gln Thr Arg Ala Pro 245 250 255Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg Ile Ser Phe Gly 260 265 270Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu Asn Cys His 275 280 285His His Ala Ser Arg Val Ala Arg Glu Phe Gly Ser Glu Leu Glu Ala 290 295 300Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys305 310 315 320Glu Asn Ser Phe Glu Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala 325 330 335Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln 340 345 350Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu 355 360 365Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile 370 375 380Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala385 390 395 400Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg 405 410 415Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly 420 425 430Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala 435 440 445Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr 450 455 460Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu465 47055541PRTArtificial SequenceFusion protein HBx-preS1-TStx-18sCD40L 55Met Lys Leu Thr Met Gly Ser Ser His His His His His His Ser Ser1 5 10 15Gly Leu Val Pro Arg Gly Ser His Met Ala Ala Arg Met Cys Cys Gln 20 25 30Leu Asp Pro Ala Arg Asp Val Leu Cys Leu Arg Pro Val Gly Ala Glu 35 40 45Ser Arg Gly Arg Pro Leu Pro Gly Pro Leu Gly Ala Leu Pro Pro Ser 50 55 60Ser Ala Ser Ala Val Pro Ala Asp His Gly Ser His Leu Ser Leu Arg65 70 75 80Gly Leu Pro Val Cys Ser Phe Ser Ser Ala Gly Pro Cys Ala Leu Arg 85 90 95Phe Thr Ser Ala Arg Arg Met Glu Thr Thr Val Asn Ala Pro Trp Ser 100 105 110Leu Pro Thr Val Leu His Lys Arg Thr Ile Gly Leu Ser Gly Arg Ser 115 120 125Met Thr Trp Ile Glu Glu Tyr Ile Lys Asp Cys Val Phe Lys Asp Trp 130 135 140Glu Glu Leu Gly Glu Glu Ile Arg Leu Lys Val Phe Val Leu Gly Gly145 150 155 160Cys Arg His Lys Leu Val Cys Ser Pro Ala Pro Cys Asn Phe Phe Thr 165 170 175Ser Ala Arg Gly Arg Ala Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala 180 185 190Ala Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Ala Glu 195 200 205Phe Met Gly Gly Trp Ser Ser Lys Pro Arg Gln Gly Met Gly Thr Asn 210 215 220Leu Ser Val Pro Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp225 230 235 240Pro Ala Phe Gly Ala Asn Ser Asn Asn Pro Asp Trp Asp Phe Asn Pro 245 250 255Asn Lys Asp His Trp Pro Glu Ala Asn Gln Val Gly Ala Gly Ala Phe 260 265 270Gly Pro Gly Phe Thr Pro Pro His Gly Gly Leu Leu Gly Trp Ser Pro 275 280 285Gln Ala Gln Gly Ile Leu Thr Thr Val Pro Ala Ala Pro Pro Pro Ala 290 295 300Ser Thr Asn Arg Gln Ser Gly Arg Gln Pro Thr Pro Ile Glu Leu Thr305 310 315 320Arg Ala Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg Ile 325 330

335Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu 340 345 350Asn Cys His His His Ala Ser Arg Val Ala Arg Glu Phe Gly Ser Glu 355 360 365Leu Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala 370 375 380Ala Ala Lys Glu Asn Ser Phe Glu Met Gln Lys Gly Asp Gln Asn Pro385 390 395 400Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser 405 410 415Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu 420 425 430Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu 435 440 445Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser 450 455 460Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg465 470 475 480Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys 485 490 495Pro Cys Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln 500 505 510Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser 515 520 525His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu 530 535 540566PRTArtificial SequenceRigid linker 1 with n=1 56Glu Ala Ala Ala Ala Lys1 5

Claims

1. 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.

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-terminal 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, said PE translocation peptide comprising 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-terminal 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, said Stx translocation peptide comprising 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 permits removal of the CD40-binding domain away 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 the 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 CD40 ligand (CD40L) or a functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 19, the CD40L or the functional fragment thereof having 154-261 amino acid residues in length.

11. The fusion protein of claim 1, wherein the CD40-binding domain is a CD40-specific antibody or a binding fragment thereof, or a single chain variable fragment (scFv), said CD40-specific antibody or scFv comprising a V.sub.H and a V.sub.L, wherein: (a) the V.sub.H comprises the amino acid sequence of SEQ ID NO: 22; and (b) the V.sub.L comprises the amino acid sequence of SEQ ID NO: 23.

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

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

14. The fusion protein of claim 1, wherein the antigen is an antigen of a pathogen selected from the group consisting of 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-associated coronavirus (SARS-CoV), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), Epstein-Barr virus (EBV), Zika Virus, Rabies Virus, Variola virus, Chikungunya Virus, West Nile virus, Poliovirus, Measles virus, Rubella virus, Hantavirus, Japanese encephalitis virus, Coxsackievirus, Echovirus, Enterovirus, Mumps virus, Varicella-zoster virus (VZV), Cercopithecine herpesvirus-1 (CHV-1), Yellow fever virus (YFV), Rift Valley Fever Virus, Lassa virus, Marburg virus, Ebolavirus, 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 Botulinum, Corynebcicterium diphtheriae, Salmonella enterica serovar Typhi, Salmonella enterica serovar Paratyphi A, Shiga toxin-producing E. coli (STEC), Shigella dysenteriae, Shigella flexneri, Shigella boydii, Shigella sonnei, Entamoeba histolytica, Vibrio cholerae, Mycobacterium tuberculosis, Neisseria meningitidis, Bordetella pertusis, Haemophilus influenzae type B (HiB), Clostridium letani, Listeria monocytogenes and Streptococcus pneumoniae.

15. A method for eliciting an antigen-specific cell-mediated immune response, comprising: administering a therapeutically effective amount of the fusion protein of claim 1 to a subject in need thereof, and thereby eliciting an antigen-specific cell-mediated immune response in the subject in need thereof.

16. A method for treating a tumor in a subject in need thereof, comprising: administering to the subject in need thereof a therapeutically effective amount of the fusion protein of claim 1, wherein the antigen of the fusion protein is a tumor antigen, and thereby treating the subject in need thereof.

17. A method for treating a disease caused by a pathogen in a subject in need thereof, comprising: administering to the subject in need thereof a therapeutically effective amount of the fusion protein of claim 1, wherein the antigen of the fusion protein is an antigen of the pathogen, and thereby treating the disease caused by the pathogen.

18. The fusion protein of claim 2, further comprising a CD28-activating peptide located between the CD40-binding domain and the furin and/or cathepsin L cleavage site, wherein the CD28-activating peptide has a length of 28-53 amino acid residues and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 35, 36 and 37.

19. The fusion protein of claim 2, wherein the PE translocation peptide comprises an amino acid sequence selected from the group consisting of 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 the group consisting of SEQ ID NOs: 12, 13, 14, 15 and 16.