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.

What is claimed is: 
     
         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 H  and a V L , wherein:   (a) the V H  comprises the amino acid sequence of SEQ ID NO: 22; and   (b) the V 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 H  and a V L , the V H  comprising V H  CDR1, V H  CDR2 and V H  CDR3; and the V L  comprising V L  CDR1, V L  CDR2 and V L  CDR3, wherein:
 (i) the V H  CDR1, V H  CDR2 and V H  CDR3 comprises the amino acid sequence of SEQ ID NOs: 24, 25 and 26, respectively; and   (ii) the V L  CDR1, V L  CDR2 and V 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.